EPA/600/R-04/126E September 29, 2009 

Standardized Analytical Methods

for Environmental Restoration 

Following Homeland Security Events 

REVISION 5.0 

SCIENCE 

Office of Research and Development

National Homeland Security Research Center 

EPA/600/R-04/126E| September 2009 | www.epa.gov/sam 

Standardized Analytical Methods 

for Environmental Restoration 

Following Homeland Security 

Events – Revision 5.0 

UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

Cincinnati, OH 45268 

Office of Research and Development 

National Homeland Security Research Center, Response Capability Enhancement 
Disclaimer 

Disclaimer 

The Environmental Protection Agency (EPA) through its Office of Research and Development funded 

and managed the research described here under Contract EP-W-06-046 to Computer Sciences 

Corporation (CSC). This document has been subjected to the Agency’s review and has been approved for 

publication. Note that approval does not signify that the contents necessarily reflect the views of the 

Agency. 

Mention of trade names or commercial products in this document or in the methods referenced in this 

document does not constitute endorsement or recommendation for use. 

Questions concerning this document or its application should be addressed to: 

Rob Rothman 

National Homeland Security Research Center 

Office of Research and Development (NG16) 

U.S. Environmental Protection Agency
 

26 West Martin Luther King Drive 

Cincinnati, OH 45268 

(513) 569-7187 

rothman.rob@epa.gov 

Questions concerning this document and its information also can be submitted using the SAM Web site: 

www.epa.gov/sam. 

 SAM Revision 5.0 ii September 29, 2009 
Use of This Document 

Use of This Document 

The information contained in this document represents the latest step in an ongoing effort of the 

Environmental Protection Agency’s (EPA’s) National Homeland Security Research Center 

(NHSRC) to provide standardized analytical methods for use by those laboratories tasked with 

performing confirmatory analyses of environmental samples in support of EPA restoration efforts 

following a homeland security incident. The information also can be found on the SAM Web site 

(www.epa.gov/sam), which provides searchable links to supporting information based on SAM 

analytes and the analytical methods listed. 

Although at this time, some of the methods listed have not been fully validated for a particular 

analyte (e.g., analytes not explicitly identified in the method) or sample type, the methods are 

considered to contain the most appropriate currently available techniques. Unless a published 

method listed in this document states specific applicability to the analyte/sample type for which it 

has been selected, it should be assumed that method testing is needed, and adjustments may be 

required to accurately account for variations in analyte/sample type characteristics, environmental 

samples, analytical interferences, and target risk levels. 

Many of the SAM analytes have only recently become an environmental concern. EPA is actively 

pursuing development and validation of Standard Analytical Protocols (SAPs) based on the 

methods listed, including optimization of procedures for measuring target analytes or agents. In 

those cases where method procedures are determined to be insufficient for a particular situation, 

EPA will provide guidlines regarding appropriate actions. This will be an ongoing process as EPA 

will strive to establish a consistent level of validation for all listed analytes. 

 SAM Revision 5.0 iii September 29, 2009 
Foreword 

Foreword

The U.S. Environmental Protection Agency (EPA) is charged by Congress to protect human health and 

the environment and specifically, to protect the Nation’s land, air, and water resources. Since 1970, EPA 

has been working toward a cleaner and healthier environment for the American people. To help meet this 

mandate, EPA’s research program provides data and technical support for solving environmental 

problems today and for building the scientific base necessary to manage our ecological resources wisely, 

understand how pollutants affect our health and prevent or reduce environmental risks in the future. 

Following the terrorist attacks of September 11, 2001, and the subsequent mailing of anthrax-tainted 

letters, EPA’s role with respect to homeland security was expanded. Presidential Directives identified 

EPA both as the primary federal agency responsible for the country’s water supplies and for 

decontamination following a chemical, biological, and/or radiological (CBR) attack. To provide 

scientific and technical support to help EPA meet this expanded role, EPA’s National Homeland Security 

Research Center (NHSRC) was established. The NHSRC research program is focused on developing and 

delivering reliable, responsive expertise and products based on scientific research and evaluations of 

technology. NHSRC’s research is conducted to help protect the country’s water infrastructure and to 

facilitate decontamination of indoor and outdoor areas in the event of CBR attacks. 

One specific goal of NHSRC’s research is to support the Environmental Response Laboratory Network 

(ERLN), a nationwide network of federal, state and local environmental laboratories. Toward this end, 

NHSRC has undertaken research designed to provide appropriate, effective, and verified technologies and 

methods to understand the risks posed by CBR agents and to enhance EPA’s ability to detect, contain, and 

clean up in the event of an incident involving such agents. This document provides a compendium of 

methods that can be used when laboratories are faced with analytical demands associated with an 

environmental restoration crisis involving CBR contaminants. Additionally, this document can be used as 

a tool to identify analytes that require further method development and verification to ensure desired 

performance. 

This publication represents the fifth version and update to the analytical methods available for use in an 

environmental crisis event requiring decontamination and restoration. The information contained within 

this document will continue to be revised as our research progress and new information becomes 

available. 

Cynthia Sonich-Mullin, Acting Director 

 National Homeland Security Research Center 

 SAM Revision 5.0 iv September 29, 2009 
Abbreviations and Acronyms

Abbreviations and Acronyms 

AdV40 Adenovirus 40 

AdV41 Adenovirus 41 

AEM Applied and Environmental Microbiology 

amp-ELISA Amplified-enzyme-linked immunosorbent assay 

APHA American Public Health Association 

APHL Association of Public Health Laboratories 

AOAC AOAC International (formerly the Association of Official Analytical Chemists) 

ASM American Society for Microbiology 

ASTM ASTM International (formerly the American Society for Testing and Materials) 

AWWA American Water Works Association 

BCYE Buffered charcoal yeast extract 

BGMK Buffalo green monkey kidney 

BHT Butylated hydroxytoluene 

BS Bismuth sulfite 

BLEB Buffered Listeria enrichment broth 

BMBL Biosafety in Microbiological and Biomedical Laboratories 

BSA Bovine serum albumin 

BSL Biosafety Level 

BZ Quinuclidinyl benzilate 

°C Degrees Celsius 

CA Chocolate agar 

CAS RN Chemical Abstracts Service Registry Number 

CBR Chemical, biological, and/or radiological 

CCID Coordinating Center for Infectious Diseases 

CDC Centers for Disease Control and Prevention 

cDNA Complementary deoxyribonucleic acid 

CFR Code of Federal Regulations 

CFSAN Center for Food Safety and Applied Nutrition 

CFU Colony forming unit 

CIEIA Competitive inhibition enzyme immunoassay 

CLLE Continuous liquid-liquid extraction 

CLP Contract Laboratory Program 

CPE Cytopathic effect 

cps Counts per second 

CRL Chicago Regional Laboratory 

CVAA Cold vapor atomic absorption 

2-CVAA 2-Chlorovinylarsonous acid 

CVAFS Cold vapor atomic fluorescence spectrometry 

2,4-D 2,4-Dichlorophenoxyacetic acid 

DAPI 4',6-Diamidino-2-phenylindole 

DAS Diacetoxyscirpenol 

DAS-HG-HSA Diacetoxyscirpenol hemiglutarate human serum albumin 

DAS-HS-HRP Diacetoxyscirpenol hemisuccinate horseradish peroxidase conjugate 

DB-1 100% Dimethylpolysiloxane 

DBPR Division of Bioterrorism Preparedness and Response 

DHS U.S. Department of Homeland Security 

DIC Differential interference contrast 

DIG-ELISA Digoxigenin labeled enzyme-linked immunosorbent assay 

DIMP Diisopropyl methylphosphonate 

DNA Deoxyribonucleic acid 

2,4-DNPH 2,4-Dinitrophenylhydrazine 

 SAM Revision 5.0 v September 29, 2009 
Abbreviations and Acronyms

DoD U.S. Department of Defense 

DOE U.S. Department of Energy 

DOT U.S. Department of Transportation 

DPD N,N-Diethyl-p-phenylenediamine 

DQO Data quality objective 

DTPA Diethylenetriamine-pentaacetate 

EA2192 Diisopropylaminoethyl methylthiolophosphonate 

ECD Electron capture detector 

e-CFR Electronic Code of Federal Regulations 

ECL Electrochemiluminescence 

ED Ethyldichloroarsine 

EDEA N-Ethyldiethanolamine 

EDL Estimated detection limit 

EDTA Ethylenediaminetetraacetic acid 

EDXA Energy dispersive X-ray analysis 

EEB Enterohemorrhagic E. coli enrichment broth 

EIA Enzyme immunoassay 

ELISA Enzyme-Linked Immunosorbent Assay 

EMC Emission Measurement Center 

EMJH Ellinghausen-McCullough Johnson Harris Formulation 

EML Environmental Measurements Laboratory 

EMMI Environmental Monitoring Methods Index 

EMPA Ethyl methylphosphonic acid 

EMSL Environmental Monitoring and Support Laboratory 

EPA U.S. Environmental Protection Agency 

EQL Estimated quantitation limit 

ESI Electrospray ionization 

ETV Environmental Technology Verification 

FA Fluorescence assay 

FBI U.S. Federal Bureau of Investigation 

FDA U.S. Food and Drug Administration 

FEMS Federation of European Microbiological Societies 

FGI Fluorescein derivative of Conus geographus α-conotoxin 

FID Flame ionization detector 

FL Fluorescence detector 

FPD Flame photometric detector 

FRET Fluorescence resonance energy transfer 

FRMAC Federal Radiological Monitoring and Assessment Center 

FSIS Food Safety and Inspection Service 

GA Tabun 

GB Sarin 

GC Gas chromatograph or Gas chromatography 

GC-ECD Gas chromatography – electron capture detector 

GC-FID Gas chromatography – flame ionization detector 

GC-FPD Gas chromatography – flame photometric detector 

GC-MS Gas chromatography – mass spectrometry 

GC-MD Gas chromatography – multi-detector 

GC-NPD Gas chromatography – nitrogen-phosphorus detector 

GD Soman 

GE 1-Methylethyl ester ethylphosphonofluoridic acid 

Ge Germanium 

Ge(Li) Germanium (Lithium) 

 SAM Revision 5.0 vi September 29, 2009 
Abbreviations and Acronyms

GESTIS A German database (Gefahrstoffdaten banken) containing data and information on 

hazardous substances and products 

GF Cyclohexyl sarin 

GFAA Graphite furnace atomic absorption spectrophotometer or Graphite furnace atomic 

absorption spectrophotometry 

GTC Guanidinium thiocyanate 

HAdV Human adenoviruses 

HASL	 Health and Safety Laboratory, currently known as Environmental Measurements 

Laboratory (EML) 

HAV Hepatitis A virus 

HCoV Human coronavirus 

HEV Hepatitis E virus 

HD	 Sulfur mustard / mustard gas; bis(2-chloroethyl) sulfide 

HHS	 U.S. Health and Human Services 

HMTD Hexamethylenetriperoxidediamine 

HMX Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine 

HN-1 Nitrogen mustard 1; bis(2-chloroethyl)ethylamine 

HN-2 Nitrogen mustard 2; 2,2'-dichloro-N-methyldiethylamine N,N-bis(2-

chloroethyl)methylamine 

HN-3 Nitrogen mustard 3; tris(2-chloroethyl)amine 

HPGe High purity Germanium 

HPLC High performance liquid chromatography 

HPLC-FL	 High performance liquid chromatography – fluorescence 

HPLC-MS	 High performance liquid chromatography – mass spectrometery 

HPLC-MS-MS High performance liquid chromatography tandem mass spectrometry 

HPLC-PDA High performance liquid chromatography – photodiode array detector 

HPLC-UV High performance liquid chromatography – ultraviolet 

HPLC-vis High performance liquid chromatography – visible 

HRP Horseradish peroxidase 

HV High volume 

IC Ion chromatograph or Ion chromatography 

IC 20	 Inhibitory concentration – Concentration to inhibit 20% 

IC 50	 Inhibitory concentration – Concentration to inhibit 50% 

ICP Inductively coupled plasma 

ICP-AES Inductively coupled plasma – atomic emission spectrometry 

ICP-MS Inductively coupled plasma – mass spectrometry 

ICR Information Collection Requirements Rule 

ID50 A dose which would be infectious to 50% of the population 

IDL Instrument detection limit 

ILM Inorganic Laboratory Method 

IMPA Isopropyl methylphosphonic acid 

IMS Immunomagnetic separation 

INCHEM	 INCHEM is a means of rapid access to internationally peer reviewed information on 

chemicals commonly used throughout the world, which may also occur as contaminants 

in the environment and food. It consolidates information from a number of 

intergovernmental organizations whose goal it is to assist in the sound management of 

chemicals. http://www.inchem.org/ 

IO Inorganic 

i.p. Intraperitoneally 

IRIS Integrated Risk Information System (EPA) 

ISE Ion specific electrode 

ISG Impregnated silica gel 

ISO International Organization for Standardization 

 SAM Revision 5.0	 vii September 29, 2009 
Abbreviations and Acronyms

L-1 Lewisite 1; 2-Chlorovinyldichloroarsine 

L-2 Lewisite 2; bis(2-Chlorovinyl)chloroarsine 

L-3 Lewisite 3; tris(2-Chlorovinyl)arsine 

LB-M Lim Benyesh-Melnick 

LC Liquid chromatograph or Liquid chromatography 

LC/APCI-MS Liquid chromatography / atmospheric pressure chemical ionization – mass spectrometry 

LC/ESI-MS Liquid chromatography / electrospray ionization – mass spectrometry 

LC-MS Liquid chromatography – mass spectrometry 

LC-MS-MS Liquid chromatography tandem mass spectrometry 

LC-TSP Liquid chromatography – thermospray 

LFD Lateral flow device 

LIA Lysine iron agar 

LLD Lower limit of detection 

LOD Limit of detection 

LRN Laboratory Response Network 

LSE Liquid-solid extraction 

Ltd. A private company limited by shares 

mAbs Monoclonal antibodies 

MARLAP Multi-Agency Radiological Laboratory Analytical Protocols (EPA/402/3-04/001A, B, C) 

MCAWW Methods for Chemical Analysis of Water and Waste (EPA/600/4-79/020) 

MDL Method detection limit 

MF Membrane filtration 

MIC Methyl isocyanate 

MLD Minimum lethal dose 

MOPS Morpholinepropanesulfonic acid 

MPA Methylphosphonic acid 

MPN Most probable number 

MRM Multiple reaction monitoring 

mRNA Messenger ribonucleic acid 

MS Mass spectrometer or Mass spectrometry 

MS-MS Tandem mass spectrometry 

MS/MSD Matrix spike/Matrix spike duplicate 

MSE Microscale solvent extraction 

MSRV Modified semisolid Rappaport-Vassiliadis 

MTBE Methyl tert-butyl ether 

MW Molecular weight 

NA Not applicable 

nAchR Nicotinic acetylcholine receptor 

NaI(Tl) Thallium-activated sodium iodide 

NBD chloride 7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole 

NBD-F 7-Fluoro-4-nitro-2,1,3-benzoxadiazole 

NCPDCID National Center for the Prevention, Detection, and Control of Infectious Diseases 

NCRP National Council on Radiation Protection and Measurements 

NCTC National collection of type cultures 

NEMI National Environmental Methods Index 

NERL National Exposure Research Laboratory 

NHSRC EPA National Homeland Security Research Center 

NIOSH National Institute for Occupational Safety and Health 

NIST National Institute of Standards and Technology 

nM Nanomolar 

NMAM NIOSH Manual of Analytical Methods 

NNSA National Nuclear Security Administration 

NoV Norovirus 

 SAM Revision 5.0 viii September 29, 2009 
Abbreviations and Acronyms

NOS	 Not otherwise specified 

NPD Nitrogen-phosphorus detector 

NRC	 U.S. Nuclear Regulatory Commission 

NRMRL EPA National Risk Management Research Laboratory 

nS nano Siemens 

NTIS National Technical Information Service 

NTU Nephelometric turbidity units 

OAQPS	 EPA Office of Air Quality Planning and Standards 

OAR EPA Office of Air and Radiation 

ORAU	 Oak Ridge Associated Universities 

ORD	 EPA Office of Research and Development 

ORF	 Open reading frame 

ORIA Office of Radiation and Indoor Air 

ORISE	 Oak Ridge Institute for Science and Education 

OSWER	 EPA Office of Solid Waste and Emergency Response 

OSHA Occupational Safety and Health Administration 

OVS	 OSHA versatile sampler 

OW EPA Office of Water 

OXA	 Oxford medium 

PBS Phosphate buffered saline 

PCDDs Polychlorinated dibenzo-p-dioxins 

PCDFs Polychlorinated dibenzofurans 

PCR	 Polymerase chain reaction 

PDA Photodiode array detector 

PEL Permissible exposure limit 

PETN Pentaerythritol tetranitrate 

PFE Pressurized fluid extraction 

PID 50	 50% Pig infectious dose 

PLET Polymyxin, lysozyme, EDTA, thallous acetate 

PMPA Pinacolyl methyl phosphonic acid 

1,2-PP	 1-(2-pyridyl)piperazine 

PubMED PubMED is a service of the U.S. National Library of Medicine (http://www.pubmed.gov), 

containing citations from scientific journals 

PUF Polyurethane foam 

PVC Polyvinyl chloride 

QA Quality assurance 

QAP	 Quality assessment program 

QC Quality control 

qPCR Quantitative polymerase chain reaction 

® Registered trademark 

R 33	 Methylphosphonothioic acid, S-[2-(diethylamino)ethyl] O-2-methylpropyl ester (VR) 

RCRA Resource Conservation and Recovery Act 

RDX Hexahydro-1,3,5-trinitro-1,3,5-triazine 

RLAB Regional laboratory 

RNA Ribonucleic acid 

rpm Revolutions per minute 

rRNA Ribosomal ribonucleic acid 

RTECS Registry of Toxic Effects of Chemical Substances 

RT-PCR	 Reverse transcription polymerase chain reaction 

SAED	 Select area electron diffraction 

SAM Standardized Analytical Methods for Environmental Restoration Following Homeland 

Security Events 

SAP Standard Analytical Protocol 

 SAM Revision 5.0	 ix September 29, 2009 
Abbreviations and Acronyms

SARS Severe acute respiratory syndrome 

SaV Sapovirus 

SBA Sheep blood agar 

SIM Selective ion monitoring 

SM Standard Methods for the Examination of Water and Wastewater 

SPE Solid-phase extraction 

spp. Species (plural) 

SRM Single reaction monitoring 

STEC Shiga-toxigenic E. coli 

STEL Short term exposure limit 

STX Saxitoxin 

Stx-1 Shiga toxin Type 1 

Stx-2 Shiga toxin Type 2 

SW Solid Waste 

TATP Triacetone triperoxide 

TBD To be determined 

TCBS Thiosulfate-citrate-bile salts-sucrose 

TC-SMAC Tellurite Cefixime-Sorbitol MacConkey Agar 

TCLP Toxicity Characteristic Leaching Procedure 

TDG Thiodiglycol 

TEA Triethanolamine 

TEM Transmission electron microscope or Transmission electron microscopy 

TETR Touchdown enzyme time release 

TFA Trifluoroacetic acid 

TM Unregistered trademark 

TM Thayer-Martin 

1,3,5-TNB 1,3,5-Trinitrobenzene 

2,4,6-TNT 2,4,6-Trinitrotoluene 

TO Toxic Organic 

TOFMS Time-of-flight mass spectrometry 

TOXNET Toxicology Data Network 

TP-S-1 Trypticase-panmede liver digest-serum 

TRU Transuranic 

TSB Tryptic soy broth 

TSAye TrypticaseTM soy agar with yeast extract 

TSI Triple sugar iron 

TSP Thermospray 

TSP-MS Thermospray –mass spectrometry 

TTN Technical Transfer Network 

TTX Tetrodotoxin 

TYI-S-33 Trypticase-yeast-iron-serum 

UF Ultrafiltration 

U.S. United States 

USDA U.S. Department of Agriculture 

USGS U.S. Geological Survey 

UV Ultraviolet 

UVM University of Vermont 

VCSB Voluntary Consensus Standard Body 

VE Phosphonothioic acid, ethyl-, S-(2-(diethylamino)ethyl) O-ethyl ester 

VG Phosphonothioic acid, S-(2-(diethylamino)ethyl) O,O-diethyl ester 

vis Visible detector 

VM Phosphonothioic acid, methyl-,S-(2-(diethylamino)ethyl) O-ethyl ester 

VOCs Volatile organic compounds 

 SAM Revision 5.0 x September 29, 2009 
Abbreviations and Acronyms

VR Methylphosphonothioic acid, S-[2-(diethylamino)ethyl] O-2-methylpropyl ester (R 33) 

VX O-ethyl-S-(2-diisopropylaminoethyl)methylphosphonothiolate 

WEF Water Environment Federation 

WHO World Health Organization 

XLD Xylose lysine deoxycholate 

 SAM Revision 5.0 xi September 29, 2009 
Abbreviations and Acronyms

 SAM Revision 5.0 xii September 29, 2009 
C

Acknowledgments 

Acknowledgments 

Contributions of the following individuals and organizations to the development of SAM Revision 5.0 are 

gratefully acknowledged. Please refer to older versions of SAM for historical acknowledgments. 

United States Environmental Protection Agency (EPA) 

Office of Research and Development, National Homeland Security Research Center

 Kathy Hall 

 Romy Lee 

 Alan Lindquist 

 Matthew Magnuson 

 Tonya Nichols 

 Eugene Rice 

 Rob Rothman 

 Frank Schaefer 

 Sanjiv Shah 

 Sarah Taft 

 Oba Vincent 

Stuart Willison 

•	 Office of Research and Development, National Exposure Research Laboratory (NERL), Las 

Vegas 

Stephen Pia (Environmental Sciences Division) 

•	 Office of Research and Development, National Exposure Research Laboratory (NERL), 

Cincinnati
 

Gerard Stelma (Microbiological and Chemical Exposure Assessment Research Division) 

Ann Grimm (Microbiological and Chemical Exposure Assessment Research Division) 

•	 Office of Solid Waste and Emergency Response 

Michele Burgess (Office of Emergency Management)
 

Jeanelle Martinez (Office of Emergency Management)
 

Marissa Mullins (Office of Emergency Management)
 

Terry Smith (Office of Emergency Management) 

•	 Office of Air and Radiation, Office of Radiation and Indoor Air 

George Dilbeck (National Air and Radiation Environmental Laboratory)
 

John Griggs (National Air and Radiation Environmental Laboratory) 

Daniel Mackney (National Air and Radiation Environmental Laboratory)
 

•	 Office of Water, Office of Ground Water and Drinking Water 

Malik Raynor (Water Security Division) 

•	 Office of Prevention, Pesticides, and Toxic Substances, Office of Pesticide Programs 

Elizabeth Flynt (Stennis Space Center) 

•	 Office of Enforcement and Compliance Assurance, Office of Criminal Enforcement, 

Forensics and Training
 

Don Smith (National Enforcement Investigations Center) 

 SAM Revision 5.0	 xiii September 29, 2009 
Acknowledgments 

• EPA Regions 

Katie Adams (Region 10) 

Jack Berges (Region 9) 

Diane Gregg (Region 6) 

Ted Haigh (Region 5) 

Stephanie Harris (Region 10) 

Jesse Kiernan (Region 8) 

Ed O’Neill (Region 6) 

Steve Reimer (Region 10) 

Michael Wasko (Region 4)
 

Larry Zintek (Region 5) 

United States Department of Health and Human Services 

• Centers for Disease Control and Prevention (CDC) 

Kevin Ashley (National Institute for Occupational Safety and Health) 

Clayton B’Hymer (National Institute for Occupational Safety and Health) 

Raymond Biagini (National Institute for Occupational Safety and Health) 

Barun De (National Center for Zoonotic, Vector-Borne, and Enteric Diseases) 

Jay Gee (National Center for Zoonotic, Vector-Borne, and Enteric Diseases) 

Mindy Glass (National Center for Zoonotic, Vector-Borne, and Enteric Diseases) 

Vincent Hill (National Center for Zoonotic, Vector-Borne, and Enteric Diseases) 

Alex Hoffmaster (National Center for Zoonotic, Vector-Borne, and Enteric Diseases) 

Rudolph Johnson (National Center for Environmental Health) 

Jennifer Links (National Center for Environmental Health) 

Chung Marston (National Center for Zoonotic, Vector-Borne, and Enteric Diseases) 

Stephen Morse (National Center for Preparedness, Detection and Control of Infectious Diseases) 

Michele Parsons (National Center for Zoonotic, Vector-Borne, and Enteric Diseases) 

Laura Rose (National Center for Preparedness, Detection and Control of Infectious Diseases) 

Richard Wang (National Center for Environmental Health) 

Betsy Weirich (National Center for Preparedness, Detection and Control of Infectious Diseases) 

Lihua Xiao (National Center for Zoonotic, Vector-Borne, and Enteric Diseases)
 

• United States Food and Drug Administration 

Eric Garber 

Michael McLaughlin 

Socrates Trujillo 

United States Department of Agriculture 

Mark Jenkins 

Robert Phillips 

United States Department of Commerce 

Peter Moeller (National Oceanic & Atmospheric Administration) 

United States Department of Defense 

Jon Davis (U.S. Army, Medical Research Institute of Infectious Diseases) 

Paul Grasso (U.S. Army) 

Johnathan Kiel (U.S. Air Force) 

Jose-Luis Sagripanti (U.S. Army Research, Development, and Engineering Command - Edgewood 

Chemical Biological Center) 

Elaine Strauss (U.S. Navy, Naval Surface Warfare Center Dahlgren Division) 

Ronald Swatski (U.S. Army, Center for Health Promotion and Preventive Medicine) 

 SAM Revision 5.0 xiv September 29, 2009 
Acknowledgments 

United States Department of Energy 

Steve Goldberg (New Brunswick Laboratory) 

United States Geological Survey 

 Donna Francy 

State Agencies 

Jack Bennett (State of Connecticut Department of Public Health) 

Timothy Fitzpatrick (Florida Department of Environmental Protection) 

Liang Lin (Florida Department of Environmental Protection) 

Christopher Retarides (Virginia State Department of General Services) 

Colin Wright (Florida Department of Environmental Protection) 

National Laboratories 

Staci Kane (Lawrence Livermore National Laboratory) 

Carolyn Koester (Lawrence Livermore National Laboratory) 

Timothy Straub (Pacific Northwest National Laboratory) 

Environmental Management Support, Inc. 

Anna Berne 

G. Fred Lee & Associates 

Fred Lee 

Hamilton Sundstrand 

 Richard Trubey 

Texas A&M University – Corpus Christi

 Paul Zimba 

Computer Sciences Corporation

 Eric Boring 

 Joan Cuddeback 

 Danielle Garozzo 

 Melody Jensen 

 Larry Umbaugh 

 Joshua Vinson 

 SAM Revision 5.0 xv September 29, 2009 
Acknowledgments 

 SAM Revision 5.0 xvi September 29, 2009 
Table of Contents

Standardized Analytical Methods for Environmental Restoration 

Following Homeland Security Events
 

Revision 5.0 

September 29, 2009 

Contents 

Disclaimer ....................................................................................................................................................ii 

Use of This Document................................................................................................................................iii 

Foreword.....................................................................................................................................................iv 

Abbreviations and Acronyms ....................................................................................................................v 

Acknowledgments ....................................................................................................................................xiii 

Section 1.0: Introduction ...........................................................................................................................1 

Section 2.0: Background............................................................................................................................3 

Section 3.0: Scope and Application ..........................................................................................................7 

Section 4.0: Points of Contact .................................................................................................................11 

Section 5.0: Selected Chemical Methods................................................................................................13 

5.1 General Guidelines ....................................................................................................................14 

5.1.1 Standard Operating Procedures for Identifying Chemical Methods .....................................14 

5.1.2 General Quality Control (QC) Guidelines for Chemical Methods........................................33 

5.1.3 Safety and Waste Management .............................................................................................34 

5.2 Method Summaries....................................................................................................................35 

5.2.1	 EPA Method 8: Determination of Sulfuric Acid and Sulfur Dioxide Emissions from
 

Stationary Sources.................................................................................................................35 

5.2.2 EPA Method 200.7: Determination of Metals and Trace Elements in Waters and Wastes by 

Inductively Coupled Plasma-Atomic Emission Spectrometry..............................................35 

5.2.3 EPA Method 200.8: Determination of Trace Elements in Waters and Wastes by Inductively
 

Coupled Plasma-Mass Spectrometry ....................................................................................36 

5.2.4	 EPA Method 245.1: Determination of Mercury in Water by Cold Vapor Atomic Absorption 

Spectrometry (CVAA) ..........................................................................................................38
 

5.2.5 EPA Method 252.2: Osmium (Atomic Absorption, Furnace Technique).............................38 

5.2.6	 EPA Method 300.1, Revision 1.0: Determination of Inorganic Anions in Drinking Water by
 

Ion Chromatography .............................................................................................................39 

5.2.7 EPA Method 335.4: Determination of Total Cyanide by Semi-Automated Colorimetry .....40 

5.2.8 EPA Method 350.1: Nitrogen, Ammonia (Colorimetric, Automated Phenate) ....................40 

5.2.9	 EPA Method 507: Determination of Nitrogen- and Phosphorus-Containing Pesticides in 

Water by Gas Chromatography with a Nitrogen-Phosphorus Detector................................41 

 SAM Revision 5.0	 xvii September 29, 2009 
Table of Contents

5.2.10 EPA Method 524.2: Measurement of Purgeable Organic Compounds in Water by Capillary
 

Column Gas Chromatography / Mass Spectrometry.............................................................42 

5.2.11	 EPA Method 525.2: Determination of Organic Compounds in Drinking Water by LiquidSolid Extraction and Capillary Column Gas Chromatography / Mass Spectrometry ...........42 

5.2.12 EPA Method 531.2: Measurement of N-Methylcarbamoyloximes and N-Methylcarbamates 

in Water by Direct Aqueous Injection HPLC with Postcolumn Derivatization....................43 

5.2.13	 EPA Method 549.2: Determination of Diquat and Paraquat in Drinking Water by LiquidSolid Extraction and High Performance Liquid Chromatography with Ultraviolet Detection
 

...............................................................................................................................................44 

5.2.14	 EPA Method 551.1: Determination of Chlorination Disinfection Byproducts, Chlorinated 

Solvents, and Halogenated Pesticides/Herbicides in Drinking Water by Liquid-Liquid 

Extraction and Gas Chromatography with Electron-Capture Detection ...............................44 

5.2.15	 EPA Method 614: The Determination of Organophosphorus Pesticides in Municipal and 

Industrial Wastewater............................................................................................................45 

5.2.16	 EPA Method 3031 (SW-846): Acid Digestion of Oils for Metals Analysis by Atomic 

Absorption or ICP Spectrometry...........................................................................................46 

5.2.17 EPA Method 3050B (SW-846): Acid Digestion of Sediments, Sludges, and Soils..............46 

5.2.18 EPA Method 3520C (SW-846): Continuous Liquid-Liquid Extraction................................48 

5.2.19 EPA Method 3535A (SW-846): Solid-Phase Extraction ......................................................49 

5.2.20 EPA Method 3541 (SW-846): Automated Soxhlet Extraction .............................................52 

5.2.21 EPA Method 3545A (SW-846): Pressurized Fluid Extraction (PFE)...................................54 

5.2.22 EPA Method 3570 (SW-846): Microscale Solvent Extraction (MSE) .................................57 

5.2.23	 EPA Method 3571 (SW-846): Extraction of Solid and Aqueous Samples for Chemical 

Agents ...................................................................................................................................61 

5.2.24 EPA Method 3580A (SW-846): Waste Dilution................................................................... 61 

5.2.25 EPA Method 3585 (SW-846): Waste Dilution for Volatile Organics...................................64 

5.2.26 EPA Method 5030C (SW-846): Purge-and-Trap for Aqueous Samples...............................65 

5.2.27 EPA Method 5035A (SW-846): Closed-System Purge-and-Trap and Extraction for Volatile 

Organics in Soil and Waste Samples.....................................................................................66 

5.2.28	 EPA Method 6010C (SW-846): Inductively Coupled Plasma - Atomic Emission 

Spectrometry .........................................................................................................................67 

5.2.29 EPA Method 6020A (SW-846): Inductively Coupled Plasma - Mass Spectrometry............68 

5.2.30 EPA Method 7010 (SW-846): Graphite Furnace Atomic Absorption Spectrophotometry...69 

5.2.31	 EPA Method 7470A (SW-846): Mercury in Liquid Wastes (Manual Cold-Vapor Technique)
 

...............................................................................................................................................69 

5.2.32	 EPA Method 7471B (SW-846): Mercury in Solid or Semisolid Wastes (Manual Cold-Vapor 

Technique).............................................................................................................................70 

5.2.33 EPA Method 7473 (SW-846): Mercury in Solids and Solutions by Thermal Decomposition, 

Amalgamation, and Atomic Absorption Spectrophotometry................................................71 

5.2.34	 EPA Method 7580 (SW-846): White Phosphorus (P4) by Solvent Extraction and Gas 

Chromatography....................................................................................................................71 

5.2.35 EPA Method 8015C (SW-846): Nonhalogenated Organics Using GC/FID.........................72 

5.2.36 EPA Method 8260C (SW-846): Volatile Organic Compounds by Gas ChromatographyMass Spectrometry (GC/MS)................................................................................................73 

5.2.37 EPA Method 8270D (SW-846): Semivolatile Organic Compounds by Gas 

Chromatography/Mass Spectrometry (GC-MS) ...................................................................74 

5.2.38	 EPA Method 8290A, Appendix A (SW-846): Procedure for the Collection, Handling, 

Analysis, and Reporting of Wipe Tests Performed within the Laboratory ...........................77 

5.2.39	 EPA Method 8315A (SW-846): Determination of Carbonyl Compounds by High
 

Performance Liquid Chromatography (HPLC).....................................................................81 

5.2.40	 EPA Method 8316 (SW-846): Acrylamide, Acrylonitrile and Acrolein by High Performance 

Liquid Chromatography (HPLC) ..........................................................................................81 

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5.2.41	 EPA Method 8318A (SW-846): N-Methylcarbamates by High Performance Liquid 

Chromatography (HPLC)......................................................................................................82 

5.2.42 EPA Method 8321B (SW-846): Solvent-Extractable Nonvolatile Compounds by High 

Performance Liquid Chromatography-Thermospray-Mass Spectrometry (HPLC-TS-MS) or 

Ultraviolet (UV) Detection....................................................................................................83 

5.2.43	 EPA Method 8330B (SW-846): Nitroaromatics and Nitramines by High Performance 

Liquid Chromatography (HPLC) ..........................................................................................84 

5.2.44	 EPA CRL MS014: Analysis of Aldicarb, Bromadiolone, Carbofuran, Oxamyl and 

Methomyl in Water by Multiple Reaction Monitoring Liquid Chromatography / Tandem
 

Mass Spectrometry (LC/MS/MS) .........................................................................................85 

5.2.45 EPA CRL MS015: Analysis of Thiodiglycol in Water by Single Reaction Monitoring 

Liquid Chromatography / Tandem Mass Spectrometry (LC/MS/MS)..................................86 

5.2.46	 EPA CRL MS016: Analysis of Diethanolamine, Triethanolamine, n-Methyldiethanolamine 

and n-Ethyldiethanolamine in Water by Single Reaction Monitoring Liquid 

Chromatography / Tandem Mass Spectrometry (LC/MS/MS) .............................................86 

5.2.47	 EPA CRL MS017: Analysis of Diisopropyl Methylphosphonate, Ethyl Hydrogen
 

Dimethylamidophosphate, Isopropyl Methylphosphonic Acid, Methylphosphonic Acid and 

Pinacolyl Methylphosphonic Acid in Water by Multiple Reaction Monitoring Liquid 

Chromatography / Tandem Mass Spectrometry (LC/MS/MS) .............................................87 

5.2.48 EPA ILM05.3 Cyanide: Analytical Methods for Total Cyanide Analysis............................88 

5.2.49	 EPA Region 7 RLAB Method 3135.2I: Cyanide, Total and Amenable in Aqueous and Solid
 

Samples Automated Colorimetric with Manual Digestion ...................................................88 

5.2.50	 IO [Inorganic] Compendium Method IO-3.1: Selection, Preparation, and Extraction of Filter 

Material .................................................................................................................................89 

5.2.51	 IO [Inorganic] Compendium Method IO-3.4: Determination of Metals in Ambient 

Particulate Matter Using Inductively Coupled Plasma (ICP) Spectroscopy .........................90 

5.2.52	 IO [Inorganic] Compendium Method IO-3.5: Determination of Metals in Ambient 

Particulate Matter Using Inductively Coupled Plasma/Mass Spectrometry (ICP-MS) ........ 91 

5.2.53	 IO [Inorganic] Compendium Method IO-5: Sampling and Analysis for Vapor and Particle 

Phase Mercury in Ambient Air Utilizing Cold Vapor Atomic Fluorescence Spectrometry
 

(CVAFS) ...............................................................................................................................92 

5.2.54	 EPA Air Method, Toxic Organics - 10A (TO-10A): Determination of Pesticides and 

Polychlorinated Biphenyls in Ambient Air Using Low Volume Polyurethane Foam (PUF) 

Sampling Followed by Gas Chromatographic/Multi-Detector Detection (GC/MD)............93 

5.2.55 EPA Air Method, Toxic Organics - 15 (TO-15): Determination of Volatile Organic 

Compounds (VOCs) in Air Collected in Specially-Prepared Canisters and Analyzed by Gas 

Chromatography/Mass Spectrometry (GC/MS)....................................................................95 

5.2.56 NIOSH Method 1612: Propylene Oxide ...............................................................................96 

5.2.57 NIOSH Method 2016: Formaldehyde ...................................................................................97 

5.2.58 NIOSH Method 2513: Ethylene Chlorohydrin .....................................................................97 

5.2.59 NIOSH Method 3510: Monomethylhydrazine......................................................................98 

5.2.60 NIOSH Method 5600: Organophosphorus Pesticides...........................................................98 

5.2.61 NIOSH Method 5601: Organonitrogen Pesticides................................................................99 

5.2.62 NIOSH Method 6001: Arsine .............................................................................................100 

5.2.63 NIOSH Method 6002: Phosphine .......................................................................................100 

5.2.64 NIOSH Method 6004: Sulfur Dioxide ................................................................................101 

5.2.65 NIOSH Method 6010: Hydrogen Cyanide..........................................................................101 

5.2.66 NIOSH Method 6013: Hydrogen Sulfide............................................................................102 

5.2.67 NIOSH Method 6015: Ammonia ........................................................................................102 

5.2.68 NIOSH Method 6402: Phosphorus Trichloride...................................................................103 

5.2.69 NIOSH Method 7903: Acids, Inorganic .............................................................................103 

5.2.70 NIOSH Method 7905: Phosphorus .....................................................................................104 

5.2.71 NIOSH Method 7906: Fluorides, Aerosol and Gas, by IC .................................................104 

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5.2.72 NIOSH Method 9102: Elements on Wipes.........................................................................105 

5.2.73 NIOSH Method S301-1: Fluoroacetate Anion....................................................................106 

5.2.74 OSHA Method 40: Methylamine ........................................................................................106 

5.2.75 OSHA Method 54: Methyl Isocyanate (MIC).....................................................................107 

5.2.76 OSHA Method 61: Phosgene ..............................................................................................107 

5.2.77 OSHA Method ID-211: Sodium Azide and Hydrazoic Acid in Workplace Atmospheres .108 

5.2.78	 OSHA Method ID216SG: Boron Trifluoride (BF3)............................................................108 

5.2.79 OSHA Method PV2004: Acrylamide ................................................................................109 

5.2.80 OSHA Method PV2103: Chloropicrin...............................................................................109 

5.2.81	 ASTM Method D5755-03: Standard Test Method for Microvacuum Sampling and Indirect 

Analysis of Dust by Transmission Electron Microscopy for Asbestos Structure Number 

Surface Loading ..................................................................................................................110 

5.2.82	 ASTM Method D6480-05: Standard Test Method for Wipe Sampling of Surfaces, Indirect 

Preparation, and Analysis for Asbestos Structure Number Concentration by Transmission 

Electron Microscopy ...........................................................................................................110 

5.2.83	 ISO Method 10312:1995: Ambient Air - Determination of Asbestos Fibres - Direct-transfer 

Transmission Electron Microscopy Method .......................................................................111 

5.2.84 Standard Method 4500-NH3 B: Nitrogen (Ammonia) Preliminary Distillation Step .........111 

5.2.85 Standard Method 4500-NH3 G: Nitrogen (Ammonia) Automated Phenate Method ..........112 

5.2.86 Standard Method 4500-Cl G: DPD Colorimetric Method ..................................................112 

5.2.87 Literature Reference for Chlorine (Analyst, 1999. 124: 1853–1857) .................................113 

5.2.88 Literature Reference for Fluoroacetate salts (Analytical Letters, 1994. 27 (14): 2703–2718)
 

.............................................................................................................................................113 

5.2.89 Literature Reference for Methamidophos (Chromatographia. 2006. 63(5/6): 233–237)....114 

5.2.90	 Literature Reference for Methamidophos (Journal of Chromatography A, 2007. 1154: 3–25)
 

.............................................................................................................................................114 

5.2.91 Literature Reference for Fluoroacetamide (Journal of Chromatography B, 2008. 876(1): 

103–108) .............................................................................................................................115 

5.2.92 Literature Reference for Sodium Azide (Journal of Forensic Sciences, 1998. 43(1): 200–
 

202) .....................................................................................................................................116 

Section 6.0: Selected Radiochemical Methods.....................................................................................117 

6.1 General Guidelines .................................................................................................................118 

6.1.1 Standard Operating Procedures for Identifying Radiochemical Methods...........................118 

6.1.2 General QC Guidelines for Radiochemical Methods..........................................................121 

6.1.3 Safety and Waste Management ...........................................................................................122
 

6.2 Method Summaries..................................................................................................................123 

6.2.1	 EPA Method 111: Determination of Polonium-210 Emissions from Stationary Sources ..123 

6.2.2 EPA Method 900.0: Gross Alpha and Gross Beta Radioactivity in Drinking Water..........123 

6.2.3 EPA Method 901.1: Gamma Emitting Radionuclides in Drinking Water..........................124 

6.2.4 EPA Method 903.0: Alpha-Emitting Radium Isotopes in Drinking Water.........................125 

6.2.5 EPA Method 903.1: Radium-226 in Drinking Water – Radon Emanation Technique .......126 

6.2.6 EPA Method 906.0: Tritium in Drinking Water .................................................................126 

6.2.7 EPA Method 908.0: Uranium in Drinking Water – Radiochemical Method ......................127 

6.2.8	 EPA Method EMSL-19: Determination of Radium-226 and Radium-228 in Water, Soil, Air 

and Biological Tissue..........................................................................................................127 

6.2.9	 EPA Method EMSL-33: Isotopic Determination of Plutonium, Uranium, and Thorium in 

Water, Soil, Air, and Biological Tissue ..............................................................................128 

6.2.10 EML HASL-300 Method Am-01-RC: Americium in Soil .................................................129 

6.2.11 EML HASL-300 Method Am-02-RC: Americium-241 in Soil-Gamma Spectrometry......129 

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6.2.12 EML HASL-300 Method Am-04-RC: Americium in QAP Water and Air Filters - Eichrom’s 

TRU Resin...........................................................................................................................130 

6.2.13 EML HASL-300 Method Ga-01-R: Gamma Radioassay ...................................................130 

6.2.14 EML HASL-300 Method Po-02-RC: Polonium in Water, Vegetation, Soil, and Air Filters
 

.............................................................................................................................................131 

6.2.15	 EML HASL-300 Method Pu-12-RC: Plutonium and/or Americium in Soil or Sediments.132 

6.2.16 EML HASL-300 Method Sr-03-RC: Strontium-90 in Environmental Samples .................132 

6.2.17	 EML HASL-300 Method Tc-02-RC: Technetium-99 in Water – TEVA® Resin ...............133 

6.2.18 FRMAC Method Volume 2, Page 33: Gross Alpha and Beta in Air ..................................133 

6.2.19 ORISE Method AP1: Gross Alpha and Beta for Various Matrices ....................................134 

6.2.20 ORISE Method AP2: Determination of Tritium.................................................................135 

6.2.21 ORISE Method AP5: Determination of Technetium-99.....................................................136 

6.2.22	 ORISE Method AP11: Sequential Determination of the Actinides in Environmental Samples 

Using Total Sample Dissolution and Extraction Chromatography .....................................136 

6.2.23 ORISE Method Procedure #9: Determination of I-125 in Environmental Samples ...........137 

6.2.24 ASTM Method D3084-05: Standard Practice for Alpha Spectrometry in Water ...............138 

6.2.25	 ASTM Method D3972-02: Standard Test Method for Isotopic Uranium in Water by
 

Radiochemistry ...................................................................................................................138 

6.2.26	 Standard Method 7110 B: Gross Alpha and Gross Beta Radioactivity (Total, Suspended, 

and Dissolved).....................................................................................................................139 

6.2.27 Standard Method 7120: Gamma-Emitting Radionuclides ..................................................140 

6.2.28 Standard Method 7500-Ra B: Radium: Precipitation Method ............................................141 

6.2.29 Standard Method 7500-Ra C: Radium: Emanation Method ...............................................141 

6.2.30	 Standard Method 7500-Sr B: Total Radioactive Strontium and Strontium-90: Precipitation 

Method ................................................................................................................................142 

6.2.31 Standard Method 7500-U B: Uranium: Radiochemical Method.........................................142 

6.2.32 Standard Method 7500-U C: Uranium: Isotopic Method....................................................143 

Section 7.0: Selected Pathogen Methods ..............................................................................................145 

7.1 General Guidelines .................................................................................................................146 

7.1.1 Standard Operating Procedures for Identifying Pathogen Methods....................................147 

7.1.2 General QC Guidelines for Pathogen Methods...................................................................148 

7.1.3 Safety and Waste Management ...........................................................................................149
 

7.1.4 Laboratory Response Network (LRN)................................................................................150 

7.2 Method Summaries for Bacteria.............................................................................................151 

7.2.1	 Bacillus anthracis [Anthrax] – BSL-3 ................................................................................151 

7.2.1.1 Literature Reference for B. anthracis (Public Health Reports. 1977. 92(2): 176–186)
 

................................................................................................................................152 

7.2.2	 Brucella spp. [Brucellosis] – BSL-3 ...................................................................................152 

7.2.2.1	 ASM Sentinel Laboratory Guidelines for Suspected Agents of Bioterrorism: 

Brucella species .....................................................................................................153 

7.2.3	 Burkholderia mallei [Glanders] – BSL-3 and Burkholderia pseudomallei [Melioidosis] – 

BSL-3 ..................................................................................................................................153 

7.2.3.1	 ASM Sentinel Laboratory Guidelines for Suspected Agents of Bioterrorism: 

Burkholderia mallei and Burkholderia pseudomallei ............................................153 

7.2.4	 Campylobacter jejuni [Campylobacteriosis] – BSL-2 ........................................................154 

7.2.4.1	 Standard Method 9260 G: Campylobacter jejuni ..................................................154 

7.2.4.2 Literature Reference for Campylobacter jejuni (Molecular and Cellular Probes. 

2006. 20(5): 269–279)............................................................................................155 

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7.2.5	 Chlamydophila psittaci [Psittacosis] (formerly known as Chlamydia psittaci) – BSL-2; 

BSL-3 for aerosols and large volumes................................................................................156 

7.2.5.1 Literature Reference for Chlamydophila psittaci (Journal of Clinical Microbiology. 

2000. 38(3): 1085–1093)........................................................................................156 

7.2.6	 Coxiella burnetii [Q-fever] – BSL- 3..................................................................................157 

7.2.7	 Escherichia coli O157:H7 – BSL-2 ....................................................................................157 

7.2.7.1	 Standard Method 9260 F: Pathogenic Escherichia coli .........................................157 

7.2.7.2 Literature Reference for Escherichia coli O157:H7 (Applied and Environmental 

Microbiology. 2003. 69(10): 6327–6333)..............................................................158 

7.2.8	 Francisella tularensis [Tularemia] – BSL-3.......................................................................158 

7.2.8.1 CDC, ASM, APHL: Basic Protocols for Level A Laboratories for the Presumptive 

Identification of Francisella tularensis..................................................................159 

7.2.9	 Leptospira interrogans [Leptospirosis] – BSL-2 ................................................................159 

7.2.9.1	 Standard Method 9260 I: Leptospira .....................................................................159 

7.2.9.2 Literature Reference for Leptospira spp. (Diagnostic Microbiology and Infectious
 

Diseases. 2009. 64(3): 247–255)............................................................................160 

7.2.10	 Listeria monocytogenes [Listeriosis] – BSL-2....................................................................161 

7.2.10.1	 USDA Laboratory Guidebook: “FSIS Procedure for the Use of a Listeria 

monocytogenes Polymerase Chain Reaction (PCR) Screening Test.” MLG 8A.03. 

2007........................................................................................................................161
 

7.2.10.2	 U.S. FDA Bacteriological Analytical Manual, Chapter 10, 2003: Listeria 

monocytogenes .......................................................................................................162 

7.2.11 Non-typhoidal Salmonella (Not applicable to S. Typhi) [Salmonellosis] – BSL-2 ............162 

7.2.11.1 EPA Method 1682: Salmonella spp. ......................................................................163 

7.2.11.2	 Literature Reference for Non-Typhoidal Salmonella (Journal of Applied 

Microbiology. 2007. 102(2): 516–530)..................................................................163 

7.2.12	 Salmonella Typhi [Typhoid fever] – BSL-2; BSL-3 for aerosol release ............................164 

7.2.12.1 CDC Laboratory Assay: “Triplex PCR for Detection of S. Typhi Using 

SmartCycler®”.......................................................................................................164 

7.2.12.2	 Standard Method 9260 B: General Qualitative Isolation and Identification 

Procedures for Salmonella .....................................................................................165 

7.2.13	 Shigella spp. [Shigellosis] – BSL-2 ....................................................................................165 

7.2.13.1 CDC Laboratory Assay: “Detection of Diarrheagenic Escherichia coli and Shigella
 

Using LightCycler®” .............................................................................................165 

7.2.13.2	 Standard Method 9260 E: Shigella.........................................................................166 

7.2.14	 Staphylococcus aureus – BSL-2 .........................................................................................167 

7.2.14.1	 Standard Method 9213 B: Staphylococcus aureus.................................................167 

7.2.15	 Vibrio cholerae [Cholera] – BSL-2.....................................................................................167 

7.2.15.1 CDC Laboratory Assay: “TaqMan Assays for Detection of V. cholerae ctxA, O1 

rfb, and O139 rfb.” .................................................................................................168 

7.2.15.2	 Standard Method 9260 H: Vibrio cholerae ............................................................168 

7.2.16	 Yersinia pestis [Plague] – BSL-3 ........................................................................................169 

7.2.16.1	 ASM Sentinel Laboratory Guidelines for Suspected Agents of Bioterrorism: 

Yersinia pestis ........................................................................................................169 

7.3 Method Summaries for Viruses.............................................................................................. 170 

7.3.1 Adenoviruses: Enteric and non-enteric (A-F) – BSL-2.......................................................170 

7.3.1.1 Literature Reference for Adenoviruses (Applied and Environmental Microbiology. 

2005. 71(6): 3131–3136)........................................................................................170 

7.3.2 Astroviruses – BSL not specified........................................................................................171 

7.3.2.1 Literature Reference for Astroviruses (Canadian Journal of Microbiology. 2004. 50: 

269–278) ................................................................................................................171 

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7.3.3 Caliciviruses: Noroviruses – BSL-2....................................................................................171
 

7.3.3.1 Literature Reference for Noroviruses (Journal of Clinical Microbiology. 2004. 

42(10): 4679–4685)................................................................................................172 

7.3.4 Caliciviruses: Sapovirus – BSL-2 .......................................................................................172 

7.3.4.1 Literature Reference for Sapoviruses (Journal of Medical Virology. 2006. 78(10): 

1347–1353) ............................................................................................................172 

7.3.5 Coronaviruses: SARS-associated human coronavirus – BSL-2; BSL-3 for propagation ...173 

7.3.5.1 Literature Reference for Coronaviruses (SARS) (Journal of Virological Methods. 

2004. 122: 29–36) ..................................................................................................173 

7.3.6 Hepatitis E virus (HEV) – BSL-2 .......................................................................................174
 

7.3.6.1 Literature Reference for Hepatitis E Virus (Journal of Virological Methods. 2006. 

131(1): 65–71)........................................................................................................174 

7.3.7 Influenza H5N1 virus – BSL-3 ........................................................................................... 174 

7.3.7.1 Literature Reference for Influenza H5N1 (Emerging Infectious Diseases. 2005. 

11(8): 1303–1305)..................................................................................................175 

7.3.8 Picornaviruses: Enteroviruses – BSL-2...............................................................................175 

7.3.8.1 USEPA Manual of Methods for Virology, EPA/600/4-84/013, April 2001 .........175 

7.3.8.2 Literature Reference for Enteric Viruses (Applied and Environmental Microbiology. 

2003. 69(6): 3158–3164)........................................................................................176 

7.3.9 Picornaviruses: Hepatitis A virus (HAV) – BSL-2.............................................................177 

7.3.10 Reoviruses: Rotavirus (Group A) – BSL not specified.......................................................177 

7.3.10.1 Literature Reference for Reoviruses: Rotavirus (Group A) (Journal of Virological 

Methods. 2009. 155: 126–131) ..............................................................................177 

7.4 Method Summaries for Protozoa............................................................................................178 

7.4.1 Cryptosporidium spp. [Cryptosporidiosis] – BSL-2 ...........................................................178 

7.4.1.1 EPA Method 1622: Cryptosporidium in Water by Filtration/IMS/FA ..................178 

7.4.1.2 EPA Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA
 

................................................................................................................................179 

7.4.1.3 Literature Reference for Cryptosporidium spp. (Applied and Environmental 

Microbiology. 1999. 65(9): 3936–3941)................................................................180 

7.4.1.4 Literature Reference for Cryptosporidium spp. (Applied and Environmental 

Microbiology. 2007. 73(13): 4218–4225)..............................................................180 

7.4.2 Entamoeba histolytica – BSL-2 ..........................................................................................181 

7.4.2.1 Literature Reference for Entamoeba histolytica (Journal of Parasitology. 1972. 

58(2): 306–310)......................................................................................................181 

7.4.2.2 Literature Reference for Entamoeba histolytica (Journal of Clinical Microbiology. 

2005. 43(11): 5491–5497)......................................................................................182 

7.4.3 Giardia spp. [Giardiasis] – BSL-2 ......................................................................................182 

7.4.3.1 Literature Reference for Giardia spp. (Transactions of the Royal Society of 

Tropical Medicine and Hygiene. 1983. 77(4): 487–488).......................................182 

7.4.4 Toxoplasma gondii [Toxoplasmosis] – BSL-2....................................................................183 

7.4.4.1 Literature Reference for Toxoplasma gondii (Emerging Infectious Diseases. 2006. 

12(2): 326–329)......................................................................................................183 

7.4.4.2 Literature Reference for Toxoplasma gondii (Applied and Environmental 

Microbiology. 2004. 70(7): 4035–4039)................................................................184 

7.5 Method Summaries for Helminths .........................................................................................184
 

7.5.1 Baylisascaris procyonis [Raccoon roundworm fever] – BSL-2..........................................184 

7.5.1.1 USEPA Environmental Regulations and Technology, Control of Pathogens and 

Vector Attraction in Sewage Sludge EPA/625/R-92/013, July 2003: Baylisascaris 

procyonis................................................................................................................185 

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Section 8.0: Selected Biotoxin Methods................................................................................................187 

8.1 General Guidelines .................................................................................................................188 

8.1.1 Standard Operating Procedures for Identifying Biotoxin Methods.....................................189 

8.1.2 General QC Guidelines for Biotoxin Methods....................................................................190 

8.1.3 Safety and Waste Management ...........................................................................................191
 

8.1.4 Laboratory Response Network (LRN)................................................................................192 

8.2 Method Summaries for Protein Biotoxins..............................................................................193 

8.2.1 Abrin ...................................................................................................................................193 

8.2.1.1 Literature Reference for Abrin (Journal of Food Protection. 2008. 71(9): 1868–
 

1874) ......................................................................................................................193 

8.2.1.2 Literature Reference for Abrin and Ricin (Analytical Biochemistry. 2008. 378(1): 

87-89).....................................................................................................................194 

8.2.1.3 Literature Reference for Abrin and Shiga and Shiga-like Toxins (Pharmacology
 

Toxicology. 2001. 88(5): 255–260) .......................................................................195 

8.2.2 Botulinum neurotoxins (Serotypes A, B, E, F) ...................................................................195 

8.2.2.1	 U.S. FDA, Bacteriological Analytical Manual Online, Chapter 17, 2001: Botulinum
 

Neurotoxins............................................................................................................196 

8.2.2.2 Lateral Flow Immunoassay Kits ..........................................................................196 

8.2.3 Ricin (Ricinine)...................................................................................................................197 

8.2.3.1 Literature Reference for Ricin (Journal of AOAC International. 2008. 91(2): 376–
 

382) ........................................................................................................................197
 

8.2.3.2 Literature Reference for Ricin by Ricinine detection (Journal of Analytical 

Toxicology. 2005. 29(3): 149–155) .......................................................................198 

8.2.4 Shiga and Shiga-like toxins (Stx, Stx-1, Stx-2)...................................................................199 

8.2.4.1 U.S. FDA, Bacteriological Analytical Manual Online, Appendix 1, 2001: Rapid 

Methods for Detecting Foodborne Pathogens........................................................199 

8.2.4.2 Literature Reference for Shiga and Shiga-like Toxin (Journal of Clinical 

Microbiology. 2007. 45(10): 3377–3380)..............................................................199 

8.2.5 Staphylococcal enterotoxins (SEA, SEB, SEC)..................................................................200 

8.2.5.1 AOAC Official Method 993.06: Staphylococcal Enterotoxins in Selected Foods 200 

8.3 Method Summaries for Small Molecule Biotoxins ................................................................ 200 

8.3.1 Aflatoxin (Type B1)............................................................................................................201 

8.3.1.1 AOAC Official Method 991.31: Aflatoxins in Corn, Raw Peanuts, and Peanut 

Butter......................................................................................................................201
 

8.3.2 α-Amanitin ..........................................................................................................................201 

8.3.2.1 Literature Reference for α-Amanitin (Journal of Chromatography B. 1991. 563(2): 

299–311) ................................................................................................................201 

8.3.2.2 Literature Reference for α-Amanitin, T-2 Mycotoxin (Journal of Food Protection. 

2005. 68(6): 1294–1301)........................................................................................202 

8.3.3 Anatoxin-a...........................................................................................................................203 

8.3.3.1 Literature Reference for Anatoxin-a (Biomedical Chromatography. 1996. 10(1): 

46–47) ....................................................................................................................203 

8.3.4 Brevetoxins (B form) ..........................................................................................................203 

8.3.4.1 Literature Reference for Brevetoxins (Environmental Health Perspectives. 2002. 

110(2): 179–185)....................................................................................................203 

8.3.4.2 Literature Reference for Brevetoxins (Toxicon. 2004. 43(4): 455–465) ..............204 

8.3.5 α-Conotoxin ........................................................................................................................204 

8.3.5.1 Literature Reference for α-Conotoxin (Biochemical Journal. 1997. 328(1): 245–
 

250) ........................................................................................................................204
 

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8.3.5.2 Literature Reference for α-Conotoxin (Journal of Medicinal Chemistry. 2004. 

47(5): 1234–1241)..................................................................................................205 

8.3.6 Cylindrospermopsin ............................................................................................................206 

8.3.6.1 Literature Reference for Cylindrospermopsin (FEMS Microbiology Letters. 2002. 

216(2): 159–164)....................................................................................................206 

8.3.6.2 ELISA Kits for Cylindrospermopsin.....................................................................206 

8.3.7 Diacetoxyscirpenol (DAS)..................................................................................................207 

8.3.7.1 Literature Reference for Diacetoxyscirpenol (DAS) (International Journal of Food 

Microbiology. 1988. 6(1): 9–17)............................................................................207 

8.3.7.2 Literature Reference for Diacetoxyscirpenol (DAS) and T-2 Mycotoxin (Rapid 

Communications in Mass Spectrometry. 2006. 20(9): 1422–1428) ......................207 

8.3.8 Microcystins (Principal isoforms: LA, LR, LW, RR, YR) .................................................208 

8.3.8.1 Literature Reference for Microcystins (Journal of AOAC International. 2001. 

84(4): 1035–1044)..................................................................................................208 

8.3.8.2 Literature Reference for Microcystins (Analyst. 1994. 119(7): 1525–1530)........208 

8.3.9 Picrotoxin ............................................................................................................................209 

8.3.9.1 Literature Reference for Picrotoxin (Journal of Pharmaceutical & Biomedical 

Analysis. 1989. 7(3): 369–375)..............................................................................209 

8.3.10 Saxitoxins (Principal isoforms: STX, NEOSTX, GTX, dcGTX, dcSTX)..........................210 

8.3.10.1 Literature Reference for Saxitoxin (Journal of AOAC International. 1995. 78(2): 

528–532) ................................................................................................................210 

8.3.10.2 ELISA Kits for Saxitoxins.....................................................................................210 

8.3.11 T-2 Mycotoxin ....................................................................................................................211 

8.3.12 Tetrodotoxin........................................................................................................................211 

8.3.12.1	 Literature Reference for Tetrodotoxin (Analytical Biochemistry. 2001. 290(1): 10–
 

17) ..........................................................................................................................211 

8.3.12.2	 Literature Reference for Tetrodotoxin (Journal of Clinical Laboratory Analysis. 

1992. 6(2): 65–72)..................................................................................................212 

Section 9.0: Conclusions ........................................................................................................................213 

Appendices 

Appendix A: Selected Chemical Methods............................................................................................A-1 

Appendix B: Selected Radiochemical Methods................................................................................... B-1 

Appendix C: Selected Pathogen Methods............................................................................................ C-1 

Appendix D: Selected Biotoxin Methods .............................................................................................D-1 

Figures 

Figure 1-1. Environmental Evaluation Analytical Process Roadmap for Homeland Security Events .........2 

Figure 2-1. SAM Method Selection Process.................................................................................................5 

 SAM Revision 5.0	 xxv September 29, 2009 
Table of Contents

Tables 

Table 5-1. Chemical Methods and Corresponding Text Section Numbers................................................14 

Table 5-2. Sources of Chemical Methods..................................................................................................32 

Table 6-1. Radiochemical Methods and Corresponding Text Section Numbers.....................................118 

Table 6-2. Sources of Radiochemical Methods .......................................................................................120 

Table 7-1. Sources of Pathogen Methods ................................................................................................147 

Table 8-1. Sources of Biotoxin Methods.................................................................................................189 

 SAM Revision 5.0 xxvi September 29, 2009 
Section 1 – Introduction 

Section 1.0: Introduction 

After the terrorist attacks of September 11, 2001 and the anthrax attacks in the fall of 2001, federal and 

state personnel provided response, recovery, and remediation under trying circumstances, including 

unprecedented demand on their capabilities to analyze environmental samples. In reviewing these events, 

the Environmental Protection Agency (EPA) identified several areas where the country could better 

prepare itself in the event of future terrorist incidents. The need to improve the nation’s laboratory 

capacity and capability to analyze environmental samples following a homeland security event (i.e., 

chemical, biological, and/or radiological [CBR] crime/attack) was one of the most important areas 

identified. 

In response, EPA formed the Homeland Security Laboratory Capacity Workgroup to identify and 

implement opportunities for near-term improvements and to develop recommendations for addressing 

longer-term laboratory issues. The EPA Homeland Security Laboratory Capacity Workgroup consists of 

representatives from the Office of Research and Development (ORD), Office of Air and Radiation 

(OAR), Office of Water (OW), Office of Solid Waste and Emergency Response (OSWER), Office of 

Environmental Information, Office of Pollution Prevention and Toxics, and several EPA regional offices. 

A critical area identified by the workgroup was the need for a list of analytical methods to be used by all 

laboratories when analyzing homeland security event samples and, in particular, when analysis of many 

samples is required over a short period of time. Having standardized methods would reduce confusion, 

permit sharing of sample load between laboratories, improve data comparability, and simplify the task of 

outsourcing analytical support to the commercial laboratory sector. Standardized methods would also 

improve the follow-up activities of validating results, evaluating data, and making decisions. To this end, 

workgroup members formed an Analytical Methods Subteam to address homeland security methods 

issues. 

The Analytical Methods Subteam recognized that widely different analytical methods are required for 

various phases of environmental sample analyses in support of homeland security preparation and 

response: (1) ongoing surveillance and monitoring; (2) response and rapid screening for determining 

whether an event has occurred; (3) preliminary site characterizations to determine the extent and type of 

contamination; and (4) confirmatory laboratory analyses to plan, implement, and evaluate the 

effectiveness of site remediation. Figure 1-1 represents these analytical phases. EPA’s Standardized 

Analytical Methods for Environmental Restoration Following Homeland Security Events (SAM) provides 

information for analytical methods to be applied during the “Site Remediation” phase. 

 SAM Revision 5.0 1 September 29, 2009 
Section 1 – Introduction 

Figure 1-1. Environmental Evaluation Analytical Process Roadmap for Homeland 

Security Events 

Surveillance and Monitoring 

Immediate Response/ 

Credibility Determination 

Preliminary Site 

Characterization 

SAM 

(Standardized Analytical Methods for Environmental 

Restoration Following Homeland Security Events) 

Site Remediation 

Assessment Cleanup Clearance 

 SAM Revision 5.0 2 September 29, 2009 
Section 2 – Background

Section 2.0: Background 

In support of this document, EPA periodically assembles methods experts from within EPA and other 

federal agencies to review methods and, if necessary, revise the methods listed. SAM identifies a single 

method or method group per analyte/sample type to ensure a consistent analytical approach across 

multiple laboratories when analyzing environmental samples following an event. Method selection is 

based on consideration of specific criteria that emphasize method performance and include existing 

laboratory capabilities, laboratory capacity, method applicability to multiple environmental sample types, 

and method applicability to multiple SAM analytes. For some analytes, the preferred method is a clear 

choice; for others, competing criteria make the choice more difficult. Final method selections are based 

on technical recommendations from the SAM work groups. For analytes where limited laboratory 

testing/experience exists, such as chemical warfare agents, methods were selected based on their 

applicability to similar chemicals (e.g., nerve agents and some pesticides). In these cases, laboratory 

studies to test the ability of the selected method to measure the target analyte(s) are either underway or 

planned. Figure 2-1 summarizes steps and provides the criteria used during the SAM method selection 

process. It is important to note that the method selection criteria included in this figure are listed in nonhierarchical order and, in some cases, only a subset of the criteria was considered. 

Since 2004, EPA’s National Homeland Security Research Center (NHSRC) has brought together experts 

from across EPA and its sister agencies to develop this compendium of analytical methods to be used 

when analyzing environmental samples, and to address site characterization, remediation and clearance 

following future homeland security events. Participants have included representatives from EPA program 

offices, EPA regions, EPA laboratories, Centers for Disease Control and Prevention (CDC), Food and 

Drug Administration (FDA), Department of Homeland Security (DHS), Federal Bureau of Investigation 

(FBI), Department of Defense (DoD), Department of Agriculture (USDA), and U.S. Geological Survey 

(USGS). Methodologies were considered for chemical and biological agents of concern in the types of 

environmental samples that would be anticipated. The primary objective of this effort was to identify 

appropriate SAM Analytical Methods Subteam consensus methods that represent a balance between 

providing existing, documented, determinative techniques and providing consistent and valid analytical 

results. 

A survey of available confirmatory analytical methods for approximately 120 biological and chemical 

analytes was conducted using existing resources including the following: 

C National Environmental Methods Index (NEMI) and NEMI for Chemical, Biological, and 

Radiological Methods (NEMI-CBR) 

C Environmental Monitoring Method Index (EMMI) 

C EPA Test Methods Index 

C EPA Office of Solid Waste SW-846 Methods 

C EPA Microbiological Methods 

C National Institute for Occupational Safety and Health (NIOSH) Manual of Analytical Methods 

(NMAM) 

C Occupational Safety and Health Administration (OSHA) Index of Sampling and Analytical 

Methods 

C AOAC International 

C ASTM International 

C International Organization for Standardization (ISO) methods 

C Standard Methods for the Examination of Water and Wastewater 

C PubMED Literature Database 

In September 2004, EPA published Standardized Analytical Methods for Use During Homeland Security 

Events, Revision 1.0 (SAM, Revision 1.0, EPA/600/R-04/126), which provided a list of analytical and 

sample preparation methods that were selected for measurement of 82 chemical analytes in 

 SAM Revision 5.0 3 September 29, 2009 
Section 2 – Background

aqueous/liquid, solid, oily solid, and air samples, and 27 biological analytes in water, dust, and aerosol 

samples. During 2005, SAM was expanded to include radioisotopes, several persistent chemical warfare 

agent degradation products, a drinking water sample type, methods for determination of the viability of 

biological organisms, and a separate section for biotoxin analytes. Where necessary, the methods 

included in SAM Revision 1.0 were updated to reflect more recent or appropriate methodologies. Similar 

efforts to those used for method selection during development of SAM Revision 1.0 were undertaken to 

select and include methods for measurement of radioisotopes and chemical warfare agent degradation 

products in all sample types, for measurement of CBR analytes in drinking water, and to determine the 

viability of biological organisms. These additional analytes and the corresponding methods selected were 

included in SAM Revision 2.0. 

During 2006, SAM was revised further to incorporate analytes included on updated federal agency lists, 

provide additional or more current method listings for target analytes, incorporate explosives into the 

chemical analytes listing, combine identification and viability methods information for pathogens, and 

address comments from EPA Science Advisory Board’s Homeland Security Advisory Committee1

 to 

clarify the intended use of the document. These changes were included in SAM Revision 3.0 

(Standardized Analytical Methods for Environmental Restoration Following Homeland Security Events, 

February 2007 / EPA/600/R-07/015). SAM Revision 3.0 included a new title to emphasize the intended 

use of SAM methods for analysis during environmental restoration activities. Following publication of 

SAM Revision 3.0, SAM workgroups updated the document to include the addition of several chemical 

analytes, one radionuclide, and one biotoxin, along with corresponding selected methods, and provided 

the updated documents as SAM Revision 3.1 (November 2007 / EPA/600/R-07/136). In 2007, NHSRC 

also developed a Web-based version of the SAM document to allow users and other stakeholders to 

search for specific needs and to submit questions and comments regarding the information. 

NHSRC is continuing to convene SAM Technical Workgroups at least once per year, to evaluate and, if 

necessary, update the analytes and methods that are listed. SAM Revision 4.0 included the addition of a 

wipe sample type for chemical analytes and several polymerase chain reaction (PCR) methods for 

pathogens. SAM Revision 5.0 reflects the addition of a drinking water sample type for biotoxins. Both 

Revisions 4.0 and 5.0 also reflect the addition of several chemical and radiochemical analytes. 

1

EPA Science Advisory Board’s Homeland Security Advisory Committee: 

http://yosemite.epa.gov/sab/sabpeople.nsf/WebCommittees/BOARD 

 SAM Revision 5.0 4 September 29, 2009 
Step 2 

Step 1 

Is there an EPA 

published method for
 

measurement of the 

analyte in the sample 

type of interest? 

Step 3 

Step 4 

Is there a method that has been 

developed and published by another 

federal agency or Voluntary Consensus 

Standard Body (VCSB) for measurement 

of the analyte in the sample type of 

interest? 

Repeat Steps 1 - 4 to identify methods that measure 

analytes similar to the analyte of concern 

NO 

NO 

NO 

NO 

YES 

YES 

YES 

If no methods are available, prioritize 

for further research 

Is there an EPA, 

federal, or VCSB method 

that has been developed for 

measurement of the analyte 

in another environmental 

sample type? 

Are there procedures 

described and supported by 

data in a peer-reviewed 

journal article for 

measurement of the analyte 

in the sample type of 

interest? 

YES 

Evaluate method against 

selection criteria 

Use the following criteria as guidelines to assess which method is most 

appropriate for inclusion in SAM: 

• Has the method been tested/approved by issuing program office? 

• Has the method been evaluated based on reliability, performance criteria 

(e.g., sensitivity, specificity, false positives/false negatives, precision, 

recovery)? 

• Is the method appropriate for measurement of this analyte in the sample 

type of interest to assess extent of contamination and decontamination 

effectiveness? 

• Has the method been tested for the specific intended use? 

• Is the existing lab capacity (i.e., equipment, number of labs, cost) suitable 

for implementation of the method? 

• Is the required equipment readily available? 

• Is the method capable of determining viability of an organism? 

• What is the time required for analysis? 

• Are reagents, standards, controls, etc., available and accessible? 

• Is specific and/or unique training required? 

• Are large sample volumes required? 

• Are analytical costs high? 

• Has the method already been selected for other SAM analytes? 

• Are modifications needed to accommodate the analytes or sample types? 

Select method for inclusion in SAM 

Figure 2-1. SAM Method Selection Process 

Section 2 – Background

 SAM Revision 5.0 5 September 29, 2009 
Section 2 – Background

 SAM Revision 5.0 6 September 29, 2009 
Section 3 – Scope and Application

Section 3.0: Scope and Application 

The premise and purpose of this document is to standardize the analytical methods that will be used in 

cases when multiple laboratories are called on to analyze environmental samples following a homeland 

security event (i.e., CBR crime/attack). The document also is intended as a tool that will be available to 

assist state and local laboratories in planning for and analyzing environmental samples following a 

homeland security event. The methods presented in this document should be used to: 

•	 Determine the extent of site contamination (assumes early responders have identified contaminants 

prior to EPA’s remediation effort), and 

•	 Confirm effectiveness of decontamination in support of site clearance decisions. 

The methods provided are limited to those that would be used to determine, to the extent possible within 

analytical limitations, the presence of chemical, radiochemical, pathogen, and biotoxin analytes of 

concern and their concentrations in environmental media. The methods include detailed laboratory 

procedures for confirming the identification of analytes and determining their concentrations in 

environmental samples. The methods, therefore, are not designed to be used for rapid or immediate 

response or for conducting an initial evaluation (triage or screening) of suspected material to determine if 

it poses an immediate danger or should be analyzed in specially designed, highly secure facilities. This 

document also is not intended to provide information regarding sample collection activities or equipment. 

Methods for addressing these needs are and will be the subject of other efforts. 

Methods are provided in this document as corresponding to specific analyte/sample type combinations 

that are listed in Appendices A (chemical), B (radiochemical), C (pathogen), and D (biotoxin). 

Summaries of each method are provided in numerical order by the developing agency, throughout 

Sections 5.2 (chemical methods), 6.2 (radiochemical methods), 7.2 (pathogen methods), and 8.2 (biotoxin 

methods). 

It is important to note that, in some cases, the methods included in this document have not been fully 

validated for the analyte/sample type combination(s) for which they have been selected. The 

information contained in this document represents the latest step in an ongoing effort by EPA’s 

NHSRC to provide standardized analytical methods for use by those laboratories tasked with 

performing confirmatory analyses on environmental samples in support of EPA restoration efforts 

following a homeland security incident. The information also can be found on the SAM Web site 

(www.epa.gov/sam), which provides searchable links to supporting information based on SAM 

analytes and the analytical methods listed. 

Although at this time, some of the methods listed have not been fully validated for a particular 

analyte (e.g., analytes not explicitly identified in the method) or sample type, the methods are 

considered to contain the most appropriate currently available techniques. Unless a published 

method listed in this document states specific applicability to the analyte/sample type combination 

for which it has been selected, it should be assumed that method testing is needed, and adjustments 

may be required to accurately account for variations in analyte characteristics, environmental 

samples, analytical interferences, and target risk levels. 

Many of the SAM analytes have only recently become an environmental concern. EPA is actively 

pursuing development and validation of Standard Analytical Protocols (SAPs) based on the methods 

listed, including optimization of procedures for measuring target analytes or agents. In those cases 

where method procedures are determined to be insufficient for a particular situation, EPA will 

provide guidelines regarding appropriate actions. This will be an ongoing process as EPA will strive 

to establish a consistent level of validation for all listed analytes. 

 SAM Revision 5.0	 7 September 29, 2009 
Section 3 – Scope and Application

EPA recognizes that specification of a single method may limit laboratory capacity and techniques that 

may be needed to evaluate difficult samples. In those cases where method procedures are determined to 

be insufficient for a particular situation, EPA will provide guidelines regarding appropriate actions (see 

list of contacts in Section 4). Where further development and testing are necessary, EPA is developing 

and validating SAPs based on the methods that are listed in this document. Once validation is complete, 

data regarding the resulting method performance and data quality objectives will be available. The SAM 

document and corresponding SAPs will be reviewed frequently. EPA plans to continue to update the 

SAM document to address the needs of homeland security, to reflect improvements in analytical 

methodology and new technologies, and to incorporate changes in analytes based on needs. EPA also 

anticipates that addenda may be generated to provide guidelines regarding issues that currently are not 

addressed by this document. Any deviations from the methods referenced in this document should be 

coordinated with the appropriate point(s) of contact identified in Section 4. 

Participants in the chemical, radiochemical, pathogen, and biotoxin work groups, including 

representatives from the EPA, CDC, FDA, DHS, FBI, DoD, USDA, and USGS evaluated the suitability 

of existing methodologies and selected this set of methods for use by those laboratories that support EPA 

environmental restoration efforts in an emergency. EPA recognizes that this advanced selection of such 

methods may pose potential risks, including the following: 

•	 Selecting technologies that may not be the most cost-effective technologies currently available for 

addressing the particular situation at hand; 

•	 Selecting methodologies that may not be appropriate for use in responding to a particular 

emergency because EPA did not anticipate having to analyze for a particular analyte or 

analyte/sample type combination; and 

•	 Preventing development and adoption of new and better measurement technologies. 

To address these potential risks as soon as possible, EPA plans to take several steps. These include the 

following: 

•	 Developing and specifying measurement quality objectives for all analyte/sample type 

combinations listed in this document. This includes required minimum standards of accuracy (bias 

and precision) and sensitivity for the analysis of samples that support the data quality needs of the 

particular stage of the emergency response/recovery process); 

•	 Specifying guidelines for ensuring the analytical methods listed provide results that are consistent 

with and support their intended use as indicated in SAM; 

•	 Working with other government agencies and the private sector to establish a laboratory network to 

ensure that laboratories, selected to assist EPA and its federal, state, and local partners in 

responding to homeland security events, have the requisite expertise and systems to perform this 

type of testing; and 

•	 Continuing to work with multiple agencies and stakeholders to update SAM and supporting 

documents periodically. 

Public officials must accurately assess all of the activities that are needed concerning site contamination 

following an emergency situation. These activities include initial assessment of potential site 

contamination for determination of immediate public and environmental risk, determination of the extent 

of contamination, and full remediation of the site. EPA recognizes that having data of known and 

documented quality is critical in making proper decisions during each of these activities. Data quality 

 SAM Revision 5.0	 8 September 29, 2009 
Section 3 – Scope and Application

objectives (DQOs) must be established for each response activity2. These DQOs are based upon needs 

for both quality and response time. During initial assessments, time is of utmost importance and DQOs 

must be established that weigh the need for rapid analytical response (e.g., using screening methods) 

against the need for very high quality data (confirmational methods such as those listed in SAM). Many 

of the methods listed in this document include quality control (QC) requirements for collecting and 

analyzing samples. EPA will assess these QC requirements to ensure analytical data quality supports 

decisions concerning site remediation and release. These QC requirements may be adjusted as necessary 

to maximize data and decision quality. Specific QC considerations and recommendations for analysis of 

samples for chemical, radiochemical, pathogen, and biotoxin analytes are provided in each corresponding 

section of this document (i.e., Sections 5.1.2, 6.1.2, 7.1.2, and 8.1.2, respectively). 

2

 Information regarding EPA’s DQO process, considerations, and planning is available at: 

http://www.epa.gov/QUALITY/dqos.html. 

 SAM Revision 5.0 9 September 29, 2009 
Section 3 – Scope and Application

 SAM Revision 5.0 10 September 29, 2009 
Section 4 – Points of Contact 

Section 4.0: Points of Contact 

Questions concerning this document, or the methods identified in this document, should be addressed to 

the appropriate point(s) of contact identified below. These contacts should be consulted regarding any 

method deviations or modifications, sample problems or interferences, QC requirements, or the use of 

potential alternative methods. As previously indicated, any deviations from the recommended method(s) 

should be reported immediately to ensure data comparability is maintained when responding to homeland 

security events. In addition, general questions and comments can be submitted via the SAM Web site 

(www.epa.gov/sam). 

General 

Rob Rothman - Primary 

National Homeland Security Research Center 

U.S. EPA ORD (NG16) 

26 West Martin Luther King Jr. Drive 

Cincinnati, OH 45268 

(513) 569-7187 

rothman.rob@epa.gov 

Romy Lee - Alternate 

National Homeland Security Research Center 

U.S. EPA ORD (NG16) 

26 West Martin Luther King Jr. Drive 

Cincinnati, OH 45268 

(513) 569-7016 

lee.romy@epa.gov 

Chemical Methods 

Steve Reimer - Primary 

U.S. EPA Region 10 - Manchester Laboratory 

7411 Beach Drive East 

Port Orchard, WA 98366 

(360) 871-8718 

reimer.steve@epa.gov 

Matthew Magnuson - Alternate 

National Homeland Security Research Center 

U.S. EPA ORD (NG16) 

26 West Martin Luther King Jr. Drive 

Cincinnati, OH 45268 

(513) 569-7321 

magnuson.matthew@epa.gov 

Radiochemical Methods 

John Griggs - Primary 

U.S. EPA Office of Radiation and Indoor Air 

Environmental Laboratory 

540 South Morris Avenue 

Montgomery, AL 36115-2601 

(334) 270-3450 

griggs.john@epa.gov 

Kathy Hall - Alternate 

National Homeland Security Research Center 

U.S. EPA ORD (NG16) 

26 West Martin Luther King Jr. Drive 

Cincinnati, OH 45268 

(513) 379-5260 

hall.kathy@epa.gov 

Pathogen Methods 

Michele Burgess - Primary 

U.S. EPA OSWER 

Ariel Rios Building - 5104A 

1200 Pennsylvania Avenue, NW 

Washington, DC 20460 

(202) 564-8006 

burgess.michele@epa.gov 

Sanjiv Shah - Alternate 

National Homeland Security Research Center 

U.S. EPA ORD - 8801RR 

1200 Pennsylvania Avenue, NW 

Washington, DC 20460 

(202) 564-9522 

shah.sanjiv@epa.gov 

Biotoxins Methods 

Matthew Magnuson - Primary 

National Homeland Security Research Center 

U.S. EPA ORD (NG16) 

26 West Martin Luther King Jr. Drive 

Cincinnati, OH 45268 

(513) 569-7321 

magnuson.matthew@epa.gov 

Sanjiv Shah - Alternate 

National Homeland Security Research Center 

U.S. EPA ORD - 8801RR 

1200 Pennsylvania Avenue, NW 

Washington, DC 20460 

(202) 564-9522 

shah.sanjiv@epa.gov 

 SAM Revision 5.0 11 September 29, 2009 
Section 4 – Points of Contact 

 SAM Revision 5.0 12 September 29, 2009 
Section 5 – Selected Chemical Methods

Section 5.0: Selected Chemical Methods 

Appendix A provides a list of methods to be used in analyzing environmental samples for chemical 

contaminants during remediation activities that result from a homeland security event. Methods are listed 

for each analyte and for each sample type that potentially may need to be measured and analyzed when 

responding to an environmental emergency. Procedures from peer-reviewed journal articles are listed for 

those analyte-sample type combinations where methods are not available. Once standard procedures are 

available, the literature references will be replaced. 

Please note: This section provides guidance for selecting chemical methods that have a high likelihood 

of assuring analytical consistency when laboratories are faced with a large scale environmental 

restoration crisis. Not all methods have been verified for the analyte/sample type combination listed in 

Appendix A. Please refer to the specified method to identify analyte/sample type combinations that 

have been verified. Any questions regarding information discussed in this section should be addressed 

to the appropriate contact(s) listed in Section 4. 

Appendix A is sorted alphabetically by analyte and includes the following information: 

•	 Analyte(s). The component, contaminant, or constituent of interest. 

•	 Chemical Abstracts Service Registration Number (CAS RN). A unique identifier for chemical 

substances that provides an unambiguous way to identify a chemical or molecular structure when 

there are many possible systematic, generic, or trivial names. 

•	 Determinative technique. An analytical instrument or technique used to determine the quantity and 

identification of compounds or components in a sample. 

•	 Method type. Two method types (sample preparation and determinative) are used to complete 

sample analysis. In some cases, a single method contains information for both sample preparation 

and determinative procedures. In most instances, however, two separate methods may need to be 

used in conjunction. 

•	 Solid samples. The recommended method/procedure to identify and measure the analyte of interest 

in solid phase samples. 

•	 Non-aqueous liquid/organic solid samples. The recommended method/procedure to identify and 

measure the analyte of interest in non-aqueous liquid/organic phase samples. An organic solid 

sample is a solid that completely dissolves in an organic solvent and leaves no solid residue. 

•	 Aqueous liquid samples. The recommended method/procedure to identify and measure the analyte 

of interest in aqueous liquid phase samples. 

•	 Drinking water samples. The recommended method/procedure to identify and measure the analyte 

of interest in drinking water samples. 

•	 Air samples. The recommended method/procedure to identify and measure the analyte of interest in 

air samples. 

•	 Wipe samples. The recommended method/procedure to identify and measure the analyte of interest 

in wipes used to collect a sample from a surface. 

Following a homeland security event, it is assumed that only those areas with contamination greater than 

pre-existing/naturally prevalent levels commonly found in the environment would be subject to 

remediation. Dependent on site- and event-specific goals, investigation of background levels using 

methods listed in Appendix A is recommended. 

 SAM Revision 5.0	 13 September 29, 2009 
Section 5 – Selected Chemical Methods

5.1 General Guidelines 

This section provides a general overview of how to identify the appropriate chemical method(s) for a 

given analyte-sample type combination, as well as recommendations for QC procedures. 

For additional information on the properties of the chemicals listed in Appendix A, TOXNET 

(http://toxnet.nlm.nih.gov/index.html), a cluster of databases on toxicology, hazardous chemicals, and 

related areas maintained by the National Library of Medicine, is an excellent resource. Additional 

resources include: 

•	 SRC’s PHYSPROP (http://srcinc.com/what-we-do/product.aspx?id=133) and CHEMFATE, part of 

the Environmental Fate Database supported by EPA (http://srcinc.com/what-wedo/product.aspx?id=132&terms=Environmental+Fate+and+Exposure). 

•	 INCHEM at http://www.inchem.org/ contains both chemical and toxicity information. 

•	 The Registry of Toxic Effects of Chemical Substances (RTECS) database can be accessed via the 

NIOSH Web site at http://www.cdc.gov/niosh/rtecs/default.html for toxicity information. 

•	 EPA’s Integrated Risk Information System (IRIS): http://www.epa.gov/iris/ contains toxicity 

information (searchable on TOXNET). 

•	 Forensic Science and Communications published by the Laboratory Division of the FBI. 

http://www.fbi.gov/hq/lab/fsc/current/backissu.htm. 

•	 Joint Research Centre/Institute for Health & Consumer Protection: http://ecb.jrc.it and 

http://ecb.jrc.it/testing-methods/ containing information regarding European Directive 67/548/EEC 

and Annex V. 

Additional research on chemical contaminants is ongoing within EPA. Databases to manage this 

information are currently under development. 

5.1.1 Standard Operating Procedures for Identifying Chemical Methods 

To determine the appropriate method to be used on an environmental sample, locate the analyte of 

concern under the “Analyte(s)” column in Appendix A: Chemical Methods under. After locating the 

analyte of concern, continue across the table to identify the appropriate determinative technique (e.g., 

high performance liquid chromatography [HPLC], gas chromatography – mass spectrometry [GC-MS]), 

then identify the appropriate sample preparation and determinative method(s) for the sample type of 

interest (solid, non-aqueous liquid/organic solid, aqueous liquid, drinking water, air, or wipe). In some 

cases, two methods (sample preparation and determinative) are needed to complete sample analysis. 

Sections 5.2.1 through 5.2.92 below provide summaries of the sample preparation and determinative 

methods listed in Appendix A. Once a method has been identified in Appendix A, Table 5-1 can be used 

to locate the method summary. 

Table 5-1. Chemical Methods and Corresponding Text Section Numbers 

Analyte CAS RN Method Section 

Acephate 30560-19-1 

Chromatographia. 2006. 63(5/6): 

233–237 5.2.89 

Journal of Chromatography A. 

2007. 1154(1): 3–25 5.2.90 

 SAM Revision 5.0	 14 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte CAS RN Method Section 

Acrylamide 79-06-1 

3570 (EPA SW-846) 5.2.22 

8290A Appendix A (EPA SW-846) 5.2.38 

Acrylonitrile 107-13-1 8316 (EPA SW-846) 5.2.40 

PV2004 (OSHA) 5.2.79 

531.2 (EPA OW) 5.2.12 

Aldicarb (Temik) 116-06-3 3570 (EPA SW-846) 5.2.22 

Aldicarb sulfone 1646-88-4 8290A Appendix A (EPA SW-846) 5.2.38 

8318A (EPA SW-846) 5.2.41 

Aldicarb sulfoxide 1646-87-3 MS014 (EPA CRL) 5.2.44 

5601 (NIOSH) 5.2.61 

3585 (EPA SW-846) 5.2.25 

5030C (EPA SW-846) 5.2.26 

Allyl alcohol 107-18-6 5035A (EPA SW-846) 5.2.27 

8260C (EPA SW-846) 5.2.36 

TO-15 (EPA ORD) 5.2.55 

3535A (EPA SW-846) 5.2.19 

4-Aminopyridine 504-24-5 3570 (EPA SW-846) 5.2.22 

8290A Appendix A (EPA SW-846) 5.2.38 

8330B (EPA SW-846) 5.2.43 

350.1 (EPA OW) 5.2.8 

Ammonia 7664-41-7 6015 (NIOSH) 5.2.67 

4500-NH3 B (SM) 5.2.84 

4500-NH3 G (SM) 5.2.85 

200.7 (EPA OW) 5.2.2 

200.8 (EPA OW) 5.2.3 

3031 (EPA SW-846) 5.2.16 

Ammonium metavanadate 7803-55-6 3050B (EPA SW-846) 5.2.17 

Arsenic, Total 7440-38-2 6010C (EPA SW-846) 5.2.28 

6020A (EPA SW-846) 5.2.29 

Arsenic trioxide 1327-53-3 IO-3.1 (EPA ORD) 5.2.50 

IO-3.4 (EPA ORD) 5.2.51 

IO-3.5 (EPA ORD) 5.2.52 

9102 (NIOSH) 5.2.72 

200.7 (EPA OW) 5.2.2 

200.8 (EPA OW) 5.2.3 

Arsine 7784-42-1 3050B (EPA SW-846) 5.2.17 

7010 (EPA SW-846) 5.2.30 

6001 (NIOSH) 5.2.62 

9102 (NIOSH) 5.2.72 

D5755-03 (ASTM) 5.2.81 

Asbestos 1332-21-4 D6480-05 (ASTM) 5.2.82 

10312:1995 (ISO) 5.2.83 

Boron trifluoride 7637-07-2 ID216SG (OSHA) 5.2.78 

 SAM Revision 5.0 15 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte CAS RN Method Section 

3520C (EPA SW-846) 5.2.18 

3535A (EPA SW-846) 5.2.19 

3541 (EPA SW-846) 5.2.20 

Brodifacoum 56073-10-0 3545A (EPA SW-846) 5.2.21 

3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8290A Appendix A (EPA SW-846) 5.2.38 

8321B (EPA SW-846) 5.2.42 

3520C (EPA SW-846) 5.2.18 

3535A (EPA SW-846) 5.2.19 

3541 (EPA SW-846) 5.2.20 

3545A (EPA SW-846) 5.2.21 

Bromadiolone 28772-56-7 3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8290A Appendix A (EPA SW-846) 5.2.38 

8321B (EPA SW-846) 5.2.42 

MS014 (EPA CRL) 5.2.44 

3520C (EPA SW-846) 5.2.18 

3535A (EPA SW-846) 5.2.19 

3541 (EPA SW-846) 5.2.20 

3545A (EPA SW-846) 5.2.21 

BZ [Quinuclidinyl benzilate] 6581-06-2 3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8290A Appendix A (EPA SW-846) 5.2.38 

8321B (EPA SW-846) 5.2.42 

TO-10A (EPA ORD) 5.2.54 

200.7 (EPA OW) 5.2.2 

200.8 (EPA OW) 5.2.3 

3031 (EPA SW-846) 5.2.16 

3050B (EPA SW-846) 5.2.17 

Calcium arsenate 7778-44-1 6010C (EPA SW-846) 5.2.28 

6020A (EPA SW-846) 5.2.29 

IO-3.1 (EPA ORD) 5.2.50 

IO-3.4 (EPA ORD) 5.2.51 

IO-3.5 (EPA ORD) 5.2.52 

9102 (NIOSH) 5.2.72 

531.2 (EPA OW) 5.2.12 

3570 (EPA SW-846) 5.2.22 

Carbofuran (Furadan) 1563-66-2 8290A Appendix A (EPA SW-846) 5.2.38 

8318A (EPA SW-846) 5.2.41 

MS014 (EPA CRL) 5.2.44 

5601 (NIOSH) 5.2.61 

 SAM Revision 5.0 16 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte CAS RN Method Section 

3520C (EPA SW-846) 5.2.18 

3535A (EPA SW-846) 5.2.19 

3541 (EPA SW-846) 5.2.20 

Carfentanil 59708-52-0 3545A (EPA SW-846) 5.2.21 

3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8290A Appendix A (EPA SW-846) 5.2.38 

8321B (EPA SW-846) 5.2.42 

524.2 (EPA OW) 5.2.10 

3585 (EPA SW-846) 5.2.25 

Carbon disulfide 75-15-0 5030C (EPA SW-846) 5.2.26 

5035A (EPA SW-846) 5.2.27 

8260C (EPA SW-846) 5.2.36 

TO-15 (EPA ORD) 5.2.55 

3520C (EPA SW-846) 5.2.18 

3535A (EPA SW-846) 5.2.19 

3541 (EPA SW-846) 5.2.20 

3545A (EPA SW-846) 5.2.21 

Chlorfenvinphos 470-90-6 3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

TO-10A (EPA ORD) 5.2.54 

Chlorine 7782-50-5 4500-Cl G (SM) 5.2.86 

Analyst. 1999. 124: 1853–1857 5.2.87 

3585 (EPA SW-846) 5.2.25 

5030C (EPA SW-846) 5.2.26 

2-Chloroethanol 107-07-3 5035A (EPA SW-846) 5.2.27 

8260C (EPA SW-846) 5.2.36 

2513 (NIOSH) 5.2.58 

3520C (EPA SW-846) 5.2.18 

3535A (EPA SW-846) 5.2.19 

3541 (EPA SW-846) 5.2.20 

3545A (EPA SW-846) 5.2.21 

3-Chloro-1,2-propanediol 96-24-2 3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

TO-10A (EPA ORD) 5.2.54 

551.1 (EPA OW) 5.2.14 

3545A (EPA SW-846) 5.2.21 

3570 (EPA SW-846) 5.2.22 

Chloropicrin 76-06-2 3580A (EPA SW-846) 5.2.24 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

PV2103 (OSHA) 5.2.80 

 SAM Revision 5.0 17 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte CAS RN Method Section 

3520C (EPA SW-846) 5.2.18 

3535A (EPA SW-846) 5.2.19 

3541 (EPA SW-846) 5.2.20 

Chlorosarin 1445-76-7 3545A (EPA SW-846) 5.2.21 

Chlorosoman 7040-57-5 

3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

TO-10A (EPA ORD) 5.2.54 

200.7 (EPA OW) 5.2.2 

200.8 (EPA OW) 5.2.3 

3031 (EPA SW-846) 5.2.16 

3050B (EPA SW-846) 5.2.17 

2-Chlorovinylarsonous acid (2-CVAA) 85090-33-1 6010C (EPA SW-846) 5.2.28 

6020A (EPA SW-846) 5.2.29 

IO-3.1 (EPA ORD) 5.2.50 

IO-3.4 (EPA ORD) 5.2.51 

IO-3.5 (EPA ORD) 5.2.52 

9102 (NIOSH) 5.2.72 

3520C (EPA SW-846) 5.2.18 

3535A (EPA SW-846) 5.2.19 

3541 (EPA SW-846) 5.2.20 

Chlorpyrifos 

Chlorpyrifos oxon 

2921-88-2 

5598-15-2 

3545A (EPA SW-846) 5.2.21 

3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

TO-10A (EPA ORD) 5.2.54 

3535A (EPA SW-846) 5.2.19 

3545A (EPA SW-846) 5.2.21 

Crimidine 535-89-7 3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

Cyanide, Amenable to chlorination NA RLAB Method 3135.2I 5.2.49 

335.4 (EPA OW) 5.2.7 

Cyanide, Total 57-12-5 ILM05.3 CN (EPA CLP) 5.2.48 

6010 (NIOSH) 5.2.65 

3585 (EPA SW-846) 5.2.25 

5030C (EPA SW-846) 5.2.26 

Cyanogen chloride 506-77-4 5035A (EPA SW-846) 5.2.27 

8260C (EPA SW-846) 5.2.36 

TO-15 (EPA ORD) 5.2.55 

 SAM Revision 5.0 18 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte CAS RN Method Section 

3520C (EPA SW-846) 5.2.18 

3535A (EPA SW-846) 5.2.19 

3541 (EPA SW-846) 5.2.20 

3545A (EPA SW-846) 5.2.21 

Cyclohexyl sarin (GF) 329-99-7 3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

TO-10A (EPA ORD) 5.2.54 

524.2 (EPA OW) 5.2.10 

3585 (EPA SW-846) 5.2.25 

1,2-Dichloroethane 107-06-2 5030C (EPA SW-846) 5.2.26 

5035A (EPA SW-846) 5.2.27 

8260C (EPA SW-846) 5.2.36 

TO-15 (EPA ORD) 5.2.55 

525.2 (EPA OW) 5.2.11 

3535A (EPA SW-846) 5.2.19 

3545A (EPA SW-846) 5.2.21 

Dichlorvos 62-73-7 3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

TO-10A (EPA ORD) 5.2.54 

3535A (EPA SW-846) 5.2.19 

3545A (EPA SW-846) 5.2.21 

3570 (EPA SW-846) 5.2.22 

Dicrotophos 141-66-2 3580A (EPA SW-846) 5.2.24 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

TO-10A (EPA ORD) 5.2.54 

3520C (EPA SW-846) 5.2.18 

3535A (EPA SW-846) 5.2.19 

3541 (EPA SW-846) 5.2.20 

Diesel range organics NA 3545A (EPA SW-846) 5.2.21 

3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8015C (EPA SW-846) 5.2.35 

8290A Appendix A (EPA SW-846) 5.2.38 

3535A (EPA SW-846) 5.2.19 

3545A (EPA SW-846) 5.2.21 

3570 (EPA SW-846) 5.2.22 

Diisopropyl methylphosphonate (DIMP) 1445-75-6 3580A (EPA SW-846) 5.2.24 

8290A Appendix A (EPA SW-846) 5.2.38 

8321B (EPA SW-846) 5.2.42 

MS017 (EPA CRL) 5.2.47 

TO-10A (EPA ORD) 5.2.54 

 SAM Revision 5.0 19 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte CAS RN Method Section 

3535A (EPA SW-846) 5.2.19 

3545A (EPA SW-846) 5.2.21 

3570 (EPA SW-846) 5.2.22 

Dimethylphosphite 868-85-9 3580A (EPA SW-846) 5.2.24 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

TO-10A (EPA ORD) 5.2.54 

3535A (EPA SW-846) 5.2.19 

3545A (EPA SW-846) 5.2.21 

3570 (EPA SW-846) 5.2.22 

Dimethylphosphoramidic acid 33876-51-6 3580A (EPA SW-846) 5.2.24 

8290A Appendix A (EPA SW-846) 5.2.38 

8321B (EPA SW-846) 5.2.42 

TO-10A (EPA ORD) 5.2.54 

3520C (EPA SW-846) 5.2.18 

3535A (EPA SW-846) 5.2.19 

3541 (EPA SW-846) 5.2.20 

Diphacinone 82-66-6 3545A (EPA SW-846) 5.2.21 

3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8290A Appendix A (EPA SW-846) 5.2.38 

8321B (EPA SW-846) 5.2.42 

525.2 (EPA OW) 5.2.11 

3541 (EPA SW-846) 5.2.20 

Disulfoton 298-04-4 

3545A (EPA SW-846) 5.2.21 

3570 (EPA SW-846) 5.2.22 

Disulfoton sulfoxide 2497-07-6 3580A (EPA SW-846) 5.2.24 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

5600 (NIOSH) 5.2.60 

3535A (EPA SW-846) 5.2.19 

3545A (EPA SW-846) 5.2.21 

1,4-Dithiane 505-29-3 3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

3535A (EPA SW-846) 5.2.19 

3545A (EPA SW-846) 5.2.21 

EA2192 [Diisopropylaminoethyl methylthiolophosphonate] 73207-98-4 

3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8290A Appendix A (EPA SW-846) 5.2.38 

8321B (EPA SW-846) 5.2.42 

TO-10A (EPA ORD) 5.2.54 

 SAM Revision 5.0 20 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte CAS RN Method Section 

3535A (EPA SW-846) 5.2.19 

3545A (EPA SW-846) 5.2.21 

3570 (EPA SW-846) 5.2.22 

Ethyl methylphosphonic acid (EMPA) 1832-53-7 3580A (EPA SW-846) 5.2.24 

8290A Appendix A (EPA SW-846) 5.2.38 

8321B (EPA SW-846) 5.2.42 

MS017 (EPA CRL) 5.2.47 

TO-10A (EPA ORD) 5.2.54 

3535A (EPA SW-846) 5.2.19 

3545A (EPA SW-846) 5.2.21 

Ethyldichloroarsine (ED) 598-14-1 3580A (EPA SW-846) 5.2.24 

8270D (EPA SW-846) 5.2.37 

TO-15 (EPA ORD) 5.2.55 

9102 (NIOSH) 5.2.72 

3520C (EPA SW-846) 5.2.18 

3535A (EPA SW-846) 5.2.19 

3541 (EPA SW-846) 5.2.20 

3545A (EPA SW-846) 5.2.21 

N-Ethyldiethanolamine (EDEA) 139-87-7 3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8290A Appendix A (EPA SW-846) 5.2.38 

8321B (EPA SW-846) 5.2.42 

MS016 (EPA CRL) 5.2.46 

TO-10A (EPA ORD) 5.2.54 

3585 (EPA SW-846) 5.2.25 

5030C (EPA SW-846) 5.2.26 

Ethylene oxide 75-21-8 5035A (EPA SW-846) 5.2.27 

8260C (EPA SW-846) 5.2.36 

TO-15 (EPA ORD) 5.2.55 

525.2 (EPA OW) 5.2.11 

3520C (EPA SW-846) 5.2.18 

3535A (EPA SW-846) 5.2.19 

3541 (EPA SW-846) 5.2.20 

Fenamiphos 22224-92-6 3545A (EPA SW-846) 5.2.21 

3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

TO-10A (EPA ORD) 5.2.54 

3520C (EPA SW-846) 5.2.18 

3535A (EPA SW-846) 5.2.19 

3541 (EPA SW-846) 5.2.20 

Fentanyl 437-38-7 3545A (EPA SW-846) 5.2.21 

3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8290A Appendix A (EPA SW-846) 5.2.38 

8321B (EPA SW-846) 5.2.42 

 SAM Revision 5.0 21 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte CAS RN Method Section 

Fluoride 16984-48-8 300.1, Rev 1.0 (EPA OW) 5.2.6 

Fluoroacetamide 640-19-7 Journal of Chromatography B. 

2008. 876, 103–108 5.2.91 

300.1, Rev 1.0 (EPA OW) 5.2.6 

3570 (EPA SW-846) 5.2.22 

Fluoroacetic acid and fluoroacetate salts NA 8290A Appendix A (EPA SW-846) 5.2.38 

S301-1 (NIOSH) 5.2.73 

Analytical Letters. 1994. 27(14): 

2703–2718 5.2.88 

3585 (EPA SW-846) 5.2.25 

5030C (EPA SW-846) 5.2.26 

2-Fluoroethanol 371-62-0 5035A (EPA SW-846) 5.2.27 

8260C (EPA SW-846) 5.2.36 

2513 (NIOSH) 5.2.58 

3570 (EPA SW-846) 5.2.22 

Formaldehyde 50-00-0 8290A Appendix A (EPA SW-846) 5.2.38 

8315A (EPA SW-846) 5.2.39 

2016 (NIOSH) 5.2.57 

3570 (EPA SW-846) 5.2.22 

3585 (EPA SW-846) 5.2.25 

Gasoline range organics NA 5030C (EPA SW-846) 5.2.26 

5035A (EPA SW-846) 5.2.27 

8015C (EPA SW-846) 5.2.35 

8290A Appendix A (EPA SW-846) 5.2.38 

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) 121-82-4 

3535A (EPA SW-846) 5.2.19 

3570 (EPA SW-846) 5.2.22 

Hexamethylenetriperoxidediamine (HMTD) 283-66-9 8290A Appendix A (EPA SW-846) 5.2.38 

8330B (EPA SW-846) 5.2.43 

Hydrogen bromide 

Hydrogen chloride 

10035-10-6 

7647-01-0 7903 (NIOSH) 5.2.69 

Hydrogen cyanide 74-90-8 6010 (NIOSH) 5.2.65 

Hydrogen fluoride 7664-39-3 7903 (NIOSH) 5.2.69 

Hydrogen sulfide 7783-06-4 6013 (NIOSH) 5.2.66 

3535A (EPA SW-846) 5.2.19 

3545A (EPA SW-846) 5.2.21 

3570 (EPA SW-846) 5.2.22 

Isopropyl methylphosphonic acid (IMPA) 1832-54-8 3580A (EPA SW-846) 5.2.24 

8290A Appendix A (EPA SW-846) 5.2.38 

8321B (EPA SW-846) 5.2.42 

MS017 (EPA CRL) 5.2.47 

TO-10A (EPA ORD) 5.2.54 

3570 (EPA SW-846) 5.2.22 

3585 (EPA SW-846) 5.2.25 

Kerosene 64742-81-0 5030C (EPA SW-846) 5.2.26 

5035A (EPA SW-846) 5.2.27 

8015C (EPA SW-846) 5.2.35 

8290A Appendix A (EPA SW-846) 5.2.38 

 SAM Revision 5.0 22 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte CAS RN Method Section 

200.7 (EPA OW) 5.2.2 

Lead arsenate 7645-25-2 200.8 (EPA OW) 5.2.3 

Lewisite 1 (L-1) [2-chlorovinyldichloroarsine] 541-25-3 

3031 (EPA SW-846) 5.2.16 

3050B (EPA SW-846) 5.2.17 

Lewisite 2 (L-2) [bis(2­ 40334-69-8 6010C (EPA SW-846) 5.2.28 

chlorovinyl)chloroarsine] 6020A (EPA SW-846) 5.2.29 

Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine] 40334-70-1 IO-3.1 (EPA ORD) 5.2.50 

IO-3.4 (EPA ORD) 5.2.51 

Lewisite oxide 1306-02-1 IO-3.5 (EPA ORD) 5.2.52 

9102 (NIOSH) 5.2.72 

245.1 (EPA OW) 5.2.4 

Mercuric chloride 7487-94-7 7473 (EPA SW-846) 5.2.33 

9102 (NIOSH) 5.2.72 

245.1 (EPA OW) 5.2.4 

Mercury, Total 7439-97-6 7473 (EPA SW-846) 5.2.33 

IO-5 (EPA ORD) 5.2.53 

9102 (NIOSH) 5.2.72 

Methamidophos 10265-92-6 

Chromatographia. 2006. 63(5/6): 

233–237 5.2.89 

Journal of Chromatography A. 

2007. 1154(1): 3–25 5.2.90 

531.2 (EPA OW) 5.2.12 

3570 (EPA SW-846) 5.2.22 

Methomyl 16752-77-5 8290A Appendix A (EPA SW-846) 5.2.38 

8318A (EPA SW-846) 5.2.41 

MS014 (EPA CRL) 5.2.44 

5601 (NIOSH) 5.2.61 

245.1 (EPA OW) 5.2.4 

Methoxyethylmercuric acetate 151-38-2 7473 (EPA SW-846) 5.2.33 

IO-5 (EPA ORD) 5.2.53 

9102 (NIOSH) 5.2.72 

3570 (EPA SW-846) 5.2.22 

Methyl acrylonitrile 126-98-7 8290A Appendix A (EPA SW-846) 5.2.38 

8316 (EPA SW-846 5.2.40 

PV2004 (OSHA) 5.2.79 

300.1, Rev 1.0 (EPA OW) 5.2.6 

3570 (EPA SW-846) 5.2.22 

Methyl fluoroacetate 453-18-9 8290A Appendix A (EPA SW-846) 5.2.38 

S301-1 (NIOSH) 5.2.73 

Analytical Letters. 1994. 27(14): 

2703–2718 5.2.88 

 SAM Revision 5.0 23 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte CAS RN Method Section 

3520C (EPA SW-846) 5.2.18 

3535A (EPA SW-846) 5.2.19 

3541 (EPA SW-846) 5.2.20 

3545A (EPA SW-846) 5.2.21 

Methyl hydrazine 60-34-4 3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

3510 (NIOSH) 5.2.59 

Methyl isocyanate 624-83-9 OSHA 54 5.2.75 

3535A (EPA SW-846) 5.2.19 

3545A (EPA SW-846) 5.2.21 

Methyl paraoxon 

Methyl parathion 

950-35-6 

298-00-0 

3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

TO-10A (EPA ORD) 5.2.54 

Methylamine 74-89-5 OSHA 40 5.2.74 

3520C (EPA SW-846) 5.2.18 

3535A (EPA SW-846) 5.2.19 

3541 (EPA SW-846) 5.2.20 

3545A (EPA SW-846) 5.2.21 

N-Methyldiethanolamine (MDEA) 105-59-9 3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8290A Appendix A (EPA SW-846) 5.2.38 

8321B (EPA SW-846) 5.2.42 

MS016 (EPA CRL) 5.2.46 

TO-10A (EPA ORD) 5.2.54 

3520C (EPA SW-846) 5.2.18 

3535A (EPA SW-846) 5.2.19 

3541 (EPA SW-846) 5.2.20 

1-Methylethyl ester ethylphosphonofluoridic 

acid (GE) 1189-87-3 

3545A (EPA SW-846) 5.2.21 

3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

TO-10A (EPA ORD) 5.2.54 

3535A (EPA SW-846) 5.2.19 

3545A (EPA SW-846) 5.2.21 

3570 (EPA SW-846) 5.2.22 

Methylphosphonic acid (MPA) 993-13-5 3580A (EPA SW-846) 5.2.24 

8290A Appendix A (EPA SW-846) 5.2.38 

8321B (EPA SW-846) 5.2.42 

MS017 (EPA CRL) 5.2.47 

TO-10A (EPA ORD) 5.2.54 

 SAM Revision 5.0 24 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte CAS RN Method Section 

525.2 (EPA OW) 5.2.11 

3535A (EPA SW-846) 5.2.19 

3545A (EPA SW-846) 5.2.21 

Mevinphos 7786-34-7 3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

TO-10A (EPA ORD) 5.2.54 

3535A (EPA SW-846) 5.2.19 

3545A (EPA SW-846) 5.2.21 

3570 (EPA SW-846) 5.2.22 

Monocrotophos 6923-22-4 3580A (EPA SW-846) 5.2.24 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

TO-10A (EPA ORD) 5.2.54 

3520C (EPA SW-846) 5.2.18 

Mustard, nitrogen (HN-1) [bis(2-chloroethyl)­

ethylamine] 538-07-8 

3535A (EPA SW-846) 5.2.19 

3541 (EPA SW-846) 5.2.20 

Mustard, nitrogen (HN-2) [2,2’-dichloro-Nmethyldiethylamine N,N-bis(2-chloroethyl)- 

methylamine] 

51-75-2 

3545A (EPA SW-846) 5.2.21 

3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

Mustard, nitrogen (HN-3) [tris(2-chloroethyl)- 

amine] 555-77-1 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

TO-10A (EPA ORD) 5.2.54 

3570 (EPA SW-846) 5.2.22 

3571 (EPA SW-846) 5.2.23 

Mustard, sulfur / Mustard gas (HD) 505-60-2 8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

TO-10A (EPA ORD) 5.2.54 

3535A (EPA SW-846) 5.2.19 

3545A (EPA SW-846) 5.2.21 

Nicotine compounds 54-11-5 3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

3535A (EPA SW-846) 5.2.19 

Octahydro-1,3,5,7-tetranitro-1,3,5,7­

tetrazocine (HMX) 2691-41-0 3570 (EPA SW-846) 5.2.22 

8290A Appendix A (EPA SW-846) 5.2.38 

8330B (EPA SW-846) 5.2.43 

507 (EPA OW) 5.2.9 

614 (EPA OW) 5.2.15 

3541 (EPA SW-846) 5.2.20 

3545A (EPA SW-846) 5.2.21 

Organophosphate pesticides, NOS NA 3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

5600 (NIOSH) 5.2.60 

 SAM Revision 5.0 25 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte CAS RN Method Section 

252.2 (EPA OW) 5.2.5 

3050B (EPA SW-846) 5.2.17 

Osmium tetroxide 20816-12-0 6010C (EPA SW-846) 5.2.28 

IO-3.1 (EPA ORD) 5.2.50 

IO-3.4 (EPA ORD) 5.2.51 

9102 (NIOSH) 5.2.72 

531.2 (EPA OW) 5.2.12 

3570 (EPA SW-846) 5.2.22 

Oxamyl 23135-22-0 8290A Appendix A (EPA SW-846) 5.2.38 

8318A (EPA SW-846) 5.2.41 

MS014 (EPA CRL) 5.2.44 

5601 (NIOSH) 5.2.61 

Paraquat 4685-14-7 549.2 (EPA OW) 5.2.13 

3520C (EPA SW-846) 5.2.18 

3535A (EPA SW-846) 5.2.19 

3541 (EPA SW-846) 5.2.20 

Paraoxon 311-45-5 3545A (EPA SW-846) 5.2.21 

Parathion 56-38-2 

3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

TO-10A (EPA ORD) 5.2.54 

3535A (EPA SW-846) 5.2.19 

Pentaerythritol tetranitrate (PETN) 78-11-5 3570 (EPA SW-846) 5.2.22 

8290A Appendix A (EPA SW-846) 5.2.38 

8330B (EPA SW-846) 5.2.43 

3535A (EPA SW-846) 5.2.19 

3545A (EPA SW-846) 5.2.21 

3570 (EPA SW-846) 5.2.22 

Phencyclidine 77-10-1 3580A (EPA SW-846) 5.2.24 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

TO-10A (EPA ORD) 5.2.54 

3520C (EPA SW-846) 5.2.18 

3535A (EPA SW-846) 5.2.19 

3541 (EPA SW-846) 5.2.20 

3545A (EPA SW-846) 5.2.21 

Phenol 108-95-2 3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

TO-10A (EPA ORD) 5.2.54 

 SAM Revision 5.0 26 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte CAS RN Method Section 

3535A (EPA SW-846) 5.2.19 

Phorate 298-02-2 3545A (EPA SW-846) 5.2.21 

3570 (EPA SW-846) 5.2.22 

Phorate sulfone 2588-04-7 3580A (EPA SW-846) 5.2.24 

Phorate sulfoxide 2588-03-6 8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

TO-10A (EPA ORD) 5.2.54 

Phosgene 75-44-5 OSHA 61 5.2.76 

3520C (EPA SW-846) 5.2.18 

3535A (EPA SW-846) 5.2.19 

3541 (EPA SW-846) 5.2.20 

3545A (EPA SW-846) 5.2.21 

Phosphamidon 13171-21-6 3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

TO-10A (EPA ORD) 5.2.54 

Phosphine 7803-51-2 6002 (NIOSH) 5.2.63 

Phosphorus trichloride 7719-12-2 6402 (NIOSH) 5.2.68 

3535A (EPA SW-846) 5.2.19 

3545A (EPA SW-846) 5.2.21 

3570 (EPA SW-846) 5.2.22 

Pinacolyl methyl phosphonic acid (PMPA) 616-52-4 3580A (EPA SW-846) 5.2.24 

8290A Appendix A (EPA SW-846) 5.2.38 

8321B (EPA SW-846) 5.2.42 

MS017 (EPA CRL) 5.2.47 

TO-10A (EPA ORD) 5.2.54 

3585 (EPA SW-846) 5.2.25 

5030C (EPA SW-846) 5.2.26 

Propylene oxide 75-56-9 5035A (EPA SW-846) 5.2.27 

8260C (EPA SW-846) 5.2.36 

1612 (NIOSH) 5.2.56 

3520C (EPA SW-846) 5.2.18 

3535A (EPA SW-846) 5.2.19 

3541 (EPA SW-846) 5.2.20 

R 33 (VR) [methylphosphonothioic acid, S­

[2-(diethylamino)ethyl] O-2-methylpropyl 

ester] 

159939-87-4 

3545A (EPA SW-846) 5.2.21 

3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

TO-10A (EPA ORD) 5.2.54 

3570 (EPA SW-846) 5.2.22 

3571 (EPA SW-846) 5.2.23 

Sarin (GB) 107-44-8 8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

TO-10A (EPA ORD) 5.2.54 

 SAM Revision 5.0 27 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte CAS RN Method Section 

525.2 (EPA OW) 5.2.11 

3520C (EPA SW-846) 5.2.18 

3535A (EPA SW-846) 5.2.19 

3541 (EPA SW-846) 5.2.20 

Semivolatile organic compounds, NOS NA 3545A (EPA SW-846) 5.2.21 

3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

TO-10A (EPA ORD) 5.2.54 

200.7 (EPA OW) 5.2.2 

200.8 (EPA OW) 5.2.3 

3031 (EPA SW-846) 5.2.16 

3050B (EPA SW-846) 5.2.17 

Sodium arsenite 7784-46-5 6010C (EPA SW-846) 5.2.28 

6020A (EPA SW-846) 5.2.29 

IO-3.1 (EPA ORD) 5.2.50 

IO-3.4 (EPA ORD) 5.2.51 

IO-3.5 (EPA ORD) 5.2.52 

9102 (NIOSH) 5.2.72 

300.1, Rev 1.0 (EPA OW) 5.2.6 

Sodium azide 26628-22-8 

3580A (EPA SW-846) 5.2.24 

ID-211 (OSHA) 5.2.77 

Journal of Forensic Sciences. 

1998. 43(1): 200–202 5.2.92 

3535A (EPA SW-846) 5.2.19 

3545A (EPA SW-846) 5.2.21 

3570 (EPA SW-846) 5.2.22 

Soman (GD) 96-64-0 3580A (EPA SW-846) 5.2.24 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

TO-10A (EPA ORD) 5.2.54 

3535A (EPA SW-846) 5.2.19 

3545A (EPA SW-846) 5.2.21 

Strychnine 57-24-9 3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

Sulfur dioxide 7446-09-5 6004 (NIOSH) 5.2.64 

Sulfur trioxide 7446-11-9 Method 8 (EPA) 5.2.1 

3535A (EPA SW-846) 5.2.19 

3545A (EPA SW-846) 5.2.21 

3570 (EPA SW-846) 5.2.22 

Tabun (GA) 77-81-6 3580A (EPA SW-846) 5.2.24 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

TO-10A (EPA ORD) 5.2.54 

 SAM Revision 5.0 28 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte CAS RN Method Section 

3535A (EPA SW-846) 5.2.19 

3545A (EPA SW-846) 5.2.21 

3570 (EPA SW-846) 5.2.22 

Tetraethyl pyrophosphate 107-49-3 3580A (EPA SW-846) 5.2.24 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

TO-10A (EPA ORD) 5.2.54 

3520C (EPA SW-846) 5.2.18 

3535A (EPA SW-846) 5.2.19 

3541 (EPA SW-846) 5.2.20 

3545A (EPA SW-846) 5.2.21 

Tetramethylenedisulfotetramine 80-12-6 3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

TO-10A (EPA ORD) 5.2.54 

200.7 (EPA OW) 5.2.2 

200.8 (EPA OW) 5.2.3 

3031 (EPA SW-846) 5.2.16 

3050B (EPA SW-846) 5.2.17 

Thallium sulfate 10031-59-1 6010C (EPA SW-846) 5.2.28 

6020A (EPA SW-846) 5.2.29 

IO-3.1 (EPA ORD) 5.2.50 

IO-3.4 (EPA ORD) 5.2.51 

IO-3.5 (EPA ORD) 5.2.52 

9102 (NIOSH) 5.2.72 

3535A (EPA SW-846) 5.2.19 

3545A (EPA SW-846) 5.2.21 

3570 (EPA SW-846) 5.2.22 

Thiodiglycol (TDG) 111-48-8 3580A (EPA SW-846) 5.2.24 

8290A Appendix A (EPA SW-846) 5.2.38 

8321B (EPA SW-846) 5.2.42 

MS015 (EPA CRL) 5.2.45 

TO-10A (EPA ORD) 5.2.54 

531.2 (EPA OW) 5.2.12 

3520C (EPA SW-846) 5.2.18 

3535A (EPA SW-846) 5.2.19 

3541 (EPA SW-846) 5.2.20 

Thiofanox 39196-18-4 3545A (EPA SW-846) 5.2.21 

3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8290A Appendix A (EPA SW-846) 5.2.38 

8321B (EPA SW-846) 5.2.42 

5601 (NIOSH) 5.2.61 

 SAM Revision 5.0 29 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte CAS RN Method Section 

3535A (EPA SW-846) 5.2.19 

3545A (EPA SW-846) 5.2.21 

1,4-Thioxane 15980-15-1 3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

3050B (EPA SW-846) 5.2.17 

Titanium tetrachloride 7550-45-0 6010C (EPA SW-846) 5.2.28 

6020A (EPA SW-846) 5.2.29 

9102 (NIOSH) 5.2.72 

3520C (EPA SW-846) 5.2.18 

3535A (EPA SW-846) 5.2.19 

3541 (EPA SW-846) 5.2.20 

3545A (EPA SW-846) 5.2.21 

Triethanolamine (TEA) 102-71-6 3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8290A Appendix A (EPA SW-846) 5.2.38 

8321B (EPA SW-846) 5.2.42 

MS016 (EPA CRL) 5.2.46 

TO-10A (EPA ORD) 5.2.54 

3535A (EPA SW-846) 5.2.19 

3545A (EPA SW-846) 5.2.21 

3570 (EPA SW-846) 5.2.22 

Trimethyl phosphite 121-45-9 3580A (EPA SW-846) 5.2.24 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

TO-10A (EPA ORD) 5.2.54 

1,3,5-Trinitrobenzene (1,3,5-TNB) 99-35-4 

3535A (EPA SW-846) 5.2.19 

3570 (EPA SW-846) 5.2.22 

2,4,6-Trinitrotoluene (2,4,6-TNT) 118-96-7 8290A Appendix A (EPA SW-846) 5.2.38 

8330B (EPA SW-846) 5.2.43 

200.7 (EPA OW) 5.2.2 

200.8 (EPA OW) 5.2.3 

3031 (EPA SW-846) 5.2.16 

3050B (EPA SW-846) 5.2.17 

Vanadium pentoxide 1314-62-1 6010C (EPA SW-846) 5.2.28 

6020A (EPA SW-846) 5.2.29 

IO-3.1 (EPA ORD) 5.2.50 

IO-3.4 (EPA ORD) 5.2.51 

IO-3.5 (EPA ORD) 5.2.52 

9102 (NIOSH) 5.2.72 

 SAM Revision 5.0 30 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte CAS RN Method Section 

VE [phosphonothioic acid, ethyl-, S-(2­

(diethylamino)ethyl) O-ethyl ester] 

VG [phosphonothioic acid, S-(2­

(diethylamino)ethyl) O,O-diethyl ester] 

VM [phosphonothioic acid, methyl-,S-(2­

(diethylamino)ethyl) O-ethyl ester] 

21738-25-0 

78-53-5 

21770-86-5 

3520C (EPA SW-846) 5.2.18 

3535A (EPA SW-846) 5.2.19 

3541 (EPA SW-846) 5.2.20 

3545A (EPA SW-846) 5.2.21 

3570 (EPA SW-846) 5.2.22 

3580A (EPA SW-846) 5.2.24 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

TO-10A (EPA ORD) 5.2.54 

VX [O-ethyl-S-(2­

diisopropylaminoethyl)methylphosphonothiolate] 50782-69-9 

3570 (EPA SW-846) 5.2.22 

3571 (EPA SW-846) 5.2.23 

8270D (EPA SW-846) 5.2.37 

8290A Appendix A (EPA SW-846) 5.2.38 

TO-10A (EPA ORD) 5.2.54 

White phosphorus 12185-10-3 

3570 (EPA SW-846) 5.2.22 

7580 (EPA SW-846) 5.2.34 

8290A Appendix A (EPA SW-846) 5.2.38 

7905 (NIOSH) 5.2.70 

The following analytes should be prepared and/or analyzed by the following methods only if problems (e.g., 

insufficient recovery, interferences) occur when using the sample preparation/determinative techniques identified 

for these analytes in Appendix A. 

Allyl alcohol 107-18-6 TO-10A (EPA ORD) 5.2.54 

BZ [Quinuclidinyl benzilate] 6581-06-2 8270D (EPA SW-846) 5.2.37 

3-Chloro-1,2-propanediol 96-24-2 TO-15 (EPA ORD) 5.2.55 

Chlorosarin 

Chlorosoman 

1445-76-7 

7040-57-5 TO-15 (EPA ORD) 5.2.55 

Crimidine 535-89-7 8321B (EPA SW-846) 5.2.42 

Diisopropyl methylphosphonate (DIMP) 1445-75-6 8270D (EPA SW-846) 5.2.37 

TO-15 (EPA ORD) 5.2.55 

Dimethylphosphoramidic acid 33876-51-6 8270D (EPA SW-846) 5.2.37 

EA2192 [Diisopropylaminoethyl methylthiolophosphonate] 

Ethyl methylphosphonic acid (EMPA) 

73207-98-4 

1832-53-7 

8270D (EPA SW-846) 5.2.37 

Hydrogen fluoride 7664-39-3 7906 (NIOSH) 5.2.71 

Isopropyl methylphosphonic acid (IMPA) 1832-54-8 8270D (EPA SW-846) 5.2.37 

Mercuric chloride 

Mercury, Total 

7487-94-7 

7439-97-6 

7470A (EPA SW-846) 5.2.31 

7471B (EPA SW-846) 5.2.32 

Methamidophos 10265-92-6 5600 (NIOSH) 5.2.60 

Methoxyethylmercuric acetate 151-38-2 7470A (EPA SW-846) 5.2.31 

7471B (EPA SW-846) 5.2.32 

1-Methylethyl ester ethylphosphonofluoridic 

acid (GE) 1189-87-3 TO-15 (EPA ORD) 5.2.55 

Methylphosphonic acid (MPA) 993-13-5 8270D (EPA SW-846) 5.2.37 

Pinacolyl methyl phosphonic acid (PMPA) 616-52-4 8270D (EPA SW-846) 5.2.37 

Sarin (GB) 

Soman (GD) 

107-44-8 

96-64-0 TO-15 (EPA ORD) 5.2.55 

 SAM Revision 5.0 31 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte CAS RN Method Section 

3585 (EPA SW-846) 5.2.25 

1,4-Thioxane 15980-15-1 5030C (EPA SW-846) 5.2.26 

5035A (EPA SW-846) 5.2.27 

8260C (EPA SW-846) 5.2.36 

Method summaries are listed in order of method selection hierarchy (see Figure 2-1), starting with EPA 

methods, followed by methods from other federal agencies, voluntary consensus standard bodies 

(VCSBs), and literature references. Methods are listed in numerical order under each publisher. Where 

available, a direct link to the full text of the method is provided in the method summary. For additional 

information on preparation procedures and methods available through consensus standards organizations, 

please use the contact information provided in Table 5-2. 

Table 5-2. Sources of Chemical Methods 

Name Publisher Reference 

NEMI EPA, USGS http://www.nemi.gov 

EPA OW Methods EPA OW http://www.epa.gov/safewater/methods/ 

sourcalt.html 

EPA SW-846 Methods EPA OSWER http://www.epa.gov/epaoswer/hazwaste 

/test/main.htm 

EPA ORD Methods EPA ORD http://www.epa.gov/ttnamti1/ 

EPA Air Toxics Methods EPA OAR http://www.epa.gov/ttn/amtic/airtox.html 

OSHA Methods OSHA http://www.osha.gov/dts/sltc/methods/in 

dex.html 

NIOSH Methods NIOSH http://www.cdc.gov/niosh/nmam/ 

Standard Methods for the Examination 

of Water and Wastewater (SM), 21st 

Edition, 2005* 

American Public Health 

Association (APHA), 

American Water Works 

Association (AWWA), and 

Water Environment 

Federation (WEF) 

http://www.standardmethods.org 

Annual Book of ASTM Standards* ASTM International http://www.astm.org 

GESTIS Substance Database BGIA http://www.dguv.de/bgia/en/gestis/stoffd 

b/index.jsp 

ISO Methods* ISO http://www.iso.org 

Official Methods of Analysis of AOAC 

International* 

AOAC International http://www.aoac.org 

Analyst Royal Society of Chemistry http://www.rsc.org/Publishing/Journals/ 

AN/ 

Analytical Letters* Taylor & Francis http://www.informaworld.com/smpp/title 

~content=t713597227 

Journal of Chromatography A* Elsevier Science Publishers http://www.elsevier.com/ 

Journal of Forensic Sciences* ASTM International http://www.astm.org 

Chromatographia Vieweg+Teubner http://www.chromatographia.de/ 

* Subscription and/or purchase required. 

 SAM Revision 5.0 32 September 29, 2009 
Section 5 – Selected Chemical Methods

5.1.2 General QC Guidelines for Chemical Methods 

Having analytical data of appropriate quality requires that laboratories: (1) conduct the necessary QC 

activities to ensure that measurement systems are in control and operating correctly; (2) properly 

document results of the analyses; and (3) properly document measurement system evaluation of the 

analysis-specific QC, including corrective actions. In addition to the laboratories being capable of 

generating accurate and precise data during an emergency situation, they must be able to deliver results in 

a timely and efficient manner. Therefore, laboratories must be prepared with calibrated instruments, the 

proper standards, standard analytical procedures, standard operating procedures, and qualified and trained 

staff. Moreover, laboratories also must be capable of providing rapid turnaround of sample analyses and 

data reporting. 

The level or amount of QC needed during sample analysis and reporting depends on the intended purpose 

of the data that are generated (e.g., the decision(s) to be made). The specific needs for data generation 

should be identified. QC requirements and data quality objectives should be derived based on those 

needs, and should be applied consistently across laboratories when multiple laboratories are used. For 

almost all of the chemical warfare agents, most laboratories will not have access to analytical standards 

for calibration and QC. Use of these agents is strictly controlled by the DoD and access is limited. For 

information regarding purchase and distribution of ultradilute agents for laboratory use, please contact 

Terry Smith, EPA's Office of Emergency Management, at (202) 564-2908. 

A minimum set of analytical QC procedures should be planned, documented, and conducted for all 

chemical testing. Some method-specific QC requirements are described in many of the individual 

methods that are cited in this document and will be referenced in any SAPs developed to address specific 

analytes and sample types of concern. Individual methods, sampling and analysis protocols, or 

contractual statements of work should also be consulted to determine if any additional QC might be 

needed. Analytical QC requirements generally consist of analysis of laboratory control samples to 

document whether the analytical system is in control; matrix spikes to identify and quantify measurement 

system accuracy for the media of concern and, at the levels of concern, various blanks as a measure of 

freedom from contamination; as well as matrix spike duplicates or sample replicates to assess data 

precision. 

In general, for measurement of chemical analytes, appropriate QC includes an initial demonstration of 

measurement system capability, as well as ongoing analysis of standards and other samples to ensure the 

continued reliability of the analytical results. Examples of appropriate QC include: 

•	 Demonstration that the measurement system is operating properly: 

< Initial calibration; and 

< Method blanks. 

•	 Demonstration of analytical method suitability for intended use: 

< Detection and quantitation limits; 

< Precision and recovery (verify measurement system has adequate accuracy); and 

< Analyte/matrix/level of concern-specific QC samples (verify that measurement system has 

adequate sensitivity at levels of concern). 

•	 Demonstration of continued analytical method reliability: 

< Matrix spike/matrix spike duplicates (MS/MSDs) (recovery and precision); 

< QC samples (system accuracy and sensitivity at levels of concern); 

< Surrogate spikes (where appropriate); 

< Continuing calibration verification; and 

< Method blanks. 

 SAM Revision 5.0	 33 September 29, 2009 
Section 5 – Selected Chemical Methods

QC tests should be consistent with EPA’s Good Laboratory Practice Standards 

(http://www.epa.gov/oecaerth/monitoring/programs/fifra/glp.html) and be run as frequently as necessary 

to ensure the reliability of analytical results. As with the identification of needed QC samples, the 

frequency of QC sampling should be established based on an evaluation of data quality objectives. The 

type and frequency of QC tests can be refined over time. 

Ensuring data quality also requires that laboratory results are properly assessed and documented. The 

results of the data quality assessment are transmitted to decision makers. This evaluation is as important 

as the data for ensuring informed and effective decisions. While some degree of data evaluation is 

necessary in order to be able to confirm data quality, 100% verification and/or validation is neither 

necessary nor conducive to efficient decision making in emergency situations. The level of such reviews 

should be determined based on the specific situation being assessed and on the corresponding data quality 

objectives. In every case, the levels of QC and data review necessary to support decision making should 

be determined as much in advance of data collection as possible. 

Please note: The appropriate point of contact identified in Section 4 should be consulted regarding 

appropriate quality assurance (QA) and QC procedures prior to sample analysis. These contacts will 

consult with the EPA OSWER coordinator responsible for laboratory activities during the specific event 

to ensure QA/QC procedures are performed consistently across laboratories. OSWER is planning to 

develop QA/QC guidance for laboratory support. EPA program offices will be responsible for ensuring 

that the QA/QC practices are implemented. 

5.1.3 Safety and Waste Management 

It is imperative that safety precautions are used during collection, processing, and analysis of 

environmental samples. Laboratories should have a documented health and safety plan for handling 

samples that may contain the target CBR contaminants. Laboratory staff should be trained in, and need to 

implement, the safety procedures included in the plan. In addition, many of the methods summarized or 

cited in Section 5.2 contain some specific requirements, guidelines, or information regarding safety 

precautions that should be followed when handling or processing environmental samples and reagents. 

These methods also provide information regarding waste management. Other resources that can be 

consulted for additional information include the following: 

C	 CDC – Title 42 of the Code of Federal Regulations part 72 (42 CFR 72). Interstate Shipment of 

Etiologic Agents. 

C	 CDC – 42 CFR part 73. Select Agents and Toxins. 

C	 Department of Transportation (DOT) – 49 CFR part 172. Hazardous Materials Table, Special 

Provisions, Hazardous Materials Communications, Emergency Response Information, and Training 

Requirements. 

C	 EPA – 40 CFR part 260. Hazardous Waste Management System: General. 

C	 EPA – 40 CFR part 270. EPA Administered Permit Programs: The Hazardous Waste Permit 

Program. 

C	 OSHA – 29 CFR part 1910.1450. Occupational Exposure to Hazardous Chemicals in Laboratories. 

C	 OSHA – 29 CFR part 1910.120. Hazardous Waste Operations and Emergency Response. 

Please note that the Electronic Code of Federal Regulations (e-CFR) is available at 

http://ecfr.gpoaccess.gov/. 

 SAM Revision 5.0	 34 September 29, 2009 
Section 5 – Selected Chemical Methods

5.2 Method Summaries 

Summaries for the analytical methods listed in Appendix A are provided in Sections 5.2.1 through 5.2.92. 

These sections contain summary information only, extracted from the selected methods. Each method 

summary contains a table identifying the contaminants in Appendix A to which the method applies, a 

brief description of the analytical method, and a link to, or source for, obtaining a full version of the 

method. The full version of the method should be consulted prior to sample analysis. 

5.2.1 EPA Method 8: Determination of Sulfuric Acid and Sulfur Dioxide Emissions from 

Stationary Sources 

Analyte(s) CAS RN 

Sulfur Trioxide 7446-11-9 

Analysis Purpose: Sample preparation and analysis 

Sample Preparation Technique: Isokinetic extraction 

Determinative Technique: Titrimetry 

Method Developed for: Sulfuric acid, sulfur trioxide, and sulfur dioxide in air 

Method Selected for: SAM lists this method for preparation and analysis of air samples. 

Detection and Quantitation: Collaborative tests have shown that the minimum detectable limit of the 

method is 0.06 mg/m3

 for sulfuric acid. 

Description of Method: A gas sample is extracted isokinetically. Sulfuric acid and sulfur dioxide are 

separated, and both fractions are measured separately by the barium-thorin titration method. Sulfur 

trioxide is measured by the analysis of sulfuric acid. Possible interfering agents include fluorides, free 

ammonia, and dimethyl aniline. 

Special Considerations: Possible interfering agents include fluorides, free ammonia, and dimethyl
 

aniline. 

Source: EPA Emission Measurement Center (EMC) of the Office of Air Quality Planning and Standards 

(OAQPS). “Method 8: Determination of Sulfuric Acid and Sulfur Dioxide Emissions from Stationary 

Sources.” http://www.epa.gov/sam/pdfs/EPA-Method8.pdf
 

5.2.2 EPA Method 200.7: Determination of Metals and Trace Elements in Waters and 

Wastes by Inductively Coupled Plasma-Atomic Emission Spectrometry 

Analyte(s) CAS RN 

Ammonium metavanadate 7803-55-6 

Arsenic, Total 7440-38-2 

Arsenic trioxide 1327-53-3 

Arsine 7784-42-1 

Calcium arsenate 7778-44-1 

2-Chlorovinylarsonous acid (2-CVAA) 85090-33-1 

Lead arsenate 7645-25-2 

 SAM Revision 5.0	 35 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte(s) CAS RN 

Lewisite 1 (L-1) [2-chlorovinyldichloroarsine] 541-25-3 

Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine] 40334-69-8 

Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine] 40334-70-1 

Lewisite oxide 1306-02-1 

Sodium arsenite 7784-46-5 

Thallium sulfate 10031-59-1 

Vanadium pentoxide 1314-62-1 

Analysis Purpose: Sample preparation and analysis
 

Sample Preparation Technique: Acid digestion
 

Determinative Technique: Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES) 

Method Developed for: Determination of metals in solution. This method is a consolidation of existing
 

methods for water, wastewater, and solid wastes. 

Method Selected for: SAM lists this method for preparation and analysis of aqueous liquid and drinking 

water samples. 

Detection and Quantitation: Method detection limits (MDLs) in aqueous samples have been found to
 

be 0.008 mg/L for arsenic, 0.003 mg/L for vanadium, and 0.001 mg/L for thallium.
 

Description of Method: This method will determine metal-containing compounds only as the total metal 

(e.g., total arsenic) in aqueous samples. An aliquot of a well-mixed, homogeneous sample is accurately
 

weighed or measured for sample processing. For total recoverable analysis of a sample containing 

undissolved material, analytes are first solubilized by gentle refluxing with nitric and hydrochloric acids. 

After cooling, the sample is made up to volume, mixed, and centrifuged or allowed to settle overnight 

prior to analysis. For determination of dissolved analytes in a filtered aqueous sample aliquot, or for the 

“direct analysis” total recoverable determination of analytes in drinking water where sample turbidity is < 

1 nephelometric turbidity units (NTU), the sample is made ready for analysis by the addition of nitric 

acid, and then diluted to a predetermined volume and mixed before analysis. The prepared sample is 

analyzed using ICP-AES. Specific analytes targeted by Method 200.7 are listed in Section 1.1 of the 

method. 

Special Considerations: Laboratory testing is currently underway for speciation of lewisite 1 using GCMS techniques. 

Source: EPA. 1994. “Method 200.7: Determination of Metals and Trace Elements in Water and Wastes 

by Inductively Coupled Plasma-Atomic Emission Spectrometry,” Revision 4.4.
 

http://www.epa.gov/sam/pdfs/EPA-200.7.pdf
 

5.2.3 EPA Method 200.8: Determination of Trace Elements in Waters and Wastes by 

Inductively Coupled Plasma-Mass Spectrometry 

Analyte(s) CAS RN 

Ammonium metavanadate 7803-55-6 

Arsenic, Total 7440-38-2 

Arsenic trioxide 1327-53-3 

 SAM Revision 5.0	 36 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte(s) CAS RN 

Arsine 7784-42-1 

Calcium arsenate 7778-44-1 

2-Chlorovinylarsonous acid (2-CVAA) 85090-33-1 

Lead arsenate 7645-25-2 

Lewisite 1 (L-1) [2-chlorovinyldichloroarsine] 541-25-3 

Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine] 40334-69-8 

Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine] 40334-70-1 

Lewisite oxide 1306-02-1 

Sodium arsenite 7784-46-5 

Thallium sulfate 10031-59-1 

Vanadium pentoxide 1314-62-1 

Analysis Purpose: Sample preparation and analysis
 

Sample Preparation Technique: Acid digestion
 

Determinative Technique: Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) 

Method Developed for: Dissolved and total elements in ground water, surface water, drinking water, 

wastewater, sludges, and soils. 

Method Selected for: SAM lists this method for preparation and analysis of aqueous liquid and drinking 

water samples. 

Detection and Quantitation: MDLs for arsenic in aqueous samples have been found to be 1.4 μg/L in
 

scanning mode, and 0.4 μg/L in selected ion monitoring mode. The recommended calibration range is 10 

to 200 μg/L. 

Description of Method: This method will determine metal-containing compounds only as the total metal 

(e.g., total arsenic). An aliquot of a well-mixed, homogeneous sample is accurately weighed or measured 

for sample processing. For total recoverable analysis of a sample containing undissolved material, 

analytes are first solubilized by gentle refluxing with nitric and hydrochloric acids. After cooling, the 

sample is made up to volume, mixed, and centrifuged or allowed to settle overnight prior to analysis. For 

determination of dissolved analytes in a filtered aqueous sample aliquot, or for the “direct analysis” total 

recoverable determination of analytes in drinking water where sample turbidity is < 1 NTU, the sample is
 

made ready for analysis by the addition of nitric acid, and then diluted to a predetermined volume and 

mixed before analysis. The prepared sample is analyzed using ICP-MS. Specific analytes targeted by
 

Method 200.8 are listed in Section 1.1 of the method. 

Special Considerations: Laboratory testing is currently underway for speciation of lewisite 1 using GCMS techniques. 

Source: EPA. 1994. “Method 200.8: Determination of Trace Elements in Waters and Wastes by
 

Inductively Coupled Plasma-Mass Spectrometry,” Revision 5.4. http://www.epa.gov/sam/pdfs/EPA200.8.pdf
 

 SAM Revision 5.0 37 September 29, 2009 
Section 5 – Selected Chemical Methods

5.2.4 EPA Method 245.1: Determination of Mercury in Water by Cold Vapor Atomic 

Absorption Spectrometry (CVAA) 

Analyte(s) CAS RN 

Mercuric chloride 7487-94-7 

Mercury, Total 7439-97-6 

Methoxyethylmercuric acetate 151-38-2 

Analysis Purpose: Sample preparation and analysis
 

Sample Preparation Technique: Acid digestion 

Determinative Technique: Cold vapor atomic absorption (CVAA) 

Method Developed for: Mercury in surface waters. It may be applicable to saline waters, wastewaters, 

effluents, and domestic sewages providing potential interferences are not present.
 

Method Selected for: SAM lists this method for preparation and analysis of drinking water samples. 

Detection and Quantitation: Applicable concentration range is 0.2 to 10.0 μg Hg/L. The detection limit 

for this method is 0.2 μg Hg/L. 

Description of Method: This method will determine mercuric chloride and methoxyethylmercuric 

acetate as total mercury. If dissolved mercury is targeted, the sample is filtered prior to acidification. To 

detect total mercury (inorganic and organic mercury), the sample is treated with potassium permanganate 

and potassium persulfate to oxidize organic mercury compounds prior to analysis. Inorganic mercury is 

reduced to the elemental state (using stannous chloride) and aerated from solution. The mercury vapor
 

passes through a cell positioned in the light path of a CVAA spectrophotometer. The concentration of 

mercury is measured using the CVAA spectrophotometer. 

Source: EPA. 1994. “Method 245.1: Determination of Mercury in Water by Cold Vapor Atomic 

Absorption Spectrometry CVAA).” http://www.epa.gov/sam/pdfs/EPA-245.1.pdf
 

5.2.5 EPA Method 252.2: Osmium (Atomic Absorption, Furnace Technique) 

Analyte(s) CAS RN 

Osmium tetroxide 20816-12-0 

Analysis Purpose: Sample preparation and analysis 

Sample Preparation Technique: Direct aspiration 

Determinative Technique: Graphite furnace atomic absorption spectrophotometry (GFAA) 

Method Developed for: Osmium in drinking, surface, and saline waters, and domestic and industrial 

wastes 

Method Selected for: SAM lists this method for preparation and analysis of aqueous liquid and drinking 

water samples. 

Detection and Quantitation: Detection limit for osmium is 20 μg/L. The optimal applicable 

concentration range is 50 to 500 μg/L. 

Description of Method: This method will determine osmium tetroxide as osmium. Method 252.2 is not 

a stand-alone method in that sections of the method reference Methods of Chemical Analysis of Water and 

Waste, EPA/600/4-79/020, March 1983 (MCAWW). Samples are prepared according to the “direct 

aspiration method” (See Section 9.1 of the Atomic Absorption Methods section of MCAWW, 

http://www.epa.gov/sam/pdfs/MCAWW-9.1.pdf) except that the addition of sulfuric acid is omitted in the 

 SAM Revision 5.0 38 September 29, 2009 
Section 5 – Selected Chemical Methods

final adjustment. If only dissolved osmium is determined, the sample is filtered before acidification with 

nitric acid. For total osmium, the sample is digested with nitric and hydrochloric acids and made up to 

volume. Samples are analyzed according to the “furnace procedure” (see Section 9.3 of the Atomic 

Absorption Methods section of MCAWW, http://www.epa.gov/sam/pdfs/MCAWW-9.3.pdf), using 

GFAA. A representative aliquot of sample is placed in the graphite tube in the furnace, evaporated to 

dryness, chaffed, and atomized. Radiation from an excited element is passed through the vapor 

containing ground state atoms of the element. The intensity of the transmitted radiation decreases in 

proportion to the amount of the ground state element in the vapor. A monochromator isolates the 

characteristic radiation from the hollow cathode lamp and a photosensitive device measures the attenuated 

transmitted radiation. 

Special Considerations: Concerns have been raised regarding the use of nitric acid when analyzing 

samples for osmium tetroxide; hydrochloric acid should be considered and evaluated as a possible 

alternative. 

Source: EPA. 1978. “Method 252.2: Osmium (Atomic Absorption, Furnace Technique).” 

http://www.epa.gov/sam/pdfs/EPA-252.2.pdf 

5.2.6 EPA Method 300.1, Revision 1.0: Determination of Inorganic Anions in Drinking 

Water by Ion Chromatography 

Analyte(s) CAS RN 

Fluoride 16984-48-8 

Fluoroacetic acid and fluoroacetate salts NA 

Methyl fluoroacetate 453-18-9 

Sodium azide (analyze for hydrazoic acid) 26628-22-8 

Analysis Purpose: Sample preparation and/or analysis 

Sample Preparation Technique: For fluoride, use direct injection. For fluoroacetic acid, fluoroacetate 

salts, and methyl fluoroacetate, use Analytical Letters, 1994, 27(14): 2703-2718 (solid and non-aqueous 

liquid/organic solid samples), NIOSH Method S301-1 (air samples), and EPA SW-846 Method 

3570/8290A Appendix A (wipe samples). For sodium azide, use water extraction, filtration, and 

acidification steps from the Journal of Forensic Science, 1998. 43(1):200-202 (solid samples), filtration 

and acidification steps from this journal (aqueous liquid and drinking water samples), and the 

acidification step from the journal with EPA SW-846 Method 3580A (non-aqueous liquid/organic solid 

samples). 

Determinative Technique: Ion chromatography (IC) 

Method Developed for: Inorganic anions in reagent water, surface water, ground water, and finished 

drinking water 

Method Selected for: SAM lists this method for preparation and analysis of aqueous liquid and drinking 

water samples for fluoride, fluoroacetic acid, fluoroacetate salts, and methyl fluoroacetate. It also should 

be used for analysis of solid and non-aqueous liquid/organic solid, air, and/or wipe samples for 

fluoroacetic acid, fluoroacetate salts, methyl fluoroacetate, and sodium azide when appropriate sample 

preparation techniques have been applied. 

Detection and Quantitation: The detection limit for fluoride in reagent water is 0.009 mg/L. The MDL 

varies depending upon the nature of the sample and the specific instrumentation employed. The estimated 

calibration range should not extend over more than 2 orders of magnitude in concentration over the 

expected concentration range of the samples. 

 SAM Revision 5.0	 39 September 29, 2009 
Section 5 – Selected Chemical Methods

Description of Method: This method was developed for analysis of aqueous samples, and can be 

adapted for analysis of prepared non-aqueous liquid/organic solid, solid, and air samples when 

appropriate sample preparation techniques have been applied (see Appendix A). A small volume of an 

aqueous liquid sample (10 μL or 50 μL) is introduced into an ion chromatograph. The volume selected 

depends on the concentration of fluoroacetate ion in the sample. The anions of interest are separated and 

measured, using a system comprising a guard column, analytical column, suppressor device, and 

conductivity detector. The separator columns and guard columns, as well as eluent conditions, are 

identical. To achieve comparable detection limits, an ion chromatographic system must use suppressed 

conductivity detection, be properly maintained, and be capable of yielding a baseline with no more than 5 

nS noise/drift per minute of monitored response over the background conductivity. 

Special Considerations: For sodium azide, if analyses are problematic, refer to column manufacturer 

for alternate conditions. 

Source: EPA. 1997. “Method 300.1: Determination of Inorganic Anions in Drinking Water by Ion 

Chromatography,” Revision 1.0. http://www.epa.gov/sam/pdfs/EPA-300.1.pdf 

5.2.7 EPA Method 335.4: Determination of Total Cyanide by Semi-Automated 

Colorimetry 

Analyte(s) CAS RN 

Cyanide, Total 57-12-5 

Analysis Purpose: Sample preparation and analysis
 

Sample Preparation Technique: Reflux-distillation
 

Determinative Technique: Spectrophotometry
 

Method Developed for: Cyanide in drinking, ground, surface, and saline waters, and domestic and 

industrial wastes 

Method Selected for: SAM lists this method for preparation and analysis of drinking water samples. 

Detection and Quantitation: The applicable range is 5 to 500 μg/L. 

Description of Method: Cyanide is released from cyanide complexes as hydrocyanic acid by manual 

reflux-distillation, and absorbed in a scrubber containing sodium hydroxide solution. The cyanide ion in
 

the absorbing solution is converted to cyanogen chloride by reaction with chloramine-T, which 

subsequently reacts with pyridine and barbituric acid to give a red-colored complex. 

Special Considerations: Some interferences include aldehydes, nitrate-nitrite, and oxidizing agents, 

such as chlorine, thiocyanate, thiosulfate, and sulfide. These interferences can be eliminated or reduced 

by distillation.
 

Source: EPA. 1993. “Method 335.4: Determination of Total Cyanide by Semi-Automated Colorimetry,” 

Revision 1.0. http://www.epa.gov/sam/pdfs/EPA-335.4.pdf
 

5.2.8 EPA Method 350.1: Nitrogen, Ammonia (Colorimetric, Automated Phenate) 

Analyte(s) CAS RN 

Ammonia 7664-41-7 

 SAM Revision 5.0	 40 September 29, 2009 
Section 5 – Selected Chemical Methods

Analysis Purpose: Sample preparation and analysis 

Sample Preparation Technique: Distillation 

Determinative Technique: Spectrophotometry 

Method Developed for: Ammonia in drinking, ground, surface, and saline waters, and domestic and 

industrial wastes 

Method Selected for: SAM lists this method for preparation and analysis of drinking water samples. 

Detection and Quantitation: The working range for ammonia is 0.01 to 2.0 mg/L. 

Description of Method: This method identifies and determines the concentration of ammonia in 

drinking water samples by spectrophotometry. Samples are buffered at a pH of 9.5 with borate buffer to 

decrease hydrolysis of cyanates and organic nitrogen compounds, and are distilled into a solution of boric 

acid. Alkaline phenol and hypochlorite react with ammonia to form indophenol blue that is proportional 

to the ammonia concentration. The blue color formed is intensified with sodium nitroprusside and 

measured spectrophotometrically. 

Source: EPA. 1993. “Method 350.1: Nitrogen, Ammonia (Colorimetric, Automated Phenate),” Revision 

2.0. http://www.epa.gov/sam/pdfs/EPA-350.1.pdf 

5.2.9 EPA Method 507: Determination of Nitrogen- and Phosphorus-Containing 

Pesticides in Water by Gas Chromatography with a Nitrogen-Phosphorus Detector 

Analyte(s) CAS RN 

Organophosphate pesticides, NOS NA 

Analysis Purpose: Sample preparation and analysis 

Sample Preparation Technique: Solvent extraction 

Determinative Technique: Gas chromatography –nitrogen-phosphorus detector (GC-NPD) 

Method Developed for: Nitrogen- and phosphorus-containing pesticides in ground water and finished 

drinking water 

Method Selected for: SAM lists this method for preparation and analysis of drinking water samples. 

Detection and Quantitation: Estimated detection limits (EDLs) and MDLs differ depending on the 

specific pesticide. 

Description of Method: A 1-L sample is extracted with methylene chloride by shaking in a separatory 

funnel or mechanical tumbling in a bottle. The methylene chloride extract is isolated, dried, and 

concentrated to a volume of 5 mL during a solvent exchange to methyl tert-butyl ether (MTBE). The 

concentrations of pesticides in the extract are measured using a capillary column gas chromatography 

(GC) system equipped with a nitrogen-phosphorus detector (NPD). Specific analytes targeted by Method 

507 are listed in Section 1.1 of the method. 

Special Considerations: The presence of organophosphate pesticides should be confirmed by either a 

secondary GC column or by an MS. 

Source: EPA. 1995. “Method 507: Determination of Nitrogen- and Phosphorus-Containing Pesticides in 

Water by Gas Chromatography with a Nitrogen-Phosphorus Detector,” Revision 2.1. 

http://www.epa.gov/sam/pdfs/EPA-507.pdf 

 SAM Revision 5.0	 41 September 29, 2009 
Section 5 – Selected Chemical Methods

5.2.10 EPA Method 524.2: Measurement of Purgeable Organic Compounds in Water by 

Capillary Column Gas Chromatography / Mass Spectrometry 

Analyte(s) CAS RN 

Carbon disulfide 75-15-0 

1,2-Dichloroethane 107-06-2 

Analysis Purpose: Sample preparation and analysis 

Sample Preparation Technique: Purge-and-trap 

Determinative Technique: GC-MS 

Method Developed for: Purgeable volatile organic compounds (VOCs) in surface water, ground water, 

and drinking water in any stage of treatment 

Method Selected for: SAM lists this method for preparation and analysis of drinking water samples. 

Detection and Quantitation: Detection levels for carbon disulfide and 1,2-dichloroethane in reagent 

water have been found to be 0.093 μg/L and 0.02 μg/L, respectively. The applicable concentration range 

of this method is primarily column and matrix dependent, and is approximately 0.02 to 200 μg/L when a 

wide-bore thick-film capillary column is used. Narrow-bore thin-film columns may have a lower 

capacity, which limits the range to approximately 0.02 to 20 μg/L. 

Description of Method: VOCs and surrogates with low water solubility are extracted (purged) from the 

sample matrix by bubbling an inert gas through the aqueous sample. Purged sample components are 

trapped in a tube containing suitable sorbent materials. When purging is complete, the sorbent tube is 

heated and backflushed with helium to desorb the trapped sample components into a capillary GC column 

interfaced to a mass spectrometer (MS). The column is temperature programmed to facilitate the 

separation of the method analytes, which are then detected with the MS. Specific analytes targeted by 

Method 524.2 are listed in Section 1.1 of the method. 

Source: EPA. 1992. “Method 524.2: Measurement of Purgeable Organic Compounds in Water by 

Capillary Column Gas Chromatography/Mass Spectrometry,” Revision 4.0. 

http://www.epa.gov/sam/pdfs/EPA-524.2.pdf 

5.2.11 EPA Method 525.2: Determination of Organic Compounds in Drinking Water by 

Liquid-Solid Extraction and Capillary Column Gas Chromatography / Mass 

Spectrometry 

Analyte(s) CAS RN 

Dichlorvos 62-73-7 

Disulfoton 298-04-4 

Disulfoton sulfoxide 2497-07-6 

Fenamiphos 22224-92-6 

Mevinphos 7786-34-7 

Semivolatile organic compounds, NOS NA 

Analysis Purpose: Sample preparation and analysis 

Sample Preparation Technique: Liquid-solid extraction (LSE) or solid-phase extraction (SPE) 

Determinative Technique: GC-MS 

 SAM Revision 5.0 42 September 29, 2009 
Section 5 – Selected Chemical Methods

Method Developed for: Organic compounds in finished drinking water, source water, or drinking water 

in any treatment stage 

Method Selected for: SAM lists this method for preparation and analysis of aqueous liquid and/or 

drinking water samples. 

Detection and Quantitation: The applicable concentration range for most analytes is 0.1 to 10 μg/L. 

Description of Method: Organic compounds, internal standards, and surrogates are extracted from a 

water sample by passing 1 L of sample through a cartridge or disk containing a solid matrix with 

chemically bonded C18 organic phase (LSE or SPE). The organic compounds are eluted from the LSE 

(SPE) cartridge or disk with small quantities of ethyl acetate followed by methylene chloride. The 

resulting extract is concentrated further by evaporation of some of the solvent. Sample components are 

separated, identified, and measured by injecting an aliquot of the concentrated extract into a high 

resolution fused silica capillary column of a GC-MS system. Specific analytes targeted by Method 525.2 

are listed in Section 1.1 of the method. 

Special Considerations: SPE using C18 resin may not work for certain compounds having high water 

solubility. In these cases, other sample preparation techniques or different SPE resins may be required. 

Source: EPA. 1995. “Method 525.2: Determination of Organic Compounds in Drinking Water by 

Liquid-Solid Extraction and Capillary Column Gas Chromatography/Mass Spectrometry,” Revision 2.0. 

http://www.epa.gov/sam/pdfs/EPA-525.2.pdf 

5.2.12 EPA Method 531.2: Measurement of N-Methylcarbamoyloximes and NMethylcarbamates in Water by Direct Aqueous Injection HPLC with Postcolumn 

Derivatization 

Analyte(s) CAS RN 

Aldicarb (Temik) 116-06-3 

Aldicarb sulfone 1646-88-4 

Aldicarb sulfoxide 1646-87-3 

Carbofuran (Furadan) 1563-66-2 

Methomyl 16752-77-5 

Oxamyl 23135-22-0 

Thiofanox 39196-18-4 

Analysis Purpose: Sample preparation and analysis 

Sample Preparation Technique: Direct injection 

Determinative Technique: HPLC 

Method Developed for: N-methylcarbamoyloximes and N-methylcarbamates in finished drinking 

water 

Method Selected for: SAM lists this method for preparation and analysis of drinking water samples. 

Detection and Quantitation: Detection limits range from 0.026 to 0.115 μg/L. The concentration 

range for target analytes in this method was evaluated between 0.2 μg/L and 10 μg/L. 

Description of Method: An aliquot of sample is measured in a volumetric flask. Samples are preserved, 

spiked with appropriate surrogates and then filtered. Analytes are chromatographically separated by 

injecting a sample aliquot (up to 1000 μL) into a HPLC system equipped with a reverse phase (C18) 

column. After elution from the column, the analytes are hydrolyzed in a post column reaction to form 

 SAM Revision 5.0 43 September 29, 2009 
Section 5 – Selected Chemical Methods

methylamine, which is in turn reacted to form a fluorescent isoindole that is detected by a fluorescence 

(FL) detector. Analytes also are quantitated using the external standard technique. 

Source: EPA. 2001. “Method 531.2: Measurement of N-Methylcarbamoyloximes and NMethylcarbamates in Water by Direct Aqueous Injection HPLC with Postcolumn Derivatization,” 

Revision 1.0. http://www.epa.gov/sam/pdfs/EPA-531.2.pdf 

5.2.13 EPA Method 549.2: Determination of Diquat and Paraquat in Drinking Water by 

Liquid-Solid Extraction and High Performance Liquid Chromatography with 

Ultraviolet Detection 

Analyte(s) CAS RN 

Paraquat 4685-14-7 

Analysis Purpose: Sample preparation and analysis 

Sample Preparation Technique: LSE or SPE 

Determinative Technique: HPLC 

Method Developed for: Diquat and paraquat in drinking water sources and finished drinking water 

Method Selected for: SAM lists this method for preparation and analysis of aqueous liquid and drinking 

water samples. 

Detection and Quantitation: MDL for paraquat is 0.68 μg/L. The analytical range depends on the 

sample matrix and the instrumentation used. 

Description of Method: A 250-mL sample is extracted using a C8 LSE cartridge or a C8 disk that has 

been specially prepared for the reversed-phase, ion-pair mode. The LSE disk or cartridge is eluted with 

acidic aqueous solvent to yield the eluate/extract. An ion-pair reagent is added to the eluate/extract. The 

concentrations of paraquat in the eluate/extract are measured using a HPLC system equipped with an 

ultraviolet (UV) absorbance detector. A photodiode array detector is used to provide simultaneous 

detection and confirmation of the method analytes. 

Source: EPA. 1997. “Method 549.2: Determination of Diquat and Paraquat in Drinking Water by 

Liquid-Solid Extraction and High Performance Liquid Chromatography with Ultraviolet Detection,” 

Revision 1.0. http://www.epa.gov/sam/pdfs/EPA-549.2.pdf 

5.2.14 EPA Method 551.1: Determination of Chlorination Disinfection Byproducts, 

Chlorinated Solvents, and Halogenated Pesticides/Herbicides in Drinking Water by 

Liquid-Liquid Extraction and Gas Chromatography with Electron-Capture 

Detection 

Analyte(s) CAS RN 

Chloropicrin 76-06-2 

Analysis Purpose: Sample preparation and analysis 

Sample Preparation Technique: Solvent extraction 

Determinative Technique: Gas chromatography –electron capture detector (GC-ECD) 

Method Developed for: Chlorination disinfection byproducts, chlorinated solvents, and halogenated 

pesticides/herbicides in finished drinking water, drinking water during intermediate stages of treatment, 

and raw source water 

 SAM Revision 5.0	 44 September 29, 2009 
Section 5 – Selected Chemical Methods

Method Selected for: SAM lists this method for preparation and analysis of aqueous liquid and drinking 

water samples. 

Detection and Quantitation: The EDL using MTBE and ammonium chloride-preserved reagent water 

on a 100% dimethylpolysiloxane (DB-1) column has been found to be 0.014 μg/L. 

Description of Method: This is a GC-ECD method applicable to the determination of halogenated 

analytes in finished drinking water, drinking water during intermediate stages of treatment, and raw 

source water. A 50-mL sample aliquot is extracted with 3 mL of MTBE or 5 mL of pentane. Two μL of 

the extract is then injected into a GC equipped with a fused silica capillary column and linearized ECD 

for separation and analysis. This liquid/liquid extraction technique efficiently extracts a wide boiling 

range of non-polar and polar organic components of the sample. Thus, confirmation is quite important, 

particularly at lower analyte concentrations. A confirmatory column is suggested for this purpose. 

Special Considerations: The presence of chloropicrin should be confirmed by either a secondary GC 

column or by an MS. 

Source: EPA. 1995. “Method 551.1: Determination of Chlorination Disinfection Byproducts, 

Chlorinated Solvents, and Halogenated Pesticides/Herbicides in Drinking Water by Liquid-Liquid 

Extraction and Gas Chromatography with Electron-Capture Detection,” Revision 1.0.
 

http://www.epa.gov/sam/pdfs/EPA-551.1.pdf
 

5.2.15 EPA Method 614: The Determination of Organophosphorus Pesticides in 

Municipal and Industrial Wastewater 

Analyte(s) CAS RN 

Organophosphate pesticides, NOS NA 

Analysis Purpose: Sample preparation and analysis 

Sample Preparation Technique: Solvent extraction 

Determinative Technique: Gas chromatography –flame photometric detector (GC-FPD) 

Method Developed for: Organophosphorus pesticides in municipal and industrial wastewater 

Method Selected for: SAM lists this method for preparation and analysis of aqueous liquid samples. 

Description of Method: This is a GC method applicable to the determination of organophosphate 

pesticides in industrial and municipal discharges using a GC with a phosphorus-specific flame 

photometric detector (FPD) or thermionic bead detector in the nitrogen mode. A measured volume of 

sample, approximately 1 L, is extracted with 15% methylene chloride in hexane using a separatory funnel. 

The extract is dried and concentrated to a volume of 10 mL or less. GC conditions are described for the 

separation and measurement of the compounds in the extract by flame photometric or thermionic bead 

GC. Specific analytes targeted by Method 614 are listed in Section 1.1 of the method. 

Special Considerations: The presence of organophosphate pesticides should be confirmed by either a 

secondary GC column or by an MS. 

Source: EPA. “Method 614: The Determination of Organophosphorus Pesticides in Municipal and 

Industrial Wastewater.” http://www.epa.gov/sam/pdfs/EPA-614.pdf 

 SAM Revision 5.0 45 September 29, 2009 
Section 5 – Selected Chemical Methods

5.2.16 EPA Method 3031 (SW-846): Acid Digestion of Oils for Metals Analysis by Atomic 

Absorption or ICP Spectrometry 

Analyte(s) CAS RN 

Ammonium metavanadate 7803-55-6 

Arsenic, Total 7440-38-2 

Arsenic trioxide 1327-53-3 

Calcium arsenate 7778-44-1 

2-Chlorovinylarsonous acid (2-CVAA) 85090-33-1 

Lead arsenate 7645-25-2 

Lewisite 1 (L-1) [2-chlorovinyldichloroarsine] 541-25-3 

Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine] 40334-69-8 

Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine] 40334-70-1 

Lewisite oxide 1306-02-1 

Sodium arsenite 7784-46-5 

Thallium sulfate 10031-59-1 

Vanadium pentoxide 1314-62-1 

Analysis Purpose: Sample preparation 

Sample Preparation Technique: Acid digestion 

Determinative Technique: EPA SW-846 Method 6010C or Method 6020A 

Method Developed for: Metals in oils, oil sludges, tars, waxes, paints, paint sludges and other viscous 

petroleum products 

Method Selected for: SAM lists this method for preparation of non-aqueous liquid/organic solid 

samples. 

Description of Method: This method is used to prepare samples for the determination of arsenic 

trioxide, lewisite, lewisite degradation products, calcium and lead arsenate, and sodium arsenite as total 

arsenic; thallium sulfate as total thallium; and ammonium metavanadate and vanadium pentoxide as total 

vanadium. A 0.5-g sample of oil, oil sludge, tar, wax, paint, or paint sludge is mixed with potassium 

permanganate and sulfuric acid. The mixture is then treated with nitric and hydrochloric acids, filtered, 

and diluted to volume. Excess manganese may be removed with ammonium hydroxide. Digestates are 

analyzed by Method 6020A or 6010C (SW-846). 

Source: EPA. 1996. “Method 3031 (SW-846): Acid Digestion of Oils for Metals Analysis by Atomic 

Absorption or ICP Spectrometry,” Revision 0. http://www.epa.gov/sam/pdfs/EPA-3031.pdf 

5.2.17 EPA Method 3050B (SW-846): Acid Digestion of Sediments, Sludges, and Soils 

Analyte(s) CAS RN 

Ammonium metavanadate 7803-55-6 

Arsenic, Total 7440-38-2 

 SAM Revision 5.0 46 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte(s) CAS RN 

Arsenic trioxide 1327-53-3 

Arsine 7784-42-1 

Calcium arsenate 7778-44-1 

2-Chlorovinylarsonous acid (2-CVAA) 85090-33-1 

Lead arsenate 7645-25-2 

Lewisite 1 (L-1) [2-chlorovinyldichloroarsine] 541-25-3 

Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine] 40334-69-8 

Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine] 40334-70-1 

Lewisite oxide 1306-02-1 

Osmium tetroxide 20816-12-0 

Sodium arsenite 7784-46-5 

Thallium sulfate 10031-59-1 

Titanium tetrachloride 7550-45-0 

Vanadium pentoxide 1314-62-1 

Analysis Purpose: Sample preparation 

Sample Preparation Technique: Acid digestion 

Determinative Technique: EPA SW-846 Method 6010C, Method 6020A, or Method 7010. Refer to 

Appendix A for which of these determinative methods should be used for a particular analyte. 

Method Developed for: Metals in sediments, sludges, and soil samples 

Method Selected for: SAM lists this method for preparation of solid samples. 

Description of Method: This method is used to prepare samples for the determination of arsenic 

trioxide, arsine, lewisite, lewisite degradation products, calcium and lead arsenate, and sodium arsenite as 

total arsenic; thallium sulfate as total thallium; titanium tetrachloride as titanium; osmium tetroxide as 

osmium; and ammonium metavanadate and vanadium pentoxide as total vanadium. A 1-g to 2-g sample 

is digested with nitric acid and hydrogen peroxide. Sample volumes are reduced, then brought up to a 

final volume of 100 mL. Samples are analyzed for total arsenic, total thallium, total titanium, or total 

vanadium by Method 6010C or 6020A (SW-846); use Method 6010C (SW-846) for total osmium; use 

Method 7010 (SW-846) for arsine. 

Special Considerations: Concerns have been raised regarding the use of nitric acid when analyzing 

samples for osmium tetroxide; hydrochloric acid should be considered and evaluated as a possible 

alternative. 

Source: EPA. 1996. “Method 3050B (SW-846): Acid Digestion of Sediments, Sludges, and Soils,” 

Revision 2. http://www.epa.gov/sam/pdfs/EPA-3050b.pdf 

 SAM Revision 5.0 47 September 29, 2009 
Section 5 – Selected Chemical Methods

5.2.18 EPA Method 3520C (SW-846): Continuous Liquid-Liquid Extraction 

Analyte(s) CAS RN 

Brodifacoum 56073-10-0 

Bromadiolone 28772-56-7 

BZ [Quinuclidinyl benzilate] 6581-06-2 

Carfentanil 59708-52-0 

Chlorfenvinphos 470-90-6 

3-Chloro-1,2-propanediol 96-24-2 

Chlorosarin 1445-76-7 

Chlorosoman 7040-57-5 

Chlorpyrifos 2921-88-2 

Chlorpyrifos oxon 5598-15-2 

Cyclohexyl sarin (GF) 329-99-7 

Diesel range organics NA 

Diphacinone 82-66-6 

N-Ethyldiethanolamine (EDEA) 139-87-7 

Fenamiphos 22224-92-6 

Fentanyl 437-38-7 

Methyl hydrazine 60-34-4 

N-Methyldiethanolamine (MDEA) 105-59-9 

1-Methylethyl ester ethylphosphonofluoridic acid (GE) 1189-87-3 

Mustard, nitrogen (HN-1) [bis(2-chloroethyl)ethylamine] 538-07-8 

Mustard, nitrogen (HN-2) [2,2’-dichloro-Nmethyldiethylamine N,N-bis(2-chloroethyl)methylamine] 51-75-2 

Mustard, nitrogen (HN-3) [tris(2-chloroethyl)amine] 555-77-1 

Paraoxon 311-45-5 

Parathion 56-38-2 

Phenol 108-95-2 

Phosphamidon 13171-21-6 

R 33 (VR) [methylphosphonothioic acid, S-[2­

(diethylamino)ethyl] O-2-methylpropyl ester] 159939-87-4 

Semivolatile organic compounds, NOS NA 

Tetramethylenedisulfotetramine 80-12-6 

 SAM Revision 5.0 48 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte(s) CAS RN 

Thiofanox 39196-18-4 

Triethanolamine (TEA) 102-71-6 

VE [phosphonothioic acid, ethyl-, S-(2­

(diethylamino)ethyl) O-ethyl ester] 21738-25-0 

VG [phosphonothioic acid, S-(2-(diethylamino)ethyl) 

O,O-diethyl ester] 78-53-5 

VM [phosphonothioic acid, methyl-, S-(2­

(diethylamino)ethyl) O-ethyl ester] 21770-86-5 

Analysis Purpose: Sample preparation
 

Sample Preparation Technique: Continuous liquid-liquid extraction (CLLE) 

Determinative Technique: EPA SW-846 Method 8015C, Method 8270D, or Method 8321B. Refer to 

Appendix A for which of these determinative methods should be used for a particular analyte. 

Method Developed for: Organic compounds in aqueous samples 

Method Selected for: SAM lists this method for preparation of aqueous liquid and/or drinking water 

samples. Please note: Drinking water samples for fenamiphos and semivolatile organic compounds 

should be prepared and analyzed by EPA Method 525.2; drinking water samples for thiofanox should be 

prepared and analyzed by EPA Method 531.2; aqueous/liquid samples for bromadiolone should be 

analyzed using EPA CRL MS014; aqueous liquid samples for EDEA, MDEA, and TEA should be 

analyzed using EPA CRL MS016. All other drinking water and aqueous liquid samples should be 

prepared using this method (SW-846 Method 3520C). 

Description of Method: This method is applicable to the isolation and concentration of water-insoluble 

and slightly soluble organics in preparation for a variety of chromatographic procedures. A measured 

volume of sample, usually 1 L, is placed into a continuous liquid-liquid extractor, adjusted, if necessary,
 

to a specific pH and extracted with organic solvent for 18 to 24 hours. The extract is filtered through 

sodium sulfate to remove residual moisture, concentrated, and exchanged as necessary into a solvent 

compatible with the cleanup or determinative procedure used for analysis. 

Special Considerations: Some of the target compounds will hydrolyze in water, with hydrolysis rates 

dependant on various factors such as sample pH and temperature. For more information on the 

preparation and analysis of thiofanox, see application note: 

http://www.pickeringlabs.com/catalog/pdfs/MA112%20expanded%20Carbamates.pdf 

Source: EPA. 1996. “Method 3520C (SW-846): Continuous Liquid-Liquid Extraction,” Revision 3. 

http://www.epa.gov/sam/pdfs/EPA-3520c.pdf 

5.2.19 EPA Method 3535A (SW-846): Solid-Phase Extraction 

Analyte(s) CAS RN 

4-Aminopyridine 504-24-5 

Brodifacoum 56073-10-0 

Bromadiolone 28772-56-7 

BZ [Quinuclidinyl benzilate] 6581-06-2 

 SAM Revision 5.0 49 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte(s) CAS RN 

Carfentanil 59708-52-0 

Chlorfenvinphos 470-90-6 

3-Chloro-1,2-propanediol 96-24-2 

Chlorosarin 1445-76-7 

Chlorosoman 7040-57-5 

Chlorpyrifos 2921-88-2 

Chlorpyrifos oxon 5598-15-2 

Crimidine 535-89-7 

Cyclohexyl sarin (GF) 329-99-7 

Dichlorvos 62-73-7 

Dicrotophos 141-66-2 

Diesel range organics NA 

Diisopropyl methylphosphonate (DIMP) 1445-75-6 

Dimethylphosphite 868-85-9 

Dimethylphosphoramidic acid 33876-51-6 

Diphacinone 82-66-6 

1,4-Dithiane 505-29-3 

EA2192 [Diisopropylaminoethyl 

methylthiolophosphonate] 73207-98-4 

Ethyl methylphosphonic acid (EMPA) 1832-53-7 

Ethyldichloroarsine (ED) 598-14-1 

N-Ethyldiethanolamine (EDEA) 139-87-7 

Fenamiphos 22224-92-6 

Fentanyl 437-38-7 

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) 121-82-4 

Hexamethylenetriperoxidediamine (HMTD) 283-66-9 

Isopropyl methylphosphonic acid (IMPA) 1832-54-8 

Methyl hydrazine 60-34-4 

Methyl paraoxon 950-35-6 

Methyl parathion 298-00-0 

N-Methyldiethanolamine (MDEA) 105-59-9 

1-Methylethyl ester ethylphosphonofluoridic acid (GE) 1189-87-3 

 SAM Revision 5.0 50 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte(s) CAS RN 

Methylphosphonic acid (MPA) 993-13-5 

Mevinphos 7786-34-7 

Monocrotophos 6923-22-4 

Mustard, nitrogen (HN-1) [bis(2-chloroethyl)ethylamine] 538-07-8 

Mustard, nitrogen (HN-2) [2,2’-dichloro-Nmethyldiethylamine N,N-bis(2-chloroethyl)methylamine] 51-75-2 

Mustard, nitrogen (HN-3) [tris(2-chloroethyl)amine] 555-77-1 

Nicotine compounds 54-11-5 

Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) 2691-41-0 

Paraoxon 311-45-5 

Parathion 56-38-2 

Pentaerythritol tetranitrate (PETN) 78-11-5 

Phencyclidine 77-10-1 

Phenol 108-95-2 

Phorate 298-02-2 

Phorate sulfone 2588-04-7 

Phorate sulfoxide 2588-03-6 

Phosphamidon 13171-21-6 

Pinacolyl methyl phosphonic acid (PMPA) 616-52-4 

R 33 (VR) [methylphosphonothioic acid, S-[2­

(diethylamino)ethyl] O-2-methylpropyl ester] 159939-87-4 

Semivolatile organic compounds, NOS NA 

Soman (GD) 96-64-0 

Strychnine 57-24-9 

Tabun (GA) 77-81-6 

Tetraethyl pyrophosphate 107-49-3 

Tetramethylenedisulfotetramine 80-12-6 

Thiodiglycol (TDG) 111-48-8 

Thiofanox 39196-18-4 

1,4-Thioxane 15980-15-1 

Triethanolamine (TEA) 102-71-6 

Trimethyl phosphite 121-45-9 

1,3,5-Trinitrobenzene (1,3,5-TNB) 99-35-4 

 SAM Revision 5.0 51 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte(s) CAS RN 

2,4,6-Trinitrotoluene (2,4,6-TNT) 118-96-7 

VE [phosphonothioic acid, ethyl-, S-(2­

(diethylamino)ethyl) O-ethyl ester] 21738-25-0 

VG [phosphonothioic acid, S-(2-(diethylamino)ethyl) 

O,O-diethyl ester] 78-53-5 

VM [phosphonothioic acid, methyl-, S-(2­

(diethylamino)ethyl) O-ethyl ester] 21770-86-5 

Analysis Purpose: Sample preparation 

Sample Preparation Technique: SPE 

Determinative Technique: EPA SW-846 Method 8015C, Method 8270D, Method 8321B, or Method 

8330B. Refer to Appendix A for which of these determinative methods should be used for a particular 

analyte. 

Method Developed for: Organic compounds in ground water, wastewater, and Toxicity Characteristic 

Leaching Procedure (TCLP, Method 1311) leachates 

Method Selected for: SAM lists this method for preparation of aqueous liquid and/or drinking water 

samples. Please note: Drinking water samples for dichlorvos, fenamiphos, mevinphos, and semivolatile 

organic compounds NOS should be prepared and analyzed by EPA Method 525.2; drinking water 

samples for thiofanox should be prepared and analyzed by EPA Method 531.2; aqueous liquid samples 

for DIMP, EMPA, IMPA, MPA, and PMPA should be prepared and analyzed using EPA CRL MS017; 

aqueous liquid samples for EDEA, MDEA and TEA should be prepared and analyzed using EPA CRL 

MS016; aqueous liquid samples for bromadiolone should be prepared and analyzed using EPA CRL 

MS014; aqueous liquid samples for thiodiglycol should be prepared and analyzed using EPA CRL 

MS015. All other drinking water samples and all aqueous liquid samples should be prepared using this 

method (SW-846 Method 3535A). 

Description of Method: This method describes a procedure for isolating target organic analytes from 

aqueous and liquid samples using SPE media. Sample preparation procedures vary by analyte group. 

Following any necessary pH adjustment, a measured volume of sample is extracted by passing it through 

the SPE medium (disks or cartridges), which is held in an extraction device designed for vacuum filtration 

of the sample. Target analytes are eluted from the solid-phase media using an appropriate solvent which 

is collected in a receiving vessel. The resulting solvent extract is dried using sodium sulfate and 

concentrated, as needed. 

Special Considerations: Tetramethylenedisulfotetramine may require SPE extraction using acetone or 

methyl ethylketone. Water samples that contain a high level of particulates or a large amount of humic 

products may not be extractable by SPE. Some of the target compounds will hydrolyze in water, with 

hydrolysis rates dependant on various factors such as sample pH and temperature. 

Source: EPA. 1998. “Method 3535A (SW-846): Solid-Phase Extraction (SPE),” Revision 1. 

http://www.epa.gov/sam/pdfs/EPA-3535a.pdf 

5.2.20 EPA Method 3541 (SW-846): Automated Soxhlet Extraction 

Analyte(s) CAS RN 

Brodifacoum 56073-10-0 

Bromadiolone 28772-56-7 

 SAM Revision 5.0 52 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte(s) CAS RN 

BZ [Quinuclidinyl benzilate] 6581-06-2 

Carfentanil 59708-52-0 

Chlorfenvinphos 470-90-6 

3-Chloro-1,2-propanediol 96-24-2 

Chlorosarin 1445-76-7 

Chlorosoman 7040-57-5 

Chlorpyrifos 2921-88-2 

Chlorpyrifos oxon 5598-15-2 

Cyclohexyl sarin (GF) 329-99-7 

Diesel range organics NA 

Diphacinone 82-66-6 

Disulfoton 298-04-4 

Disulfoton sulfoxide 2497-07-6 

N-Ethyldiethanolamine (EDEA) 139-87-7 

Fenamiphos 22224-92-6 

Fentanyl 437-38-7 

Methyl hydrazine 60-34-4 

N-Methyldiethanolamine (MDEA) 105-59-9 

1-Methylethyl ester ethylphosphonofluoridic acid (GE) 1189-87-3 

Mustard, nitrogen (HN-1) [bis(2-chloroethyl)ethylamine] 538-07-8 

Mustard, nitrogen (HN-2) [2,2’-dichloro-Nmethyldiethylamine N,N-bis(2-chloroethyl)methylamine] 51-75-2 

Mustard, nitrogen (HN-3) [tris(2-chloroethyl)amine] 555-77-1 

Organophosphate pesticides, NOS NA 

Paraoxon 311-45-5 

Parathion 56-38-2 

Phenol 108-95-2 

Phosphamidon 13171-21-6 

R 33 (VR) [methylphosphonothioic acid, S-[2­

(diethylamino)ethyl] O-2-methylpropyl ester] 159939-87-4 

Semivolatile organic compounds, NOS NA 

Tetramethylenedisulfotetramine 80-12-6 

 SAM Revision 5.0 53 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte(s) CAS RN 

Thiofanox 39196-18-4 

Triethanolamine (TEA) 102-71-6 

VE [phosphonothioic acid, ethyl-, S-(2­

(diethylamino)ethyl) O-ethyl ester] 21738-25-0 

VG [phosphonothioic acid, S-(2-(diethylamino)ethyl) 

O,O-diethyl ester] 78-53-5 

VM [phosphonothioic acid, methyl-, S-(2­

(diethylamino)ethyl) O-ethyl ester] 21770-86-5 

Analysis Purpose: Sample preparation 

Sample Preparation Technique: Automated Soxhlet extraction 

Determinative Technique: EPA SW-846 Method 8015C, Method 8270D, or Method 8321B. Refer to 

Appendix A for which of these determinative methods should be used for a particular analyte. 

Method Developed for: Organic compounds in soil, sediment, sludges, and waste solids 

Method Selected for: SAM lists this method for preparation of solid samples. 

Description of Method: Approximately 10 g of solid sample is mixed with an equal amount of 

anhydrous sodium sulfate and placed in an extraction thimble or between two plugs of glass wool. After 

adding the appropriate surrogate amount, the sample is extracted using an appropriate solvent in an 

automated Soxhlet extractor. The extract is dried with sodium sulfate to remove residual moisture, 

concentrated and exchanged, as necessary, into a solvent compatible with the cleanup or determinative 

procedure for analysis. 

Special Considerations: Some of the target compounds will hydrolyze in water, with hydrolysis rates 

dependant on various factors such as sample pH and temperature. 

Source: EPA. 1994. “Method 3541 (SW-846): Automated Soxhlet Extraction,” Revision 0. 

http://www.epa.gov/sam/pdfs/EPA-3541.pdf 

5.2.21 EPA Method 3545A (SW-846): Pressurized Fluid Extraction (PFE) 

Analyte(s) CAS RN 

Brodifacoum 56073-10-0 

Bromadiolone 28772-56-7 

BZ [Quinuclidinyl benzilate] 6581-06-2 

Carfentanil 59708-52-0 

Chlorfenvinphos 470-90-6 

3-Chloro-1,2-propanediol 96-24-2 

Chloropicrin 76-06-2 

Chlorosarin 1445-76-7 

Chlorosoman 7040-57-5 

 SAM Revision 5.0 54 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte(s) CAS RN 

Chlorpyrifos 2921-88-2 

Chlorpyrifos oxon 5598-15-2 

Crimidine 535-89-7 

Cyclohexyl sarin (GF) 329-99-7 

Dichlorvos 62-73-7 

Dicrotophos 141-66-2 

Diesel range organics NA 

Diisopropyl methylphosphonate (DIMP) 1445-75-6 

Dimethylphosphite 868-85-9 

Dimethylphosphoramidic acid 33876-51-6 

Diphacinone 82-66-6 

Disulfoton 298-04-4 

Disulfoton sulfoxide 2497-07-6 

1,4-Dithiane 505-29-3 

EA2192 [Diisopropylaminoethyl 

methylthiolophosphonate] 73207-98-4 

Ethyl methylphosphonic acid (EMPA) 1832-53-7 

Ethyldichloroarsine (ED) 598-14-1 

N-Ethyldiethanolamine (EDEA) 139-87-7 

Fenamiphos 22224-92-6 

Fentanyl 437-38-7 

Isopropyl methylphosphonic acid (IMPA) 1832-54-8 

Methyl hydrazine 60-34-4 

Methyl paraoxon 950-35-6 

Methyl parathion 298-00-0 

N-Methyldiethanolamine (MDEA) 105-59-9 

1-Methylethyl ester ethylphosphonofluoridic acid (GE) 1189-87-3 

Methylphosphonic acid (MPA) 993-13-5 

Mevinphos 7786-34-7 

Monocrotophos 6923-22-4 

Mustard, nitrogen (HN-1) [bis(2-chloroethyl)ethylamine] 538-07-8 

 SAM Revision 5.0 55 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte(s) CAS RN 

Mustard, nitrogen (HN-2) [2,2’-dichloro-Nmethyldiethylamine N,N-bis(2-chloroethyl)methylamine] 51-75-2 

Mustard, nitrogen (HN-3) [tris(2-chloroethyl)amine] 555-77-1 

Nicotine compounds 54-11-5 

Organophosphate pesticides, NOS NA 

Paraoxon 311-45-5 

Parathion 56-38-2 

Phencyclidine 77-10-1 

Phenol 108-95-2 

Phorate 298-02-2 

Phorate sulfone 2588-04-7 

Phorate sulfoxide 2588-03-6 

Phosphamidon 13171-21-6 

Pinacolyl methyl phosphonic acid (PMPA) 616-52-4 

R 33 (VR) [methylphosphonothioic acid, S-[2­

(diethylamino)ethyl] O-2-methylpropyl ester] 159939-87-4 

Semivolatile organic compounds, NOS NA 

Soman (GD) 96-64-0 

Strychnine 57-24-9 

Tabun (GA) 77-81-6 

Tetraethyl pyrophosphate 107-49-3 

Tetramethylenedisulfotetramine 80-12-6 

Thiodiglycol (TDG) 111-48-8 

Thiofanox 39196-18-4 

1,4-Thioxane 15980-15-1 

Triethanolamine (TEA) 102-71-6 

Trimethyl phosphite 121-45-9 

VE [phosphonothioic acid, ethyl-, S-(2­

(diethylamino)ethyl) O-ethyl ester] 21738-25-0 

VG [phosphonothioic acid, S-(2-(diethylamino)ethyl) 

O,O-diethyl ester] 78-53-5 

VM [phosphonothioic acid, methyl-, S-(2­

(diethylamino)ethyl) O-ethyl ester] 21770-86-5 

 SAM Revision 5.0 56 September 29, 2009 
Section 5 – Selected Chemical Methods

Analysis Purpose: Sample preparation
 

Sample Preparation Technique: Pressurized Fluid Extraction (PFE) 

Determinative Technique: EPA SW-846 Method 8015C, Method 8270D, or Method 8321B. Refer to 

Appendix A for which of these determinative methods should be used for a particular analyte. 

Method Developed for: Organic compounds in soils, clays, sediments, sludges, and waste solids 

Method Selected for: SAM lists this method for preparation of solid samples. 

Detection and Quantitation: This method has been validated for solid matrices containing 250 to 

12,500 μg/kg of semivolatile organic compounds, 250 to 2500 μg/kg of organophosphorus pesticides, 5 to 

250 μg/kg of organochlorine pesticides, 50 to 5000 μg/kg of chlorinated herbicides, and 1 to 2500 ng/kg 

of polychlorinated dibenzo-p-dioxins (PCDDs) / polychlorinated dibenzofurans (PCDFs). 

Description of Method: Approximately 10 to 30 g of soil sample is prepared for extraction either by air 

drying the sample, or by mixing the sample with anhydrous sodium sulfate or pelletized diatomaceous 

earth. The sample is then ground and loaded into the extraction cell. The extraction cell containing the 

sample is heated to the extraction temperature, pressurized with the appropriate solvent system, and 

extracted for 5 minutes (or as recommended by the instrument manufacturer). The extract may be 

concentrated, if necessary, and exchanged into a solvent compatible with the cleanup or determinative 

step being employed. 

Special Considerations: Sodium sulfate can cause clogging, and air-drying or palletized diatomaceous 

earth may be preferred. Phencyclidine and VX require extraction with 5% triethylamine in ethyl acetate.
 

Some of the target compounds will hydrolyze in water, with hydrolysis rates dependant on various factors 

such as sample pH and temperature. 

Source: EPA. 1998. “Method 3545A (SW-846): Pressurized Fluid Extraction (PFE),” Revision 1. 

http://www.epa.gov/sam/pdfs/EPA-3545a.pdf 

5.2.22 EPA Method 3570 (SW-846): Microscale Solvent Extraction (MSE) 

Analyte(s) CAS RN 

Acrylamide 79-06-1 

Acrylonitrile 107-13-1 

Aldicarb (Temik) 116-06-3 

Aldicarb sulfone 1646-88-4 

Aldicarb sulfoxide 1646-87-3 

4-Aminopyridine 504-24-5 

BZ [Quinuclidinyl benzilate] 6581-06-2 

Brodifacoum 56073-10-0 

Bromadiolone 28772-56-7 

Carfentanil 59708-52-0 

Carbofuran (Furadan) 1563-66-2 

Chlorfenvinphos 470-90-6 

3-Chloro-1,2-propanediol 96-24-2 

Chloropicrin 76-06-2 

 SAM Revision 5.0 57 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte(s) CAS RN 

Chlorosarin 1445-76-7 

Chlorosoman 7040-57-5 

Chlorpyrifos 2921-88-2 

Chlorpyrifos oxon 5598-15-2 

Crimidine 535-89-7 

Cyclohexyl sarin (GF) 329-99-7 

Dichlorvos 62-73-7 

Dicrotophos 141-66-2 

Diesel range organics NA 

Diisopropyl methylphosphonate (DIMP) 1445-75-6 

Dimethylphosphite 868-85-9 

Dimethylphosphoramidic acid 33876-51-6 

Diphacinone 82-66-6 

Disulfoton 298-04-4 

Disulfoton sulfoxide 2497-07-6 

1,4-Dithiane 505-29-3 

EA2192 [Diisopropylaminoethyl 

methylthiolophosphonate] 73207-98-4 

Ethyl methylphosphonic acid (EMPA) 1832-53-7 

N-Ethyldiethanolamine (EDEA) 139-87-7 

Fenamiphos 22224-92-6 

Fentanyl 437-38-7 

Fluoroacetic acid and fluoroacetate salts NA 

Formaldehyde 50-00-0 

Gasoline range organics NA 

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) 121-82-4 

Hexamethylenetriperoxidediamine (HMTD) 283-66-9 

Isopropyl methylphosphonic acid (IMPA) 1832-54-8 

Kerosene 64742-81-0 

Methomyl 16752-77-5 

Methyl acrylonitrile 126-98-7 

Methyl fluoroacetate 453-18-9 

 SAM Revision 5.0 58 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte(s) CAS RN 

Methyl hydrazine 60-34-4 

Methyl paraoxon 950-35-6 

Methyl parathion 298-00-0 

N-Methyldiethanolamine (MDEA) 105-59-9 

1-Methylethyl ester ethylphosphonofluoridic acid (GE) 1189-87-3 

Methylphosphonic acid (MPA) 993-13-5 

Mevinphos 7786-34-7 

Monocrotophos 6923-22-4 

Mustard, nitrogen (HN-1) [bis(2-chloroethyl)ethylamine] 538-07-8 

Mustard, nitrogen (HN-2) [2,2’-dichloro-Nmethyldiethylamine N,N-bis(2-chloroethyl)methylamine] 51-75-2 

Mustard, nitrogen (HN-3) [tris(2-chloroethyl)amine] 555-77-1 

Mustard, sulfur / Mustard gas (HD) 505-60-2 

Nicotine compounds 54-11-5 

Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) 2691-41-0 

Organophosphate pesticides, NOS NA 

Oxamyl 23135-22-0 

Paraoxon 311-45-5 

Parathion 56-38-2 

Pentaerythritol tetranitrate (PETN) 78-11-5 

Phencyclidine 77-10-1 

Phenol 108-95-2 

Phorate 298-02-2 

Phorate sulfone 2588-04-7 

Phorate sulfoxide 2588-03-6 

Phosphamidon 13171-21-6 

Pinacolyl methyl phosphonic acid (PMPA) 616-52-4 

R 33 (VR) [methylphosphonothioic acid, S-[2­

(diethylamino)ethyl] O-2-methylpropyl ester] 159939-87-4 

Sarin (GB) 107-44-8 

Semivolatile organic compounds, NOS NA 

Soman (GD) 96-64-0 

 SAM Revision 5.0 59 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte(s) CAS RN 

Strychnine 57-24-9 

Tabun (GA) 77-81-6 

Tetraethyl pyrophosphate 107-49-3 

Tetramethylenedisulfotetramine 80-12-6 

Thiodiglycol (TDG) 111-48-8 

Thiofanox 39196-18-4 

1,4-Thioxane 15980-15-1 

Triethanolamine (TEA) 102-71-6 

Trimethyl phosphite 121-45-9 

1,3,5-Trinitrobenzene (1,3,5-TNB) 99-35-4 

2,4,6-Trinitrotoluene (2,4,6-TNT) 118-96-7 

VE [phosphonothioic acid, ethyl-, S-(2­

(diethylamino)ethyl) O-ethyl ester] 21738-25-0 

VG [phosphonothioic acid, S-(2-(diethylamino)ethyl) 

O,O-diethyl ester] 78-53-5 

VM [phosphonothioic acid, methyl-, S-(2­

(diethylamino)ethyl) O-ethyl ester] 21770-86-5 

VX [O-ethyl-S-(2-diisopropylaminoethyl)methylphosphonothiolate] 50782-69-9 

White phosphorus 12185-10-3 

Analysis Purpose: Sample preparation 

Sample Preparation Technique: MSE 

Determinative Technique: EPA OW Method 300.1 Revision 1.0; EPA SW-846 Methods 7580, 8015C, 

8270D, 8315A, 8316, 8318A, 8321B, and 8330B. Refer to Appendix A for which of these determinative 

methods should be used for a particular analyte. 

Method Developed for: Extracting volatile, semivolatile, and nonvolatile organic compounds from 

solids such as soils, sludges, and wastes 

Method Selected for: SAM lists this method for preparation of wipe samples. 

Description of Method: Samples are prepared by shake extraction with an organic solvent in sealed 

extraction tubes. Careful manipulation of the sample, solvent, drying agent, and spiking solutions during 

the procedure minimizes loss of volatile compounds while maximizing extraction of volatile, 

semivolatile, and nonvolatile compounds. Sample extracts are collected, dried, and concentrated using a 

modification of the Kuderna-Danish concentration method or other appropriate concentration technique. 

By increasing the number of theoretical plates and reducing the distillation temperature, extracts are 

concentrated without loss of volatile constituents. Samples should be prepared one at a time to the point 

of solvent addition (i.e., do not pre-weigh a number of samples then add the solvent). Samples should be 

extracted as soon after collection as possible, and exposure to air before sample extraction is minimized 

as much as possible. 

 SAM Revision 5.0 60 September 29, 2009 
Section 5 – Selected Chemical Methods

Source: EPA. 2002. “Method 3570 (SW-846): Microscale Solvent Extraction (MSE),” Revision 0. 

http://www.epa.gov/sam/pdfs/EPA-3570.pdf 

5.2.23 EPA Method 3571 (SW-846): Extraction of Solid and Aqueous Samples for 

Chemical Agents 

Analyte(s) CAS RN 

Mustard, sulfur / Mustard gas (HD) 505-60-2 

Sarin (GB) 107-44-8 

VX [O-ethyl-S-(2-diisopropylaminoethyl)methylphosphonothiolate] 50782-69-9 

Analysis Purpose: Sample preparation 

Sample Preparation Technique: MSE 

Determinative Technique: EPA SW-846 Method 8270D 

Method Developed for: HD, GB, and VX in concrete, charcoal, wood, water, brine, ash, coral, sand, and 

soil 

Method Selected for: SAM lists this method for preparation of solid, non-aqueous liquid/organic solid, 

aqueous liquid, and drinking water samples. 

Description of Method: This method provides procedures for sample collection and extraction of the 

referenced compounds from solids and aqueous samples. A separate extract is required for each agent to 

be measured. Glacial acetic acid is added as a preservative to samples being assayed for GB and glacial 

acetic acid/sodium chloride is a preservative for samples assayed for HD. No preservative is added for 

VX. Samples are extracted with 10% isopropanol in dichloromethane by vortex mixing and filtered, if 

necessary. An optional water wash is included for VX that back-extracts the compound from heavy 

organics that could interfere with the assay. An optional column cleanup procedure is described to 

separate GB from heavy organics, if needed. Solvents are used to elute the extract first through the 

Carboprep90 column, then the silica column. 

Source: EPA. 2007. “Method 3571 (SW-846): Extraction of Solid and Aqueous Samples for Chemical 

Agents,” Revision 0. http://www.epa.gov/sam/pdfs/EPA-3571.pdf
 

5.2.24 EPA Method 3580A (SW-846): Waste Dilution 

Analyte(s) CAS RN 

Brodifacoum 56073-10-0 

Bromadiolone 28772-56-7 

BZ [Quinuclidinyl benzilate] 6581-06-2 

Carfentanil 59708-52-0 

Chlorfenvinphos 470-90-6 

3-Chloro-1,2-propanediol 96-24-2 

Chloropicrin 76-06-2 

 SAM Revision 5.0 61 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte(s) CAS RN 

Chlorosarin 1445-76-7 

Chlorosoman 7040-57-5 

Chlorpyrifos 2921-88-2 

Chlorpyrifos oxon 5598-15-2 

Crimidine 535-89-7 

Cyclohexyl sarin (GF) 329-99-7 

Dichlorvos 62-73-7 

Dicrotophos 141-66-2 

Diesel range organics NA 

Diisopropyl methylphosphonate (DIMP) 1445-75-6 

Dimethylphosphite 868-85-9 

Dimethylphosphoramidic acid 33876-51-6 

Diphacinone 82-66-6 

Disulfoton 298-04-4 

Disulfoton sulfoxide 2497-07-6 

1,4-Dithiane 505-29-3 

EA2192 [Diisopropylaminoethyl 

methylthiolophosphonate] 73207-98-4 

Ethyl methylphosphonic acid (EMPA) 1832-53-7 

Ethyldichloroarsine (ED) 598-14-1 

N-Ethyldiethanolamine (EDEA) 139-87-7 

Fenamiphos 22224-92-6 

Fentanyl 437-38-7 

Isopropyl methylphosphonic acid (IMPA) 1832-54-8 

Methyl hydrazine 60-34-4 

Methyl paraoxon 950-35-6 

Methyl parathion 298-00-0 

N-Methyldiethanolamine (MDEA) 105-59-9 

1-Methylethyl ester ethylphosphonofluoridic acid (GE) 1189-87-3 

Methylphosphonic acid (MPA) 993-13-5 

Mevinphos 7786-34-7 

Monocrotophos 6923-22-4 

 SAM Revision 5.0 62 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte(s) CAS RN 

Mustard, nitrogen (HN-1) [bis(2-chloroethyl)ethylamine] 538-07-8 

Mustard, nitrogen (HN-2) [2,2’-dichloro-Nmethyldiethylamine N,N-bis(2-chloroethyl)methylamine] 51-75-2 

Mustard, nitrogen (HN-3) [tris(2-chloroethyl)amine] 555-77-1 

Nicotine compounds 54-11-5 

Organophosphate pesticides, NOS NA 

Paraoxon 311-45-5 

Parathion 56-38-2 

Phencyclidine 77-10-1 

Phenol 108-95-2 

Phorate 298-02-2 

Phorate sulfone 2588-04-7 

Phorate sulfoxide 2588-03-6 

Phosphamidon 13171-21-6 

Pinacolyl methyl phosphonic acid (PMPA) 616-52-4 

R 33 (VR) [methylphosphonothioic acid, S-[2­

(diethylamino)ethyl] O-2-methylpropyl ester] 159939-87-4 

Semivolatile organic compounds, NOS NA 

Sodium azide (analyze for hydrazoic acid) 26628-22-8 

Soman (GD) 96-64-0 

Strychnine 57-24-9 

Tabun (GA) 77-81-6 

Tetraethyl pyrophosphate 107-49-3 

Tetramethylenedisulfotetramine 80-12-6 

Thiodiglycol (TDG) 111-48-8 

Thiofanox 39196-18-4 

1,4-Thioxane 15980-15-1 

Triethanolamine (TEA) 102-71-6 

Trimethyl phosphite 121-45-9 

VE [phosphonothioic acid, ethyl-, S-(2­

(diethylamino)ethyl) O-ethyl ester] 21738-25-0 

VG [phosphonothioic acid, S-(2-(diethylamino)ethyl) 

O,O-diethyl ester] 78-53-5 

 SAM Revision 5.0 63 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte(s) CAS RN 

VM [phosphonothioic acid, methyl-, S-(2­

(diethylamino)ethyl) O-ethyl ester] 21770-86-5 

Analysis Purpose: Sample preparation 

Sample Preparation Technique: Waste dilution 

Determinative Technique: EPA SW-846 Method 8015C, Method 8270D, or Method 8321B. Refer to 

Appendix A for which of these determinative methods should be used for a particular analyte. 

Method Developed for: Organic compounds in non-aqueous waste samples 

Method Selected for: SAM lists this method for preparation of non-aqueous liquid/organic solid 

samples. 

Description of Method: This method describes solvent dilution of a non-aqueous waste sample prior to 

cleanup and/or analysis. One gram of sample is weighed into a capped tube and the sample is diluted to 

10.0 mL with an appropriate solvent. 

Special Considerations: The method is designed for wastes that may contain organic chemicals at a 

concentration greater than 20,000 mg/kg and that are soluble in the dilution solvent. 

Source: EPA. 1992. “Method 3580A (SW-846): Waste Dilution,” Revision 1. 

http://www.epa.gov/sam/pdfs/EPA-3580a.pdf 

5.2.25 EPA Method 3585 (SW-846): Waste Dilution for Volatile Organics 

Analyte(s) CAS RN 

Allyl alcohol 107-18-6 

Carbon disulfide 75-15-0 

2-Chloroethanol 107-07-3 

Cyanogen chloride 506-77-4 

1,2-Dichloroethane 107-06-2 

Ethylene oxide 75-21-8 

2-Fluoroethanol 371-62-0 

Gasoline range organics NA 

Kerosene 64742-81-0 

Propylene oxide 75-56-9 

The following analytes should be prepared by this method (and determined by the corresponding SW-846 Method 

8260C) only if problems (e.g., insufficient recovery, interferences) occur when using the sample 

preparation/determinative techniques identified for these analytes in Appendix A. 

1,4-Thioxane 15980-15-1 

Analysis Purpose: Sample preparation 

Sample Preparation Technique: Waste dilution 

 SAM Revision 5.0 64 September 29, 2009 
Section 5 – Selected Chemical Methods

Determinative Technique: EPA SW-846 Method 8015C or Method 8260C. Refer to Appendix A for 

which of these determinative methods should be used for a particular analyte. 

Method Developed for: VOCs in non-aqueous waste 

Method Selected for: SAM lists this method for preparation of non-aqueous liquid/organic solid 

samples. 

Description of Method: This method describes solvent dilution of a non-aqueous waste sample prior to 

direct injection analysis. It is designed for use in conjunction with GC or GC-MS analysis of wastes that 

may contain organic chemicals at a concentration greater than 1 mg/kg and that are soluble in the dilution 

solvent. Highly contaminated or highly complex samples may be diluted prior to analysis for volatiles 

using direct injection. One gram of sample is weighed into a capped tube or volumetric flask. The 

sample is diluted to 2.0 to 10.0 mL with n-hexadecane or other appropriate solvent. Diluted samples are 

injected into the GC or GC-MS for analysis. 

Special Considerations: For use in analysis of wastes that may contain organic chemicals at a 

concentration greater than 1 mg/kg and that are soluble in the dilution solvent. 

Source: EPA. 1996. “Method 3585 (SW-846): Waste Dilution for Volatile Organics,” Revision 0. 

http://www.epa.gov/sam/pdfs/EPA-3585.pdf 

5.2.26 EPA Method 5030C (SW-846): Purge-and-Trap for Aqueous Samples 

Analyte(s) CAS RN 

Allyl alcohol 107-18-6 

Carbon disulfide 75-15-0 

2-Chloroethanol 107-07-3 

Cyanogen chloride 506-77-4 

1,2-Dichloroethane 107-06-2 

Ethylene oxide 75-21-8 

2-Fluoroethanol 371-62-0 

Gasoline range organics NA 

Kerosene 64742-81-0 

Propylene oxide 75-56-9 

The following analytes should be prepared by this method (and determined by the corresponding SW-846 Method 

8260C) only if problems (e.g., insufficient recovery, interferences) occur when using the sample 

preparation/determinative techniques identified for these analytes in Appendix A. 

1,4-Thioxane 15980-15-1 

Analysis Purpose: Sample preparation 

Sample Preparation Technique: Purge-and-trap 

Determinative Technique: EPA SW-846 Method 8015C or Method 8260C. Refer to Appendix A for 

which of these determinative methods should be used for a particular analyte. 

 SAM Revision 5.0 65 September 29, 2009 
Section 5 – Selected Chemical Methods

Method Developed for: VOCs in aqueous and water miscible liquid samples 

Method Selected for: SAM lists this method for preparation of aqueous liquid and/or drinking water 

samples. For carbon disulfide and 1,2-dichloroethane, EPA Method 524.2 (rather than Method 5030C) 

should be used for preparation of drinking water samples. 

Description of Method: This method describes a purge-and-trap procedure for the analysis of VOCs in 

aqueous liquid samples and water miscible liquid samples. An inert gas is bubbled through a portion of 

the aqueous liquid sample at ambient temperature, and the volatile components are transferred from the 

aqueous liquid phase to the vapor phase. The vapor is swept through a sorbent column where the volatile 

components are adsorbed. After purging is completed, the sorbent column is heated and backflushed with 

inert gas to desorb the components onto a GC column. 

Special Considerations: Heated purge may be required for poor-purging analytes. 

Source: EPA. 2003. “Method 5030C (SW-846): Purge-and-Trap for Aqueous Samples, Revision 3. 

http://www.epa.gov/sam/pdfs/EPA-5030c.pdf 

5.2.27 EPA Method 5035A (SW-846): Closed-System Purge-and-Trap and Extraction for 

Volatile Organics in Soil and Waste Samples 

Analyte(s) CAS RN 

Allyl alcohol 107-18-6 

Carbon disulfide 75-15-0 

2-Chloroethanol 107-07-3 

Cyanogen chloride 506-77-4 

1,2-Dichloroethane 107-06-2 

Ethylene oxide 75-21-8 

2-Fluoroethanol 371-62-0 

Gasoline range organics NA 

Kerosene 64742-81-0 

Propylene oxide 75-56-9 

The following analytes should be prepared by this method (and determined by the corresponding SW-846 Method 

8260C) only if problems (e.g., insufficient recovery, interferences) occur when using the sample 

preparation/determinative techniques identified for these analytes in Appendix A. 

1,4-Thioxane 15980-15-1 

Analysis Purpose: Sample preparation 

Sample Preparation Technique: Purge-and-trap 

Determinative Technique: EPA SW-846 Method 8015C or Method 8260C. Refer to Appendix A for 

which of these determinative methods should be used for a particular analyte. 

Method Developed for: VOCs in solid materials (e.g., soils, sediments, and solid waste) and oily wastes 

Method Selected for: SAM lists this method for preparation of solid samples. 

Description of Method: This method describes a closed-system purge-and-trap process for analysis of 

VOCs in solid samples containing low levels (0.5 to 200 μg/kg) of VOCs. The method also provides 

 SAM Revision 5.0 66 September 29, 2009 
Section 5 – Selected Chemical Methods

specific procedures for preparation of samples containing high levels (>200 μg/kg ) of VOCs. For lowlevel VOCs, a 5-g sample is collected into a vial that is placed into an autosampler device. Reagent 

water, surrogates, and internal standards are added automatically, and the vial is heated to 40°C. The 

volatiles are purged into an appropriate trap using an inert gas combined with sample agitation. When 

purging is complete, the trap is heated and backflushed with helium to desorb the trapped sample 

components into a GC for analysis. For high-level VOCs, samples are either collected into a vial that 

contains a water-miscible organic solvent or a portion of sample is removed from the vial and dispersed in 

a water-miscible solvent. An aliquot of the solvent is added to reagent water, along with surrogates and 

internal standards, then purged and analyzed using an appropriate determinative method (e.g., Method 

8015C or 8260C (SW-846)). 

Source: EPA. 2002. “Method 5035A (SW-846): Closed-System Purge-and-Trap and Extraction for 

Volatile Organics in Soil and Waste Samples,” Draft Revision 1. http://www.epa.gov/sam/pdfs/EPA5035a.pdf 

5.2.28 EPA Method 6010C (SW-846): Inductively Coupled Plasma - Atomic Emission 

Spectrometry 

Analyte(s) CAS RN 

Ammonium metavanadate 7803-55-6 

Arsenic, Total 7440-38-2 

Arsenic trioxide 1327-53-3 

Calcium arsenate 7778-44-1 

2-Chlorovinylarsonous acid (2-CVAA) 85090-33-1 

Lead arsenate 7645-25-2 

Lewisite 1 (L-1) [2-chlorovinyldichloroarsine] 541-25-3 

Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine] 40334-69-8 

Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine] 40334-70-1 

Lewisite oxide 1306-02-1 

Osmium tetroxide 20816-12-0 

Sodium arsenite 7784-46-5 

Thallium sulfate 10031-59-1 

Titanium tetrachloride 7550-45-0 

Vanadium pentoxide 1314-62-1 

Analysis Purpose: Analysis 

Sample Preparation Technique: EPA SW-846 Method 3050B (solid samples), Method 3031 (nonaqueous liquid/organic solid samples), and NIOSH Method 9102 (wipe samples) 

Determinative Technique: ICP-AES 

Method Developed for: Trace elements in solution 

Method Selected for: SAM lists this method for analysis of solid, non-aqueous liquid/organic solid, and 

wipe samples. Osmium tetroxide and titanium tetrachloride are not of concern in non-aqueous 

liquid/organic solid samples. 

 SAM Revision 5.0 67 September 29, 2009 
Section 5 – Selected Chemical Methods

Detection and Quantitation: Detection limits vary with each analyte. Estimated instrument detection 

limits (IDLs) for arsenic and titanium are 30 μg/L and 5.0 μg/L, respectively. The upper end of the 

analytical range may be extended by sample dilution. 

Description of Method: This method determines arsenic trioxide, lewisite, lewisite degradation 

products, calcium and lead arsenate, and sodium arsenite as total arsenic; osmium tetroxide as osmium; 

thallium sulfate as thallium; titanium tetrachloride as titanium; and ammonium metavanadate and 

vanadium pentoxide as total vanadium. Soil samples (prepared using SW-846 Method 3050B), nonaqueous liquid/organic solid samples (prepared using SW-846 Method 3031), and wipe samples (prepared 

using NIOSH Method 9102) are analyzed by ICP-AES. 

Special Considerations: Laboratory testing is currently underway for speciation of lewisite 1 using GCMS techniques. 

Source: EPA. 2007. “Method 6010C (SW-846): Inductively Coupled Plasma-Atomic Emission 

Spectrometry,” Revision 3. http://www.epa.gov/sam/pdfs/EPA-6010c.pdf 

5.2.29 EPA Method 6020A (SW-846): Inductively Coupled Plasma - Mass Spectrometry 

Analyte(s) CAS RN 

Ammonium metavanadate 7803-55-6 

Arsenic, Total 7440-38-2 

Arsenic trioxide 1327-53-3 

Calcium arsenate 7778-44-1 

2-Chlorovinylarsonous acid (2-CVAA) 85090-33-1 

Lead arsenate 7645-25-2 

Lewisite 1 (L-1) [2-chlorovinyldichloroarsine] 541-25-3 

Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine] 40334-69-8 

Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine] 40334-70-1 

Lewisite oxide 1306-02-1 

Sodium arsenite 7784-46-5 

Thallium sulfate 10031-59-1 

Titanium tetrachloride 7550-45-0 

Vanadium pentoxide 1314-62-1 

Analysis Purpose: Analysis 

Sample Preparation Technique: EPA SW-846 Method 3050B (solid samples), Method 3031 (nonaqueous liquid/organic solid samples), and NIOSH Method 9102 (wipe samples) 

Determinative Technique: ICP-MS 

Method Developed for: Elements in water samples and in waste extracts or digests 

Method Selected for: SAM lists this method for analysis of solid, non-aqueous liquid/organic solid, and 

wipe samples. Titanium tetrachloride is not of concern in non-aqueous liquid/organic solid samples. 

 SAM Revision 5.0 68 September 29, 2009 
Section 5 – Selected Chemical Methods

Detection and Quantitation: In relatively simple sample types, detection limits will generally be below 

0.1 μg/L. Less sensitive elements, such as arsenic, may have detection limits of 1.0 μg/L or higher. The 

upper end of the analytical range may be extended by sample dilution. 

Description of Method: This method will determine arsenic trioxide, lewisite, lewisite degradation 

products, calcium and lead arsenate, and sodium arsenite as total arsenic. The method also will determine 

thallium sulfate as total thallium, titanium tetrachloride as titanium, and ammonium metavanadate and 

vanadium pentoxide as total vanadium. Soil samples (prepared using SW-846 Method 3050B), nonaqueous liquid/organic solid samples (prepared using SW-846 Method 3031), and wipe samples (prepared 

using NIOSH Method 9102) are analyzed by ICP-MS. IDLs, sensitivities, and linear ranges vary with 

sample type, instrumentation, and operation conditions. 

Special Considerations: Laboratory testing is currently underway for speciation of lewisite 1 using GCMS techniques. 

Source: EPA. 1998. “Method 6020A (SW-846): Inductively Coupled Plasma-Mass Spectrometry,” 

Revision 1. http://www.epa.gov/sam/pdfs/EPA-6020a.pdf 

5.2.30 EPA Method 7010 (SW-846): Graphite Furnace Atomic Absorption 

Spectrophotometry 

Analyte(s) CAS RN 

Arsine 7784-42-1 

Analysis Purpose: Analysis
 

Sample Preparation Technique: EPA SW-846 Method 3050B (solid samples) and NIOSH Method 

9102 (wipe samples) 

Determinative Technique: GFAA 

Method Developed for: Metals in environmental samples including, but not limited to, ground water, 

domestic and industrial wastes, extracts, soils, sludges, sediments, and similar wastes 

Method Selected for: SAM lists this method for analysis of solid and wipe samples. 

Detection and Quantitation: Detection limits vary with each sample type and instrument used. The 

analytical range may be extended by sample dilution. 

Description of Method: This method determines arsine as total arsenic in environmental samples. Soil 

samples (prepared using SW-846 Method 3050B) and wipe samples (prepared using NIOSH Method 

1902) are analyzed by GFAA. A representative aliquot of the sample is placed in the graphite tube in the
 

furnace, evaporated to dryness, charred, and atomized. 

Source: EPA. 1998. “Method 7010 (SW-846): Graphite Furnace Atomic Absorption 

Spectrophotometry,” Revision 0. http://www.epa.gov/sam/pdfs/EPA-7010.pdf
 

5.2.31 EPA Method 7470A (SW-846): Mercury in Liquid Wastes (Manual Cold-Vapor 

Technique) 

Analyte(s) CAS RN 

Mercuric chloride 7487-94-7 

Mercury, Total 7439-97-6 

Methoxyethylmercuric acetate 151-38-2 

 SAM Revision 5.0 69 September 29, 2009 
Section 5 – Selected Chemical Methods

Analysis Purpose: Sample preparation and/or analysis 

Sample Preparation Technique: Acid digestion (solid and aqueous liquid samples) and NIOSH Method 

9102 (wipe samples) 

Determinative Technique: CVAA 

Method Developed for: Mercury in mobility-procedure extracts, aqueous wastes, and ground waters 

Method Selected for: SAM lists this method for use if problems occur when using EPA SW-846 

Method 7473 for these analytes during preparation and analysis of aqueous liquid samples. (See Footnote 

13 of Appendix A.) 

Detection and Quantitation: The detection limit for the method is 0.2 μg/L. 

Description of Method: A 100-mL aqueous sample is digested with acids, permanganate solution, 

persulfate solution, and heat. The sample is cooled and reduced with hydroxylamine-sodium chloride 

solution. Just prior to analysis, the sample is treated with Sn(II), reducing the mercury to Hg(0). The 

reduced sample is sparged and the mercury vapor is analyzed by CVAA. 

Special Considerations: Chloride and copper are potential interferences. 

Source: EPA. 1994. “Method 7470A (SW-846): Mercury in Liquid Waste (Manual Cold-Vapor 

Technique),” Revision 1. http://www.epa.gov/sam/pdfs/EPA-7470a.pdf 

5.2.32 EPA Method 7471B (SW-846): Mercury in Solid or Semisolid Wastes (Manual ColdVapor Technique) 

Analyte(s) CAS RN 

Mercuric chloride 7487-94-7 

Mercury, Total 7439-97-6 

Methoxyethylmercuric acetate 151-38-2 

Analysis Purpose: Sample preparation and/or analysis 

Sample Preparation Technique: Acid digestion (solid and aqueous liquid samples) and NIOSH Method 

9102 (wipe samples) 

Determinative Technique: CVAA 

Method Developed for: Total mercury in soils, sediments, bottom deposits, and sludge-type materials 

Method Selected for: SAM lists this method for use if problems occur when using EPA SW-846 

Method 7473 for these analytes during preparation and analysis of solid and wipe samples. (See Footnote 

13 of Appendix A.) 

Description of Method: A 0.5-g to 0.6-g sample is digested with aqua regia, permanganate solution, and 

heat. The sample is cooled and reduced with hydroxylamine-sodium chloride solution. Just prior to 

analysis, the sample is treated with Sn(II), reducing the mercury to Hg(0). The reduced sample is sparged 

and the mercury vapor is analyzed by CVAA. 

Special Considerations: Chloride and copper are potential interferences. 

Source: EPA. 1998. “Method 7471B (SW-846): Mercury in Solid or Semisolid Waste (Manual ColdVapor Technique),” Revision 2. http://www.epa.gov/sam/pdfs/EPA-7471b.pdf 

 SAM Revision 5.0 70 September 29, 2009 
Section 5 – Selected Chemical Methods

5.2.33 EPA Method 7473 (SW-846): Mercury in Solids and Solutions by Thermal 

Decomposition, Amalgamation, and Atomic Absorption Spectrophotometry 

Analyte(s) CAS RN 

Mercuric chloride 7487-94-7 

Mercury, Total 7439-97-6 

Methoxyethylmercuric acetate 151-38-2 

Analysis Purpose: Sample preparation and/or analysis 

Sample Preparation Technique: Thermal decomposition (solid and aqueous liquid samples) and 

NIOSH Method 9102 (wipe samples) 

Determinative Technique: Spectrophotometry 

Method Developed for: Total mercury in solids, aqueous samples, and digested solutions 

Method Selected for: SAM lists this method for preparation and analysis of solid, aqueous liquid, and 

wipe samples. 

Detection and Quantitation: The IDL is 0.01 ng total mercury. The typical working range for this 

method is 0.05 to 600 ng. 

Description of Method: Controlled heating in an oxygenated decomposition furnace is used to liberate 

mercury from solid and aqueous samples. The sample is dried and then thermally and chemically 

decomposed within the furnace. The decomposition products are carried by flowing oxygen to the 

catalytic section of the furnace, where oxidation is completed and halogens and nitrogen/sulfur oxides are 

trapped. The remaining decomposition products are then carried to an amalgamator that selectively traps 

mercury. After the system is flushed with oxygen to remove any remaining gases or decomposition 

products, the amalgamator is rapidly heated, releasing mercury vapor. Flowing oxygen carries the 

mercury vapor through absorbance cells positioned in the light path of a single wavelength atomic 

absorption spectrophotometer. Absorbance (peak height or peak area) is measured at 253.7 nm as a 

function of mercury concentration. 

Special Considerations: If equipment is not available, use CVAA Methods 7471B (EPA SW-846) for 

solid samples and 7470A (EPA SW-846) for aqueous liquid samples. 

Source: EPA. 1998. “Method 7473 (SW-846): Mercury in Solids and Solutions by Thermal 

Decomposition, Amalgamation, and Atomic Absorption Spectrophotometry,” Revision 0. 

http://www.epa.gov/sam/pdfs/EPA-7473.pdf 

5.2.34 EPA Method 7580 (SW-846): White Phosphorus (P4) by Solvent Extraction and Gas 

Chromatography 

Analyte(s) CAS RN 

White phosphorus 12185-10-3 

Analysis Purpose: Sample preparation and/or analysis 

Sample Preparation Technique: Solvent extraction (solid, non-aqueous liquid/organic solid, aqueous 

liquid, and drinking water samples) and EPA SW-846 Method 3570/8290A Appendix A (wipe samples) 

Determinative Technique: GC-NPD 

 SAM Revision 5.0 71 September 29, 2009 
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Method Developed for: White phosphorus in soil, sediment, and water 

Method Selected for: SAM lists this method for preparation and analysis of solid, non-aqueous 

liquid/organic solid, aqueous liquid, drinking water, and wipe samples. 

Detection and Quantitation: MDLs for reagent water, well water, and pond water were calculated to be 

0.008, 0.009, 0.008 μg/L, respectively. MDLs for sand, a sandy loam soil (Lebanon soil), and soil from 

the Rocky Mountain Arsenal (U.S. Army Environmental Center soil) were calculated to be 0.02, 0.43, 

0.07 μg/kg, respectively. This procedure provides sensitivity on the order of 0.01 μg/L for water samples 

and 1 μg/kg for soil samples. 

Description of Method: Method 7580 may be used to determine the concentration of white phosphorus 

in soil, sediment, and water samples using solvent extraction and GC. Water samples are extracted by 

one of two procedures, depending on the sensitivity required. For the more sensitive procedure, a 500-

mL water sample is extracted with 50 mL of diethyl ether. The extract is concentrated by back extraction 

with reagent water, yielding a final extract volume of approximately 1.0 mL. A 1.0 μL aliquot of this 

extract is injected into a GC equipped with an NPD. Wet soil or sediment samples are analyzed by 

extracting a 40 g wet-weight aliquot of the sample with a mixture of 10.0 mL degassed reagent water and 

10.0 mL isooctane. The extraction is performed in a glass jar on a platform shaker for 18 hours. A 1.0 

μL aliquot of the extract is analyzed by GC-NPD. 

Special Considerations: The presence of white phosphorus should be confirmed by either a secondary 

GC column or by an MS. 

Source: EPA. 1996. “Method 7580 (SW-846): White Phosphorus (P4) by Solvent Extraction and Gas 

Chromatography,” Revision 0. http://www.epa.gov/sam/pdfs/EPA-7580.pdf 

5.2.35 EPA Method 8015C (SW-846): Nonhalogenated Organics Using GC/FID 

Analyte(s) CAS RN 

Diesel range organics NA 

Gasoline range organics NA 

Kerosene 64742-81-0 

Analysis Purpose: Analysis
 

Sample Preparation Technique: EPA SW-846 Method 3545A/3541 or Method 5035A (solid samples), 

Method 3580A or Method 3585 (non-aqueous liquid/organic solid samples), Method 3535A or 5030C 

(aqueous liquid and drinking water samples), and Method 3570/8290A Appendix A (wipe samples). 

Refer to Appendix A for which of these preparation methods should be used for a particular 

analyte/sample type combination. 

Determinative Technique: Gas chromatograph –flame ionization detector (GC-FID) 

Method Developed for: Various nonhalogenated VOCs and semivolatile organic compounds in water 

samples 

Method Selected for: SAM lists this method for analysis of solid, non-aqueous liquid/organic solid, 

aqueous liquid, drinking water, and wipe samples. 

Detection and Quantitation: The estimated MDLs vary with each analyte and range between 2 and 48 

μg/L for aqueous liquid samples. The MDLs in other matrices have not been evaluated. The analytical 

range depends on the target analyte(s) and the instrument used. 

Description of Method: This method provides GC conditions for the detection of certain 

nonhalogenated volatile and semivolatile organic compounds. Depending on the analytes of interest, 

 SAM Revision 5.0 72 September 29, 2009 
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samples may be introduced into the GC by a variety of techniques including purge-and-trap, direct 

injection of aqueous liquid samples, and solvent extraction. An appropriate column and temperature 

program are used in the GC to separate the organic compounds. Detection is achieved by a flame 

ionization detector (FID). The method allows the use of packed or capillary columns for the analysis and 

confirmation of the non-halogenated individual analytes. 

Special Considerations: The presence of the analytes listed in the table above should be confirmed by 

either a secondary GC column or by an MS. 

Source: EPA. 2000. “Method 8015C (SW-846): Nonhalogenated Organics Using GC/FID,” Revision 3. 

http://www.epa.gov/sam/pdfs/EPA-8015c.pdf 

5.2.36 EPA Method 8260C (SW-846): Volatile Organic Compounds by Gas 

Chromatography-Mass Spectrometry (GC/MS) 

Analyte(s) CAS RN 

Allyl alcohol 107-18-6 

Carbon disulfide 75-15-0 

2-Chloroethanol 107-07-3 

Cyanogen chloride 506-77-4 

1,2-Dichloroethane 107-06-2 

Ethylene oxide 75-21-8 

2-Fluoroethanol 371-62-0 

Propylene oxide 75-56-9 

The following analytes should be determined by this method (and corresponding sample preparation methods) 

only if problems (e.g., insufficient recovery, interferences) occur when using the sample preparation/determinative 

techniques identified for these analytes in Appendix A. 

1,4-Thioxane 15980-15-1 

Analysis Purpose: Analysis
 

Sample Preparation Technique: EPA SW-846 Method 5035A (solid samples), Method 3585 (nonaqueous liquid/organic solid samples), and Method 5030C (aqueous liquid and drinking water samples) 

Determinative Technique: GC-MS 

Method Developed for: Applicable to nearly all types of samples, regardless of water content, including 

various air sampling trapping media, ground and surface water, aqueous sludges, caustic liquors, acid 

liquors, waste solvents, oily wastes, mousses (emulsified oil), tars, fibrous wastes, polymeric emulsions, 

filter cakes, spent carbons, spent catalysts, soils, and sediments. 

Method Selected for: SAM lists this method for analysis of solid, non-aqueous liquid/organic solid, 

aqueous liquid, and drinking water samples. For carbon disulfide and 1,2-dichloroethane only, EPA 

Method 524.2 (rather than 8260C) should be used for analysis of drinking water samples. 

Detection and Quantitation: Using standard quadrupole instrumentation and the purge-and-trap i 

Description of Method: Volatile compounds are introduced into a GC by purge-and-trap or other 

procedures (see Section 1.2 in Method 8260C). The analytes can be introduced directly to a wide-bore 

capillary column or cryofocused on a capillary pre-column before being flash evaporated to a narrow-bore 

capillary for analysis. Alternatively, the effluent from the trap is sent to an injection port operating in the 

split mode for injection to a narrow-bore capillary column. The column is temperature-programmed to 

 SAM Revision 5.0 73 September 29, 2009 
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separate the analytes, which are then detected with a MS interfaced to the GC. Analytes eluted from the 

capillary column are introduced into the MS via a jet separator or a direct connection. 

Source: EPA. 2006. “Method 8260C (SW-846): Volatile Organic Compounds by Gas 

Chromatography/Mass Spectrometry (GC/MS),” Revision 3. http://www.epa.gov/sam/pdfs/EPA8260c.pdf 

5.2.37 EPA Method 8270D (SW-846): Semivolatile Organic Compounds by Gas 

Chromatography/Mass Spectrometry (GC-MS) 

Analyte(s) CAS RN 

Chlorfenvinphos 470-90-6 

3-Chloro-1,2-propanediol1

 96-24-2 

Chloropicrin2 76-06-2 

Chlorosarin 1445-76-7 

Chlorosoman 7040-57-5 

Chlorpyrifos 2921-88-2 

Chlorpyrifos oxon 5598-15-2 

Crimidine3

 535-89-7 

Cyclohexyl sarin (GF) 329-99-7 

Dichlorvos 62-73-7 

Dicrotophos 141-66-2 

Dimethylphosphite 868-85-9 

Disulfoton 298-04-4 

Disulfoton sulfoxide 2497-07-6 

1,4-Dithiane 505-29-3 

Ethyldichloroarsine (ED) 598-14-1 

Fenamiphos 22224-92-6 

Methyl hydrazine 60-34-4 

Methyl paraoxon 950-35-6 

Methyl parathion 298-00-0 

1-Methylethyl ester ethylphosphonofluoridic acid (GE) 1189-87-3 

Mevinphos 7786-34-7 

Monocrotophos 6923-22-4 

Mustard, nitrogen (HN-1) [bis(2-chloroethyl)ethylamine] 538-07-8 

 SAM Revision 5.0 74 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte(s) CAS RN 

Mustard, nitrogen (HN-2) [2,2’-dichloro-Nmethyldiethylamine N,N-bis(2-chloroethyl)methylamine] 51-75-2 

Mustard, nitrogen (HN-3) [tris(2-chloroethyl)amine] 555-77-1 

Mustard, sulfur / Mustard gas (HD)4 505-60-2 

Nicotine compounds 54-11-5 

Organophosphate pesticides, NOS NA 

Paraoxon 311-45-5 

Parathion 56-38-2 

Phencyclidine 77-10-1 

Phenol 108-95-2 

Phorate 298-02-2 

Phorate sulfone 2588-04-7 

Phorate sulfoxide 2588-03-6 

Phosphamidon 13171-21-6 

R 33 (VR) [methylphosphonothioic acid, S-[2­

(diethylamino)ethyl] O-2-methylpropyl ester] 159939-87-4 

Sarin (GB)4 107-44-8 

Semivolatile organic compounds, NOS NA 

Soman (GD) 96-64-0 

Strychnine 57-24-9 

Tabun (GA) 77-81-6 

Tetraethyl pyrophosphate 107-49-3 

Tetramethylenedisulfotetramine2

 80-12-6 

1,4-Thioxane5

 15980-15-1 

Trimethyl phosphite2 121-45-9 

VE [phosphonothioic acid, ethyl-, S-(2­

(diethylamino)ethyl) O-ethyl ester] 21738-25-0 

VG [phosphonothioic acid, S-(2-(diethylamino)ethyl) 

O,O-diethyl ester] 78-53-5 

VM [phosphonothioic acid, methyl-, S-(2­

(diethylamino)ethyl) O-ethyl ester] 21770-86-5 

VX [O-ethyl-S-(2-diisopropylaminoethyl)methylphosphonothiolate]4 50782-69-9 

 SAM Revision 5.0 75 September 29, 2009 
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Analyte(s) CAS RN 

The following analyte should be determined by this method only if liquid chromatography-mass spectrometry (LCMS) [electrospray] procedures are not available to the laboratory. Sample preparation methods should remain the 

same. 

BZ [Quinuclidinyl benzilate]1

 6581-06-2 

Diisopropyl methylphosphonate (DIMP) 1445-75-6 

Dimethylphosphoramidic acid1

 33876-51-6 

EA2192 [Diisopropylaminoethyl 

methylthiolophosphonate]1 73207-98-4 

Ethyl methylphosphonic acid (EMPA)1

 1832-53-7 

Isopropyl methylphosphonic acid (IMPA)1

 1832-54-8 

Methylphosphonic acid (MPA)1 993-13-5 

Pinacolyl methyl phosphonic acid (PMPA)1

 616-52-4 

1 For this analyte, SW-846 Method 8270D must be modified to include a derivatization step.
 2 If problems occur with analyses, lower the injection temperature. 

3 If problems occur when using this method, it is recommended that SW-846 Method 8321B be used. Sample 

preparation methods should remain the same. 

4 For this analyte, refer to EPA SW-846 Method 8271 for GC-MS conditions. 

5 If problems occur when using this method, it is recommended that SW-846 Method 8260C and appropriate 

corresponding sample preparation procedures (i.e., Method 5035A for solid samples, Method 3585 for non-aqueous 

liquid/organic solid samples, and Method 5030C for aqueous liquid and drinking water samples) be used. 

Analysis Purpose: Analysis 

Sample Preparation Technique: EPA SW-846 Method 3545A/3541 (solid samples), Method 3580A 

(non-aqueous liquid/organic solid samples), Method 3520C/3535A (aqueous liquid and drinking water 

samples), and Method 3570/8290A Appendix A or NIOSH 9102 (wipe samples). Refer to Appendix A 

for which of these preparation methods should be used for a particular analyte/sample type combination. 

Determinative Technique: GC-MS 

Method Developed for: Semivolatile organic compounds in extracts prepared from many types of solid 

waste matrices, soils, air sampling media and water samples 

Method Selected for: SAM lists this method for analysis of solid, non-aqueous liquid/organic solid, 

aqueous liquid, drinking water, and/or wipe samples. Please note: drinking water samples for dichlorvos, 

disulfoton, disulfoton sulfoxide, fenamiphos, mevinphos, and semivolatile organic compounds NOS 

should be prepared and analyzed by EPA Method 525.2; aqueous liquid and drinking water samples for 

organophosphate pesticides NOS should be prepared and analyzed by EPA Methods 614 and 507, 

respectively; aqueous liquid and drinking water samples for chloropicrin should be prepared and analyzed 

by EPA Method 551.1; all other analyte/sample type combinations should be analyzed by this method 

(SW-846 8270D). 

Detection and Quantitation: The EDLs vary with each analyte and range between 10 and 1000 μg/L for 

aqueous liquid samples and 660 and 3300 μg/kg for soil samples. The analytical range depends on the 

target analyte(s) and the instrument used. 

Description of Method: Samples are prepared for analysis by GC-MS using the appropriate sample 

preparation and, if necessary, sample cleanup procedures. Semivolatile compounds are introduced into 

the GC-MS by injecting the sample extract into a GC with a narrow-bore fused-silica capillary column. 

The GC column is temperature-programmed to separate the analytes, which are then detected with a MS 

connected to the GC. Analytes eluted from the capillary column are introduced into the MS. For the 

 SAM Revision 5.0 76 September 29, 2009 
Section 5 – Selected Chemical Methods

determination of 3-chloro-1,2-propanediol, dimethylphosphoramidic acid, EA2192, EMPA, IMPA, MPA, 

and pinacolyl methyl phosphonic acid, a derivatization step is required prior to injection into the GC-MS. 

The phosphonic acids require derivatization with a trimethylsilyl agent and 3-chloro-1,2-propanediol 

requires derivatization with a heptafluorobutyryl agent. 

Special Considerations: Refer to footnotes provided in analyte table above for special considerations 

that should be applied when measuring specific analytes. Procedures for derivatization are described in 

the following references: 

Black et al. 1994. “Application of gas chromatography-mass spectrometry and gas chromatographytandem mass spectrometry to the analysis of chemical warfare samples, found to contain residues of the 

nerve agent sarin, sulphur mustard and their degradation products.” Journal of Chromatography A. 

662(2): 301–321. http://www.sciencedirect.com/science/journal/00219673 

Brereton, P., Kelly, J., Crews, C., Honour, S., and Wood, R. 2001. “Determination of 3-Chloro-1,2-

Propanediol in Foods and Food Ingredients by Gas Chromatography with Mass Spectrometric Detection: 

Collaborative Study.” Journal of AOAC International. 84(2): 455–465. http://www.atyponlink.com/AOAC/doi/abs/10.5555/jaoi.2001.84.2.455 

Divinova, V., Svejkovska, B., Dolezal, M., and Velisek, J. 2004. “Determination of Free and Bound 3-

Chloropropane-1,2-diol by Gas Chromatography with Mass Spectrometric Detection using Deuterated 3-

Chloropropane-1,2-diol as Internal Standard.” Czech Journal of Food Sciences. 22(5): 182–189. 

http://www.epa.gov/sam/pdfs/Czech_J_Food_Sci-22(5)_pg182-189.pdf 

Retho, C., and Blanchard, F. 2005. “Determination of 3-chloropropane-1,2-diol as its 1,3-dioxolane 

derivative at the μg kg-1 level: Application to a wide range of foods.” Food Additives & Contaminants: 

Part A Chemistry, Analysis, Control, Exposure & Risk Assessment. 22(12): 1189–1197. 

http://www.informaworld.com/smpp/content~db=all~content=a727751832 

White et al. 1992. “Determination of 3-Quinuclidinyl Benzilate (QNB) and Its Major Methoabolites in 

Urine by Isotope Dilution Gas Chromatography/Mass Spectrometry.” Journal of Analytical Toxicology. 

16: 182–187. http://www.jatox.com/shop/shopexd.asp?id=4062 

Source: EPA. 1998. “Method 8270D (SW-846): Semivolatile Organic Compounds by Gas 

Chromatography/Mass Spectrometry (GC/MS),” Revision 4. http://www.epa.gov/sam/pdfs/EPA8270d.pdf 

5.2.38 EPA Method 8290A, Appendix A (SW-846): Procedure for the Collection, Handling,

Analysis, and Reporting of Wipe Tests Performed within the Laboratory 

Analyte(s) CAS RN 

Acrylamide 79-06-1 

Acrylonitrile 107-13-1 

Aldicarb (Temik) 116-06-3 

Aldicarb sulfone 1646-88-4 

Aldicarb sulfoxide 1646-87-3 

4-Aminopyridine 504-24-5 

BZ [Quinuclidinyl benzilate] 6581-06-2 

Brodifacoum 56073-10-0 

 SAM Revision 5.0	 77 September 29, 2009 
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Analyte(s) CAS RN 

Bromadiolone 28772-56-7 

Carfentanil 59708-52-0 

Carbofuran (Furadan) 1563-66-2 

Chlorfenvinphos 470-90-6 

3-Chloro-1,2-propanediol 96-24-2 

Chloropicrin 76-06-2 

Chlorosarin 1445-76-7 

Chlorosoman 7040-57-5 

Chlorpyrifos 2921-88-2 

Chlorpyrifos oxon 5598-15-2 

Crimidine 535-89-7 

Cyclohexyl sarin (GF) 329-99-7 

Dichlorvos 62-73-7 

Dicrotophos 141-66-2 

Diesel range organics NA 

Diisopropyl methylphosphonate (DIMP) 1445-75-6 

Dimethylphosphite 868-85-9 

Dimethylphosphoramidic acid 33876-51-6 

Diphacinone 82-66-6 

Disulfoton 298-04-4 

Disulfoton sulfoxide 2497-07-6 

1,4-Dithiane 505-29-3 

EA2192 [Diisopropylaminoethyl 

methylthiolophosphonate] 73207-98-4 

Ethyl methylphosphonic acid (EMPA) 1832-53-7 

N-Ethyldiethanolamine (EDEA) 139-87-7 

Fenamiphos 22224-92-6 

Fentanyl 437-38-7 

Fluoroacetic acid and fluoroacetate salts NA 

Formaldehyde 50-00-0 

Gasoline range organics NA 

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) 121-82-4 

 SAM Revision 5.0 78 September 29, 2009 
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Analyte(s) CAS RN 

Hexamethylenetriperoxidediamine (HMTD) 283-66-9 

Isopropyl methylphosphonic acid (IMPA) 1832-54-8 

Kerosene 64742-81-0 

Methomyl 16752-77-5 

Methyl acrylonitrile 126-98-7 

Methyl fluoroacetate 453-18-9 

Methyl hydrazine 60-34-4 

Methyl paraoxon 950-35-6 

Methyl parathion 298-00-0 

N-Methyldiethanolamine (MDEA) 105-59-9 

1-Methylethyl ester ethylphosphonofluoridic acid (GE) 1189-87-3 

Methylphosphonic acid (MPA) 993-13-5 

Mevinphos 7786-34-7 

Monocrotophos 6923-22-4 

Mustard, nitrogen (HN-1) [bis(2-chloroethyl)ethylamine] 538-07-8 

Mustard, nitrogen (HN-2) [2,2’-dichloro-Nmethyldiethylamine N,N-bis(2-chloroethyl)methylamine] 51-75-2 

Mustard, nitrogen (HN-3) [tris(2-chloroethyl)amine] 555-77-1 

Mustard, sulfur / Mustard gas (HD) 505-60-2 

Nicotine compounds 54-11-5 

Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) 2691-41-0 

Organophosphate pesticides, NOS NA 

Oxamyl 23135-22-0 

Paraoxon 311-45-5 

Parathion 56-38-2 

Pentaerythritol tetranitrate (PETN) 78-11-5 

Phencyclidine 77-10-1 

Phenol 108-95-2 

Phorate 298-02-2 

Phorate sulfone 2588-04-7 

Phorate sulfoxide 2588-03-6 

Phosphamidon 13171-21-6 

 SAM Revision 5.0 79 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte(s) CAS RN 

Pinacolyl methyl phosphonic acid (PMPA) 616-52-4 

R 33 (VR) [methylphosphonothioic acid, S-[2­

(diethylamino)ethyl] O-2-methylpropyl ester] 159939-87-4 

Sarin (GB) 107-44-8 

Semivolatile organic compounds, NOS NA 

Soman (GD) 96-64-0 

Strychnine 57-24-9 

Tabun (GA) 77-81-6 

Tetraethyl pyrophosphate 107-49-3 

Tetramethylenedisulfotetramine 80-12-6 

Thiodiglycol (TDG) 111-48-8 

Thiofanox 39196-18-4 

1,4-Thioxane 15980-15-1 

Triethanolamine (TEA) 102-71-6 

Trimethyl phosphite 121-45-9 

1,3,5-Trinitrobenzene (1,3,5-TNB) 99-35-4 

2,4,6-Trinitrotoluene (2,4,6-TNT) 118-96-7 

VE [phosphonothioic acid, ethyl-, S-(2­

(diethylamino)ethyl) O-ethyl ester] 21738-25-0 

VG [phosphonothioic acid, S-(2-(diethylamino)ethyl) 

O,O-diethyl ester] 78-53-5 

VM [phosphonothioic acid, methyl-, S-(2­

(diethylamino)ethyl) O-ethyl ester] 21770-86-5 

VX [O-ethyl-S-(2-diisopropylaminoethyl)methylphosphonothiolate] 50782-69-9 

White phosphorus 12185-10-3 

Analysis Purpose: Sample preparation 

Sample Preparation Technique: Solvent extraction 

Determinative Technique: EPA OW Method 300.1 Revision 1.0; EPA SW-846 Methods 7580, 8015C, 

8270D, 8315A, 8316, 8318A, 8321B, and 8330B. Refer to Appendix A for which of these determinative 

methods should be used for a particular analyte. 

Method Developed for: Evaluation of surface contamination by 2,3,7,8-substituted PCDD and PCDF 

congeners 

Method Selected for: SAM lists this method for preparation of wipe samples. 

 SAM Revision 5.0 80 September 29, 2009 
Section 5 – Selected Chemical Methods

Description of Method: A surface area of 2 inches by 1 foot is wiped with glass fiber paper saturated 

with distilled-in-glass acetone. One wipe is used per designated area. Wipes are combined into a single 

composite sample in an extraction jar and solvent extracted using a wrist action shaker. 

Special Considerations: The solvent systems described in this method extraction have been evaluated 

for PCDD and PCDF congeners only. Other analytes may require different solvent systems for optimal 

sample extraction. 

Source: EPA. 2007. “Method 8290A, Appendix A (SW-846): Procedure for the Collection, Handling, 

Analysis, and Reporting of Wipe Tests Performed within the Laboratory,” Revision 1. 

http://www.epa.gov/sam/pdfs/EPA-8290a.pdf 

5.2.39 EPA Method 8315A (SW-846): Determination of Carbonyl Compounds by High 

Performance Liquid Chromatography (HPLC) 

Analyte(s) CAS RN 

Formaldehyde 50-00-0 

Analysis Purpose: Sample preparation and/or analysis
 

Sample Preparation Technique: Solvent extraction (solid, aqueous liquid, and drinking water samples) 

and EPA SW-846 Method 3570/8290A Appendix A (wipe samples) 

Determinative Technique: HPLC 

Method Developed for: Free carbonyl compounds in aqueous, soil, waste, and stack samples 

Method Selected for: SAM lists this method for preparation and analysis of solid, aqueous liquid, 

drinking water, and wipe samples. 

Detection and Quantitation: The MDL for formaldehyde varies depending on sample conditions and 

instrumentation, but is approximately 6.2 μg/L for aqueous liquid samples. 

Description of Method: A measured volume of aqueous liquid sample (approximately 100 mL), or an 

appropriate amount of solids extract (approximately 25 g), is buffered to pH 3 and derivatized with 2,4-

dinitrophenylhydrazine (2,4-DNPH). Using the appropriate extraction technique, the derivatives are 

extracted using methylene chloride and the extracts are exchanged with acetonitrile prior to HPLC 

analysis. HPLC conditions are described permitting the separation and measurement of various carbonyl
 

compounds in the extract by absorbance detection at 360 nm. If formaldehyde is the only analyte of 

interest, the aqueous liquid sample and/or solid sample extract should be buffered to pH 5.0 to minimize 

the formation of artifact formaldehyde. 

Source: EPA. 1996. “Method 8315A (SW-846): Determination of Carbonyl Compounds by High 

Performance Liquid Chromatography (HPLC),” Revision 1. http://www.epa.gov/sam/pdfs/EPA8315a.pdf
 

5.2.40 EPA Method 8316 (SW-846): Acrylamide, Acrylonitrile and Acrolein by High 

Performance Liquid Chromatography (HPLC) 

Analyte(s) CAS RN 

Acrylamide 79-06-1 

Acrylonitrile 107-13-1 

Methyl acrylonitrile 126-98-7 

 SAM Revision 5.0 81 September 29, 2009 
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Analysis Purpose: Sample preparation and/or analysis
 

Sample Preparation Technique: Direct injection (aqueous liquid and drinking water samples), water 

extraction (solid and non-aqueous liquid/organic solid samples), and EPA SW-846 Method 3570/8290A 

Appendix A (wipe samples) 

Determinative Technique: HPLC 

Method Developed for: Acrylamide, acrylonitrile, and acrolein in water samples 

Method Selected for: SAM lists this method for preparation and/or analysis of solid, non-aqueous 

liquid/organic solid, aqueous liquid, drinking water, and wipe samples. 

Detection and Quantitation: Acrylamide has an MDL of 10 μg/L; acrylonitrile has an MDL of 20 μg/L. 

Description of Method: Samples are analyzed by HPLC. A 200-μL aliquot is injected onto a C18
 

reverse-phase column, and compounds in the effluent are detected with a UV detector. Solid and nonaqueous liquid/organic solid samples should be water extracted prior to injection. Aqueous liquid and 

drinking water samples can be directly injected. 

Source: EPA. 1994. “Method 8316 (SW-846): Acrylamide, Acrylonitrile and Acrolein by High 

Performance Liquid Chromatography (HPLC),” Revision 0. http://www.epa.gov/sam/pdfs/EPA-8316.pdf
 

5.2.41 EPA Method 8318A (SW-846): N-Methylcarbamates by High Performance Liquid 

Chromatography (HPLC) 

Analyte(s) CAS RN 

Aldicarb (Temik) 116-06-3 

Aldicarb sulfone 1646-88-4 

Aldicarb sulfoxide 1646-87-3 

Carbofuran (Furadan) 1563-66-2 

Methomyl 16752-77-5 

Oxamyl 23135-22-0 

Analysis Purpose: Sample preparation and/or analysis
 

Sample Preparation Technique: Solvent extraction (solid, non-aqueous liquid/organic solid samples), 

and EPA SW-846 Method 3570/8290A Appendix A (wipe samples) 

Determinative Technique: HPLC 

Method Developed for: N-methylcarbamates in soil, water, and waste matrices
 

Method Selected for: SAM lists this method for preparation and/or analysis of solid, non-aqueous 

liquid/organic solid, and wipe samples. 

Detection and Quantitation: The estimated MDLs vary with each analyte and range from 1.7 to 9.4 

μg/L for aqueous samples and 10 to 50 μg/kg for soil samples. 

Description of Method: N-methylcarbamates are extracted from aqueous liquid samples with methylene 

chloride, and from soils, oily solid waste, and oils with acetonitrile. The extract solvent is exchanged to 

methanol/ethylene glycol, and the extract is cleaned using a C18 cartridge, filtered, and eluted on a C18
 

analytical column. After separation, the target analytes are hydrolyzed and derivatized post-column, then 

quantified fluorometrically. The sensitivity of the method usually depends on the level of interferences 

present, rather than on instrument conditions. Waste samples with a high level of extractable fluorescent 

compounds are expected to yield significantly higher detection limits. 

 SAM Revision 5.0 82 September 29, 2009 
Section 5 – Selected Chemical Methods

Source: EPA. 2000. “Method 8318A (SW-846): N-Methylcarbamates by High Performance Liquid 

Chromatography (HPLC),” Revision 1. http://www.epa.gov/sam/pdfs/EPA-8318a.pdf 

5.2.42 EPA Method 8321B (SW-846): Solvent-Extractable Nonvolatile Compounds by

High Performance Liquid Chromatography-Thermospray-Mass Spectrometry

(HPLC-TS-MS) or Ultraviolet (UV) Detection 

Analyte(s) CAS RN 

Brodifacoum 56073-10-0 

Bromadiolone 28772-56-7 

BZ [Quinuclidinyl benzilate]1

 6581-06-2 

Carfentanil 59708-52-0 

Diisopropyl methylphosphonate (DIMP)2 1445-75-6 

Dimethylphosphoramidic acid1

 33876-51-6 

Diphacinone 82-66-6 

EA2192 [Diisopropylaminoethyl 

methylthiolophosphonate]1 73207-98-4 

Ethyl methylphosphonic acid (EMPA)1

 1832-53-7 

N-Ethyldiethanolamine (EDEA) 139-87-7 

Fentanyl 437-38-7 

Isopropyl methylphosphonic acid (IMPA)1

 1832-54-8 

N-Methyldiethanolamine (MDEA) 105-59-9 

Methylphosphonic acid (MPA)1

 993-13-5 

Pinacolyl methyl phosphonic acid (PMPA)1

 616-52-4 

Thiodiglycol (TDG) 111-48-8 

Thiofanox 39196-18-4 

Triethanolamine (TEA) 102-71-6 

The following analyte should be determined by this method only if problems (e.g., insufficient recovery, 

interferences) occur when using SW-846 Method 8270D. Sample preparation methods should remain the same as 

those listed in Appendix A. 

Crimidine3

 535-89-7 

1	 LC-MS (electrospray) procedures are preferred for these analytes; however, if this technique is not available to the 

laboratory, GC-MS procedures using derivatization based on SW-846 Method 8270D may be used. Sample 

preparation methods should remain the same. Both electrospray LC-MS and GC-MS derivatization procedures are 

currently under development. 2 If problems occur with the analysis of DIMP using EPA SW-846 Method 8321B, use SW-846 Method 8270D. 3 This analyte needs to be determined using a wavelength of 230 nm. 

Analysis Purpose: Analysis 

Sample Preparation Technique: EPA SW-846 Method 3545A/3541 (solid samples), 3580A (nonaqueous liquid/organic solid samples), 3520C/3535A (aqueous liquid and drinking water samples), and 

 SAM Revision 5.0	 83 September 29, 2009 
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Method 3570/8290A Appendix A (wipe samples). For thiofanox, EPA Method 531.2 (rather than 

Method 3520C/3535A) should be used for preparation of drinking water samples. Refer to Appendix A 

for which of these preparation methods should be used for a particular analyte/sample type combination. 

Determinative Technique: HPLC 

Method Developed for: Solvent-extractable nonvolatile compounds, including dyes, organophosphorus 

compounds, phenoxyacid herbicides, and carbamates in solid, water, aqueous, and non-aqueous samples 

Method Selected for: SAM lists this method for analysis of solid, non-aqueous liquid/organic solid, 

aqueous liquid, drinking water, and wipe samples. Aqueous liquid samples for DIMP, EMPA, IMPA, 

MPA, and PMPA should be analyzed using EPA Chicago Regional Laboratory (CRL) MS017; aqueous 

liquid samples for EDEA, MDEA, and TEA should be prepared and analyzed using EPA CRL MS016; 

aqueous liquid samples for bromadiolone should be prepared and analyzed using EPA CRL MS014; 

aqueous liquid samples for thiodiglycol should be prepared and analyzed using EPA CRL MS015. 

Description of Method: This method provides reversed-phase HPLC, thermospray (TSP) MS, and UV 

conditions for detection of the target analytes. Sample extracts can be analyzed by direct injection into 

the TSP or onto a LC-TSP interface. A gradient elution program is used to separate the compounds. 

Primary analysis may be performed by UV detection; however, positive results should be confirmed by 

TSP-MS. Quantitative analysis may be performed by either TSP-MS or UV detection, using either an 

external or internal standard approach. TSP-MS detection may be performed in either a negative 

ionization (discharge electrode) mode or a positive ionization mode, with a single quadrupole MS. The 

use of MS-MS techniques is an option. The analytical range and detection limits vary depending on the 

target analyte and instrument used. 

Special Considerations: Refer to footnotes provided in analyte table above for special considerations 

that should be applied when measuring specific analytes. 

Source: EPA. 1998. “Method 8321B (SW-846): Solvent-Extractable Nonvolatile Compounds by High 

Performance Liquid Chromatography-Thermospray-Mass Spectrometry (HPLC-TSP-MS) or Ultraviolet 

(UV) Detection,” Revision 2. http://www.epa.gov/sam/pdfs/EPA-8321b.pdf 

5.2.43 EPA Method 8330B (SW-846): Nitroaromatics and Nitramines by High Performance 

Liquid Chromatography (HPLC) 

Analyte(s) CAS RN 

4-Aminopyridine 504-24-5 

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) 121-82-4 

Hexamethylenetriperoxidediamine (HMTD) 283-66-9 

Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) 2691-41-0 

Pentaerythritol tetranitrate (PETN) 78-11-5 

1,3,5-Trinitrobenzene (1,3,5-TNB) 99-35-4 

2,4,6-Trinitrotoluene (2,4,6-TNT) 118-96-7 

Analysis Purpose: Sample preparation and/or analysis 

Sample Preparation Technique: Solvent extraction or direct injection (solid and non-aqueous 

liquid/organic solid samples), EPA SW-846 Method 3535A (aqueous liquid and drinking water samples), 

and EPA SW-846 Method 3570/8290A Appendix A (wipe samples) 

Determinative Technique: HPLC 

 SAM Revision 5.0 84 September 29, 2009 
Section 5 – Selected Chemical Methods

Method Developed for: Trace analysis of explosives and propellant residues in water, soil, or sediment 

Method Selected for: SAM lists this method for preparation and/or analysis of solid, non-aqueous 

liquid/organic solid, aqueous liquid, drinking water, and wipe samples. Aqueous liquid and drinking 

water samples are prepared using Methods 3535A or 8330B prior to analysis. 

Detection and Quantitation: The detection limits, ranges, and interferences depend on the target 

compound 

Description of Method: This method is intended for the trace analysis of explosives and propellant 

residues by HPLC using a dual wavelength UV detector in a water, soil, or sediment matrix. All of the 

compounds listed in this method are either used in the manufacture of explosives or propellants, or they 

are the degradation products of compounds used for that purpose. Samples are prepared for analysis by 

high performance liquid chromatography – ultraviolet (HPLC-UV) using the appropriate sample 

preparation technique (solid phase extraction by 3535A or solvent extraction by 8330B) and, if necessary, 

sample cleanup procedures. Direct injection of diluted and filtered water samples can be used for water 

samples of higher concentration. Soil and sediment samples are extracted using acetonitrile in an 

ultrasonic bath, filtered and chromatographed. 

Source: EPA. 2006. “Method 8330B (SW-846): Nitroaromatics, Nitramines, and Nitrate Esters by High 

Performance Liquid Chromatography (HPLC),” Revision 2. http://www.epa.gov/sam/pdfs/EPA8330b.pdf 

5.2.44 EPA CRL MS014: Analysis of Aldicarb, Bromadiolone, Carbofuran, Oxamyl and 

Methomyl in Water by Multiple Reaction Monitoring Liquid Chromatography / 

Tandem Mass Spectrometry (LC/MS/MS) 

Analyte(s) CAS RN 

Aldicarb (Temik) 116-06-3 

Aldicarb sulfone 1646-88-4 

Aldicarb sulfoxide 1646-87-3 

Bromadiolone 28772-56-7 

Carbofuran (Furadan) 1563-66-2 

Methomyl 16752-77-5 

Oxamyl 23135-22-0 

Analysis Purpose: Sample preparation and/or analysis 

Sample Preparation Technique: Solvent extraction 

Determinative Technique: Liquid Chromatography Tandem Mass Spectrometry (LC-MS-MS) 

Method Developed for: Determination of aldicarb, bromadiolone, carbofuran, oxamyl and methomyl in 

water 

Method Selected for: SAM lists this method for preparation and analysis of aqueous liquid samples. 

Detection and Quantitation: The limit of detection for aldicarb, bromadiolone, carbofuran, methomyl 

and oxamyl is 100 ng/L. The reporting limit for aldicarb, carbofuran, methomyl and oxamyl is 1 μg/L. 

Description of Method: Target compounds are analyzed by direct injection without derivatization by 

LC-MS-MS. Samples are shipped to the laboratory at 4°C (± 2°C), spiked with surrogates, filtered using 

a syringe driven filter unit, and analyzed directly by LC-MS-MS within 7 days. If stored prior to analysis, 

samples must be stored at ≤ 4°C (± 2°C). The target compounds are identified by comparing the sample 

 SAM Revision 5.0 85 September 29, 2009 
Section 5 – Selected Chemical Methods

primary and confirmatory multiple reaction monitoring (MRM) transitions to the known standard primary 

and confirmatory MRM transitions. The retention time for the analytes of interest must also fall within 

the retention time of the standard by ± 5%. The target compounds are quantitated using the primary 

single reaction monitoring (SRM) transition and external standard calibration. 

Source: EPA, Chicago Regional Laboratory (CRL). 2008. “MS014: Analysis of Aldicarb, 

Bromadiolone, Carbofuran, Oxamyl and Methomyl in Water by Multiple Reaction Monitoring Liquid 

Chromatography/ Tandem Mass Spectrometry (LC/MS/MS).” http:\\www.epa.gov/sam/pdfs/EPAMS014.pdf 

5.2.45 EPA CRL MS015: Analysis of Thiodiglycol in Water by Single Reaction Monitoring 

Liquid Chromatography / Tandem Mass Spectrometry (LC/MS/MS) 

Analyte(s) CAS RN 

Thiodiglycol 111-48-8 

Analysis Purpose: Sample preparation and/or analysis 

Sample Preparation Technique: Solvent extraction 

Determinative Technique: LC-MS-MS 

Method Developed for: Determination of thiodiglycol in water samples 

Method Selected for: SAM lists this method for preparation and analysis of aqueous liquid samples. 

Detection and Quantitation: The limit of detection for thiodiglycol is 50 μg/L; the reporting limit is 250 

μg/L. 

Description of Method: Thiodiglycol is analyzed by direct injection without derivatization by LC-MSMS. Samples are shipped to the laboratory at 4°C (± 2°C), spiked with surrogates, filtered using a syringe 

driven filter unit and analyzed directly by LC-MS-MS within 7 days. The target compound is identified 

by comparing the sample primary SRM transition to the known standard SRM transition. The retention 

time must fall within the retention time of the standard by ± 5%. Thiodiglycol is quantitated using the 

primary SRM transition utilizing an external standard calibration. 

Source: EPA, CRL. 2008. “MS015: Analysis of Thiodiglycol in Water by Single Reaction Monitoring 

Liquid Chromatography / Tandem Mass Spectrometry (LC/MS/MS).” 

http:\\www.epa.gov/sam/pdfs/EPA-MS015.pdf
 

5.2.46 EPA CRL MS016: Analysis of Diethanolamine, Triethanolamine, nMethyldiethanolamine and n-Ethyldiethanolamine in Water by Single Reaction 

Monitoring Liquid Chromatography / Tandem Mass Spectrometry (LC/MS/MS) 

Analyte(s) CAS RN 

N-Ethyldiethanolamine (EDEA) 139-87-7 

N-Methyldiethanolamine (MDEA) 105-59-9 

Triethanolamine (TEA) 102-71-6 

Analysis Purpose: Sample preparation and/or analysis 

Sample Preparation Technique: Solvent extraction 

Determinative Technique: LC-MS-MS 

 SAM Revision 5.0 86 September 29, 2009 
Section 5 – Selected Chemical Methods

Method Developed for: Determination of diethanolamine, triethanolamine, n-methyldiethanolamine and 

n-ethyldiethanolamine in water samples 

Method Selected for: SAM lists this method for preparation and analysis of aqueous liquid samples. 

Detection and Quantitation: The limit of detection for n-ethyldiethanolamine and triethanolamine is 5 

μg/L; the reporting limit is 25 μg/L. The limit of detection for n-methyldiethanolamine is 20 μg/L, with a 

reporting limit of 25 μg/L. 

Description of Method: Target compounds are analyzed by direct injection without derivatization by 

LC-MS-MS. Samples are shipped to the laboratory at 4°C (± 2°C), spiked with surrogates, filtered using 

a syringe driven filter unit and analyzed directly by LC-MS-MS within 7 days. Target compounds are 

identified by comparing sample SRM transitions to the known standard SRM transitions. The retention 

time for the analytes of interest must also fall within the retention time of the standard by ± 5%. The 

target compounds are quantitated using the SRM transition and external standard calibration. 

Source: EPA, CRL. 2008. “MS016: Analysis of Diethanolamine, Triethanolamine, nMethyldiethanolamine and n-Ethyldiethanolamine in Water by Single Reaction Monitoring Liquid 

Chromatography / Tandem Mass Spectrometry (LC/MS/MS).” http:\\www.epa.gov/sam/pdfs/EPAMS016.pdf 

5.2.47 EPA CRL MS017: Analysis of Diisopropyl Methylphosphonate, Ethyl Hydrogen 

Dimethylamidophosphate, Isopropyl Methylphosphonic Acid, Methylphosphonic 

Acid and Pinacolyl Methylphosphonic Acid in Water by Multiple Reaction 

Monitoring Liquid Chromatography / Tandem Mass Spectrometry (LC/MS/MS) 

Analyte(s) CAS RN 

Diisopropyl methylphosphonate (DIMP) 1445-75-6 

Ethyl methylphosphonic acid (EMPA) 1832-53-7 

Isopropyl methylphosphonic acid (IMPA) 1832-54-8 

Methylphosphonic acid (MPA) 993-13-5 

Pinacolyl methyl phosphonic acid (PMPA) 616-52-4 

Analysis Purpose: Sample preparation and/or analysis 

Sample Preparation Technique: Solvent extraction 

Determinative Technique: LC-MS-MS 

Method Developed for: Determination of diisopropyl methylphosphonate, ethyl hydrogen 

dimethylamidophosphate, isopropyl methylphosphonic acid, methylphosphonic acid and pinacolyl 

methylphosphonic acid in water 

Method Selected for: SAM lists this method for preparation and analysis of aqueous liquid samples. 

Detection and Quantitation: The limits of detection and reporting limits for this method vary for each 

analyte and range between 0.10 to 20 μg/L and 5 to 100 μg/L, respectively. 

Description of Method: Target compounds are analyzed by direct injection without derivatization by 

LC-MS-MS. Samples are shipped to the laboratory at 4°C (± 2°C), spiked with surrogates, filtered using 

a syringe driven filter unit and analyzed directly by LC-MS-MS within 1 day. The target compounds are 

identified by comparing the sample SRM transitions to the known standard SRM transitions. The 

retention time for the analytes of interest must also fall within the retention time of the standard by ± 5%. 

Target compounds are quantitated using the SRM transition of the target compounds and external 

standard calibration. 

 SAM Revision 5.0 87 September 29, 2009 
Section 5 – Selected Chemical Methods

Source: EPA, CRL. 2008. “MS017: Analysis of Diisopropyl Methylphosphonate, Ethyl Hydrogen 

Dimethylamidophosphate, Isoproyl Methylphosphonic Acid, Methylphosphonic Acid and Pinacolyl 

Methylphosphonic Acid in Water by Multiple Reaction Monitoring Liquid Chromatography / Tandem 

Mass Spectrometry (LC/MS/MS).” http:\\www.epa.gov/sam/pdfs/EPA-MS017.pdf 

5.2.48 EPA CLP ILM05.3 Cyanide: Analytical Methods for Total Cyanide Analysis 

Analyte(s) CAS RN 

Cyanide, Total 57-12-5 

Analysis Purpose: Sample preparation and/or analysis 

Sample Preparation Technique: Acid digestion followed by distillation 

Determinative Technique: Spectrophotometry 

Method Developed for: Metals in water, sediment, sludge, and soil 

Method Selected for: SAM lists this method for preparation and/or analysis of solid, aqueous liquid, and 

wipe samples. 

Detection and Quantitation: The method quantitation limits are 10 μg/L or 2.5 mg/kg 

Description of Method: The method allows for either large volume (500-mL aqueous liquid samples or 

1-g to 5-g solid samples mixed with 500 mL of reagent water) or medium volume (50-mL aqueous liquid 

samples or 1-g solid samples mixed with 50 mL of reagent water) sample preparation. Aqueous liquid 

samples are tested for sulfides and oxidizing agents prior to preparation. Sulfides are removed with 

cadmium carbonate or lead carbonate. Samples are treated with sulfuric acid and magnesium chloride 

and distilled into a sodium hydroxide solution. The solution is treated with color agents and the cyanide 

determined as an ion complex by visible spectrophotometry. 

Special Considerations: Surfactants may interfere with the distillation procedure. 

Source: EPA Contract Laboratory Program (CLP). “ILM05.3: Exhibit D – Part D: Analytical Methods 

for Total Cyanide Analysis.” http://www.epa.gov/sam/pdfs/EPA-ILM05.3.pdf 

5.2.49 EPA Region 7 RLAB Method 3135.2I: Cyanide, Total and Amenable in Aqueous 

and Solid Samples Automated Colorimetric with Manual Digestion 

Analyte(s) CAS RN 

Cyanide, Amenable to chlorination NA 

Analysis Purpose: Sample preparation and analysis 

Sample Preparation Technique: Acid digestion followed by distillation 

Determinative Technique: Spectrophotometry 

Method Developed for: Cyanide in drinking, ground, and surface waters, domestic and industrial waste 

waters, sediments and solid waste 

Method Selected for: SAM lists this method for preparation and analysis of solid, aqueous liquid, 

drinking water, and wipe samples. 

Detection and Quantitation: The applicable range is 0.003 to 0.500 mg/L cyanide in the distillate. This 

range can be expanded by sample dilution, either by using less sample for distillation or diluting the 

distillate. 

 SAM Revision 5.0	 88 September 29, 2009 
Section 5 – Selected Chemical Methods

Description of Method: This method detects inorganic cyanides that are present as either simple soluble 

salts or complex radicals. It may be used to determine values for both total cyanide and cyanide 

amenable to chlorination (also known as available cyanide). Cyanide in the sample released as 

hydrocyanic acid by refluxing the sample with strong acid. The hydrocyanic acid is distilled and 

collected in an absorber-scrubber containing sodium hydroxide solution. The cyanide ion in the 

absorbing solution is then determined by automated colorimetry. For determination of cyanide amenable 

to chlorination, a portion of the sample is chlorinated using sodium hypochlorite at a pH > 11 to 

decompose the cyanide. Cyanide levels are then determined in both the chlorinated sample portion of the 

sample and a portion of the sample that has not been chlorinated using the total cyanide method. 

Cyanides amenable to chlorination are then calculated by difference between unchlorinated and the 

chlorinated aliquots of the sample. 

Special Considerations: Alternate cyanide analyzer equipment may be used, provided it is used 

according to the procedures described and the laboratory can demonstrate equivalent performance. 

Source: EPA Region 7. 2008. “RLAB Method 3135.2I: Cyanide, Total and Amenable in Aqueous and 

Soil Samples Automated Colorimetric with Manual Digestion.” http://www.epa.gov/sam/pdfs/EPA3135.2I.pdf 

5.2.50 IO [Inorganic] Compendium Method IO-3.1: Selection, Preparation, and Extraction 

of Filter Material 

Analyte(s) CAS RN 

Ammonium metavanadate 7803-55-6 

Arsenic, Total 7440-38-2 

Arsenic trioxide 1327-53-3 

Calcium arsenate 7778-44-1 

2-Chlorovinylarsonous acid (2-CVAA) 85090-33-1 

Lead arsenate 7645-25-2 

Lewisite 1 (L-1) [2-chlorovinyldichloroarsine] 541-25-3 

Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine] 40334-69-8 

Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine] 40334-70-1 

Lewisite oxide 1306-02-1 

Osmium tetroxide 20816-12-0 

Sodium arsenite 7784-46-5 

Thallium sulfate 10031-59-1 

Vanadium pentoxide 1314-62-1 

Analysis Purpose: Sample preparation
 

Sample Preparation Technique: Acid extraction 

Determinative Technique: EPA Method IO-3.4 or Method IO-3.5. Osmium tetroxide should be 

analyzed by Method IO-3.4. 

Method Developed for: Particulate metals in air. 

Method Selected for: SAM lists this method for preparation of air samples. 

 SAM Revision 5.0 89 September 29, 2009 
Section 5 – Selected Chemical Methods

Description of Method: This method supports determination of arsenic trioxide, lewisite, lewisite 

degradation products, calcium and lead arsenate, and sodium arsenite as total arsenic. Thallium sulfate is 

determined as total thallium and ammonium metavanadate and vanadium pentoxide are determined as 

total vanadium. A subsample (one-ninth of the overall filter) is obtained by cutting a strip from the filter 

used to collect the sample. The filter strip is extracted using a hydrochloric/nitric acid mix and 

microwave or hotplate heating. The extract is filtered, worked up to 20 mL, and analyzed using either 

Method IO-3.4 or Method IO-3.5. 

Source: EPA. 1999. “IO Compendium Method IO-3.1: Compendium of Methods for the Determination 

of Inorganic Compounds in Ambient Air: Selection, Preparation and Extraction of Filter Material.” 

http://www.epa.gov/sam/pdfs/EPA-IO-3.1.pdf 

5.2.51 IO [Inorganic] Compendium Method IO-3.4: Determination of Metals in Ambient 

Particulate Matter Using Inductively Coupled Plasma (ICP) Spectroscopy 

Analyte(s) CAS RN 

Ammonium metavanadate 7803-55-6 

Arsenic, Total 7440-38-2 

Arsenic trioxide 1327-53-3 

Calcium arsenate 7778-44-1 

2-Chlorovinylarsonous acid (2-CVAA) 85090-33-1 

Lead arsenate 7645-25-2 

Lewisite 1 (L-1) [2-chlorovinyldichloroarsine] 541-25-3 

Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine] 40334-69-8 

Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine] 40334-70-1 

Lewisite oxide 1306-02-1 

Osmium tetroxide 20816-12-0 

Sodium arsenite 7784-46-5 

Thallium sulfate 10031-59-1 

Vanadium pentoxide 1314-62-1 

Analysis Purpose: Analysis 

Sample Preparation Technique: EPA Method IO-3.1 

Determinative Technique: ICP-AES 

Method Developed for: Metals in ambient particulate matter 

Method Selected for: SAM lists this method for analysis of air samples. 

Description of Method: This method determines arsenic trioxide, lewisite, lewisite degradation 

products, calcium and lead arsenate, and sodium arsenite as total arsenic. Osmium tetroxide is 

determined as total osmium, thallium sulfate is determined as total thallium, and ammonium
 

metavanadate and vanadium pentoxide are determined as total vanadium. Ambient air is sampled by
 

high-volume filters using Method IO-2.1 (a sampling method) and the filters are extracted by Method IO3.1. Detection limits, ranges, and interference corrections are dependent on the analyte and the 

instrument used. 

 SAM Revision 5.0 90 September 29, 2009 
Section 5 – Selected Chemical Methods

Special Considerations: Laboratory testing is currently underway for speciation of lewisite 1 using GCMS techniques. Concerns have been raised regarding the use of nitric acid when analyzing samples for 

osmium tetroxide; hydrochloric acid should be considered and evaluated as a possible alternative. 

Source: EPA. 1999. “IO Compendium Method IO-3.4: Compendium of Methods for the Determination 

of Inorganic Compounds in Ambient Air: Determination of Metals in Ambient Particulate Matter Using 

Inductively Coupled Plasma (ICP) Spectroscopy.” http://www.epa.gov/sam/pdfs/EPA-IO-3.4.pdf 

EPA. 1999. “IO Compendium Method IO-2.1: Compendium of Methods for the Determination of 

Inorganic Compounds in Ambient Air: Sampling of Ambient Air for Total Suspended Particulate Matter 

(SPM) and PM10 Using High Volume (HV) Sampler.” http://www.epa.gov/sam/pdfs/EPA-IO-2.1.pdf 

5.2.52 IO [Inorganic] Compendium Method IO-3.5: Determination of Metals in Ambient 

Particulate Matter Using Inductively Coupled Plasma/Mass Spectrometry (ICP-MS) 

Analyte(s) CAS RN 

Ammonium metavanadate 7803-55-6 

Arsenic, Total 7440-38-2 

Arsenic trioxide 1327-53-3 

Calcium arsenate 7778-44-1 

2-Chlorovinylarsonous acid (2-CVAA) 85090-33-1 

Lead arsenate 7645-25-2 

Lewisite 1 (L-1) [2-chlorovinyldichloroarsine] 541-25-3 

Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine] 40334-69-8 

Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine] 40334-70-1 

Lewisite oxide 1306-02-1 

Sodium arsenite 7784-46-5 

Thallium sulfate 10031-59-1 

Vanadium pentoxide 1314-62-1 

Analysis Purpose: Analysis 

Sample Preparation Technique: EPA Method IO-3.1 

Determinative Technique: ICP-MS 

Method Developed for: Metals in ambient particulate matter 

Method Selected for: SAM lists this method for analysis of air samples. 

Description of Method: This method determines arsenic trioxide, lewisite, lewisite degradation 

products, calcium and lead arsenate, and sodium arsenite as total arsenic. Thallium sulfate is determined 

as total thallium and ammonium metavanadate and vanadium pentoxide are determined as total vanadium.
 

Ambient air is sampled by high-volume filters using Method IO-2.1 (a sampling method). The filters are 

extracted by Method IO-3.1. Detection limits, ranges, and interference corrections are dependent on the 

analyte and the instrument used. 

 SAM Revision 5.0 91 September 29, 2009 
Section 5 – Selected Chemical Methods

Special Considerations: Laboratory testing is currently underway for speciation of lewisite 1 using GCMS techniques. 

Source: EPA. 1999. “IO Compendium Method IO-3.5: Compendium of Methods for the Determination 

of Inorganic Compounds in Ambient Air: Determination of Metals in Ambient Particulate Matter Using 

Inductively Coupled Plasma/Mass Spectrometry (ICP/MS).” http://www.epa.gov/sam/pdfs/EPA-IO3.5.pdf 

EPA. 1999. “IO Compendium Method IO-2.1: Compendium of Methods for the Determination of 

Inorganic Compounds in Ambient Air: Sampling of Ambient Air for Total Suspended Particulate Matter 

(SPM) and PM10 Using High Volume (HV) Sampler.” http://www.epa.gov/sam/pdfs/EPA-IO-2.1.pdf 

5.2.53 IO [Inorganic] Compendium Method IO-5: Sampling and Analysis for Vapor and 

Particle Phase Mercury in Ambient Air Utilizing Cold Vapor Atomic Fluorescence 

Spectrometry (CVAFS) 

Analyte(s) CAS RN 

Mercury, Total 7439-97-6 

Methoxyethylmercuric acetate 151-38-2 

Analysis Purpose: Sample preparation and analysis 

Sample Preparation Technique: Acid digestion for particulate mercury 

Determinative Technique: Cold vapor atomic fluorescence spectrometry (CVAFS) 

Method Developed for: Vapor and particle phase mercury in ambient air 

Method Selected for: SAM lists this method for preparation and analysis of air samples. 

Detection and Quantitation: The detection limits are 30 pg/m3 for particulate mercury and 45 pg/m3 for 

vapor phase mercury. Detection limits, analytical range, and interferences are dependent on the 

instrument used. 

Description of Method: Vapor phase mercury is collected using gold-coated glass bead traps at a flow 

rate of 0.3 L/min. The traps are directly desorbed onto a second (analytical) trap. The mercury desorbed 

from the analytical trap is determined by Atomic Fluorescence Spectrometry. Particulate mercury is 

sampled on glass-fiber filters at a flow rate of 30 L/min. The filters are extracted with nitric acid and 

microwave heating. The extract is oxidized with bromine chloride, then reduced with stannous chloride 

and purged from solution onto a gold-coated glass bead trap. This trap is desorbed onto a second trap, the 

second trap is desorbed, and the mercury is determined by CVAFS. 

Special Considerations: There are no known positive interferences at 253.7 nm wavelength. Water 

vapor will cause a negative interference. 

Source: EPA. 1999. “IO Compendium Method IO-5: Compendium of Methods for the Determination of 

Inorganic Compounds in Ambient Air: Sampling and Analysis for Vapor and Particle Phase Mercury in 

Ambient Air Utilizing Cold Vapor Atomic Fluorescence Spectrometry (CVAFS).” 

http://www.epa.gov/sam/pdfs/EPA-IO-5.pdf 

 SAM Revision 5.0 92 September 29, 2009 
Section 5 – Selected Chemical Methods

5.2.54 EPA Air Method, Toxic Organics - 10A (TO-10A): Determination of Pesticides and 

Polychlorinated Biphenyls in Ambient Air Using Low Volume Polyurethane Foam 

(PUF) Sampling Followed by Gas Chromatographic/Multi-Detector Detection 

(GC/MD) 

Analyte(s) CAS RN 

BZ [Quinuclidinyl benzilate]1

 6581-06-2 

Chlorfenvinphos 470-90-6 

3-Chloro-1,2-propanediol2

 96-24-2 

Chlorosarin2 1445-76-7 

Chlorosoman2

 7040-57-5 

Chlorpyrifos 2921-88-2 

Chlorpyrifos oxon 5598-15-2 

Cyclohexyl sarin (GF) 329-99-7 

Dichlorvos 62-73-7 

Dicrotophos 141-66-2 

Diisopropyl methylphosphonate (DIMP)2 1445-75-6 

Dimethylphosphite 868-85-9 

Dimethylphosphoramidic acid1

 33876-51-6 

EA2192 [Diisopropylaminoethyl 

methylthiolophosphonate]1 73207-98-4 

Ethyl methylphosphonic acid (EMPA)1

 1832-53-7 

N-Ethyldiethanolamine (EDEA) 139-87-7 

Fenamiphos 22224-92-6 

Isopropyl methylphosphonic acid (IMPA)1

 1832-54-8 

Methyl paraoxon 950-35-6 

Methyl parathion 298-00-0 

N-Methyldiethanolamine (MDEA) 105-59-9 

1-Methylethyl ester ethylphosphonofluoridic acid (GE)2

 1189-87-3 

Methylphosphonic acid (MPA)1

 993-13-5 

Mevinphos 7786-34-7 

Monocrotophos 6923-22-4 

Mustard, nitrogen (HN-1) [bis(2-chloroethyl)ethylamine] 538-07-8 

Mustard, nitrogen (HN-2) [2,2’-dichloro-Nmethyldiethylamine N,N-bis(2-chloroethyl)methylamine] 51-75-2 

Mustard, nitrogen (HN-3) [tris(2-chloroethyl)amine] 555-77-1 

 SAM Revision 5.0 93 September 29, 2009 
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Analyte(s) CAS RN 

Mustard, sulfur / Mustard gas (HD) 505-60-2 

Paraoxon 311-45-5 

Parathion 56-38-2 

Phencyclidine 77-10-1 

Phenol 108-95-2 

Phorate 298-02-2 

Phorate sulfone 2588-04-7 

Phorate sulfoxide 2588-03-6 

Phosphamidon 13171-21-6 

Pinacolyl methyl phosphonic acid (PMPA)1

 616-52-4 

R 33 (VR) [methylphosphonothioic acid, S-[2­

(diethylamino)ethyl] O-2-methylpropyl ester] 159939-87-4 

Sarin (GB)2

 107-44-8 

Semivolatile organic compounds, NOS NA 

Soman (GD)2

 96-64-0 

Tabun (GA) 77-81-6 

Tetraethyl pyrophosphate 107-49-3 

Tetramethylenedisulfotetramine 80-12-6 

Thiodiglycol (TDG) 111-48-8 

Triethanolamine (TEA) 102-71-6 

Trimethyl phosphite 121-45-9 

VE [phosphonothioic acid, ethyl-, S-(2­

(diethylamino)ethyl) O-ethyl ester] 21738-25-0 

VG [phosphonothioic acid, S-(2-(diethylamino)ethyl) 

O,O-diethyl ester] 78-53-5 

VM [phosphonothioic acid, methyl-, S-(2­

(diethylamino)ethyl) O-ethyl ester] 21770-86-5 

VX [O-ethyl-S-(2-diisopropylaminoethyl)methylphosphonothiolate] 50782-69-9 

The following analyte should be determined by this method only if problems (e.g., insufficient recovery, 

interferences) occur when using Method TO-15. 

Allyl alcohol 107-18-6 

1

 For this analyte, HPLC is the preferred technique; however, if problems occur, Method TO-10A must be modified to 

include a derivatization step prior to analysis by GC-MS. 

2

 If problems occur when using this method, it is recommended that the canister Method TO-15 be used.
 

 SAM Revision 5.0 94 September 29, 2009 
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Analysis Purpose: Sample preparation and analysis 

Sample Preparation Technique: Solvent extraction 

Determinative Technique: GC-MS or HPLC 

Method Developed for: Pesticides and polychlorinated biphenyls in ambient air 

Method Selected for: SAM lists this method for preparation and analysis of air samples. 

Detection and Quantitation: The limit of detection (LOD) will depend on the specific compounds 

measured, the concentration level, and the degree of specificity required. This method is applicable to 

multicomponent atmospheres, 0.001 to 50 μg/m3

 concentrations, and 4 to 24-hour sampling periods. 

Description of Method: A low-volume (1 to 5 L/min) sample collection rate is used to collect vapors on 

a sorbent cartridge containing PUF in combination with another solid sorbent. Airborne particles also are 

collected, but the sampling efficiency is not known. Pesticides and other chemicals are extracted from the 

sorbent cartridge with 5% diethyl ether in hexane and determined by GC-MS. For common pesticides, 

HPLC coupled with a UV detector or electrochemical detector is preferable. If analyzed by GC-MS, BZ, 

dimethylphosphoramidic acid, EA2192, EMPA, IMPA, MPA, and PMPA require derivatization with a 

trimethylsilyl agent prior to injection into the GC. 

Special Considerations: Refer to footnotes provided in analyte table above for special considerations 

that should be applied when measuring specific analytes. See Special Considerations in Section 5.2.37 

for information regarding derivatization of compounds. 

Source: EPA. 1999. “Air Method, Toxic Organics-10A (TO-10A): Compendium of Methods for the 

Determination of Inorganic Compounds in Ambient Air: Determination of Pesticides and Polychlorinated 

Biphenyls in Ambient Air Using Low Volume Polyurethane Foam (PUF) Sampling Followed by Gas 

Chromatographic/Multi-Detector Detection (GC/MD).” http://www.epa.gov/sam/pdfs/EPA-TO-10a.pdf 

5.2.55 EPA Air Method, Toxic Organics - 15 (TO-15): Determination of Volatile Organic 

Compounds (VOCs) in Air Collected in Specially-Prepared Canisters and Analyzed 

by Gas Chromatography/Mass Spectrometry (GC/MS) 

Analyte(s) CAS RN 

Allyl alcohol 107-18-6 

Carbon disulfide 75-15-0 

Cyanogen chloride 506-77-4 

1,2-Dichloroethane 107-06-2 

Ethyldichloroarsine (ED) 598-14-1 

Ethylene oxide 75-21-8 

The following analytes should be determined by this method only if problems (e.g., insufficient recovery, 

interferences) occur when using Method TO-10A. 

3-Chloro-1,2-propanediol 96-24-2 

Chlorosarin 1445-76-7 

Chlorosoman 7040-57-5 

Diisopropyl methylphosphonate (DIMP) 1445-75-6 

1-Methylethyl ester ethylphosphonofluoridic acid (GE) 1189-87-3 

 SAM Revision 5.0 95 September 29, 2009 
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Analyte(s) CAS RN 

Sarin (GB) 107-44-8 

Soman (GD) 96-64-0 

Analysis Purpose: Sample preparation and analysis 

Sample Preparation Technique: Samples are collected using canisters. 

Determinative Technique: GC-MS 

Method Developed for: VOCs in air 

Method Selected for: SAM lists this method for preparation and analysis of air samples. 

Detection and Quantitation: This method applies to ambient concentrations of VOCs above 0.5 ppbv 

and typically requires VOC enrichment by concentrating up to 1 L of a sample volume; however, when 

using current technologies, quantifications of approximately 100 pptv have been achieved with 0.5-L 

sample volumes. 

Description of Method: The atmosphere is sampled by introduction of air into a specially prepared 

stainless steel canister (electropolished or silica-coated). A sample of air is drawn through a sampling 

train comprising components that regulate the rate and duration of sampling into the pre-evacuated and 

passivated canister. Grab samples also may be collected. After the air sample is collected, the canister 

valve is closed, an identification tag is attached to the canister, and the canister is transported to the 

laboratory for analysis. To analyze the sample, a known volume of sample is directed from the canister 

through a solid multisorbent concentrator. Recovery of less volatile compounds may require heating the 

canister. 

After the concentration and drying steps are completed, VOCs are thermally desorbed, entrained in a 

carrier gas stream, and then focused in a small volume by trapping on a cryo-focusing (ultra-low 

temperature) trap or small volume multisorbent trap. The sample is then released by thermal desorption 

and analyzed by GC-MS. 

Special Considerations: If problems occur when using this method for determination of allyl alcohol, it 

is recommended that Method TO-10A be used. 

Source: EPA. 1999. “Air Method, Toxic Organics-15 (TO-15): Compendium of Methods for the 

Determination of Toxic Organic Compounds in Ambient Air, Second Edition: Determination of Volatile 

Organic Compounds (VOCs) in Air Collected in Specially-Prepared Canisters and Analyzed by Gas 

Chromatography/Mass Spectrometry (GC/MS).” http://www.epa.gov/sam/pdfs/EPA-TO-15.pdf 

5.2.56 NIOSH Method 1612: Propylene Oxide 

Analyte(s) CAS RN 

Propylene oxide 75-56-9 

Analysis Purpose: Sample preparation and analysis
 

Sample Preparation Technique: Coconut shell charcoal solid sorbent tube 

Determinative Technique: GC-FID 

Method Developed for: Propylene oxide in air 

Method Selected for: SAM lists this method for preparation and analysis of air samples. 

Detection and Quantitation: The working range is between 8 and 295 ppm for air samples of 5 L. 

 SAM Revision 5.0 96 September 29, 2009 
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Description of Method: A sample tube containing coconut shell charcoal is used for sample collection 

with a flow rate of 0.01 to 0.2 L/min. One milliliter of carbon disulfide is added to the vial and allowed to 

sit for 30 minutes prior to analysis with occasional agitation. Analysis is performed on a GC-FID. No 

interferences have been found. 

Special Considerations: The presence of propylene oxide should be confirmed by either a secondary 

GC column or by an MS. 

Source: NIOSH. 1994. “Method 1612: Propylene Oxide,” Issue 2. 

http://www.epa.gov/sam/pdfs/NIOSH-1612.pdf 

5.2.57 NIOSH Method 2016: Formaldehyde 

Analyte(s) CAS RN 

Formaldehyde 50-00-0 

Analysis Purpose: Sample preparation and analysis 

Sample Preparation Technique: Solvent extraction 

Determinative Technique: HPLC 

Method Developed for: Formaldehyde in air 

Method Selected for: SAM lists this method for preparation and analysis of air samples. 

Detection and Quantitation: The detection limit for formaldehyde is 0.07 μg/sample. The working 

range is 0.015 to 2.5 mg/m3

 (0.012 to 2.0 ppm) for a 15-L sample. 

Description of Method: This method can be used for the determination of formaldehyde using HPLC 

with a UV detector. Air is sampled onto a cartridge containing silica gel coated with 2,4-DNPH, at a rate 

of 0.03 to 1.5 L/min. The cartridge is extracted with 10 mL of acetonitrile and analyzed by HPLC-UV at 

a wavelength of 360 nm. Ozone has been observed to consume the 2,4-DNPH reagent and to degrade the 

formaldehyde derivative. Ketones and other aldehydes can react with 2,4-DNPH; the derivatives 

produced, however, are separated chromatographically from the formaldehyde derivative. 

Source: NIOSH. 2003. “Method 2016: Formaldehyde,” Issue 2. 

http://www.epa.gov/sam/pdfs/NIOSH-2016.pdf 

5.2.58 NIOSH Method 2513: Ethylene Chlorohydrin 

Analyte(s) CAS RN 

2-Chloroethanol 107-07-3 

2-Fluoroethanol 371-62-0 

Analysis Purpose: Sample preparation and analysis 

Sample Preparation Technique: Solvent desorption 

Determinative Technique: GC-FID 

Method Developed for: Ethylene chlorohydrin (2-chloroethanol) in air 

Method Selected for: SAM lists this method for preparation and analysis of air samples. 

Detection and Quantitation: The working range of the method is 0.5 to 15 ppm for a 20-L air sample. 

 SAM Revision 5.0 97 September 29, 2009 
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Description of Method: Samples are drawn into a tube containing petroleum charcoal at a rate of 0.01 to 

0.2 L/min and transferred into vials containing eluent (carbon disulfide, 2-propanol, and n-pentadiene as 

an internal standard). Vials must sit for 30 minutes prior to analysis by GC-FID. No interferences have 

been identified. Humidity may decrease the breakthrough volume during sample collection. 

Special Considerations: The presence of 2-chloroethanol should be confirmed by either a secondary 

GC column or by an MS. 

Source: NIOSH. 1994. “Method 2513: Ethylene Chlorohydrin,” Issue 2. 

http://www.epa.gov/sam/pdfs/NIOSH-2513.pdf 

5.2.59 NIOSH Method 3510: Monomethylhydrazine 

Analyte(s) CAS RN 

Methyl hydrazine (monomethylhydrazine) 60-34-4 

Analysis Purpose: Sample preparation and analysis
 

Sample Preparation Technique: Samples are collected into a bubbler containing hydrochloric acid. 

Determinative Technique: Spectrophotometry
 

Method Developed for: Monomethylhydrazine in air 

Method Selected for: SAM lists this method for preparation and analysis of air samples. 

Detection and Quantitation: The working range of the method is 0.027 to 2.7 ppm for a 20-L air 

sample. 

Description of Method: Samples are collected into a bubbler containing hydrochloric acid using a flow 

rate of 0.5 to 1.5 L/min. Samples are transferred to a 25-mL flask, mixed with phosphomolybdic acid 

solution, diluted to the mark with 0.1 M hydrochloric acid, and then transferred to a large test tube for 

spectrophotometric analysis. Positive interferences include other hydrazines, as well as stannous ion, 

ferrous ion, zinc, sulfur dioxide, and hydrogen sulfide. Negative interferences may occur by oxidation of 

the monomethylhydrazine by halogens, oxygen (especially in the presence of copper (I) ions) and 

hydrogen dioxide. 

Source: NIOSH. 1994. “Method 3510: Monomethylhydrazine,” Issue 1. 

http://www.epa.gov/sam/pdfs/NIOSH-3510.pdf 

5.2.60 NIOSH Method 5600: Organophosphorus Pesticides 

Analyte(s) CAS RN 

Disulfoton 298-04-4 

Disulfoton sulfoxide 2497-07-6 

Organophosphate pesticides, NOS NA 

Analysis Purpose: Sample preparation and analysis 

Sample Preparation Technique: Solvent desorption 

Determinative Technique: GC-FPD 

Method Developed for: Organophosphorus pesticides in air 

Method Selected for: SAM lists this method for preparation and analysis of air samples. 

 SAM Revision 5.0 98 September 29, 2009 
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Detection and Quantitation: The detection limit depends on the compound being measured. The 

working range for each analyte is provided in Table 5 of the method. These ranges cover from 0.1 to 2 

times the OSHA Permissible Exposure Limits (PELs). 

Description of Method: This method is used for the detection of organophosphorus pesticides using GC 

with a FPD. Samples are prepared by desorbing the XAD-2 resin with 2 mL of toluene/acetone (90/10 

v/v) solution. The method also may be applicable to the determination of other organophosphorus 

compounds after evaluation for desorption efficiency, sample capacity, sample stability, and precision and 

accuracy. The working range for each analyte is provided in Table 5 of the method. These ranges cover 

from 0.1 to 2 times the OSHA PELs (see Table 5 of the method). The method also is applicable to Short 

Term Exposure Limit (STEL) measurements using 12-L samples. 

Special Considerations: Several organophosphates may co-elute with either target analytes or internal 

standards causing integration errors. These include other pesticides, and the following: tributyl 

phosphate, tris-(2-butoxy ethyl) phosphate, tricresyl phosphate, and triphenyl phosphate. 

Source: NIOSH. 1994. “Method 5600: Organophosphorus Pesticides,” Issue 1. 

http://www.epa.gov/sam/pdfs/NIOSH-5600.pdf 

5.2.61 NIOSH Method 5601: Organonitrogen Pesticides 

Analyte(s) CAS RN 

Aldicarb (Temik) 116-06-3 

Aldicarb sulfone 1646-88-4 

Aldicarb sulfoxide 1646-87-3 

Carbofuran (Furadan) 1563-66-2 

Methomyl 16752-77-5 

Oxamyl 23135-22-0 

Thiofanox 39196-18-4 

Analysis Purpose: Sample preparation and analysis 

Sample Preparation Technique: Solvent desorption 

Determinative Technique: HPLC 

Method Developed for: Organonitrogen pesticides in air 

Method Selected for: SAM lists this method for preparation and analysis of air samples. 

Detection and Quantitation: The detection limit for aldicarb is 1.2 μg per sample and 0.6 μg per sample 

for carbofuran, methomyl, and oxamyl. The working ranges for aldicarb, carbofuran, and oxamyl are 

listed in Table 2 of the method, and range from 0.5 to 10 times the OSHA PEL. 

Description of Method: This method can be used for the determination of organonitrogen pesticides 

using HPLC with a UV detector. Samples are prepared by desorbing the XAD-2 resin with 2 mL of 

triethylamine-phosphate solution, rotating end-over-end for 45 minutes, and filtering. The method also 

may be applicable to the determination of other organonitrogen compounds and to a broad range of 

pesticides having UV chromophores, e.g., acetanilides, acid herbicides, organophosphates, phenols, 

pyrethroids, sulfonyl ureas, sulfonamides, triazines, and uracil pesticides. Because of the broad response 

of the UV detector at shorter wavelengths, there are many potential interferences. Those tested include 

solvents (chloroform and toluene), antioxidants (butylated hydroxytoluene [BHT]), plasticizers (dialkyl 

 SAM Revision 5.0 99 September 29, 2009 
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phthalates), nitrogen compounds (nicotine and caffeine), impurities in HPLC reagents (e.g., in 

triethylamine), other pesticides (2,4-Dichlorophenoxyacetic acid [2,4-D], atrazine, parathion, etc.), and 

pesticide hydrolysis products (1-naphthol). Confirmation techniques are recommended when analyte 

identity is uncertain. 

Special Considerations: The presence of the analytes listed in the table above should be confirmed by 

either a secondary HPLC column or by an MS. 

Source: NIOSH. 1998. “Method 5601: Organonitrogen Pesticides,” Issue 1. 

http://www.epa.gov/sam/pdfs/NIOSH-5601.pdf 

5.2.62 NIOSH Method 6001: Arsine 

Analyte(s) CAS RN 

Arsine 7784-42-1 

Analysis Purpose: Sample preparation and analysis
 

Sample Preparation Technique: Coconut shell charcoal solid sorbent tube
 

Determinative Technique: GFAA 

Method Developed for: Arsine in air 

Method Selected for: SAM lists this method for preparation and analysis of air samples. 

Detection and Quantitation: The working range of the method is 0.001 to 0.2 mg/m3 for a 10-L sample. 

Description of Method: Arsine is determined as arsenic. A 0.1- to 10-L volume of air is drawn through 

a sorbent tube containing activated charcoal. The sorbent is extracted with a nitric acid solution, and 

arsenic is determined by GFAA. 

Special Considerations: The method is subject to interferences from other arsenic compounds. 

Source: NIOSH. 1994. “Method 6001: Arsine,” Issue 2. http://www.epa.gov/sam/pdfs/NIOSH-6001.pdf 

5.2.63 NIOSH Method 6002: Phosphine 

Analyte(s) CAS RN 

Phosphine 7803-51-2 

Analysis Purpose: Sample preparation and analysis
 

Sample Preparation Technique: Solvent desorption with hot acidic permanganate solution
 

Determinative Technique: Spectrophotometry
 

Method Developed for: Phosphine in air 

Method Selected for: SAM lists this method for preparation and analysis of air samples. 

Detection and Quantitation: The working range of the method is 0.02 to 0.9 mg/m3 for a 16-L sample. 

Description of Method: In this method, phosphine is determined as phosphate. A volume of 1 to 16 L 

of air is drawn through a sorbent tube containing silica gel coated with mercuric cyanide. The sorbent is 

extracted with a potassium permanganate/sulfuric acid solution and washed with reagent water. 

Following treatment with the color agent and extraction into organic solvent, phosphate is determined by
 

visible spectrometry.
 

 SAM Revision 5.0 100 September 29, 2009 
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Special Considerations: The method is subject to interferences from phosphorus trichloride, 

phosphorus pentachloride, and organic phosphorus compounds. 

Source: NIOSH. 1994. “Method 6002: Phosphine,” Issue 2. 

http://www.epa.gov/sam/pdfs/NIOSH-6002.pdf 

5.2.64 NIOSH Method 6004: Sulfur Dioxide 

Analyte(s) CAS RN 

Sulfur dioxide 7446-09-5 

Analysis Purpose: Sample preparation and analysis
 

Sample Preparation Technique: Treated filter extracted with carbonate/bicarbonate solution 

Determinative Technique: IC 

Method Developed for: Sulfur dioxide in air 

Method Selected for: SAM lists this method for preparation and analysis of air samples. 

Detection and Quantitation: The working range of the method is 0.5 to 20 mg/m3 for a 100-L sample. 

Description of Method: In this method, sulfur dioxide is determined as sulfite plus sulfate. A volume of 

40 to 200 L of air is drawn through a sodium carbonate-treated filter that is preceded by a 0.8 μm filter to 

remove particulates and sulfuric acid. The treated filter is extracted with a carbonate/bicarbonate solution 

and the extract analyzed by IC for sulfite and sulfate. The sulfur dioxide is present as sulfite on the filter; 

however, because sulfite oxidizes to sulfate, both ions must be determined and the results summed. 

Special Considerations: The method is subject to interference from sulfur trioxide in dry conditions. 

Source: NIOSH. 1994. “Method 6004: Sulfur Dioxide,” Issue 2. 

http://www.epa.gov/sam/pdfs/NIOSH-6004.pdf 

5.2.65 NIOSH Method 6010: Hydrogen Cyanide 

Analyte(s) CAS RN 

Cyanide, Total 57-12-5 

Hydrogen cyanide 74-90-8 

Analysis Purpose: Sample preparation and analysis
 

Sample Preparation Technique: Solvent desorption 

Determinative Technique: Spectrophotometry
 

Method Developed for: Hydrogen cyanide in air 

Method Selected for: SAM lists this method for preparation and analysis of air samples. 

Detection and Quantitation: The working range of the method is 3 to 260 mg/m3 for a 3-L sample. 

Description of Method: Hydrogen cyanide is determined as a cyanide ion complex by this method. A 

volume of 0.6 to 90 L of air is drawn through a soda lime sorbent tube. A glass-fiber filter is used to 

remove particulate cyanides prior to the sorbent tube. Cyanide is extracted from the sorbent with reagent 

water treated with sodium hydroxide. The extract is pH adjusted with hydrochloric acid, oxidized with Nchlorosuccinimide/succinimide, and treated with the coupling-color agent (barbituric acid/pyridine). The 

cyanide ion is determined by visible spectrophotometry using a wavelength of 580 nm. 

 SAM Revision 5.0 101 September 29, 2009 
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Special Considerations: The method is subject to interference from high concentrations of hydrogen 

sulfide. Two liters is the minimum volume required to measure concentration of 5 ppm. 

Source: NIOSH. 1994. “Method 6010: Hydrogen Cyanide,” Issue 2. 

http://www.epa.gov/sam/pdfs/NIOSH-6010.pdf 

5.2.66 NIOSH Method 6013: Hydrogen Sulfide 

Analyte(s) CAS RN 

Hydrogen sulfide 7783-06-4 

Analysis Purpose: Sample preparation and analysis 

Sample Preparation Technique: Solvent extraction 

Determinative Technique: IC 

Method Developed for: Hydrogen sulfide in air 

Method Selected for: SAM lists this method for preparation and analysis of air samples. 

Detection and Quantitation: The working range of the method is 0.9 to 20 mg/m3 for a 20-L sample. 

Description of Method: Hydrogen sulfide is determined as sulfate by this method. A volume of 15 to 

40 L of air is drawn through charcoal sorbent. A prefilter is used to remove particulates. The sorbent 

portions are extracted with an ammonium hydroxide/hydrogen peroxide solution and the extract is 

analyzed for sulfate by IC. 

Special Considerations: The method is subject to interference from sulfur dioxide. 

Source: NIOSH. 1994. “Method 6013: Hydrogen Sulfide,” Issue 1. 

http://www.epa.gov/sam/pdfs/NIOSH-6013.pdf 

5.2.67 NIOSH Method 6015: Ammonia 

Analyte(s) CAS RN 

Ammonia 7664-41-7 

Analysis Purpose: Sample preparation and analysis 

Sample Preparation Technique: Water extraction 

Determinative Technique: Spectrophotometry 

Method Developed for: Ammonia in air 

Method Selected for: SAM lists this method for preparation and analysis of air samples. 

Detection and Quantitation: The working range of the method is 0.15 to 300 mg/m3 for a 10-L sample. 

Twice the recommended sample volume should be collected in order to achieve an action level of 70 

μg/m3

. 

Description of Method: Ammonia is determined as indophenol blue by this method. A volume of 0.1 to
 

96 L of air is drawn through a sulfuric acid-treated silica gel sorbent. A prefilter is used to remove 

particulates. The sorbent is extracted with reagent water, the pH adjusted, and reagents are added to 

generate the indophenol blue compound in the presence of ammonium. The extract is analyzed by visible 

spectrophotometry. No interferences have been identified. 

 SAM Revision 5.0 102 September 29, 2009 
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Source: NIOSH. 1994. “Method 6015: Ammonia,” Issue 2. 

http://www.epa.gov/sam/pdfs/NIOSH-6015.pdf 

5.2.68 NIOSH Method 6402: Phosphorus Trichloride 

Analyte(s) CAS RN 

Phosphorus trichloride 7719-12-2 

Analysis Purpose: Sample preparation and analysis
 

Sample Preparation Technique: Add reagent to samples in bubbler solution and heat
 

Determinative Technique: Spectrophotometry
 

Method Developed for: Phosphorus trichloride in air 

Method Selected for: SAM lists this method for preparation and analysis of air samples. 

Detection and Quantitation: The working range of the method is 1.2 to 80 mg/m3 for a 25-L sample. 

Description of Method: In this method, phosphorus trichloride is determined as phosphate. A volume 

of 11 to 100 L of air is drawn through a bubbler containing reagent water. The resulting phosphorus acid 

solution is oxidized with bromine to phosphoric acid and color agent (sodium molybdate) and reducing 

agent (hydrazine sulfate) are added. The solution is analyzed for the resulting molybdenum blue complex 

by visible spectrophotometry. Phosphorus (V) compounds do not interfere. Sample solutions are stable 

to oxidation by air during sampling. 

Source: NIOSH. 1994. “Method 6402: Phosphorus Trichloride,” Issue 2. 

http://www.epa.gov/sam/pdfs/NIOSH-6402.pdf 

5.2.69 NIOSH Method 7903: Acids, Inorganic 

Analyte(s) CAS RN 

Hydrogen bromide 10035-10-6 

Hydrogen chloride 7647-01-0 

Hydrogen fluoride 7664-39-3 

Analysis Purpose: Sample preparation and analysis 

Sample Preparation Technique: Solvent desorption 

Determinative Technique: IC 

Method Developed for: Inorganic acids in air 

Method Selected for: SAM lists this method for preparation and analysis of air samples. 

Detection and Quantitation: The working range of this method is 0.01 to 5 mg/m3 for a 50-L sample. 

Description of Method: Acids are analyzed as bromide, chloride, and fluoride. A volume of 3 to 100 L 

of air is drawn through a silica gel sorbent. The sorbent portions are extracted with a buffered
 

carbonate/bicarbonate solution and the extract is analyzed by IC. 

Special Considerations: Particulate salts of the acids are an interference (trapped on the glass wool 

filter plug in the sorbent tube). Chlorine and bromine are also interferences. Acetate, formate, and 

propionate interferences may be reduced by use of a weaker eluent. If problems occur when using this 

method for analysis of hydrogen fluoride, it is recommended that NIOSH Method 7906 be used. 

 SAM Revision 5.0 103 September 29, 2009 
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Source: NIOSH. 1994. “Method 7903: Acids, Inorganic,” Issue 2. 

http://www.epa.gov/sam/pdfs/NIOSH-7903.pdf 

5.2.70 NIOSH Method 7905: Phosphorus 

Analyte(s) CAS RN 

White phosphorus 12185-10-3 

Analysis Purpose: Sample preparation and analysis
 

Sample Preparation Technique: GC solid sorbent tube and solvent extracted (desorbed) 

Determinative Technique: GC-FPD 

Method Developed for: Phosphorus in air 

Method Selected for: SAM lists this method for preparation and analysis of air samples. 

Detection and Quantitation: The LOD for samples analyzed by GC-FPD is 0.005 μg per sample. The 

working range for samples analyzed by GC-FPD is 0.056 to 0.24 mg/m3

 for a 12-L sample. 

Description of Method: This method identifies and determines the concentration of white phosphorus in 

air by using a GC-FPD. Five to 100 L of air is drawn through a GC solid sorbent tube, and the sorbent is 

extracted (desorbed) with xylene. The method is applicable to vapor-phase phosphorus only; if
 

particulate phosphorus is expected, a filter could be used in the sampling train. 

Special Considerations: The presence of white phosphorus should be confirmed by either a secondary
 

GC column or by an MS. 

Source: NIOSH. 1994. “Method 7905: Phosphorus,” Issue 2. 

http://www.epa.gov/sam/pdfs/NIOSH-7905.pdf 

5.2.71 NIOSH Method 7906: Fluorides, Aerosol and Gas, by IC 

Analyte(s) CAS RN 

Hydrogen fluoride 7664-39-3 

Analysis Purpose: Sample preparation and analysis 

Sample Preparation Technique: Water extraction 

Determinative Technique: IC 

Method Developed for: Fluorides in aerosol and gas 

Method Selected for: SAM lists this method for use if problems occur when using NIOSH Method 7903 

for the analysis of hydrogen fluoride during preparation and analysis of air samples. (See Footnote 11 of 

Appendix A.) 

Detection and Quantitation: The working range of the method is 0.04 to 8 mg/m3 for 250-L samples. 

Description of Method: Hydrogen fluoride is determined as fluoride ion by this method. A volume of 1 

to 800 L of air is drawn through a 0.8-μm cellulose ester membrane (to trap particulate fluorides) and a 

cellulose pad treated with sodium carbonate (to trap gaseous fluoride). The pad is extracted with reagent 

water and the extract is analyzed for fluoride by IC. 

Special Considerations: If other aerosols are present, gaseous fluoride may be slightly underestimated 

due to adsorption onto or reaction with particles, with concurrent overestimation of particulate/gaseous 

fluoride ratio. 

 SAM Revision 5.0 104 September 29, 2009 
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Source: NIOSH. 1994. “Method 7906: Fluorides, Aerosol and Gas by IC,” Issue 1. 

http://www.epa.gov/sam/pdfs/NIOSH-7906.pdf 

5.2.72 NIOSH Method 9102: Elements on Wipes 

Analyte(s) CAS RN 

Ammonium metavanadate 7803-55-6 

Arsenic, Total 7440-38-2 

Arsenic trioxide 1327-53-3 

Arsine 7784-42-1 

Calcium arsenate 7778-44-1 

2-Chlorovinylarsonous acid (2-CVAA) 85090-33-1 

Ethyldichloroarsine (ED) 598-14-1 

Lead arsenate 7645-25-2 

Lewisite 1 (L-1) [2-chlorovinyldichloroarsine] 541-25-3 

Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine] 40334-69-8 

Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine] 40334-70-1 

Lewisite oxide 1306-02-1 

Mercuric chloride 7487-94-7 

Mercury, Total 7439-97-6 

Methoxyethylmercuric acetate 151-38-2 

Osmium tetroxide 20816-12-0 

Sodium arsenite 7784-46-5 

Thallium sulfate 10031-59-1 

Titanium tetrachloride 7550-45-0 

Vanadium pentoxide 1314-62-1 

Analysis Purpose: Sample preparation
 

Sample Preparation Technique: Acid digestion
 

Determinative Technique: EPA SW-846 Methods 6010C, 6020A, 7010, and 7473. Refer to Appendix 

A for which of these determinative methods should be used for a particular analyte. 

Method Developed for: Measurement of metals on wipe surfaces using ICP-AES 

Method Selected for: SAM lists this method for preparation of wipe samples. 

Detection and Quantitation: The range for arsenic is 0.261 to105 μg/wipe; for thallium 0.136 to 50.0 

μg/wipe; for vanadium 0.0333 to 25.0 μg/wipe. 

Description of Method: Surface wipe samples are transferred to a clean beaker, followed by the addition 

of concentrated nitric and perchloric acids. The beaker contents are held at room temperature for 30 

minutes, then heated at 150°C for 8 hours. Additional nitric acid is added until the wipe media is 

 SAM Revision 5.0 105 September 29, 2009 
Section 5 – Selected Chemical Methods

completely destroyed. The sample is then taken to near dryness and the residue dissolved and diluted 

before being analyzed. 

Special Considerations: ICP-MS may also be used for the analysis of wipe samples; however, at this 

time, this technique has not been evaluated for wipes. 

Source: NIOSH. 2003. “Method 9102, Issue 1: Elements on Wipes.” 

http://www.epa.gov/sam/pdfs/NIOSH-9102.pdf 

5.2.73 NIOSH Method S301-1: Fluoroacetate Anion 

Analyte(s) CAS RN 

Fluoroacetic acid and fluoroacetate salts NA 

Methyl fluoroacetate 453-18-9 

Analysis Purpose: Sample preparation 

Sample Preparation Technique: Water extraction 

Determinative Technique: EPA Method 300.1 Rev 1.0 

Method Developed for: Fluoroacetate anion in air 

Method Selected for: SAM lists this method for preparation of air samples. 

Detection and Quantitation: The detection limit is estimated to be 20 ng of sodium fluoroacetate per 

injection, corresponding to a 100-μL aliquot of a 0.2-μg/mL standard. The analytical range of this 

method is estimated to be 0.01 to 0.16 mg/m3

. 

Description of Method: This method was developed specifically for sodium fluoroacetate, but also may 

be applicable to other fluoroacetate salts. The method determines fluoroacetate salts as fluoroacetate 

anion. A known volume of air (e.g., 480 L was used in validation of this method) is drawn through a 

cellulose ester membrane filter to collect sodium fluoroacetate. Sodium fluoroacetate is extracted from 

the filter with 5 mL of deionized water, and the resulting sample is analyzed by IC using electrolytic 

conductivity detection. 

Special Considerations: When analyzing samples for methyl fluoroacetate (as fluoroacetate ion), 

addition of base is required to assist dissociation into fluoroacetate anion. 

Source: NIOSH. 1977. “Method S301-1: Sodium Fluoroacetate.” 

http://www.epa.gov/sam/pdfs/NIOSH-S301-1.pdf 

5.2.74 OSHA Method 40: Methylamine 

Analyte(s) CAS RN 

Methylamine 74-89-5 

Analysis Purpose: Sample preparation and analysis 

Sample Preparation Technique: Solvent desorption 

Determinative Technique: HPLC 

Method Developed for: Methylamine in air 

Method Selected for: SAM lists this method for preparation and analysis of air samples. 

 SAM Revision 5.0 106 September 29, 2009 
Section 5 – Selected Chemical Methods

Detection and Quantitation: The detection limit of the overall procedure is 0.35 μg per sample (28 ppb 

or 35 μg/m3

). Quantitation limits of 28 ppb (35 μg/m3) have been achieved. This is the smallest amount 

of methylamine that can be quantified within the requirements of a recovery of at least 75% and a 

precision (standard deviation of 1.96) of ± 25% or better. 

Description of Method: This method is used for detection of methylamine using HPLC with a FL or 

visible (vis) detector. Samples are collected by drawing 10-L volumes of air at a rate of 0.2 L/min 

through standard size sampling tubes containing XAD-7 resin coated with 10% 7-chloro-4-nitrobenzo-2-

oxa-1,3-diazole (NBD chloride) by weight. Samples are desorbed with 5% (w/v) NBD chloride in 

tetrahydrofuran (with a small amount of sodium bicarbonate present), heated in a hot water bath, and 

analyzed by high performance liquid chromatography – fluorescence (HPLC-FL) or high performance 

liquid chromatography – visible (HPLC-vis). 

Source: OSHA. 1982. “Method 40: Methylamine.” Method originally obtained from www.osha.gov, 

but is provided here for reference. http://www.epa.gov/sam/pdfs/OSHA-Method40.pdf 

5.2.75 OSHA Method 54: Methyl Isocyanate (MIC) 

Analyte(s) CAS RN 

Methyl isocyanate 624-83-9 

Analysis Purpose: Sample preparation and analysis 

Sample Preparation Technique: Solvent desorption 

Determinative Technique: HPLC 

Method Developed for: Methyl isocyanate in air 

Method Selected for: SAM lists this method for preparation and analysis of air samples. 

Description of Method: This method determines the concentration of methyl isocyanate in air by using
 

HPLC with a FL or UV detector. Samples are collected by drawing a known volume of air through 

XAD-7 tubes coated with 0.3 mg of 1-(2-pyridyl)piperazine (1-2PP). Samples are desorbed with 

acetonitrile and analyzed by HPLC using a FL or UV detector. 

Source: OSHA. 1985. “Method 54: Methyl Isocyanate (MIC).” Method originally obtained from
 

www.osha.gov, but is provided here for reference. http://www.epa.gov/sam/pdfs/OSHA-Method54.pdf
 

5.2.76 OSHA Method 61: Phosgene 

Analyte(s) CAS RN 

Phosgene 75-44-5 

Analysis Purpose: Sample preparation and analysis 

Sample Preparation Technique: Solvent desorption 

Determinative Technique: GC-NPD 

Method Developed for: Phosgene in air samples 

Method Selected for: SAM lists this method for preparation and analysis of air samples. 

Description of Method: This method determines the concentration of phosgene in air by using GC with 

an NPD. Air samples are collected by drawing known volumes of air through sampling tubes containing 

 SAM Revision 5.0 107 September 29, 2009 
Section 5 – Selected Chemical Methods

XAD-2 adsorbent that has been coated with 2-(hydroxymethyl)piperidine. The samples are desorbed with 

toluene and then analyzed by GC using an NPD. 

Special Considerations: The presence of phosgene should be confirmed by either a secondary GC 

column or by MS 

Source: OSHA. 1986. “Method 61: Phosgene.” Method originally obtained from www.osha.gov, but is 

provided here for reference. http://www.epa.gov/sam/pdfs/OSHA-Method61.pdf 

5.2.77 OSHA Method ID-211: Sodium Azide and Hydrazoic Acid in Workplace 

Atmospheres 

Analyte(s) CAS RN 

Sodium azide 26628-22-8 

Analysis Purpose: Sample preparation and analysis 

Sample Preparation Technique: Buffer desorption 

Determinative Technique: IC 

Method Developed for: Sodium azide and hydrazoic acid in workplace atmospheres 

Method Selected for: SAM lists this method for preparation and analysis of air and wipe samples. 

Detection and Quantitation: The detection limit was found to be 0.001 ppm as hydrazoic acid (HN3) or 

0.003 mg/m3

 as sodium azide (NaN3) for a 5-L air sample. The quantitation limit was found to be 0.004 

ppm as HN3 or 0.011 mg/m3

 as NaN3 for a 5-L air sample. 

Description of Method: This method describes sample collection and analysis of airborne azides [as 

NaN3 and hydrazoic acid HN3]. Particulate NaN3 is collected on a PVC filter or in the glass wool plug of 

the sampling tube. Gaseous HN3 is collected and converted to NaN3 by the impregnated silica gel (ISG) 

sorbent within the sampling tube. The collected azide on either media is desorbed in a weak buffer 

solution, and the resultant anion (N3

-

) is analyzed by IC using a variable wavelength UV detector at 210 

nm. A gravimetric conversion is used to calculate the amount of NaN3 or HN3 collected. 

Source: OSHA. 1992. “Method ID-211: Sodium Azide and Hydrazoic Acid in Workplace 

Atmospheres.” http://www.epa.gov/sam/pdfs/OSHA-ID-211.pdf 

5.2.78 OSHA Method ID216SG: Boron Trifluoride (BF3) 

Analyte(s) CAS RN 

Boron trifluoride 7637-07-2 

Analysis Purpose: Sample preparation and analysis 

Sample Preparation Technique: Sample collected in bubbler (no sample preparation required) 

Determinative Technique: Ion specific electrode (ISE) 

Method Developed for: Boron trifluoride in air samples 

Method Selected for: SAM lists this method for preparation and analysis of air samples. 

Detection and Quantitation: The detection limit is 10 μg in a 30-L sample. 

 SAM Revision 5.0 108 September 29, 2009 
Section 5 – Selected Chemical Methods

Description of Method: Boron trifluoride is determined as fluoroborate. A volume of 30 to 480 L of air 

is drawn through a bubbler containing 0.1 M ammonium fluoride. The solution is diluted and analyzed 

with a fluoroborate ISE. 

Source: OSHA. 1989. “Method ID216SG: Boron Trifluoride (BF3).” Method originally obtained from 

www.osha.gov, but is provided here for reference. http://www.epa.gov/sam/pdfs/OSHA-ID216SG.pdf 

5.2.79 OSHA Method PV2004: Acrylamide 

Analyte(s) CAS RN 

Acrylamide 79-06-1 

Acrylonitrile 107-13-1 

Methyl acrylonitrile 126-98-7 

Analysis Purpose: Sample preparation and analysis 

Sample Preparation Technique: Solvent desorption 

Determinative Technique: HPLC 

Method Developed for: Acrylamide in air 

Method Selected for: SAM lists this method for preparation and analysis of air samples. 

Detection and Quantitation: The detection limit was found to be 0.7 μg/mL (0.006 mg/m3 for a 1-mL
 

desorption volume or 0.029 mg/m3

 for a 5-mL desorption volume based on a 120-L air volume). 

Applicable working ranges for a 1-mL and 5-mL desorption volume are 0.017 - 1.5 mg/m3

 and 0.083 - 7.5 

mg/m3

, respectively.
 

Description of Method: This method determines the concentration of acrylamide in air by using HPLC 

with a UV detector. Samples are collected by drawing known volumes of air through OSHA versatile 

sampler (OVS-7) tubes, each containing a glass fiber filter and two sections of XAD-7 adsorbent. 

Samples are desorbed with a solution of 5% methanol/95% water and analyzed by HPLC using a UV 

detector. 

Source: OSHA. 1991. “Method PV2004: Acrylamide.” http://www.epa.gov/sam/pdfs/OSHAPV2004.pdf
 

5.2.80 OSHA Method PV2103: Chloropicrin 

Analyte(s) CAS RN 

Chloropicrin 79-06-2 

Analysis Purpose: Sample preparation and analysis 

Sample Preparation Technique: Solvent desorption 

Determinative Technique: GC-ECD 

Method Developed for: Chloropicrin in air 

Method Selected for: SAM lists this method for preparation and analysis of air samples. 

Detection and Quantitation: The detection limit is 0.01 ng, with a 1-μL injection volume. This is the 

smallest amount that could be detected under normal operating conditions. The working range is 33.2 to 

1330 μg/m3

. 

 SAM Revision 5.0 109 September 29, 2009 
Section 5 – Selected Chemical Methods

Description of Method: This method determines the concentration of chloropicrin in air by GC-ECD. 

Samples are collected by drawing a known volume of air through two XAD-4 tubes in series. Samples 

are desorbed with ethyl acetate and analyzed by GC-ECD. 

Special Considerations: The presence of chloropicrin should be confirmed by either a secondary GC 

column or by an MS. Chloropicrin is light sensitive, and samples should be protected from light. 

Source: OSHA. 1991. “Method PV2103: Chloropicrin.” http://www.epa.gov/sam/pdfs/OSHAPV2103.pdf 

5.2.81 ASTM Method D5755-03: Standard Test Method for Microvacuum Sampling and 

Indirect Analysis of Dust by Transmission Electron Microscopy for Asbestos 

Structure Number Surface Loading 

Analyte(s) CAS RN 

Asbestos 1332-21-4 

Analysis Purpose: Sample preparation and analysis 

Sample Preparation Technique: Direct transfer 

Determinative Technique: Transmission electron microscopy (TEM) 

Method Developed for: Asbestos in dust 

Method Selected for: SAM lists this method for preparation and analysis of solid (e.g., soft surfacesmicrovac) samples. 

Description of Method: This method describes procedures to identify asbestos in dust and provide an 

estimate of the surface loading of asbestos reported as the number of asbestos structures per unit area of 

sampled surface. The sample is collected by vacuuming a known surface area with a standard 25- or 37-

mm air sampling cassette using a plastic tube that is attached to the inlet orifice, which acts as a nozzle. 

The sample is transferred from inside the cassette to an aqueous suspension of known volume. Aliquots 

of the suspension are then filtered through a membrane, and a section of the membrane is prepared and 

transferred to a TEM grid using a direct transfer method. The asbestiform structures are identified, sized, 

and counted by TEM, using select area electron diffraction (SAED) and energy dispersive X-ray analysis 

(EDXA) at a magnification of 15,000 to 20,000X. 

Source: ASTM. 2003. “Method D5755-03: Standard Test Method for Microvacuum Sampling and 

Indirect Analysis of Dust by Transmission Electron Microscopy for Asbestos Structure Number Surface 

Loading.” http://www.astm.org/Standards/D5755.htm 

5.2.82 ASTM Method D6480-05: Standard Test Method for Wipe Sampling of Surfaces, 

Indirect Preparation, and Analysis for Asbestos Structure Number Concentration 

by Transmission Electron Microscopy 

Analyte(s) CAS RN 

Asbestos 1332-21-4 

Analysis Purpose: Sample preparation and analysis 

Sample Preparation Technique: Direct transfer 

Determinative Technique: TEM 

 SAM Revision 5.0 110 September 29, 2009 
Section 5 – Selected Chemical Methods

Method Developed for: Asbestos in samples wiped from surfaces 

Method Selected for: SAM lists this method for preparation and analysis of wipe (e.g., hard surfaceswipes) samples. 

Description of Method: This method describes a procedure to identify asbestos in samples wiped from 

surfaces and to provide an estimate of the concentration of asbestos reported as the number of asbestos 

structures per unit area of sampled surface. A sample is collected by wiping a surface of known area with 

a wipe material. The sample is transferred from the wipe material to an aqueous suspension of known 

volume. Aliquots of the suspension are then filtered through a membrane filter, and a section of the 

membrane filter is prepared and transferred to a TEM grid, using the direct transfer method. The 

asbestiform structures are identified, sized, and counted by TEM, using electron diffraction and EDXA at 

a magnification from 15,000 to 20,000X. 

Source: ASTM. 2005. “Method D6480-05: Standard Test Method for Wipe Sampling of Surfaces, 

Indirect Preparation, and Analysis for Asbestos Structure Number Concentration by Transmission 

Electron Microscopy.” http://www.astm.org/Standards/D6480.htm 

5.2.83 ISO Method 10312:1995: Ambient Air - Determination of Asbestos Fibres - Directtransfer Transmission Electron Microscopy Method 

Analyte(s) CAS RN 

Asbestos 1332-21-4 

Analysis Purpose: Sample preparation and analysis 

Sample Preparation Technique: Direct transfer 

Determinative Technique: TEM 

Method Developed for: Asbestos in ambient air 

Method Selected for: SAM lists this method for preparation and analysis of air samples. 

Detection and Quantitation: In a 4000-L air sample with approximately 10 pg/m3 (typical of clean or 

rural atmospheres), an analytical sensitivity of 0.5 structure/L can be obtained. This is equivalent to a 

detection limit of 1.8 structure/L when an area of 0.195 mm of the TEM specimen is examined. The 

range of concentrations that can be determined is 50 to 7,000 structures/mm2

 on the filter. 

Description of Method: This method determines the type(s) of asbestos fibers present, but cannot 

discriminate between individual fibers of the asbestos and non-asbestos analogues of the same amphibole 

mineral. The method is defined for polycarbonate capillan/pore filters or cellulose ester (either mixed 

esters of cellulose or cellulose nitrate) filters through which a known volume of air has been drawn. The 

method is suitable for determination of asbestos in both exterior and building atmospheres. 

Source: ISO. 2005. “Method 10312: 1995: Ambient Air - Determination of Asbestos Fibres - Direct 

Transfer Transmission Electron Microscopy Method.” 

http://www.iso.org/iso/iso_catalogue/catalogue_tc/catalogue_detail.htm?csnumber=18358 

5.2.84 Standard Method 4500-NH3 B: Nitrogen (Ammonia) Preliminary Distillation Step 

Analyte(s) CAS RN 

Ammonia 7664-41-7 

Analysis Purpose: Sample preparation 

Sample Preparation Technique: Distillation 

 SAM Revision 5.0 111 September 29, 2009 
Section 5 – Selected Chemical Methods

Determinative Technique: Standard Method 4500-NH3 G 

Method Developed for: Nitrogen (ammonia) in drinking waters, clean surface or groundwater, and 

good-quality nitrified wastewater effluent 

Method Selected for: SAM lists this method for preparation of aqueous liquid samples. 

Description of Method: A 0.5- to 1-L sample is dechlorinated, buffered, adjusted to pH 9.5, and distilled 

into a sulfuric acid solution. The distillate is brought up to volume, neutralized with sodium hydroxide, 

and analyzed by Method 4500-NH3 G. 

Source: APHA, AWWA, and WEF. 2005. “Method 4500-NH3 B: Nitrogen (Ammonia) Preliminary 

Distillation Step.” Standard Methods for the Examination of Water and Wastewater. 21st Edition. 

http://www.standardmethods.org/ 

5.2.85 Standard Method 4500-NH3 G: Nitrogen (Ammonia) Automated Phenate Method 

Analyte(s) CAS RN 

Ammonia 7664-41-7 

Analysis Purpose: Analysis 

Sample Preparation Technique: Standard Method 4500-NH3 B 

Determinative Technique: Spectrophotometry 

Method Developed for: Nitrogen (ammonia) in drinking waters, clean surface or groundwater, and 

good-quality nitrified wastewater effluent 

Method Selected for: SAM lists this method for analysis of aqueous liquid samples. 

Detection and Quantitation: The range of the method is 0.02 to 2.0 mg/L. 

Description of Method: Ammonia is determined as indophenol blue by this method. A portion of the 

neutralized sample distillate (from procedure 4500-NH3 B) is run through a manifold. The ammonium in 

the distillate reacts with solutions of disodium ethylenediaminetetraacetic acid (EDTA), sodium phenate, 

sodium hypochlorite, and sodium nitroprusside. The resulting indophenol blue is detected by colorimetry 

in a flow cell. Photometric measurement is made between the wavelengths of 630 and 660 nm. 

Source: APHA, AWWA, and WEF. 2005. “Method 4500-NH3 G: Nitrogen (Ammonia) Automated 

Phenate Method.” Standard Methods for the Examination of Water and Wastewater. 21st Edition.
 

http://www.standardmethods.org/ 

5.2.86 Standard Method 4500-Cl G: DPD Colorimetric Method 

Analyte(s) CAS RN 

Chlorine 7782-50-5 

Analysis Purpose: Sample preparation and/or analysis 

Sample Preparation Technique: Water samples are buffered and colorimetric agent is added. 

Procedures in Analyst, 1999. 124: 1853–1857 are used for preparation of air samples.
 

Determinative Technique: Spectrophotometry
 

 SAM Revision 5.0 112 September 29, 2009 
Section 5 – Selected Chemical Methods

Method Developed for: Chlorine in water and wastewater 

Method Selected for: SAM lists this method for preparation and analysis of aqueous liquid and drinking 

water samples. It also should be used for analysis of air samples when appropriate sample preparation 

techniques have been applied. 

Detection and Quantitation: The method can detect 10 μg/L chlorine. 

Description of Method: A portion of aqueous liquid sample is buffered and reacted with N,N-diethyl-pphenylenediamine (DPD) color agent. The resulting free chlorine is determined by colorimetry. If total 

chlorine (including chloroamines and nitrogen trichloride) is to be determined, potassium iodide crystals 

are added. Results for chromate and manganese are blank corrected using thioacetamide solution. 

Special Considerations: Organic contaminants and strong oxidizers may cause interference. 

Source: APHA, AWWA, and WEF. 2005. “Method 4500-Cl G: DPD Colorimetric Method.” Standard 

Methods for the Examination of Water and Wastewater. 21st Edition. http://www.standardmethods.org/ 

5.2.87 Literature Reference for Chlorine (Analyst, 1999. 124: 1853–1857) 

Analyte(s) CAS RN 

Chlorine 7782-50-5 

Analysis Purpose: Sample preparation 

Sample Preparation Technique: Buffered water extraction
 

Determinative Technique: Standard Method 4500-Cl G 

Method Developed for: Active chlorine in air 

Method Selected for: SAM lists this procedure for preparation of air samples. 

Detection and Quantitation: Detection limit of 0.1 μg of chlorine; the collection efficiency was >90%; 

recovery of chlorine spikes from 0.05-g aliquots of the sorbent was not quantitative (~60%) but was 

reproducible.
 

Description of Method: A procedure is described for determination of total combined gas-phase active 

chlorine (i.e., Cl2, hypochlorous acid [HOCl], and chloramines) and is based on a sulfonamidefunctionalized silica gel sorbent. For determination of the collected chlorine, a modified version of the 

DPD colorimetric procedure is used, which yielded a detection limit of 0.1 μg of chlorine. At flow rates 

ranging from 31 to 294 mL/min, the collection efficiency was >90% based on breakthrough analysis. 

Recovery of chlorine spikes from 0.05-g aliquots of the sorbent was not quantitative (~60%) but was 

reproducible; the recovery is accounted for in samples by adding weighed amounts of sorbent to the 

standards. 

Source: Johnson, B.J., Emerson, D.W., Song, L., Floyd, J., and Tadepalli, B. 1999. “Determination of 

active chlorine in air by bonded phase sorbent collection and spectrophotometric analysis.” Analyst.
 

124(12): 1853–1857. www.epa.gov/sam/pdfs/Analyst124_pg1853-1857.pdf
 

5.2.88 Literature Reference for Fluoroacetate salts (Analytical Letters, 1994. 27 (14): 

2703–2718) 

Analyte(s) CAS RN 

Fluoroacetic acid and fluoroacetate salts NA 

Methyl fluoroacetate 453-18-9 

 SAM Revision 5.0 113 September 29, 2009 
Section 5 – Selected Chemical Methods

Analysis Purpose: Sample preparation 

Sample Preparation Technique: Ultrasonic extraction 

Determinative Technique: EPA Method 300.1, Revision 1.0 

Method Developed for: Sodium fluoroacetate in soil 

Method Selected for: SAM lists this procedure for preparation of solid and non-aqueous liquid/organic 

solid samples. 

Description of Method: Sodium fluoroacetate is determined at sub-microgram per gram concentrations 

in small (~1 g) soil samples. Samples are ultrasonically extracted with water, filtered, and analyzed by 

Method 300.1. 

Source: Tomkins, B.A. 1994. “Screening-Procedure for Sodium Fluoroacetate (Compound 1080) at 

Sub-Microgram/Gram Concentrations in Soils.” Analytical Letters. 27(14): 2703–2718. 

http://www.informaworld.com/smpp/content~content=a747219004~db=all~order=page 

5.2.89 Literature Reference for Methamidophos (Chromatographia. 2006. 63(5/6): 233– 

237) 

Analyte(s) CAS RN 

Acephate 30560-19-1 

Methamidophos 10265-92-6 

Analysis Purpose: Sample preparation and analysis 

Sample Preparation Technique: SPE 

Determinative Technique: LC-MS-MS 

Method Developed for: Pesticides (methamidophos) in water samples 

Method Selected for: SAM lists this procedure for preparation and analysis of aqueous liquid and 

drinking water samples. 

Detection and Quantitation: The limit of detection for this limit is 30 μg/L. 

Description of Method: A multi-residue analytical method is described for monitoring polar pesticides, 

such as acephate and methamidophos, in water with SPE (solid-phase extraction) and LC-MS-MS. 

Samples are analyzed using a C18 analytical column (150 mm x 3.2 mm I.D., 5μm particle size) coupled 

with a C18 guard cartridge system (4 mm x 3.0 mm I.D.).
 

Special Considerations: The procedure described above has been developed for the analysis of various 

pesticides (methamidophos) in reagent water by LC-MS-MS. Modifications may be needed for 

application to drinking water samples. 

Source: Liu, F., Bischoff, G., Pestemer, W., Xu, W., and Kofoet, A. 2006. “Multi-residue Analysis of 

Some Polar Pesticides in Water Samples with SPE and LC/MS/MS.” Chromatographia. 63(5/6): 233–
 

237. http://www.epa.gov/sam/pdfs/Chromatographia-63_pg233-237.pdf 

5.2.90 Literature Reference for Methamidophos (Journal of Chromatography A, 2007. 

1154: 3–25) 

Analyte(s) CAS RN 

Acephate 30560-19-1 

 SAM Revision 5.0 114 September 29, 2009 
Section 5 – Selected Chemical Methods

Analyte(s) CAS RN 

Methamidophos 10265-92-6 

Analysis Purpose: Sample preparation and analysis 

Sample Preparation Technique: Solvent extraction 

Determinative Technique: LC-MS-MS 

Method Developed for: Pesticides (methamidophos) in crops 

Method Selected for: SAM lists this procedure for preparation and analysis of solid, non-aqueous 

liquid/organic solid, air, and wipe samples. 

Detection and Quantitation: The limit of detection for this method is 0.01 mg/kg. 

Description of Method: A liquid chromatography–tandem quadrupole mass spectrometry (LC-MS-MS) 

multi-residue method for the simultaneous target analysis of a wide range of pesticides and metabolites in 

fruit, vegetables and cereals is described. Gradient elution has been used in conjunction with positive 

mode electrospray ionization tandem mass spectrometry to detect up to 171 pesticides and/or metabolites 

in different crop matrices using a single chromatographic run. Pesticide residues are extracted/partitioned 

from the samples with acetone/dichloromethane/light petroleum. Samples are analyzed by LC-MS-MS 

using a C18 analytical column (150 mm x 3.2 mm I.D., 5μm particle size) coupled with a C18 guard
 

cartridge system (4 mm x 3.0 mm I.D.).
 

Special Considerations: The procedure has been developed for the analysis of various pesticides 

(methamidophos) in crops using LC-MS-MS; modifications will be needed for application to 

environmental samples such as soils, wipes, and air samples collected on sorbent/filters. 

Source: Hiemstra, M., de Kok, A. 2007. “Comprehensive Multi-residue Method for the Target Analysis 

of Pesticides in Crops Using Liquid Chromatography-tandem Mass Spectrometry.” Journal of
 

Chromatography A. 1154(1): 3–25. http://www.sciencedirect.com/science/journal/00219673
 

5.2.91 Literature Reference for Fluoroacetamide (Journal of Chromatography B, 2008. 

876(1): 103–108) 

Analyte(s) CAS RN 

Fluoroacetamide 640-19-7 

Analysis Purpose: Sample preparation and analysis 

Sample Preparation Technique: Water extraction 

Determinative Technique: GC/MS 

Method Developed for: Fluoroacetamide and tetramine in blood, urine and stomach contents 

Method Selected for: SAM lists this procedure for preparation and analysis of solid, non-aqueous 

liquid/organic solid, aqueous liquid, drinking water, air, and wipe samples. 

Detection and Quantitation: The detection limit of this method for fluoroacetamide is 0.01 μg/g. 

Description of Method: Samples are extracted by microscale liquid-liquid extraction using acetonitrile, 

ENVI-CARB, and sodium chloride. Samples are analyzed by GC/MS using a 30-m DB-5MS capillary
 

column (or equivalent) coupled with a 1.5 m Innowax capillary column (or equivalent) by a quartz 

capillary column connector. If analyzing for fluoroacetamide alone, only the Innowax capillary column is 

needed. 

 SAM Revision 5.0 115 September 29, 2009 
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Special Considerations: The procedure has been developed for the analysis of fluoroacetamide and 

tetramine in blood, urine and stomach fluid samples; modifications will be needed for application to 

environmental samples. 

Source: Xu, X., Song, G., Zhu, Y., Zhang, J., Zhao, Y., Shen, H., Cai, Z., Han, J., and Ren, Y. 2008. 

“Simultaneous Determination of two Accute Poisoning Rodenticides Tetramine and Fluoroacetamide 

with a Coupled Column in Poisoning Cases.” Journal of Chromatography B. 876(1): 103–108. 

http://www.sciencedirect.com/science/journal/15700232 

5.2.92 Literature Reference for Sodium Azide (Journal of Forensic Sciences, 1998. 43(1): 

200–202) 

Analyte(s) CAS RN 

Sodium azide 26628-22-8 

Analysis Purpose: Sample preparation 

Sample Preparation Technique: Water extraction, filtration, and/or acidification 

Determinative Technique: EPA Method 300.1, Revision 1.0 

Method Developed for: Sodium azide in blood 

Method Selected for: SAM lists this procedure for preparation of solid, aqueous liquid, and drinking 

water samples. 

Detection and Quantitation: This method can routinely quantify to at least 100 μg/L, and the detection 

limit is estimated to be 30 μg/L. 

Description of Method: Samples are analyzed by IC using suppressed conductivity detection. Water 

extraction and filtration steps should be used for the preparation of solid samples. Filtration steps should 

be used for preparation of aqueous liquid and drinking water samples. An acidification step may be 

required prior to using EPA SW-846 Method 3580A for preparation of non-aqueous liquid/organic solid 

samples. 

Special Considerations: The procedure described above has been developed for the analysis of sodium 

azide in blood samples. 

Source: Kruszyna, R., Smith, R.P., and Kruszyna, H. 1998. “Determining Sodium Azide Concentration 

in the Blood by Ion Chromatography.” Journal of Forensic Sciences. 43(1): 200–202. 

http://www.astm.org/JOURNALS/FORENSIC/PAGES/2933.htm 

 SAM Revision 5.0 116 September 29, 2009 
Section 6 – Selected Radiochemical Methods

Section 6.0: Selected Radiochemical Methods 

A list of analytical methods to be used in analyzing environmental samples for radiochemical 

contaminants during homeland security events is provided in Appendix B. Methods are listed for each 

isotope and for each sample type that potentially may need to be measured and analyzed when responding 

to an environmental emergency. 

Please note: This section provides guidance for selecting radiochemical methods that have a high 

likelihood of assuring analytical consistency when laboratories are faced with a large scale 

environmental restoration crisis. Not all methods have been verified for the analyte/sample type 

combination listed in Appendix B. Please refer to the specified method to identify analyte/sample type 

combinations that have been verified. Any questions regarding information discussed in this section 

should be addressed to the appropriate contact(s) listed in Section 4. 

Appendix B is sorted alphabetically by analyte and includes the following information: 

•	 Analyte(s). The radionuclide(s) or contaminant(s) of interest. 

•	 CAS RN. A unique identifier for chemical substances that provides an unambiguous way to identify 

a chemical or molecular structure when there are many possible systematic, generic, or trivial names. 

In this section (Section 6.0) and Appendix B, the CAS RNs correspond to the specific radionuclide 

identified. 

•	 Determinative technique. An analytical instrument or technique used for qualitative and 

confirmatory determination of compounds or components in a sample. 

•	 Drinking water sample methods. The recommended methods/procedures for sample preparation 

and analysis to measure the analyte of interest in drinking water samples. Methods have been 

identified for qualitative and confirmatory determination. 

•	 Aqueous and liquid phase sample methods. The recommended methods/procedures for sample 

preparation and analysis to measure the analyte of interest in aqueous and/or non-aqueous liquid 

phase samples. Methods have been identified for qualitative and confirmatory determination. 

•	 Soil and sediment phase sample methods. The recommended methods/procedures for sample 

preparation and analysis to measure the analyte of interest in soil and sediment samples. Methods 

have been identified for qualitative and confirmatory determination. 

•	 Surface wipe sample methods. The recommended methods/procedures for sample preparation and 

analysis to measure the analyte of interest in surface wipe samples. Methods have been identified for 

qualitative and confirmatory determination. 

•	 Air filter sample methods. The recommended methods/procedures for sample preparation and 

analysis to measure the analyte of interest in air filter samples. Methods have been identified for 

qualitative and confirmatory determination. 

•	 Qualitative determination method identifier. A unique identifier or number assigned to an 

analytical method by the method publisher. The identified method is intended to determine the 

presence of a radiological element or isotope. These methods are less precise than confirmatory 

methods, and are used when greater sample throughput and more rapid reporting of results is 

required. 

•	 Confirmatory method identifier. A unique identifier or number assigned to an analytical method by 

the method publisher. The identified method is for measurement of the activity from a particular 

radioisotope per unit of mass, volume, or area sampled. 

Following a homeland security event, it is assumed that only those areas with contamination greater than 

pre-existing/naturally prevalent levels commonly found in the environment would be subject to 

 SAM Revision 5.0	 117 September 29, 2009 
Section 6 – Selected Radiochemical Methods

remediation. Dependent on site- and event-specific goals, investigation of background levels using 

methods listed in Appendix B is recommended. 

6.1 General Guidelines 

The guidelines summarized in this section provide a general overview of how to identify the appropriate 

radiochemical method(s) for a given analyte-sample type combination, as well as recommendations for 

QC procedures. 

For additional information on the properties of the radionuclides listed in Appendix B, TOXNET 

(http://toxnet.nlm.nih.gov/index.html), a cluster of databases on toxicology, hazardous chemicals, and 

related areas maintained by the National Library of Medicine, is an excellent resource. EPA’s Radiation 

Protection (http://www.epa.gov/radiation/radionuclides/index.html) and the Multi-Agency Radiological 

Laboratory Analytical Protocols Manual (MARLAP) (http://www.epa.gov/radiation/marlap/manual.html) 

Web sites provide some additional information pertaining to radionuclides of interest and selection of 

radiochemical methods. Emergency response documents recently developed by EPA’s Office of Radiation 

and Indoor Air (ORIA) may be found at http://www.epa.gov/narel/incident_guides.html. 

6.1.1 Standard Operating Procedures for Identifying Radiochemical Methods 

To determine the appropriate method to be used on an environmental sample, locate the analyte of 

concern in Appendix B: Radiochemical Methods under the “Analyte Class” or “Analyte(s)” column. 

After locating the analyte of concern, continue across the table to identify the appropriate determinative 

technique (e.g., alpha spectrometry), then identify the appropriate qualitative and/or confirmatory method 

for the sample type of interest (drinking water, aqueous and liquid phase, soil and sediment, surface 

wipes, and air filters) for the particular analyte. 

Sections 6.2.1 through 6.2.32, below, provide summaries of the qualitative and confirmatory methods 

listed in Appendix B. Once a method has been identified in Appendix B, Table 6-1 can be used to locate 

the method summary. 

Table 6-1. Radiochemical Methods and Corresponding Text Section Numbers 

Analyte / Analyte Class CAS RN Method Section 

900.0 (EPA) 6.2.2 

Gross Alpha NA FRMAC, Vol 2, pg. 33 6.2.18 

Gross Beta NA AP1 (ORISE) 6.2.19 

7110 B (SM) 6.2.26 

Gamma 

Select Mixed Fission Products* NA 901.1 (EPA) 6.2.3 

Ga-01-R (HASL-300) 6.2.13 

Am-01-RC (HASL-300) 6.2.10 

Am-02-RC (HASL-300) 6.2.11 

Americium-241 14596-10-2 Am-04-RC (HASL-300) 6.2.12 

Pu-12-RC (HASL-300) 6.2.15 

AP11 (ORISE) 6.2.22 

D3084-05 (ASTM) 6.2.24 

* Please note that this category does not cover all fission products. 

 SAM Revision 5.0 118 September 29, 2009 
Section 6 – Selected Radiochemical Methods

Analyte / Analyte Class CAS RN Method Section 

Am-01-RC (HASL-300) 6.2.10 

Am-04-RC (HASL-300) 6.2.12 

Californium-252 13981-17-4 Pu-12-RC (HASL-300) 6.2.15 

AP11 (ORISE) 6.2.22 

D3084-05 (ASTM) 6.2.24 

Cesium-137 10045-97-3 901.1 (EPA) 6.2.3 

Cobalt-60 10198-40-0 

Ga-01-R (HASL-300) 6.2.13 

7120 (SM) 6.2.27 

Am-01-RC (HASL-300) 6.2.10 

Am-04-RC (HASL-300) 6.2.12 

Curium-244 13981-15-2 Pu-12-RC (HASL-300) 6.2.15 

AP11 (ORISE) 6.2.22 

D3084-05 (ASTM) 6.2.24 

901.1 (EPA) 6.2.3 

Europium-154 15585-10-1 Ga-01-R (HASL-300) 6.2.13 

7120 (SM) 6.2.27 

Iodine-125 14158-31-7 Procedure #9 (ORISE) 6.2.23 

Iodine-131 10043-66-0 901.1 (EPA) 6.2.3 

Ga-01-R (HASL-300) 6.2.13 

901.1 (EPA) 6.2.3 

Iridium-192 14694-69-0 Ga-01-R (HASL-300) 6.2.13 

7120 (SM) 6.2.27 

Molybdenum-99 14119-15-4 901.1 (EPA) 6.2.3 

Ga-01-R (HASL-300) 6.2.12 

Plutonium-238 13981-16-3 EMSL-33 (EPA) 6.2.9 

Plutonium-239 15117-48-3 

AP11 (ORISE) 6.2.22 

D3084-05 (ASTM) 6.2.24 

Polonium-210 13981-52-7 Method 111 (EPA) 6.2.1 

Po-02-RC (HASL-300) 6.2.14 

903.0 (EPA) 6.2.4 

903.1 (EPA) 6.2.5 

Radium-226 13982-63-3 EMSL-19 (EPA) 6.2.8 

D3084-05 (ASTM) 6.2.24 

7500-Ra B (SM) 6.2.28 

7500-Ra C (SM) 6.2.29 

Ruthenium-103 13968-53-1 901.1 (EPA) 6.2.3 

Ruthenium-106 13967-48-1 Ga-01-R (HASL-300) 6.2.13 

Selenium-75 14265-71-5 7120 (SM) 6.2.27 

Strontium-90 10098-97-2 Sr-03-RC (HASL-300) 6.2.16 

7500-Sr B (SM) 6.2.30 

 SAM Revision 5.0 119 September 29, 2009 
Section 6 – Selected Radiochemical Methods 

Analyte / Analyte Class CAS RN Method Section 

Technetium-99 14133-76-7 Tc-02-RC (HASL-300) 6.2.17 

AP5 (ORISE) 6.2.21 

Tritium (Hydrogen-3) 10028-17-8 906.0 (EPA) 6.2.6 

AP2 (ORISE) 6.2.20 

908.0 (EPA) 6.2.7 

Uranium-234 13966-29-5 EMSL-33 (EPA) 6.2.9 

AP11 (ORISE) 6.2.22 

Uranium-235 15117-96-1 D3084-05 (ASTM) 6.2.24 

Uranium-238 7440-61-1 D3972-02 (ASTM) 6.2.25 

7500-U B (SM) 6.2.31 

7500-U C (SM) 6.2.32 

The method summaries are listed in order of method selection hierarchy (see Figure 2-1), starting with 

EPA methods, followed by methods from other federal agencies and VCSBs. Methods are listed in 

numerical order under each publisher. Where available, a direct link to the full text of the selected 

analytical method is provided in the method summary. For additional information regarding sample 

preparation and analysis procedures and on methods available through consensus standards organizations, 

please use the contact information provided in Table 6-2. 

Table 6-1. Sources of Radiochemical Methods 

Name Publisher Reference 

NEMI EPA, USGS http://www.nemi.gov 

CFR Promulgated Test 

Methods 

EPA, Technical Transfer Network 

(TTN) EMC 

http://www.epa.gov/ttn/emc/promgate.html 

Prescribed Procedures for 

Measurement of Radioactivity 

in Drinking Water (EPA-600 4-

80-032, August 1980) 

EPA, ORD, Environmental 

Monitoring and Support Laboratory 

(EMSL) 

http://www.sld.state.nm.us/Documents/for 

ewd.pdf 

Also available from National Technical 

Information Service (NTIS)*, U.S. 

Department of Commerce, 5285 Port 

Royal Road, Springfield, VA 22161, (703) 

605-6000. 

Radiochemical Analytical 

Procedures for Analysis of 

Environmental Samples, March 

1979. EMSL-LV-0539-17 

EPA, EMSL Available NTIS*, U.S. Department of 

Commerce, 5285 Port Royal Road, 

Springfield, VA 22161, (703) 605-6000. 

EML Procedures Manual, 

Health and Safety Laboratory 

(HASL-300), 28th Edition, 

February, 1997 

Department of Energy (DOE), 

Environmental Measurements 

Laboratory (EML) / Now DHS 

http://www.eml.st.dhs.gov/publications/pro 

cman.cfm 

Also available from NTIS*, U.S. 

Department of Commerce, 5285 Port 

Royal Road, Springfield, VA 22161, (703) 

605-6000. 

Federal Radiological Monitoring 

and Assessment Center 

(FRMAC) Laboratory Manual 

DOE, National Nuclear Security 

Administration (NNSA) 

http://www.nv.doe.gov/nationalsecurity/ho 

melandsecurity/frmac/manuals.aspx 

Oak Ridge Institute for Science 

and Education (ORISE) 

Laboratory Procedures Manual 

ORISE, Independent 

Environmental Assessment and 

Verification 

http://orise.orau.gov/ieav/surveyprojects/lab-manual.htm 

SAM Revision 5.0 120 September 29, 2009 
Section 6 – Selected Radiochemical Methods

Name Publisher Reference 

Annual Book of ASTM 

Standards, Vol. 11.02* 

ASTM International http://www.astm.org 

Standard Methods for the 

Examination of Water and 

Wastewater, 21st Edition, 2005* 

APHA, AWWA, and WEF http://www.standardmethods.org 

* Subscription and/or purchase required. 

6.1.2 General QC Guidelines for Radiochemical Methods 

Having data of known and documented quality is critical so that public officials can accurately assess the 

activities that may be needed in responding to emergency situations. Having such data requires that 

laboratories: (1) conduct the necessary QC to ensure that measurement systems are in control and 

operating correctly; (2) properly document results of the analyses; and (3) properly document 

measurement system evaluation of the analysis-specific QC. Ensuring data quality also requires that 

laboratory results are properly evaluated and the results of the data quality evaluation are transmitted to 

decision makers. 

The level or amount of QC needed often depends on the intended purpose of the data that are generated. 

Various levels of QC may be required if the data are generated during contaminant presence/absence 

qualitative determinations versus confirmatory analyses. The specific needs for data generation should be 

identified. QC requirements and data quality objectives should be derived based on those needs, and 

should be applied consistently across laboratories when multiple laboratories are used. For example, 

during rapid sample screening analyses, minimal QC samples (e.g., blanks, duplicates) and 

documentation might be required to ensure data quality. Implementation of the analytical methods for 

evaluation of environmental samples during site assessment through site clearance, such as those 

identified in this document, might require increased QC. 

Some method-specific QC requirements are described in many of the individual methods that are cited in 

this manual. QC requirements will be referenced in SAPs developed to address specific analytes and 

sample types of concern. Individual methods, sampling and analysis protocols, or contractual statements 

of work also should be consulted to determine any additional QC that may be needed. 

QC samples are required to assess the precision, bias, and reliability of sample results. All QC results are 

tracked on control charts and reviewed for acceptability and trends in analysis or instrument operation. 

QC parameters are measured as required per method at the prescribed frequency. QC of laboratory 

analyses using radiochemical methods includes ongoing analysis of QC samples and tracking QC 

parameters including, but not limited to the following: 

• Method blanks; 

• Calibration checks; 

• Sample and sample duplicates; 

• Laboratory control sample recoveries; 

• MS/MSD recoveries; and 

• Tracer and/or carrier yield. 

Please note: The appropriate point of contact identified in Section 4 should be consulted regarding 

appropriate QA/QC procedures prior to sample analysis. These contacts will consult with the EPA 

coordinator responsible for laboratory activities during the specific event to ensure QA/QC procedures are 

performed consistently across laboratories. EPA program offices will be responsible for ensuring that the 

QA/QC practices are implemented. 

 SAM Revision 5.0 121 September 29, 2009 
Section 6 – Selected Radiochemical Methods

6.1.3 Safety and Waste Management 

It is imperative that safety precautions be used during collection, processing, and analysis of 

environmental samples. Laboratories should have a documented health and safety plan for handling 

samples that may contain target CBR contaminants, and laboratory staff should be trained in and 

implement the safety procedures included in the plan. In addition, many of the methods summarized or 

cited in Section 6.2 contain specific requirements, guidelines, or information regarding safety precautions 

that should be followed when handling or processing environmental samples and reagents. These 

methods may also provide information regarding waste management. Laboratories should consult with 

the responsible government agencies prior to disposal of waste materials. Other resources that can be 

consulted for additional information include the following: 

•	 OSHA - 29 CFR part 1910.1450. Occupational Exposure to Hazardous Chemicals in Laboratories. 

http://www.access.gpo.gov/nara/cfr/waisidx_06/29cfr1910a_06.html 

•	 EPA - 40 CFR part 260. Hazardous Waste Management System: General. 

http://www.access.gpo.gov/nara/cfr/waisidx_07/40cfr260_07.html 

•	 EPA - 40 CFR part 270. EPA Administered Permit Programs: The Hazardous Waste Permit Program. 

http://www.access.gpo.gov/nara/cfr/waisidx_07/40cfr270_07.html 

•	 NRC - 10 CFR part 20. Standards for Protection Against Radiation 

http://www.access.gpo.gov/nara/cfr/waisidx_00/10cfr20_00.html 

•	 DOE. Order O 435.1: Radioactive Waste Management. July 1, 1999. Available at: 

www.directives.doe.gov/pdfs/doe/doetext/neword/435/o4351.html 

•	 DOE. M 435.1-1. Radioactive Waste Management Manual. Office of Environmental Management. 

July 9, 1999. Available at: http://www.directives.doe.gov/pdfs/doe/doetext/neword/435/m4351-

1.html 

•	 DOE. Compendium of EPA-Approved Analytical Methods for Measuring Radionuclides in Drinking 

Water. Prepared by the Office of Environmental Policy and Assistance Air, Water and Radiation 

Division (EH-412). June 1998. Available at: 

http://www.orau.org/ptp/PTP%20Library/library/DOE/Misc/radmeth3.pdf 

•	 EPA. 1996. Profile and Management Options for EPA Laboratory Generated Mixed Waste. Office 

of Radiation and Indoor Air, Washington, DC. EPA 402-R-96-015. Available at: 

http://www.epa.gov/rpdweb00/docs/mixed-waste/402-r-96-015.pdf 

•	 EPA. 2001. Changes to 40 CFR 266 (Storage, Treatment, Transportation, and Disposal of Mixed 

Waste), Federal Register 66:27217-27266, May 16. Available at: 

http://frwebgate.access.gpo.gov/cgi-bin/getdoc.cgi?dbname=2001_register&docid=01-11408-

filed.pdf 

•	 EPA. 2008. Resource Conservation and Recovery Act (RCRA) Orientation Manual. OSWER, 

Washington, DC. EPA530-R-02-016. 259 pp. Available at: 

http://www.epa.gov/osw/inforesources/pubs/orientat/ 

•	 MARLAP Manual. 2004. Chapter 17. Waste Management in a Radioanalytical Laboratory. 

Available at: http://www.epa.gov/rpdweb00/docs/marlap/402-b-04-001b-17-final.pdf 

•	 National Research Council. 1995. Prudent Practices in the Laboratory; Handling and Disposal of 

Chemicals, National Academy Press, Washington, DC. Available at: 

http://books.nap.edu/openbook.php?isbn=0309052297 

•	 National Council on Radiation Protection and Measurements (NCRP). 2002. Risk-Based 

Classification of Radioactive and Hazardous Chemical Wastes, Report Number 139. 7910 

Woodmont Avenue, Suite 400, Bethesda, MD 20814–3095 

•	 Nuclear Regulatory Commission (NRC) / EPA. 1995. Joint Nuclear Regulatory 

Commission/Environmental Protection Agency Guidance on the Storage of Mixed Radioactive and 

Hazardous Waste., Federal Register 60:40204-40211 

 SAM Revision 5.0	 122 September 29, 2009 
Section 6 – Selected Radiochemical Methods

6.2 Method Summaries 

Summaries for the analytical methods listed in Appendix B are provided in Sections 6.2.1 through 6.2.32. 

These summaries contain information that has been extracted from the selected methods. Each method 

summary contains a table identifying the contaminants in Appendix B to which the method applies, a 

brief description of the analytical method, and a link to the full version of the method or a source for 

obtaining a full version of the method. The full version of the method should be consulted prior to 

sample analysis. 

6.2.1 EPA Method 111: Determination of Polonium-210 Emissions from Stationary 

Sources 

Analyte(s) CAS RN

Polonium-210 13981-52-7

Analysis Purpose: Qualitative and confirmatory determination 

Determinative Technique: Alpha spectrometry 

Method Developed for: Polonium-210 in particulate matter samples collected from stationary source 

exhaust stacks 

Method Selected for: SAM lists this method for qualitative and confirmatory analysis of surface wipes 

and air filters. 

Description of Method: This method covers the determination of polonium-210 in particulate matter 

samples collected from stationary sources such as exhaust stacks. Polonium-210 in the sample is put in 

solution, deposited on a metal disc, and the radioactive disintegration rate measured. Polonium in acid 

solution spontaneously deposits on surface metals that are more electropositive than polonium. 

Source: EPA EMC, prepared by the OAQPS. 2000. “Method 111: Determination of Polonium-210 

Emissions from Stationary Sources.” http://www.epa.gov/sam/pdfs/EPA-111.pdf 

6.2.2 EPA Method 900.0: Gross Alpha and Gross Beta Radioactivity in Drinking Water 

Analysis Purpose: Gross alpha and gross beta determination 

Determinative Technique: Alpha/Beta counting 

Method Developed for: Gross alpha and gross beta particle activities in drinking water 

Method Selected for: SAM lists this method for gross alpha and gross beta determination in drinking 

water samples. 

Description of Method: The method provides an indication of the presence of alpha and beta emitters, 

including the following SAM analytes: 

• Americium-241 (CAS RN 14596-10-2) Alpha emitter 

• Californium-252 (CAS RN 13981-17-4) Alpha emitter 

• Cesium-137 (CAS RN 10045-97-3) Beta emitter 

• Cobalt-60 (CAS RN 10198-40-0) Beta emitter 

• Curium-244 (CAS RN 13981-15-2) Alpha emitter 

• Europium-154 (CAS RN 15585-10-1) Beta emitter 

• Iridium-192 (CAS RN 14694-69-0) Beta emitter 

• Plutonium-238 (CAS RN 13981-16-3) Alpha emitter 

• Plutonium-239 (CAS RN 15117-48-3) Alpha emitter 

 SAM Revision 5.0	 123 September 29, 2009 
Section 6 – Selected Radiochemical Methods

• Polonium-210 (CAS RN 13981-52-7) Alpha emitter 

• Radium-226 (CAS RN 13982-63-3) Alpha emitter 

• Ruthenium-103 (CAS RN 13968-53-1) Beta emitter 

• Ruthenium-106 (CAS RN 13967-48-1) Beta emitter 

• Strontium-90 (CAS RN 10098-97-2) Beta emitter 

• Uranium-234 (CAS RN 13966-29-5) Alpha emitter 

• Uranium-235 (CAS RN 15117-96-1) Alpha emitter 

• Uranium-238 (CAS RN 7440-16-1) Alpha emitter 

An aliquot of a preserved drinking water sample is evaporated to a small volume (3 to 5 mL) and 

transferred quantitatively to a tarred 2-inch planchet. The aliquot volume is determined based on a 

maximum total solids content of 100 mg. The sample aliquot is evaporated to dryness in the planchet to a 

constant weight, cooled, and counted using a gas proportional or scintillation counting system. The 

counting system is calibrated with thorium-230 for gross alpha, and with strontium-90 for gross beta 

analysis3

. A traceable standards-based efficiency curve must be developed for each calibration nuclide 

(Th-230 and Sr-90) based on a range of total solids content in the 2-inch planchet from 0 to 100 mg (see 

method for specific recommendations and requirements for the use of cesium-137). 

Special Considerations: Long counting time and increased sample size may be required to meet 

detection limits. Sensitivity is limited by the concentration of solids in the sample. 

Source: EPA, EMSL. 1980. “Method 900.0: Gross Alpha and Gross Beta Radioactivity in Drinking 

Water.” Prescribed Procedures for Measurement of Radioactivity in Drinking Water, EPA/600/4/80/032. 

http://www.epa.gov/sam/pdfs/EPA-900.0.pdf 

6.2.3 EPA Method 901.1: Gamma Emitting Radionuclides in Drinking Water 

Analyte(s) CAS RN 

Cesium-137 10045-97-3 

Cobalt-60 10198-40-0 

Europium-154 15585-10-1 

Iodine-131 10043-66-0 

Iridium-192 14694-69-0 

Molybdenum-99 14119-15-4 

Ruthenium-103 13968-53-1 

Ruthenium-106 13967-48-1 

Selenium-75 14265-71-5 

Analysis Purpose: Qualitative and confirmatory analysis 

Determinative Technique: Gamma spectrometry 

Method Developed for: Gamma emitting radionuclides in drinking water 

Method Selected for: SAM lists this method for qualitative and confirmatory analysis of select gamma 

emitters in drinking water samples. 

3

 EPA lists standards for use when analyzing drinking water in the table at 40 CFR 141.25 (footnote 11). 

 SAM Revision 5.0 124 September 29, 2009 
Section 6 – Selected Radiochemical Methods

Description of Method: This method is applicable for analysis of water samples that contain 

radionuclides that emit gamma photons with energies ranging from approximately 60 to 2000 keV. The 

method uses gamma spectroscopy for measurement of gamma photons emitted from radionuclides 

without separating them from the sample matrix. A homogeneous aliquot of water is placed into a 

standard geometry (normally a Marinelli beaker) for gamma counting, typically using a high purity 

germanium (HPGe) detector. Detectors such as Germanium (Lithium) (Ge(Li)) or thallium-activated 

sodium iodide (NaI(Tl)) also can be used. Sample aliquots are counted long enough to meet the required 

sensitivity of measurement. To reduce adsorbance of radionuclides on the walls of the counting 

container, the sample is acidified at collection time. Due to its lower resolution, significant interference 

can occur using the NaI(Tl) detector when counting a sample containing radionuclides that emit gamma 

photons of similar energies. When using this method, shielding is needed to reduce background 

interference. Detection limits are dependent on sample volume, geometry (physical shape), and counting 

time. 

Source: EPA, EMSL. 1980. “Method 901.1: Gamma Emitting Radionuclides in Drinking Water.” 

Prescribed Procedures for Measurement of Radioactivity in Drinking Water, EPA/600/4/80/032. 

http://www.epa.gov/sam/pdfs/EPA-901.1.pdf 

6.2.4 EPA Method 903.0: Alpha-Emitting Radium Isotopes in Drinking Water 

Analyte(s) CAS RN 

Radium-226 13982-63-3 

Analysis Purpose: Qualitative determination 

Determinative Technique: Alpha counting 

Method Developed for: Total soluble alpha emitting radioisotopes of radium, namely radium-223, 

radium-224 and radium-226 in drinking water 

Method Selected for: SAM lists this method for qualitative determination in drinking water samples. 

Description of Method: This method covers measurement of the total soluble alpha emitting 

radioisotopes of radium, namely radium-223, radium-224 and radium-226 in drinking water. The method 

does not give an accurate measurement of radium-226 content in the sample when other alpha emitters are 

present. If radium-223 and radium-224 are present, the results can be used to provide a gross 

determination of radium-226. When the total radium alpha activity of a drinking water sample is greater 

than 5 pCi/L, use of Method 903.1 (Radium-226 in Drinking Water) is preferred. Radium in the water 

sample is collected by coprecipitation with barium and lead sulfate, and purified by re-precipitation from
 

EDTA solution. Citric acid is added to ensure that complete interchange occurs before the first 

precipitation step. The final barium sulfate precipitate is alpha counted to determine the total 

disintegration rate of the radium isotopes. By making a correction for the ingrowth of radon and its alpha 

emitting progeny for the elapsed time after separation, one can determine radium activity in the sample. 

Presence of significant natural barium in the sample can result in a falsely high yield. Based on a 1000-

mL sample and 100-minute counting time, the minimum detectable level for this method is 0.5 pCi/L. 

Source: EPA, EMSL. 1980. “Method 903.0: Alpha-Emitting Radium Isotopes in Drinking Water.”
 

Prescribed Procedures for Measurement of Radioactivity in Drinking Water, EPA/600/4/80/032. 

http://www.epa.gov/sam/pdfs/EPA-903.0.pdf 

 SAM Revision 5.0 125 September 29, 2009 
Section 6 – Selected Radiochemical Methods

6.2.5 EPA Method 903.1: Radium-226 in Drinking Water – Radon Emanation Technique 

Analyte(s) CAS RN 

Radium-226 13982-63-3 

Analysis Purpose: Confirmatory analysis 

Determinative Technique: Alpha counting 

Method Developed for: Radium-226 in drinking water 

Method Selected for: SAM lists this method for confirmatory analysis of drinking water samples. 

Description of Method: This method is specific for radium-226, and is based on the emanation and 

scintillation counting of radon-222, a daughter product of radium-226. Radium-226 is concentrated and 

separated from the water sample by coprecipitation on barium sulfate. The precipitate is dissolved in 

EDTA reagent, placed in a sealed bubbler and stored for ingrowth of radon-222. After ingrowth, the 

radon-222 gas is purged into a scintillation cell. When the short-lived radon-222 daughters are in 

equilibrium with the parent (after ~4h), the scintillation cell is counted for activity. The absolute 

measurement of radium-226 is effected by calibrating the scintillation cell system with a standard solution 

of the nuclide. There are no radioactive interferences in this method. Based on a 1000-mL sample and 

100-minute counting time, the minimum detectable level for this method is 0.5 pCi/L. 

Source: EPA, EMSL. 1980. “Method 903.1: Radium-226 in Drinking Water – Radon Emanation 

Technique.” Prescribed Procedures for Measurement of Radioactivity in Drinking Water, 

EPA/600/4/80/032. http://www.epa.gov/sam/pdfs/EPA-903.1.pdf
 

6.2.6 EPA Method 906.0: Tritium in Drinking Water 

Analyte(s) CAS RN 

Tritium (Hydrogen-3) 10028-17-8 

Analysis Purpose: Qualitative and confirmatory analysis 

Determinative Technique: Liquid scintillation 

Method Developed for: Tritium (as T2O or HTO) in drinking water 

Method Selected for: SAM lists this method for qualitative and confirmatory analysis of drinking water 

and aqueous/liquid phase samples. 

Description of Method: An unpreserved 100-mL aliquot of a drinking water sample is distilled after 

adjusting pH with a small amount of sodium hydroxide and adding potassium permanganate. The 

alkaline treatment prevents other radionuclides, such as radioiodine and radiocarbon, from distilling with 

the tritium. The permanganate treatment oxidizes trace organics that may be present in the sample and 

prevents their appearance in the distillate. To determine the concentration of tritium, the middle fraction 

of the distillate is used, because the early and late fractions are more apt to contain materials interfering 

with the liquid scintillation counting process. A portion of this collected fraction is added to a liquid 

scintillator cocktail, and the solution is mixed, dark adapted and counted for beta particle activity. The 

efficiency of the system can be determined by the use of prepared tritiated water standards having the 

same density and color as the sample. 

Source: EPA, EMSL. 1980. “Method 906.0: Tritium in Drinking Water.” Prescribed Procedures for 

Measurement of Radioactivity in Drinking Water, EPA/600/4/80/032. http://www.epa.gov/sam/pdfs/EPA906.0.pdf 

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6.2.7 EPA Method 908.0: Uranium in Drinking Water – Radiochemical Method 

Analyte(s) CAS RN 

Uranium-234 13966-29-5 

Uranium-235 15117-96-1 

Uranium-238 7440-61-1 

Analysis Purpose: Qualitative determination 

Determinative Technique: Alpha counting 

Method Developed for: Total uranium alpha particle activity in drinking water 

Method Selected for: SAM lists this method for qualitative determination in drinking water samples. 

Description of Method: This method measures total uranium alpha activity of a sample, without doing
 

an isotopic uranium analysis. The sample is acidified with hydrochloric acid and boiled to eliminate 

carbonate and bicarbonate ions. Uranium is coprecipitated with ferric hydroxide and separated from the 

sample. The uranium is then separated from other radionuclides that were carried down with the ferric 

hydroxide by dissolving the hydroxide precipitate in hydrochloric acid, putting the solution through an
 

anion exchange column, washing the column with hydrochloric acid, and finally eluting the uranium with 

hydrochloric acid. The uranium eluate is evaporated and the uranium chemical form is converted to 

nitrate. The residue is transferred to a stainless steel planchet, dried, flamed, and counted for alpha 

particle activity. Since uranium is a naturally occurring radionuclide, reagents must be checked for 

uranium contamination by analyzing a complete reagent blank by the same procedure as used for the 

samples. Based on a 1000- mL sample and 100-minute counting time in a single laboratory study, the 

minimum detectable level for this method is 1.0 pCi/L. 

Special Considerations: If it is suspected that the sample exists in refractory form (i.e., non-digestible 

or dissolvable material after normal digestion methods) or if there is a matrix interference problem, use 

ORISE Method AP11. 

Source: EPA, EMSL. 1980. “Method 908.0: Uranium in Drinking Water – Radiochemical Method.”
 

Prescribed Procedures for Measurement of Radioactivity in Drinking Water, EPA/600/4/80/032. 

http://www.epa.gov/sam/pdfs/EPA-908.0.pdf
 

6.2.8 EPA Method EMSL-19: Determination of Radium-226 and Radium-228 in Water, 

Soil, Air and Biological Tissue 

Analyte(s) CAS RN 

Radium-226 13982-63-3 

Analysis Purpose: Confirmatory analysis 

Determinative Technique: Alpha counting 

Method Developed for: Radium-226 and radium-228 in water, soil, air, biological tissues, and 

biological fluids 

Method Selected for: SAM lists this method for confirmatory analysis of soil/sediment, surface wipe, 

and air filter samples. 

Description of Method: Following acid digestion and filtration of soil, sediment, surface wipe, or air 

filter samples, radium is precipitated with barium sulfate. Barium-radium-sulfate is dissolved in a 

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pentasodium diethylenetriamine-pentaacetate (DTPA) solution and transferred to an emanation tube. The 

radon is allowed to come to equilibrium for approximately 30 days. Radium-226 decays by alpha 

emission to radon-222. Radon-222 is separated and collected from the liquid by a de-emanation 

technique. The radon is counted by alpha scintillation 4.5 hours after de-emanation, at which time the 

short-lived progeny have reached 97% of equilibrium. An applicable measurement range has not been 

determined; however, samples that contain 0.1 pCi of Radium-226 have been analyzed. 

Source: EPA, EMSL. 1979. “EMSL-19: Determination of Radium-226 and Radium-228 in Water, Soil, 

Air and Biological Tissue.” Radiochemical Analytical Procedures for Analysis of Environmental 

Samples. http://www.epa.gov/sam/pdfs/EPA-EMSL-19.pdf 

6.2.9 EPA Method EMSL-33: Isotopic Determination of Plutonium, Uranium, and 

Thorium in Water, Soil, Air, and Biological Tissue 

Analyte(s) CAS RN 

Plutonium-238 13981-16-3 

Plutonium-239 15117-48-3 

Uranium-234 13966-29-5 

Uranium-235 15117-96-1 

Uranium-238 7440-61-1 

Analysis Purpose: Confirmatory analysis
 

Determinative Technique: Alpha spectrometry
 

Method Developed for: Isotopic plutonium, uranium, and thorium, together or individually, in soil, 

water, air filters, urine, or ashed residues of vegetation, animal tissues, and bone
 

Method Selected for: SAM lists this method for confirmatory analysis of drinking water, 

aqueous/liquid, soil/sediment, surface wipe, and/or air filter samples. 

Description of Method: This method is appropriate for the analysis of isotopic plutonium, uranium, and 

thorium, together or individually, by alpha spectrometry. Plutonium-236, uranium-232, and thorium-234 

tracer standards are added for the determination of chemical yields. Samples are decomposed by nitrichydrofluoric acid digestion or ignition to assure that all of the plutonium is dissolved and chemically
 

separated from the sample by coprecipitation with sodium and ammonium hydroxide, anion exchange, 

and electrodeposition. The residues are dissolved in dilute nitric acid and successive sodium and 

ammonium hydroxide precipitations are performed in the presence of boric acid to remove fluoride and 

soluble salts. The hydroxide precipitate is dissolved, the solution is pH-adjusted with hydrochloric acid, 

and plutonium and uranium are adsorbed on an anion exchange column, separating them from thorium.
 

Plutonium is eluted with hydrobromic acid. The actinides are electrodeposited on stainless steel discs 

from an ammonium sulfate solution and subsequently counted by alpha spectrometry. This method is 

designed to detect environmental levels of activity as low as 0.02 pCi per sample. To avoid possible 

cross-contamination, sample aliquot activities should be limited to 25 pCi or less. 

Special Considerations: If it is suspected that the sample exists in refractory form (i.e., non-digestible 

or dissolvable material after normal digestion methods) or if there is a matrix interference problem, use 

ORISE Method AP11. 

Source: EPA, EMSL. 1979. “EMSL-33: Isotopic Determination of Plutonium, Uranium, and Thorium in 

Water, Soil, Air, and Biological Tissue.” Radiochemical Analytical Procedures for Analysis of 

Environmental Sample. http://www.epa.gov/sam/pdfs/EPA-EMSL-33.pdf
 

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6.2.10 EML HASL-300 Method Am-01-RC: Americium in Soil 

Analyte(s) CAS RN 

Americium-241 14596-10-2 

Californium-252 13981-17-4 

Curium-244 13981-15-2 

Analysis Purpose: Confirmatory analysis 

Determinative Technique: Alpha spectrometry
 

Method Developed for: Americium in soil 

Method Selected for: SAM lists this method for confirmatory analysis of soil/sediment samples. 

Description of Method: This method uses alpha spectrometry for determination of americium-241 in 

soil, and also can be applied for determination of californium. Americium is leached from soil with nitric 

acid and hydrochloric acid. Americium-243 is added as a tracer to determine chemical yield. The soil is 

processed through the plutonium separation steps using ion exchange resin according to Method Pu-11-

RC. Americium is collected with a calcium oxalate precipitation and finally isolated and purified by ion 

exchange. Californium-252 and curium-244 are eluted with americium as americium is stripped off the 

column. After source preparation by microprecipitation, americium-241, californium-252, and curium244 are determined by alpha spectrometry analysis. The counting period chosen depends on the 

sensitivity required of the measurement and the degree of uncertainty in the result that is acceptable. The 

lower limit of detection (LLD) for americium-241 is 0.5 mBq when counted for 1000 minutes. In cases 

where less than 100 g of sample is available, use of Pu-12-RC is recommended. 

Special Considerations: If it is suspected that the sample exists in refractory form (i.e., non-digestible 

or dissolvable material after normal digestion methods) or if there is a matrix interference problem, use 

ORISE Method AP11. 

Source: EML, DOE (EML is currently part of the DHS). 1997. “HASL-300 Method Am-01-RC: 

Americium in Soil.” EML Procedures Manual, HASL-300, 28th Edition. 

http://www.epa.gov/sam/pdfs/EML-Am-01-RC.pdf 

6.2.11 EML HASL-300 Method Am-02-RC: Americium-241 in Soil-Gamma Spectrometry 

Analyte(s) CAS RN 

Americium-241 14596-10-2

Analysis Purpose: Qualitative determination
 

Determinative Technique: Gamma spectrometry
 

Method Developed for: Americium-241 in large volume soil samples 

Method Selected for: SAM lists this method for qualitative determination in soil/sediment samples. 

Description of Method: This method uses gamma spectrometry for determination of americium-241 in 

soil. Americium-241 decays with the emission of a gamma ray at 59.5 keV with a decay frequency
 

(abundance or yield) of 35.9%. The sample is placed into an appropriately sized standard geometry
 

(normally a Marinelli beaker) after drying and grinding the sample for homogenization. Gamma-ray
 

attenuation corrections are required if the calibration source and the sample are in a different matrix or are 

 SAM Revision 5.0 129 September 29, 2009 
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of different densities. The LLD for 600 to 800 g of soil in a Marinelli beaker is 0.74 mBq for a 1000-

minute count. 

Source: EML, DOE (EML is currently part of the DHS). 1997. “HASL-300 Method Am-02-RC: 

Americium-241 in Soil-Gamma Spectrometry.” EML Procedures Manual, HASL-300, 28th Edition. 

http://www.epa.gov/sam/pdfs/EML-Am-02-RC.pdf 

6.2.12 EML HASL-300 Method Am-04-RC: Americium in QAP Water and Air Filters - 

Eichrom’s TRU Resin 

Analyte(s) CAS RN 

Americium-241 14596-10-2 

Californium-252 13981-17-4 

Curium-244 13981-15-2 

Analysis Purpose: Confirmatory analysis 

Determinative Technique: Alpha spectrometry 

Method Developed for: Americium (but not lanthanides) in water and air filters 

Method Selected for: SAM lists this method for confirmatory analysis of drinking water, aqueous/liquid 

samples, surface wipes, and air filters. 

Description of Method: This method is specific to measurement of americium isotopes in samples that 

do not contain lanthanides, but also can be used for measurement of californium and curium. The method 

uses microprecipitation and determination by alpha spectrometry. Americium-243 is added to the sample 

to determine chemical yield. The sample is processed through separation steps using ion exchange resins. 

The eluate from the ion exchange column containing americium (and all other ions, except plutonium) is 

evaporated, redissolved, and loaded onto a Transuranic (TRU) Resin extraction column. Americium (and 

curium and californium, if present) is separated and purified on the column and finally stripped with 

dilute nitric acid stripping solution. Microprecipitation is used to prepare for alpha spectrometry. The 

method involves sample preparation steps from EML HASL-300 Method Pu-10-RC for water samples. 

The LLD for total americium is 0.3 mBq when counted for 1000 minutes. 

Special Considerations: If it is suspected that the sample exists in refractory form (i.e., non-digestible 

or dissolvable material after normal digestion methods) or if there is a matrix interference problem, use 

ORISE Method AP11. 

Source: EML, DOE (EML is currently part of the DHS). 1997. “HASL-300 Method Am-04-RC: 

Americium in QAP Water and Air Filters - Eichrom’s TRU Resin.” EML Procedures Manual, HASL300, 28th Edition. http://www.epa.gov/sam/pdfs/EML-Am-04-RC.pdf 

6.2.13 EML HASL-300 Method Ga-01-R: Gamma Radioassay 

Analyte(s) CAS RN 

Cesium-137 10045-97-3 

Cobalt-60 10198-40-0 

Europium-154 15585-10-1 

Iodine-131 10043-66-0 

 SAM Revision 5.0 130 September 29, 2009 
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Analyte(s) CAS RN 

Iridium-192 14694-69-0 

Molybdenum-99 14119-15-4 

Ruthenium-103 13968-53-1 

Ruthenium-106 13967-48-1 

Selenium-75 14265-71-5 

Analysis Purpose: Qualitative and confirmatory analysis or gross gamma determination 

Determinative Technique: Gamma spectrometry 

Method Developed for: Gamma-ray emitting radionuclides in a variety of environmental matrices 

Method Selected for: SAM lists this method for qualitative and/or confirmatory analysis of select 

gamma emitters in aqueous/liquid, soil/sediment, surface wipes, and/or air filter samples. 

Description of Method: This method uses gamma spectroscopy for the measurement of gamma photons 

emitted from radionuclides without separating them from the sample matrix. Samples are placed into a 

standard geometry for gamma counting, typically using an HPGe detector. Detectors such as Ge(Li) or 

NaI(Tl) also can be used. The sample is placed into a standard geometry for gamma counting. Soil 

samples and sludge are placed into an appropriately sized Marinelli beaker after drying and grinding the 

sample for homogenization. Air filters and surface wipes can be counted directly or pressed into a 

planchet and counted. Samples are counted long enough to meet the required sensitivity of measurement. 

For typical counting systems and sample types, activity levels of approximately 40 Bq are measured, and 

sensitivities as low as 0.002 Bq can be achieved for many nuclides. Because of electronic limitations, 

count rates higher than 2000 counts per second (cps) should be avoided. High activity samples may be 

diluted, reduced in size, or moved away from the detector (a limited distance) to reduce the count rate and 

allow for analysis. The method is applicable for analysis of samples that contain radionuclides emitting 

gamma photons with energies above approximately 20 keV for germanium (Ge) (both HPGe and GeLi) 

detectors and above 50 keV for NaI(Tl) detectors. 

Source: EML, DOE (EML is currently part of the DHS). 1997. “HASL-300 Method Ga-01-R: Gamma 

Radioassay.” EML Procedures Manual, HASL-300, 28th Edition. http://www.epa.gov/sam/pdfs/EML-Ga01-R.pdf 

6.2.14 EML HASL-300 Method Po-02-RC: Polonium in Water, Vegetation, Soil, and Air 

Filters 

Analyte(s) CAS RN 

Polonium-210 1-3981-52-7

Analysis Purpose: Qualitative and confirmatory analysis 

Determinative Technique: Alpha spectrometry
 

Method Developed for: Polonium in water, vegetation, soil, and air filters 

Method Selected for: SAM lists this method for qualitative and confirmatory analysis of drinking water, 

aqueous/liquid, and soil/sediment samples. 

Description of Method: This method uses alpha spectrometry for determination of polonium in water, 

vegetation, soil, and air filter samples. Polonium equilibrated with Po-208 or Po-209 tracer is isolated 

from most other elements by coprecipitation with lead sulfide. The sulfide precipitate is dissolved in 

 SAM Revision 5.0 131 September 29, 2009 
Section 6 – Selected Radiochemical Methods

weak hydrochloric acid solution. Polonium is quantitatively deposited on a nickel disc, and the plated 

disc is counted on an alpha spectrometer to measure chemical yield and activity of the sample. The 

solution from the deposition may be retained and analyzed for Pb-210. When counted for 1000 minutes, 

the LLD for polonium is 1.0 mBq for water and 1.3 mBq for vegetation, soil and filters. 

Source: EML, DOE (EML is currently part of the DHS). 1997. “HASL-300 Method Po-02-RC: 

Polonium in Water, Vegetation, Soil, and Air Filters.” EML Procedures Manual, HASL-300, 28th Edition. 

http://www.epa.gov/sam/pdfs/EML-Po-02-RC.pdf 

6.2.15 EML HASL-300 Method Pu-12-RC: Plutonium and/or Americium in Soil or 

Sediments 

Analyte(s) CAS RN 

Americium-241 14596-10-2 

Californium-252 13981-17-4 

Curium-244 13981-15-2 

Analysis Purpose: Confirmatory analysis 

Determinative Technique: Alpha spectrometry 

Method Developed for: Plutonium and americium in soil 

Method Selected for: This method is listed in SAM for use when small soil and sediment sample sizes 

(≤100 g) will be analyzed. 

Description of Method: A sample of soil of up to 100 g in size is equilibrated with Am-243 tracer. 

Contaminant isotopes are leached with nitric and hydrochloric acid. Plutonium is removed by ion 

exchange. The eluent from the plutonium separation is saved for determination of americium, curium, 

and californium. Americium, curium, and californium are collected with a calcium oxalate 

coprecipitation, isolated and purified by extraction chromatography. Microprecipitation is used to prepare 

the sample for analysis by alpha spectrometry of americium, curium, and californium. The LLD for 

americium is 0.5 mBq when counted for 1000 minutes. 

Special Considerations: In cases where only small sample sizes (≤100 g) will be analyzed, this method 

is recommended for confirmatory analysis. If it is suspected that the sample exists in refractory form (i.e., 

non-digestible or dissolvable material after normal digestion methods) or if there is a matrix interference 

problem, use ORISE Method AP11. 

Source: EML, DOE (EML is currently part of the DHS). 1997. “HASL-300 Method Pu-12-RC: 

Plutonium and/or Americium in Soil or Sediments.” EML Procedures Manual, HASL-300, 28th Edition. 

http://www.epa.gov/sam/pdfs/EML-Pu-12-RC.pdf 

6.2.16 EML HASL-300 Method Sr-03-RC: Strontium-90 in Environmental Samples 

Analyte(s) CAS RN 

Strontium-90 10098-97-2

Analysis Purpose: Qualitative and confirmatory analysis 

Determinative Technique: Beta counting 

 SAM Revision 5.0 132 September 29, 2009 
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Method Developed for: Strontium-90 in vegetation, water, air filters and soil 

Method Selected for: SAM lists this method for qualitative and confirmatory analysis of soil/sediment, 

surface wipe, and air filter samples. 

Description of Method: Strontium is separated from calcium, other fission products, and natural 

radioactive elements. Fuming nitric acid separations remove the calcium and most other interfering ions. 

Radium, lead and barium are removed with barium chromate. Traces of other fission products are 

scavenged with iron hydroxide. After strontium-90 and yttrium-90 equilibrium has been attained, yttrium90 is precipitated as the hydroxide and converted to oxalate for counting on a low-background gas 

proportional beta counter. Chemical yield is determined with strontium-85 tracer by counting in a gamma 

well detector.
 

Source: EML, DOE (EML is currently part of the DHS). 1997. “HASL-300 Method Sr-03-RC: 

Strontium-90 in Environmental Samples.” EML Procedures Manual, HASL-300, 28th Edition. 

http://www.epa.gov/sam/pdfs/EML-Sr-03-RC.pdf 

6.2.17 EML HASL-300 Method Tc-02-RC: Technetium-99 in Water – TEVA® Resin 

Analyte(s) CAS RN 

Technetium-99 14133-76-7

Analysis Purpose: Qualitative and confirmatory analysis 

Determinative Technique: Liquid scintillation 

Method Developed for: Technetium-99 (Tc-99) in water 

Method Selected for: SAM lists this method for qualitative and confirmatory analysis of drinking water 

and aqueous/liquid phase samples. 

Description of Method: The sample containing Tc-99 is mixed with Technetium-95m (Tc-95m) added 

as a gamma-emitting tracer. The two isotopes of technetium are brought to an isotopic equilibrium and 

separated from other elements by ferrous and ferric hydroxide coprecipitation. The precipitate is dissolved 

with dilute nitric acid and passed through a commercially available resin column (TEVA® Resin) which is 

highly specific for technetium in the pertechnatate form. The resin is washed with dilute nitric acid to 

remove possible interferences and then it is extruded directly into a suitable liquid scintillation cocktail. 

The sample is typically counted for 1 hour to simultaneously determine Tc-99 activity and the Tc-95m 

radiochemical yield. Quench/efficiency calibration curves need to be established for the liquid 

scintillation spectrometer for both Tc-95m and Tc-99. 

Source: EML, DOE (EML is currently part of the DHS). 1997. “HASL-300 Method Tc-02-RC: 

Technetium-99 in Water – TEVA® Resin.” EML Procedures Manual, HASL-300, 28th Edition. 

http://www.epa.gov/sam/pdfs/EML-Tc-02-RC.pdf 

6.2.18 FRMAC Method Volume 2, Page 33: Gross Alpha and Beta in Air 

Analysis Purpose: Gross alpha and gross beta determination
 

Determinative Technique: Alpha/Beta counting 

Method Developed for: Gross alpha and beta in air
 

Method Selected for: SAM lists this method for gross alpha and gross beta determination in air filters, 

and for direct counting of surface wipes. 

 SAM Revision 5.0 133 September 29, 2009 
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Description of Method: A thin-window gas-flow proportional counter is used for counting gross alpha 

and beta radioactivity. The method supplies an approximation of the alpha and beta activity present in the 

air or the removable surface activity dependent on the sample type. The method provides an indication of 

the presence of alpha and beta emitters, including the following SAM analytes: 

• Americium-241 (CAS RN 14596-10-2) Alpha emitter 

• Californium-252 (CAS RN 13981-17-4) Alpha emitter 

• Cesium-137 (CAS RN 10045-97-3) Beta emitter 

• Cobalt-60 (CAS RN 10198-40-0) Beta emitter 

• Curium-244 (CAS RN 13981-15-2) Alpha emitter 

• Europium-154 (CAS RN 15585-10-1) Beta emitter 

• Iridium-192 (CAS RN 14694-69-0) Beta emitter 

• Plutonium-238 (CAS RN 13981-16-3) Alpha emitter 

• Plutonium-239 (CAS RN 15117-48-3) Alpha emitter 

• Polonium-210 (CAS RN 13981-52-7) Alpha emitter 

• Radium-226 (CAS RN 13982-63-3) Alpha emitter 

• Ruthenium-103 (CAS RN 13968-53-1) Beta emitter 

• Ruthenium-106 (CAS RN 13967-48-1) Beta emitter 

• Strontium-90 (CAS RN 10098-97-2) Beta emitter 

• Uranium-234 (CAS RN 13966-29-5) Alpha emitter 

• Uranium-235 (CAS RN 15117-96-1) Alpha emitter 

• Uranium-238 (CAS RN 7440-16-1) Alpha emitter 

For this application, the procedure requires the use of thorium-230 for alpha counting efficiency and 

cesium-137 for beta counting efficiency in the calibration of the detector. An air filter or swipe sample is 

placed onto a planchet then counted for alpha and beta radioactivity. Activity is reported in activity units 

per volume of air sampled, as units of activity per surface area sampled, or as total units of activity in 

cases where sample collection information is not available. 

Source: FRMAC. 1998. “Gross Alpha and Beta in Air.” FRMAC Monitoring and Analysis Manual – 

Sample Preparation and Analysis - Volume 2, DOE/NV/11718-181 Vol. 2, UC-707, p. 33. 

http://www.epa.gov/sam/pdfs/FRMAC-Vol2-pg33.pdf 

6.2.19 ORISE Method AP1: Gross Alpha and Beta for Various Matrices 

Analysis Purpose: Gross alpha and gross beta determination 

Determinative Technique: Alpha/Beta counting 

Method Developed for: Gross alpha and beta in water, soil, vegetation, and other solids 

Method Selected for: SAM lists this method for gross alpha and gross beta determination in 

soil/sediment samples. 

Description of Method: This method provides an indication of the presence of alpha and beta emitters, 

including the following SAM analytes: 

• Americium-241 (CAS RN 14596-10-2) Alpha emitter 

• Californium-252 (CAS RN 13981-17-4) Alpha emitter 

• Cesium-137 (CAS RN 10045-97-3) Beta emitter 

• Cobalt-60 (CAS RN 10198-40-0) Beta emitter 

• Curium-244 (CAS RN 13981-15-2) Alpha emitter 

• Europium-154 (CAS RN 15585-10-1) Beta emitter 

• Iridium-192 (CAS RN 14694-69-0) Beta emitter 

• Plutonium-238 (CAS RN 13981-16-3) Alpha emitter 

• Plutonium-239 (CAS RN 15117-48-3) Alpha emitter 

 SAM Revision 5.0 134 September 29, 2009 
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• Polonium-210 (CAS RN 13981-52-7) Alpha emitter 

• Radium-226 (CAS RN 13982-63-3) Alpha emitter 

• Ruthenium-103 (CAS RN 13968-53-1) Beta emitter 

• Ruthenium-106 (CAS RN 13967-48-1) Beta emitter 

• Strontium-90 (CAS RN 10098-97-2) Beta emitter 

• Uranium-234 (CAS RN 13966-29-5) Alpha emitter 

• Uranium-235 (CAS RN 15117-96-1) Alpha emitter 

• Uranium-238 (CAS RN 7440-16-1) Alpha emitter 

This procedure provides screening measurements to indicate whether specific chemical analyses are 

required for water, soil, vegetation, and other solids. Liquid samples are acidified, concentrated, dried in 

a planchet, and counted in a low-background proportional counter. Solid samples are dried and processed 

to provide homogeneity, and a known quantity is transferred to a planchet and counted in a lowbackground proportional counter. 

Special Considerations: Volatile radionuclides will not be accurately determined using this procedure. 

Source: ORISE, Oak Ridge Associated Universities (ORAU). 2001. “Method AP1: Gross Alpha and 

Beta for Various Matrices.” Laboratory Procedures Manual for the Environmental Survey and Site 

Assessment Program. http://www.epa.gov/sam/pdfs/ORISE-AP1.pdf 

6.2.20 ORISE Method AP2: Determination of Tritium 

Analyte(s) CAS RN

Tritium (Hydrogen-3) 10028-17-8 

Analysis Purpose: Qualitative and confirmatory analysis 

Determinative Technique: Liquid scintillation 

Method Developed for: Tritium in soil, sediment, animal tissue, vegetation, smears, and water samples 

Method Selected for: SAM lists this method for qualitative and confirmatory analysis of soil/sediment 

and surface wipe samples. 

Description of Method: The tritium in aqueous and solid samples is distilled using an Allihn condenser. 

For solid samples, an appropriate volume of water is added to facilitate distillation. Certain solid samples 

may be refluxed to ensure distribution of any tritium that may be in the sample. The sample may be 

spiked with a standard tritium solution to evaluate quenching and counting efficiency. After the sample 

has been distilled, an aliquot of the distillate is added to a scintillation cocktail and the sample is counted 

using a liquid scintillation analyzer. 

Special Considerations: Other volatile radionuclides such as iodine and carbon isotopes may interfere 

and may require that the sample be made alkaline using solid sodium hydroxide before distillation. 

Organic impurities may interfere and may require the addition of an oxidizing agent to the sample as well 

as spiking the samples with a standard tritium solution. The addition of a standard tritium solution to 

each sample allows for counting efficiencies to be calculated for each individual sample. 

Source: ORISE, ORAU. 2001. “Method AP2: Determination of Tritium.” Laboratory Procedures 

Manual for the Environmental Survey and Site Assessment Program. 

http://www.epa.gov/sam/pdfs/ORISE-AP2.pdf 

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6.2.21 ORISE Method AP5: Determination of Technetium-99 

Analyte(s) CAS RN

Technetium-99 14133-76-7

Analysis Purpose: Qualitative and confirmatory analysis 

Determinative Technique: Liquid scintillation 

Method Developed for: Technetium-99 in sediment, soil, smears, and water at environmental levels 

Method Selected for: SAM lists this method for qualitative and confirmatory analysis of soil/sediment, 

surface wipe, and air filter samples. 

Description of Method: Solid samples are leached with dilute nitric acid. The leachates are passed 

through a commercially available resin column (TEVA® resin) which is highly specific for technetium in 

the pertechnatate form. The technetium is absorbed onto the extraction resin. The resin is added to a 

scintillation vial containing an appropriate cocktail and counted using a liquid scintillation analyzer. 

Most interfering beta emitting radionuclides (including C-14, P-32, S-35, Sr-90, Y-90, and Th-234) are 

effectively removed using TEVA® resin under the conditions in this procedure. 

Special Considerations: Tritium may follow technetium due to the absorption of some tritium-labeled 

compounds by the resin. Possible tritium interferences are eliminated by setting the technetium counting 

window above the maximum energy of tritium beta particles. 

Source: ORISE, ORAU. 2001. “Method AP5: Determination of Technetium-99.” Laboratory 

Procedures Manual for the Environmental Survey and Site Assessment Program. 

http://www.epa.gov/sam/pdfs/ORISE-AP5.pdf 

6.2.22 ORISE Method AP11: Sequential Determination of the Actinides in Environmental 

Samples Using Total Sample Dissolution and Extraction Chromatography 

Analyte(s) CAS RN 

Americium-241 14596-10-2 

Californium-252 13981-17-4 

Curium-244 13981-15-2 

Plutonium-238 13981-16-3 

Plutonium-239 15117-48-3 

Uranium-234 13966-29-5 

Uranium-235 15117-96-1 

Uranium-238 7440-61-1 

Analysis Purpose: Qualitative and confirmatory analysis 

Determinative Technique: Alpha spectrometry 

Method Developed for: Americium, curium, plutonium, neptunium, thorium, and/or uranium in water 

and solid samples 

Method Selected for: SAM recommends this method for confirmatory analysis when a sample exists in 

a refractory form (i.e., non-digestible or dissolvable material after normal digestion methods) or if there is 

a matrix interference problem. In the event of refractory radioactive material, SAM recommends this 

 SAM Revision 5.0 136 September 29, 2009 
Section 6 – Selected Radiochemical Methods

method for both qualitative determination and confirmatory analysis of drinking water, aqueous/liquid, 

soil/sediment, surface wipes, and air filter samples. 

Description of Method: Solid and unfiltered aqueous samples are dissolved completely by a 

combination of potassium hydrogen fluoride and pyrosulfate fusions. Filtered aqueous samples are 

evaporated to dryness followed by a pyrosulfate fusion. The fusion cake is dissolved and, for analyses 

requiring uranium only, two barium sulfate precipitations are performed, and the uranium is separated 

using EDTA. For all other analyses, one barium sulfate precipitation is performed and all alpha emitters 

are coprecipitated on barium sulfate. The barium sulfate is dissolved and the actinides are separated by 

extraction chromatography. An optional section is presented for the separation of americium from the 

lanthanides. All actinides are coprecipitated on cerium fluoride and counted with an alpha spectrometer 

system. 

Source: ORISE, ORAU. 2001. “Method AP11: Sequential Determination of the Actinides in 

Environmental Samples Using Total Sample Dissolution and Extraction Chromatography.” Laboratory 

Procedures Manual for the Environmental Survey and Site Assessment Program. 

http://www.epa.gov/sam/pdfs/ORISE-AP11.pdf 

6.2.23 ORISE Method Procedure #9: Determination of I-125 in Environmental Samples 

Analyte(s) CAS RN

Iodine-125 14158-31-7

Analysis Purpose: Qualitative and confirmatory analysis 

Determinative Technique: Gamma spectrometry
 

Method Developed for: Iodine-125 in environmental samples, such as soil, sediment, vegetation, water, 

milk, filters (air or water), etc. 

Method Selected for: SAM lists this method for qualitative and confirmatory analysis of drinking water, 

aqueous/liquid, soil/sediment, surface wipe, and air filter samples. 

Description of Method: In this method a direct comparison is made between the sample and a source 

prepared from a National Institute of Standards and Technology (NIST) traceable standard. If it is 

known, either by the sample preparation procedure or by a qualitative analysis on some device (high 

resolution intrinsic planar detector) that I-125 is the only radionuclide contributing to the observed peak, 

then this method can be used as a rapid quantitative method. 

The sample is prepared by matrix specific techniques and the final sample is placed in a 16 millimeter 

culture tube and counted in a 3” x 3” thin window sodium iodide (NaI) well detector attached to a pulse 

height analyzer. I-125 gamma counting rate is determined in the 25 to 35 keV energy range by pulse 

height analysis. NIST traceable liquid standards are also counted in the same geometric configuration as 

the samples to determine I-125 counting efficiency. 

Special Considerations: Due to the low photon energy of I-125, the Compton scattering and x-ray 

photons from other radionuclides may cause significant interferences in this procedure. 

Source: ORISE, ORAU. 1995. “Procedure #9: Determination of I-125 in Environmental Samples.”
 

Laboratory Procedures Manual for the Environmental Survey and Site Assessment Program. 

http://www.epa.gov/sam/pdfs/ORISE-Procedure9-1995.pdf
 

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Section 6 – Selected Radiochemical Methods

6.2.24 ASTM Method D3084-05: Standard Practice for Alpha Spectrometry in Water 

Analyte(s) CAS RN 

Americium-241 14596-10-2 

Californium-252 13981-17-4 

Curium-244 13981-15-2 

Plutonium-238 13981-16-3 

Plutonium-239 15117-48-3 

Radium-226 13982-63-3 

Uranium-234 13966-29-5 

Uranium-235 15117-96-1 

Uranium-238 7440-61-1 

Analysis Purpose: Qualitative determination 

Determinative Technique: Alpha spectrometry
 

Method Developed for: Alpha particle spectra in water 

Method Selected for: SAM lists this method for qualitative determination in drinking water, 

aqueous/liquid, soil and sediment, surface wipes, and/or air filter samples. 

Description of Method: This standard practice covers the process that is required to obtain wellresolved alpha spectra from water samples and discusses the associated problems. This practice is 

typically preceded with specific chemical separations and mounting techniques that are included in 

referenced methods. A chemical procedure is required to isolate and purify the radionuclides (see ASTM 

Methods D3865, Standard Test Method for Plutonium in Water and D3972, Standard Test Method for 

Isotopic Uranium in Water by Radiochemistry), and a radioactive tracer is added to determine yield. A 

source is prepared by employing electrodeposition, microprecipitation, or evaporation (depositing the 

solution onto a stainless steel or platinum disc). Electrodeposition and microprecipitation are preferred. 

The source’s radioactivity is then measured in an alpha spectrometer according to manufacturer’s
 

operating instructions. The counting period chosen depends on the sensitivity required of the 

measurement and the degree of uncertainty in the result that is acceptable. 

Special Considerations: If it is suspected that the sample exists in refractory form (i.e., non-digestible 

or dissolvable material after normal digestion methods) or if there is a matrix interference problem, use 

ORISE Method AP11 for sample preparation instead of the methods referenced in ASTM Method D3084.
 

Source: ASTM. 2005. “Method D3084-05: Standard Practice for Alpha Spectrometry in Water.” Annual 

Book of ASTM Standards, Vol. 11.02. http://www.astm.org/Standards/D3084.htm
 

6.2.25 ASTM Method D3972-02: Standard Test Method for Isotopic Uranium in Water by 

Radiochemistry 

Analyte(s) CAS RN 

Uranium-234 13966-29-5 

Uranium-235 15117-96-1 

Uranium-238 7440-61-1 

 SAM Revision 5.0 138 September 29, 2009 
Section 6 – Selected Radiochemical Methods

Analysis Purpose: Confirmatory analysis 

Determinative Technique: Alpha spectrometry 

Method Developed for: Alpha-particle-emitting isotopes of uranium in water
 

Method Selected for: SAM lists this method for confirmatory analysis of drinking water samples. 

Description of Method: Uranium is chemically separated from a water sample by coprecipitation with 

ferrous hydroxide followed by anion exchange, and electrodeposition. When suspended matter is present, 

an acid dissolution step is added to ensure that all of the uranium dissolves. The sample is acidified, and 

uranium-232 is added as an isotopic tracer to determine chemical yield. Uranium is coprecipitated from
 

the sample with ferrous hydroxide. This precipitate is dissolved in concentrated hydrochloric acid, or is 

subjected to acid dissolution with concentrated nitric and hydrofluoric acids, if the hydrochloric acid fails 

to dissolve the precipitate. Uranium is separated from other radionuclides by adsorption on anion 

exchange resin, followed by elution with hydrochloric acid. The uranium is finally electrodeposited onto
 

a stainless steel disc and counted using alpha spectrometry.
 

Special Considerations: If it is suspected that the sample exists in refractory form (i.e., non-digestible 

or dissolvable material after normal digestion methods) or if there is a matrix interference problem, use 

ORISE Method AP11. 

Source: ASTM. 2002. “Method D3972-02: Standard Test Method for Isotopic Uranium in Water by
 

Radiochemistry.” Annual Book of ASTM Standards, Vol. 11.02.
 

http://www.astm.org/DATABASE.CART/HISTORICAL/D3972-02.htm
 

6.2.26 Standard Method 7110 B: Gross Alpha and Gross Beta Radioactivity (Total, 

Suspended, and Dissolved) 

Analysis Purpose: Gross alpha and gross beta determination 

Determinative Technique: Alpha/Beta counting 

Method Developed for: Gross alpha and gross beta activity in water 

Method Selected for: SAM lists this method for gross alpha and gross beta determination in 

aqueous/liquid samples. 

Description of Method: This method allows for measurement of gross alpha and gross beta radiation in 

water samples. The method provides an indication of the presence of alpha and beta emitters, including 

the following SAM analytes: 

• Americium-241 (CAS RN 14596-10-2) Alpha emitter 

• Californium-252 (CAS RN 13981-17-4) Alpha emitter 

• Cesium-137 (CAS RN 10045-97-3) Beta emitter 

• Cobalt-60 (CAS RN 10198-40-0) Beta emitter 

• Curium-244 (CAS RN 13981-15-2) Alpha emitter 

• Europium-154 (CAS RN 15585-10-1) Beta emitter 

• Iridium-192 (CAS RN 14694-69-0) Beta emitter 

• Plutonium-238 (CAS RN 13981-16-3) Alpha emitter 

• Plutonium-239 (CAS RN 15117-48-3) Alpha emitter 

• Polonium-210 (CAS RN 13981-52-7) Alpha emitter 

• Radium-226 (CAS RN 13982-63-3) Alpha emitter 

• Ruthenium-103 (CAS RN 13968-53-1) Beta emitter 

• Ruthenium-106 (CAS RN 13967-48-1) Beta emitter 

• Strontium-90 (CAS RN 10098-97-2) Beta emitter 

 SAM Revision 5.0 139 September 29, 2009 
Section 6 – Selected Radiochemical Methods

• Uranium-234 (CAS RN 13966-29-5) Alpha emitter 

• Uranium-235 (CAS RN 15117-96-1) Alpha emitter 

• Uranium-238 (CAS RN 7440-16-1) Alpha emitter 

This method recommends using a thin-window gas-flow proportional counter for counting gross alpha 

and beta radioactivity. An internal proportional or Geiger counter may also be used. An aliquot of 

sample is evaporated to a small volume and transferred to a tared counting pan. The sample residue is 

dried to constant weight, cooled, and reweighed to determine dry residue weight, then counted for alpha 

and beta radioactivity. 

Special Considerations: Ground water samples containing elevated levels of dissolved solids will 

require use of smaller sample volumes. 

Source: APHA, AWWA, and WEF. 2005. “Method 7110 B: Gross Alpha and Gross Beta Radioactivity 

(Total, Suspended, and Dissolved).” Standard Methods for the Examination of Water and Wastewater. 

21st Edition. http://www.standardmethods.org/ 

6.2.27 Standard Method 7120: Gamma-Emitting Radionuclides 

Analyte(s) CAS RN 

Cesium-137 10045-97-3 

Cobalt-60 10198-40-0 

Europium-154 15585-10-1 

Iridium-192 14694-69-0 

Ruthenium-103 13968-53-1 

Ruthenium-106 13967-48-1 

Selenium-75 14265-71-5 

Analysis Purpose: Qualitative and confirmatory determination 

Determinative Technique: Gamma spectrometry 

Method Developed for: Gamma emitting radionuclides in water 

Method Selected for: SAM lists this method for qualitative and confirmatory analysis of select gamma 

emitters in aqueous/liquid samples. 

Description of Method: The method uses gamma spectroscopy using either Ge detectors or NaI(Tl) 

crystals for the measurement of gamma photons emitted from radionuclides present in water. The method 

can be used for qualitative and confirmatory determinations with Ge detectors or semi-qualitative and 

semi-quantitative determinations (using NaI(Tl) detectors). Exact confirmation using NaI is possible for 

single nuclides or when the gamma emissions are limited to a few well-separated energies. A 

homogeneous water sample is placed into a standard geometry (normally a Marinelli beaker) for gamma 

counting. Sample portions are counted long enough to meet the required sensitivity of measurement. A 

standard containing a mixture of gamma energies from approximately 100 to 2000 keV is used for energy 

calibration. 

Source: APHA, AWWA, and WEF. 2005. “Method 7120: Gamma-Emitting Radionuclides.” Standard 

Methods for the Examination of Water and Wastewater. 21st Edition. http://www.standardmethods.org/ 

 SAM Revision 5.0 140 September 29, 2009 
Section 6 – Selected Radiochemical Methods

6.2.28 Standard Method 7500-Ra B: Radium: Precipitation Method 

Analyte(s) CAS RN 

Radium-226 13982-63-3

Analysis Purpose: Qualitative determination 

Determinative Technique: Alpha counting 

Method Developed for: Alpha-emitting isotopes of radium in water 

Method Selected for: SAM lists this method for qualitative determination in aqueous/liquid samples. 

Description of Method: This method is for determination of all alpha-emitting radium isotopes by alpha 

decay analysis. Lead and barium carriers are added to the sample containing alkaline citrate, then sulfuric 

acid is added to precipitate radium, barium, and lead as sulfates. The precipitate is purified by washing 

with nitric acid, dissolving in alkaline EDTA, and re-precipitating as radium-barium sulfate after pH 

adjustment to 4.5. This slightly acidic EDTA keeps other naturally occurring alpha-emitters and the lead 

carrier in solution. Radium-223, -224, and -226 are identified by the rate of ingrowth of their daughter 

products in barium sulfate precipitate. The results are corrected by the rate of ingrowth of daughter 

products to determine radium activity. This method involves alpha counting by a gas-flow internal 

proportional counter, scintillation counter, or thin end-window gas-flow proportional counter.
 

Source: APHA, AWWA, and WEF. 2005. “Method 7500-Ra B: Radium: Precipitation Method.”
 

Standard Methods for the Examination of Water and Wastewater. 21st Edition.
 

http://www.standardmethods.org/ 

6.2.29 Standard Method 7500-Ra C: Radium: Emanation Method 

Analyte(s) CAS RN 

Radium-226 13982-63-3

Analysis Purpose: Confirmatory determination 

Determinative Technique: Alpha counting 

Method Developed for: Soluble, suspended, and total radium-226 in water
 

Method Selected for: SAM lists this method for confirmatory analysis of aqueous/liquid samples. 

Description of Method: Radium in water is concentrated and separated from sample solids by
 

coprecipitation with a relatively large amount of barium as the sulfate. The precipitate is treated to 

remove silicates, if present, and to decompose insoluble radium compounds, fumed with phosphoric acid 

to remove sulfite, and dissolved in hydrochloric acid. The completely dissolved radium is placed in a 

bubbler, which is then closed and stored for a period of several days to 4 weeks for ingrowth of radon. 

The bubbler is connected to an evacuation system and the radon gas is removed from the liquid by
 

aeration and helium, dried with a desiccant, and collected in a counting cell. Four hours after radon 

collection, the cell is counted. The activity of the radon is equal to the radium concentration. The 

minimum detectable concentration depends on counter characteristics, background-counting rate of 

scintillation cell, cell efficiency, length of counting period, and contamination of apparatus and 

environment by radium-226. Without reagent purification, the overall reagent blank (excluding
 

background) should be between 0.03 and 0.05 pCi radium-226, which may be considered the minimum 

detectable amount under routine conditions.
 

 SAM Revision 5.0 141 September 29, 2009 
Section 6 – Selected Radiochemical Methods

Source: APHA, AWWA, and WEF. 2005. “Method 7500-Ra C: Radium: Emanation Method.” Standard 

Methods for the Examination of Water and Wastewater. 21st Edition. http://www.standardmethods.org/ 

6.2.30 Standard Method 7500-Sr B: Total Radioactive Strontium and Strontium-90: 

Precipitation Method 

Analyte(s) CAS RN 

Strontium-90 10098-97-2

Analysis Purpose: Qualitative and confirmatory analysis 

Determinative Technique: Beta counting 

Method Developed for: Strontium-90 or total radioactive strontium in drinking water or filtered raw 

water 

Method Selected for: SAM lists this method for qualitative and confirmatory analysis of drinking water 

and aqueous/liquid samples. 

Description of Method: A known amount of inactive strontium ions, in the form of strontium nitrate, is 

added as a “carrier.” The carrier, alkaline earths, and rare earths are precipitated as the carbonate to 

concentrate the radiostrontium. The carrier, along with the radionuclides of strontium, is separated from 

other radioactive elements and inactive sample solids by precipitation as strontium nitrate using fuming 

nitric acid solution. The carrier and radionuclides of strontium are precipitated as strontium carbonate, 

which is dried, weighed to determine recovery of carrier, and measured for radioactivity. The activity of 

the final precipitate is due to radioactive strontium only, because all other radioactive elements have been 

removed. Because it is impossible to separate the isotopes of strontium-89 and strontium-90 by any 

chemical procedure, the amount of strontium-90 is determined by separating and measuring the activity of 

yttrium-90, its daughter product. This method involves beta counting by a gas-flow internal proportional 

counter or thin end-window low-background proportional counter. A correction is applied to compensate 

for loss of carriers and activity during the various purification steps. 

Source: APHA, AWWA, and WEF. 2005. “Method 7500-Sr B: Total Radioactive Strontium and 

Strontium-90: Precipitation Method.” Standard Methods for the Examination of Water and Wastewater. 

21st Edition. http://www.standardmethods.org/ 

6.2.31 Standard Method 7500-U B: Uranium: Radiochemical Method 

Analyte(s) CAS RN 

Uranium-234 13966-29-5 

Uranium-235 15117-96-1 

Uranium-238 7440-61-1 

Analysis Purpose: Qualitative determination 

Determinative Technique: Alpha counting 

Method Developed for: Total uranium alpha activity in water 

Method Selected for: SAM lists this method for qualitative determination in aqueous/liquid samples. 

Description of Method: The sample is acidified with hydrochloric or nitric acid and boiled to eliminate 

carbonate and bicarbonate ions. Uranium is coprecipitated with ferric hydroxide and subsequently
 

separated. The ferric hydroxide is dissolved, passed through an anion-exchange column, and washed with 

 SAM Revision 5.0 142 September 29, 2009 
Section 6 – Selected Radiochemical Methods

acid, and the uranium is eluted with dilute hydrochloric acid. The acid eluate is evaporated to near 

dryness, the residual salt is converted to nitrate, and the alpha activity is counted by a gas-flow 

proportional counter or alpha scintillation counter. 

Special Considerations: If it is suspected that the sample exists in refractory form (i.e., non-digestible 

or dissolvable material after normal digestion methods) or if there is a matrix interference problem, use 

ORISE Method AP11. 

Source: APHA, AWWA, and WEF. 2005. “Method 7500-U B: Uranium: Radiochemical Method.” 

Standard Methods for the Examination of Water and Wastewater. 21st Edition. 

http://www.standardmethods.org/ 

6.2.32 Standard Method 7500-U C: Uranium: Isotopic Method 

Analyte(s) CAS RN 

Uranium-234 13966-29-5 

Uranium-235 15117-96-1 

Uranium-238 7440-61-1 

Analysis Purpose: Confirmatory determination 

Determinative Technique: Alpha spectrometry 

Method Developed for: Isotopic content of the uranium alpha activity; determining the differences 

among naturally occurring, depleted, and enriched uranium in water 

Method Selected for: SAM lists this method for confirmatory analysis of aqueous/liquid samples. 

Description of Method: This method is a radiochemical procedure for determination of the isotopic 

content of uranium alpha activity. The sample is acidified with hydrochloric or nitric acid and uranium232 is added as an isotopic tracer. Uranium is coprecipitated with ferric hydroxide and subsequently 

separated from the sample. The ferric hydroxide precipitate is dissolved and the solution passed through 

an anion-exchange column. The uranium is eluted with dilute hydrochloric acid. The acid eluate is 

evaporated to near dryness, and the residual salt is converted to nitrate and electrodeposited onto a 

stainless steel disc and counted by alpha spectrometry. 

Special Considerations: If it is suspected that the sample exists in refractory form (i.e., non-digestible 

or dissolvable material after normal digestion methods) or if there is a matrix interference problem, use 

ORISE Method AP11. 

Source: APHA, AWWA, and WEF. 2005. “Method 7500-U C: Uranium: Isotopic Method.” Standard 

Methods for the Examination of Water and Wastewater. 21st Edition. http://www.standardmethods.org/ 

 SAM Revision 5.0 143 September 29, 2009 
Section 6 – Selected Radiochemical Methods

 SAM Revision 5.0 144 September 29, 2009 
Section 7– Selected Pathogen Methods 

Section 7.0: Selected Pathogen Methods 

A list of the most appropriate methods currently available for use in analyzing environmental samples for 

pathogens is provided in Appendix C. This list represents an initial effort towards the goal of providing 

standardized analytical procedures. These methods should be used to support remediation activities (site 

assessment through clearance) following a homeland security event. The purpose of this section is to 

provide summary information regarding the procedures listed. Methods are listed for each pathogen that 

may need to be measured and analyzed following an event. Appendix C is sorted alphabetically within 

pathogen categories (i.e., bacteria, viruses, protozoa, and helminths). 

Protocols from peer-reviewed journal articles are listed where standardized methods for pathogens are not 

currently available. Future steps include the development and validation of standardized methods. The 

literature references will be replaced as standardized, validated protocols become available. 

Pathogens that are categorized as Biosafety Level 4 (BSL-4), such as hemorrhagic fever viruses and 

smallpox, will be handled only by reference laboratories with BSL-4 capability and are not included in 

this document. All other pathogens are to be handled using BSL-2 or BSL-3 containment and practices 

(as appropriate) using SAM procedures. If known, the BSL classification for each pathogen is provided 

in the method summaries in Sections 7.2 through 7.5. Pathogens that are considered to be solely of 

agricultural concern (i.e., animal and plant pathogens) are not currently included. Such pathogens may be 

considered for possible inclusion in future document revisions. Following a homeland security event, it is 

assumed that only those areas with contamination greater than pre-existing levels commonly found in the 

environment would be subject to remediation. Dependent on site- and event-specific goals, investigation 

of background levels, using methods listed in Appendix C, should be performed. 

Selection of methods from Appendix C should be based on specific data and information needs, including 

whether there is a need to determine either the presence of a pathogen, the viability of a pathogen, or both. 

In addition to analytical methods, Appendix C lists commercially available spore strips. Commercially 

available spore strips may be used as general indicators that a decontamination process (e.g., fumigation) 

has been successful. Spore strips, however, cannot replace negative-culture results as an indicator of 

decontamination efficacy. Culture-based methods have been selected for many of the pathogens; 

however, due to technical difficulty and time constraints, molecular techniques such as PCR will likely be 

used for viruses. Prior to the start of site decontamination, viability may not be an issue and rapid mass 

screening techniques, such as PCR, may be more appropriate. After decontamination, viability may be 

required to evaluate the efficacy of decontamination procedures; thus, a technique that determines 

viability should be chosen. Methods that combine rapid sample processing and viability determination, 

such as culture confirmed by PCR, should be considered for processing large numbers of samples in a 

timely manner. Viability procedures are listed for each pathogen where available. 

Users of this document should be aware that analysis of environmental samples poses specific problems, 

and it is likely that a single analytical procedure will not be applicable to all sample types. The methods 

listed in this document attempt to address a wide range of environmental samples (e.g., drinking water, 

soil), each with specific physical and biological properties (e.g., pH, inhibitory substances, background 

microorganisms). Within each sample type, a high level of variability may also exist. For example, soils 

sampled in one geographical area may have qualities affecting analysis that are not present in soils from 

other areas. Sample preparatory techniques, such as immunomagnetic separation (IMS), may be useful 

for reducing the impact of these variables. Analyzing representative quantities of environmental samples 

also presents a problem and can result in reduced analytical sensitivity. In these cases, sample 

concentration techniques, such as membrane filtration (MF) and ultrafiltration (UF), may be useful. In 

addition to removing background microorganisms and inhibitory substances, IMS may also be useful for 

concentrating sample aliquots to ensure that the full sample volume can be assayed. This may increase 

the sensitivity of methods such as PCR. 

 SAM Revision 5.0 145 September 29, 2009 
Section 7– Selected Pathogen Methods 

Appendix C includes the following information: 

•	 Pathogen(s). A specific causative agent (e.g., viruses, bacteria) of disease. 

•	 Viability. Ability to reproduce. 

•	 Analytical technique. An analytical instrument or procedure used to determine the identity, 

quantity, and/or viability of a pathogen. 

•	 Analytical method. The unique identifier or number assigned to an analytical method by the method 

publisher. 

•	 Solid (soil, powder). The recommended method/procedure for the pathogen of interest in solid 

samples such as soil and powders. 

•	 Particulate (swabs, wipes, filters). The recommended method/procedure to measure the pathogen of 

interest in particulate sample collection devices such as swabs, wipes and dust-collecting socks used 

with vacuum collection. 

•	 Liquid/water (filter, grab). The recommended method/procedure for the pathogen of interest in 

liquid or aqueous samples that have been concentrated or grab samples. 

•	 Drinking water (filter, grab). The recommended method/procedure for the pathogen of interest in 

potable water samples that have been concentrated or grab samples. 

•	 Aerosol (growth media, filter, liquid). The recommended method/procedure for the pathogen of 

interest in air sample collectors such as growth media, filters, or liquid. 

Some of the methods in Appendix C include multiple analytical techniques by inference. The analytical 

technique listed in Appendix C is intended to be a description of the predominant technique that is 

required to provide the data quality parameter (viability or detection and identification). This description 

does not preclude the use of other techniques that are within or referenced by the method. For example, a 

viability test listed as “culture” may include immunochemical or PCR based assays for the identification 

of isolates. 

Several of the methods listed in Appendix C also include options such as the potential for use of multiple 

cell culture media for primary isolation, allowance for selection of a defined subset of a larger number of 

biochemical tests for biochemical testing, or use of alternative devices for sample concentration. The 

method may provide guidelines as to which options should be used under particular circumstances, or this 

may be left to the discretion of the laboratory. 

7.1 General Guidelines 

This section provides a general overview of how to identify the appropriate pathogen method(s) for a 

given pathogen as well as recommendations for QC procedures. 

For additional information on the properties of the pathogens listed in Appendix C, TOXNET 

(http://toxnet.nlm.nih.gov/index.html), a cluster of databases on toxicology, hazardous chemicals, and 

related areas maintained by the National Library of Medicine, is an excellent resource. Also informative 

are CDC’s Emergency Preparedness and Response Web site (http://www.bt.cdc.gov/) and the FDA 

Center for Food Safety and Applied Nutrition (CFSAN) “Bad Bug Book” 

(http://www.cfsan.fda.gov/~mow/intro.html). Further research on pathogens is ongoing within EPA. 

Databases to manage this information are currently under development. 

 SAM Revision 5.0	 146 September 29, 2009 
Section 7– Selected Pathogen Methods 

7.1.1 Standard Operating Procedures for Identifying Pathogen Methods 

To determine the appropriate method that is to be used on an environmental sample, locate the pathogen 

in Appendix C: Pathogen Methods under the “Pathogen(s)” column. After locating the pathogen, 

continue across the table and select an analytical technique. After an analytical technique has been 

chosen (e.g., culture, PCR, immunoassay), select the analytical method applicable to the sample type of 

interest (solid, particulate, liquid/drinking water or aerosol). 

Once a method has been identified in Appendix C, the corresponding method summary can be found in 

Sections 7.2.1 through 7.5. Method summaries are listed first by alphabetical order within each pathogen 

subcategory (i.e., bacteria, virus, protozoa, helminths) and then in order of method selection hierarchy 

(see Figure 2-1), starting with EPA methods, followed by methods from other federal agencies, VCSBs, 

and journal articles. Where available, a direct link to the full text of the method is provided with the 

method summary. For additional information regarding sample preparation and analysis procedures 

available through consensus standards organizations, other federal agencies, and journals please use the 

contact information provided in Table 7-1. 

Table 7-1. Sources of Pathogen Methods 

Name Publisher Reference 

NEMI EPA, USGS http://www.nemi.gov 

EPA Microbiology Home Page EPA http://www.epa.gov/microbes/ 

Information Collection Requirements 

Rule (ICR) Microbial Laboratory Manual 

EPA ORD http://www.epa.gov/nerlcwww/icrmi 

cro.pdf 

EPA Manual of Methods for Virology EPA http://www.epa.gov/nerlcwww/abo 

ut.htm 

Environmental Regulations and 

Technology: Control of Pathogens and 

Vector Attraction in Sewage and Sludge 

EPA, National Risk Management 

Research Laboratory (NRMRL) 

http://www.epa.gov/nrmrl/pubs/625 

r92013/625r92013.htm 

CDC Laboratory Assays HHS, CDC eija.trees@cdc.hhs.gov 

USDA / Food Safety and Inspection 

Service (FSIS) Microbiology Laboratory 

Guidebook 

USDA FSIS http://www.fsis.usda.gov/Science/ 

Microbiological_Lab_Guidebook/in 

dex.asp 

Bacteriological Analytical Manual FDA, CFSAN http://www.cfsan.fda.gov/~ebam/b 

am-toc.html 

OSHA Methods OSHA http://www.osha.gov 

NIOSH Methods NIOSH http://www.cdc.gov/niosh/nmam/ 

Standard Methods for the Examination 

of Water and Wastewater, 21st Edition, 

2005* 

APHA, AWWA, and WEF http://www.standardmethods.org 

Annual Book of ASTM Standards* ASTM International http://www.astm.org 

Applied and Environmental Microbiology 

(AEM)* 

American Society for Microbiology 

(ASM) 

http://aem.asm.org/ 

Journal of Clinical Microbiology* ASM http://jcm.asm.org/ 

Clinical Microbiology Procedures 

Handbook, 2nd Edition, 2004* 

ASM http://estore.asm.org/viewItemDeta 

ils.asp?ItemID=323 

Molecular and Cellular Probes* Elsevier http://www.elsevier.com 

 SAM Revision 5.0 147 September 29, 2009 
Section 7– Selected Pathogen Methods 

Name Publisher Reference 

Canadian Journal of Microbiology* NRC Research Press http://pubs.nrc-cnrc.gc.ca/ 

Journal of Medical Virology* Wiley InterScience http://www3.interscience.wiley.com 

/cgi-bin/home 

Journal of Virological Methods* Elsevier http://www.elsevier.com 

Diagnostics Microbiology and Infectious 

Disease 

Elsevier http://www.elsevier.com 

Emerging Infectious Diseases CDC http://www.cdc.gov/ncidod/EID/ 

Journal of Parasitology* American Society of 

Parasitologists 

http://www.bioone.org 

Transactions of the Royal Society of 

Tropical Medicine and Hygiene* 

The Royal Society of Tropical 

Medicine and Hygiene 

http://www.rstmh.org/ 

Diagnostic Procedures in Veterinary 

Bacteriology and Mycology 

Academic Press http://www.pubmedcentral.nih.gov/ 

articlerender.fcgi?artid=1481267 

Sentinel Level Clinical Microbiology 

Laboratory Guidelines 

ASM http://www.asm.org/index.php?opti 

on=com_content&view=article&id= 

6342 

Journal of Applied Microbiology* Blackwell Publishing http://www.blackwellpublishing.co 

m/journal.asp?ref=1364­

5072&site=1 

* Subscription and/or purchase required. ASM does not require a subscription or purchase 6 months after the 

publication date. 

7.1.2 General QC Guidelines for Pathogen Methods 

Generation of analytical data of known and documented quality is a critical factor in the accurate 

assessment of and appropriate response to emergency situations. The generation of data of sufficient 

quality requires that analytical laboratories: (1) have appropriately trained personnel; (2) acquire and 

maintain required supplies, equipment, and reagents; (3) conduct the appropriate QC procedures to ensure 

that all measurement systems are in control and operating properly; (4) properly document all analytical 

results; and (5) properly document analytical QC procedures and corrective actions. 

The level or amount of QC needed depends on the intended purpose of the data generated. Various levels 

of QC may be required if the data are generated for presence/absence determinations versus quantitative 

results. Specific data needs should be identified and QC requirements, based on those needs, applied 

consistently across laboratories when multiple laboratories are used. The individual methods listed, 

sampling and analytical protocols, or contractual statements of work should be consulted to determine if 

additional QC procedures are required. 

Method-specific QC requirements are described in many of the methods cited in this manual and will be 

included in protocols developed to address specific pathogen/sample type combinations of concern. In 

general, analytical QC requirements for pathogen methods include an initial demonstration of 

measurement system capability, as well as the capability of the laboratory and the analyst to perform the 

method with the required precision and accuracy. 

 SAM Revision 5.0 148 September 29, 2009 
Section 7– Selected Pathogen Methods 

Ongoing analysis of control samples to ensure the continued reliability of the analytical results should 

also be performed. At a minimum, the following QC analyses should be conducted on an ongoing basis: 

•	 Media and reagent sterility checks; 

•	 Positive and negative controls; 

•	 Method blanks; 

•	 Reference matrix spikes to evaluate initial and ongoing method/analyst performance, if available; 

•	 Matrix spikes to evaluate method performance in the sample type of interest; 

•	 MSD and/or sample replicates to assess method precision; and 

•	 Instrument calibration checks and temperature controls. 

QC procedures and proper maintenance of ancillary laboratory equipment (e.g., thermometers, 

autoclaves) should be performed as frequently as necessary to ensure the reliability of analytical results. 

Please note: The appropriate point of contact identified in Section 4 should be consulted regarding 

appropriate QA/QC procedures prior to sample analysis. These contacts will consult with the EPA 

OSWER coordinator responsible for laboratory activities during the specific event to ensure QA/QC 

procedures are performed consistently across laboratories. OSWER is planning to develop QA/QC 

guidance for laboratory support. EPA program offices will be responsible for ensuring that the QA/QC 

practices are implemented. 

7.1.3 Safety and Waste Management 

It is imperative that safety precautions be used during collection, processing, and analysis of 

environmental samples. Laboratories should have a documented health and safety plan for handling 

samples that may contain target CBR contaminants, and laboratory staff should be trained in and 

implement the safety procedures included in the plan. Pathogens in samples taken from areas 

contaminated as the result of a homeland security event may be more hazardous than naturally occurring 

pathogens of the same genus and species. The pathogens may have been manufactured, engineered, or 

treated in such a manner as to enhance dispersion or virulence characteristics. These conditions may 

warrant special handling for samples arising from intentional contamination incidents. A laboratory must 

be made aware of these potential circumstances, and should carefully consider implementing additional 

safety measures before agreeing to accept these samples. 

In addition, many of the methods listed in Appendix C and summarized or cited in Section 7.2 contain 

specific requirements, guidelines, or information regarding safety precautions that should be followed 

when handling or processing environmental samples and reagents. BSL-2 is suitable for work involving 

agents that pose moderate hazards to personnel and the environment. BSL-3 is applicable when 

performing manipulations of indigenous or exotic agents that may cause serious or potentially lethal 

disease and also have the potential for aerosol transmission. BSLs are provided in the method summaries 

in Section 7.2 whenever available. It is important to note, however, that some pathogens that are 

normally handled at BSL-2 may require BSL-3 procedures and facilities if large volumes, high 

concentrations, or potential aerosols are expected as a part of the analytical process. For more 

information on BSL practices and procedures, the following references should be consulted: 

•	 Biosafety in Microbiological and Biomedical Laboratories (BMBL), 5th Edition, found at 

http://www.cdc.gov/OD/ohs/biosfty/bmbl5/bmbl5toc.htm 

•	 “Laboratory Security and Emergency Response Guidance for Laboratories Working with Select 

Agents,” Morbidity and Mortality Weekly Report, Vol. 51, No. RR-19, 1–6, December 6, 2002, found 

at http://www.cdc.gov/mmwr/pdf/rr/rr5119.pdf. 

•	 Microbiology Biosafety for Level A Laboratories, found at 

http://www.bt.cdc.gov/documents/PPTResponse/table3bbiosafety.pdf 

 SAM Revision 5.0	 149 September 29, 2009 
Section 7– Selected Pathogen Methods 

•	 Select Agent Rules and Regulations found at the National Select Agent Registry 

(http://www.selectagents.gov/)and http://ecfr.gpoaccess.gov/cgi/t/text/textidx?c=ecfr&tpl=/ecfrbrowse/Title09/9cfr121_main_02.tpl 

The following sources provide information regarding waste management: 

•	 EPA – Hazardous Waste Management (40 CFR parts 260) and EPA Administered Permit Programs 

(40 CFR part 270), found at http://ecfr.gpoaccess.gov/cgi/t/text/textidx?sid=cac9da30cd241fa70d461e4a917eb75e&c=ecfr&tpl=/ecfrbrowse/Title40/40tab_02.tpl 

Other resources that can be consulted for additional information include the following: 

•	 OSHA – Hazardous Waste Operations and Emergency Response (29 CFR part 1910.120) found at 

http://www.osha.gov/pls/oshaweb/owadisp.show_document?p_table=STANDARDS&p_id=9765 

•	 OSHA – Occupational Exposure to Hazardous Chemicals in Laboratories (29 CFR part 1910.1450) 

found at 

http://www.osha.gov/pls/oshaweb/owadisp.show_document?p_table=STANDARDS&p_id=10106 

•	 OSHA – Respiratory Protection (29 CFR part 1910.134) found at 

http://www.osha.gov/pls/oshaweb/owadisp.show_document?p_id=12716&p_table=STANDARDS 

•	 DOT Hazardous Materials Shipment and Packaging (49 CFR parts 171–180) 

http://ecfr.gpoaccess.gov/cgi/t/text/textidx?sid=585c275ee19254ba07625d8c92fe925f&c=ecfr&tpl=/ecfrbrowse/Title49/49cfrv2_02.tpl 

7.1.4 Laboratory Response Network (LRN) 

The LRN was created in accordance with Presidential Directive 39, which established terrorism 

preparedness responsibilities for federal agencies. The LRN is primarily a national network of local, 

state, federal, military, food, agricultural, veterinary, and environmental laboratories; however, additional 

LRN laboratories are located in strategic international locations. The CDC provides technical and 

scientific support to member laboratories as well as secure access to standardized procedures and reagents 

for rapid (within 4–6 hours) presumptive detection of biothreat agents and emerging infectious disease 

agents. These rapid presumptive assays are part of sample type/analyte specific algorithms of assays 

which lead to a confirmed result. The algorithm for a confirmed result is often a combination of one or 

more presumptive positive results from a rapid assay in combination with a positive result from one of the 

“gold standard” methods, such as culture. The standardized procedures, reagents, and agent-specific 

algorithms are considered to be sensitive and are available only to LRN member laboratories. Thus, these 

procedures are not available to the general public and are not discussed in this document. 

It is important to note that, in some cases, the procedures may not be fully developed or validated for each 

environmental sample type/pathogen combination listed in Appendix C, nor are all LRN member 

laboratories necessarily capable of analyzing all of the sample type/pathogen combinations. Additional 

LRN comparable assays (e.g., PCR) also are being developed or acquired that may be used in place of 

LRN assays. Except for Coxiella burnetii, culture methods are available for all of these pathogens as 

Sentinel Laboratory Guidelines 

(http://www.asm.org/index.php?option=com_content&view=article&id=6342). 

The agents identified below and listed in Appendix C are included in the HHS/USDA select agent list and 

should be analyzed in accordance with appropriate regulatory compliance (42 CFR parts 72 and 73, and 9 

CFR part 121) and safety and BSL requirements (see CDC’s BMBL, 5th Edition, 

http://www.cdc.gov/OD/ohs/biosfty/bmbl5/bmbl5toc.htm). 

 SAM Revision 5.0	 150 September 29, 2009 
Section 7– Selected Pathogen Methods 

Pathogen(s) [Disease] Agent Category 

Bacillus anthracis [Anthrax] Bacteria 

Brucella spp. [Brucellosis] Bacteria 

Burkholderia mallei [Glanders] Bacteria 

Burkholderia pseudomallei [Melioidosis] Bacteria 

Coxiella burnetii [Q-fever] Bacteria 

Francisella tularensis [Tularemia] Bacteria 

Yersinia pestis [Plague] Bacteria 

For additional information on the LRN, including selection of a laboratory capable of receiving and 

processing the specified sample type/pathogen, please use the contact information provided below or visit 

http://www.bt.cdc.gov/lrn/. 

Centers for Disease Control and Prevention 

Laboratory Response Branch 

Division of Bioterrorism Preparedness and Response (DBPR) 

National Center for the Prevention, Detection, and Control of Infectious Diseases (NCPDCID) 

Coordinating Center for Infectious Diseases (CCID) 

Centers for Disease Control and Prevention (CDC) 

1600 Clifton Road NE, Mailstop C-18 

Atlanta, GA 30333 

Telephone: (404) 639-2790 

E-mail: lrn@cdc.gov 

Local public health laboratories, private laboratories, and commercial laboratories with questions about 

the LRN should contact their state public health laboratory director or the Association of Public Health 

Laboratories (APHL) (contact information provided below). 

Association of Public Health Laboratories 

8515 Georgia Avenue, Suite 700 

Silver Spring, MD 20910 Telephone: (240) 485-2745 

Fax: (240) 485-2700 

Web site: www.aphl.org 

E-mail: info@aphl.org 

7.2 Method Summaries for Bacteria 

Summaries of the analytical methods for bacteria listed in Appendix C are provided in Sections 7.2.1 

through 7.2.16. Each summary contains a brief description of the method, intended method application, 

performance data (if available), and a link to, or source for, obtaining a full version of the method. 

7.2.1 Bacillus anthracis [Anthrax] – BSL-3 

Method Analytical Technique Section 

LRN Real-time PCR/Immunoassay 7.1.4 

Public Health Reports. 1977. 92(2): 176–186. Culture 7.2.1.1 

 SAM Revision 5.0 151 September 29, 2009 
Section 7– Selected Pathogen Methods 

7.2.1.1 Literature Reference for B. anthracis (Public Health Reports. 1977. 92(2): 

176–186) 

Analysis Purpose: Detection and viability 

Sample Preparation: Solid samples should be prepared according to EPA Method 1680; 

particulate samples should be prepared according to “Evaluation of a Macrofoam Swab Protocol 

for the Recovery of Bacillus anthracis Spores from a Steel Surface,” Hodges, L.R., Rose, L.J., 

Peterson, A., Nobel-Wang, J., and Arduino, M.J. 2006. Applied and Environmental 

Microbiology. 72(6): 4429–4430 (www.epa.gov/sam/pdfs/AEM-72(6)-pgs4429-4430.pdf). 

Analytical Technique: Culture 

Method Developed for: B. anthracis in soil and water samples 

Method Selected for: SAM lists this protocol for detection and viability assessment in solid, 

particulate, aerosol, liquid, and water samples. Further research is needed to develop and 

standardize the procedures for environmental sample types. 

Description of Method: This method describes procedures for analysis of water and solid 

samples and may be adapted for assessment of particulate and aerosol samples. After dilution 

and suspension in phosphate buffer with Tween® 80, soil samples are centrifuged at low speed 

(400 × g) for 5 minutes to remove sediment, followed by a high-speed (6,000 × g) centrifugation 

of the supernatant for 30 minutes to pellet spores. The heat-shocked pellet is plated directly on 

Polymyxin, Lysozyme, EDTA, Thallous acetate (PLET) and sheep blood agar (SBA). Water 

samples are subjected only to the low speed centrifugation, followed by passing the supernatant 

through a 0.45 μm filter. The filter is placed in buffer, heat-shocked, and the supernatant plated 

on PLET and SBA. PLET and SBA plates are examined after 24 and 48 hours incubation at 37°C 

for colonies with typical morphology. Cultures (isolates) that cannot be ruled out as B. anthracis
 

based on the characteristics noted above should be referred to an appropriate reference laboratory
 

for confirmation. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. 

Special Considerations: B. anthracis is a select agent requiring regulatory compliance (42 CFR 

parts 72 and 73, and 9 CFR part 121); appropriate safety and BSL requirements should be 

followed (see CDC’s BMBL, 5th Edition, 

http://www.cdc.gov/OD/ohs/biosfty/bmbl5/bmbl5toc.htm). 

Source: Rees, H.B., Smith, M.A., Spendlove, J.C., Fraser, R.S., Fukushima, T., Barbour, A.G., 

Schoenfeld, F.J. 1977. “Epidemiologic and Laboratory Investigations of Bovine Anthrax in Two 

Utah Counties in 1975.” Public Health Reports. 92(2): 176–186. 

http://www.epa.gov/sam/pdfs/PHR-92(2)-pgs176-186.pdf 

7.2.2 Brucella spp. [Brucellosis] – BSL-3 

Method Analytical Technique Section 

LRN Real-time PCR/Immunoassay 7.1.4 

ASM Sentinel Laboratory Guidelines for Suspected 

Agents of Bioterrorism: Brucella species Culture 7.2.2.1 

 SAM Revision 5.0	 152 September 29, 2009 
Section 7– Selected Pathogen Methods 

7.2.2.1 ASM Sentinel Laboratory Guidelines for Suspected Agents of 

Bioterrorism: Brucella species 

Analysis Purpose: Detection and viability 

Sample Preparation: Solid samples should be prepared according to EPA Method 1680; 

particulate samples should be prepared according to “Evaluation of a Macrofoam Swab Protocol 

for the Recovery of Bacillus anthracis Spores from a Steel Surface,” Hodges, L.R., Rose, L.J., 

Peterson, A., Nobel-Wang, J., and Arduino, M.J. 2006. Applied and Environmental 

Microbiology. 72(6): 4429–4430 (www.epa.gov/sam/pdfs/AEM-72(6)-pgs4429-4430.pdf). 

Analytical Technique: Culture 

Method Developed for: Brucella spp. in clinical samples 

Method Selected for: SAM lists these guidelines for detection and viability assessment in solid, 

particulate, aerosol, liquid, and water samples. Further research is needed to develop and 

standardize the procedures for environmental sample types. 

Description of Method: This method describes procedures for analysis of clinical samples and 

may be adapted for assessment of solid, particulate, and liquid samples. Samples are plated 

directly on chocolate agar (CA) or SBA and incubated at 35°C (5–10% carbon dioxide) for up to 

7 days. Colonies are punctuate (minute), non-pigmented, and non-hemolytic at 48 hours. 

Presumptive identification is made by culture examination, microscopy, and biochemical testing. 

Brucella spp. are small, non-motile, Gram-negative coccobacilli and are catalase-, oxidase-, and 

urease-positive. Cultures (isolates) that cannot be ruled out as Brucella spp. based on the 

characteristics noted above may be referred to an appropriate reference laboratory for 

confirmation. Confirmation is performed using a battery of biochemical tests specific for 

Brucella spp. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. 

Special Considerations: Brucella spp. are select agents requiring regulatory compliance (42 

CFR parts 72 and 73, and 9 CFR part 121); appropriate safety and BSL requirements should also 

be followed (see CDC’s BMBL, 5th Edition, 

http://www.cdc.gov/OD/ohs/biosfty/bmbl5/bmbl5toc.htm). 

Source: ASM. 2004. “Sentinel Laboratory Guidelines for Suspected Agents of Bioterrorism: 

Brucella species.” http://www.epa.gov/sam/pdfs/ASM-Brucella.pdf 

7.2.3 Burkholderia mallei [Glanders] – BSL-3 and Burkholderia pseudomallei 

[Melioidosis] – BSL-3 

Method Analytical Technique Section 

LRN Real-time PCR/Immunoassay 7.1.4 

ASM Sentinel Laboratory Guidelines for Suspected 

Agents of Bioterrorism: Burkholderia mallei and 

Burkholderia pseudomallei 

Culture 7.2.3.1 

7.2.3.1 ASM Sentinel Laboratory Guidelines for Suspected Agents of 

Bioterrorism: Burkholderia mallei and Burkholderia pseudomallei 

Analysis Purpose: Detection and viability 

Sample Preparation: Solid samples should be prepared according to EPA Method 1680; 

particulate samples should be prepared according to “Evaluation of a Macrofoam Swab Protocol 

 SAM Revision 5.0	 153 September 29, 2009 
Section 7– Selected Pathogen Methods 

for the Recovery of Bacillus anthracis Spores from a Steel Surface,” Hodges, L.R., Rose, L.J., 

Peterson, A., Nobel-Wang, J., and Arduino, M.J. 2006. Applied and Environmental 

Microbiology. 72(6): 4429–4430 (www.epa.gov/sam/pdfs/AEM-72(6)-pgs4429-4430.pdf). 

Analytical Technique: Culture 

Method Developed for: B. mallei and B. pseudomallei in clinical samples 

Method Selected for: SAM lists these guidelines for detection and viability assessment in solid, 

particulate, aerosol, liquid, and water samples. Further research is needed to develop and 

standardize the procedures for environmental sample types. 

Description of Method: This method describes procedures for analysis of clinical samples and 

may be adapted for assessment of solid, particulate, and liquid samples. Samples are plated 

directly on SBA and incubated at 35°C–37°C. At 48 hours, B. mallei forms gray, translucent 

colonies and B. pseudomallei forms small, smooth, creamy colonies that gradually change to dry,
 

wrinkled colonies. Presumptive identification is made by culture examination, microscopy, 

motility testing, and biochemical testing. B. mallei are small, non-motile, Gram-negative 

coccobacilli or small rods and are oxidase-variable, catalase-positive, colistin-resistant, indolenegative, nitrate reductase-positive, and arginine dihydrolase-positive. B. pseudomallei are small, 

motile, Gram-negative rods and are oxidase-positive, catalase-positive, colistin-resistant, indolenegative, nitrate reductase-positive, and arginine dihydrolase-positive. Cultures (isolates) that
 

cannot be ruled out as B. mallei or B. pseudomallei based on the characteristics noted above may
 

be referred to an appropriate reference laboratory for confirmation. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. 

Special Considerations: B. mallei and B. pseudomallei are select agents requiring regulatory 

compliance (42 CFR parts 72 and 73, and 9 CFR part 121); appropriate safety and BSL 

requirements should also be followed (see CDC’s BMBL, 5th Edition, 

http://www.cdc.gov/OD/ohs/biosfty/bmbl5/bmbl5toc.htm). 

Source: ASM. 2008. “Sentinel Laboratory Guidelines for Suspected Agents of Bioterrorism: 

Burkholderia mallei and Burkholderia pseudomallei.” http://www.epa.gov/sam/pdfs/ASMBmallei-Bpseudomallei.pdf 

7.2.4 Campylobacter jejuni [Campylobacteriosis] – BSL-2 

Method Analytical Technique Section 

SM 9260 G Culture and Immunoassay 7.2.4.1 

Molecular and Cellular Probes. 2006. 20: 269–279 Real-time PCR 7.2.4.2 

7.2.4.1 Standard Method 9260 G: Campylobacter jejuni 

Analysis Purpose: Detection and viability 

Sample Preparation: Solid samples should be prepared according to EPA Method 1680; 

particulate samples should be prepared according to “Evaluation of a Macrofoam Swab Protocol 

for the Recovery of Bacillus anthracis Spores from a Steel Surface,” Hodges, L.R., Rose, L.J., 

Peterson, A., Nobel-Wang, J., and Arduino, M.J. 2006. Applied and Environmental 

Microbiology. 72(6): 4429–4430 (www.epa.gov/sam/pdfs/AEM-72(6)-pgs4429-4430.pdf). 

Analytical Technique: Culture and immunoassay 

 SAM Revision 5.0 154 September 29, 2009 
Section 7– Selected Pathogen Methods 

Method Developed for: Campylobacter jejuni in water 

Method Selected for: SAM lists this method for detection and viability assessment in solid, 

particulate, liquid, and water samples. Further research is needed to develop and standardize the 

procedures for environmental sample types other than water. 

Description of Method: This method describes procedures for analysis of water samples and 

may be adapted for assessment of solid, particulate, and liquid samples. Water samples (1 to 

several liter volumes) are filtered using a cellulose nitrate membrane filter. Filters are placed on
 

Skirrow’s medium and incubated for 24 hours at 42°C under microaerophilic conditions. 

Alternatively, samples are enriched in Campylobacter broth supplemented with antibiotics and 

lysed horse blood under microaerophilic conditions at 37°C for 4 hours, then at 42°C for 24–48 

hours prior to streaking on Skirrow’s medium. Identification is made by culture examination, 

microscopy, motility testing, and biochemical testing. Biochemical tests include oxidase, 

catalase, nitrite and nitrate reduction, hydrogen sulfide production, and hippurate hydrolysis. 

Confirmation is performed using commercially available rapid serological test kits. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. 

Special Considerations: Skirrow’s and other selective media containing antibiotics 

(trimethoprim, vancomycin, polymixin) may prevent the growth of injured organisms. 

Source: APHA, AWWA, and WEF. 2005. “Method 9260 G: Campylobacter jejuni.” Standard 

Methods for the Examination of Water and Wastewater. 21st Edition. 

http://www.standardmethods.org/ 

7.2.4.2 Literature Reference for Campylobacter jejuni (Molecular and Cellular 

Probes. 2006. 20(5): 269–279) 

Analysis Purpose: Detection, not suitable for viability 

Sample Preparation: Solid samples should be prepared according to “Quantification of Bias 

Related to the Extraction of DNA Directly from Soils,” Frostegard, A., Courtois, S., Ramisse, V., 

Clerc, S., Bernillon, D., Le Gall, F., Jeannin, P., Nesme, X., and Simonet, P. 1999. Applied and 

Environmental Microbiology. 65(12): 5409–5420 (www.epa.gov/sam/pdfs/AEM-65(12)-

pgs5409-5420.pdf). 

Analytical Technique: Real-time PCR 

Method Developed for: Campylobacter jejuni in clinical samples 

Method Selected for: SAM lists these procedures for detection in solid, particulate, aerosol, 

liquid, and water samples. Further research is needed to develop and standardize the procedures 

for environmental sample types. 

Description of Method: Procedures are described for analysis of clinical samples and may be 

adapted for assessment of solid, particulate, aerosol, liquid, and water samples. The method uses 

real-time quantitative PCR (qPCR) for identification of C. jejuni that can be used in conjunction 

with either the ABI Prism® 7700 or 7900 HT Thermal Cyclers. A high through-put method 

using guanidinium thiocyanate (GTC) and glass beads is used for extraction of deoxyribonucleic 

acid (DNA). C. jejuni uses the CJ F primer, CJ R primer, and CJ probe for real-time quantitative 

PCR. Results are evaluated against standard curves made with a 10-fold dilution series of C. 

jejuni national collection of type cultures (NCTC) 11168 DNA in a background of cecum
 

(intestinal) DNA. 

 SAM Revision 5.0	 155 September 29, 2009 
Section 7– Selected Pathogen Methods 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. PCR QC checks should be 

performed according to EPA Draft Quality Assurance/Quality Control Guidance for Laboratories 

Performing PCR Analyses on Environmental Samples document www.epa.gov/sam/pdfs/EPAQA-QC_PCR_Oct2004.pdf or consult the point of contact identified in Section 4. 

Source: Skanseng, B., Kaldhusdal, M., and Rudi, K. 2006. “Comparison of Chicken Gut 

Colonization by the Pathogens Campylobacter jejuni and Clostridium perfringens by the Realtime Quantitative PCR.” Molecular and Cellular Probes. 20(5): 269–279. 

http://www.sciencedirect.com/science/journal/08908508 

7.2.5 Chlamydophila psittaci [Psittacosis] (formerly known as Chlamydia psittaci) – 

BSL-2; BSL-3 for aerosols and large volumes 

Method Analytical Technique Section 

Journal of Clinical Microbiology. 2000. 38(3): 1085– 

1093 Tissue culture and PCR 7.2.5.1 

7.2.5.1 Literature Reference for Chlamydophila psittaci (Journal of Clinical 

Microbiology. 2000. 38(3): 1085–1093) 

Analysis Purpose: Detection and viability 

Sample Preparation: Solid samples should be prepared according to “Quantification of Bias 

Related to the Extraction of DNA Directly from Soils,” Frostegard, A., Courtois, S., Ramisse, V., 

Clerc, S., Bernillon, D., Le Gall, F., Jeannin, P., Nesme, X., and Simonet, P. 1999. Applied and 

Environmental Microbiology. 65(12): 5409–5420 (www.epa.gov/sam/pdfs/AEM-65(12)-

pgs5409-5420.pdf). 

Analytical Technique: Tissue culture and PCR 

Method Developed for: Chlamydophila psittaci in clinical samples 

Method Selected for: SAM lists these procedures for detection and viability assessment in solid, 

particulate, aerosol, liquid, and water samples. Further research is needed to develop and 

standardize the procedures for environmental sample types. 

Description of Method: Procedures are described for analysis of clinical samples and may be 

adapted for assessment of solid, particulate, aerosol, liquid, and water samples. The method uses 

touchdown enzyme time release (TETR)-PCR for detection and identification of Chlamydophila 

psittaci. DNA is extracted from chlamydia cultures (Buffalo green monkey kidney [BGMK]
 

cells), minced clinical tissues, and respiratory samples by mixing with a chelating resin, heating 

first to 56°C for 15–30 minutes, then 100°C for 8–10 minutes. Primer sets specific for C. psittaci
 

are designed based on the DNA sequences of the 16S ribosomal ribonucleic acid (rRNA) and 

16S-23S rRNA genes (CPS 100/101). PCR is conducted on a Perkin-Elmer 480 with products 

separated by electrophoresis in 12% polyacrylamide gels with Tris-borate-EDTA buffer and 

visualized with ethidium bromide. Chlamydophila psittaci samples are to be handled with BSL-3 

containment and practices.
 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. PCR QC checks should be
 

performed according to EPA Draft Quality Assurance/Quality Control Guidance for Laboratories 

Performing PCR Analyses on Environmental Samples document www.epa.gov/sam/pdfs/EPAQA-QC_PCR_Oct2004.pdf or consult the point of contact identified in Section 4.
 

 SAM Revision 5.0	 156 September 29, 2009 
Section 7– Selected Pathogen Methods 

Source: Madico, G., Quinn, T.C., Boman, J., and Gaydos, C.A. 2000. “Touchdown Enzyme 

Time Release-PCR for Detection and Identification of Chlamydia trachomatis, C. pneumoniae, 

and C. psittaci Using the 16S and 16S-23S Spacer rRNA Genes.” Journal of Clinical 

Microbiology. 38(3): 1085–1093. http://www.epa.gov/sam/pdfs/JCM-38(3)-pgs1085-1093.pdf 

7.2.6 Coxiella burnetii [Q-fever] – BSL- 3 

Method Analytical Technique Section 

LRN (Sentinel Laboratory Guideline not available) Culture and Real-time 

PCR/Immunoassay 7.1.4 

7.2.7 Escherichia coli O157:H7 – BSL-2 

Method Analytical Technique Section 

SM 9260 F Culture and Immunoassay 7.2.7.1 

Applied and Environmental Microbiology. 2003. 

69(10): 6327–6333 Real-time PCR 7.2.7.2 

7.2.7.1 Standard Method 9260 F: Pathogenic Escherichia coli 

Analysis Purpose: Detection and viability 

Sample Preparation: Solid samples should be prepared according to EPA Method 1680; 

particulate samples should be prepared according to “Evaluation of a Macrofoam Swab Protocol 

for the Recovery of Bacillus anthracis Spores from a Steel Surface,” Hodges, L.R., Rose, L.J., 

Peterson, A., Nobel-Wang, J., and Arduino, M.J. 2006. Applied and Environmental 

Microbiology. 72(6): 4429–4430 (www.epa.gov/sam/pdfs/AEM-72(6)-pgs4429-4430.pdf). 

Analytical Technique: Culture and immunoassay 

Method Developed for: Escherichia coli O157:H7 in water and apple juice 

Method Selected for: SAM lists this method for detection and viability assessment in solid, 

particulate, liquid, and water samples. Further research is needed to develop and standardize the 

procedures for environmental sample types other than water. 

Description of Method: This method describes procedures for analysis of water and apple juice 

samples and may be adapted for assessment of solid, particulate, and liquid samples. The method 

allows for two options, one being a modification of SM 9221B followed by plating and 

biochemical identification. The second option, modification of a food method, allows for the 

analysis of large sample volumes. A 200-mL water sample is centrifuged; the pellet is 

resuspended in E. coli enrichment broth (EEB) and incubated for 6 hours. Tellurite CefiximeSorbitol MacConkey (TC-SMAC) plates are inoculated with the enriched EEB culture, and 

incubated for up to 24 hours. Colorless colonies on TC-SMAC are tested for indole production. 

Presumptive positive colonies are then subjected to biochemical characterization. Confirmation 

is through serological testing. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. 

Source: APHA, AWWA, and WEF. 2005. “Method 9260 F: Pathogenic Escherichia coli.” 

Standard Methods for the Examination of Water and Wastewater. 21st Edition. 

http://www.standardmethods.org/ 

 SAM Revision 5.0 157 September 29, 2009 
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7.2.7.2 Literature Reference for Escherichia coli O157:H7 (Applied and 

Environmental Microbiology. 2003. 69(10): 6327–6333) 

Analysis Purpose: Detection, not suitable for viability 

Sample Preparation: Solid samples should be prepared according to “Quantification of Bias 

Related to the Extraction of DNA Directly from Soils,” Frostegard, A., Courtois, S., Ramisse, V., 

Clerc, S., Bernillon, D., Le Gall, F., Jeannin, P., Nesme, X., and Simonet, P. 1999. Applied and 

Environmental Microbiology. 65(12): 5409–5420 (www.epa.gov/sam/pdfs/AEM-65(12)-

pgs5409-5420.pdf). 

Analytical Technique: Real-time PCR 

Method Developed for: E. coli O157:H7 in cultures or isolates 

Method Selected for: SAM lists these procedures for detection in solid, particulate, aerosol, 

liquid, and water samples. Further research is needed to develop and standardize the procedures 

for environmental sample types. 

Description of Method: Procedures are described for analysis of isolates and may be adapted 

for assessment of solid, particulate, aerosol, liquid, and water samples. The method uses 

multiplex real-time PCR performed on a Smart Cycler® with fluorogenic probes for 

identification of cultured E. coli O157:H7 isolates. Sample template (0.5 μL) is added to a total 

reaction volume of 25 μL. Primers and probes were designed to target the stx1 and stx2 (Shiga 

toxin) genes and the single-nucleotide polymorphism at position 93 of the uidA (ß-glucoronidase) 

gene, using 6-carboxy-X-rhodamine (ROX), 6-caraboxyfluorescein (FAM), and 6-carboxy4,7,2’,7’-tetrachlorofluorescein (TET) as respective fluorogens. The method was tested with 138 

isolates, including 52 E. coli O157:H7 and two E. coli O157:H7/H-

 organisms. Specificity was 

100% for all three target genes. Sensitivities were 98.6%, 100%, and 100% for stx1, stx2, and 

uidA O157:H7–

 targets, respectively. This assay did not detect two strains of the closely related 

E. coli O55:H7/H–

 serotype. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. PCR quality control checks 

should be performed according to EPA Draft Quality Assurance/Quality Control Guidance for 

Laboratories Performing PCR Analyses on Environmental Samples document, 

www.epa.gov/sam/pdfs/EPA-QA-QC_PCR_Oct2004.pdf, or consult the point of contact 

identified in Section 4. 

Source: Jinneman, K.C., Yoshitomi, K.J., and Weagant, S.D. 2003. “Multiplex Real-Time PCR 

Method to Identify Shiga Toxin Genes stx1 and stx2 and Escherichia coli O157:H7/H– Serotype.” 

Applied and Environmental Microbiology. 69(10): 6327–6333. 

http://www.epa.gov/sam/pdfs/AEM-69(10)-pgs6327-6333.pdf. 

7.2.8 Francisella tularensis [Tularemia] – BSL-3 

Method Analytical Technique Section 

LRN Real-time PCR /Immunoassay 7.1.4 

CDC, ASM, APHL Basic Protocols for Level A 

Laboratories for the Presumptive Identification of 

Francisella tularensis 

Culture 7.2.8.1 

 SAM Revision 5.0	 158 September 29, 2009 
Section 7– Selected Pathogen Methods 

7.2.8.1 CDC, ASM, APHL: Basic Protocols for Level A Laboratories for the 

Presumptive Identification of Francisella tularensis 

Analysis Purpose: Detection and viability 

Sample Preparation: Solid samples should be prepared according to EPA Method 1680; 

particulate samples should be prepared according to “Evaluation of a Macrofoam Swab Protocol 

for the Recovery of Bacillus anthracis Spores from a Steel Surface,” Hodges, L.R., Rose, L.J., 

Peterson, A., Nobel-Wang, J., and Arduino, M.J. 2006. Applied and Environmental 

Microbiology. 72(6): 4429–4430 (www.epa.gov/sam/pdfs/AEM-72(6)-pgs4429-4430.pdf). 

Analytical Technique: Culture 

Method Developed for: F. tularensis in clinical samples 

Method Selected for: SAM lists this protocol for detection and viability assessment in solid, 

particulate, aerosol, liquid, and water samples. Further research is needed to develop and 

standardize the procedures for environmental sample types. 

Description of Method: This method describes procedures for analysis of clinical samples and 

may be adapted for assessment of solid, particulate, and liquid samples. Samples are plated 

directly on a cysteine-supplemented media such as CA, Thayer-Martin (TM) agar, or buffered 

charcoal yeast extract (BCYE) agar. After incubation at 35°C–37°C for at least 48 hours, F. 

tularensis forms small, gray-white to opaque colonies. Presumptive identification is made by
 

culture examination, microscopy, motility testing, and biochemical testing. F. tularensis is a 

minute, pleomorphic, faint-straining, Gram-negative coccobacillus and is weakly catalasepositive, oxidase-negative, ß-lactamase-positive, and urease-negative. Cultures (isolates) that
 

cannot be ruled out as F. tularensis based on the characteristics noted above may be referred to an 

appropriate reference laboratory for confirmation. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. 

Special Considerations: F. tularensis is a select agent requiring regulatory compliance (42 

CFR parts 72 and 73, and 9 CFR part 121); appropriate safety and BSL requirements should also 

be followed (see CDC’s BMBL, 5th Edition, 

http://www.cdc.gov/OD/ohs/biosfty/bmbl5/bmbl5toc.htm). 

Source: CDC, ASM, and APHL. 2001. “Basic Protocols for Level A Laboratories for the 

Presumptive Identification of Francisella tularensis.” http://www.epa.gov/sam/pdfs/CDCFtularemia.pdf 

7.2.9 Leptospira interrogans [Leptospirosis] – BSL-2 

Method Analytical Technique Section 

SM 9260 I Culture and Immunoassay 7.2.9.1 

Diagnostic Microbiology and Infectious Disease. 

2009. 64(3): 247–255 Real-time PCR 7.2.9.2 

7.2.9.1 Standard Method 9260 I: Leptospira 

Analysis Purpose: Detection and viability
 

Sample Preparation: Solid samples should be prepared according to EPA Method 1680; 

particulate samples should be prepared according to “Evaluation of a Macrofoam Swab Protocol 

for the Recovery of Bacillus anthracis Spores from a Steel Surface,” Hodges, L.R., Rose, L.J., 

Peterson, A., Nobel-Wang, J., and Arduino, M.J. 2006. Applied and Environmental 

Microbiology. 72(6): 4429–4430 (www.epa.gov/sam/pdfs/AEM-72(6)-pgs4429-4430.pdf). 

 SAM Revision 5.0	 159 September 29, 2009 
Section 7– Selected Pathogen Methods 

Analytical Technique: Culture and immunoassay 

Method Developed for: Leptospira interrogans in water 

Method Selected for: SAM lists this method for detection and viability assessment in solid, 

particulate, liquid, and water samples. Further research is needed to develop and standardize the 

procedures for environmental sample types other than water. 

Description of Method: This method describes procedures for analysis of water samples and 

may be adapted for assessment of solid, particulate, and liquid samples. Filter samples through a 

0.22-μm filter, retaining the filtrate as inoculum. If the sample is turbid, a succession of filters of 

decreasing pore size may be used prior to the 0.22-μm filter. A tube of Leptospira Medium Base 

(Ellinghausen-McCullough Johnson Harris formulation [EMJH]) supplemented either with 

leptospire antibody free serum or with bovine serum albumin (BSA) and polysorbates, is 

inoculated with the sample and incubated at 30°C for up to 6 weeks. Alternatively, the sample is
 

added directly to the media, incubated overnight, passed through a 0.22-μm membrane filter, and 

incubation continued for up to 6 weeks. Cultures are examined by darkfield microscopy for 

motile leptospires. Confirmation is performed by microscopic agglutination test using reference 

antisera. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. 

Source: APHA, AWWA, and WEF. 2005. “Method 9260 I: Leptospira.” Standard Methods for 

the Examination of Water and Wastewater. 21st Edition. http://www.standardmethods.org/
 

7.2.9.2 Literature Reference for Leptospira spp. (Diagnostic Microbiology and 

Infectious Diseases. 2009. 64(3): 247–255) 

Analysis Purpose: Detection, not suitable for viability 

Sample Preparation: Solid samples should be prepared according to “Quantification of Bias 

Related to the Extraction of DNA Directly from Soils,” Frostegard, A., Courtois, S., Ramisse, V., 

Clerc, S., Bernillon, D., Le Gall, F., Jeannin, P., Nesme, X., and Simonet, P. 1999. Applied and 

Environmental Microbiology. 65(12): 5409–5420 (www.epa.gov/sam/pdfs/AEM-65(12)-

pgs5409-5420.pdf). 

Analytical Technique: Real-time PCR 

Method Developed for: Leptospira spp. in clinical samples 

Method Selected for: SAM lists these procedures for detection in solid, particulate, aerosol, 

liquid, and water samples. Further research is needed to develop and standardize the procedures 

for environmental sample types. 

Description of Method: Procedures are described for analysis of clinical samples and may be 

adapted for assessment of solid, particulate, aerosol, liquid, and water samples. A TaqMan® 

probe for the lipL32 gene, which is present only in pathogenic strains of Leptospira, is used to 

identify pathogenic leptospires and to exclude intermediate and non-pathogenic Leptospira
 

species. The assay correctly identified 32 pathogenic strains and excluded 6 intermediate and 13 

non-pathogenic strains. Clinical samples (blood and urine) were spiked with L. interrogans
 

serovar Icterohaemorrhagiae cultures to provide a final concentration of 1 × 106

 leptospires/mL 

sample. Samples were serially diluted and DNA extracted from 200 μL of the sample using a 

QIAmp DNA blood minikit (QIAGEN). DNA eluted into water was analyzed on both the 

Lightcycler® 1.2 and the ABI 7500, and the protocol was found to be 100% sensitive and specific 

for pathogenic Leptospira spp. The assay was able to detect as low as 1 × 101

 leptospires/mL 

whole blood with a Ct value of 40.55 and down to 1 × 102

 leptospires/mL in plasma with a Ct 

value of 38.88. Using DNA from L. interrogans serovar Icterohaemorrhagiae, the lower limit of 

 SAM Revision 5.0	 160 September 29, 2009 
Section 7– Selected Pathogen Methods 

detection was found to be 20 (Lightcycler® 1.2) and 50 (ABI 7500) genomic equivalents per 

reaction using the concentration at which 95% or more of the replicated reactions yielded a 

positive result. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. PCR QC checks should be 

performed according to EPA Draft Quality Assurance/Quality Control Guidance for Laboratories 

Performing PCR Analyses on Environmental Samples document www.epa.gov/sam/pdfs/EPAQA-QC_PCR_Oct2004.pdf or consult the point of contact identified in Section 4. 

Source: Stoddard, R.A., Gee, J.E., Wilkins, P.P., McCaustland, K., and A.R. Hoffmaster. 2009. 

“Detection of Pathogenic Leptospira spp. Through TaqMan Polymerase Chain Reaction 

Targeting the LipL32 Gene.” Diagnostic Microbiology and Infectious Disease. 64(3): 247–255. 

http://www.sciencedirect.com/science/journal/07328893 

7.2.10 Listeria monocytogenes [Listeriosis] – BSL-2 

Method Analytical Technique Section 

USDA Laboratory Guidebook MLG 8A.03 Real-time PCR 7.2.10.1 

FDA/Bacteriological Analytical Manual Chapter 10, 

2003 Culture and Immunoassay 7.2.10.2 

7.2.10.1 USDA Laboratory Guidebook: “FSIS Procedure for the Use of a Listeria 

monocytogenes Polymerase Chain Reaction (PCR) Screening Test.” MLG 

8A.03. 2007. 

Analysis Purpose: Detection, not suitable for viability 

Sample Preparation: Solid samples should be prepared according to “Quantification of Bias 

Related to the Extraction of DNA Directly from Soils,” Frostegard, A., Courtois, S., Ramisse, V., 

Clerc, S., Bernillon, D., Le Gall, F., Jeannin, P., Nesme, X., and Simonet, P. 1999. Applied and 

Environmental Microbiology. 65(12): 5409–5420 (www.epa.gov/sam/pdfs/AEM-65(12)-

pgs5409-5420.pdf). 

Analytical Technique: Real-time PCR 

Method Developed for: L. monocytogenes in food samples 

Method Selected for: SAM lists these procedures for detection in solid, particulate, aerosol, 

liquid, and water samples. Further research is needed to develop and standardize the procedures 

for environmental sample types. 

Description of Method: Procedures are described for analysis of food samples and may be 

adapted for assessment of solid, particulate, aerosol, liquid, and water samples. The method uses 

real-time PCR for identification of L. monocytogenes. Samples are homogenized in modified 

University of Vermont (UVM) broth and incubated at 30.0°C ± 2.0°C for 8–24 hours. PCR is 

performed with the BAX® System. Additional enrichment may be required at 35.0°C ± 2.0°C 

for 18–24 hours, using morpholinepropanesulfonic acid (MOPS) buffered Listeria enrichment 

broth (BLEB). LODs were determined to be better than 1 colony forming unit (CFU)/g in a 25 g 

meat or poultry sample, approximately 4.5 CFU/g in a 25 g pasteurized liquid whole egg blend
 

sample, and 1.0 × 10-2 CFU/mL in a 500 mL brine sample. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. PCR QC checks should be
 

performed according to EPA Draft Quality Assurance/Quality Control Guidance for Laboratories 

 SAM Revision 5.0	 161 September 29, 2009 
Section 7– Selected Pathogen Methods 

Performing PCR Analyses on Environmental Samples document www.epa.gov/sam/pdfs/EPAQA-QC_PCR_Oct2004.pdf or consult the point of contact identified in Section 4. 

Source: USDA, FSIS. 2007. “FSIS Procedure for the Use of a Listeria monocytogenes 

Polymerase Chain Reaction (PCR) Screening Test.” Laboratory Guidebook MLG 8A.03. 

http://www.epa.gov/sam/pdfs/USDA-MLG-8A.03.pdf 

7.2.10.2 FDA Bacteriological Analytical Manual, Chapter 10, 2003: Listeria 

monocytogenes 

Analysis Purpose: Detection and viability 

Sample Preparation: Solid samples should be prepared according to EPA Method 1680; 

particulate samples should be prepared according to “Evaluation of a Macrofoam Swab Protocol 

for the Recovery of Bacillus anthracis Spores from a Steel Surface,” Hodges, L.R., Rose, L.J., 

Peterson, A., Nobel-Wang, J., and Arduino, M.J. 2006. Applied and Environmental 

Microbiology. 72(6): 4429–4430 (www.epa.gov/sam/pdfs/AEM-72(6)-pgs4429-4430.pdf). 

Analytical Technique: Culture and immunoassay 

Method Developed for: Listeria monocytogenes in food 

Method Selected for: SAM lists this manual for detection and viability assessment in solid, 

particulate, aerosol, liquid, and water samples. Further research is needed to develop and 

standardize the procedures for environmental sample types. 

Description of Method: Procedures are described for analysis of food samples and may be 

adapted for assessment of solid, particulate, aerosol, liquid, and water samples. Prepared samples 

are incubated for 4 hours in BLEB without selective agents. Cycloheximide is added and 

incubation continued. At 24 and 48 hours, BLEB cultures are streaked onto esculin-containing 

selective isolation agar (i.e., Oxford Medium [OXA]) and incubated for an additional 24 to 48
 

hours. An L. monocytogenes-L. ivanovii differential selective agar, such as Biosynth Chromogen 

Medium (BCM® Listeria monocytes Detection System), is streaked at 48 hours. Presumptive 

Listeria colonies are black with a black halo on esculin-containing media and blue on Biosynth 

Chromogen Medium plates. Isolated colonies are streaked onto TrypticaseTM soy agar with yeast 

extract (TSAye), incubated for 24 to 48 hours, and examined for morphological and biochemical 

characteristics. L. monocytogenes is a rod-shaped Gram-positive, motile bacterium. It is 

catalase-positive, ramnose-positive, and mannitol- and xylose-negative. Purified isolates may be 

rapidly identified using commercially available biochemical typing kits. Confirmation is 

performed with commercially available sera. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. 

Source: FDA, CFSAN. 2003. “Chapter 10 – Detection and Enumeration of Listeria 

monocytogenes in Foods.” Bacteriological Analytical Manual Online. 

http://www.epa.gov/sam/pdfs/FDA-BAM-Chap10.pdf
 

7.2.11 Non-typhoidal Salmonella (Not applicable to S. Typhi) [Salmonellosis] – BSL-2 

Method Analytical Technique Section 

EPA Method 1682 Culture and Immunoassay 7.2.11.1 

Journal of Applied Microbiology. 2007. 102(2): 

516–530 Real-time PCR 7.2.11.2 

 SAM Revision 5.0	 162 September 29, 2009 
Section 7– Selected Pathogen Methods 

7.2.11.1 EPA Method 1682: Salmonella spp. 

Analysis Purpose: Detection and viability 

Sample Preparation: Solid samples should be prepared according to EPA Method 1680; 

particulate samples should be prepared according to “Evaluation of a Macrofoam Swab Protocol 

for the Recovery of Bacillus anthracis Spores from a Steel Surface,” Hodges, L.R., Rose, L.J., 

Peterson, A., Nobel-Wang, J., and Arduino, M.J. 2006. Applied and Environmental 

Microbiology. 72(6): 4429–4430 (www.epa.gov/sam/pdfs/AEM-72(6)-pgs4429-4430.pdf). 

Analytical Technique: Culture and immunoassay 

Method Developed for: Non-typhoidal Salmonella in biosolids 

Method Selected for: SAM lists this method for detection and viability assessment in solid, 

particulate, liquid, and water samples. Further research is needed to develop and standardize the 

procedures for environmental sample types other than biosolids. 

Description of Method: This method describes procedures for analysis of solid samples 

(biosolids) and may be adapted for assessment of water, liquid, particulate, and aerosol samples. 

Prepared samples are inoculated into tubes of tryptic soy broth (TSB) and incubated for 24 hours. 

Positive (turbid) tubes are spotted onto plates of modified semisolid Rappaport-Vassiliadis 

(MSRV) medium and incubated at 42°C for 16 to 18 hours. The MSRV medium uses novobiocin 

and malachite green to inhibit non-Salmonella species, while allowing most Salmonella species to 

grow. Presumptive colonies are isolated on xylose lysine deoxycholate (XLD) agar and 

confirmed using lysine iron agar (LIA), triple sugar iron (TSI) agar, and urea broth, followed by
 

serological typing using polyvalent O antisera. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, matrix spike and blank. Ongoing analysis of QC 

samples to ensure reliability of the analytical results should also be performed. 

Special Considerations: This method will not detect Salmonella Typhi. MSRV and the 

elevated incubation temperature (42°C) are inhibitory for S. Typhi.
 

Source: EPA. 2006. “Method 1682: Salmonella in Sewage Sludge (Biosolids) by MSRV 

Medium.” http://www.epa.gov/sam/pdfs/EPA-1682.pdf
 

7.2.11.2 Literature Reference for Non-Typhoidal Salmonella (Journal of Applied 

Microbiology. 2007. 102(2): 516–530) 

Analysis Purpose: Detection, not suitable for viability 

Sample Preparation: Solid samples should be prepared according to “Quantification of Bias 

Related to the Extraction of DNA Directly from Soils,” Frostegard, A., Courtois, S., Ramisse, V., 

Clerc, S., Bernillon, D., Le Gall, F., Jeannin, P., Nesme, X., and Simonet, P. 1999. Applied and 

Environmental Microbiology. 65(12): 5409–5420 (www.epa.gov/sam/pdfs/AEM-65(12)-

pgs5409-5420.pdf). 

Analytical Technique: Real-time PCR 

Method Developed for: Non-typhoidal Salmonella from cultures or isolates 

Method Selected for: SAM lists these procedures for detection in solid, particulate, aerosol, 

liquid, and water samples. Further research is needed to develop and standardize the procedures 

for environmental sample types. 

Description of Method: Procedures are described for analysis of isolates and may be adapted 

for assessment of solid, particulate, aerosol, liquid, and water samples. The method uses realtime PCR for identification of Salmonella spp. PCR templates are prepared from 1.0 mL of 

overnight bacterial cultures propagated overnight in TSB at 37.0°C. Cells are pelleted, washed
 

 SAM Revision 5.0	 163 September 29, 2009 
Section 7– Selected Pathogen Methods 

twice with physiological saline, resuspended in molecular grade water, and boiled for 10 minutes. 

PCR is performed on a SmartCycler® II System using primers and probes designed for the stn 

gene. The protocol has been evaluated against 353 isolates, including 255 S. enterica 

representing 158 serotypes, 14 S. bongori representing 12 serotypes, and 84 non-Salmonella 

representing 56 species from 31 genera. The PCR method had 100% inclusivity, 96.4% 

exclusivity, and a level of detection of 3 CFUs per reaction for cultured Salmonella spp. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. PCR QC checks should be 

performed according to EPA Draft Quality Assurance/Quality Control Guidance for Laboratories 

Performing PCR Analyses on Environmental Samples document www.epa.gov/sam/pdfs/EPAQA-QC_PCR_Oct2004.pdf or consult the point of contact identified in Section 4. 

Source: Moore, M.M., and Feist, M.D. 2007. “Real-time PCR Method for Salmonella spp. 

Targeting the stn Gene.” Journal of Applied Microbiology. 102(2): 516–530. 

http://www3.interscience.wiley.com/journal/118490299/abstract 

7.2.12 Salmonella Typhi [Typhoid fever] – BSL-2; BSL-3 for aerosol release 

Method Analytical Technique Section 

CDC Laboratory Assay: S. Typhi Real-time PCR 7.2.12.1 

SM 9260 B Culture and Immunoassay 7.2.12.2 

7.2.12.1 CDC Laboratory Assay: “Triplex PCR for Detection of S. Typhi Using 

SmartCycler®” 

Analysis Purpose: Detection, not suitable for viability 

Sample Preparation: Solid samples should be prepared according to “Quantification of Bias 

Related to the Extraction of DNA Directly from Soils,” Frostegard, A., Courtois, S., Ramisse, V., 

Clerc, S., Bernillon, D., Le Gall, F., Jeannin, P., Nesme, X., and Simonet, P. 1999. Applied and 

Environmental Microbiology. 65(12): 5409–5420 (www.epa.gov/sam/pdfs/AEM-65(12)-

pgs5409-5420.pdf). 

Analytical Technique: Real-time PCR 

Method Developed for: S. Typhi from cultures or isolates 

Method Selected for: SAM lists these procedures for detection in solid, particulate, aerosol, 

liquid, and water samples. Further research is needed to develop and standardize the procedures 

for environmental sample types. 

Description of Method: Procedures are described for analysis of isolates and may be adapted 

for assessment of solid, particulate, aerosol, liquid, and water samples. The assay uses real-time
 

PCR for identification of S. Typhi. Cell lysate templates are prepared by suspending a portion of 

a colony in 300 μL of distilled water and boiling for 10 minutes. After centrifugation for 2 

minutes, 1 μL of the supernatant is used in the PCR reaction. Alternatively, DNA may be 

purified using a commercially available kit or automated DNA extraction system. PCR is 

performed on a SmartCycler® using primers and probes designed for the Vi capsular gene (viaB), 

the H antigen gene (fliC-d), and the tyvelose epimerase gene (tyv). This assay is also available
 

for the LightCycler® platform as three single target PCRs. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. PCR QC checks should be
 

performed according to EPA Draft Quality Assurance/Quality Control Guidance for Laboratories 

 SAM Revision 5.0	 164 September 29, 2009 
Section 7– Selected Pathogen Methods 

Performing PCR Analyses on Environmental Samples document www.epa.gov/sam/pdfs/EPAQA-QC_PCR_Oct2004.pdf or consult the point of contact identified in Section 4. 

Source: HHS, CDC, Laboratory Assay. “Triplex PCR for Detection of S. Typhi Using 

SmartCycler®.” Contact: Dr. Eija Trees, CDC, email: eija.trees@cdc.hhs.gov. 

7.2.12.2 Standard Method 9260 B: General Qualitative Isolation and Identification 

Procedures for Salmonella 

Analysis Purpose: Detection and viability 

Sample Preparation: Solid samples should be prepared according to EPA Method 1680; 

particulate samples should be prepared according to “Evaluation of a Macrofoam Swab Protocol 

for the Recovery of Bacillus anthracis Spores from a Steel Surface,” Hodges, L.R., Rose, L.J., 

Peterson, A., Nobel-Wang, J., and Arduino, M.J. 2006. Applied and Environmental 

Microbiology. 72(6): 4429–4430 (www.epa.gov/sam/pdfs/AEM-72(6)-pgs4429-4430.pdf). 

Analytical Technique: Culture and immunoassay 

Method Developed for: Salmonella Typhi in water 

Method Selected for: SAM lists this method for detection and viability assessment in solid, 

particulate, liquid, and water samples. Further research is needed to develop and standardize the 

procedures for environmental sample types other than water. 

Description of Method: This method describes procedures for analysis of water samples and 

may be adapted for assessment of solid, particulate, and liquid samples. Concentrated samples 

are enriched in either selenite cystine, selenite-F, or tetrathionate broths and incubated at 35°C to 

37°C for up to 5 days. An aliquot from each turbid tube is streaked onto bismuth sulfite (BS) 

plates and incubated at 35°C to 37°C for 24–48 hours. Presumptive positive colonies are then 

subjected to biochemical characterization. Confirmation is through serological testing using
 

polyvalent O and Vi antiserum. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. 

Special Considerations: This method is not preferred for non-typhoidal Salmonella. 

Source: APHA, AWWA, and WEF. 2005. “Method 9260 B: General Qualitative Isolation and 

Identification Procedures for Salmonella.” Standard Methods for the Examination of Water and 

Wastewater. 21st Edition. http://www.standardmethods.org/ 

7.2.13 Shigella spp. [Shigellosis] – BSL-2 

Method Analytical Technique Section 

CDC Laboratory Assay: Shigella Real-time PCR 7.2.13.1 

SM 9260 E Culture and Immunoassay 7.2.13.2 

7.2.13.1 CDC Laboratory Assay: “Detection of Diarrheagenic Escherichia coli and 

Shigella Using LightCycler®” 

Analysis Purpose: Detection, not suitable for viability 

Sample Preparation: Solid samples should be prepared according to “Quantification of Bias 

Related to the Extraction of DNA Directly from Soils,” Frostegard, A., Courtois, S., Ramisse, V., 

Clerc, S., Bernillon, D., Le Gall, F., Jeannin, P., Nesme, X., and Simonet, P. 1999. Applied and 

 SAM Revision 5.0	 165 September 29, 2009 
Section 7– Selected Pathogen Methods 

Environmental Microbiology. 65(12): 5409–5420 (www.epa.gov/sam/pdfs/AEM-65(12)-

pgs5409-5420.pdf). 

Analytical Technique: Real-time PCR 

Method Developed for: Shigella from cultures or isolates 

Method Selected for: SAM lists these procedures for detection in solid, particulate, aerosol, 

liquid, and water samples. Further research is needed to develop and standardize the procedures 

for environmental sample types. 

Description of Method: Procedures are described for analysis of isolates and may be adapted 

for assessment of solid, particulate, aerosol, liquid, and water samples. The assay uses real-time
 

PCR for identification of Shigella. Cell lysate templates are prepared by suspending a portion of 

a colony in 300 μL of distilled water and boiling for 10 minutes. After centrifugation for 2 

minutes, 1 μL of the supernatant is used in the PCR reaction. Alternatively, DNA may be 

purified using a commercially available kit or automated DNA extraction system. PCR is 

performed on a LightCycler® using primers and probes designed for the ipaH-plasmid, which 

encodes the invasive plasmid antigen H. This gene can be found on both the chromosome and a 

plasmid for Shigella spp. An alternative multiplex PCR is described targeting both ipaH and Stx1
 

(Shiga toxin) genes using TaqMan® chemistry and SmartCycler®.
 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. PCR QC checks should be
 

performed according to EPA Draft Quality Assurance/Quality Control Guidance for Laboratories 

Performing PCR Analyses on Environmental Samples document www.epa.gov/sam/pdfs/EPAQA-QC_PCR_Oct2004.pdf or consult the point of contact identified in Section 4.
 

Source: HHS, CDC, Laboratory Assay. “Detection of Diarrheagenic Escherichia coli and 

Shigella Using LightCycler®.” Contact: Dr. Eija Trees, CDC, email: eija.trees@cdc.hhs.gov. 

7.2.13.2 Standard Method 9260 E: Shigella 

Analysis Purpose: Detection and viability 

Sample Preparation: Solid samples should be prepared according to EPA Method 1680; 

particulate samples should be prepared according to “Evaluation of a Macrofoam Swab Protocol 

for the Recovery of Bacillus anthracis Spores from a Steel Surface,” Hodges, L.R., Rose, L.J., 

Peterson, A., Nobel-Wang, J., and Arduino, M.J. 2006. Applied and Environmental 

Microbiology. 72(6): 4429–4430 (www.epa.gov/sam/pdfs/AEM-72(6)-pgs4429-4430.pdf). 

Analytical Technique: Culture and immunoassay 

Method Developed for: Shigella spp. in water and solids 

Method Selected for: SAM lists this method for detection and viability assessment in solid, 

particulate, liquid, and water samples. Further research is needed to develop and standardize the 

procedures for environmental sample types other than water. 

Description of Method: This method describes procedures for analysis of water samples and 

may be adapted for assessment of solid, particulate, and liquid samples. This method contains 

two options for sample concentration: MF (liquid samples) and centrifugation (liquid and solid 

samples) for analyses. Both options include inoculation of an enrichment medium (Selenite F
 

broth). Isolation of the target pathogen is achieved by plating onto XLD and/or MacConkey agar. 

Biochemical identification consists of inoculating TSI and LIA slants. Confirmation is performed 

by slide agglutination tests using polyvalent antisera.
 

 SAM Revision 5.0 166 September 29, 2009 
Section 7– Selected Pathogen Methods 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. 

Source: APHA, AWWA, and WEF. 2005. “Method 9260 E: Shigella.” Standard Methods for 

the Examination of Water and Wastewater. 21st Edition. http://www.standardmethods.org/ 

7.2.14 Staphylococcus aureus – BSL-2 

Method Analytical Technique Section 

SM 9213 B Culture 7.2.14.1 

7.2.14.1 Standard Method 9213 B: Staphylococcus aureus 

Analysis Purpose: Detection and viability 

Sample Preparation: Solid samples should be prepared according to EPA Method 1680; 

particulate samples should be prepared according to “Evaluation of a Macrofoam Swab Protocol 

for the Recovery of Bacillus anthracis Spores from a Steel Surface,” Hodges, L.R., Rose, L.J., 

Peterson, A., Nobel-Wang, J., and Arduino, M.J. 2006. Applied and Environmental 

Microbiology. 72(6): 4429–4430 (www.epa.gov/sam/pdfs/AEM-72(6)-pgs4429-4430.pdf). 

Analytical Technique: Culture 

Method Developed for: Staphylococcus aureus in water 

Method Selected for: SAM lists this method for detection and viability assessment in solid, 

particulate, aerosol, liquid, and water samples. Further research is needed to develop and 

standardize the procedures for environmental sample types other than water. 

Description of Method: Procedures are described for analysis of water samples and may be 

adapted for assessment of solid, liquid, particulate, and aerosol samples. Prepared samples are 

inoculated into tubes of M-staphylococcus broth and incubated for 24 hours. Positive (turbid)
 

tubes are streaked onto plates of Baird-Parker agar and incubated for 48 hours. Presumptive S. 

aureus colonies are tested for mannitol fermentation by the addition of a drop of bromthymol 

blue, a pH indicator. Isolated colonies are examined for morphological and biochemical 

characteristics. S. aureus is a Gram-positive coccus. Biochemical characterizations include 

catalase-positive, coagulase-positive, fermentation of mannitol, and anaerobic fermentation of 

glucose. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. 

Source: APHA, AWWA, and WEF. 2005. “Method 9213 B: Staphylococcus aureus.” Standard 

Methods for the Examination of Water and Wastewater. 21st Edition. 

http://www.standardmethods.org/ 

7.2.15 Vibrio cholerae [Cholera] – BSL-2 

Method Analytical Technique Section 

CDC Laboratory Assay: V. cholerae Real-time PCR 7.2.15.1 

SM 9260 H Culture and Immunoassay 7.2.15.2 

 SAM Revision 5.0 167 September 29, 2009 
Section 7– Selected Pathogen Methods 

7.2.15.1 CDC Laboratory Assay: “TaqMan Assays for Detection of V. cholerae 

ctxA, O1 rfb, and O139 rfb.” 

Analysis Purpose: Detection, not suitable for viability 

Sample Preparation: Solid samples should be prepared according to “Quantification of Bias 

Related to the Extraction of DNA Directly from Soils,” Frostegard, A., Courtois, S., Ramisse, V., 

Clerc, S., Bernillon, D., Le Gall, F., Jeannin, P., Nesme, X., and Simonet, P. 1999. Applied and 

Environmental Microbiology. 65(12): 5409–5420 (www.epa.gov/sam/pdfs/AEM-65(12)-

pgs5409-5420.pdf). 

Analytical Technique: Real-time PCR 

Method Developed for: V. cholerae O1 and O139 from cultures or isolates 

Method Selected for: SAM lists these procedures for detection in solid, particulate, aerosol, 

liquid, and water samples. Further research is needed to develop and standardize the procedures 

for environmental sample types. 

Description of Method: Procedures are described for analysis of isolates and may be adapted 

for assessment of solid, particulate, aerosol, liquid, and water samples. The assay uses real-time
 

PCR for identification of V. cholerae. Cell lysate templates are prepared by suspending a portion 

of a colony in 300 μL of 0.01M Tris-EDTA (TE) buffer and boiling for 10 minutes. After 

centrifugation for 2 minutes, 1 μL of the supernatant is used in the PCR reaction. Alternatively, 

DNA may be purified using a commercially available kit or automated DNA extraction system. 

PCR is performed on a LightCycler® as single target assays or on a SmartCycler® as multiplex 

PCR using primers and probes designed for the cholera toxin ctx gene, the O1 antigen O1rfb 

gene, and the O139 antigen O139 rfb gene. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. PCR QC checks should be
 

performed according to EPA Draft Quality Assurance/Quality Control Guidance for Laboratories 

Performing PCR Analyses on Environmental Samples document www.epa.gov/sam/pdfs/EPAQA-QC_PCR_Oct2004.pdf or consult the point of contact identified in Section 4.
 

Source: HHS, CDC, Laboratory Assay. “TaqMan Assays for Detection of V. cholerae ctxA, O1 

rfb, and O139 rfb.” Contact: Dr. Eija Trees, CDC, email: eija.trees@cdc.hhs.gov. 

7.2.15.2 Standard Method 9260 H: Vibrio cholerae 

Analysis Purpose: Detection and viability 

Sample Preparation: Solid samples should be prepared according to EPA Method 1680; 

particulate samples should be prepared according to “Evaluation of a Macrofoam Swab Protocol 

for the Recovery of Bacillus anthracis Spores from a Steel Surface,” Hodges, L.R., Rose, L.J., 

Peterson, A., Nobel-Wang, J., and Arduino, M.J. 2006. Applied and Environmental 

Microbiology. 72(6): 4429–4430 (www.epa.gov/sam/pdfs/AEM-72(6)-pgs4429-4430.pdf). 

Analytical Technique: Culture and immunoassay 

Method Developed for: Vibrio cholerae in water 

Method Selected for: SAM lists this method for detection and viability assessment in solid, 

particulate, liquid, and water samples. Further research is needed to develop and standardize the 

procedures for environmental sample types other than water. 

Description of Method: This method describes procedures for analysis of water samples and 

may be adapted for assessment of solid, particulate, and liquid samples. Samples are enriched in 

alkaline peptone broth by incubation for up to 8 hours. Thiosulfate-citrate-bile salts-sucrose 

(TCBS) agar plates are inoculated with the incubated broth and incubated for 24 hours. Yellow 

 SAM Revision 5.0	 168 September 29, 2009 
Section 7– Selected Pathogen Methods 

sucrose-fermenting colonies are presumptive for V. cholerae and are plated on tryptic soy agar 

with 0.5% sodium chloride. Presumptive positive colonies are subjected to biochemical 

characterization. Confirmation is performed using slide agglutination assays for serological 

identification. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. 

Source: APHA, AWWA, and WEF. 2005. “Method 9260 H: Vibrio cholerae.” Standard 

Methods for the Examination of Water and Wastewater. 21st Edition. 

http://www.standardmethods.org/ 

7.2.16 Yersinia pestis [Plague] – BSL-3 

Method Analytical Technique Section 

LRN Real-time PCR/Immunoassay 7.1.4 

ASM Sentinel Laboratory Guidelines for Suspected 

Agents of Bioterrorism: Yersinia pestis Culture 7.2.16.1 

7.2.16.1 ASM Sentinel Laboratory Guidelines for Suspected Agents of 

Bioterrorism: Yersinia pestis 

Analysis Purpose: Detection and viability 

Sample Preparation: Solid samples should be prepared according to EPA Method 1680; 

particulate samples should be prepared according “Evaluation of a Macrofoam Swab Protocol for 

the Recovery of Bacillus anthracis Spores from a Steel Surface,” Hodges, L.R., Rose, L.J., 

Peterson, A., Nobel-Wang, J., and Arduino, M.J. 2006. Applied and Environmental 

Microbiology. 72(6): 4429–4430 (www.epa.gov/sam/pdfs/AEM-72(6)-pgs4429-4430.pdf). 

Analytical Technique: Culture 

Method Developed for: Y. pestis in clinical samples 

Method Selected for: SAM lists these guidelines for detection and viability assessment in solid, 

particulate, aerosol, liquid, and water samples. Further research is needed to develop and 

standardize the procedures for environmental sample types. 

Description of Method: This method describes procedures for analysis of clinical samples and 

may be adapted for assessment of solid, particulate, and liquid samples. Samples are either plated 

directly on SBA or first enriched in a nutrient broth (e.g., TSB) prior to plating. Incubation is for 

at least 3 days at 28°C–30°C. Y. pestis produces 1.0–2.0 mm, gray-white to opaque colonies on 

SBA at 24 hours, with a “fried egg” appearance with longer incubation. There is little to no 

hemolysis on SBA. In broth, Y. pestis grows in flocculent clumps. Presumptive identification is 

made by culture examination, microscopy, and biochemical testing. Y. pestis is a bi-polar 

staining, Gram-negative rod and is oxidase-negative, catalase-positive, urease-negative, and 

indole-negative. Cultures (isolates) that cannot be ruled out as Y. pestis based on the 

characteristics noted above are referred to an appropriate reference laboratory for confirmation. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. 

Special Considerations: Y. pestis is a select agent requiring regulatory compliance (42 CFR 

parts 72 and 73, and 9 CFR part 121); appropriate safety and BSL requirements should also be 

followed (see CDC’s BMBL, 5th Edition, 

http://www.cdc.gov/OD/ohs/biosfty/bmbl5/bmbl5toc.htm). 

 SAM Revision 5.0	 169 September 29, 2009 
Section 7– Selected Pathogen Methods 

Source: ASM. 2005. “Sentinel Laboratory Guidelines for Suspected Agents of Bioterrorism: 

Yersinia pestis.” http://www.epa.gov/sam/pdfs/ASM-Ypestis.pdf 

7.3 Method Summaries for Viruses 

Summaries of the analytical methods for viruses listed in Appendix C are provided in Sections 7.3.1 

through 7.3.10. Each summary contains sample preparation information, intended method application, a 

brief description of the method, performance data (if available), and a link to, or source for, obtaining a 

full version of the method. 

7.3.1 Adenoviruses: Enteric and non-enteric (A-F) – BSL-2 

Method Analytical Technique Section 

Applied and Environmental Microbiology. 2005. 

71(6): 3131–3136 Tissue culture and Real-time PCR 7.3.1.1 

7.3.1.1 Literature Reference for Adenoviruses (Applied and Environmental 

Microbiology. 2005. 71(6): 3131–3136) 

Analysis Purpose: Detection and viability
 

Sample Preparation: Samples should be prepared according to the procedures described in the 

USEPA Manual of Methods for Virology (EPA/600/4-84/013, April 2001). 

Analytical Technique: Tissue culture and real-time PCR 

Method Developed for: Human adenoviruses (HAdV) in cell culture lysate samples 

Method Selected for: SAM lists these procedures for detection and viability assessment in solid, 

particulate, aerosol, liquid, and water samples. Further research is needed to develop and 

standardize the procedures for environmental sample types. 

Description of Method: Procedures are described for analysis of cell culture lysates and may be 

adapted for assessment of solid, particulate, aerosol, liquid, and water samples. The detection 

procedure uses a broadly reactive fluorogenic 5' nuclease (TaqMan®) quantitative real-time PCR 

assay for the detection of all six species (A–F) of HAdV on a R.A.P.I.D.® PCR system. 

Sensitive detection and discrimination of adenovirus F species (Adenovirus 40 [AdV40] and 

Adenovirus 41 [AdV41]) can be achieved by using a real-time fluorescence resonance energy
 

transfer (FRET)-based PCR assay. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. PCR QC checks should be
 

performed according to EPA Draft Quality Assurance/Quality Control Guidance for Laboratories 

Performing PCR Analyses on Environmental Samples document www.epa.gov/sam/pdfs/EPAQA-QC_PCR_Oct2004.pdf or consult the point of contact identified in Section 4.
 

Special Considerations: For the viability assessment of adenovirus 40 and 41, given that they 

can be difficult to grow in culture, cell lines such as G293 (Journal of Medical Virology, 11(3): 

215–231) or CaCo-2 (Journal of Medical Virology. 1994. 44(3): 310–315) may be considered 

when these viruses are suspected to be present. As detection of adenovirus in environmental 

samples can be difficult, additional methods such as described in Effect of Adenovirus Resistance 

on UV Disinfection Experiments: A Report on the State of Adenovirus Science (J. AWWA. 2006. 

98(6):93–106) also may be useful. 

Source: Jothikumar, N., Cromeans, T.L., Hill, V.R., Lu, X., Sobsey, M., and Erdman, D.D. 

2005. “Quantitative Real-Time PCR Assays for Detection of Human Adenoviruses and 

 SAM Revision 5.0	 170 September 29, 2009 
Section 7– Selected Pathogen Methods 

Identification of Serotypes 40 and 41.” Applied and Environmental Microbiology. 71(6): 3131– 

3136. http://www.epa.gov/sam/pdfs/AEM-71(6)-pgs3131-3136.pdf 

7.3.2 Astroviruses – BSL not specified 

Method Analytical Technique Section 

Canadian Journal of Microbiology. 2004. 50: 269– 

278 

Integrated cell culture/Real-time 

reverse transcription-PCR 7.3.2.1 

7.3.2.1 Literature Reference for Astroviruses (Canadian Journal of Microbiology. 

2004. 50: 269–278) 

Analysis Purpose: Detection and viability
 

Sample Preparation: Samples should be prepared according to the procedures described in the 

USEPA Manual of Methods for Virology (EPA/600/4-84/013, April 2001). 

Analytical Technique: Integrated cell culture/real-time reverse transcription-PCR 

Method Developed for: Astroviruses in clinical samples 

Method Selected for: SAM lists these procedures for detection and viability assessment in solid, 

particulate, aerosol, liquid, and water samples. Further research is needed to develop and 

standardize the procedures for environmental sample types. 

Description of Method: Procedures are described for analysis of clinical samples and may be 

adapted for assessment of solid, particulate, aerosol, liquid, and water samples. The method 

detects eight astrovirus serotypes. The method is a real-time reverse transcription-PCR procedure 

optimized for use in a real-time PCR assay using an ABI Prism® 7000 and can be integrated with 

sample-cell culture (CaCo-2 cells) to enhance sensitivity. Water samples are collected by
 

filtration (1MDS filter), and viruses are eluted using a beef extract solution (1.5%, pH 9.5). 

Viruses are concentrated using Celite® adsorption (pH 4.0), filtration, and Celite®-elution with 

sodium phosphate (0.15 M, pH 9.0), followed by further concentration and processing to remove 

inhibitors (ultracentrifugation, solvent extraction, and molecular weight [MW]-exclusion 

filtration). Concentrated samples are analyzed directly or indirectly (following cell culture) by a 

two-step real-time reverse transcription-PCR (i.e., reverse transcription followed by real-time 

PCR) assay using astrovirus-specific primer sets. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. PCR QC checks should be
 

performed according to EPA Draft Quality Assurance/Quality Control Guidance for Laboratories 

Performing PCR Analyses on Environmental Samples document www.epa.gov/sam/pdfs/EPAQA-QC_PCR_Oct2004.pdf or consult the point of contact identified in Section 4.
 

Source: Grimm, A.C., Cashdollar, J.L., Williams, F.P., and Fout, G.S. 2004. “Development of 

an Astrovirus RT-PCR Detection Assay for Use with Conventional, Real-Time, and Integrated 

Cell Culture/RT-PCR.” Canadian Journal of Microbiology. 50(4): 269–278. http://pubs.nrccnrc.gc.ca/rp-ps/inDetail.jsp?jcode=cjm&lang=eng&vol=50&is=4 

7.3.3 Caliciviruses: Noroviruses – BSL-2 

Method Analytical Technique Section 

Journal of Clinical Microbiology. 2004. 42(10): 4679– 

4685 

Real-time reverse transcriptionPCR 7.3.3.1 

 SAM Revision 5.0	 171 September 29, 2009 
Section 7– Selected Pathogen Methods 

7.3.3.1 Literature Reference for Noroviruses (Journal of Clinical Microbiology. 

2004. 42(10): 4679–4685) 

Analysis Purpose: Detection, not suitable for viability
 

Sample Preparation: Samples should be prepared according to procedures described in the 

USEPA Manual of Methods for Virology (EPA/600/4-84/013, April 2001). 

Analytical Technique: Real-time reverse transcription-PCR 

Method Developed for: Noroviruses in clinical samples 

Method Selected for: SAM lists these procedures for detection in solid, particulate, aerosol, 

liquid, and water samples. Further research is needed to develop and standardize the procedures 

for environmental sample types. 

Description of Method: Procedures are described for analysis of clinical samples and may be 

adapted for assessment of solid, particulate, aerosol, liquid, and water samples. This method is an 

assay for the detection and quantitation of norovirus using LightCycler® real-time reverse 

transcription-PCR technology. Viral ribonucleic acid (RNA) is extracted using either a 

commercial kit or a silica-based method. For Norovirus G-1, primers based on the capsid gene 

sequence are used, and for Norovirus G-II, primers based on the polymerase gene sequence are 

used. A SYBR® Green I system is used in the reaction for visualization. External standard 

curves for the quantification of norovirus are established using RNA transcripts from strains S5 

and S19, corresponding to G-I/4 and G-II/12, respectively. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. PCR QC checks should be
 

performed according to EPA Draft Quality Assurance/Quality Control Guidance for Laboratories 

Performing PCR Analyses on Environmental Samples document www.epa.gov/sam/pdfs/EPAQA-QC_PCR_Oct2004.pdf or consult the point of contact identified in Section 4.
 

Source: Pang, X., Lee, B., Chui, L., Preiksaitis, J.K., and Monroe, S.S. 2004. “Evaluation and 

Validation of Real-Time Reverse Transcription-PCR Assay Using the LightCycler System for 

Detection and Quantitation of Norovirus.” Journal of Clinical Microbiology. 42(10): 4679–4685. 

http://www.epa.gov/sam/pdfs/JCM-42(10)-pgs4679-4685.pdf 

7.3.4 Caliciviruses: Sapovirus – BSL-2 

Method Analytical Technique Section 

Journal of Medical Virology, 2006. 78 (10): 1347– 

1353 

Real-time reverse transcriptionPCR 7.3.4.1 

7.3.4.1 Literature Reference for Sapoviruses (Journal of Medical Virology. 2006. 

78(10): 1347–1353) 

Analysis Purpose: Detection, not suitable for viability
 

Sample Preparation: Samples should be prepared according to procedures described in the 

USEPA Manual of Methods for Virology (EPA/600/4-84/013, April 2001).
 

Analytical Technique: Real-time reverse transcription-PCR 

Method Developed for: Sapoviruses in clinical samples 

Method Selected for: SAM lists these procedures for detection in solid, particulate, aerosol, 

liquid, and water samples. Further research is needed to develop and standardize the procedures 

for environmental sample types. 

 SAM Revision 5.0	 172 September 29, 2009 
Section 7– Selected Pathogen Methods 

Description of Method: Procedures are described for analysis of clinical samples and may be 

adapted for assessment of solid, particulate, aerosol, liquid, and water samples. The method is a 

TaqMan®-based real-time reverse transcriptase PCR assay that uses an ABI 7500 system. The 

assay has the ability to detect four of the five distinct sapovirus (SaV) genogroups (GI–GV). Sets 

of primers, based on the multiple alignment of 27 gene sequences for the polymerase-capsid 

junction in open reading frame 1 (ORF1), are used to detect human SaV GI, GII, GIV, and GV 

sequences in a single tube. Sensitivity using control plasmids range from 2.5 X 101

 to 2.5 X 107 

copies per tube. No cross-reactivity is observed against other enteric viruses, including norovirus 

(NoV), rotavirus, astrovirus, and adenovirus. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. PCR QC checks should be 

performed according to EPA Draft Quality Assurance/Quality Control Guidance for Laboratories 

Performing PCR Analyses on Environmental Samples document www.epa.gov/sam/pdfs/EPAQA-QC_PCR_Oct2004.pdf or consult the point of contact identified in Section 4. 

Source: Oka, T., Katayama, K., Hansman, G.S., Kageyama, T., Ogawa, S., Wu, F.T., White, 

P.A., and Takeda, N. 2006. “Detection of Human Sapovirus by Real-time Reverse TranscriptionPolymerase Chain Reaction.” Journal of Medical Virology. 78(10): 1347–1353. 

http://cat.inist.fr/?aModele=afficheN&cpsidt=18099754 

7.3.5 Coronaviruses: SARS-associated human coronavirus – BSL-2; BSL-3 for 

propagation 

Method Analytical Technique Section 

Journal of Virological Methods. 2004. 122: 29–36 Reverse transcription-PCR 7.3.5.1 

7.3.5.1 Literature Reference for Coronaviruses (SARS) (Journal of Virological 

Methods. 2004. 122: 29–36) 

Analysis Purpose: Detection, not suitable for viability 

Sample Preparation: Samples should be prepared according to procedures described in the 

USEPA Manual of Methods for Virology (EPA/600/4-84/013, April 2001). 

Analytical Technique: Reverse transcription-PCR 

Method Developed for: Severe acute respiratory syndrome (SARS)-associated human 

coronavirus (HCoV) in clinical samples 

Method Selected for: SAM lists these procedures for detection in solid, particulate, aerosol, 

liquid, and water samples. Further research is needed to develop and standardize the procedures 

for environmental sample types. 

Description of Method: Procedures are described for analysis of clinical samples and may be 

adapted for assessment of solid, particulate, aerosol, liquid, and water samples. This method uses 

a conventional single-tube reverse transcription-PCR procedure conducted on a Stratagene 

Robocycler® and based on consensus primer sequences targeting conserved regions of 

coronavirus genome sequences. End-point amplicon analysis is by electrophoresis and 

subsequent visualization. The assay can detect the SARS-HCoV as well as several other human 

respiratory coronaviruses (HCoV-OC43 and HCoV-229E). Species identification is provided by 

sequencing the amplicon, although rapid screening can be performed by restriction enzyme 

analysis. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

 SAM Revision 5.0	 173 September 29, 2009 
Section 7– Selected Pathogen Methods 

ensure reliability of the analytical results should also be performed. PCR QC checks should be 

performed according to EPA Draft Quality Assurance/Quality Control Guidance for Laboratories 

Performing PCR Analyses on Environmental Samples document www.epa.gov/sam/pdfs/EPAQA-QC_PCR_Oct2004.pdf or consult the point of contact identified in Section 4. 

Source: Adachi, D., Johnson. G., Draker, R., Ayers, M., Mazzulli, T., Talbot, P.J., and Tellier, 

R. 2004. “Comprehensive Detection and Identification of Human Coronaviruses, Including the 

SARS-associated Coronavirus, with a Single RT-PCR Assay.” Journal of Virological Methods. 

122: 29–36. http://www.sciencedirect.com/science/journal/01660934 

7.3.6 Hepatitis E virus (HEV) – BSL-2 

Method Analytical Technique Section 

Journal of Virological Methods. 2006. 131(1): 65–71 Real-time reverse transcriptionPCR 7.3.6.1 

7.3.6.1 Literature Reference for Hepatitis E Virus (Journal of Virological 

Methods. 2006. 131(1): 65–71) 

Analysis Purpose: Detection, not suitable for viability 

Sample Preparation: Samples should be prepared according to procedures described in the 

USEPA Manual of Methods for Virology (EPA/600/4-84/013, April 2001). 

Analytical Technique: Real-time reverse transcription-PCR 

Method Developed for: Hepatitis E virus (HEV) in spiked water samples 

Method Selected for: SAM lists these procedures for detection in solid, particulate, aerosol, 

liquid, and water samples. Further research is needed to develop and standardize the procedures 

for environmental sample types other than water. 

Description of Method: Procedures are described for analysis of spiked water samples and may 

be adapted for assessment of solid, particulate, aerosol, and liquid samples. The method uses a 

TaqMan® real-time reverse transcriptase-PCR assay using the R.A.P.I.D.® PCR systems to 

detect and quantitate all four major HEV genotypes that may be present in clinical and 

environmental samples. Primers and probes are based on the multiple sequence alignments of 27 

gene sequences for the ORF3 region. Thirteen HEV isolates representing genotypes 1–4 were 

used to assess assay specificity, all thirteen isolates were positive. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. PCR QC checks should be 

performed according to EPA Draft Quality Assurance/Quality Control Guidance for Laboratories 

Performing PCR Analyses on Environmental Samples document www.epa.gov/sam/pdfs/EPAQA-QC_PCR_Oct2004.pdf or consult the point of contact identified in Section 4. 

Source: Jothikumar, N., Cromeans, T.L., Robertson, B.H., Meng, X.J., and Hill, V.R. 2006. “A 

Broadly Reactive One-step Real-time RT-PCR Assay for Rapid and Sensitive Detection of 

Hepatitis E Virus.” Journal of Virological Methods, 131(1): 65–71. 

http://cat.inist.fr/?aModele=afficheN&cpsidt=17367357 

7.3.7 Influenza H5N1 virus – BSL-3 

Method Analytical Technique Section 

Emerging Infectious Diseases. 2005. 11(8): 

1303–1305 

Real-time reverse transcriptionPCR 7.3.7.1 

 SAM Revision 5.0	 174 September 29, 2009 
Section 7– Selected Pathogen Methods 

7.3.7.1 Literature Reference for Influenza H5N1 (Emerging Infectious Diseases. 

2005. 11(8): 1303–1305) 

Analysis Purpose: Detection, not suitable for viability
 

Sample Preparation: Samples should be prepared according to procedures described in the 

USEPA Manual of Methods for Virology (EPA/600/4-84/013, April 2001).
 

Analytical Technique: Real-time reverse transcription-PCR 

Method Developed for: Influenza H5N1 virus in clinical samples 

Method Selected for: SAM lists these procedures for detection in solid, particulate, aerosol, 

liquid, and water samples. Further research is needed to develop and standardize the procedures 

for environmental sample types. 

Description of Method: Procedures are described for analysis of clinical samples and may be 

adapted for assessment of solid, particulate, aerosol, liquid, and water samples. This is a twostep, real-time reverse transcriptase-PCR multiplex assay. It employs a mixture of two sets of 

primers and dual-labeled fluorescent probes that specifically target two different regions of the
 

HA gene of H5N1. Viral ribonucleic acid (RNA) is extracted using a commercial viral RNA 

extraction kit and reverse transcribed with random hexamers. Five microliters of the 

complementary (cDNA) is used for PCR which is conducted on a LightCycler®. At the end of 

each annealing step, the fluorescent signal of each reaction is measured at a wavelength of 530 

nm with the fluorimeter. The assay is specific for the H5 subtype. Influenza H5N1 virus samples 

are to be handled with BSL-3 containment and practices. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. PCR QC checks should be
 

performed according to EPA Draft Quality Assurance/Quality Control Guidance for Laboratories 

Performing PCR Analyses on Environmental Samples document www.epa.gov/sam/pdfs/EPAQA-QC_PCR_Oct2004.pdf or consult the point of contact identified in Section 4.
 

Source: Ng, E.K.O., Cheng, P.K.C., Ng, A.Y.Y., Hoang, T.L., and Lim, W.W.L. 2005. 

“Influenza A H5N1 Detection.” Emerging Infectious Diseases. 11(8): 1303–1305. 

http://www.epa.gov/sam/pdfs/EID-11(8)-pgs1303-1305.pdf 

7.3.8 Picornaviruses: Enteroviruses – BSL-2 

Method Analytical Technique Section 

USEPA Manual of Methods for Virology 

EPA/600/4–84/013, 2001 Tissue culture 7.3.8.1 

Applied and Environmental Microbiology. 2003. 

69(6): 3158–3164 Reverse transcription-PCR 7.3.8.2 

7.3.8.1 USEPA Manual of Methods for Virology, EPA/600/4-84/013, April 2001 

Analysis Purpose: Detection and viability
 

Sample Preparation: Samples should be prepared according to the procedures described in the 

USEPA Manual of Methods for Virology (EPA/600/4-84/013, April 2001).
 

Analytical Technique: Tissue culture with serum neutralization 

Method Developed for: Enteroviruses in water 

Method Selected for: SAM lists this manual for detection and viability assessment in solid, 

particulate, aerosol, liquid, and water samples. Further research is needed to develop and 

standardize the procedures for environmental sample types other than water. 

 SAM Revision 5.0	 175 September 29, 2009 
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Description of Method: This manual describes procedures for determining the infectivity of 

enteroviruses, including a neutralization test used to identify these viruses. The test uses 

reference-typing sera directed against isolated waterborne viruses, and consists of simultaneously 

inoculating virus and antiserum into a microtiter plate, incubating the virus-antibody mixture for 

2 hours, adding a suspension of host cells to the mixture, incubating the host cells-virus-antibody 

mixture for 3 days, and then examining the cells daily for 5 more days for the presence/absence of 

a cytopathic effect (CPE). The test uses the Lim Benyesh-Melnick (LB-M) antiserum pools, 

which consist of 61 equine antisera, including LB-M antiserum pools A–H for the identification 

of 41 enteroviruses. Chapters 7 and 14 in this manual describe procedures for the collection and 

preparation of virus samples. Sample preparation procedures described include concentration and 

processing of waterborne viruses by positively charged 1MDS cartridge filters and flocculation. 

These general procedures can be used for many viruses and may be adapted for analysis of 

particulate, liquid, water, and aerosol samples. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. 

Special Considerations: This manual also describes procedures for preparation of samples for 

adenovirus, astrovirus, norovirus, sapovirus, coronavirus – SARS, hepatitis E virus, influenza 

H5N1 virus, picornaviruses (enterovirus and hepatitis A virus), and reovirus (rotavirus). 

Source: EPA. 2001. “Chapter 12 – Identification of Enteroviruses.” USEPA Manual of Methods 

for Virology, EPA/600/4-84/013. http://www.epa.gov/sam/pdfs/EPA-600-4-84-013.pdf 

7.3.8.2 Literature Reference for Enteric Viruses (Applied and Environmental 

Microbiology. 2003. 69(6): 3158–3164) 

Analysis Purpose: Detection of Enteroviruses and Hepatitis A virus (HAV); Detection and 

viability of Rotoviruses 

Sample Preparation: Samples should be prepared according to procedures described in the 

USEPA Manual of Methods for Virology (EPA/600/4-84/013, April 2001). 

Analytical Technique: Reverse transcription-PCR for detection and tissue culture for viability 

Method Developed for: Enteroviruses, HAV, and Group A Rotaviruses in water 

Method Selected for: SAM lists these procedures for detection of enterovirus and HAV in solid, 

particulate, aerosol, liquid, and water samples, but the procedures are not suitable for determining 

viability of these pathogens. These procedures should also be used for detection and viability 

assessment of rotavirus (Group A) in liquid and water samples. Further research is needed to 

develop and standardize the procedures for environmental sample types other than water. 

Description of Method: Procedures are described for analysis of water samples and may be 

adapted for assessment of solid, particulate, aerosol, and liquid samples. The method is used to 

detect human enteric viruses (enteroviruses, HAV, rotavirus) in ground water samples. It is a 

multiplex reverse-transcription PCR procedure optimized for the simultaneous detection of 

enteroviruses, HAV, reoviruses, and rotaviruses. Water samples are collected by filtration and 

viruses are eluted using a beef extract solution (1.5%, pH 9.5). Viruses are concentrated using 

Celite® adsorption (pH 4.0), filtration, and Celite®-elution with sodium phosphate (0.15 M, pH 

9.0), followed by further concentration and processing to remove inhibitors (ultracentrifugation, 

solvent extraction, and MW-exclusion filtration). Concentrated samples are analyzed by a twostep multiplex reverse transcription-PCR using virus-specific primer sets. Detection of amplicons 

is by gel electrophoresis with subsequent confirmation by hybridization (dot-blot) using 

digoxigenin-labeled internal (nested) probes. 

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At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. PCR QC checks should be 

performed according to EPA Draft Quality Assurance/Quality Control Guidance for Laboratories 

Performing PCR Analyses on Environmental Samples document www.epa.gov/sam/pdfs/EPAQA-QC_PCR_Oct2004.pdf or consult the point of contact identified in Section 4. 

Source: Fout, G.S., Martinson, B.C., Moyer, M.W.N., and Dahling, D.R. 2003. “A Multiplex 

Reverse Transcription-PCR Method for Detection of Human Enteric Viruses in Groundwater.” 

Applied and Environmental Microbiology. 69(6): 3158–3164. 

http://www.epa.gov/sam/pdfs/AEM-69(6)-pgs3158-3164.pdf 

7.3.9 Picornaviruses: Hepatitis A virus (HAV) – BSL-2 

Method Analytical Technique Section 

Applied and Environmental Microbiology. 2003. 

69(6): 3158–3164 Reverse transcription-PCR 7.3.8.2 

See Section 7.3.8.2 for information on reverse transcription-PCR procedures for Picornaviruses: 

Hepatitis A virus. 

7.3.10 Reoviruses: Rotavirus (Group A) – BSL not specified 

Method Analytical Technique Section 

Applied and Environmental Microbiology. 2003. 

69(6): 3158–3164 Tissue culture 7.3.8.2 

Journal of Virological Methods. 2009. 155: 126– 

131 

Real-time reverse transcriptionPCR 7.3.10.1 

7.3.10.1 Literature Reference for Reoviruses: Rotavirus (Group A) (Journal of 

Virological Methods. 2009. 155(2): 126–131) 

Analysis Purpose: Detection 

Sample Preparation: Samples should be prepared according to procedures described in Journal 

of Virological Methods. 2009. 155: 126–131. 

Analytical Technique: Real-time reverse transcription-PCR 

Method Developed for: Group A Rotaviruses in water 

Method Selected for: SAM lists these procedures for detection of rotavirus (Group A) in solid, 

particulate, aerosol, and drinking water samples, but the procedures are not suitable for 

determining viability of these pathogens. Further research is needed to develop and standardize 

the procedures for environmental sample types other than drinking water. 

Description of Method: Procedures are described for analysis of drinking water samples and 

may be adapted for assessment of solid, particulate, and aerosol samples. The method is used to 

detect rotavirus in drinking water samples using one-step real-time reverse-transcription PCR. 

Water samples (100 L) are concentrated up to 400 mL by UF and 100-mL volumes are further 

concentrated using a 30-kDa Centricon® Plus-70 unit. Concentrated samples (approximately 1 

mL each) are subjected to nucleic acid extraction using a guanidine thiocyantate-based buffer. 

Ten percent of the RNA extract (corresponding to 2.5 L of the original water sample) is analyzed 

using RT-PCR performed on an iCylcer iQTM Real-Time Detection System with primers and 

probe specific for the rotavirus NSP3 gene. The sensitivity of the assay was found to be six virus 

particles per reaction (nuclease-free water) as determined using quantified rotavirus stocks and a 

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plasmid DNA stock. Specificity testing did not identify any cross-reactivity of the assay with a 

panel of 36 non-rotavirus enteric viruses. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. PCR QC checks should be 

performed according to EPA Draft Quality Assurance/Quality Control Guidance for Laboratories 

Performing PCR Analyses on Environmental Samples document www.epa.gov/sam/pdfs/EPAQA-QC_PCR_Oct2004.pdf or consult the point of contact identified in Section 4. 

Special Considerations: Complex matrices such as solid, particulate, source water, and aerosol 

samples may require more extensive inhibitor removal procedures than those described in this 

method. Sample preparation procedures as described by Fout et al. (Section 7.3.8.2) may be 

used. 

Source: Jothikumar, N., Kang, G., and V.R. Hill. 2009. “Broadly Reactive TaqMan® Assay for 

Real-time RT-PCR Detection of Rotavirus in Clinical and Environmental Samples.” Journal of 

Virological Methods. 155(2): 126–131. http://www.sciencedirect.com/science/journal/01660934 

7.4 Method Summaries for Protozoa 

Summaries of the analytical methods for protozoa listed in Appendix C are provided in Sections 7.4.1 

through 7.4.4. Each summary contains sample type for which the method was developed, sample type to 

which the method applies, sample preparation information, a brief description of the method, performance 

data (if available), and a link to, or source for, obtaining a full version of the method. 

7.4.1 Cryptosporidium spp. [Cryptosporidiosis] – BSL-2 

Method Analytical Technique Section 

EPA Method 1622 IMS/FA 7.4.1.1 

EPA Method 1623 IMS/FA 7.4.1.2 

Applied and Environmental Microbiology. 1999. 

65(9): 3936–3941 Tissue culture 7.4.1.3 

Applied and Environmental Microbiology. 2007. 

73(13): 4218–4225 Real-time PCR 7.4.1.4 

7.4.1.1 EPA Method 1622: Cryptosporidium in Water by Filtration/IMS/FA 

Analysis Purpose: Detection, not suitable for viability 

Sample Preparation: Samples should be prepared according to procedures in EPA Method 

1622. 

Analytical Technique: IMS and fluorescence assay (FA) microscopy 

Method Developed for: Cryptosporidium in surface water 

Method Selected for: SAM lists this method for detection in solid, particulate, liquid, and water 

samples. Further research is needed to develop and standardize the procedures for environmental 

sample types other than surface water. 

Description of Method: This method describes procedures for analysis of drinking water 

samples and may be adapted for analysis of solid and particulate samples. A water sample is 

filtered and the oocysts and extraneous materials are retained on the filter. Materials on the filter 

are eluted, the eluate is centrifuged to pellet the oocysts, and the supernatant fluid is aspirated. A 

solution containing anti-Cryptosporidium antibodies conjugated to magnetic beads is added to the 

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pellet and mixed. The oocyst magnetic bead complex is separated from the extraneous materials 

using a magnet, and the extraneous materials are discarded. The magnetic bead complex is then 

detached from the oocysts. The oocysts are stained on well slides with fluorescently labeled 

monoclonal antibodies (mAbs) and 4',6-diamidino-2-phenylindole (DAPI). The stained sample is 

examined using fluorescence and differential interference contrast (DIC) microscopy. Qualitative 

analysis is performed by scanning each slide well for objects that meet the size, shape, and 

fluorescence characteristics of Cryptosporidium oocysts. Quantitative analysis is performed by 

counting the total number of objects on the slide confirmed as oocysts. This method is not 

intended to determine viability, species, or infectivity of the oocysts. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, MS/MSD and blank. Ongoing analysis of QC 

samples to ensure reliability of the analytical results should also be performed. 

Source: EPA. 2005. “Method 1622: Cryptosporidium in Water by Filtration/IMS/FA.” 

http://www.epa.gov/sam/pdfs/EPA-1622.pdf 

7.4.1.2 EPA Method 1623: Cryptosporidium and Giardia in Water by 

Filtration/IMS/FA 

Analysis Purpose: Detection, not suitable for viability 

Sample Preparation: Samples should be prepared according to procedures in EPA Method 

1623. 

Analytical Technique: IMS and FA microscopy 

Method Developed for: Cryptosporidium and Giardia in surface water 

Method Selected for: SAM lists this method for the detection of Cryptosporidium spp. and 

Giardia spp. in solid, particulate, liquid, and water samples. Further research is needed to 

develop and standardize the procedures for environmental sample types other than surface water. 

Description of Method: This method describes procedures for analysis of water samples and 

may be adapted for assessment of solid, particulate, and liquid samples. A water sample is 

filtered and the oocysts and cysts and extraneous materials are retained on the filter. Materials on 

the filter are eluted, the eluate is centrifuged to pellet the oocysts and cysts, and the supernatant 

fluid is aspirated. A solution containing anti-Cryptosporidium and anti-Giardia antibodies 

conjugated to magnetic beads is added to the pellet and mixed. The oocyst and cyst magnetic 

bead complex is separated from the extraneous materials using a magnet, and the extraneous 

materials are discarded. The magnetic bead complex is then detached from the oocysts and cysts. 

The oocysts and cysts are stained on well slides with fluorescently labeled mAbs and DAPI. The 

stained sample is examined using fluorescence and DIC microscopy. Qualitative analysis is 

performed by scanning each slide well for objects that meet the size, shape, and fluorescence 

characteristics of Cryptosporidium oocysts and Giardia cysts. Quantitative analysis is performed 

by counting the total number of objects on the slide confirmed as oocysts or cysts. This method 

is not intended to determine viability, species, or infectivity of the parasites. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, MS/MSD and blank. Ongoing analysis of QC 

samples to ensure reliability of the analytical results should also be performed. 

Source: EPA. 2001. “Method 1623: Cryptosporidium and Giardia in Water by 

Filtration/IMS/FA.” http://www.epa.gov/sam/pdfs/EPA-1623.pdf 

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7.4.1.3 Literature Reference for Cryptosporidium spp. (Applied and 

Environmental Microbiology. 1999. 65(9): 3936–3941) 

Analysis Purpose: Detection and viability
 

Sample Preparation: Samples should be prepared according to the procedures in Applied and 

Environmental Microbiology. 1999. 65(9): 3939–3941. 

Analytical Technique: Tissue culture 

Method Developed for: Cryptosporidium in animal samples 

Method Selected for: SAM lists these procedures for detection and viability assessment in solid, 

particulate, liquid, and water samples. Further research is needed to develop and standardize the 

procedures for environmental sample types. 

Description of Method: Procedures are described for analysis of animal samples and may be 

adapted for assessment of solid, particulate, liquid, and water samples. A cell culture infectivity 

assay capable of detecting infectious oocysts is used to quantify viable oocysts through sporozoite 

invasion and clustering of foci. Oocysts diluted in a standard 5- or 10-fold multiple dilution 

format are inoculated onto human ileocecal adenocarcinoma (HCT-8) cell monolayers. After 

incubation for 48 hours, anti-sporozoite polyclonal antibody is used to detect sporozoite invasion, 

and microscopy is used to confirm replication (life stages present). Levels of infection and 

clustering are used to determine the most probable number (MPN) of infectious oocysts in the
 

stock suspension. For oocysts less than 30 days of age, the correlation between the initial oocyst 

inoculum and the MPN calculation is 0.9726. The relationship between the oocyst inoculum and 

the MPN diverge as the oocysts age. The 50% infective dose (ID50) in the cell culture system is
 

approximately 10 oocysts.
 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. 

Source: Slifko, T.R., Huffman, D.E., and Rose, J.B. 1999. “A Most-Probable-Number Assay for 

Enumeration of Infectious Cryptosporidium parvum Oocysts.” Applied and Environmental 

Microbiology. 65(9): 3936–3941. http://www.epa.gov/sam/pdfs/AEM-65(9)-pgs3936-3941.pdf
 

7.4.1.4 Literature Reference for Cryptosporidium spp. (Applied and 

Environmental Microbiology. 2007. 73(13): 4218–4225) 

Analysis Purpose: Detection, not suitable for viability 

Sample Preparation: Solid samples should be prepared according to “Quantification of Bias 

Related to the Extraction of DNA Directly from Soils,” Frostegard, A., Courtois, S., Ramisse, V., 

Clerc, S., Bernillion, D., Le Gall, F., Jeannin, P., Nesme, X., and Simonet, P. 1999. Applied and 

Environmental Microbiology. 65(12): 5409–5420 (www.epa.gov/sam/pdfs/AEM-65(12)-

pgs5409-5420.pdf). 

Analytical Technique: Real-time PCR 

Method Developed for: Cryptosporidium spp. in drinking water samples 

Method Selected for: SAM lists this protocol for detection in solid, particulate, liquid, and 

water samples. Further research is needed to develop and standardize the procedures for 

environmental sample types other than drinking water. 

Description of Method: Procedures are described for analysis of drinking water samples and 

may be adapted for assessment of solid, particulate, aerosol, and liquid samples. The method 

uses real-time PCR for identification of Cryptosporidium spp. C. parvum oocysts are seeded into 

100-L water samples at an average concentration of 590,000 ± 84,000 oocysts. One hundred 

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liters of the seeded drinking water is concentrated by hollow-fiber UF, with the filter eluted with 

a surfactant solution that is added to the retentate. A portion of the combined retentate is further 

concentrated with a 0.2 μm filter and one quarter of the filter used for PCR analysis. DNA for 

PCR analyses is recovered by bead beating, lysis of cells with a guanidine thiocyanate based 

buffer, and recovery and concentration of nucleic acids using spin columns. Real-time PCR is 

performed on an iCycler iQ4 detection system using primer sequences and a TaqMan® probe 

specific for Cryptosporidium spp. Samples from 8 sites were examined, with an average recovery 

efficiency of between 81% and 98%, with a cross-site average and standard deviation of 88% and 

10% respectively. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. 

Special Considerations: This method is only for genus-specific determination of 

Cryptosporidium and will not determine if the microorganism is infectious to humans. 

Source: Hill, V.R., Kahler, A.M., Jothikumar, N., Johnson, T.B., Hahn, D., and Cromeans, T.L. 

2007. “Multistate Evaluation of an Ultrafiltration-Based Procedure for Simultaneous Recovery of 

Enteric Microbes in 100-Liter Tap Water Samples.” Applied and Environmental Microbiology. 

73(13): 4218–4225. http://www.epa.gov/sam/pdfs/AEM-73(13)-pgs4218-4225.pdf 

7.4.2 Entamoeba histolytica – BSL-2 

Method Analytical Technique Section 

Journal of Parasitology. 1972. 58(2): 306–310 Culture 7.4.2.1 

Journal of Clinical Microbiology. 2005. 43(11): 

5491–5497 Real-time PCR 7.4.2.2 

7.4.2.1 Literature Reference for Entamoeba histolytica (Journal of Parasitology. 

1972. 58(2): 306–310) 

Analysis Purpose: Detection and viability
 

Sample Preparation: Samples should be prepared according to the procedures in Journal of
 

Parasitology. 1972. 58(2): 306–310. 

Analytical Technique: Culture 

Method Developed for: Entamoeba histolytica in clinical samples 

Method Selected for: SAM lists these procedures for detection and viability assessment in solid, 

particulate, liquid, and water samples. Further research is needed to develop and standardize the 

procedures for environmental sample types. 

Description of Method: Procedures are described for analysis of clinical samples and may be 

adapted for assessment of solid, particulate, liquid, and water samples. Entamoeba histolytica
 

cysts are placed in a modified trypticase-panmede liver digest-serum (TP-S-1) medium and 

incubated for 10 hours. Live amoebae excyst through a rupture in the cyst wall, whereas nonviable amoebae remain encysted. Microscopic examination of an aliquot of the incubated 

excystation culture allows calculation of the percent of empty (live) cysts and full (dead) cysts in 

a population. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. 

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Source: Stringert, R.P. 1972. “New Bioassay System for Evaluating Percent Survival of 

Entamoeba histolytica Cysts.” The Journal of Parasitology. 58(2): 306–310. 

http://www.epa.gov/sam/pdfs/JP-58(2)-pgs306-310.pdf 

7.4.2.2 Literature Reference for Entamoeba histolytica (Journal of Clinical 

Microbiology. 2005. 43(11): 5491–5497) 

Analysis Purpose: Detection, not suitable for viability 

Sample Preparation: Samples should be prepared according to the procedures in Journal of 

Clinical Microbiology. 2005. 43(11): 5491–5497. 

Analytical Technique: Real-time PCR 

Method Developed for: Entamoeba histolytica in clinical (fecal and liver abscess) samples 

Method Selected for: SAM lists these procedures for detection in solid, particulate, liquid, and 

water samples. Further research is needed to develop and standardize the procedures for 

environmental sample types. 

Description of Method: Procedures are described for analysis of clinical samples and may be 

adapted for assessment of solid, particulate, liquid, and water samples. The method is a real-time 

PCR assay that targets the 18S rRNA gene sequence of E. histolytica. DNA is extracted using 

cell disruption and a commercial DNA extraction kit, with a second commercial kit used to 

remove potential PCR inhibitors. TaqMan® real-time PCR is conducted on a GeneAmp® 9700 

Thermal Cycler the purified product. The LOD is 1 cell per mL of sample (SD ± 4). The method 

differentiates between E. histolytica and E. dispar. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. PCR QC checks should be 

performed according to EPA Draft Quality Assurance/Quality Control Guidance for Laboratories 

Performing PCR Analyses on Environmental Samples document www.epa.gov/sam/pdfs/EPAQA-QC_PCR_Oct2004.pdf or consult the point of contact identified in Section 4. 

Source: Qvarnstrom, Y., James, C., Xayavong, M., Holloway, B.P., Visvesvara, G.S., Sriram, 

R., and da Silva, A.J. 2005. “Comparison of Real-time PCR Protocols for Differential Laboratory 

Diagnosis of Amebiasis.” Journal of Clinical Microbiology. 43(11): 5491–5497. 

http://www.epa.gov/sam/pdfs/JCM-43(11)-pgs5491-5497.pdf 

7.4.3 Giardia spp. [Giardiasis] – BSL-2 

Method Analytical Technique Section 

EPA Method 1623 IMS/FA 7.4.1.2 

Transactions of the Royal Society of Tropical 

Medicine and Hygiene. 1983. 77(4): 487–488 Culture 7.4.3.1 

7.4.3.1 Literature Reference for Giardia spp. (Transactions of the Royal Society 

of Tropical Medicine and Hygiene. 1983. 77(4): 487–488) 

Analysis Purpose: Detection and viability 

Analytical Technique: Culture 

Method Developed for: Giardia in cell culture samples 

Method Selected for: SAM lists these procedures for detection and viability assessment in solid, 

particulate, liquid, and water samples. Further research is needed to develop and standardize the 

procedures for environmental sample types. 

 SAM Revision 5.0	 182 September 29, 2009 
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Description of Method: Procedures are described for analysis of cell culture samples and may 

be adapted for assessment of solid, particulate, liquid, and water samples. Trypticase-yeast-ironserum (TYI-S-33) medium supplemented with bovine bile and additional cysteine is used to 

isolate and culture Giardia lamblia. G. lamblia is incubated for intervals of 72 and 96 hours at 

36°C in borosilicate glass tubes. The cells form a dense, adherent monolayer on the surface of 

the glass. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. 

Source: Keister, D. 1983. “Axenic Culture of Giardia lamblia in TYI-S-33 Medium 

Supplemented with Bile.” Transactions of the Royal Society of Tropical Medicine and Hygiene. 

77(4): 487–488. http://www.sciencedirect.com/science/journal/00359203 

7.4.4 Toxoplasma gondii [Toxoplasmosis] – BSL-2 

Method Analytical Technique Section 

Emerging Infectious Diseases. 2006. 12(2): 326–329 Animal infectivity 7.4.4.1 

Applied and Environmental Microbiology. 2004. 

70(7): 4035–4039 Real-time PCR 7.4.4.2 

7.4.4.1 Literature Reference for Toxoplasma gondii (Emerging Infectious 

Diseases. 2006. 12(2): 326–329) 

Analysis Purpose: Detection and viability
 

Sample Preparation: Samples should be prepared according to the procedures in Emerging 

Infectious Diseases 12(2): 326–329. 

Analytical Technique: Animal infectivity
 

Method Developed for: Toxoplasma gondii in water 

Method Selected for: SAM lists these procedures for detection and viability assessment in solid, 

particulate, liquid, and water samples. Further research is needed to develop and standardize the 

procedures for environmental sample types other than water. 

Description of Method: Procedures are described for analysis of water samples and may be 

adapted for assessment of solid, particulate, liquid, and water samples. Water samples are filtered 

through fluoropore membrane filters and concentrated by centrifugation. The filters can be 

assayed by any of three methods. The first method involves performing a bioassay in T. gondiiseronegative chickens. Serum samples are tested by enzyme-linked immunosorbent assay
 

(ELISA) and/or modified agglutination test until seroconversion, with the organs from
 

seropositive animals examined microscopically for T. gondii. Mice are injected with brain and 

heart tissue of seropositive chickens, with parasites found in the lungs of mice being confirmatory
 

for T. gondii. The second method is a similar bioassay with pigs and cats. For the third assay, 

DNA is extracted from the fluoropore membranes for PCR identification of isolates. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. 

Source: de Moura, L., Bahia-Oliveira, L.M.G., Wada, M.Y., Jones, J.L., Tuboi, S.H., Carmo, 

E.H., Ramalho, W.M., Camargo, N.J., Trevisan, R., Graca, R.M.T., da Silva, A.J., Moura, I., 

Dubey, J.P., and Garrett, D.O. 2006. “Waterborne Toxoplasmosis, Brazil, from Field to Gene.”
 

 SAM Revision 5.0	 183 September 29, 2009 
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Emerging Infectious Diseases. 12(2): 326–329. http://www.epa.gov/sam/pdfs/EID-12(2)-pgs326-

329.pdf 

7.4.4.2 Literature Reference for Toxoplasma gondii (Applied and Environmental 

Microbiology. 2004. 70(7): 4035–4039) 

Analysis Purpose: Detection, not suitable for viability
 

Sample Preparation: Samples should be prepared according to the procedures in Applied and 

Environmental Microbiology 70(7): 4035–4039. 

Analytical Technique: Real-time PCR
 

Method Developed for: Toxoplasma gondii in water 

Method Selected for: SAM lists these procedures for detection of in solid, particulate, liquid, 

and water samples. Further research is needed to develop and standardize the procedures for 

environmental sample types other than water. 

Description of Method: Procedures are described for analysis of water samples and may be 

adapted for assessment of solid, particulate, and liquid samples. The method uses a fluorogenic 5'
 

nuclease (TaqMan®) real-time PCR assay for the detection of T. gondii oocyst DNA using genespecific (B1 gene) primers and probe. The assay uses an iCycler Real-Time PCR Detection 

System. Water samples (10 to 100 L) are filtered to concentrate oocysts. Filters are eluted and 

recovered oocysts are further purified and concentrated by differential flotation and 

centrifugation. Final sample pellets are split and subjected to PCR detection and mouse bioassay. 

In experimental seeding assays, a parasite density of 10 oocysts/L is detectable in 100% of the 

cases, and a density of 1 oocyst/L is observable in 60% of the cases. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. PCR QC checks should be
 

performed according to EPA Draft Quality Assurance/Quality Control Guidance for Laboratories 

Performing PCR Analyses on Environmental Samples document www.epa.gov/sam/pdfs/EPAQA-QC_PCR_Oct2004.pdf or consult the point of contact identified in Section 4.
 

Source: Villena, I., Aubert, D., Gomis, P., Ferte, H., Inglard, J-C., Denise-Bisiaux, H., Dondon, 

J-M., Pisano, E., Ortis, N., and Pinon, J.M. 2004. “Evaluation of a Strategy for Toxoplasma 

gondii Oocyst Detection in Water.” Applied and Environmental Microbiology. 70(7): 4035–4039. 

http://www.epa.gov/sam/pdfs/AEM-70(7)-pgs4035-4039.pdf 

7.5 Method Summaries for Helminths 

A summary of the analytical method for helminths listed in Appendix C is provided in Section 7.5.1. The 

summary contains sample preparation information, intended method application, a brief description of the 

method, performance data (if available), and a link to, or source for, obtaining a full version of the 

method. 

7.5.1 Baylisascaris procyonis [Raccoon roundworm fever] – BSL-2 

Method Analytical Technique Section 

EPA/625/R92/013 Embryonation of eggs and 

microscopy 7.5.1.1 

 SAM Revision 5.0	 184 September 29, 2009 
Section 7– Selected Pathogen Methods 

7.5.1.1 USEPA Environmental Regulations and Technology, Control of 

Pathogens and Vector Attraction in Sewage Sludge EPA/625/R-92/013, 

July 2003: Baylisascaris procyonis 

Analysis Purpose: Detection and viability 

Sample Preparation: Samples should be prepared according to the procedures in EPA/625/R92/013. 

Analytical Technique: Microscopy and embryonation of eggs 

Method Developed for: Baylisascaris procyonis in wastewater, sludge, and compost samples 

Method Selected for: SAM lists these protocols for detection and viability assessment of 

Baylisascaris procyonis in solid, particulate, liquid, and water samples. Further research is 

needed to develop and standardize the procedures for environmental sample types other than 

wastewater, sludge, and compost. 

Description of Method: The protocol describes procedures for analysis of solid and wastewater 

samples and may be adapted to analysis of particulate, liquid, water, and aerosol samples. 

Samples are processed by blending with buffered water containing a surfactant. The blend is 

screened to remove large particles, the solids in the screened portion are allowed to settle out, and 

the supernatant is decanted. The sediment is subjected to density gradient centrifugation using 

magnesium sulfate. This flotation procedure yields a layer likely to contain Ascaris and other 

parasite ova, if present in the sample. Small particulates are removed by a second screening on a 

small mesh size screen. The resulting concentrate is incubated until control Ascaris eggs are fully 

embryonated. The concentrate is then microscopically examined for the categories of Ascaris ova 

on a counting chamber. 

At a minimum, the following QC checks should be performed and evaluated when using this 

protocol: positive control, negative control, and blank. Ongoing analysis of QC samples to 

ensure reliability of the analytical results should also be performed. 

Source: EPA. 2003. “Appendix I: Test Method for Detecting, Enumerating, and Determining 

the Viability of Ascaris Ova in Sludge.” USEPA Environmental Regulations and Technology: 

Control of Pathogens and Vector Attractions in Sewage Sludge, EPA/625/R-92/013. 

http://www.epa.gov/sam/pdfs/EPA-625-R-92-013.pdf 

 SAM Revision 5.0	 185 September 29, 2009 
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 SAM Revision 5.0 186 September 29, 2009 
Section 8– Selected Biotoxin Methods 

Section 8.0: Selected Biotoxin Methods 

A list of methods or procedures to be used in analyzing environmental samples for biotoxin contaminants 

is provided in Appendix D. These methods should be used to support remediation activities (site 

assessment through clearance) following a homeland security event. Procedures have been compiled for 

each biotoxin that may need to be identified and/or quantified following a contamination incident. 

Analytical procedures are not currently available for all the analyte-sample type combinations included in 

this document. Future research needs include identification of additional methods and development and 

validation of the procedures listed. Appendix D is sorted alphabetically by analyte, within each of two 

analyte types (i.e., protein and small molecule). 

Please note: This section provides guidance for selecting biotoxin methods that have a high likelihood 

of assuring analytical consistency when laboratories are faced with a large-scale environmental 

restoration crisis. Not all methods have been verified for the analyte/sample type combination listed in 

Appendix D. Please refer to the specified method to identify analyte/sample type combinations that 

have been verified. Any questions regarding information discussed in this section should be addressed 

to the appropriate contact(s) listed in Section 4. 

Appendix D provides the following information: 

•	 Analyte(s). The compound or compound(s) of interest. 

•	 CAS RN / Description. A unique identifier for substances that provides an unambiguous way to 

identify a toxin or toxin isoform when there are many possible systematic, generic, or trivial names 

and/or a brief statement describing the toxin. 

•	 Analysis type. Tests are either for presumptive identification, confirmatory identification, or 

biological activity determination. 

•	 Analytical Technique. An analytical instrument or technique used to determine the quantity and 

identification of compounds or components in a sample. 

•	 Analytical Method. The recommended method or procedure, and the corresponding publisher. 

•	 Aerosol (filter/cassette or liquid impinger). The recommended method/procedure to measure the 

analyte of interest in air sample collection media such as filter cassettes and liquid impingers. 

•	 Solid (soil, powder). The recommended method/procedure to measure the analyte of interest in solid 

samples such as soil and powders. 

•	 Particulate (swabs, wipes, filters). The recommended method/procedure to measure the analyte of 

interest in particulate sample collection media such as swabs, wipes and dust-collecting socks used 

with vacuum collection. 

•	 Liquid/water. The recommended method/procedure to measure the analyte of interest in liquid and 

water samples. 

•	 Drinking water. The recommended method/procedure to measure the analyte of interest in drinking 

water samples. 

Following a homeland security event, it is assumed that only those areas with contamination greater than 

pre-existing, naturally prevalent levels commonly found in the environment would be subject to 

remediation. Dependent on site- and event-specific goals, investigation of background levels using 

methods listed in Appendix D is recommended. 

Procedures listed in Appendix D for protein biotoxins are intended to address presumptive, confirmatory, 

and biological activity determinations. Because the confirmatory procedures listed for the small molecule 

biotoxins involve a determination of intact compound structure (an indication of biological activity 

capability), only presumptive and confirmatory methods are listed for these biotoxins. In terms of this 

document, presumptive methods, or methods that support a reasonable basis for accurate results, should 

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Section 8– Selected Biotoxin Methods 

be used in situations that require a large number of samples to be processed. Most of the presumptive 

methods listed in Appendix D use immunoassay techniques and are designed for large scale sample 

processing. The confirmatory method, or the method that corroborates the presumptive results, may be 

used when presumptive analysis indicates the presence of the biotoxin. Several techniques are listed in 

Appendix D as confirmatory and generally are more time consuming and expensive. The use of these 

terms in this document is not intended to redefine LRN usage of these terms. The terms presumptive and 

confirmatory as used by the LRN are described in Section 8.2.1. If it is necessary to determine the 

biological activity of a toxin, either an assay (for proteins) or a technique such as HPLC that determines 

whether the structure of the biotoxin is intact and likely to be biologically active (for small molecules) 

may be used. Biological activity analysis should be applied on an as-needed basis following analysis with 

the confirmatory technique. 

Numerous analytical techniques using a variety of instrumentation (e.g., high performance liquid 

chromatography – mass spectrometer [HPLC-MS], HPLC-FL, immunoassay [ELISA], immunoassay 

[lateral flow device (LFD)], etc.) have been cited in Appendix D. It is expected that a reduced number of 

these analytical techniques and instrumentation will be necessary after method verification and validation. 

In addition, it is recognized that new reports detailing advances in biotoxin analysis appear in the 

literature frequently. Accordingly, the individual techniques and methods listed in Appendix D are to be 

regarded as a starting point; after thoughtful consideration of current technologies at the time of 

remediation and consultation with the authority in charge of the remediation activity, these techniques and 

methods can be modified as necessary for analysis of a particular sample. 

The presence of disinfectants (e.g., chlorine) and/or preservatives added during water sample collection to 

slow degradation (e.g., pH adjustors, de-chlorinating agents) could possibly affect analytical results. 

When present, the impact of these agents on method performance should be evaluated if not previously 

determined. EPA’s NHSRC is working on a sample collection document that is intended as a companion 

to SAM. This sample collection document will provide information regarding sampling container/media, 

preservation, holding time, sample size, and shipping and is intended to complement the laboratory 

analytical methods that are the focus of the SAM document. 

8.1 General Guidelines 

This section provides a general overview of how to identify the appropriate method(s) for a given 

biotoxin as well as recommendations for QC procedures. 

For additional information on the properties of the biotoxins listed in Appendix D, TOXNET 

(http://toxnet.nlm.nih.gov/index.html), a cluster of databases on toxicology, hazardous chemicals, and 

related areas maintained by the National Library of Medicine, is an excellent resource. 

Additional resources include: 

•	 Defense Against Toxin Weapons, published by the U.S. Army Medical Research Institute of 

Infectious Diseases (http://www.usamriid.army.mil/education/defensetox/toxdefbook.pdf) contains 

information regarding sample collection, toxin analysis and identification, as well as decontamination 

and water treatment. 

•	 Select Agent Rules and Regulations found at the National Select Agent Registry 

(http://www.selectagents.gov/) 

•	 The CDC has additional information regarding select agent toxins at the following Web site: 

http://www.cdc.gov/od/sap/sap/toxinamt.htm 

•	 SRC’s PHYSPROP and Chemfate, part of the Environmental Fate Database supported by EPA. See 

http://srcinc.com/what-we-do/product.aspx?id=133. 

•	 INCHEM at http://www.inchem.org/ contains both chemical and toxicity information. 

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Section 8– Selected Biotoxin Methods 

•	 The RTECS database can be accessed via the NIOSH Web site at 

http://www.cdc.gov/niosh/rtecs/default.html for toxicity information. 

•	 The Forensic Science and Communications Journal published by the Laboratory Division of the FBI. 

See http://www.fbi.gov/hq/lab/fsc/current/backissu.htm. 

Additional research on biotoxin contaminants is ongoing within EPA. 

8.1.1 Standard Operating Procedures for Identifying Biotoxin Methods 

To determine the appropriate method that is to be used on an environmental sample, locate the biotoxin of 

concern in Appendix D: Biotoxin Methods under the “Analyte(s)” column. After locating the biotoxin, 

continue across the table and identify the appropriate analysis type. After an analysis type has been 

chosen, find the analytical technique (e.g., immunoassay) and analytical method applicable to the sample 

type of interest (solid, particulate, liquid/drinking water, or aerosol) corresponding to that particular 

analyte. 

Once a method has been identified in Appendix D, the corresponding method summary can be found in 

Sections 8.2.1 through 8.3.12. Method summaries are listed first by alphabetical order within each 

biotoxin subcategory (i.e., protein and small molecule) and then in order of method selection hierarchy 

(see Figure 2-1), starting with EPA methods, followed by methods from other federal agencies, VCSBs, 

and journal articles. Where available, a direct link to the full text of the method is provided with the 

method summary. For additional information on sample preparation procedures and methods available 

through consensus standards organizations, please use the contact information provided in Table 8-1. 

Table 8-1. Sources of Biotoxin Methods 

Name Publisher Reference 

FDA, Bacteriological Analytical Manual 

Online 

FDA http://www.cfsan.fda.gov/~ebam/bamtoc.html 

Official Methods of Analysis of AOAC 

International* 

AOAC International http://www.aoac.org 

NEMI EPA, USGS http://www.nemi.gov/ 

Pharmacology & Toxicology* Blackwell Synergy http://www.blackwell-synergy.com/loi/pto 

Analytical Biochemistry* Science Direct http://www.sciencedirect.com/ 

Biochemical Journal* Portland Press Ltd. http://www.biochemj.org/ 

Journal of Medicinal Chemistry* American Chemical Society http://www.acs.org/ 

Journal of Food Protection* International Association for 

Food Protection 

http://www.foodprotection.org/ 

Journal of Chromatography B* Elsevier Science Publishers http://www.elsevier.com/ 

Biomedical Chromatography* John Wiley And Sons Ltd http://www.wiley.com/ 

Environmental Health Perspectives* National Institute of 

Environmental Health 

Sciences 

http://www.niehs.nih.gov/ 

Toxicon* Elsevier Science Publishers http://www.elsevier.com/ 

Federation of European Microbiological 

Societies (FEMS) Microbiology Letters* 

Wiley-Blackwell http://www.wiley.com/ 

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Section 8– Selected Biotoxin Methods 

Name Publisher Reference 

International Journal of Food 

Microbiology* 

Elsevier Science Publishers http://www.elsevier.com/ 

Rapid Communications in Mass 

Spectrometry * 

John Wiley And Sons Ltd. http://www.wiley.com/ 

Journal of AOAC International* AOAC International http://www.aoac.org 

Analyst* Royal Society of Chemistry http://www.rsc.org/ 

Journal of Pharmaceutical and 

Biomedical Analysis* 

Elsevier Science Publishers http://www.elsevier.com/ 

Journal of Clinical Microbiology ASM http://www.asm.org/ 

Journal of Clinical Laboratory Analysis* John Wiley And Sons Ltd. http://www.wiley.com/ 

Journal of Analytical Toxicology* S. Tinsley Preston http://www.jatox.com/ 

Lateral Flow Immunoassay Kits Environmental Technology 

Verification (ETV) Program 

http://www.epa.gov/etv/ 

* Subscription and/or purchase required. 

8.1.2 General QC Guidelines for Biotoxin Methods 

Having data of known and documented quality is critical so that public officials can accurately assess the 

activities that may be needed in remediating a site during and following emergency situations. Having 

such data requires that laboratories: (1) conduct the necessary QC to ensure that measurement systems are 

in control and operating properly; (2) properly document results of the analyses; and (3) properly 

document measurement system evaluation of the analysis-specific QC. Ensuring data quality also 

requires that laboratory results are properly evaluated and the results of the data quality evaluation are 

transmitted to decision makers. 

The level or amount of QC needed often depends on the intended purpose of the data that are generated. 

Various levels of QC may be required if the data are generated during presence/absence determinations 

versus confirmatory analyses. The specific needs for data generation should be identified. QC 

requirements and data quality objectives should be derived based on those needs and should be applied 

consistently across laboratories when multiple laboratories are used. For example, during rapid sample 

screening, minimal QC samples (e.g., blanks, duplicates) and documentation might be required to ensure 

data quality. Sample analyses for environmental evaluation during site assessment through site clearance, 

such as those identified in this document, might require increased QC (e.g., demonstrations of method 

sensitivity, precision, and accuracy). 

While method-specific QC requirements may be included in many of the procedures that are cited in this 

document, and will be referenced in any SAPs developed to address specific analytes and sample types of 

concern, the following describes a minimum set of QC samples and procedures that should be conducted 

for all analyses. Individual methods, sampling and analysis protocols, or contractual statements of work 

also should be consulted to determine any additional QC that may be needed. QC tests should be run as 

frequently as necessary to ensure the reliability of analytical results. In general, sufficient QC includes an 

initial demonstration of measurement system capability as well as ongoing assessments to ensure the 

continued reliability of the analytical results. 

Examples of sufficient QC for the presumptive tests listed in Appendix D include: 

• Method blanks; 

• Positive test samples / negative test samples; 

• Calibration check samples; 

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Section 8– Selected Biotoxin Methods 

•	 Use of test kits and reagents prior to expiration; and 

•	 Accurate temperature controls. 

Examples of sufficient QC for the confirmatory tests listed in Appendix D include: 

•	 Demonstration that the measurement system is operating properly 

< Initial calibration 

< Method blanks 

•	 Demonstration of measurement system suitability for intended use 

< Precision and recovery (verify measurement system has adequate accuracy) 

< Analyte/sample type/level of concern-specific QC samples (verify that measurement system has 

adequate sensitivity at levels of concern) 

•	 Demonstration of continued measurement system reliability 

< MS/MSDs (recovery and precision) 

< QC samples (system accuracy and sensitivity at levels of concern) 

< Continuing calibration verification 

< Method blanks 

Please note: The appropriate point of contact identified in Section 4 should be consulted regarding 

appropriate QA/QC procedures prior to sample analysis. These contacts will consult with the EPA 

OSWER coordinator responsible for laboratory activities during the specific event to ensure QA/QC 

procedures are performed consistently across laboratories. OSWER is planning to develop QA/QC 

guidance for laboratory support. EPA program offices will be responsible for ensuring that the QA/QC 

practices are implemented. 

8.1.3 Safety and Waste Management 

It is imperative that safety precautions be used during collection, processing, and analysis of 

environmental samples. Laboratories should have a documented health and safety plan for handling 

samples that may contain target CBR contaminants, and laboratory staff should be trained in and 

implement the safety procedures included in the plan. In addition, many of the methods summarized or 

cited in Section 8.2 contain some specific requirements, guidelines, or information regarding safety 

precautions that should be followed when handling or processing environmental samples and reagents. 

These methods also provide information regarding waste management. Other resources that can be 

consulted for additional information include the following: 

•	 American Biological Safety Association, Risk Group Classifications for Infectious Agents, available 

at http://www.absa.org/riskgroups/index.html. 

•	 BMBL, 5th Edition, found at http://www.cdc.gov/OD/ohs/biosfty/bmbl5/bmbl5toc.htm. 

•	 Biological Safety: Principles and Practices, 4th Ed. ASMPress (http://estore.asm.org/). 

•	 CDC – 42 CFR part 72. Interstate Shipment of Etiologic Agents. 

•	 CDC – 42 CFR part 73. Select Agents and Toxins. 

•	 DOT – 49 CFR part 172. Hazardous Materials Table, Special Provisions, Hazardous Materials 

Communications, Emergency Response Information, and Training Requirements. 

•	 EPA – 40 CFR part 260. Hazardous Waste Management System: General. 

•	 EPA – 40 CFR part 270. EPA Administered Permit Programs: The Hazardous Waste Permit 

Program. 

•	 OSHA – 29 CFR part 1910.1450. Occupational Exposure to Hazardous Chemicals in Laboratories. 

 SAM Revision 5.0	 191 September 29, 2009 
Section 8– Selected Biotoxin Methods 

• OSHA – 29 CFR part 1910.120. Hazardous Waste Operations and Emergency Response. 

• USDA – 9 CFR part 121. Possession, Use, and Transfer of Select Agents and Toxins. 

Please note that the e-CFR is available at http://ecfr.gpoaccess.gov/. 

8.1.4 Laboratory Response Network (LRN) 

The agents and sample types identified below and listed in Appendix D are included in the HHS/USDA 

select agent list and should be analyzed in accordance with the appropriate LRN protocols. Additional 

information on select agents and regulations may be obtained at the National Select Agent Registry at: 

http://www.selectagents.gov/. 

The LRN was created in accordance with Presidential Directive 39, which established terrorism 

preparedness responsibilities for federal agencies. The LRN is primarily a national network of local, 

state, federal, military, food, agricultural, veterinary, and environmental laboratories; however, additional 

LRN laboratories are located in strategic international locations. The CDC provides technical and 

scientific support to member laboratories as well as secure access to standardized procedures and reagents 

for rapid (within 4 to 6 hours) presumptive detection of biothreat agents and emerging infectious disease 

agents. These rapid presumptive assays are part of agent-specific algorithms of assays which lead to a 

confirmed result. The algorithm for a confirmed result is often a combination of one or more presumptive 

positive results from a rapid assay in combination with a positive result from one of the “gold standard” 

methods, such as culture. The standardized procedures, reagents, and agent-specific algorithms are 

considered to be sensitive and are available only to LRN member laboratories. Thus, these procedures are 

not available to the general public and are not discussed in this document. 

It is important to note that, in some cases, the procedures may not be fully developed or validated for each 

environmental sample type/analyte combination listed in Appendix D, nor are all LRN member 

laboratories necessarily capable of analyzing all of the sample type/analyte combinations. 

Analyte(s) Sample Type CAS RN / Description 

Botulinum neurotoxins (Serotypes A, B, E, F) Solid, Particulate, 

Liquid/Water, Drinking Water 

Protein composed of ~100 kDa 

heavy chain and ~50 kDa light 

chain; can be complexed with 

hemagglutinin and nonhemagglutinin components for total 

MW of ~900 kDa 

Ricin Solid, Particulate, 

Liquid/Water, Drinking Water 

9009-86-3 (ricin) / 60 kDa 

glycoprotein composed of two 

subunits (~32 kDa A chain and ~34 

kDa B chain); an agglutinin of MW 

120 kDa may be present in crude 

preparations 

Staphylococcal enterotoxin B (SEB) Solid, Particulate, 

Liquid/Water, Drinking Water 

39424-53-8 (SEB) / Monomeric 

protein of almost 28 kDa 

Please note: Not all methods have been verified for the biotoxin/sample type combination listed in 

Appendix D. Please refer to the agent-specific method to identify the biotoxin/sample type combinations 

that have been validated. Any questions regarding information discussed in this section should be 

referred to the appropriate contact(s) listed in Section 4. 

 SAM Revision 5.0 192 September 29, 2009 
Section 8– Selected Biotoxin Methods 

For additional information on the LRN, including selection of a laboratory capable of receiving and 

processing the specified sample type/analyte, please use the contact information provided below or visit 

http://www.bt.cdc.gov/lrn/. 

Centers for Disease Control and Prevention 

Laboratory Response Branch 

Division of Bioterrorism Preparedness and Response (DBPR) 

National Center for Prevention, Detection, and Control of Infectious Diseases (NCPDCID) 

Coordinating Center for Infectious Diseases (CCID) 

Centers for Disease Control and Prevention (CDC) 

1600 Clifton Road NE, Mailstop C-18 

Atlanta, GA 30333 

Telephone: (404) 639-2790 

E-mail: lrn@cdc.gov 

Local public health laboratories, private, and commercial laboratories with questions about the LRN 

should contact their state public health laboratory director or the APHL (contact information provided 

below). 

Association of Public Health Laboratories 

8515 Georgia Avenue, Suite 700 

Silver Spring, MD 20910 

Telephone: (240) 485-2745 

Fax: (240) 485-2700 

Web site: www.aphl.org 

E-mail: info@aphl.org 

8.2 Method Summaries for Protein Biotoxins 

Summaries of the analytical methods for protein biotoxins listed in Appendix D are provided in Sections 

8.2.1 through 8.2.5. These sections contain summary information only, extracted from the selected 

methods. The full version of the method should be consulted prior to sample analysis. 

Each summary contains a brief description of the method, intended method application, performance data 

(if available), and a link to or source for obtaining a full version of the method. 

8.2.1 Abrin 

CAS RN: 1393-62-0. 

Description: Glycoprotein consisting of a deadenylase (25–32 kDa A chain) and lectin (35 kDa 

B chain); an agglutinin (A2B2) may be present in crude preparations. 

Method Analytical Technique Section 

Journal of Food Protection. 2008. 71(9): 1868–1874 Immunoassay 8.2.1.1 

Pharmacology & Toxicology. 2001. 88(5): 255–260 Ribosome inactivation assay 8.2.1.2 

Analytical Biochemistry. 2008. 378: 87–89 Enzyme activity 8.2.1.3 

8.2.1.1 Literature Reference for Abrin (Journal of Food Protection. 2008. 71(9): 

1868–1874) 

Analysis Purpose: Presumptive 

Analytical Technique: Immunoassay 

 SAM Revision 5.0	 193 September 29, 2009 
Section 8– Selected Biotoxin Methods 

Method Developed for: Abrin in food 

Method Selected for: SAM lists these procedures for presumptive analysis in aerosol, solid, 

particulate, liquid, and water samples. Further research is needed to develop and standardize the 

procedures for environmental sample types. 

Description of Method: Procedures are described for using mouse mAbs and rabbit-derived 

polyclonal antibodies prepared against a mixture of abrin isozymes for three separate ELISA and 

electrochemiluminescence (ECL)-based assays in food products. The three assays vary by use of 

antibody combination (e.g, assay configuration): (1) polyclonal (capture)/polyclonal (detection) 

ELISA, (2) polyclonal/monoclonal ELISA, and (3) polyclonal/monoclonal ECL assay. The 

LODs, with purified Abrin C and various abrin extracts in buffer, are between 0.1 and 0.5 ng/mL
 

for all three assays. The LOD for abrin spiked into food products ranged from 0.1 to 0.5 ng/mL, 

using the ECL assay. The LOD for abrin spiked into food products for the ELISA assays ranged 

between 0.5 and 10 ng/mL depending on the antibody combination. In all cases, the LODs were
 

less than the concentration at which abrin may pose a health concern.
 

Special Considerations: Crude preparations of abrin may also contain agglutinins that are 

unique to rosary peas and that can cross-react in the immunoassays. Addition of non-fat milk 

powder to the sample buffer may eliminate false-positive results (Dayan-Kenigsberg, J., 

Bertocchi, A., and Garber, E.A. 2008. “Rapid Detection of Ricin in Cosmetics and Elimination of 

Artifacts Associated with Wheat Lectin.” Journal of Immunological Methods. 336(2): 251–254). 

http://www.sciencedirect.com/science/journal/00221759 

Source: Garber, E.A., Walker, J.L., and O’Brien, T.W. 2008. “Detection of Abrin in Foods 

Using Enzyme-Linked Immunosorbent Assay and Electrochemiluminescence Technologies.” 

Journal of Food Protection. 71(9): 1868–1874. 

http://www.ingentaconnect.com/content/iafp/jfp/2008/00000071/00000009/art00015 

8.2.1.2 Literature Reference for Abrin and Ricin (Analytical Biochemistry. 2008. 

378(1): 87-89) 

Analysis Purpose: Biological activity 

Analytical Technique: Enzyme activity 

Method Developed for: Jequirity seed (abrin) and castor bean (ricin) extracts in buffer 

Method Selected for: SAM lists these procedures for biological activity analysis in aerosol, 

solid, particulate, liquid, and water samples. Further research is needed to develop and 

standardize the procedures for environmental sample types. 

Description of Method: This in vitro assay is an RNA N-glycosidase enzyme activity assay for 

the detection of purified abrin and ricin toxins (Types I and II) or in jequirity seed (abrin) and 

castor bean (ricin) extracts. Synthetic biotinylated RNA substrates with varied loop sequences 

are cleaved by either the ricin or abrin toxin and the RNA products are hybridized to 

ruthenylated-oligodeoxynucleotides to generate an ECL signal. Assays require incubation for 2 

hours at 48°C. Commercially available ECL-based reagents and RNase inactivators are used. 

Control experiments for the jequirity seed experiments and the distinct GdAA/GdAGA ratio for 

the castor bean assay demonstrate lack of non-specific cleavage for the assay. The undiluted 

castor bean extract contained 22.0 ± 0.7 mg/mL total protein and 4.1 ± 0.3 mg/mL ricin 

equivalents as determined by standard protein determination and by ECL immunoassay assays 

respectively. The undiluted jequirity seed extract was similarly assayed, with a resultant 21.6 ± 

0.6 mg/mL total protein and 3.7 ± 0.3 μg/mL equivalents of toxin. Dilutions were performed to 

determine effective signal-to-background ratio and the linear range for calculation of toxin 

activity. Resultant calculations for ricin activity equivalents in the undiluted castor bean extract 

were equivalent to those obtained with the ECL immunoassays: 4.4 ± 0.2 mg/mL activity 

 SAM Revision 5.0	 194 September 29, 2009 
Section 8– Selected Biotoxin Methods 

equivalents. In contrast, the undiluted jequirity seed extract contained a calculated level of 740 ± 

50 μg/mL activity equivalents, which greatly exceeded the immunoassay-based value. 

Special Considerations: This enzyme activity assay does not test for cell binding; cell culture 

assays are being developed to test for cell binding but are not currently available. The only 

readily available assay to test for both the cell binding and enzymatic activity of the intact 

(whole) toxin is the mouse bioassay. 

Source: Keener, W.K., Rivera, V.R., Cho, C.R., Hale, M.L., Garber, E.A.E., and Poli, M.A. 

2008. “Identification of the RNA N-glycosidase Activity of Ricin in Castor bean extracts by an 

Electrochemiluminescence-based Assay.” Analytical Biochemistry. 378(1): 87–89. 

http://www.sciencedirect.com/science/journal/00032697 

8.2.1.3 Literature Reference for Abrin and Shiga and Shiga-like Toxins 

(Pharmacology Toxicology. 2001. 88(5): 255–260) 

Analysis Purpose: Confirmatory for abrin; biological activity for shiga and shiga-like toxins 

Analytical Technique: Ribosome inactivation assay 

Method Developed for: Abrin in phosphate buffered saline (PBS) 

Method Selected for: SAM lists these procedures for confirmatory analysis in aerosol, solid, 

particulate, liquid, and water samples. Further research is needed to develop and standardize the 

procedures for environmental sample types. 

Description of Method: Procedures are described for measuring the biological activity of 

ribosome-inactivating proteins using a microtiter plate format for detection of abrin in PBS. 

Nuclease-treated rabbit reticulocyte lysate containing luciferase messenger ribonucleic acid 

(mRNA) is used to measure toxin activity via inhibition of protein synthesis. The relative 

biological activity is determined by comparing luminescence levels in treated samples versus 

those of untreated controls. The amount of luciferase translated, as measured by luminescence, is 

inversely proportional to the toxin concentration. Linear dose response curves are generated for 

abrin, with a 50% inhibition of translation at 0.5 nM. Coupling this procedure, or a modification 

of this procedure, with an immunoassay will provide more information regarding the specificity 

and toxicity of the target biotoxin.
 

Special Considerations: For abrin, as well as shiga and shiga-like toxins, this assay does not 

test for cell binding; cell culture assays are being developed to test for cell binding but are not 

currently available. The only readily available assay to test for both the cell binding and 

enzymatic activity of the intact (whole) toxin is the mouse bioassay. 

Source: Hale, M.L. 2001. “Microtiter-based Assay for Evaluating the Biological Activity of 

Ribosome-inactivation Proteins.” Pharmacology Toxicology. 88(5): 255–260. 

http://www3.interscience.wiley.com/journal/120703798/abstract
 

8.2.2 Botulinum neurotoxins (Serotypes A, B, E, F) 

Description: Protein composed of ~100 kDa heavy chain and ~50 kDa light chain; can be 

complexed with hemagglutinin and non-hemagglutinin components for total MW of ~900 kDa. 

Method Analytical Technique Section 

LRN Immunoassay, Immunoassay 

(ELISA) and Mouse bioassay 8.1.4 

FDA, Bacteriological Analytical Manual Online, January 

2001, Chapter 17, Clostridium botulinum 

Immunoassay (ELISA) and 

Mouse bioassay 8.2.2.1 

Lateral Flow Immunoassay Kits Immunoassay 8.2.2.2 

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8.2.2.1 FDA, Bacteriological Analytical Manual Online, Chapter 17, 2001: 

Botulinum Neurotoxins 

Analysis Purpose: Confirmatory and biological activity 

Analytical Technique: Immunoassay (ELISA) and mouse bioassay 

Method Developed for: Botulinum neurotoxins (Serotypes A, B, E, F) in food 

Method Selected for: SAM lists this procedure for confirmation and biological activity 

assessment in aerosol samples. Further research is needed to develop and standardize the 

procedures for environmental sample types. 

Description of Method: An amplified-enzyme-linked immunosorbent assay (amp-ELISA) and a 

digoxigenin-labeled enzyme-linked immunosorbent assay (DIG-ELISA) are described for the 

detection of Types A, B, E, and F botulinum neurotoxins in food products. The amp-ELISA 

method uses goat anti-A or E, rabbit anti-B, or horse anti-F serum to capture the toxins in a 96-

well plate, and a corresponding biotinylated goat antitoxin to detect the toxin. Visualization is 

with streptavidin-alkaline phosphatase. The DIG-ELISA method is a modification of the ampELISA method, with digoxigenin-labeled antitoxin IgG’s substituted for the streptavidin-alkaline 

phosphatase. Toxin can be detected at approximately 10 minimum lethal doses (MLD)/mL (0.12 

to 0.25 ng/mL). High concentration samples (greater than 10,000 MLD/mL) may give a positive 

absorbance for more than one toxin type. Further dilution of the sample will remove crossreactivity. 

The mouse bioassay detects biologically active toxin using a three part approach: toxin screening; 

toxin titer; and finally, toxin neutralization using monovalent antitoxin sera. Samples are 

prepared by centrifugation for suspended solids under refrigeration, or solids are extracted with 

an equal volume of pH 6.2 gel-phosphate buffer and then centrifuged. Toxins from 

nonproteolytic strains of C. botulinum may need trypsin activation to be detected. Serial dilutions 

of untreated and trypsin-treated sample fluids are injected in separate pairs of mice 

intraperitoneally (i.p.). Mice are also injected with heated, untreated, undiluted sample. Death of 

mice, along with symptoms of botulism, confirms presence of botulinum toxin. After calculation 

of an MLD, dilute monovalent antitoxin sera types A, B, E, and F are injected into mice 30 

minutes to 1 hour before challenging them with the i.p. injection of each dilution that gave the 

highest MLD from the toxic preparation. 

Special Considerations: Immunoassays with botulinum toxins may produce variable results 

with uncomplexed form of toxin. 

Source: FDA, CFSAN. 2001. “Chapter 17 – Clostridium botulinum.” Bacteriological Analytical 

Manual Online. http://www.epa.gov/sam/pdfs/FDA-BAM-Chap17.pdf 

8.2.2.2 Lateral Flow Immunoassay Kits 

Analysis Purpose: Presumptive 

Analytical Technique: Immunoassay 

Method Developed for: Botulinum neurotoxins (Types A, B) and ricin in buffer or water 

samples 

Method Selected for: SAM lists these procedures for presumptive analysis in aerosol samples. 

Further research is needed to develop and standardize the procedures for environmental sample 

types other than water. 

Description of Method: Test strips are self-contained, qualitative assays for screening 

environmental samples for the presence of botulinum toxin and ricin. After the sample is 

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collected, it is transferred onto the test strip where dye-labeled antibodies detect trace amounts of 

the contaminant, as indicated by the presence of two bands in the test result window. After 15 

minutes, the results are read visually. Botulinum neurotoxin Type A can be detected at 5 mg/L 

and Type B at 4 mg/L, 33% of the time. Ricin toxin can be detected at 20 mg/L, with no crossreactivity to certain substances (i.e., lectin from soybeans). 

An alternative lateral flow immunochromatographic device also can be used. This device uses 

two antibodies in combination to specifically detect target antigen in solution. When a sufficient 

amount of target antigen is present, the colloidal gold label accumulates in the sample window on 

a test strip, forming a visible reddish-brown colored line. The presence of two bands indicates a 

positive reading. Botulinum neurotoxin Type A can be detected at 0.01 mg/L and Type B at 0.5 

mg/L, with no false negatives detected when interferents are present. Ricin toxin can be detected 

at 0.035 mg/L, with 88% accuracy. 

These two lateral flow immunoassay kits have been evaluated by the EPA ETV Program 

(http://www.epa.gov/sam/pdfs/ETV-BADD091904.pdf and http://www.epa.gov/sam/pdfs/ETVBioThreat092104.pdf) for the detection of botulinum neurotoxins Types A and B and ricin. 

Information regarding the evaluation of test strips can be accessed at these sites. 

Special Considerations: Immunoassays with botulinum toxins may produce variable results with 

uncomplexed form of toxin. Addition of non-fat milk powder to the sample buffer may eliminate 

false-positive results (Dayan-Kenigsberg, Bertocchi, J.A., and Garber, E.A.E. 2008. “Rapid 

Detection of Ricin in Cosmetics and Elimination of Artifacts Associated with Wheat Lectin.” 

Journal of Immunological Methods. 336(2): 251–254). 

http://www.sciencedirect.com/science/journal/00221759 

Source: ETV. 2006. http://www.epa.gov/etv/ 

8.2.3 Ricin (Ricinine) 

Ricin – CAS RN: 9009-86-3. 

Description: 60 kDa glycoprotein composed of two subunits (~32 kDa A chain and ~34 kDa B 

chain); an agglutinin of MW 120 kDa may be present in crude preparations. 

Ricinine – CAS RN: 5254-40-3. 

Description: Small molecule, ricin marker.
 

Method Analytical Technique Section 

LRN Immunoassay 8.1.4 

Analytical Biochemistry. 2008. 378: 87–89 Enzyme activity 8.2.1.3 

Lateral Flow Immunoassay Kits Immunoassay 8.2.2.2 

Journal of AOAC International. 2008. 91(2): 376–382 Immunoassay 8.2.3.1 

Journal of Analytical Toxicology. 2005. 29: 149–155 LC-MS 8.2.3.2 

8.2.3.1 Literature Reference for Ricin (Journal of AOAC International. 2008. 

91(2): 376–382) 

Analysis Purpose: Confirmatory 

Analytical Technique: Immunoassay 

Method Developed for: Ricin for food products 

Method Selected for: SAM lists these procedures for confirmatory analysis in aerosol, solid, 

particulate, liquid, and water samples. Further research is needed to develop and standardize the 

procedures for environmental sample types. 

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Description of Method: This immunoassay is for the detection of various concentrations of 

purified ricin in food products (e.g., juice, dairy products, vegetables, bakery products, 

condiments). The immunoassay uses ECL detection in a 96-well plate format with a monoclonal 

capture antibody against ricin (19A-2C6) and either a polyclonal or monoclonal detector 

antibody. The samples and detector antibodies can be added sequentially or in combination 

during the capture step. Using the polyclonal antibody, ricin was detected at concentrations as 

low as 0.04 ng/mL. Simultaneous addition of sample and detector antibody allowed for a 

shortened procedure with only a single 20 minute incubation with no false negatives caused by 

“hook” effects at high concentrations of ricin. Quantitation can be performed either with the 

sequential procedure or with the simultaneous procedure if it is know that the ricin concentration 

is not in the “hook” region. The simultaneous procedure should not be used when a sample 

contains constituents that may react with the ruthenium tag. Polyclonal/monoclonal antibodies 

are commercially available as an ELISA test kit. 

Special Considerations: Crude preparations of ricin may also contain agglutinins that are 

unique to castor beans and that can cross-react in the immunoassays. 

Source: Garber, E.A.E., and O’Brien, T. W. 2008. “Detection of Ricin in Food Using 

Electrochemiluminescence-Based Technology.” Journal of AOAC International. 91(2): 376–382. 

http://www.atypon-link.com/AOAC/doi/abs/10.5555/jaoi.91.2.376 

8.2.3.2 Literature Reference for Ricin by Ricinine detection (Journal of Analytical 

Toxicology. 2005. 29(3): 149–155) 

Analysis Purpose: Complementary presumptive for ricin 

Analytical Technique: LC-MS 

Method Developed for: Ricinine in human and rat urine samples 

Method Selected for: SAM lists these procedures for complementary presumptive analysis of 

ricin by ricinine detection in aerosol, solid, particulate, liquid, and water samples. Ricinine, an 

alkaloid component of castor beans, is found in crude preparations of ricin, and may be an 

indicator of ricin contamination. Further research is needed to develop and standardize the 

procedures for environmental sample types. 

Description of Method: Procedures are described for sample extraction by SPE, isocratic 

HPLC, followed by electrospray ionization (ESI) tandem mass spectrometry. For MS analyses, 

protonated molecular ions are selected in the multiple reaction monitoring mode and quantified 

by isotope dilution with 13C6-labeled ricinine as the internal reference. Urine pools enriched with 

ricinine at two concentrations were used as quality controls for validation of the method in urine 

samples. The calculated limit of detection was 0.04 ng/mL. In addition to the validation with 

urine samples, testing was performed on a single human urine sample (forensic), a crude ricin 

preparation, and urine samples from an animal ricinine exposure study. For the human urine 

sample, the concentration of ricinine was measured to be 4.24 ng/mL. After a series of simple 

extraction and filtration steps to provide a crude castor bean preparation, the final ricinine level 

was 502 ng/mL. For the animal exposure study, rats were injected with ricinine at 1, 5, and 10 

mg/kg, with mean 24-hour urine concentrations of 1010, 6364, and 17, 152 ng/mL, respectively. 

Mean 48-hour urine concentrations were 40, 324, and 610 mg/mL. Stability of ricinine in human 

urine was also tested, with ricinine found to be stable in human urine samples when heated at 

90°C for 1 hour and when stored at 25°C and 5°C for 3 weeks. 

Source: Johnson, R.C., Lemire, S.W., Woolfitt, Ospina, M., Preston, K.P, Olson, C.T., and Barr, 

J.R. 2005. “Quantification of Ricinine in Rat and Human Urine: A Biomarker for Ricin 

Exposure.” Journal of Analytical Toxicology. 29(3): 149–155. 

http://www.jatox.com/abstracts/2005/April/149-johnson.html 

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8.2.4 Shiga and Shiga-like toxins (Stx, Stx-1, Stx-2) 

CAS RN: 75757-64-1 (Stx). 

Description: Protein composed of one ~32 kDa A chain and five 7.7 kDa B chains. 

Method Analytical Technique Section 

Pharmacology & Toxicology. 2001. 88(5): 255–260 Ribosome inactivation assay 8.2.1.3 

FDA, Bacteriological Analytical Manual Online, 

January 2001, Appendix 1, Rapid Methods for 

Detecting Foodborne Pathogens 

Immunoassay (ELISA) 8.2.4.1 

Journal of Clinical Microbiology. 2007. 45(10): 3377– 

3380 Optical immunoassay 8.2.4.2 

8.2.4.1 FDA, Bacteriological Analytical Manual Online, Appendix 1, 2001: Rapid 

Methods for Detecting Foodborne Pathogens 

Analysis Purpose: Confirmatory
 

Analytical Technique: Immunoassay (ELISA) 

Method Developed for: Shiga and shiga-like toxins in food 

Method Selected for: SAM lists this manual for presumptive analysis in aerosol, solid, 

particulate, liquid, and water samples. Further research is needed to develop and standardize the 

procedures for environmental samples. 

Description of Method: Shiga toxin (Stx) is produced by Shigella dysenteriae and Shiga-like 

toxins (Shiga toxin Types 1 [Stx-1] and 2 [Stx-2]) are produced by various Shiga-toxigenic E. 

coli (STEC). An ELISA is described for the detection of these toxins. Diluted samples are added 

to microwells coated with an anti-Shiga toxin capture antibody. After incubation at room
 

temperature, a wash is performed to remove unbound material. A second anti-Shiga toxin
 

antibody is added for detection and incubation continued at room temperature. A wash is 

performed to remove unbound antibody. Enzyme conjugated anti-IgG visualization antibody,
 

directed against the species from which the second anti-Shiga toxin antibody was derived, is 

added and the plate incubated then rinsed. Substrate is added, and after incubation to develop the 

color, stop solution is added. The results are interpreted spectrophotometrically. 

Source: FDA, CFSAN. 2001. “Rapid Methods for Detecting Foodborne Pathogens.” 

Bacteriological Analytical Manual Online. http://www.epa.gov/sam/pdfs/FDA-BAMAppendix1.pdf
 

8.2.4.2 Literature Reference for Shiga and Shiga-like Toxin (Journal of Clinical 

Microbiology. 2007. 45(10): 3377–3380) 

Analysis Purpose: Presumptive 

Analytical Technique: Optical immunoassay 

Method Developed for: Shiga toxin in foods 

Method Selected for: SAM lists these procedures for presumptive analysis in aerosol, solid, 

particulate, liquid, and water samples. Further research is needed to develop and standardize the 

procedures for environmental sample types. 

Description of Method: Procedures are described for a rapid optical immunoassay for the 

detection of Stx-1 and Stx-2 using a commercially available kit. Fecal samples (742 specimens) 

are assayed for Shiga toxins with and without enrichment of the specimens in broth. Duplicate 

assays are applied using either the rapid optical immunoassay or a commercially available ELISA 

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kit. Samples producing positive results by immunoassay are confirmed by Vero cell cytotoxicity 

assay. Sensitivities of 96.8% are achieved for direct stool sample assays. 

Source: Teel, L.D., Daly, J.A., Jerris, R.C., Maul, D., Svanas, G., O’Brien, A.D., and Park, C.H. 

2007. “Rapid Detection of Shiga Toxin-Producing Escherichia coli by Optical Immunoassay.” 

Journal of Clinical Microbiology. 45(10): 3377–3380. www.epa.gov/sam/pdfs/JCM-45(10)-

pgs3377-3380.pdf 

8.2.5 Staphylococcal enterotoxins (SEA, SEB, SEC) 

CAS RNs: 39424-53-8 (SEB), 37337-57-8 (SEA), 39424-54-9 (SEC) 

Description: Monomeric protein of ~ 28 kDa (SEB), monomeric proteins of ~ 27–27.5 kDa 

(SEA and SEC) 

Method Analytical Technique Section 

LRN Immunoassay 8.1.4 

AOAC Official Method 993.06 Immunoassay 8.2.5.1 

8.2.5.1 AOAC Official Method 993.06: Staphylococcal Enterotoxins in Selected 

Foods 

Analysis Purpose: Presumptive 

Analytical Technique: Immunoassay 

Method Developed for: Staphylococcal enterotoxins in selected foods 

Method Selected for: SAM lists this method for presumptive analysis of staphylococcal 

enterotoxins Type B in aerosol samples, and Types A and C in aerosol, solid, particulate, liquid, 

and water samples. Further research is needed to develop and standardize the procedures for 

environmental sample types. 

Description of Method: This method is an enzyme immunoassay (EIA) using a mixture of highaffinity capture antibodies for identification of toxin(s) in food samples. Samples are prepared by 

dilution in Tris buffer, centrifugation, and filtration of the supernatant through a syringe, with 

adjustment to a final pH of 7.0 to 8.0. Samples are incubated in 96-well plates with the mixture 

of antibodies conjugated to horseradish peroxidase (HRP), and visualized with a peroxidase 

substrate. Assay results are determined visually or using a microtiter plate reader. Test is 

considered positive for staphylococcal enterotoxins if absorbance is >0.200 and is considered 

negative if absorbance is <0.200. Specific toxin serotypes are not differentiated. This method 

detects from 1.3 to 3.3 ng/mL staphylococcal enterotoxin in extracts prepared from food 

containing 4 to 10 ng/mL staphylococcal enterotoxin. 

Source: AOAC International. 1994. “Method 991.06: Staphylococcal Enterotoxins in Selected 

Foods.” Official Methods of Analysis of AOAC International. 16th Edition, 4th Revision; Vol. I. 

http://www.aoac.org/ 

8.3 Method Summaries for Small Molecule Biotoxins 

Summaries of the analytical methods for small molecule biotoxins listed in Appendix D are provided in 

Sections 8.3.1 through 8.3.12. These sections contain summary information only, extracted from the 

selected methods. The full version of the method should be consulted prior to sample analysis. 

Each summary contains a brief description of the method, intended method application, performance data 

(if available), and a link to or source for obtaining a full version of the method. 

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8.3.1 Aflatoxin (Type B1) 

CAS RN: 27261-02-5 

Method Analytical Technique Section 

AOAC Official Method 991.31 Immunoassay and HPLC-FL 8.3.1.1 

8.3.1.1 AOAC Official Method 991.31: Aflatoxins in Corn, Raw Peanuts, and 

Peanut Butter 

Analysis Purpose: Presumptive and confirmatory
 

Analytical Technique: Immunoassay and HPLC-FL 

Method Developed for: Aflatoxins (Type B1) in corn, raw peanuts, and peanut butter 

Method Selected for: SAM lists this method for presumptive and confirmatory analyses in 

aerosol, solid, particulate, liquid, and water samples. Further research is needed to develop and 

standardize the procedures for environmental sample types. 

Description of Method: This method is for the detection of aflatoxins in agricultural products. 

The sample is extracted with methanol-water (7 + 3), filtered, diluted with water, and applied to 

an affinity column containing mAbs specific for aflatoxins B1, B2 (CAS RN 22040-96-6), G1 

(CAS RN 1385-95-1), and G2 (CAS RN 7241-98-7). Antibody-bound aflatoxins are removed 

from the column with methanol. For detection and quantitation of total aflatoxins, fluorescence 

measurement after reaction with bromine solution is performed. For individual aflatoxins, 

fluorescence detection and postcolumn iodine derivatization are performed and quantitation is by 

LC. Method performance was characterized using various commodities (e.g., corn) at aflatoxin 

levels over a range of 10 to 30 ng/g. This method was originally designed for the analysis of 

aflatoxins (B1, B2, G1, and G2) in samples where cleanup was necessary to remove food 

components, such as fats and proteins; the cleanup procedure may not be necessary for analysis of 

water samples. 

Special Considerations: AOAC Official Method 994.08: Aflatoxin in Corn, Almonds, Brazil 

Nuts, Peanuts, and Pistachio Nuts, (AOAC International. 1998. Official Methods of Analysis of 

AOAC International, 16th Edition, 4th Revision, Vol. II. http://www.aoac.org/) may be used as a 

complementary HPLC-FL method in order to provide more flexibility for analyses. 

Source: AOAC International. 1994. “Method 991.31: Aflatoxins in Corn, Raw Peanuts, and 

Peanut Butter.” Official Methods of Analysis of AOAC International. 16th Edition, 4th Revision; 

Vol. II. http://www.aoac.org/ 

8.3.2 α-Amanitin 

CAS RN:	 23109-05-9 

Method Analytical Technique Section 

Journal of Chromatography B. 1991. 563(2): 299–311 HPLC amperometric detection 8.3.2.1 

Journal of Food Protection. 2005. 68(6): 1294–1301 Immunoassay 8.3.2.2 

8.3.2.1 Literature Reference for α-Amanitin (Journal of Chromatography B. 1991. 

563(2): 299–311) 

Analysis Purpose: Confirmatory 

Analytical Technique: HPLC with amperometric detection 

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Section 8– Selected Biotoxin Methods 

Method Developed for: α-Amanitin in plasma 

Method Selected for: SAM lists these procedures for confirmatory analysis in aerosol, solid, 

particulate, liquid, and water samples. Further research is needed to develop and standardize the 

procedures for environmental sample types. 

Description of Method: Procedures are described for the selective determination in human 

plasma of α-amanitin using HPLC with amperometric detection. After extraction of plasma with
 

disposable C18 silica cartridges, the extracts are separated by isocratic reversed-phase 

chromatography using a macroporous polystyrene-divinylbenzene column and a mobile phase of 

0.05 M phosphate buffer-acetonitrile (91:9) at pH 9.5. Amperometric detection is performed by 

applying an oxidation potential as low as +350 mV (vs. Ag/AgCl) to a glassy carbon electrode, in 

a thin-layer flow-cell. The linear range for alpha-amanitin is 3 to 200 ng/mL, and the relative 

LOD in plasma is 2 ng/mL at a signal-to-noise ratio of 2. The intra-assay precision has been 

evaluated at levels of 10 and 200 ng/mL. 

Source: Tagliaro, F., Schiavon, G., Bontempelli, G., Carli, G., and Marigo, M. 1991. “Improved 

High-performance Liquid Chromatographic Determination with Amperometric Detection of 

Alpha-amanitin in Human Plasma Based on its Voltammetric Study.” Journal of Chromatography 

B. 563(2): 299–311. http://www.ncbi.nlm.nih.gov/pubmed/2055993 

8.3.2.2 Literature Reference for α-Amanitin, T-2 Mycotoxin (Journal of Food 

Protection. 2005. 68(6): 1294–1301) 

Analysis Purpose: Presumptive 

Analytical Technique: Immunoassay 

Method Developed for: α-Amanitin, ricin, and T-2 mycotoxin in food and beverages 

Method Selected for: SAM lists these procedures for presumptive analysis of α-amanitin and T2 toxin in aerosol, solid, particulate, liquid, and water samples and for confirmatory analysis of 

ricin in aerosol, solid, particulate, liquid, and water samples. Further research is needed to 

develop and standardize the procedures for environmental sample types. 

Description of Method: Commercially available ELISAs are described and assessed for 

detection of ricin, amanitin, and T-2 toxin at levels below those described as a health concern in 

food samples. Solid food samples are prepared by washing the sample with sodium phosphate 

buffer followed by dilution with phosphate-buffered saline. Liquid beverage samples are 

prepared by dilution in sodium phosphate buffer. Amanitin samples are similarly prepared using 

water instead of buffer, and T-2 toxin samples are similarly prepared using 35% methanol in 

water instead of buffer. The prepared samples are used with commercially obtained ELISA kits 

according to the manufacturer’s directions, except for the incorporation of an eight-point 

calibration curve and reading the plates at both 405 and 650 nm after 26 minutes of incubation at 

37°C. This assay detects ricin in food products at 0.01 μg/mL with acceptable background levels. 

Amanitin can be detected in food products at 1 μg/g with the ELISA. Background responses 

occurred, but at less than the equivalent of 0.5 ppm for amanitin. The ELISA kit will successfully 

detect T-2 toxin at targeted levels of 0.2 μg/g. The ELISA kit successfully detects T-2 toxin at 

targeted levels of 0.2 μg/g; the immunoassay for T-2 toxin, however, shows significant 

background responses and varies up to 0.1 ppm. 

Source: Garber, E.A., Eppley, R.M., Stack, M.E., McLaughlin, M.A., and Park, D.L. 2005. 

“Feasibility of Immunodiagnostic Devices for the Detection of Ricin, Amanitin, and T-2 Toxin in 

Food.” Journal of Food Protection. 68(6): 1294–1301. 

http://www.ingentaconnect.com/content/iafp/jfp/2005/00000068/00000006/art00027 

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8.3.3 Anatoxin-a 

CAS RN: 64285-06-9 

Method Analytical Technique Section 

Biomedical Chromatography. 1996. 10: 46–47 HPLC-FL (precolumn 

derivatization) 8.3.3.1 

8.3.3.1 Literature Reference for Anatoxin-a (Biomedical Chromatography. 1996. 

10(1): 46–47) 

Analysis Purpose: Confirmatory
 

Analytical Technique: HPLC-FL (precolumn derivatization) 

Method Developed for: Anatoxin-a in potable water 

Method Selected for: SAM lists these procedures for confirmatory analysis in aerosol, solid, 

particulate, liquid, and water samples. Further research is needed to develop and standardize the 

procedures for environmental sample types other than water. 

Description of Method: Procedures are described for HPLC analysis with fluorimetric detection 

of anatoxin-a in water samples after derivatization with 7-fluoro-4-nitro-2,1,3-benzoxadiazole 

(NBD-F). Samples are extracted at pH 7 with SPE using a weak cation exchanger. The toxin is 

eluted with methanol containing 0.2% TFA. Samples are evaporated, reconstituted with 

acetonitrile, and re-evaporated prior to derivatization. This procedure detects anatoxin-a at 

concentrations of 0.1 μg/L with a good linear calibration. 

Source: James, K.J., and Sherlock, I.R. 1996. “Determination of the Cyanobacterial Neurotoxin, 

Anatoxin-a, by Derivatisation Using 7-Fluoro-4-Nitro-2,1,3-Benzoxadiazole (NBD-F) and HPLC 

Analysis with Fluorimetric Detection.” Biomedical Chromatography. 10(1): 46–47. 

http://www3.interscience.wiley.com/journal/18562/abstract
 

8.3.4 Brevetoxins (B form) 

CAS RN:	 79580-28-2 

Method Analytical Technique Section 

Environmental Health Perspectives. 2002. 110(2): 

179–185 Immunoassay 8.3.4.1 

Toxicon. 2004. 43(4): 455–465 HPLC-MS-MS 8.3.4.2 

8.3.4.1 Literature Reference for Brevetoxins (Environmental Health 

Perspectives. 2002. 110(2): 179–185) 

Analysis Purpose: Presumptive 

Analytical Technique: Immunoassay 

Method Developed for: Brevetoxins in shellfish 

Method Selected for: SAM lists these procedures for presumptive analysis in aerosol, solid, 

particulate, liquid, and water samples. Further research is needed to develop and standardize the 

procedures for environmental sample types. 

Description of Method: Procedures are described for a competitive ELISA used to detect 

brevetoxins in shellfish. The assay uses goat anti-brevetoxin antibodies in combination with a 

three-step signal amplification process: (1) secondary biotinylated antibodies; (2) streptavidinHRP conjugate; and (3) chromogenic enzyme substrate. Sample preparation for liquids is 

dilution in PBS. Sample preparation for solids (oysters) is homogenization in PBS, or extraction 

in acetone. The working range for the assay is 0.2 to 2.0 ng/mL for diluted and undiluted liquid 

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Section 8– Selected Biotoxin Methods 

samples, and 0.2 to 2.0 ng/mL for solid samples, corresponding to 0.8 to 8.0 μg brevetoxins/100.0 

g shellfish. The method has been compared to the mouse bioassay and is equivalent in 

sensitivity. 

Source: Naar, J., Bourdelais, A., Tomas, C., Kubanek, J., Whitney, P.L., Flewelling, L., 

Steidinger, K., Lancaster, J., and Badan, D.G. 2002. “A Competitive ELISA to Detect 

Brevetoxins from Karenia brevis (Formerly Gymnodinium breve) in Seawater, Shellfish, and 

Mammalian Body Fluid.” Environmental Health Perspectives. 110(2): 179–185. 

http://www.epa.gov/sam/pdfs/EHP-110(2)-pgs179-185.pdf 

8.3.4.2 Literature Reference for Brevetoxins (Toxicon. 2004. 43(4): 455–465) 

Analysis Purpose: Confirmatory
 

Analytical Technique: High performance liquid chromatography tandem mass spectrometers 

(HPLC-MS-MS) 

Method Developed for: Brevetoxins in shellfish 

Method Selected for: SAM lists these procedures for confirmatory analysis in aerosol, solid, 

particulate, liquid, and water samples. Further research is needed to develop and standardize the 

procedures for environmental sample types. 

Description of Method: Shellfish sample homogenates are extracted with acetone, and 

centrifuged. The supernatants are combined, evaporated, and re-solubilized in 80% methanol. 

Following a wash with 95% n-hexane, the methanolic layer is evaporated, and the residue resolubilized in 25% methanol and applied to a C18 SPE column. Analytes are eluted with 100% 

methanol, evaporated, and re-solubilized in methanol for analysis. Analysis of prepared samples 

is performed using HPLC-MS-MS with a mobile phase of water and acetonitrile with acetic acid. 

Analytes are detected by an MS with ESI interface. Brevetoxins are extensively metabolized,
 

with many sub-forms. This method describes multiple liquid chromatography/electrospray
 

ionization mass spectrometry (LC-ESI-MS) profiles for metabolites of brevetoxins from oysters. 

Source: Wang, Z., Plakas, S.M., El Said, K.R., Jester, E.L., Granade, H.R., and Dickey, R.W. 

2004. “LC/MS Analysis of Brevetoxin Metabolites in the Eastern Oyster (Crassostrea 

virginica).” Toxicon. 43(4): 455–465. http://cat.inist.fr/?aModele=afficheN&cpsidt=15668117
 

8.3.5 α-Conotoxin 

CAS RN:	 156467-85-5 

Method Analytical Technique Section 

Biochemical Journal. 1997. 328: 245–250 Immunoassay 8.3.5.1 

Journal of Medicinal Chemistry. 2004. 47(5): 1234– 

1241 HPLC-MS 8.3.5.2 

8.3.5.1 Literature Reference for α-Conotoxin (Biochemical Journal. 1997. 328(1): 

245–250) 

Analysis Purpose: Presumptive 

Analytical Technique: Immunoassay 

Method Developed for: Purified α-Conotoxin GI in phosphate buffer 

Method Selected for: SAM lists these procedures for presumptive analysis in aerosol, solid, 

particulate, liquid, and water samples. Further research is needed to develop and standardize the 

procedures for environmental sample types. 

 SAM Revision 5.0	 204 September 29, 2009 
Section 8– Selected Biotoxin Methods 

Description of Method: A biologically active fluorescein derivative of Conus geographus αconotoxin (FGI) is used in solution-phase-binding assays with two purified Torpedo californica 

monoclonal antibodies (mAbs) to detect the toxin in laboratory samples. For competitive liganddisplacement spin-column assays, FGI was premixed with various dilutions of unlabelled ligands 

and then incubated with the two mAbs (5A1 and 8D2) at room temperature. Fluorescence is 

measured in ratio mode using cuvettes with excitation and emission monochromators set at 

gamma = 490 nm and gamma = 525 nm, respectively. The binding of FGI to the mAbs had 

apparent dissociation constants of 10 to 100 nM. The binding specificity and epitopes recognized 

by the two mAbs against α-conotoxin GI are also characterized. Competitive displacement 

assays showed that both mAbs specifically bound α-conotoxin GI with high avidity. Crossreactivity with α-conotoxins M1 and S1 was not observed for either mAb in a direct ELISA. 

With spin-column assay, however, 5A1, but not 8D2, cross-reacted at a low level (100 – 300-fold 

less avid) with these α-conotoxins. An antibody/α-conotoxin GI molar ratio of 1:1 afforded 

complete protection in mouse lethal assays. 

Source: Ashcom, J.D., and Stiles, B.G. 1997. “Characterization of α-Conotoxin Interactions 

with the Nicotinic Acetylcholine Receptor and Monoclonal Antibodies.” Biochemical Journal. 

328(1): 245–250. http://www.epa.gov/sam/pdfs/BJ-328-pgs245-250.pdf 

8.3.5.2 Literature Reference for α-Conotoxin (Journal of Medicinal Chemistry. 

2004. 47(5): 1234–1241) 

Analysis Purpose: Confirmatory 

Analytical Technique: HPLC-MS 

Method Developed for: Conus anemone venom (α-Conotoxins AnIA, AnIB, and AnIC) in 

buffer 

Method Selected for: SAM lists these procedures for confirmatory analysis in aerosol, solid, 

particulate, liquid, and water samples. Further research is needed to develop and standardize the 

procedures for environmental sample types. 

Description of Method: Procedures are discussed for the detection of peptides within the αconotoxin molecular mass range using an HPLC-MS. A crude extract of Conus anemone venom 

sample is made using 30% acetonitrile/water acidified with 0.1% trifluoroacetic acid (TFA), with 

the insoluble portion of the sample removed by centrifugation. A portion of the sample extract is 

fractionated by size-exclusion chromatography in order to prepare a sample containing small 

peptides in the range of 1000 to 2500 Da. Chromatography conditions are elution with 30% 

acetonitrile / 0.048% TFA at a flow rate of 0.5 mL/minute, with detection at 214 nm. Three 

sulfated α-conotoxins (AnIA, AnIB, and AnIC) can be identified by LC-MS that are within the 

molecular mass range of other α-conotoxins (i.e., 1400–2200 Da). Peptides can be quantified by 

reversed-phase HPLC using an external reference standard for each peptide. 

Source: Loughnan, M.L., Nicke, A., Jones, A., Adams, D.J., Alewood, P.F., and Lewis, R.J. 

2004. “Chemical and Functional Identification and Characterization of Novel Sulfated Alphaconotoxins from the Cone Snail Conus anemone.” Journal of Medicinal Chemistry. 47(5): 1234– 

1241. http://pubs.acs.org/cgi-bin/abstract.cgi/jmcmar/2004/47/i05/abs/jm031010o.html 

 SAM Revision 5.0	 205 September 29, 2009 
Section 8– Selected Biotoxin Methods 

8.3.6 Cylindrospermopsin 

CAS RN: 143545-90-8 

Method Analytical Technique Section 

FEMS Microbiology Letters. 2002. 216: 159-164 HPLC-PDA 8.3.6.1 

ELISA Kits for Cylindrospermopsin Immunoassay 8.3.6.2 

8.3.6.1 Literature Reference for Cylindrospermopsin (FEMS Microbiology 

Letters. 2002. 216(2): 159–164) 

Analysis Purpose: Confirmatory
 

Analytical Technique: High performance liquid chromatography – Photodiode array detector 

(HPLC-PDA)
 

Method Developed for: Cylindrospermopsin in eutrophic waters 

Method Selected for: SAM lists these procedures for confirmatory analysis in aerosol, solid, 

particulate, liquid, and water samples. Further research is needed to develop and standardize the 

procedures for environmental sample types other than water. 

Description of Method: Cylindrospermopsin is detected using HPLC with photodiode array 

detector (PDA) in environmental waters. The suggested solvent range for cylindrospermopsin is 

below 50% methanol and 30% acetonitrile. Complex samples (culture medium) are purified on a 

C18 column with a linear gradient of 1 to 12% (v/v) methanol/water over 24 minutes at 40°C, with 

monitoring at 262 nm. The use of C18 columns for environmental waters is suggested for removal 

of the large number of organic compounds that may be present. This method detects and recovers 

cylindrospermopsin from spiked environmental water samples at 1 μg/L. 

Source: Metcalf, J.S., Beattie, K.A., Saker, M.L., and Codd, G.A. 2002. “Effects of Organic 

Solvents on the High Performance Liquid Chromatographic Analysis of the Cyanobacterial Toxin 

Cylindrospermopsin and Its Recovery from Environmental Eutrophic Waters by Solid Phase 

Extraction.” FEMS Microbiology Letters. 216(2): 159–164. 

http://cat.inist.fr/?aModele=afficheN&cpsidt=14002569
 

8.3.6.2 ELISA Kits for Cylindrospermopsin 

Analysis Purpose: Presumptive 

Analytical Technique: Immunoassay 

Method Developed for: Cylindrospermopsin in ground water, surface water, and well water 

Method Selected for: SAM lists these procedures for presumptive analysis in aerosol, solid, 

particulate, liquid, and water samples. Further research is needed to develop and standardize the 

procedures for environmental sample types other than water. 

Description of Method: Cylindrospermopsin is detected using a colorimetric immunoassay 

(competitive ELISA) procedure. A sample (0.05 mL), enzyme conjugate (cylindrospermopsinHRP), and an antibody solution containing rabbit anti-cylindrospermopsin antibodies are added to 

plate wells containing immobilized sheep anti-rabbit antibodies. Both the cylindrospermopsin (if 

present) in the sample and cylindrospermopsin-HRP conjugate compete in solution to bind to the 

rabbit anti-cylindrospermopsin antibodies in proportion to their respective concentrations. The 

anti-cylindrospermopsin antibody-target complexes are then bound to the immobilized sheep antirabbit antibodies on the plate. After incubation, the unbound molecules are washed and decanted. 

A specific substrate is then added which is converted from a colorless to a blue solution by the 

HRP enzyme conjugate solution. The reaction is terminated with the addition of a dilute acid. The 

concentration of cylindrospermopsin in the sample is determined photometrically by comparing 

 SAM Revision 5.0	 206 September 29, 2009 
Section 8– Selected Biotoxin Methods 

sample absorbance to the absorbance of the calibrators (standards) at a specific wavelength (450 

nm). The applicable concentration range is 0.4–2.0 μg/L, with a minimum detection level of 0.4 

μg/L. 

Source: NEMI. 2006. 

http://infotrek.er.usgs.gov/pls/apex/f?p=119:38:7526698938332159::::P38_METHOD_ID:9507 

8.3.7 Diacetoxyscirpenol (DAS) 

CAS RN:	 2270-40-8 

Method Analytical Technique Section 

International Journal of Food Microbiology. 1988. 

6(1): 9–17 Immunoassay 8.3.7.1 

Rapid Communications in Mass Spectrometry. 

2006. 20(9): 1422–1428 LC/APCI-MS 8.3.7.2 

8.3.7.1 Literature Reference for Diacetoxyscirpenol (DAS) (International Journal 

of Food Microbiology. 1988. 6(1): 9–17) 

Analysis Purpose: Presumptive 

Analytical Technique: Immunoassay 

Method Developed for: DAS in food 

Method Selected for: SAM lists these procedures for presumptive analysis in aerosol, solid, 

particulate, liquid, and water samples. Further research is needed to develop and standardize the 

procedures for environmental sample types. 

Description of Method: An ELISA is used for the detection of DAS in food samples. 

Antibodies against DAS are obtained after immunization of rabbits with DAS-hemiglutaratehuman serum albumin (DAS-HG-HSA), and a DAS-hemisuccinate-HRP conjugate (DAS-HSHRP) is prepared by an ester method for use as enzyme-labeled toxin in the competitive assay. 

The detection limit for DAS using this assay is approximately 10 pg/mL. This assay will crossreact related toxins. The relative cross-reactivities of the assay are 597.5, 5.2, 100.0, 2.5, and 

1.5% for 3 alpha-acetyl-DAS, DAS, T-2 toxin, neosolaniol, and 15-acetoxyscirpenol, 

respectively.
 

Source: Klaffer, U., Martlbauer, E., and Terplan, G. 1988. “Development of a Sensitive 

Enzyme-linked Immunosorbent Assay for the Detection of Diacetoxyscirpenol.” International 

Journal of Food Microbiology. 6(1): 9–17. 

http://www.sciencedirect.com/science/journal/01681605
 

8.3.7.2 Literature Reference for Diacetoxyscirpenol (DAS) and T-2 Mycotoxin 

(Rapid Communications in Mass Spectrometry. 2006. 20(9): 1422–1428) 

Analysis Purpose: Confirmatory
 

Analytical Technique: Liquid chromatography/atmospheric pressure chemical ionization mass 

spectrometry (LC/APCI-MS) 

Method Developed for: DAS and T-2 mycotoxin in food 

Method Selected for: SAM lists these procedures for confirmatory analysis in aerosol, solid, 

particulate, liquid, and water samples. Further research is needed to develop and standardize the 

procedures for environmental sample types. 

 SAM Revision 5.0	 207 September 29, 2009 
Section 8– Selected Biotoxin Methods 

Description of Method: A LC/APCI-MS procedure based on time-of-flight mass spectrometry 

(TOFMS), with a real-time reference mass correction, is used for simultaneous determination of 

Fusarium mycotoxins (to include DAS and T-2 mycotoxin) in foodstuffs. Mycotoxin samples are 

extracted with acetonitrile/water (85:15) and centrifuged, and the supernatant is applied to a 

column for cleanup. Prepared samples are separated by liquid chromatography with an aqueous 

mobile phase of ammonium acetate and methanol detection is provided in exact mass 

chromatograms with a mass window of 0.03 Th. The limits of detection range from 0.1 to 6.1 

ng/g in analyzed foodstuffs. 

Source: Tanaka, H., Takino, M., Sugita-Konishi, Y., and Tanaka, T. 2006. “Development of 

Liquid Chromatography/Time-of-flight Mass Spectrometric Method for the Simultaneous 

Determination of Trichothecenes, Zearalenone, and Aflatoxins in Foodstuffs.” Rapid 

Communications in Mass Spectrometry. 20(9): 1422–1428. 

http://cat.inist.fr/?aModele=afficheN&cpsidt=17697070 

8.3.8 Microcystins (Principal isoforms: LA, LR, LW, RR, YR) 

CAS RNs: 96180-79-9 (LA), 101043-37-2 (LR), 157622-02-1 (LW), 111755-37-4 (RR), 

101064-48-6 (YR) 

Method Analytical Technique Section 

Journal of AOAC International. 2001. 84(4): 1035– 

1044 

Immunoassay/Phosphatase 

assay 

8.3.8.1 

Analyst. 1994. 119(7): 1525–1530 HPLC-PDA 8.3.8.2 

8.3.8.1 Literature Reference for Microcystins (Journal of AOAC International. 

2001. 84(4): 1035–1044) 

Analysis Purpose: Presumptive 

Analytical Technique: Immunoassay/Phosphatase assay
 

Method Developed for: Microcystins-LA, -LR, -LW, -RR, -YR in algae products 

Method Selected for: SAM lists these procedures for presumptive analysis in aerosol, solid, 

particulate, liquid, and water samples. Further research is needed to develop and standardize the 

procedures for environmental sample types. 

Description of Method: ELISA and protein phosphatase inhibition assays are used to detect 

microcystins in blue-green algae products. Solid samples are prepared by homogenization in 

methanol (75% in water), with centrifugation to remove solids. Immunoassays are performed on 

the prepared samples using a commercially available ELISA test kit as described by the 

manufacturer. Samples are quantitated by comparison with a microcystins-LR standard curve. 

Quantitation with the colorimetric protein phosphatase inhibition assay is based on a comparison 

with a microcystin-LR standard curve. ELISA and phosphatase assay results agree over a 

concentration range of 0.5 to 35 μg/g. Neither assay is specific for a particular isoform.
 

Source: Lawrence, J.F., Niedzwiadek, B., Menard, C., Lau, B.P., Lewis, D., Kuper-Goodman, 

T., Carbone, S., and Holmes, C. 2001. “Comparison of Liquid Chromatography/Mass 

Spectrometry, ELISA, and Phosphatase Assay for the Determination of Microcystins in Bluegreen Algae Products.” Journal of AOAC International. 84(4): 1035–1044. 

http://cat.inist.fr/?aModele=afficheN&cpsidt=1135453
 

8.3.8.2 Literature Reference for Microcystins (Analyst. 1994. 119(7): 1525–1530) 

Analysis Purpose: Confirmatory 

Analytical Technique: HPLC-PDA 

 SAM Revision 5.0	 208 September 29, 2009 
Section 8– Selected Biotoxin Methods 

Method Developed for: Microcystins-LA, -LR, -LW, -RR, -YR in raw and treated waters 

Method Selected for: SAM lists these procedures for confirmatory analysis in aerosol, solid, 

particulate, liquid, and water samples. Further research is needed to develop and standardize the 

procedures for environmental sample types other than water. 

Description of Method: Procedures are discussed to test the presence of microcystin-LR, -LY, -

LW, -LF (CAS RN 154037-70-4), and -RR in treated and untreated water samples. 

Cyanobacterial cells are separated from the water by filtration through 110-mm glass fiber grade 

C (GF/C) discs. The cellular components collected on the discs are extracted three times with 

methanol; the collected extraction fluids are combined and dried. The residue is resuspended in 

methanol and analyzed by HPLC-PDA. The liquid portion of the filtered water sample is 

subjected to trace enrichment using a C18 SPE cartridge, followed by identification and 

determination by HPLC-PDA. This procedure can detect microcystin concentrations as low as 

250 ng/L and is the basis of the World Health Organization (WHO) method for the detection of 

microcystins. 

Source: Lawton, L.A., Edwards, C., and Codd, G.A. 1994. “Extraction and High-performance 

Liquid Chromatographic Method for the Determination of Microcystins in Raw and Untreated 

Waters.” Analyst. 119(7): 1525–1530. 

http://www.rsc.org/Publishing/Journals/AN/article.asp?doi=AN9941901525 

8.3.9 Picrotoxin 

CAS RN:	 124-87-8 

Method Analytical Technique Section 

Journal of Pharmaceutical and Biomedical Analysis. 

1989. 7(3): 369–375 HPLC 8.3.9.1 

8.3.9.1 Literature Reference for Picrotoxin (Journal of Pharmaceutical & 

Biomedical Analysis. 1989. 7(3): 369–375) 

Analysis Purpose: Confirmatory 

Analytical Technique: HPLC 

Method Developed for: Picrotoxin in serum
 

Method Selected for: SAM lists these procedures for confirmatory analysis in aerosol, solid, 

particulate, liquid, and water samples. Further research is needed to develop and standardize the 

procedures for environmental sample types. 

Description of Method: Procedures are described for quantification of the two components of 

picrotoxin (picrotin [CAS RN 21416-53-5] and picrotoxinin [CAS RN 17617-45-7]) in serum
 

samples. Serum samples are prepared by washing with n-hexane, followed by extraction with 

chloroform. The chloroform is evaporated and the sample is reconstituted in acetonitrile-1 mM 

ammonium acetate buffer (pH 6.4) 34:66 (v/v) for assay by reversed-phase HPLC. The effluent 

is monitored at 200 nm, and quantification is based on peak-height ratio of analyte to the internal 

standard. A linear response is obtained for both analytes (picrotin and picrotoxinin) in the range 

0.2 to 20.0 μg/mL. 

Source: Soto-Otero, R., Mendez-Alvarez, E., Sierra-Paredes, G., Galan-Valiente, J., AguilarVeiga, E., and Sierra-Marcuno, G. 1989. “Simultaneous Determination of the Two Components 

of Picrotoxin in Serum by Reversed-phase High-performance Liquid Chromatography with 

Application to a Pharmacokinetic Study in Rats.” Journal of Pharmaceutical & Biomedical 

Analysis. 7(3): 369–375. http://www.sciencedirect.com/science/journal/07317085 

 SAM Revision 5.0	 209 September 29, 2009 
Section 8– Selected Biotoxin Methods 

8.3.10 Saxitoxins (Principal isoforms: STX, NEOSTX, GTX, dcGTX, dcSTX) 

CAS RNs: 35523-89-8 (STX), 64296-20-4 (NEOSTX), 77462-64-7 (GTX), 58911-04-9 

(dcSTX) 

Method Analytical Technique Section 

Journal of AOAC International. 1995. 78: 528– 

532 

HPLC-FL (post column 

derivatization) 8.3.10.1 

ELISA Kits for Saxitoxin Immunoassay 8.3.10.2 

8.3.10.1 Literature Reference for Saxitoxin (Journal of AOAC International. 1995. 

78(2): 528–532) 

Analysis Purpose: Confirmatory
 

Analytical Technique: HPLC-FL (post column derivatization) 

Method Developed for: Saxitoxins (STX, NEOSTX, GTX, dcGTX, dcSTX) in shellfish 

Method Selected for: SAM lists these procedures for confirmatory analysis in aerosol, solid, 

particulate, liquid, and water samples. Further research is needed to develop and standardize the 

procedures for environmental sample types. 

Description of Method: Procedures are described to detect multiple analogues of saxitoxin in
 

shellfish using ion-interaction chromatography on a silica-based reversed-phase (C8) column with 

postcolumn periodate oxidation and FL detection. Toxin groups of different net charges are 

determined separately by isocratic elution using either sodium 1-heptanesulfonate in ammonium
 

phosphate (GTX-1, GTX-6, dcGTX2, dcGTX3) or sodium 1-heplanesulfonate in ammonium
 

phosphate and acetonitrile (STX [CAS RN 35523-89-8], neoSTX [CAS RN 64296-20-4], dcSTX 

[CAS RN 58911-04-9]). For biological sample types, a cleanup procedure using a C18 SPE 

cartridge is effective in preventing false peaks. High sensitivity with detection limits ranging 

from 20 to 110 fmol are achieved as a result of reduced band broadening and optimized reaction 

conditions. This method, when applied to low-toxicity shellfish, gives higher values than the 

standard mouse bioassay. 

Source: Oshima, Y. 1995. “Postcolumn Derivatization Liquid Chromatographic Method for 

Paralytic Shellfish Toxins.” Journal of AOAC International. 78(2): 528–532. 

http://cat.inist.fr/?aModele=afficheN&cpsidt=3469391
 

8.3.10.2 ELISA Kits for Saxitoxins 

Analysis Purpose: Presumptive 

Analytical Technique: Immunoassay 

Method Developed for: STX in water and solid samples (e.g., shellfish) 

Method Selected for: SAM lists these procedures for confirmatory analysis in aerosol, solid, 

particulate, liquid, and water samples. Further research is needed to develop and standardize the 

procedures for environmental sample types other than water. 

Description of Method: Saxitoxin is detected using a colorimetric immunoassay (competitive 

ELISA) procedure. A sample (0.05 mL), enzyme conjugate (saxitoxin-HRP), and an antibody
 

solution containing rabbit anti-saxitoxin antibodies are added to plate wells containing 

immobilized sheep anti-rabbit antibodies. Both the saxitoxin (if present) in the sample and 

saxitoxin-HRP conjugate compete in solution to bind to the rabbit anti-saxitoxin antibodies in 

proportion to their respective concentrations. The anti-saxitoxin antibody-target complexes are 

then bound to the immobilized sheep anti-rabbit antibodies on the plate. After incubation, the 

unbound molecules are washed and decanted. A specific substrate is then added which is 

 SAM Revision 5.0	 210 September 29, 2009 
Section 8– Selected Biotoxin Methods 

converted from a colorless to a blue solution by the HRP enzyme conjugate solution. The reaction 

is terminated with the addition of a dilute acid. The concentration of saxitoxin in the sample is 

determined photometrically by comparing sample absorbance to the absorbance of the calibrators 

(standards) at a specific wavelength (450 nm). The applicable concentration range is 0.015–0.4 

ng/mL, with a minimum detection level of 0.015 ng/mL. 

Special Considerations: This kit is not intended for other types of saxitoxin. Cross-reactivity is 

observed with the following saxitoxin types: dcSTX (29%), GTX 2, 3, and 5B (23%), sulfo GTX 

1 and 2 (2.0%, dcGTX 2 and 3 (1.4%), NEOSTX (1.3%), dcNEOSTX (0.6%), GTX 1 and 4 

(<0.2%). High concentrations (e.g., above 0.1 ng/mL for toxins with >20% cross-reactivity) of 

these other types of saxitoxin may produce false positive responses. 

Source: NEMI. 2006. 

http://infotrek.er.usgs.gov/pls/apex/f?p=119:38:8989971104293493::::P38_METHOD_ID:9512 

8.3.11 T-2 Mycotoxin 

CAS RN: 21259-20-1 

Method Analytical Technique Section 

Journal of Food Protection. 2005. 68(6): 1294–1301 Immunoassay 8.3.2.2 

Rapid Communications in Mass Spectrometry. 2006. 

20(9): 1422–1428 LC/APCI-MS 8.3.7.2 

See Sections 8.3.2.2 and 8.3.7.2 for information on immunoassay and LC/APCI-MS procedures 

for T-2 Mycotoxin. 

8.3.12 Tetrodotoxin 

CAS RN: 9014-39-5 

Method Analytical Technique Section 

Analytical Biochemistry. 2001. 290: 10-17 LC/ESI-MS 8.3.12.1 

Journal of Clinical Laboratory Analysis. 1992. 6: 65– 

72 Immunoassay 8.3.12.2 

8.3.12.1 Literature Reference for Tetrodotoxin (Analytical Biochemistry. 2001. 

290(1): 10–17) 

Analysis Purpose: Confirmatory 

Analytical Technique: LC/ESI-MS 

Method Developed for: Tetrodotoxin (TTX) from puffer fish and newt tissues 

Method Selected for: SAM lists these procedures for confirmatory analysis in aerosol, solid, 

particulate, liquid, and water samples. Further research is needed to develop and standardize the 

procedures for environmental sample types. 

Description of Method: Procedures are described for LC/ESI-MS analysis of TTXs in tissue 

samples from puffer fish and newts by a combination of chromatography on a reversed-phase 

column with long carbon chains (C30) and with the mobile phase containing an ion pair reagent 

(ammonium heptafluorobutyrate). The relationship between the amount of applied standard TTX 

and its peak area on the mass chromatogram (m/z 320) shows good linearity over a range of 50 to 

1000 pmol. The detection limit of TTX in the SIM mode is estimated to be 0.7 pmol, with a 

signal to noise ratio of 2:1.
 

 SAM Revision 5.0	 211 September 29, 2009 
Section 8– Selected Biotoxin Methods 

Source: Shoji, Y., Yotsu-Yamashita, M., Miyazawa, T., and Yasumoto, T. 2001. “Electrospray 

Ionization Mass Spectrometry of Tetrodotoxin and its Analogs: Liquid Chromatography/Mass 

Spectrometry, Tandem Mass Spectrometry, and Liquid Chromatography/Tandem Mass 

Spectrometry.” Analytical Biochemistry. 290(1): 10–17. 

http://www.sciencedirect.com/science/journal/00032697 

8.3.12.2 Literature Reference for Tetrodotoxin (Journal of Clinical Laboratory 

Analysis. 1992. 6(2): 65–72) 

Analysis Purpose: Presumptive 

Analytical Technique: Immunoassay 

Method Developed for: Tetrodotoxin in buffer 

Method Selected for: SAM lists these procedures for presumptive analysis in aerosol, solid, 

particulate, liquid, and water samples. Further research is needed to develop and standardize the 

procedures for environmental sample types. 

Description of Method: Procedures are described for a competitive inhibition enzyme
 

immunoassay (CIEIA) for tetrodotoxin in biological samples. An anti-TTX mAb, designated 

T20G10, is directly labeled with alkaline phosphatase for use in the assay. Sensitivities of 6 to 7 

ng/mL (IC 50) and 2 to 3 ng/mL (IC 20) are achieved. 

Source: Raybould, T.J., Bignami, G.S., Inouye, L.K., Simpson, S.B., Byrnes, J.B., Grothaus, 

P.G., and Vann, D.C. 1992. “A Monoclonal Antibody-based Immunoassay for Detecting 

Tetrodotoxin in Biological Samples.” Journal of Clinical Laboratory Analysis. 6(2): 65–72. 

http://www3.interscience.wiley.com/journal/112131435/abstract
 

 SAM Revision 5.0	 212 September 29, 2009 
Section 9 – Conclusions

Section 9.0: Conclusions
 

Methods listed in Appendix A (chemical methods), Appendix B (radiochemical methods), Appendix C 

(pathogen methods), and Appendix D (biotoxin methods) are recommended for use in assessment of the 

extent of contamination and the effectiveness of decontamination following a homeland security event. 

The primary objective of this document is not necessarily to identify the “best” analytical methods, but 

rather to identify appropriate methods that represent a balance between providing existing, documented, 

determinative techniques and providing consistent and valid analytical results. The method selected for 

each analyte/sample type combination was deemed the most general, appropriate, and broadly applicable 

of available methods. This is a living document, which can be used as a guide by EPA and EPAcontracted laboratories tasked with analysis of environmental samples following a homeland security 

event. Recommended methods are subject to change based on procedure testing and advances in 

technology. 

Any questions concerning the information in this document should be directed to the appropriate point(s) 

of contact listed in Section 4. 

 SAM Revision 5.0 213 September 29, 2009 
Section 9 – Conclusions

 SAM Revision 5.0 214 September 29, 2009 
Appendix A – Selected Chemical Methods 

Appendix A: Selected Chemical Methods 

SAM Revision 5.0 September 29, 2009 
Appendix A – Selected Chemical Methods 

SAM Revision 5.0 September 29, 2009 
Appendix A: Selected Chemical Methods 

Analyte(s) CAS RN Determinative Technique Method Type Solid Samples Non-aqueous Liquid/Organic Solid Samples1 Aqueous Liquid Samples Drinking Water Samples A

Acephate 30560-19-1 HPLC 

Sample Prep Adapted from Journal of 

Chromatography A, 

1154(1): 3-25 

Adapted from 

Journal of 

Chromatography A, 

1154(1): 3-25 

Adapted from 

Chromatographia, 

63(5/6): 233-237 

Adapted from 

Chromatographia, 

63(5/6): 233-237 

A

Chro

1

Determinative 

Acrylamide 79-06-1 HPLC 

Sample Prep Water extraction Water extraction 8316 

(EPA SW-846) 

8316 

(EPA SW-846) 

Determinative 8316 (EPA SW-846) 8316 (EPA SW-846) 

Acrylonitrile 107-13-1 HPLC 

Sample Prep Water extraction Water extraction 8316 

(EPA SW-846) 

8316 

(EPA SW-846) 

Determinative 8316 (EPA SW-846) 8316 (EPA SW-846) 

Aldicarb (Temik) 116-06-3 HPLC 

Sample Prep 8318A 

(EPA SW-846) 

8318A 

(EPA SW-846) 

MS014 

(EPA CRL) 

531.2 

(EPA OW) 

Determinative 

Aldicarb sulfone 1646-88-4 HPLC 

Sample Prep 8318A 

(EPA SW-846) 

8318A 

(EPA SW-846) 

MS014 

(EPA CRL) 

531.2 

(EPA OW) 

Determinative 

Aldicarb sulfoxide 1646-87-3 HPLC 

Sample Prep 8318A 

(EPA SW-846) 

8318A 

(EPA SW-846) 

MS014 

(EPA CRL) 

531.2 

(EPA OW) 

Determinative 

Allyl alcohol 107-18-6 GC-MS 

Sample Prep 5035A (EPA SW-846) 3585 (EPA SW-846) 5030C (EPA SW-846) 5030C (EPA SW-846) 

(

Determinative 8260C (EPA SW-846) 8260C (EPA SW-846) 8260C (EPA SW-846) 8260C (EPA SW-846) 

4-Aminopyridine 504-24-5 HPLC 

Sample Prep 8330B 

(EPA SW-846) 

8330B 

(EPA SW-846) 

3535A/8330B 

(EPA SW-846) 

3535A/8330B 

(EPA SW-846) No

Determinative 8330B (EPA SW-846) 8330B (EPA SW-846) 

Ammonia 7664-41-7 Spectrophotometry 

Sample Prep 

Not of concern Not of concern 

4500- NH3 B 

(SM) 350.1 

(EPA OW) 

Determinative 4500- NH3 G (SM) 

Ammonium metavanadate 7803-55-6 Sample Prep 3050B (EPA SW-846) 3031 (EPA SW-846) 200.7/200.8 200.7/200.8 

(

(analyze for total vanadium) ICP-AES / ICP-MS Determinative 6010C/6020A (EPA SW-846) 6010C/6020A (EPA SW-846) (EPA OW) (EPA OW) IO

(

Arsenic, Total 7440-38-2 ICP-AES / ICP-MS 

Sample Prep 3050B (EPA SW-846) 3031 (EPA SW-846) 200.7/200.8 

(EPA OW) 

200.7/200.8 

(EPA OW) 

(

Determinative 6010C/6020A (EPA SW-846) 6010C/6020A (EPA SW-846) IO

(

SAM Revision 5.0, Appendix A A-1 
Analyte(s) CAS RN Determinative Technique Method Type Solid Samples Non-aqueous Liquid/Organic Solid Samples1 Aqueous Liquid Samples Drinking Water Samples A

Arsenic trioxide 1327-53-3 Sample Prep 3050B (EPA SW-846) 3031 (EPA SW-846) 200.7/200.8 200.7/200.8 

(

(analyze for total arsenic) ICP-AES / ICP-MS Determinative 6010C/6020A (EPA SW-846) 6010C/6020A (EPA SW-846) (EPA OW) (EPA OW) IO

(

Arsine 7784-42-1 GFAA / ICP-AES / ICP-MS Sample Prep 3050B (EPA SW-846) Not of concern 200.7/200.8 (EPA OW) 200.7/200.8 (EPA OW) 

Determinative 7010 (EPA SW-846) 

Asbestos 1332-21-4 TEM 

Sample Prep D5755-03 (soft 

surfaces-microvac) 

(ASTM) 

Not of concern Not of concern Not of concern 1

Determinative 

Boron trifluoride 7637-07-2 ISE 

Sample Prep 

Not of concern Not of concern Not of concern Not of concern 

Determinative 

Brodifacoum 56073-10-0 HPLC 

Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) 3520C/3535A (EPA SW-846) 3520C/3535A (EPA SW-846) No

Determinative 8321B (EPA SW-846) 8321B (EPA SW-846) 8321B (EPA SW-846) 8321B (EPA SW-846) 

Bromadiolone 28772-56-7 HPLC / LC-MS-MS 

Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) MS014 

(EPA CRL) 

3520C/3535A 

(EPA SW-846) No

Determinative 8321B (EPA SW-846) 8321B (EPA SW-846) 8321B (EPA SW-846) 

BZ [Quinuclidinyl benzilate] 6581-06-2 HPLC 

Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) 3520C/3535A (EPA SW-846) 3520C/3535A (EPA SW-846) 

(

Determinative 8321B3 (EPA SW-846) 8321B3 (EPA SW-846) 8321B3 (EPA SW-846) 8321B3 (EPA SW-846) 

Calcium arsenate 7778-44-1 Sample Prep 3050B (EPA SW-846) 3031 (EPA SW-846) 200.7/200.8 200.7/200.8 

(

(analyze as total arsenic) ICP-AES / ICP-MS Determinative 6010C/6020A (EPA SW-846) 6010C/6020A (EPA SW-846) (EPA OW) (EPA OW) IO

(

Carbofuran (Furadan) 1563-66-2 HPLC / LC-MS-MS 

Sample Prep 8318A 

(EPA SW-846) 

8318A 

(EPA SW-846) 

MS014 

(EPA CRL) 

531.2 

(EPA OW) 

Determinative 

Carfentanil 59708-52-0 HPLC 

Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) 3520C/3535A (EPA SW-846) 3520C/3535A (EPA SW-846) No

Determinative 8321B (EPA SW-846) 8321B (EPA SW-846) 8321B (EPA SW-846) 8321B (EPA SW-846) 

Carbon disulfide 75-15-0 GC-MS 

Sample Prep 5035A (EPA SW-846) 3585 (EPA SW-846) 5030C (EPA SW-846) 524.2 

(EPA OW) 

(

Determinative 8260C (EPA SW-846) 8260C (EPA SW-846) 8260C (EPA SW-846) 

Chlorfenvinphos 470-90-6 GC-MS 

Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) 3520C/3535A (EPA SW-846) 3520C/3535A (EPA SW-846) 

(

Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 

SAM Revision 5.0, Appendix A A-2 
Analyte(s) CAS RN Determinative Technique Method Type Solid Samples Non-aqueous Liquid/Organic Solid Samples1 Aqueous Liquid Samples Drinking Water Samples A

A

Chlorine 7782-50-5 Spectrophotometry 

Sample Prep 

Not of concern Not of concern 4500-Cl G 4500-Cl G Ana

Determinative 

(SM) (SM) 

2-Chloroethanol 107-07-3 GC-MS / GC-FID 

Sample Prep 5035A (EPA SW-846) 3585 (EPA SW-846) 5030C (EPA SW-846) 5030C (EPA SW-846) 

Determinative 8260C (EPA SW-846) 8260C (EPA SW-846) 8260C (EPA SW-846) 8260C (EPA SW-846) 

3-Chloro-1,2-propanediol 96-24-2 GC-MS 

Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) 3520C/3535A (EPA SW-846) 3520C/3535A (EPA SW-846) 

(

Determinative 8270D5 (EPA SW-846) 8270D5 (EPA SW-846) 8270D5 (EPA SW-846) 8270D5 (EPA SW-846) 

Chloropicrin 76-06-2 GC-MS / GC-ECD 

Sample Prep 3545A (EPA SW-846) 3580A (EPA SW-846) 551.1 

(EPA OW) 

551.1 

(EPA OW) PV

Determinative 8270D7 (EPA SW-846) 8270D7 (EPA SW-846) 

Chlorosarin 1445-76-7 GC-MS 

Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) 3520C/3535A (EPA SW-846) 3520C/3535A (EPA SW-846) 

(

Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 

Chlorosoman 7040-57-5 GC-MS 

Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) 3520C/3535A (EPA SW-846) 3520C/3535A (EPA SW-846) 

(

Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 

2-Chlorovinylarsonous acid (2-CVAA) (degradation product of Lewisite) 

85090-33-1 ICP-AES / ICP-MS 

Sample Prep 3050B (EPA SW-846) 3031 (EPA SW-846) 200.7/200.8 

(EPA OW) 

200.7/200.8 

(EPA OW) 

(

Determinative 6010C/6020A (EPA SW-846) 6010C/6020A (EPA SW-846) IO

(

Chlorpyrifos 2921-88-2 GC-MS 

Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) 3520C/3535A (EPA SW-846) 3520C/3535A (EPA SW-846) 

(

Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 

Chlorpyrifos oxon 5598-15-2 GC-MS 

Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) 3520C/3535A (EPA SW-846) 3520C/3535A (EPA SW-846) 

(

Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 

Crimidine 535-89-7 GC-MS 

Sample Prep 3545A (EPA SW-846) 3580A (EPA SW-846) 3535A (EPA SW-846) 3535A (EPA SW-846) No

Determinative 8270D8 (EPA SW-846) 8270D8 (EPA SW-846) 8270D8 (EPA SW-846) 8270D8 (EPA SW-846) 

Cyanide, Amenable to chlorination NA Spectrophotometry 

Sample Prep 3135.2I 

(EPA RLAB) Not of concern 3135.2I (EPA RLAB) 3135.2I (EPA RLAB) No

Determinative 

SAM Revision 5.0, Appendix A A-3 
Analyte(s) CAS RN Determinative Technique Method Type Solid Samples Non-aqueous Liquid/Organic Solid Samples1 Aqueous Liquid Samples Drinking Water Samples A

Cyanide, Total 57-12-5 Spectrophotometry 

Sample Prep ILM05.3 CN 

(EPA CLP) Not of concern ILM05.3 CN (EPA CLP) 335.4 (EPA OW) 

Determinative 

Cyanogen chloride 506-77-4 GC-MS 

Sample Prep 5035A (EPA SW-846) 3585 (EPA SW-846) 5030C (EPA SW-846) 5030C (EPA SW-846) 

(

Determinative 8260C (EPA SW-846) 8260C (EPA SW-846) 8260C (EPA SW-846) 8260C (EPA SW-846) 

Cyclohexyl sarin (GF) 329-99-7 GC-MS 

Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) 3520C/3535A (EPA SW-846) 3520C/3535A (EPA SW-846) 

(

Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 

1,2-Dichloroethane 107-06-2 GC-MS Sample Prep 5035A (EPA SW-846) 3585 (EPA SW-846) 5030C (EPA SW-846) 524.2 (EPA OW) 

(

(degradation product of HD) Determinative 8260C (EPA SW-846) 8260C (EPA SW-846) 8260C (EPA SW-846) 

Dichlorvos 62-73-7 GC-MS 

Sample Prep 3545A (EPA SW-846) 3580A (EPA SW-846) 3535A (EPA SW-846) 525.2 

(EPA OW) 

(

Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 

Dicrotophos 141-66-2 GC-MS 

Sample Prep 3545A (EPA SW-846) 3580A (EPA SW-846) 3535A (EPA SW-846) 3535A (EPA SW-846) 

(

Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 

Diesel range organics NA GC-FID 

Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) 3520C/3535A (EPA SW-846) 3520C/3535A (EPA SW-846) No

Determinative 8015C (EPA SW-846) 8015C (EPA SW-846) 8015C (EPA SW-846) 8015C (EPA SW-846) 

Diisopropyl methylphosphonate (DIMP) (degradation product of GB) 

1445-75-6 HPLC / LC-MS-MS 

Sample Prep 3545A (EPA SW-846) 3580A (EPA SW-846) MS017 

(EPA CRL) 

3535A 

(EPA SW-846) 

(

Determinative 8321B9 (EPA SW-846) 8321B9 (EPA SW-846) 8321B9 (EPA SW-846) 

Dimethylphosphite 868-85-9 GC-MS 

Sample Prep 3545A (EPA SW-846) 3580A (EPA SW-846) 3535A (EPA SW-846) 3535A (EPA SW-846) 

(

Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 

Dimethylphosphoramidic acid 33876-51-6 HPLC Sample Prep 3545A (EPA SW-846) 3580A (EPA SW-846) 3535A (EPA SW-846) 3535A (EPA SW-846) 

(degradation product of GA) Determinative 8321B3 (EPA SW-846) 8321B3 (EPA SW-846) 8321B3 (EPA SW-846) 8321B3 (EPA SW-846) (

Diphacinone 82-66-6 HPLC 

Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) 3520C/3535A (EPA SW-846) 3520C/3535A (EPA SW-846) No

Determinative 8321B (EPA SW-846) 8321B (EPA SW-846) 8321B (EPA SW-846) 8321B (EPA SW-846) 

Disulfoton 298-04-4 GC-MS / GC-FPD 

Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) 525.2 

(EPA OW) 

525.2 

(EPA OW) 

Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 

SAM Revision 5.0, Appendix A A-4 
Analyte(s) CAS RN Determinative Technique Method Type Solid Samples Non-aqueous Liquid/Organic Solid Samples1 Aqueous Liquid Samples Drinking Water Samples A

Disulfoton sulfoxide 2497-07-6 GC-MS / GC-FPD 

Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) 525.2 

(EPA OW) 

525.2 

(EPA OW) 

Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 

1,4-Dithiane 505-29-3 GC-MS Sample Prep 3545A (EPA SW-846) 3580A (EPA SW-846) 3535A (EPA SW-846) 3535A (EPA SW-846) No

(degradation product of HD) Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 

EA2192 [Diisopropylaminoethyl methylthiolophosphonate] (hydrolysis product of VX) 

73207-98-4 HPLC 

Sample Prep 3545A (EPA SW-846) 3580A (EPA SW-846) 3535A (EPA SW-846) 3535A (EPA SW-846) 

(

Determinative 8321B3 (EPA SW-846) 8321B3 (EPA SW-846) 8321B3 (EPA SW-846) 8321B3 (EPA SW-846) 

Ethyl methylphosphonic acid (EMPA) (degradation product of VX) 

1832-53-7 HPLC / LC-MS-MS 

Sample Prep 3545A (EPA SW-846) 3580A (EPA SW-846) MS017 

(EPA CRL) 

3535A 

(EPA SW-846) 

(

Determinative 8321B3 (EPA SW-846) 8321B3 (EPA SW-846) 8321B3 (EPA SW-846) 

Ethyldichloroarsine (ED) 598-14-1 GC-MS 

Sample Prep 3545A (EPA SW-846) 3580A (EPA SW-846) 3535A (EPA SW-846) 3535A (EPA SW-846) 

(

Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 

N-Ethyldiethanolamine (EDEA) 139-87-7 Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) MS016 3520C/3535A (EPA SW-846) 

(degradation product of HN-1) HPLC / LC-MS-MS Determinative 8321B (EPA SW-846) 8321B (EPA SW-846) (EPA CRL) 8321B (EPA SW-846) (

Ethylene oxide 75-21-8 GC-MS 

Sample Prep 5035A (EPA SW-846) 3585 (EPA SW-846) 5030C (EPA SW-846) 5030C (EPA SW-846) 

(

Determinative 8260C (EPA SW-846) 8260C (EPA SW-846) 8260C (EPA SW-846) 8260C (EPA SW-846) 

Fenamiphos 22224-92-6 GC-MS 

Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) 3520C/3535A (EPA SW-846) 525.2 

(EPA OW) 

(

Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 

Fentanyl 437-38-7 HPLC 

Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) 3520C/3535A (EPA SW-846) 3520C/3535A (EPA SW-846) No

Determinative 8321B (EPA SW-846) 8321B (EPA SW-846) 8321B (EPA SW-846) 8321B (EPA SW-846) 

Fluoride 16984-48-8 IC 

Sample Prep 

Not of concern Not of concern 300.1, Rev 1.0 (EPA OW) 300.1, Rev 1.0 (EPA OW) No

Determinative 

Fluoroacetamide 640-19-7 GC-MS 

Sample Prep Adapted from Journal of 

Chromatography B, 

876(1): 103-108 

Adapted from 

Journal of 

Chromatography B, 

876(1): 103-108 

Adapted from 

Journal of 

Chromatography B, 

876(1): 103-108 

Adapted from 

Journal of 

Chromatography B, 

876(1): 103-108 

A

Chro

87

Determinative 

SAM Revision 5.0, Appendix A A-5 
Analyte(s) CAS RN Determinative Technique Method Type Solid Samples Non-aqueous Liquid/Organic Solid Samples1 Aqueous Liquid Samples Drinking Water Samples A

Fluoroacetic acid and fluoroacetate salts (analyze for fluoroacetate ion) NA IC 

Sample Prep 

Adapted from 

Analytical Letters, 

27(14): 2703-2718 

Adapted from 

Analytical Letters, 

27(14): 2703-2718 300.1, Rev 1.0 (EPA OW) 300.1, Rev 1.0 (EPA OW) 

Determinative 300.1, Rev 1.0 (EPA OW) 300.1, Rev 1.0 (EPA OW) 30

2-Fluoroethanol 371-62-0 GC-MS / GC-FID 

Sample Prep 5035A (EPA SW-846) 3585 (EPA SW-846) 5030C (EPA SW-846) 5030C (EPA SW-846) 

Determinative 8260C (EPA SW-846) 8260C (EPA SW-846) 8260C (EPA SW-846) 8260C (EPA SW-846) 

Formaldehyde 50-00-0 HPLC 

Sample Prep 8315A 

(EPA SW-846) Not of concern 8315A (EPA SW-846) 8315A (EPA SW-846) 

Determinative 

Gasoline range organics NA GC-FID 

Sample Prep 5035A (EPA SW-846) 3585 (EPA SW-846) 5030C (EPA SW-846) 5030C (EPA SW-846) No

Determinative 8015C (EPA SW-846) 8015C (EPA SW-846) 8015C (EPA SW-846) 8015C (EPA SW-846) 

Hexahydro-1,3,5-trinitro-1,3,5- 121-82-4 HPLC Sample Prep 8330B 8330B 3535A/8330B (EPA SW-846) 3535A/8330B (EPA SW-846) No

triazine (RDX) Determinative (EPA SW-846) (EPA SW-846) 8330B (EPA SW-846) 8330B (EPA SW-846) 

Hexamethylenetriperoxidediamine 283-66-9 HPLC Sample Prep 8330B 8330B 3535A/8330B (EPA SW-846) 3535A/8330B (EPA SW-846) No

(HMTD) Determinative (EPA SW-846) (EPA SW-846) 8330B (EPA SW-846) 8330B (EPA SW-846) 

Hydrogen bromide 10035-10-6 IC 

Sample Prep 

Not of concern Not of concern Not of concern Not of concern 

Determinative 

Hydrogen chloride 7647-01-0 IC 

Sample Prep 

Not of concern Not of concern Not of concern Not of concern 

Determinative 

Hydrogen cyanide 74-90-8 Spectrophotometry 

Sample Prep 

Not of concern Not of concern Not of concern Not of concern 

Determinative 

Hydrogen fluoride 7664-39-3 IC 

Sample Prep 

Not of concern Not of concern Not of concern Not of concern 

Determinative 

Hydrogen sulfide 7783-06-4 IC 

Sample Prep 

Not of concern Not of concern Not of concern Not of concern 

Determinative 

SAM Revision 5.0, Appendix A A-6 
Analyte(s) CAS RN Determinative Technique Method Type Solid Samples Non-aqueous Liquid/Organic Solid Samples1 Aqueous Liquid Samples Drinking Water Samples A

Isopropyl methylphosphonic acid (IMPA) (degradation product of GB) 

1832-54-8 HPLC / LC-MS-MS 

Sample Prep 3545A (EPA SW-846) 3580A (EPA SW-846) MS017 

(EPA CRL) 

3535A 

(EPA SW-846) 

(

Determinative 8321B3 (EPA SW-846) 8321B3 (EPA SW-846) 8321B3 (EPA SW-846) 

Kerosene 64742-81-0 GC-FID 

Sample Prep 5035A (EPA SW-846) 3585 (EPA SW-846) 5030C (EPA SW-846) 5030C (EPA SW-846) No

Determinative 8015C (EPA SW-846) 8015C (EPA SW-846) 8015C (EPA SW-846) 8015C (EPA SW-846) 

Lead arsenate 7645-25-2 Sample Prep 3050B (EPA SW-846) 3031 (EPA SW-846) 200.7/200.8 200.7/200.8 

(

(analyze as total arsenic) ICP-AES / ICP-MS Determinative 6010C/6020A (EPA SW-846) 6010C/6020A (EPA SW-846) (EPA OW) (EPA OW) IO

(

Lewisite 1 (L-1)11 

[2-chlorovinyldichloroarsine] (analyze for total arsenic) 

541-25-3 ICP-AES / ICP-MS 

Sample Prep 3050B (EPA SW-846) 3031 (EPA SW-846) 200.7/200.8 

(EPA OW) 

200.7/200.8 

(EPA OW) 

(

Determinative 6010C/6020A (EPA SW-846) 6010C/6020A (EPA SW-846) IO

(

Lewisite 2 (L-2) [bis(2-chlorovinyl)chloroarsine] (analyze for total arsenic) 

40334-69-8 ICP-AES / ICP-MS 

Sample Prep 3050B (EPA SW-846) 3031 (EPA SW-846) 200.7/200.8 

(EPA OW) 

200.7/200.8 

(EPA OW) 

(

Determinative 6010C/6020A (EPA SW-846) 6010C/6020A (EPA SW-846) IO

(

Lewisite 3 (L-3) [tris(2-chlorovinyl)arsine] (analyze for total arsenic) 

40334-70-1 ICP-AES / ICP-MS 

Sample Prep 3050B (EPA SW-846) 3031 (EPA SW-846) 200.7/200.8 

(EPA OW) 

200.7/200.8 

(EPA OW) 

(

Determinative 6010C/6020A (EPA SW-846) 6010C/6020A (EPA SW-846) IO

(

Lewisite oxide 1306-02-1 Sample Prep 3050B (EPA SW-846) 3031 (EPA SW-846) 200.7/200.8 200.7/200.8 

(

(degradation product of Lewisite) ICP-AES / ICP-MS Determinative 6010C/6020A (EPA SW-846) 6010C/6020A (EPA SW-846) (EPA OW) (EPA OW) IO

(

Mercuric chloride (analyze for total 7487-94-7 Spectrophotometry Sample Prep 747312 Not of concern 747312 245.1 No

mercury) / CVAA / CVAFS Determinative (EPA SW-846) (EPA SW-846) (EPA OW) 

Mercury, Total 7439-97-6 Spectrophotometry / CVAA / CVAFS Sample Prep 747312 (EPA SW-846) Not of concern 747312 (EPA SW-846) 245.1 (EPA OW) 

(

Determinative 

Methamidophos 10265-92-6 LC-MS-MS 

Sample Prep Adapted from Journal of 

Chromatography A, 

1154(1): 3-25 

Adapted from 

Journal of 

Chromatography A, 

1154(1): 3-25 

Adapted from 

Chromatographia, 

63(5/6): 233-237 

Adapted from 

Chromatographia, 

63(5/6): 233-237 

A

Chro

1

Determinative 

Methomyl 16752-77-5 HPLC / LC-MS-MS 

Sample Prep 8318A 

(EPA SW-846) 

8318A 

(EPA SW-846) 

MS014 

(EPA CRL) 

531.2 

(EPA OW) 

Determinative 

Methoxyethylmercuric acetate 151-38-2 Spectrophotometry Sample Prep 747312 Not of concern 747312 245.1 

(analyze for total mercury) / CVAA / CVAFS Determinative (EPA SW-846) (EPA SW-846) (EPA OW) (

SAM Revision 5.0, Appendix A A-7 
Analyte(s) CAS RN Determinative Technique Method Type Solid Samples Non-aqueous Liquid/Organic Solid Samples1 Aqueous Liquid Samples Drinking Water Samples A

Methyl acrylonitrile 126-98-7 HPLC 

Sample Prep Water extraction Water extraction 8316 

(EPA SW-846) 

8316 

(EPA SW-846) 

Determinative 8316 (EPA SW-846) 8316 (EPA SW-846) 

Methyl fluoroacetate (analyze for fluoroacetate ion) 453-18-9 IC 

Sample Prep 

Adapted from 

Analytical Letters, 

27(14): 2703-2718 

Adapted from 

Analytical Letters, 

27(14): 2703-2718 300.1, Rev 1.0 (EPA OW) 300.1, Rev 1.0 (EPA OW) 

Determinative 300.1, Rev 1.0 (EPA OW) 300.1, Rev 1.0 (EPA OW) 30

Methyl hydrazine 60-34-4 GC-MS / Spectrophotometry Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) 3520C/3535A (EPA SW-846) 3520C/3535A (EPA SW-846) 

Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 

Sample Prep 

Methyl isocyanate 624-83-9 HPLC Not of concern Not of concern Not of concern Not of concern 

Determinative 

Methyl paraoxon 950-35-6 GC-MS 

Sample Prep 3545A (EPA SW-846) 3580A (EPA SW-846) 3535A (EPA SW-846) 3535A (EPA SW-846) 

(

Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 

Methyl parathion 298-00-0 GC-MS 

Sample Prep 3545A (EPA SW-846) 3580A (EPA SW-846) 3535A (EPA SW-846) 3535A (EPA SW-846) 

(

Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 

Sample Prep 

Methylamine 74-89-5 HPLC Not of concern Not of concern Not of concern Not of concern 

Determinative 

N-Methyldiethanolamine (MDEA) 105-59-9 Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) MS016 3520C/3535A (EPA SW-846) (degradation product of HN-2) HPLC / LC-MS-MS Determinative 8321B (EPA SW-846) 8321B (EPA SW-846) (EPA CRL) 8321B (EPA SW-846) (

1-Methylethyl ester 1189-87-3 GC-MS Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) 3520C/3535A (EPA SW-846) 3520C/3535A (EPA SW-846) 

ethylphosphonofluoridic acid (GE) Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) (

Methylphosphonic acid (MPA) (degradation product of VX, GB, & GD) 

993-13-5 HPLC 

Sample Prep 3545A (EPA SW-846) 3580A (EPA SW-846) MS017 

(EPA CRL) 

3535A 

(EPA SW-846) 

(

Determinative 8321B3 (EPA SW-846) 8321B3 (EPA SW-846) 8321B3 (EPA SW-846) 

Mevinphos 7786-34-7 GC-MS 

Sample Prep 3545A (EPA SW-846) 3580A (EPA SW-846) 3535A (EPA SW-846) 525.2 

(EPA OW) 

(

Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 

SAM Revision 5.0, Appendix A A-8 
Analyte(s) CAS RN Determinative Technique Method Type Solid Samples Non-aqueous Liquid/Organic Solid Samples1 Aqueous Liquid Samples Drinking Water Samples A

Monocrotophos 6923-22-4 GC-MS 

Sample Prep 3545A (EPA SW-846) 3580A (EPA SW-846) 3535A (EPA SW-846) 3535A (EPA SW-846) 

(

Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 

Mustard, nitrogen (HN-1) 538-07-8 GC-MS Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) 3520C/3535A (EPA SW-846) 3520C/3535A (EPA SW-846) 

[bis(2-chloroethyl)ethylamine] Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) (

Mustard, nitrogen (HN-2) [2,2'-dichloro-N-methyldiethylamine 51-75-2 GC-MS Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) 3520C/3535A (EPA SW-846) 3520C/3535A (EPA SW-846) 

N,N-bis(2-chloroethyl)methylamine] Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) (

Mustard, nitrogen (HN-3) 555-77-1 GC-MS Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) 3520C/3535A (EPA SW-846) 3520C/3535A (EPA SW-846) 

[tris(2-chloroethyl)amine] Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) (

Mustard, sulfur / Mustard gas (HD) 505-60-2 GC-MS 

Sample Prep 3571 (EPA SW-846) 3571 (EPA SW-846) 3571 (EPA SW-846) 3571 (EPA SW-846) 

(

Determinative 8270D13 (EPA SW-846) 8270D13 (EPA SW-846) 8270D13 (EPA SW-846) 8270D13 (EPA SW-846) 

Nicotine compounds 54-11-5 GC-MS Sample Prep 3545A (EPA SW-846) 3580A (EPA SW-846) 3535A (EPA SW-846) 3535A (EPA SW-846) No

(analyze as nicotine) Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 

Octahydro-1,3,5,7-tetranitro-1,3,5,7 2691-41-0 HPLC Sample Prep 8330B 8330B 3535A/8330B (EPA SW-846) 3535A/8330B (EPA SW-846) No

tetrazocine (HMX) Determinative (EPA SW-846) (EPA SW-846) 8330B (EPA SW-846) 8330B (EPA SW-846) 

Organophosphate pesticides, NOS NA GC-MS / GC-NPD / GC-FPD Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) 614 (EPA OW) 507 (EPA OW) 

Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 

Osmium tetroxide 20816-12-0 Sample Prep 3050B (EPA SW-846) Not of concern 252.2 252.2 

(

(analyze for total osmium) ICP-AES / GFAA Determinative 6010C (EPA SW-846) (EPA OW) (EPA OW) 

(

Oxamyl 23135-22-0 HPLC / LC-MS-MS 

Sample Prep 8318A 

(EPA SW-846) 

8318A 

(EPA SW-846) 

MS014 

(EPA CRL) 

531.2 

(EPA OW) 

Determinative 

Paraquat 4685-14-7 HPLC 

Sample Prep 

Not of concern Not of concern 549.2 (EPA OW) 549.2 (EPA OW) No

Determinative 

Paraoxon 311-45-5 GC-MS 

Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) 3520C/3535A (EPA SW-846) 3520C/3535A (EPA SW-846) 

(

Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 

SAM Revision 5.0, Appendix A A-9 
Analyte(s) CAS RN Determinative Technique Method Type Solid Samples Non-aqueous Liquid/Organic Solid Samples1 Aqueous Liquid Samples Drinking Water Samples A

Parathion 56-38-2 GC-MS 

Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) 3520C/3535A (EPA SW-846) 3520C/3535A (EPA SW-846) 

(

Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 

Pentaerythritol tetranitrate (PETN) 78-11-5 HPLC 

Sample Prep 8330B 

(EPA SW-846) 

8330B 

(EPA SW-846) 

3535A/8330B 

(EPA SW-846) 

3535A/8330B 

(EPA SW-846) No

Determinative 8330B (EPA SW-846) 8330B (EPA SW-846) 

Phencyclidine 77-10-1 GC-MS 

Sample Prep 3545A (EPA SW-846) 3580A (EPA SW-846) 3535A (EPA SW-846) 3535A (EPA SW-846) 

(

Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 

Phenol 108-95-2 GC-MS 

Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) 3520C/3535A (EPA SW-846) 3520C/3535A (EPA SW-846) 

(

Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 

Phorate 298-02-2 GC-MS 

Sample Prep 3545A (EPA SW-846) 3580A (EPA SW-846) 3535A (EPA SW-846) 3535A (EPA SW-846) 

(

Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 

Phorate sulfone 2588-04-7 GC-MS 

Sample Prep 3545A (EPA SW-846) 3580A (EPA SW-846) 3535A (EPA SW-846) 3535A (EPA SW-846) 

(

Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 

Phorate sulfoxide 2588-03-6 GC-MS 

Sample Prep 3545A (EPA SW-846) 3580A (EPA SW-846) 3535A (EPA SW-846) 3535A (EPA SW-846) 

(

Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 

Sample Prep 

Phosgene 75-44-5 GC-NPD Not of concern Not of concern Not of concern Not of concern 

Determinative 

Phosphamidon 13171-21-6 GC-MS 

Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) 3520C/3535A (EPA SW-846) 3520C/3535A (EPA SW-846) 

(

Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 

Phosphine 7803-51-2 Spectrophotometry 

Sample Prep 

Not of concern Not of concern Not of concern Not of concern 

Determinative 

Phosphorus trichloride 7719-12-2 Spectrophotometry 

Sample Prep 

Not of concern Not of concern Not of concern Not of concern 

Determinative 

Pinacolyl methyl phosphonic acid (PMPA) (degradation product of GD) 

616-52-4 HPLC / LC-MS-MS 

Sample Prep 3545A (EPA SW-846) 3580A (EPA SW-846) MS017 

(EPA CRL) 

3535A 

(EPA SW-846) 

(

Determinative 8321B3 (EPA SW-846) 8321B3 (EPA SW-846) 8321B3 (EPA SW-846) 

SAM Revision 5.0, Appendix A A-10 
Analyte(s) CAS RN Determinative Technique Method Type Solid Samples Non-aqueous Liquid/Organic Solid Samples1 Aqueous Liquid Samples Drinking Water Samples A

Propylene oxide 75-56-9 GC-MS / GC-FID 

Sample Prep 5035A (EPA SW-846) 3585 (EPA SW-846) 5030C (EPA SW-846) 5030C (EPA SW-846) 

Determinative 8260C (EPA SW-846) 8260C (EPA SW-846) 8260C (EPA SW-846) 8260C (EPA SW-846) 

R 33 (VR) [methylphosphonothioic acid, S-[2- 159939-87-4 GC-MS Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) 3520C/3535A (EPA SW-846) 3520C/3535A (EPA SW-846) (diethylamino)ethyl] O-2-methylpropyl ester] Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) (

Sarin (GB) 107-44-8 GC-MS 

Sample Prep 3571 (EPA SW-846) 3571 (EPA SW-846) 3571 (EPA SW-846) 3571 (EPA SW-846) 

(

Determinative 8270D13 (EPA SW-846) 8270D13 (EPA SW-846) 8270D13 (EPA SW-846) 8270D13 (EPA SW-846) 

Semivolatile organic compounds, NA GC-MS Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) 3520C/3535A (EPA SW-846) 525.2 NOS 

Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) (EPA OW) (

Sodium arsenite 7784-46-5 Sample Prep 3050B (EPA SW-846) 3031 (EPA SW-846) 200.7/200.8 200.7/200.8 

(

(analyze for total arsenic) ICP-AES / ICP-MS Determinative 6010C/6020A (EPA SW-846) 6010C/6020A (EPA SW-846) (EPA OW) (EPA OW) IO

(

Sodium azide (analyze as azide ion) 26628-22-8 IC 

Sample Prep 

Adapted from J. of 

Forensic Sciences, 

43(1): 200-20214 

3580A14 

(EPA SW-846) 

Adapted from J. of 

Forensic Sciences, 

43(1): 200-20214 

Adapted from J. of 

Forensic Sciences, 

43(1): 200-20214 ID

Determinative 300.1, Rev 1.015 (EPA OW) 300.1, Rev 1.015 (EPA OW) 300.1, Rev 1.015 (EPA OW) 300.1, Rev 1.015 (EPA OW) 

Soman (GD) 96-64-0 GC-MS 

Sample Prep 3545A (EPA SW-846) 3580A (EPA SW-846) 3535A (EPA SW-846) 3535A (EPA SW-846) 

(

Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 

Strychnine 57-24-9 GC-MS 

Sample Prep 3545A (EPA SW-846) 3580A (EPA SW-846) 3535A (EPA SW-846) 3535A (EPA SW-846) No

Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 

Sulfur dioxide 7446-09-5 IC 

Sample Prep 

Not of concern Not of concern Not of concern Not of concern 

Determinative 

Sulfur trioxide 7446-11-9 Titrimetry 

Sample Prep 

Not of concern Not of concern Not of concern Not of concern 

(E

Determinative 

Tabun (GA) 77-81-6 GC-MS 

Sample Prep 3545A (EPA SW-846) 3580A (EPA SW-846) 3535A (EPA SW-846) 3535A (EPA SW-846) 

(

Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 

SAM Revision 5.0, Appendix A A-11 
Analyte(s) CAS RN Determinative Technique Method Type Solid Samples Non-aqueous Liquid/Organic Solid Samples1 Aqueous Liquid Samples Drinking Water Samples A

Tetraethyl pyrophosphate 107-49-3 GC-MS 

Sample Prep 3545A (EPA SW-846) 3580A (EPA SW-846) 3535A (EPA SW-846) 3535A (EPA SW-846) 

(

Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 

Tetramethylenedisulfotetramine 80-12-6 GC-MS 

Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) 3520C/3535A (EPA SW-846) 3520C/3535A (EPA SW-846) 

(

Determinative 8270D7 (EPA SW-846) 8270D7 (EPA SW-846) 8270D7 (EPA SW-846) 8270D7 (EPA SW-846) 

Thallium sulfate 10031-59-1 Sample Prep 3050B (EPA SW-846) 3031 (EPA SW-846) 200.7/200.8 200.7/200.8 

(

(analyze for total thallium) ICP-AES / ICP-MS Determinative 6010C/6020A (EPA SW-846) 6010C/6020A (EPA SW-846) (EPA OW) (EPA OW) IO

(

Thiodiglycol (TDG) 111-48-8 Sample Prep 3545A (EPA SW-846) 3580A (EPA SW-846) MS015 3535A (EPA SW-846) 

(degradation product of HD) HPLC / LC-MS-MS Determinative 8321B (EPA SW-846) 8321B (EPA SW-846) (EPA CRL) 8321B (EPA SW-846) (

Thiofanox 39196-18-4 HPLC 

Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) 3520C/3535A (EPA SW-846) 531.2 

(EPA OW) 

Determinative 8321B (EPA SW-846) 8321B (EPA SW-846) 8321B (EPA SW-846) 

1,4-Thioxane 15980-15-1 GC-MS Sample Prep 3545A (EPA SW-846) 3580A (EPA SW-846) 3535A (EPA SW-846) 3535A (EPA SW-846) No

(degradation product of HD) Determinative 8270D16 (EPA SW-846) 8270D16 (EPA SW-846) 8270D16 (EPA SW-846) 8270D16 (EPA SW-846) 

Titanium tetrachloride 7550-45-0 Sample Prep 3050B (EPA SW-846) Not of concern Not of concern Not of concern No

(analyze for total titanium) ICP-AES / ICP-MS Determinative 6010C/6020A (EPA SW-846) Triethanolamine (TEA) 102-71-6 Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) MS016 3520C/3535A (EPA SW-846) 

(degradation product of HN-3) HPLC / LC-MS-MS Determinative 8321B (EPA SW-846) 8321B (EPA SW-846) (EPA CRL) 8321B (EPA SW-846) (

Trimethyl phosphite 121-45-9 GC-MS 

Sample Prep 3545A (EPA SW-846) 3580A (EPA SW-846) 3535A (EPA SW-846) 3535A (EPA SW-846) 

(

Determinative 8270D7 (EPA SW-846) 8270D7 (EPA SW-846) 8270D7 (EPA SW-846) 8270D7 (EPA SW-846) 

1,3,5-Trinitrobenzene (1,3,5-TNB) 99-35-4 HPLC 

Sample Prep 8330B 

(EPA SW-846) 

8330B 

(EPA SW-846) 

3535A/8330B 

(EPA SW-846) 

3535A/8330B 

(EPA SW-846) No

Determinative 8330B (EPA SW-846) 8330B (EPA SW-846) 

2,4,6-Trinitrotoluene (2,4,6-TNT) 118-96-7 HPLC 

Sample Prep 8330B 

(EPA SW-846) 

8330B 

(EPA SW-846) 

3535A/8330B 

(EPA SW-846) 

3535A/8330B 

(EPA SW-846) No

Determinative 8330B (EPA SW-846) 8330B (EPA SW-846) 

Vanadium pentoxide 1314-62-1 Sample Prep 3050B (EPA SW-846) 3031 (EPA SW-846) 200.7/200.8 200.7/200.8 

(

(analyze for total vanadium) ICP-AES / ICP-MS Determinative 6010C/6020A (EPA SW-846) 6010C/6020A (EPA SW-846) (EPA OW) (EPA OW) IO

(

SAM Revision 5.0, Appendix A A-12 
Analyte(s) CAS RN Determinative Technique Method Type Solid Samples Non-aqueous Liquid/Organic Solid Samples1 Aqueous Liquid Samples Drinking Water Samples A

VE [phosphonothioic acid, ethyl-, S-

(2-(diethylamino)ethyl) O-ethyl ester] 

21738-25-0 GC-MS 

Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) 3520C/3535A (EPA SW-846) 3520C/3535A (EPA SW-846) 

(

Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 

VG [phosphonothioic acid, S-(2-(diethylamino)ethyl) O,O-diethyl ester] 

78-53-5 GC-MS 

Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) 3520C/3535A (EPA SW-846) 3520C/3535A (EPA SW-846) 

(

Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 

VM [phosphonothioic acid, methyl-, S-(2-(diethylamino)ethyl) O-ethyl ester] 

21770-86-5 GC-MS 

Sample Prep 3541/3545A (EPA SW-846) 3580A (EPA SW-846) 3520C/3535A (EPA SW-846) 3520C/3535A (EPA SW-846) 

(

Determinative 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 8270D (EPA SW-846) 

VX [O-ethyl-S-(2-diisopropylaminoethyl)methylphosphonothiolate] 50782-69-9 GC-MS 

Sample Prep 3571 (EPA SW-846) 3571 (EPA SW-846) 3571 (EPA SW-846) 3571 (EPA SW-846) 

(

Determinative 8270D13 (EPA SW-846) 8270D13 (EPA SW-846) 8270D13 (EPA SW-846) 8270D13 (EPA SW-846) 

White phosphorus 12185-10-3 GC-NPD / GC-FPD 

Sample Prep 7580 

(EPA SW-846) 

7580 

(EPA SW-846) 

7580 

(EPA SW-846) 

7580 

(EPA SW-846) 

Determinative 

Footnotes
 

1

 An organic solid sample is a solid that completely dissolves in an organic solvent and leaves no solid residue.
 

2

 If problems occur when using this method, it is recommended that TO-10A be used.
 

3 LC-MS (electrospray) procedures are preferred for these analytes; however, if this technique is not available to the laboratory, GC-MS procedures using derivatization based 

Sample preparation methods should remain the same. Both electrospray LC-MS and GC-MS derivatization procedures are currently under development.
 

4 For this analyte, HPLC is the preferred technique; however, if problems occur, Method TO-10A must be modified to include a derivatization step prior to analysis by GC-MS.
 5

 For this analyte, SW-846 Method 8270D must be modified to include a derivatization step.
 

6

 If problems occur when using this method, it is recommended that the canister Method TO-15 be used. 

7

 If problems occur with analyses, lower the injection temperature. 

8

 If problems occur when using this method, it is recommended that SW-846 Method 8321B be used as the determinative method. Sample preparation methods should remain 

9 If problems occur with the analysis of DIMP using EPA SW-846 Method 8321B, use SW-846 Method 8270D.
 10

 If problems occur when using this method, it is recommended that NIOSH Method 7906 be used.
 

11

 Laboratory testing is currently under way for speciation of Lewisite 1 using GC-MS techniques.
 

12 If equipment is not available, use CVAA Methods 7471B (EPA SW-846) for solid samples and 7470A (EPA SW-846) for aqueous liquid samples.
 13

 For this analyte, refer to EPA SW-846 Method 8271 for GC-MS conditions.
 14 Water extraction, filtration, and acidification steps from the Journal of Forensic Science, 1998. 43(1): 200-202 should be used for the preparation of solid samples. Filtration 

should be used for preparation of aqueous liquid and drinking water samples. The acidification step from the journal should be used with EPA SW-846 Method 3580A for prepasamples.
 

15

 If analyses are problematic, refer to column manufacturer for alternate conditions 

16 If problems occur when using this method, it is recommended that SW-846 Method 8260C and appropriate corresponding sample preparation procedures (i.e., 5035A for so

liquid/organic solid samples, and 5030C for aqueous liquid and drinking water samples) be used. 

SAM Revision 5.0, Appendix A A-13 
SAM Revision 5.0, Appendix A A - 14 
Appendix B – Selected Radiochemical Methods 

Appendix B: Selected Radiochemical Methods
 

SAM Revision 5.0 September 29, 2009 
Appendix B – Selected Radiochemical Methods 

SAM Revision 5.0 September 29, 2009 
Appendix B: Selected Radiochemical Methods 

Analyte Class Determinative Technique Drinking Water Samples Aqueous and Liquid Phase Samples Soil and Sediment Samples Surface Wipes 

Gross Alpha Alpha/Beta counting 900.0 (EPA) 7110 B (SM) AP1 (ORISE) FRMAC, Vol 2, pg. 3

Gross Beta Alpha/Beta counting 900.0 (EPA) 7110 B (SM) AP1 (ORISE) FRMAC, Vol 2, pg. 3

Gamma Gamma spectrometry 901.1 (EPA) Ga-01-R (HASL-300) Ga-01-R (HASL-300) Ga-01-R (HASL-300) 

Select Mixed Fission 

Products1 

Gamma 

spectrometry 901.1 (EPA) Ga-01-R (HASL-300) Ga-01-R (HASL-300) Ga-01-R (HASL-300) 

Analyte(s) CAS RN Determinative Technique 

Drinking Water Samples Aqueous and Liquid Phase Samples Soil and Sediment Samples Surface Wipes 

Qualitative 

Determination2 Confirmatory Qualitative Determination2 Confirmatory Qualitative Determination2 Confirmatory Qualitative Determination2 Confirm

Americium-2413 14596-10-2 Alpha/Gamma spectrometry D3084-05 (ASTM) Am-04-RC (HASL-300) D3084-05 (ASTM) Am-04-RC (HASL-300) Am-02-RC (HASL-300) Am-01-RC4 (HASL-300) D3084-05 (ASTM) Am-04

(HASL-

Californium-2523 13981-17-4 Alpha spectrometry D3084-05 (ASTM) Am-04-RC (HASL-300) D3084-05 (ASTM) Am-04-RC (HASL-300) D3084-05 (ASTM) Am-01-RC4 (HASL-300) D3084-05 (ASTM) Am-04

(HASL-

Cesium-137 10045-97-3 Gamma spectrometry 901.1 (EPA) 901.1 (EPA) 7120 (SM) 7120 (SM) Ga-01-R (HASL-300) Ga-01-R (HASL-300) Ga-01-R (HASL-300) Ga-0

(HASL-

Cobalt-60 10198-40-0 Gamma spectrometry 901.1 (EPA) 901.1 (EPA) 7120 (SM) 7120 (SM) Ga-01-R (HASL-300) Ga-01-R (HASL-300) Ga-01-R (HASL-300) Ga-0

(HASL-

Curium-2443 13981-15-2 Alpha spectrometry D3084-05 (ASTM) Am-04-RC (HASL-300) D3084-05 (ASTM) Am-04-RC (HASL-300) D3084-05 (ASTM) Am-01-RC4 (HASL-300) D3084-05 (ASTM) Am-04

(HASL-

Europium-154 15585-10-1 Gamma spectrometry 901.1 (EPA) 901.1 (EPA) 7120 (SM) 7120 (SM) Ga-01-R (HASL-300) Ga-01-R (HASL-300) Ga-01-R (HASL-300) Ga-0

(HASL-

Iodine-125 14158-31-7 Gamma spectrometry Procedure #9 (ORISE) Procedure #9 (ORISE) Procedure #9 (ORISE) Procedure #9 (ORISE) Procedure #9 (ORISE) Procedure #9 (ORISE) Procedure #9 (ORISE) Procedu

(ORIS

Iodine-131 10043-66-0 Gamma spectrometry 901.1 (EPA) 901.1 (EPA) Ga-01-R (HASL-300) Ga-01-R (HASL-300) Ga-01-R (HASL-300) Ga-01-R (HASL-300) Ga-01-R (HASL-300) Ga-0

(HASL-

Iridium-192 14694-69-0 Gamma spectrometry 901.1 (EPA) 901.1 (EPA) 7120 (SM) 7120 (SM) Ga-01-R (HASL-300) Ga-01-R (HASL-300) Ga-01-R (HASL-300) Ga-0

(HASL-

SAM Revision 5.0, Appendix B B - 1 
Analyte(s) CAS RN Determinative Technique 

Drinking Water Samples Aqueous and Liquid Phase Samples Soil and Sediment Samples Surface Wipes 

Qualitative 

Determination2 Confirmatory Qualitative Determination2 Confirmatory Qualitative Determination2 Confirmatory Qualitative Determination2 Confirm

Molybdenum-99 14119-15-4 Gamma spectrometry 901.1 (EPA) 901.1 (EPA) Ga-01-R (HASL-300) Ga-01-R (HASL-300) Ga-01-R (HASL-300) Ga-01-R (HASL-300) Ga-01-R (HASL-300) Ga-0

(HASL-

Plutonium-2383 13981-16-3 Alpha spectrometry D3084-05 (ASTM) EMSL-33 (EPA) D3084-05 (ASTM) EMSL-33 (EPA) D3084-05 (ASTM) EMSL-33 (EPA) D3084-05 (ASTM) EMSL

(EPA

Plutonium-2393 15117-48-3 Alpha spectrometry D3084-05 (ASTM) EMSL-33 (EPA) D3084-05 (ASTM) EMSL-33 (EPA) D3084-05 (ASTM) EMSL-33 (EPA) D3084-05 (ASTM) EMSL

(EPA

Polonium-210 13981-52-7 Alpha spectrometry Po-02-RC (HASL-300) Po-02-RC (HASL-300) Po-02-RC (HASL-300) Po-02-RC (HASL-300) Po-02-RC (HASL-300) Po-02-RC (HASL-300) Method 111 (EPA) Method 

(EPA

Radium-226 13982-63-3 Alpha counting / spectrometry 903.0 (EPA) 903.1 (EPA) 7500-Ra B (SM) 7500-Ra C (SM) D3084-05 (ASTM) EMSL-19 (EPA) D3084-05 (ASTM) EMSL

(EPA

Ruthenium-103 13968-53-1 Gamma spectrometry 901.1 (EPA) 901.1 (EPA) 7120 (SM) 7120 (SM) Ga-01-R (HASL-300) Ga-01-R (HASL-300) Ga-01-R (HASL-300) Ga-0

(HASL-

Ruthenium-106 13967-48-1 Gamma spectrometry 901.1 (EPA) 901.1 (EPA) 7120 (SM) 7120 (SM) Ga-01-R (HASL-300) Ga-01-R (HASL-300) Ga-01-R (HASL-300) Ga-0

(HASL-

Selenium-75 14265-71-5 Gamma spectrometry 901.1 (EPA) 901.1 (EPA) 7120 (SM) 7120 (SM) Ga-01-R (HASL-300) Ga-01-R (HASL-300) Ga-01-R (HASL-300) Ga-0

(HASL-

Strontium-90 10098-97-2 Beta counting 7500-Sr B (SM) 7500-Sr B (SM) 7500-Sr B (SM) 7500-Sr B (SM) Sr-03-RC (HASL-300) Sr-03-RC (HASL-300) Sr-03-RC (HASL-300) Sr-03-

(HASL-

Technetium-99 14133-76-7 Liquid scintillation Tc-02-RC (HASL-300) Tc-02-RC (HASL-300) Tc-02-RC (HASL-300) Tc-02-RC (HASL-300) AP5 (ORISE) AP5 (ORISE) AP5 (ORISE) AP

(ORIS

Tritium 

(Hydrogen-3) 10028-17-8 Liquid scintillation 906.0 (EPA) 906.0 (EPA) 906.0 (EPA) 906.0 (EPA) AP2 (ORISE) AP2 (ORISE) AP2 (ORISE) AP

(ORIS

Uranium-2343 13966-29-5 Alpha counting / spectrometry 908.07 (EPA) D3972-02 (ASTM) 7500-U B7 (SM) 7500-U C (SM) D3084-05 (ASTM) EMSL-33 (EPA) D3084-05 (ASTM) EMSL

(EPA

Uranium-2353 15117-96-1 Alpha counting / spectrometry 908.07 (EPA) D3972-02 (ASTM) 7500-U B7 (SM) 7500-U C (SM) D3084-05 (ASTM) EMSL-33 (EPA) D3084-05 (ASTM) EMSL

(EPA

SAM Revision 5.0, Appendix B B - 2 
Analyte(s) CAS RN Determinative Technique 

Drinking Water Samples Aqueous and Liquid Phase Samples Soil and Sediment Samples Surface Wipes 

Qualitative 

Determination2 Confirmatory Qualitative Determination2 Confirmatory Qualitative Determination2 Confirmatory Qualitative Determination2 Confirm

Uranium-2383 7440-61-1 Alpha counting / spectrometry 908.07 (EPA) D3972-02 (ASTM) 7500-U B7 (SM) 7500-U C (SM) D3084-05 (ASTM) EMSL-33 (EPA) D3084-05 (ASTM) EMSL

(EPA

Footnotes
 

1

 Please note that this category does not cover all fission products. 

2 In those cases where the same method is listed for qualitative determination and confirmatory analysis, qualitative determination can be performed by application of the meth

used for confirmatory analysis. 

3 If it is suspected that the sample exists in refractory form (i.e., non-digestible or dissolvable material after normal digestion methods) or if there is a matrix interference problem

qualitative determination or confirmatory analysis of alpha radioactivity.
 

4

 In cases where only small sample volumes (≤100 g) are available, use HASL-300 Method Pu-12-RC.
 

5

 This procedure should be used only for filters specifically designed for iodine.
 

6

 Because tritium is not sampled using traditional air filters, this matrix is not applicable.
 

7

 This method was developed for measurement of total uranium and does not distinguish between uranium isotopes.
 

SAM Revision 5.0, Appendix B B - 3 
SAM Revision 5.0, Appendix B B - 4 
Appendix C – Selected Pathogen Methods 

Appendix C: Selected Pathogen Methods
 

SAM Revision 5.0 September 29, 2009 
Appendix C – Selected Pathogen Methods 

SAM Revision 5.0 September 29, 2009 
Appendix C: Selected Pathogen Methods 

Not all methods have been evaluated for each pathogen/sample type/environmental matrix combination in Appendix C. Each laboratory usinoperate a formal quality assurance program and, at a minimum, analyze appropriate quality control samples (Section 7.1.2). Also, if requiredreplacement of a sample preparation method may be warranted for a specific pathogen/sample type/environmental matrix or a combination Pathogen primary and alternate points of contact should be consulted for additional guidance (Section 4.0, Points of Contact). 

Note: If viability determinations are needed (e.g., evaluation of the efficacy of disinfection), a viability-based procedure (such as culture) shotechniques (such as PCR, some immunoassays) are preferred for estimating the extent of contamination and should be used in conjunction of identification. In cases where a method is listed as "requires modification of analytical method," the analytical method should be used as as necessary for analysis of a particular sample type. 

Pathogen(s) 

[Disease] 

Analytical 

Technique 

Analytical 

Method 

Solid1 

(soil, powder) 

Particulate2 

(swabs, wipes, dust 

socks) 

Liquid/Water 

(filter, grab) 

Drin

(fil

Bacteria Bacillus anthracis [Anthrax] 

Culture Public Health Reports 92(2): 176-186 Public Health Reports 92(2): 176-186 Requires modification of analytical method Public Health Reports 92(2): 176-186 Pub

Rep

1

Real-time PCR/ 

Immunoassay LRN comparable assays 

Brucella spp. (B. abortus, B. melitensis, B. suis ) [Brucellosis] 

Culture Sentinel Laboratory Guidelines for Suspected Agents of Bioterrorism: Bruc

ASM 

Real-time PCR/ 

Immunoassay LRN comparable assays 

Burkholderia mallei [Glanders] 

Culture Sentinel Laboratory Guidelines for Suspected Agents of Bioterrorism: Burkholderia ma

ASM 

Real-time PCR/ 

Immunoassay LRN comparable assays 

Burkholderia pseudomallei [Melioidosis] 

Culture Sentinel Laboratory Guidelines for Suspected Agents of Bioterrorism: Burkholderia ma

ASM 

Real-time PCR/ 

Immunoassay LRN comparable assays 

Campylobacter jejuni [Campylobacteriosis] 

Culture SM 9260 G 

Requires 

modification of 

analytical method 

Requires 

modification of 

analytical method 

SM 9260 G SM 

Immunoassay SM 9260 G 

Requires 

modification of 

analytical method 

Requires 

modification of 

analytical method 

SM 9260 G SM 

Real-time PCR Molecular and Cellular Probes 20(5): 269-279 Requires modification of analytical method Requires modification of analytical method Requires modification of analytical method R

mod

analy

SAM Revision 5.0, Appendix C C - 1 
Pathogen(s) 

[Disease] 

Analytical 

Technique 

Analytical 

Method 

Solid1 

(soil, powder) 

Particulate2 

(swabs, wipes, dust 

socks) 

Liquid/Water 

(filter, grab) 

Drin

(fil

Chlamydophila psittaci ( formerly known as Chlamydia psittaci ) 

[Psittacosis] 

Tissue culture 

Journal of Clinical 

Microbiology 

38(3): 1085-1093 

Requires 

modification of 

analytical method 

Requires 

modification of 

analytical method 

Requires 

modification of 

analytical method 

R

mod

analy

PCR 

Journal of Clinical 

Microbiology 

38(3): 1085-1093 

Requires 

modification of 

analytical method 

Requires 

modification of 

analytical method 

Requires 

modification of 

analytical method 

R

mod

analy

Coxiella burnetii 

Culture If analysis of this pathogen is required, contact the LRN (404-639-2790) for information on the LRN 

processing the sample. In some cases, methods that apply to environmental samples may no

[Q-fever] Real-time PCR/ 

Immunoassay LRN comparable assays 

Culture SM 9260 F 

Requires 

modification of 

analytical method 

Requires 

modification of 

analytical method 

SM 9260 F SM 

Escherichia coli O157:H7 Immunoassay SM 9260 F 

Requires 

modification of 

analytical method 

Requires 

modification of 

analytical method 

SM 9260 F SM 

Real-time PCR 

Applied & 

Environmental 

Microbiology 69(10): 

6327-6333 

Requires 

modification of 

analytical method 

Requires 

modification of 

analytical method 

Requires 

modification of 

analytical method 

R

mod

analy

Francisella tularensis 

Culture Basic Protocols for Level A Laboratories for the Presumptive Identification of Fra

CDC/ASM/APHL 

[Tularemia] Real-time PCR/ 

Immunoassay LRN comparable assays 

Leptospira (L. interrogans Serovars 

Icteroheamorrhagiae, Autralis, Balum, Bataviae, Bejro, Pomona) 

Culture SM 9260 I SM 9260 I 

Requires 

modification of 

analytical method 

SM 9260 I S

Immunoassay SM 9260 I SM 9260 I 

Requires 

modification of 

analytical method 

SM 9260 I S

[Leptospirosis] 

Real-time PCR 

Diagnostic Microbiology 

and Infectious Disease 

64(3): 247-255 

Requires 

modification of 

analytical method 

Requires 

modification of 

analytical method 

Requires 

modification of 

analytical method 

R

mod

analy

SAM Revision 5.0, Appendix C C - 2 
Pathogen(s) 

[Disease] 

Analytical 

Technique 

Analytical 

Method 

Solid1 

(soil, powder) 

Particulate2 

(swabs, wipes, dust 

socks) 

Liquid/Water 

(filter, grab) 

Drin

(fil

FDA/Bacteriological FDA/Bacteriological Requires Requires R

Culture Analytical Manual Analytical Manual modification of modification of mod

Chapter 10, 2003 Chapter 10, 2003 analytical method analytical method analy

Listeria monocytogenes [Listeriosis] Immunoassay 

FDA/Bacteriological 

Analytical Manual 

Chapter 10, 2003 

FDA/Bacteriological 

Analytical Manual 

Chapter 10, 2003 

Requires 

modification of 

analytical method 

Requires 

modification of 

analytical method 

R

mod

analy

USDA Laboratory Requires Requires Requires R

Real-time PCR Guidebook modification of modification of modification of mod

MLG 8A.03 analytical method analytical method analytical method analy

Requires Requires R

Culture EPA Method 1682 EPA Method 1682 modification of modification of mod

analytical method analytical method analy

Non-typhoidal Salmonella (Not applicable to S. Typhi) 

[Salmonellosis] 

Immunoassay EPA Method 1682 EPA Method 1682 

Requires 

modification of 

analytical method 

Requires 

modification of 

analytical method 

R

mod

analy

Journal of Applied Requires Requires Requires R

Real-time PCR Microbiology modification of modification of modification of mod

102(2): 516–530 analytical method analytical method analytical method analy

Requires Requires 

Culture SM 9260 B modification of modification of SM 9260 B SM 

analytical method analytical method

Salmonella Typhi [Typhoid fever] Immunoassay SM 9260 B 

Requires 

modification of 

analytical method 

Requires 

modification of 

analytical method

 SM 9260 B SM 

Real-time PCR CDC Laboratory Assay: S. Typhi Requires modification of analytical method Requires modification of analytical method Requires modification of analytical method R

mod

analy

Requires Requires 

Culture SM 9260 E modification of modification of SM 9260 E SM 

analytical method analytical method 

Shigella spp. [Shigellosis] Immunoassay SM 9260 E 

Requires 

modification of 

analytical method 

Requires 

modification of 

analytical method 

SM 9260 E SM 

Real-time PCR CDC Laboratory Assay: Shigella Requires modification of analytical method Requires modification of analytical method Requires modification of analytical method R

mod

analy

Requires Requires 

Staphylococcus aureus Culture SM 9213 B modification of modification of SM 9213 B SM 

analytical method analytical method 

SAM Revision 5.0, Appendix C C - 3 
Pathogen(s) 

[Disease] 

Analytical 

Technique 

Analytical 

Method 

Solid1 

(soil, powder) 

Particulate2 

(swabs, wipes, dust 

socks) 

Liquid/Water 

(filter, grab) 

Drin

(fil

Vibrio cholerae O1 and O139 [Cholera] 

Culture SM 9260 H 

Requires 

modification of 

analytical method 

Requires 

modification of 

analytical method 

SM 9260 H SM 

Immunoassay SM 9260 H 

Requires 

modification of 

analytical method 

Requires 

modification of 

analytical method 

SM 9260 H SM 

Real-time PCR CDC Laboratory Assay: V. cholerae Requires modification of analytical method Requires modification of analytical method Requires modification of analytical method R

mod

analy

Yersinia pestis [Plague] 

Culture Sentinel Laboratory Guidelines for Suspected Agents of Bioterrorism: Yer

ASM 

Real-time PCR/ 

Immunoassay LRN comparable assays 

Viruses Adenoviruses: Enteric and non-enteric (A-F) 

Tissue culture3 

Applied & 

Environmental 

Microbiology 71(6): 

3131-3136 

Requires 

modification of 

analytical method4 

Requires 

modification of 

analytical method4 

Requires 

modification of 

analytical method4 

R

mod

analyt

Real-time PCR 

Applied & 

Environmental 

Microbiology 71(6): 

3131-3136 

Requires 

modification of 

analytical method4 

Requires 

modification of 

analytical method4 

Requires 

modification of 

analytical method4 

R

mod

analyt

Astroviruses 

Integrated Cell 

Culture/Reverse 

transcription-PCR 

Canadian Journal of 

Microbiology 50(4): 

269-278 

Requires 

modification of 

analytical method4 

Requires 

modification of 

analytical method4 

Requires 

modification of 

analytical method4 

R

mod

analyt

Real-time reverse 

transcription-PCR 

Canadian Journal of 

Microbiology 50(4): 

269-278 

Requires 

modification of 

analytical method4 

Requires 

modification of 

analytical method4 

Requires 

modification of 

analytical method4 

R

mod

analyt

Caliciviruses: Noroviruses Real-time reverse transcription-PCR Journal of Clinical Microbiology 42(10): 4679-4685 Journal of Clinical Microbiology Vol. 42(10): 4679-46854 Requires modification of analytical method4 Requires modification of analytical method4 R

mod

analyt

Caliciviruses: Sapovirus Real-time reverse transcription-PCR Journal of Medical Virology 78(10): 1347-1353 Requires modification of analytical method4 Requires modification of analytical method4 Requires modification of analytical method4 R

mod

analyt

Coronaviruses: SARS-associated human coronavirus 

Reverse 

transcription-PCR 

Journal of Virological 

Methods 122(1): 29-36 

Requires 

modification of 

analytical method4 

Requires 

modification of 

analytical method4 

Requires 

modification of 

analytical method4 

R

mod

analyt

Hepatitis E virus (HEV) Real-time reverse transcription-PCR Journal of Virological Methods 131(1): 65-71 Requires modification of analytical method4 Requires modification of analytical method4 Requires modification of analytical method4 R

mod

analyt

SAM Revision 5.0, Appendix C C - 4 
Pathogen(s) 

[Disease] 

Analytical 

Technique 

Analytical 

Method 

Solid1 

(soil, powder) 

Particulate2 

(swabs, wipes, dust 

socks) 

Liquid/Water 

(filter, grab) 

Drin

(fil

Influenza H5N1 virus Real-time reverse transcription-PCR Emerging Infectious Diseases 11(8): 1303-1305 Requires modification of analytical method4 Requires modification of analytical method4 Requires modification of analytical method4 R

mod

analyt

Picornaviruses: Enteroviruses 

Tissue culture 

USEPA Manual of 

Methods for Virology 

EPA/600/4-84/013, 

April 2001 

Requires 

modification of 

analytical method4 

Requires 

modification of 

analytical method4 

USEPA Manual of 

Methods for Virology 

EPA/600/4-84/013, 

April 2001 

USEP

Method

EPA/6

A

Reverse 

transcription-PCR 

Applied & 

Environmental 

Microbiology 69(6): 

3158-3164 

Requires 

modification of 

analytical method4 

Requires 

modification of 

analytical method4 

Applied & 

Environmental 

Microbiology 69(6): 

3158-31644 

A

Env

Microb

31

Picornaviruses: Hepatitis A virus (HAV) 

Reverse transcriptionPCR 

Applied & 

Environmental 

Microbiology 69(6): 

3158-3164 

Requires 

modification of 

analytical method4 

Requires 

modification of 

analytical method4 

Applied & 

Environmental 

Microbiology 69(6): 

3158-31644 

A

Env

Microb

31

Reoviruses: Rotavirus (Group A) 

Tissue culture 

Applied & 

Environmental 

Microbiology 69(6): 

3158-3164 

Requires 

modification of 

analytical method4 

Requires 

modification of 

analytical method4 

Applied & 

Environmental 

Microbiology 69(6): 

3158-31644 

A

Env

Microb

31

Real-time reverse 

transcription-PCR 

Journal of Virological 

Methods 155(2): 

126-131 

Requires 

modification of 

analytical method4 

Requires 

modification of 

analytical method4 

Applied & 

Environmental 

Microbiology 69(6): 

3158-31644 

Journa

M

155(

Protozoa Cryptosporidium spp. [Cryptosporidiosis] 

Tissue culture 

Applied & 

Environmental 

Microbiology 65(9): 

3936-3941 

Requires 

modification of 

analytical method 

Requires 

modification of 

analytical method 

Requires 

modification of 

analytical method 

R

mod

analy

IMS/FA EPA Method 1622 and/or Method 1623 Requires modification of analytical method Requires modification of analytical method EPA Method 1622 and/or Method 1623 EPA M

and/or 

Real-time PCR 

Applied & 

Environmental 

Microbiology 73(13): 

4218-4225 

Requires 

modification of 

analytical method 

Requires 

modification of 

analytical method 

Requires 

modification of 

analytical method 

A

Env

Microb

42

SAM Revision 5.0, Appendix C C - 5 
Pathogen(s) 

[Disease] 

Analytical 

Technique 

Analytical 

Method 

Solid1 

(soil, powder) 

Particulate2 

(swabs, wipes, dust 

socks) 

Liquid/Water 

(filter, grab) 

Drin

(fil

Entamoeba histolytica 

Culture Journal of Parasitology 58(2): 306-310 Requires modification of analytical method Requires modification of analytical method Requires modification of analytical method R

mod

analy

Journal of Clinical Requires Requires Requires R

Real-time PCR Microbiology modification of modification of modification of mod

43(11): 5491-5497 analytical method analytical method analytical method analy

Giardia spp. [Giardiasis] 

Culture 

Trans. R. Soc. Trop. 

Med. Hyg. 77(4): 

487-488 

Requires 

modification of 

analytical method 

Requires 

modification of 

analytical method 

Requires 

modification of 

analytical method 

R

mod

analy

IMS/FA EPA Method 1623 

Requires 

modification of 

analytical method 

Requires 

modification of 

analytical method 

EPA Method 1623 EPA M

Emerging Infectious Requires Requires Emerging Infectious Emerg

Animal infectivity Diseases 12(2): modification of modification of Diseases 12(2): Dise

Toxoplasma gondii [Toxoplasmosis] 

326-329 analytical method analytical method 326-329 3

Real-time PCR 

Applied & 

Environmental 

Microbiology 70(7): 

4035-4039 

Requires 

modification of 

analytical method 

Requires 

modification of 

analytical method 

Applied & 

Environmental 

Microbiology 70(7): 

4035-4039 

A

Env

Microb

40

Helminths Baylisascaris procyonis [Raccoon roundworm infection] 

Embryonation of eggs 

and microscopy EPA/625/R-92/013 EPA/625/R-92/013 

Requires 

modification of 

analytical method 

EPA/625/R-92/013 EPA/6

General Remediation Efficacy Biological indicator (spore) strips Culture Manufacturers' Instructions 

1 Solid samples (except those containing viruses) should be (1) prepared for culture according to EPA Method 1680, Fecal Coliforms in Sewage Sludge (Biosolids) by M

Lauryl Tryptose Broth (LTB) and EC Medium, and (2) prepared for PCR according to "Quantification of Bias Related to the Extraction of DNA Directly from Soils," FrostClerc, S., Bernillon, D., Le Gall, F., Jeannin, P., Nesme, X., and Simonet, P. 1999. Applied and Environmental Microbiology. 65(12): 5409-5420. 

2 Particulate samples (except those containing viruses) should be prepared for culture according to "Swab Materials and Bacillus anthracis Spore Recovery from Nonp

B., Peterson, A., Banerjee, S.N., and Arduino, M.J. 2004. Emerging Infectious Diseases. 10(6): 1023-1029 and "Evaluation of a Macrofoam Swab Protocol for the Reco

from a Steel Surface," Hodges, L.R., Rose, L.J., Peterson, A., Nobel-Wang, J., and Arduino, M.J. 2006. Applied and Environmental Microbiology. 72(6): 4429-4430. 

3 Given that adenovirus 40 and 41 can be difficult to grow in culture, additional cell lines such as G293 (Journal of Medical Virology. 1983. 11(3): 215-231) or Caco-2 (J

44(3): 310-315) may be considered when these viruses are suspected to be present. 

4 Samples should be prepared according to procedures found in USEPA Manual of Methods for Virology EPA/600/4-84/013, April 2001. 

SAM Revision 5.0, Appendix C C - 6 
Appendix D – Selected Biotoxin Methods 

Appendix D: Selected Biotoxin Methods
 

SAM Revision 5.0 September 29, 2009 
Appendix D – Selected Biotoxin Methods 

SAM Revision 5.0 September 29, 2009 
Appendix D: Selected Biotoxin Methods 

Note: The presence of disinfectants (e.g., chlorine) and/or preservatives added during water sample collection to slow degradation (e.g., pH adjustorcould possibly affect analytical results. When present, the impact of these agents on method performance should be evaluated if not previously det

Analyte(s) CAS RN / Description Analysis Type Analytical Technique Aerosol (filter/cassette, liquid impinger) Solid (soil, powder) Particulate (swabs, wipes, dust socks) Liqu

Protein Abrin 

1393-62-0 (abrin) / 

Glycoprotein consisting 

of a deadenylase (25–32 

kDa A chain) and lectin 

(35 kDa B chain); an 

agglutinin (A2B2) may 

be present in crude 

preparations 

Presumptive Immunoassay1 

Adapted from Journal 

of Food Protection 

71(9): 1868-1874 

Adapted from Journal 

of Food Protection 

71(9): 1868-1874 

Adapted from Journal 

of Food Protection 

71(9): 1868-1874 

Adapted 

of Food 

71(9): 

Confirmatory 

Ribosome 

inactivation 

assay 

Adapted from 

Pharmacology & 

Toxicology 

88(5): 255-260 

Adapted from 

Pharmacology & 

Toxicology 

88(5): 255-260 

Adapted from 

Pharmacology & 

Toxicology

 88(5): 255-260 

Adap

Pharm

Tox

88(5)

Biological 

Activity Enzyme activity2 

Adapted from 

Analytical 

Biochemistry 

378(1): 87-89 

Adapted from 

Analytical 

Biochemistry 

378(1): 87-89 

Adapted from 

Analytical 

Biochemistry 

378(1): 87-89 

Adap

An

Bioc

378(

Botulinum neurotoxins (Serotoypes A, B, E, F) 

Protein composed of 

~100 kDa heavy chain 

and ~50 kDa light chain; 

can be complexed with 

hemagglutinin and nonhemagglutinin 

components for total 

MW of ~900 kDa 

Presumptive Immunoassay3 Adapted from lateral flow immunassay kits 

LRN 

If analysis for this agent is required in solid, particu

the LRN at (404) 639-2790 for information of the c

of receiving and processing the sample. The terms 

as used for LRN methods are describe

Confirmatory Immunoassay3 (ELISA) 

Adapted from FDA 

Bacteriological 

Analytical Manual, 

Chapter 17 

Biological 

Activity Mouse Bioassay 

Adapted from FDA 

Bacteriological 

Analytical Manual, 

Chapter 17 

Ricin 

9009-86-3 (ricin) / 

60 kDa glycoprotein 

composed of two 

subunits (~32 kDa A 

chain and ~34 kDa B 

chain); an agglutinin of 

MW 120 kDa may be 

present in crude 

preparations 

5254-40-3 (ricinine) / 

small molecule, ricin 

marker 

Presumptive Immunoassay1 Adapted from lateral flow immunassay kits 

LRN 

If analysis for this agent is required in solid, particu

the LRN at (404) 639-2790 for information of the c

of receiving and processing the sample. The terms 

as used for LRN methods are describe

Complementary 

Presumptive 

(ricinine) 

LC-MS 

Adapted from Journal 

of Analytical 

Toxicology 

29(3): 149-155 

Adapted from Journal 

of Analytical 

Toxicology 

29(3): 149-155 

Adapted from Journal 

of Analytical 

Toxicology 

29(3): 149-155 

Adapted 

of A

Tox

29(3)

Confirmatory Immunoassay 

Adapted from Journal 

of AOAC International 

91(2): 376-382 

Adapted from Journal 

of AOAC International 

91(2): 376-382 

Adapted from Journal 

of AOAC International 

91(2): 376-382 

Adapted 

of AOAC 

91(2)

Biological 

Activity Enzyme activity2 

Adapted from 

Analytical 

Biochemistry 

378(1): 87-89 

Adapted from 

Analytical 

Biochemistry 

378(1): 87-89 

Adapted from 

Analytical 

Biochemistry 

378(1): 87-89 

Adap

An

Bioc

378(

SAM Revision 5.0, Appendix D D - 1 
Analyte(s) CAS RN / Description Analysis Type Analytical Technique Aerosol (filter/cassette, liquid impinger) Solid (soil, powder) Particulate (swabs, wipes, dust socks) Liqu

Shiga and Shiga-like Toxins (Stx, Stx-1, Stx-2) 

75757-64-1 (Stx) / 

Protein composed of 

one ~32 kDa A chain 

and five 7.7 kDa B 

chains 

Presumptive Optical immunoassay 

Adapted from Journal 

of Clinical 

Microbiology 

45(10): 3377–3380 

Adapted from Journal 

of Clinical 

Microbiology 

45(10): 3377–3380 

Adapted from Journal 

of Clinical 

Microbiology 

45(10): 3377–3380 

Adapted 

of C

Micr

45(10): 

Confirmatory Immunoassay (ELISA) 

Adapted from FDA 

Bacteriological 

Analytical Manual, 

Appendix 1 

Adapted from FDA 

Bacteriological 

Analytical Manual, 

Appendix 1 

Adapted from FDA 

Bacteriological 

Analytical Manual, 

Appendix 1 

Adapted 

Bacte

Analytic

App

Biological 

Activity 

Ribosome 

inactivation 

assay2 

Adapted from 

Pharmacology & 

Toxicology 

88(5): 255-260 

Adapted from 

Pharmacology & 

Toxicology 

88(5): 255-260 

Adapted from 

Pharmacology & 

Toxicology 

88(5): 255-260 

Adap

Pharm

Tox

88(5)

LRN 

Staphylococcal enterotoxins (SEB) 

39424-53-8 (SEB) / 

Monomeric protein of

 ~ 28 kDa 

Presumptive Immunoassay Adapted from 993.06 (AOAC) If analysis for this agent is required in solid, particu

the LRN at (404) 639-2790 for information of the c

of receiving and processing the sample. The terms 

as used for LRN methods are describe

Confirmatory TBD TBD TBD TBD T

Biological 

Activity TBD TBD TBD TBD T

Staphylococcal enterotoxins (SEA, SEC)

37337-57-8 (SEA) 

39424-54-9 (SEC) / 

Monomeric proteins of 

~ 27–27.5 kDa 

Presumptive Immunoassay Adapted from 993.06 (AOAC) Adapted from 993.06 (AOAC) Adapted from 993.06 (AOAC) Adapted 

(A

Confirmatory TBD TBD TBD TBD T

Biological 

Activity TBD TBD TBD TBD T

Small Molecule 

Presumptive Immunoassay Adapted from 991.31 (AOAC) Adapted from 991.31 (AOAC) Adapted from 991.31 (AOAC) Adapted 

(A

Aflatoxin (Type B1) 27261-02-5 

Confirmatory HPLC-FL Adapted from 991.31 (AOAC) Adapted from 991.31 (AOAC) Adapted from 991.31 (AOAC) Adapted 

(A

Adapted from Journal Adapted from Journal Adapted from Journal Adapted 

Presumptive Immunoassay of Food Protection of Food Protection of Food Protection of Food 

α-Amanitin 23109-05-9 68(6): 1294-1301 68(6): 1294-1301 68(6): 1294-1301 68(6): 

HPLC Adapted from Journal Adapted from Journal Adapted from Journal Adapted 

Confirmatory amperometric of Chromatography of Chromatography of Chromatography of Chro

detection 563(2): 299-311 563(2): 299-311 563(2): 299-311 563(2)

SAM Revision 5.0, Appendix D D - 2 
Analyte(s) CAS RN / Description Analysis Type Analytical Technique Aerosol (filter/cassette, liquid impinger) Solid (soil, powder) Particulate (swabs, wipes, dust socks) Liqu

Presumptive TBD TBD TBD TBD T

Anatoxin-a 64285-06-9

Confirmatory 

HPLC-FL 

(precolumn 

derivatization) 

Adapted from 

Biomedical 

Chromatography B 

10(1): 46-47 

Adapted from 

Biomedical 

Chromatography B 

10(1): 46-47 

Adapted from 

Biomedical 

Chromatography B 

10(1): 46-47 

Adap

Biom

Chroma

10(1

Brevetoxins (B form) 79580-28-2 

Presumptive Immunoassay 

Adapted from 

Environmental Health 

Perspectives 

110(2): 179-185 

Adapted from 

Environmental Health 

Perspectives 

110(2): 179-185 

Adapted from 

Environmental Health 

Perspectives 

110(2): 179-185 

Adap

Environm

Pers

110(2)

Confirmatory HPLC-MS-MS Adapted from Toxicon 43(4): 455-465 Adapted from Toxicon 43(4): 455-465 Adapted from Toxicon 43(4): 455-465 Adapted 

43(4)

Presumptive Immunoassay 

Adapted from 

Biochemical Journal 

328(1): 245-250 

Adapted from 

Biochemical Journal 

328(1): 245-250 

Adapted from 

Biochemical Journal 

328(1): 245-250 

Adap

Biochem

328(1)

α-Conotoxin 156467-85-5 

Confirmatory HPLC-MS 

Adapted from Journal 

of Medicinal Chemistry 

47(5): 1234-1241 

Adapted from Journal 

of Medicinal Chemistry 

47(5): 1234-1241 

Adapted from Journal 

of Medicinal Chemistry 

47(5): 1234-1241 

Adapted 

of Medicin

47(5): 

Cylindrospermopsin 143545-90-8 

Presumptive Immunoassay 

Adapted from 

ELISA kits for 

Cylindrospermopsin 

Adapted from 

ELISA kits for 

Cylindrospermopsin 

Adapted from 

ELISA kits for 

Cylindrospermopsin 

Adap

ELIS

Cylindro

Confirmatory HPLC-PDA 

Adapted from FEMS 

Microbiology Letters 

216(2): 159-164 

Adapted from FEMS 

Microbiology Letters 

216(2): 159-164 

Adapted from FEMS 

Microbiology Letters 

216(2): 159-164 

Adapted 

Microbio

216(2)

Diacetoxyscirpenol (DAS) 2270-40-8 

Presumptive Immunoassay 

Adapted from 

International Journal of 

Food Microbiology 

6(1): 9-17 

Adapted from 

International Journal of 

Food Microbiology 

6(1): 9-17 

Adapted from 

International Journal of 

Food Microbiology 

6(1): 9-17 

Adap

Internatio

Food M

6(1

Confirmatory LC/APCI-MS 

Adapted from Rapid 

Communications in 

Mass Spectrometry 

20(9): 1422-1428 

Adapted from Rapid 

Communications in 

Mass Spectrometry 

20(9): 1422-1428 

Adapted from Rapid 

Communications in 

Mass Spectrometry 

20(9): 1422-1428 

Adapted 

Commu

Mass S

20(9): 

Microcystins Principal isoforms: LA, LR, LW, 

96180-79-9 (LA) 

101043-37-2 (LR) 

157622-02-1 (LW) 

Presumptive 

Immunoassay/ 

Phosphatase 

assay 

Adapted from Journal 

of AOAC International 

84(4): 1035-1044 

Adapted from Journal 

of AOAC International 

84(4): 1035-1044 

Adapted from Journal 

of AOAC International 

84(4): 1035-1044 

Adapted 

of AOAC 

84(4): 

RR, YR 111755-37-4 (RR) 

101064-48-6 (YR) Confirmatory HPLC-PDA Adapted from Analyst 119(7): 1525-1530 Adapted from Analyst 119(7): 1525-1530 Adapted from Analyst 119(7): 1525-1530 Adapted 

119(7): 

Presumptive Immunoassay TBD TBD TBD T

Picrotoxin 124-87-8 

Confirmatory HPLC 

Adapted from Journal 

of Pharmaceutical and 

Biomedical Analysis 

7(3): 369-375 

Adapted from Journal 

of Pharmaceutical and 

Biomedical Analysis 

7(3): 369-375 

Adapted from Journal 

of Pharmaceutical and 

Biomedical Analysis 

7(3): 369-375 

Adapted 

of Pharm

Biomedi

7(3): 

SAM Revision 5.0, Appendix D D - 3 
Analyte(s) CAS RN / Description Analysis Type Analytical Technique Aerosol (filter/cassette, liquid impinger) Solid (soil, powder) Particulate (swabs, wipes, dust socks) Liqu

Saxitoxins Principal isoforms: Saxitoxin (STX) Neosaxitoxin (NEOSTX) Gonyautoxin (GTX) Decarbamoylgonyautoxin (dcGTX) 

Decarbamoylsaxitoxin (dcSTX) 

35523-89-8 (STX) 

64296-20-4 (NEOSTX) 

77462-64-7 (GTX) 

None given (dcGTX) 

58911-04-9 (dcSTX) 

Presumptive Immunoassay Adapted from ELISA kits for Saxitoxins Adapted from ELISA kits for Saxitoxins Adapted from ELISA kits for Saxitoxins Adapted 

kits for 

Confirmatory 

HPLC-FL 

(post column 

derivatization) 

Adapted from Journal 

of AOAC International 

78(2): 528-532 

Adapted from Journal 

of AOAC International 

78(2): 528-532 

Adapted from Journal 

of AOAC International 

78(2): 528-532 

Adapted 

of AOAC 

78(2)

Adapted from Journal Adapted from Journal Adapted from Journal Adapted 

Presumptive Immunoassay of Food Protection of Food Protection of Food Protection of Food 

68(6): 1294-1301 68(6): 1294-1301 68(6): 1294-1301 68(6): 

T-2 Mycotoxin 21259-20-1 

Confirmatory LC/APCI-MS 

Adapted from Rapid 

Communications in 

Mass Spectrometry 

20(9): 1422-1428 

Adapted from Rapid 

Communications in 

Mass Spectrometry 

20(9): 1422-1428 

Adapted from Rapid 

Communications in 

Mass Spectrometry 

20(9): 1422-1428 

Adapted 

Commu

Mass S

20(9): 

Tetrodotoxin 9014-39-5 

Presumptive Immunoassay 

Adapted from Journal 

of Clinical Laboratory 

Analysis 6(2): 65-72 

Adapted from Journal 

of Clinical Laboratory 

Analysis 6(2): 65-72 

Adapted from Journal 

of Clinical Laboratory 

Analysis 6(2): 65-72 

Adapted 

of Clinica

Analysis 

Confirmatory LC/ESI-MS 

Adapted from 

Analytical 

Biochemistry 

290(1): 10-17 

Adapted from 

Analytical 

Biochemistry 

290(1): 10-17 

Adapted from 

Analytical 

Biochemistry 

290(1): 10-17 

Adap

An

Bioc

290(

1 Crude preparations of ricin and abrin may also contain agglutinins that are unique to castor beans and rosary peas, respectively, and that can cross-react in the immunoassay2 This assay does not test for cell binding; cell culture assays are being developed to test for cell binding but are not currently available. The only readily available assay to test 

enzymatic activity of the intact (whole) toxin is a mouse bioassay.
 

3

 Immunoassays may produce variable results with uncomplexed form of toxin.
 

SAM Revision 5.0, Appendix D D - 4 

SCIENCE
 PRESORTED STANDARD
 

POSTAGE & FEES PAID
 

EPA
 

PERMIT NO. G-35
 

Office of Research and Development 

National Homeland Security Research Center 

Cincinnati, OH 45268