Overview of the Contaminant Candidate 

List and the Unregulated Contaminant 

Monitoring Rule 

1 

Steven C. Wendelken, Ph.D.

Office of Groundwater and 

Drinking Water

Standards and Risk 

Management Division

Technical Support Center
How Are Drinking Water Standards 

Developed?

• 1996 SDWA amendments changed the process of 

developing and reviewing NPDWS

– Contaminant Candidate List (CCL)

– Unregulated Contaminant Monitoring Rule (UCMR)

– Regulatory Determination

– Six-Year Review

2 
Contaminant Candidate List (CCL)

• SDWA requires EPA to list unregulated 

contaminants that may require a national 

drinking water regulation in the future

• Every five years CCL defines unregulated 

contaminants for which EPA needs

– Occurrence data

– Analytical methods

– Potential health effects

– Evaluation of treatment techniques

3 
UCMR Objective

• Collect occurrence data for suspected drinking 

water contaminants that do not have healthbased standards set under SDWA

• Occurrence information is used to support future 

regulatory decision-making

– Supports the Administrator’s determination of 

whether (or not) to regulate a contaminant under the 

drinking water program

4 
Three Regulatory Determination Criteria

SDWA requires EPA to consider the following criteria in 

evaluating whether to regulate a contaminant:

1) The contaminant may have an adverse effect on the 

health of persons;

2) The contaminant is known to occur or there is 

substantial likelihood that the contaminant will 

occur in public water systems with a frequency and 

at levels of public health concern; and

3) In the sole judgment of the Administrator, regulation of such 

contaminant presents a meaningful opportunity for health risk 

reduction for persons served by public water systems

*SDWA Section 1412(b)(1)

5 
Status and Next Steps for 

Regulatory Determinations 3 (RD 3)

• Published Third Contaminant Candidate List (CCL 3) in Oct 2009, which 

listed 116 contaminants:

– 12 microbes (e.g., viruses, bacteria)

– 104 chemicals (pesticides, industrial chemicals, pharmaceuticals, inorganics)

• Evaluating the health and occurrence information to identify which CCL 3 

contaminants have sufficient information to make to the preliminary 

regulatory determinations

• Expect to publish preliminary RD 3 for public comment in 2013

• After considering public comments, expect to publish final RD3 in 2014

6 
Six Year Review 3

• 1996 SDWA Amendments require EPA to review and, if 

appropriate, revise existing National Primary Drinking 

Water Regulations (NPDWRs) every six years

– In 2003, EPA completed the 1st Six Year Review of 69 NPDWRs; made 

decision to revise TCR

– In 2010, EPA completed the 2nd Six Year Review of 71 NPDWRs and made 

decisions to revise tetrachloroethylene (PCE), trichloroethylene (TCE), 

acrylamide and epichlorohydrin 

• Occurrence analysis is a key component in the Six Year 

Review process 

• Expect to complete Six Year Review 3 by 2016

7 
General Flow of SDWA Regulatory Processes

Draft CCL

Final CCL

Final Rule 

(NPDWR) 

Six Year Review of 

Existing NPDWRs

No further action if make 

decision to not to regulate (may 

develop health advisory). 

Preliminary 

Regulatory 

Determinations 

Final Regulatory 

Determinations 

Proposed Rule 

(NPDWR) 

Public review and comment

Draft UCMR

Final UCMR

UCMR Monitoring 

Results

At each stage, need increased specificity and confidence in the type 

of supporting data used (e.g. health, occurrence, treatment). 

8 
Generation of CCL 3

• EPA considered approximately 7,500 potential chemical 

and microbial contaminants

• Screening process based on a contaminant’s potential to 

occur in public water systems (PWSs) and the potential for 

public health concerns

• Further detailed evaluations, public input, and expert 

judgment and review are the final contaminant selection 

tools 

• Final CCL 3 published September 2009

– 104 chemicals or chemical groups and 12 microbiological 

contaminants 

9 
Unregulated CCL 3 Contaminants

104 Chemicals and 12 Microbes

1,1,1,2-Tetrachloroethane 

1,1-Dichloroethane 

1,2,3-Trichloropropane 

1,3-Butadiene 

1,3-Dinitrobenzene 

1,4-Dioxane 

17 alpha-Estradiol 

1-Butanol 

2-Methoxyethanol 

2-Propen-1-ol 

3-Hydroxycarbofuran (degradate) 

4,4'-Methylenedianiline 

Acephate 

Acetaldehyde 

Acetamide 

Acetochlor 

Acetochlor ethanesulfonic acid (ESA) 

Acetochlor oxanilic acid (OA) 

Acrolein 

Alachlor ethanesulfonic acid (ESA)

Alachlor oxanilic acid (OA) 

alpha-Hexachlorocyclohexane (former) 

Aniline 

Bensulide 

Benzyl chloride 

Butylated hydroxyanisole 

Captan 

Chlorate (also D-DBP) 

Chloromethane (Methyl chloride) 

Clethodim 

Cobalt 

Cumene hydroperoxide 

Cyanotoxins (3)

Dicrotophos 

Dimethipin 

Dimethoate 

Disulfoton 

Diuron 

Equilenin

Equilin 

Erythromycin 

Estradiol (17-beta) 

Estriol 

Estrone 

Ethinyl Estradiol (17-alpha) 

Ethoprop 

Ethylene glycol 

Ethylene oxide 

Ethylene thiourea 

Fenamiphos 

Formaldehyde (formerly)

Germanium 

Halon 1011 (Bromochloromethane) 

HCFC-22 

Hexane 

Hydrazine 

Mestranol 

Methamidophos 

Methyl bromide (Bromomethane)

Methyl tert-butyl ether 

Metolachlor 

Metolachlor ethanesulfonic acid (ESA)

Metolachlor oxanilic acid (OA) 

Molinate 

 

 

Molybdenum 

Nitrobenzene 

Nitroglycerin 

N-Methyl-2-pyrrolidone 

N-Nitrosodiethylamine (NDEA) 

N-nitrosodimethylamine (NDMA) 

N-Nitroso-di-n-propylamine (NDPA) 

N-Nitrosodiphenylamine 

N-nitrosopyrrolidine (NPYR) 

Norethindrone (19-Norethisterone) 

n-Propylbenzene 

o-Toluidine 

Oxirane, methylOxydemeton-methyl 

Oxyfluorfen

Perchlorate 

Perfluorooctane sulfonic acid (PFOS)

Perfluorooctanoic acid (PFOA) 

Permethrin

Profenofos 

Quinoline 

RDX 

sec-Butylbenzene 

Strontium 

Tebuconazole 

Tebufenozide 

Tellurium 

Terbufos 

Terbufos sulfone 

Thiodicarb

Thiophanate-methyl 

Toluene diisocyanate 

Tribufos 

 

Triethylamine 

Triphenyltin hydroxide (TPTH) 

Urethane 

Vanadium 

Vinclozolin 

Ziram 

Adenovirus 

Caliciviruses 

Campylobacter jejuni 

Enterovirus 

Escherichia coli (0157) 

Helicobacter pylori 

Hepatitis A virus 

Legionella pneumophila 

Mycobacterium avium 

Naegleria fowleri 

Salmonella enterica 

Shigella sonnei 

10
UCMR 3

• Final rule published May 2, 2012 

• http://water.epa.gov/lawsregs/rulesregs/sdwa/ucmr/u

cmr3/index.cfm 

• Monitoring will occur from 2013-15 

• 28 chemicals and 2 viruses

• Contaminants include hormones, perfluorinated 

compounds (e.g., PFOS/PFOA), VOCs, metals (including 

Cr-6 and total Cr), 1,4-dioxane, chlorate and pathogens

11
UCMR 3 – Monitoring Required

• Assessment monitoring (List 1)

– All systems serving >10,000 people

– 800 representative systems serving <10,000 people

• Screening Survey (List 2)

– All systems serving >100,000 people

– 320 representative systems serving 10,001 to 100,000 

people

– 480 representative systems serving <10,000 people

12
UCMR 3 – Monitoring Required (cont.)

• Pre-Screen Testing (List 3)

– Selected 800 systems serving <1000 people that do not 

disinfect. Systems with wells that are located in areas 

of karst or fractured bedrock

• EPA pays for analysis of all samples from systems 

serving <10,000 and arranges for collection of List 

3 samples

13
UCMR 3 – Contaminants:

Assessment Monitoring (“List 1”)

• Perfluorinated Chemicals (EPA Method 537) 

– perfluorooctanonic acid (PFOA)

– perfluorooctane sulfonic acid (PFOS)

– perfluoroheptanoic acid (PFHpA)

– perfluorononanoic acid (PFNA)

– perfluorobutane sulfonic acid (PFBS)

– perfluorohexane sulfonic acid (PFHxS)

• Metals (EPA Method 200.8)

– cobalt 

– molybdenum

– strontium

– vanadium

– (total) chromium

•EPA Method 218.7

-hexavalent chromium 

• Volatile Organic Compounds (EPA 

Method 524.3)

- 1,1-dichloroethane 

- 1,2,3-trichloropropane

- 1,3-butadiene 

- bromochloromethane

- chlorodifluoromethane 

- chloromethane

- methyl bromide 

• EPA Method 522

-1,4-dioxane

• EPA Method 300.1

-chlorate

14
UCMR 3 – Contaminants:

Screening Survey (“List 2”) and 

Pre-Screen Testing (“List 3”)

Hormones (EPA Method 539) – List 2

-17-α-ethynylestradiol 

-

-

-

-

-

-

17-β-estradiol

equilin 

estriol

estrone 

testosterone

 4-androstene-3,17-dion

15

Viruses – List 3

– enterovirus (qPCR & cell 

culture)

– norovirus (qPCR)

– “Indicator organisms”

• Total coliform

• E. coli

• enterococci

• coliphage

• aerobic spores
Unregulated CCL 3 Contaminants

104 Chemicals and 12 Microbes

1,1,1,2-Tetrachloroethane 

1,1-Dichloroethane 

1,2,3-Trichloropropane 

1,3-Butadiene 

1,3-Dinitrobenzene 

1,4-Dioxane 

17 alpha-Estradiol 

1-Butanol 

2-Methoxyethanol 

2-Propen-1-ol 

3-Hydroxycarbofuran (degradate) 

4,4'-Methylenedianiline 

Acephate 

Acetaldehyde 

Acetamide 

Acetochlor 

Acetochlor ethanesulfonic acid (ESA) 

Acetochlor oxanilic acid (OA) 

Acrolein 

Alachlor ethanesulfonic acid (ESA)

Alachlor oxanilic acid (OA) 

alpha-Hexachlorocyclohexane (former) 

Aniline 

Bensulide 

Benzyl chloride 

Butylated hydroxyanisole 

Captan 

Chlorate (also D-DBP) 

Chloromethane (Methyl chloride) 

Clethodim 

Cobalt 

Cumene hydroperoxide 

Cyanotoxins (3)

Dicrotophos 

Dimethipin 

Dimethoate 

Disulfoton 

Diuron 

Equilenin

Equilin 

Erythromycin 

Estradiol (17-beta) 

Estriol 

Estrone 

Ethinyl Estradiol (17-alpha) 

Ethoprop 

Ethylene glycol 

Ethylene oxide 

Ethylene thiourea 

Fenamiphos 

Formaldehyde (formerly)

Germanium 

Halon 1011 (Bromochloromethane) 

HCFC-22 

Hexane 

Hydrazine 

Mestranol 

Methamidophos 

Methyl bromide (Bromomethane)

Methyl tert-butyl ether 

Metolachlor 

Metolachlor ethanesulfonic acid (ESA)

Metolachlor oxanilic acid (OA) 

Molinate 

 

 

Molybdenum 

Nitrobenzene 

Nitroglycerin 

N-Methyl-2-pyrrolidone 

N-Nitrosodiethylamine (NDEA) 

N-nitrosodimethylamine (NDMA) 

N-Nitroso-di-n-propylamine (NDPA) 

N-Nitrosodiphenylamine 

N-nitrosopyrrolidine (NPYR) 

Norethindrone (19-Norethisterone) 

n-Propylbenzene 

o-Toluidine 

Oxirane, methyl 

Oxydemeton-methyl 

Oxyfluorfen

Perchlorate 

Perfluorooctane sulfonic acid (PFOS)

Perfluorooctanoic acid (PFOA) 

Permethrin

Profenofos 

Quinoline 

RDX 

sec-Butylbenzene 

Strontium 

Tebuconazole 

Tebufenozide 

Tellurium 

Terbufos 

Terbufos sulfone 

Thiodicarb

Thiophanate-methyl 

Toluene diisocyanate 

Tribufos 

 

Triethylamine 

Triphenyltin hydroxide (TPTH) 

Urethane 

Vanadium 

Vinclozolin 

Ziram 

Adenovirus 

Caliciviruses 

Campylobacter jejuni 

Enterovirus 

Escherichia coli (0157) 

Helicobacter pylori 

Hepatitis A virus 

Legionella pneumophila 

Mycobacterium avium 

Naegleria fowleri 

Salmonella enterica 

Shigella sonnei 

Red = No EPA Method

Blue = Current Method 

Development

16
Contaminant Candidate List 4 (CCL 4) 

• Spring 2012 - Published FR notice requesting nominations of 

contaminants to be considered for inclusion on CCL 4

• Summary of Nominations: 

• 59 unique contaminants were nominated by 10 organizations and 

individuals

• 5 microbes and 54 chemicals

• 8 contaminants were nominated more than once

• EPA is currently evaluating the new data provided for nominated 

chemicals and microbes, to determine the appropriateness of 

inclusion on the CCL 4 

• The nomination letters and web site submittals can be found in the CCL 4 

docket (EPA-HQ-OW-2012-0217) at www.regulations.gov

17
Current Status

18

• 2013 - Expect to publish Draft CCL 4 

for public review and comment

• 2014 - Expect to publish Final CCL 4
Method Development Challenges

• Lack of suitable standards, internal standards and 

surrogate compounds

• Compounds with extremely high water solubility

• Low detection limits are often required

• Timeframe imposed by continual CCL/UCMR cycle

• Lack of previous method development work

19
Office of Research and Development

National Exposure Research Laboratory

Development of LC/MS/MS Methods for the 

Analysis of Chemicals on 

U.S. EPA’s Contaminant Candidate List

Jody A. Shoemaker and Daniel R. Tettenhorst 

Disclaimer: Mention of trade names or commercial products does not constitute endorsement or 

recommendation for use.

CCL/UCMR Methods 

Webinar -2013 
Office of Research and Development

National Exposure Research Laboratory

NO 

Does Method YES

Meet DQOs? 

70-130% recovery 

with <30% RSD

Revise Technical Approach

Write Method

Use in Future UCMR 

Optimize Instrumentation

(chromatography, mass calibrate, 

tune, evaluate instrument stability)

Determine Preservatives

 (select antimicrobial & dechlor) 

Determine Best Calibration 

(linear/quadratic, internal standards)

Determine Interferences (Are DQOs 

met in various difficult matrices?)

Determine Aqueous & 

Extract Holding Times

Optimize SPE (select sorbent, surrogates, solvents, solvent 

volumes, sample volume, evaporation parameters)

Multi-Lab 

Verification 

Method Development Process
Office of Research and Development

National Exposure Research Laboratory

ORD/NERL LC/MS/MS Methods 

 LC/MS/MS Drinking Water Methods





Method 535 – 12 acetanilide degradates (6 CCL)-UCMR 2

Method 537 – 14 perfluorinated alkyl acids (2 CCL)-UCMR 3

 Method 538 – 11 chemicals (4 CCL)

 LC/MS/MS Methods Under Development







Method 540 – 12 pesticides (5 CCL) 

Method 543 – 8 pesticides (6 CCL) 

Method 544 – 8 cyanotoxins (1 CCL)

http://www.epa.gov/nerlcwww/ordmeth.htm
Office of Research and Development

National Exposure Research Laboratory

Method 538: DAI-LC/MS/MS

Contains 11 Analytes 

 

Method Analytes 

acephate aldicarb

dicrotophos aldicarb sulfone

methamidophos diisopropyl methylphosphonate (DIMP)

oxydemeton-methyl fenamiphos sulfone

quinoline fenamiphos sulfoxide

thiofanox

Analytes in red are on CCL 3

 Most method analytes are pesticides (except for quinoline and DIMP) 

with the potential to contaminate drinking water sources 

 Quinoline is an industrial starting material, a pharmaceutical (antimalarial) and a flavoring agent 

 DIMP is a chemical by-product in the production of sarin gas
Office of Research and Development

National Exposure Research Laboratory

M538 

Direct Aqueous Injection Approach

Selected Reaction Monitoring

LC Source Detector

Pos ESI 50 μL 

injection

40 mL sample 

10 μL IS PDS 

acephate, d6 quinoline, d7 

methamidophos, d6 DIMP, d14

oxydemeton-methyl, d6

Antimicrobial: 

64 mg/L sodium omadine

Remove free chlorine:

20 mM ammonium acetate

990 μL aliquot 
Office of Research and Development

National Exposure Research Laboratory

M538 Performance Data

Fortified at 0.99 – 43 μg/L (n=7)

0

20

40

60

80

100

120

mean % recovery

Groundwater source Surface water source

LCMRL range: 11-180 ng/L 

(quinoline = 1.5 μg/L)

HRLs: 250 ng/L – 175 μg/L 

(quinoline 10 ng/L)
Office of Research and Development

National Exposure Research Laboratory

Method 540: SPE-LC/MS/MS

Contains 12 Analytes 

Method Analytes on CCL 3 

3-hydroxycarbofuran fenamiphos sulfone

fenamiphos fenamiphos sulfoxide

bensulide methomyl

tebuconazole chlorpryifos oxon

tebufenozide phorate sulfone

disulfoton sulfoxide phorate sulfoxide

 

Analytes in red are on CCL 3

 all method analytes are pesticides or pesticide degradates with 

the potential to contaminate drinking water sources
Office of Research and Development

National Exposure Research Laboratory

Method 540 Analytical Procedure

inject

Elute with 5 mL MeOH

150 mg Water Oasis HLB

J.T. Baker Speedisk H2O-Philic DVB

 N2 blowdown

+ IS 

C Source Det

LC/MS/MS - Selected Reaction Monitoring

Argon

L ector 

+ ESI 

 Rinse cartridge 

with 5 mL reagent 

water after loading

Surrogates & 

preservatives

Surrogates

methomyl-13C2,

15N

tebuconazole-d6 

Preservatives

2-chloracetamide

ascorbic acid

Trizma

250 mL

sample 

Condition with 5 mL MeOH 

& 10 mL reagent water

Internal Standards

carbofuran-13C6 

bensulide-d14

phorate-d10
Office of Research and Development

National Exposure Research Laboratory

0

20

40

60

80

100

120

mean % recovery

Groundwater source

M540 Performance Data

Fortified at 12.8 – 32 ng/L (n=4)

Surface water source

LCMRL range: 0.64 – 2.0 ng/L HRLs: 420 ng/L – 210 μg/L 
Office of Research and Development

National Exposure Research Laboratory

Method 543: On-line SPE-LC/MS/MS

Contains 8 Analytes 

 

Method Analytes on CCL 3 

3-hydroxycarbofuran fenamiphos sulfone

fenamiphos fenamiphos sulfoxide

bensulide

quinoline

tebuconazole

tebufenozide Analytes in red are on CCL 3

 all method analytes are pesticides or pesticide degradates (except 

quinoline) with the potential to contaminate drinking water sources

 

 concentration, elution, separation all done by 

automation

 1-5 mL sample volume typical

 analysis time/sample is <20 min

 high throughput
Office of Research and Development

National Exposure Research Laboratory

SPE injection port

2.5 mL injections

UPLC injection port

1-10 μL range

SPE 5-mL sample loop

Analytical sample loop

10 μL 

SPE cartridge 1

Oasis HLB

SPE cartridge 2

Oasis HLB

LC/MS/MS - Selected Reaction Monitoring

Argon

UPLC Source Detector

Pos ESI

20 mL

sample vial

Internal Standards

quinoline-d7 

carbofuran-13C6 

tebuconazole-d6 

bensulide-d14

IS(s) & 

preservatives:

potassium citrate

ascorbic acid

Method 543 Preliminary Procedure
Office of Research and Development

National Exposure Research Laboratory

Method 543: On-line SPE 

Events

0 

Time

2 4 6 8 10 12

Start

Loading

3 min/5mL

SPE wash

5 min gradient elution Re-equilibration

SPE Cartridge #1

0 

Time

2 4 6 8 10 12

Start Re-equilibration

SPE Cartridge #2

Re-condition

2% NH4OH

2% FA

0.5% FA

ACN + 0.5% FA

ACN +

0.5% FA 2% FA

Quat Pump Quat Pump

Quat 

Pump Quat Pump

Binary Pump Binary Pump
Office of Research and Development

National Exposure Research Laboratory

On-line SPE-LC/MS/MS Preliminary Performance Data

Fortified at 4 – 10 ng/L (n=3)

0

20

40

60

80

100

120

mean % recovery

Surface water source 3 mg/L Ohio River NOM

LCMRL range: TBD HRLs: 10 ng/L – 210 μg/L 
Office of Research and Development

National Exposure Research Laboratory

Method 544: SPE-LC/MS/MS

Potentially 8 Cyanotoxins 

 Method Analytes

MC-LR MC-RR

MC-YR MC-LA

MC-LW MC -LF

MC-LY Nodularin

 widespread occurrence of blooms

potential for occurrence in finished drinking water

toxic – HRL is 21 ng/L for MC-LR 

unique problems in development of robust analytical methods – 

availability of multiple sources of high purity standards, lack of 

isotopically labeled IS and SUR, matrix interferences 







Analytes in red are on CCL 3
Office of Research and Development

National Exposure Research Laboratory

Preliminary

Method 544 Analytical Procedure

inject

Elute with 10 mL MeOH

Water Oasis HLB

others?

 N2 blowdown

+ IS 

LC/MS/MS - Selected Reaction Monitoring

Argon

LC Source Detector 

+ ESI 

 Rinse cartridge 

with 15 mL reagent 

water after loading

Surrogates & 

preservatives

Surrogates

TBD

e.g., leucine-enkephalin

cyclo-RADFV

Preservatives

2-chloracetamide

ascorbic acid

Trizma

250-500 mL

sample 

Condition with MeOH & 

reagent water

Internal Standards

TBD

Current : Me-MC-LR, CD3 

Lysing of the 

cyanobacterial 

cells in the 

sample will be 

investigated
Office of Research and Development

National Exposure Research Laboratory

Method 544 Preliminary Performance Data

Fortified at 200 – 400 ng/L

except MC-LA at 1000 ng/L (n=3)

0

0

0

0

0

20

40

6

8

10

12

mean % recovery

Groundwater source (Internal Cal) Surface water source(External Cal)

LCMRL range: TBD HRL MC-LR: 21 ng/L 
Office of Research and Development

National Exposure Research Laboratory

Summary of ORD/NERL LC/MS/MS Methods

 2 LC/MS/MS methods completed and ready for 

potential use in UCMR 4 – total of 9 CCL 3 

chemicals

• Method 538

• Method 540

 2 methods under development for potential 

use in UCMR 4 – one additional CCL 3 analyte

• Method 543 (optional method for Method 540 analytes)

• Method 544 (MC-LR) 
Office of Research and Development

National Exposure Research Laboratory

High water solubility

Aniline

N-Methyl-2-pyrrolidone

Triethylamine

Attempted but failed DQOs

4,4’-methylenedianiline 

Thiodicarb

Nitroglycerin

Clethodim

Not suitable for MS 

analysis

 Triphenyltin 

hydroxide

Ziram

ETU

ETU=ethylenethiourea

DQOs=data quality objectives

Aqueous instability

Captan

Hexane

Benzyl chloride

N-Nitrosodiphenylamine

Thiophanate-methyl

Toluene diisocyanate

Oxirane, methylEthylene oxide

Remaining CCL 3 Chemicals – Challenges

(as of May 2013)
Office of Research and Development

National Exposure Research Laboratory, 26 W. Martin Luther King Dr., Cincinnati, OH 45268

Photo image area measures 2” H x 6.93” W and can be masked by a 

collage strip of one, two or three images.

The photo image area is located 3.19” from left and 3.81” from top of page. 

Each image used in collage should be reduced or cropped to a maximum of 

2” high, stroked with a 1.5 pt white frame and positioned edge-to-edge with 

accompanying images.

Disclaimer: Although this work was reviewed by EPA and approved for publication, it may not necessarily reflect official agency policy. Mention of trade 

names or commercial products does not constitute endorsement or recommendation for use.

Drinking Water Methods Development 

by Gas Chromatography-Mass 

Spectrometry (GC-MS)

Paul E. Grimmett
Office of Research and Development

National Exposure Research Laboratory, 26 W. Martin Luther King Dr., Cincinnati, OH 45268 1 

Background 

• The Safe Drinking Water Act, as amended in 1996, requires EPA to execute a 

program that identifies, monitors and considers for regulation new and emerging 

chemicals that may pose a risk to drinking water consumers.

• EPA notifies the public of chemicals it is investigating by publishing the drinking 

water Contaminant Candidate List (CCL). 

• In order for EPA’s Office of Water to evaluate CCL chemicals for regulation, it is 

vital that nationwide occurrence data for these chemicals in drinking water are 

obtained. 

• Nationwide occurrence data are obtained through Unregulated Contaminant 

Monitoring (UCM). 

• Rugged, accurate, and sensitive methods are needed for UCM. 
Office of Research and Development

National Exposure Research Laboratory, 26 W. Martin Luther King Dr., Cincinnati, OH 45268

Method Development Goals

•

•

•

•

•

Determine appropriate surrogates and internal standards

Establish Data Quality Objectives (DQOs): normally 70-130% recovery 

with <30% RSD

Preservation scheme to allow for sample holding: antimicrobial, 

dechlorinating agent, and pH buffer

Establish sample and extract holding times – ideally ≥14 days

Establish detection limits (DLs) and lowest concentration minimum 

reporting levels (LCMRLs) below health reference levels (HRLs)

2 
Office of Research and Development

National Exposure Research Laboratory, 26 W. Martin Luther King Dr., Cincinnati, OH 45268

3 

Recently Published GC Methods by ORD-NERL

1. Method 522: 1,4-dioxane 

– SPE on coconut carbon sorbent, followed by GC/MS

– allows for selected ion monitoring (SIM)

– two sampling/extraction options (100 mL and 500 mL samples)

– method currently used for occurrence data in UCMR 3 (2013-2015)
Office of Research and Development

National Exposure Research Laboratory, 26 W. Martin Luther King Dr., Cincinnati, OH 45268

Recently Published GC Methods by ORD-NERL (cont.)

2. Method 525.3: Semi-volatiles 

– published in February 2012

– approx. 130 analytes (pesticides, herbicides, PCBs, PAHs, etc)

– includes 16 CCL 3 compounds and 17 regulated contaminants

 

– SPE on polymeric sorbent (DVB), followed by GC/MS

– improved preservation scheme, updated surrogates and internal 

standards, addition of SIM option, and new PCB screening 

procedure

4 
Office of Research and Development

National Exposure Research Laboratory, 26 W. Martin Luther King Dr., Cincinnati, OH 45268

5 

Current Work

Development of drinking water method for o-toluidine, quinoline, 

dimethipin, captan, and butylated hydroxyanisole (BHA) 

• o-Toluidine and quinoline (both industrial precursors) have basic structures, which 

led to poor sorbent recovery using Method 525.3 preservatives (acidic scheme) 

• BHA (a food and packaging preservative) suffered from randomly occurring 

extraction inefficiency and was removed from Method 525.3

• Dimethipin was included in EPA Method 525.3, but with limited sorbent options

• Captan was removed from Method 525.3 in the evaluation phase, due to 

hydrolysis (half life in water is less than 1 day)

• All 5 compounds are being evaluated for an EPA method using a neutral pH 

preservation scheme
Office of Research and Development

National Exposure Research Laboratory, 26 W. Martin Luther King Dr., Cincinnati, OH 45268

 Current SPE Extraction Technique - Cartridges

Add surrogates:

o-toluidine-d9 

quinoline-d7 

Add preservatives: 

ascorbic acid

Trizma buffer (pH 7.0)

EDTA

DZU

Rinse & condition:

methylene chloride

 methanol 

 water

Add 1 L water sample 

extract at ~10 mL/min 

Rinse with RW

Dry cartridge for 10 min

Elute with 

methylene chloride

Add IS(s): 

acenaphthene-d10 

phenanthrene-d10 

chrysene-d12

Dry and concentrate 

extract to 1 mL

GC/MS: 

Full scan option or 

SIM option

Multiple 

hydrophilicmodified 

sorbent 

options 
Office of Research and Development

National Exposure Research Laboratory, 26 W. Martin Luther King Dr., Cincinnati, OH 45268

7 

 GC/MS Full Scan Analysis Specs

Column: Restek RXI-5sil-MS and J&W DB-1701 

 30 m x 0.25 mm x 0.25 μm column 

Injector: 275 °C (splitless mode), 20 psi pulse

Inj. vol: 1 μL 

Flow: 1 mL/min, helium carrier gas 

Oven: 60 °C for 1 min, to 300 °C at 10 

°C/min, hold 2 min. Total time = 27 

min. 

MS Transfer Line: 275 °C 

MS: scan 50-350 amu, full scan
Office of Research and Development

National Exposure Research Laboratory, 26 W. Martin Luther King Dr., Cincinnati, OH 45268

8 

 GC/MS SIM Analysis Specs

 (Preliminary)

Column: Restek RXI-5sil-MS

Injector: 275 °C (splitless mode), 20 psi pulse

Inj. vol: 1 μL 

Flow: 1 mL/min, helium carrier gas 

Oven: 60 °C for 1 min, to 300 °C at 10 °C/min, 

 hold 2 min. Total time = 27 min. 

MS Transfer Line: 275 °C 

MS SIM: 6 windows (amu, dwell time)

WIN1, 5.00 min: (106.10, 25) (107.10, 25) (112.10, 25)(114.10, 25)

WIN2, 7.64 min: (102.00, 25) (108.10, 25) (129.10, 25)(136.10, 25)

WIN3, 10.62 min: (137.10, 25) (162.10, 25) (164.10, 25)(180.10, 25)

WIN4, 13.76 min: ( 54.10, 75) (118.00, 75) (188.10, 25)

WIN5, 17.16 min: ( 79.00, 75) (149.00, 75)

WIN6, 20.37 min: (236.20, 25) (240.20, 25)
Office of Research and Development

National Exposure Research Laboratory, 26 W. Martin Luther King Dr., Cincinnati, OH 45268

Sample Total Ion Chromatogram (TIC) 

with Mass Spectra, 5 μg/mL standard

9 

Internal Standards acenaphthene-d10, phenanthrene-d10, and chrysene-d12 labeled as ISTD 1, ISTD 2, and ISTD 3, 

respectively. 
Office of Research and Development

National Exposure Research Laboratory, 26 W. Martin Luther King Dr., Cincinnati, OH 45268

Quality Control Data at Various Concentrations, 

Drinking Water Matrices

• Three different water matrices were fortified with CCL 3 compounds of 

interest, then extracted and analyzed.

 

1. Drinking water – surface water source

2. Drinking water – groundwater source (hardness ~ 350 mg/L)

3. Natural Organic Material NOM water – lab water spiked with concentrated organic 

matter from the Ohio River (TOC @ 1.8-2.2 mg/L)

• Fortification performed at 5 μg/L, 1 μg/L, and 0.1 μg/L. 

• Each matrix was extracted in 4x replicates 

10
Office of Research and Development

National Exposure Research Laboratory, 26 W. Martin Luther King Dr., Cincinnati, OH 45268

Quality Control Data from Matrix Extracts at High Level 

Fortification (Full Scan GC/MS)

11

Lower and upper limit bars are set at 70% and 130%, respectively. 
Office of Research and Development

National Exposure Research Laboratory, 26 W. Martin Luther King Dr., Cincinnati, OH 45268

Quality Control Data from Matrix Extracts at Mid Level 

Fortification (Full Scan GC/MS)

12

Lower and upper limit bars are set at 70% and 130%, respectively. 
Office of Research and Development

National Exposure Research Laboratory, 26 W. Martin Luther King Dr., Cincinnati, OH 45268

13

Quality Control Data from Matrix Extracts at Low Level 

Fortification (Full Scan GC/MS)

Lower and upper limit bars are set at 50% and 150%, respectively. 
Office of Research and Development

National Exposure Research Laboratory, 26 W. Martin Luther King Dr., Cincinnati, OH 45268

Remaining Laboratory Work with Current Method

• Perform sample and extract holding time study 

 - GOALS:14-28 day holding time for samples; 14-28 days for extracts

 - Special attention to captan (hydrolysis issues) and o-toluidine (solvent stability issues) 

• LCMRL and MDL analysis and calculations

• Repeat sample matrix challenges, holding time studies, and LCMRL/MDL calculations in 

SIM (work has been initiated) 

- quinoline: HRL of 0.010 μg/L 

• Multi-laboratory verification

14
Office of Research and Development

National Exposure Research Laboratory, 26 W. Martin Luther King Dr., Cincinnati, OH 45268

15

Future Research and Potential Challenges

Review of Select Remaining CCL 3 Compounds:

CCL3 Compound Comments

Acetamide

Not suitable for generic SPE, highly water soluble (2000g/L); Too small 

molecular weight (59.1) for LC/MS or EI-GC/MS

Acrolein Volatile, water solubility > 10%. Stability issues in water. 

Cumene hydroperoxide Safety concerns due to high reactivity, low water solubility

Ethylene glycol

Too small molecular weight (62.1) for EI-GC/MS or LC/MS; miscible with 

water, not suitable for generic SPE

Ethylene oxide

Too small molecular weight (44.0) for EI-GC/MS or LC/MS/MS; gas at 

room temperature

Hydrazine Too small molecular weight (32.0) for EI-GC/MS or LC/MS; volatile

Methanol Too small molecular weight (32.0) for EI-GC/MS or LC/MS; volatile

Nitroglycerin

Not amenable to GC due to thermal instability; cannot ionize by positive 

ion ESI-LC/MS/MS

N-Methyl-2-pyrrolidone Miscible with water, not suitable for generic SPE

Oxirane, methyl- (propylene oxide) Too small molecular weight (58.1) for EI-GC/MS or LC/MS; volatile

Toluene diisocyanate Reacts immediately with water, stability issues

Urethane Not suitable for GC; Too small molecular weight (89.1) for LC/MS
 

A World of SolutionsTM

METHOD DEVELOPMENT PROCESS 

FOR DRINKING WATER ANALYSIS

CB&I

Alan Zaffiro
A World of SolutionsTM 1 

W HAT WE DO TO CREATE A METHOD

PLANNING 

AGENCY DIRECTION (e.g.,CCL3) 

ANALYTE GROUPING

LITERATURE SEARCH

PRIVATE PARTNERS

RESEARCH PLAN

(NEW OR UPDATE EXISTING METHOD)

EXECUTION 

METHOD DEVELOPMENT

VALIDATION 

REVIEW

PUBLICATION 
 

A World of SolutionsTM 2 

WHAT WE DO TO CREATE A METHOD

PLANNING 

AGENCY DIRECTION (e.g.,CCL3) 

ANALYTE GROUPING

LITERATURE SEARCH

PRIVATE PARTNERS

RESEARCH PLAN

(NEW OR UPDATE EXISTING METHOD)

EXECUTION 

METHOD DEVELOPMENT

VALIDATION 

REVIEW

PUBLICATION 
 

A World of SolutionsTM 3 

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









INITIAL ANALYTE GROUPING

CONSIDERATIONS

MAXIMIZE # OF COMPOUNDS ANALYZED BY A SINGLE 

TECHNIQUE

PHYSICAL-CHEMICAL PROPERTIES

REACTIVITY / FORM

MONITORING LIMITS

USE OR ENVIRONMENTAL IMPACT

RELEVANCE

EXTEND EXISTING DRINKING WATER METHOD
WHAT WE DO TO CREATE A 

PLANNING 

 METHOD

AGENCY DIRECTION (e.g.,CCL3) 

ANALYTE GROUPING

LITERATURE SEARCH

PRIVATE PARTNERS

RESEARCH PLAN

(NEW OR UPDATE EXISTING METHOD)

EXECUTION 

METHOD DEVELOPMENT

VALIDATION 

REVIEW

PUBLICATION 

A World of SolutionsTM 4 
A World of SolutionsTM 5 



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

L ITERATURE SEARCH AND PARTNERS

SOURCES

EPA METHODS PUBLISHED BY OTHER DIVISIONS

JOURNALS

METHODS PUBLISHED BY OTHER GOVERNMENT AGENCIES, e.g., 

USGS

APPLICATION NOTES

VENDORS

UNIVERSITIES 

PERSONAL COMMUNICATION

PAST TSC ATTEMPTS

PRELIMINARY LAB WORK

VOLUNTARY CONSENSUS STANDARD BODIES (e.g. ASTM, 

STANDARD METHODS)
WHAT WE DO TO CREATE A METHOD 

PLANNING 

AGENCY DIRECTION (e.g.,CCL3) 

ANALYTE GROUPING

LITERATURE SEARCH

PRIVATE PARTNERS

RESEARCH PLAN

(NEW OR UPDATE EXISTING METHOD)

EXECUTION 

METHOD DEVELOPMENT

VALIDATION 

REVIEW

PUBLICATION 

A World of SolutionsTM 6 
 

A World of SolutionsTM 7 











RESEARCH PLAN

ELEMENTS

LITERATURE SEARCH RESULTS

PROPOSED TARGET LIST

PROPOSED QC COMPOUNDS

– INTERNAL AND SURROGATE STANDARDS

PROPOSED TECHNIQUE

– PRESERVATION

– DIRECT INSTRUMENTAL ANALYSIS?

– SAMPLE PREPARATION (EXTRACTION, DERIVATIZATION, BUFFER)

PROPOSED CONDITIONS
WHAT WE DO TO CREATE A METHOD 

PLANNING 

AGENCY DIRECTION (e.g.,CCL3) 

ANALYTE GROUPING

LITERATURE SEARCH

PRIVATE PARTNERS

RESEARCH PLAN

(NEW OR UPDATE EXISTING METHOD)

EXECUTION 

METHOD DEVELOPMENT

VALIDATION 

REVIEW

PUBLICATION 

A World of SolutionsTM 8 
 

A World of SolutionsTM 9 









METHOD DEVELOPMENT

BENCH WORK TO OPTIMIZE CONDITIONS

WORK BACKWARDS ITERATIVELY

– INSTRUMENTAL DETERMINATION>Sx PPEP>PRESERVATION

– REAGENT WATER>FIELD SAMPLES

GOALS

– ROBUST, REPEATABLE, TRANSLATABLE METHOD

– 70 TO 130% RECOVERY; +30% PRECISION

– AT LEAST 14 DAYS STORAGE STABILITY

– DETECTION LIMITS MEET MONITORING / REGULATORY OBJECTIVES

MINIMIZE COST AND HAZARDOUS MATERIALS

UTILIZE GENERIC LABORATORY MATERIALS
WHAT WE DO TO CREATE A METHOD 

PLANNING 

AGENCY DIRECTION (e.g.,CCL3) 

ANALYTE GROUPING

LITERATURE SEARCH

PRIVATE PARTNERS

RESEARCH PLAN

(NEW OR UPDATE EXISTING METHOD)

EXECUTION 

METHOD DEVELOPMENT

VALIDATION

REVIEW

PUBLICATION 

A World of SolutionsTM 10
 

A World of SolutionsTM 11





DEMONSTRATION

VERIFY PERFORMANCE AT EPA AND MULTIPLE OUTSIDE 

LABORATORIES

SINGLE LABORATORY--MOST EXTENSIVE SCOPE

– LCMRL, P&A (CHALLENGING MATRIXES), MRL CONFIRMATION

– STORAGE STABILITY WITH MICROBIAL INOCULANT PRESENT

– DATA PUBLISHED IN SECTION 17 DRINKING WATER METHODS

MULTI-LABORATORY VALIDATION

– LCMRL, P&A (ONE MATRIX), MRL CONFIRMATION

– DATA PUBLISHED IN RESEARCH SUMMARY

– LABORATORIES RECOGNIZED IN METHOD
WHAT WE DO TO CREATE A METHOD 

PLANNING 

AGENCY DIRECTION (e.g.,CCL3) 

ANALYTE GROUPING

LITERATURE SEARCH

PRIVATE PARTNERS

RESEARCH PLAN

(NEW OR UPDATE EXISTING METHOD)

EXECUTION 

METHOD DEVELOPMENT

VALIDATION 

REVIEW

PUBLICATION 

A World of SolutionsTM 12
 

A World of SolutionsTM 13





REVIEW

METHOD DOCUMENT AND PERFORMANCE DATA

PEER REVIEW OF METHOD

– CONCURRENT WITH MULTI-LABORATORY VALIDATION

AGENCY

– INTERNAL (CB&I) 

– EPA PO

– EPA MANAGEMENT

– EPA HEADQUARTERS

– VERIFY PDF CONVERSION (SECTION 508 COMPLIANT)
WHAT WE DO TO CREATE A METHOD 

 World of SolutionsTM 14

PLANNING 

AGENCY DIRECTION (e.g.,CCL3) 

ANALYTE GROUPING

LITERATURE SEARCH

PRIVATE PARTNERS

RESEARCH PLAN

(NEW OR UPDATE EXISTING METHOD)

EXECUTION 

METHOD DEVELOPMENT

VALIDATION 

REVIEW

PUBLICATION

A
A World of SolutionsTM 15

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CCL3 LO W MW COMPOUNDS REMAINING

12 CANDIDATES

INITIAL ANALYTE GROUPING

Ethylene oxide, propylene oxide, methanol, 1-butanol, allyl 

alcohol, 2-methoxy ethanol, ethylene glycol, acrolein, 

hexane, o-toluidine, triethylamine, benzyl chloride

SMALL, POLAR MOLECULES POSSIBLY AMENABLE TO GC

MOST POSSIBLY ISOLATED FROM H2O USING CARBON- OR 

CMS-BASED EXTRACTION MEDIA
 

A World of SolutionsTM 16

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

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RESEARCH PLAN

12 CANDIDATES

LITERATURE SEARCH

PHYSICAL PROPERTIES

– HENRY’S LAW CONSTANT

– MW

– BOILING POINT

HRL 

METHODS PUBLISHED BY OTHER GOVERNMENT AGENCIES

TECHNIQUES REPORTED IN:

– APPLICATION NOTES

– JOURNALS

– PROCEEDINGS OF CONFERENCES

– PERSONAL COMMUNICATION
 

A World of SolutionsTM 17

RESEARCH PLAN

Table 1. Physical properties, published methods, and literature summary.

a. Reference: ethanol (1.9 x 102), benzene (1.8 x 10-1), 1-propanol (1.4 x 102), MtBE (1.7), 1,4-dioxane (2.1 X 102 ), TBA (8.4).

b. Azeotropic distillation

c. Purge and trap technique

Analyte(s) Henry’s Law 

Constant, M/atm a MW bp, °C

HRL, 

μg/L SW846 Literature techniques (author reference in parentheses)

Ethylene oxide

Propylene oxide

7.1

5.2

44

58

10.7

34

0.1

0.2

5031b, 8015D (direct aqueous 

injection—DAI) Air and pharmaceutical residual methods

Methanol 2.3 x 102 32 64.7 3,500 8015D DAI

P&T w/FID

Ether extraction (Woo)

Headspace in blood (numerous citations)

Derivatization to alkyl nitrite/extraction/GC/ECD (Nguyen)

SPME (Supelco application notes)

1-Butanol 1.2 x 102 74 118 700 5031, 5030Cc w/heat, Ether extraction (Woo)

Allyl alcohol 2 x 102 58 97 35 5031, 5030C w/heat, 8015D DAI

2-Methoxyethanol 12.1 76 125 21 Not found SPE-carbon molecular sieve (Supelco application notes)

Ethylene glycol 1.7 x 104 62 197 14,000 8015B DAI SPE-carbon molecular sieve (Supelco), DAI-GC-FID 

(Turner, Restek, Teske)

Acrolein 7 – 8 56 53 3.5 5030C, 8015D DAI, 8261A vacuum 

distillation EPA Method 603 (heated purge and trap)

Hexane 5.5 x 10-4 86 69 420 Not found P&T (524.3 Research Summary)

Ortho-toluidine Not available 107 200 0.19 8015D DAI SPME (Supelco), IC (Zhu)

Triethylamine Not available 101 90 2.3 8015D DAI SPME (Alltech application note)

Benzyl chloride 1.6 127 179 0.2 5030C P&T (524.2)

LITERATURE SEARCH SUMMARY TABLE
 

A World of SolutionsTM 18

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RESEARCH PLAN

12 CANDIDATES

INITIAL LAB WORK AND PROPOSED METHOD

PRIORITY CONTAMINANT: 2-methoxyethanol 

LIMITED INFORMATION OR EXPENSIVE INSTRUMENTS 

NEEDED

– methanol, hexane, acrolein, triethyl amine, o-toluidine (not 

practical)

INITIAL LAB INVESTIGATION REQUIRED

– EtO AND ptO: HEADSPACE, EXTRACTION, AND P&T FAILED

– THESE TWO ELIMINATED

REMAINING 5 COMPOUNDS AMENABLE TO EXTRACTION

– SIMILAR TECHNIQUE TO EPA METHOD 522

– ORD-NERL ORIGINALLY PROPOSED MULTIPLE TARGETS FOR THIS 

METHOD
 

A World of SolutionsTM 19

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RESEARCH PLAN

6 CANDIDATES

PROPOSED METHOD (CONT.)

PROPOSE EXTENDING M-522 beyond 1,4-dioxane 

NEW TARGETS: n-butanol, allyl alcohol, 2-

methoxyethanol, ethylene glycol, benzyl chloride

– CONTAINS PRIORITY CONTAMINANT

QC COMPOUNDS

– LABELED ISOTOPES OF 1,4-dioxane, n-butanol, allyl alcohol

PROPOSED CONDITIONS

– CARBON-EXTRACTION, GC-MS DETERMINATION
 

A World of SolutionsTM 20

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METHOD DEVELOPMENT

6 CANDIDATES

BENCH WORK TO OPTIMIZE CONDITIONS

ETHYLENE GLYCOL NOT RETAINED BY ANY EXTRACTION 

MEDIA EVALUATED

BENZYL CHLORIDE FAILED PRELIMINARY STORAGE 

STABILITY

ONLY ONE OF THE EXTRACTION MEDIA IN M-522 

SUITABLE FOR REMAINING COMPOUNDS

MODIFICATION OF M-522 EXTRACTION SOLVENT 

REQUIRED TO RECOVER ADDITIONAL TARGETS

1,4-dioxane, n-butanol, allyl alcohol, 2-methoxyethanol
 DEMONSTRATION/VALIDATION

A World of SolutionsTM 21

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

4 

 ANALYTES 

VERIFY PERFORMANCE AT EPA AND MULTIPLE 

OUTSIDE LABORATORIES

SUCCESSFULLY DEMONSTRATED AT EPA

METHOD CURRENTLY BEING WRITTEN

CURRENTLY RECRUITING OUTSIDE 

LABORATORIES
 

A World of SolutionsTM 22

CONCLUSION SLIDE 1

ABILITY TO MEET MONITORING GOALS

MRL’s and HRLs

Analytes MRL * CCL3 HRL

1,4-dioxane 0.2 μg/L 3.0 μg/L (cancer)

allyl alcohol 1.0 μg/L 35 μg/L

n-butanol 1.0 μg/L 700 μg/L

2-methoxyethanol 1.0 μg/L 21 μg/L

* This is the aqueous concentration equivalent to the low calibration standard.
 

A World of SolutionsTM 23

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



CONCLUSION SLIDE 2

METHOD PARAMETERS

Preservation: identical to EPA method 522

SPE: 0.1 L Sx; neutralized with 5 mL x 0.8 M NaHCO3; Supelco 

Envi-Carb Plus (400 mg); 60-min air dry in reverse direction; 

elution 20% MeOH:DCM; 2-mL extract volume

Extract analysis: 30 m x 0.25 mm i.d. x 0.5 μm df

 column 

(Agilent P/N CP9222 VF-WAXms); 1 μL injection @ 200 °C 

inlet; temperature- programmed separation; MS detection in 

SIM mode
 

A World of SolutionsTM 24

CONCLUSION SLIDE 3

ANALYTE LIST AND CALIBRATION RANGES

Analytes Range, μg/L Range, ng/mL (equivalent extract concentration)

I.S.: 1,4-dioxane-d8 added to extract 250

I.S.: chlorobenzene-d5 added to extract 250

Surr: allyl alcohol-d6 5.0 250 (also serves as isotope dilution internal standard)

Surr: n-butanol-d10 5.0 250 (also serves as isotope dilution internal standard)

1,4-dioxane 0.10 — 8.0 5.0 — 400

allyl alcohol 0.50 —20 25 — 1000

allyl alcohol* 0.50 —20 25 — 1000

n-butanol 0.50 —20 25 — 1000

n-butanol* 0.50 —20 25 — 1000

2-methoxyethanol 0.50 —20 25 — 1000

* Reported with isotope dilution quantitation
 

A World of SolutionsTM 25

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



CONCLUSION SLIDE 4

CHALLENGES

WATER MANAGEMENT

– EXTRACTION MEDIA IS DIFFICULT TO DRY

– WET EXTRACTS CAUSE RETENTION TIME SHIFTS 

(COMPROMISING QUALITATIVE ID) AND CAN AFFECT 

ANALYTE RESPONSE (QUANTITATIVE AND QC)

– EXCESSIVE DRYING CAUSES LOW RECOVERY

ONLY 1 EXTRACTION MEDIUM AVAILABLE FOR THE 

ANALYSIS OF THESE ANALYTES

LOT TO LOT VARIATION

– LOADING TIME

– DRYING TIME
 

A World of SolutionsTM

ANALYSIS OF ERYTHROMYCIN AND 

OTHER PHARMACEUTICALS BY LCMS/MS

CB&I

William A. Adams, Ph.D.
 

A World of SolutionsTM 1 



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PHARMACEUTICAL TARGET ANALYTES

Carbamazepine, Diazepam, Diclofenac (sodium salt), 

Enalapril (maleate salt), Fluoxetine (HCl), Gemfibrozil, 

Naproxen, Phenytoin, Sulfamethoxazole, Triclosan, 

Trimethoprim, and Erythromycin (measured as 

Erythromycin–H2O) 

Variety of chemically unrelated analytes

– Both ESI positive and ESI negative modes

Analysis by LC-MS/MS using an 5 mM ammonium acetate 

and methanol gradient

SPE (6 mL, 200 mg HLB cartridge)

13C-Naproxen-d3, Triclosan-d3, Carbamazepine-d10, chosen as 

internal standards; 13C-Trimethoprim-d3 and Diclofenac-d4

chosen as surrogate standards
 

A World of SolutionsTM 2 





CCL3 AND PRIORITY TARGET ANALYTES

Erythromycin and triclosan

Only erythromycin listed on CCL3

Erythromycin Triclosan 
 

A World of SolutionsTM 3 

 Erythromycin 

– At pH <7, water is 

removed and compound 

no longer exhibits 

antibiotic properties 

(Hirsch et al., 1999)

– For analysis, erythromycin 

is measured as 

erythromycin–H2O (717.0 

> 158.3 m/z) 

Erythromycin 

Erythromycin 
 

A World of SolutionsTM 4 

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Preservation

Store samples in refrigerator (6°C) 

100 mg/L ascorbic acid

– Reduces free chlorine present in tap water samples

– Easy to handle

– Solid can be added to bottles before sampling

350 mg/L EDTA

– Chelates metals in tap water samples

– Prevents metal hydrolysis

– Solid can be added to bottles before sampling

9.4 g/L potassium citrate

– Acts as a microbial inhibitor

– pH ~3.8

– Solid can be added to bottles before sampling
 

A World of SolutionsTM 5 





SAMPLE PREPARATION

Solid phase extraction (SPE)

– 6 cc, 200 mg HLB cartridges

– Vacuum manifold

– 1 liter samples extracted and eluted with 5 mL 1:1 

methanol/acetone

Dilution

– 5 mL reagent water added to extract

– 100:1 sample to extract concentration factor
 

A World of SolutionsTM 6 

INSTRUMENTAL METHOD

CHROMATOGRAPHIC CONDITIONS

ESI positive

HPLC

Column: Waters Xterra® MS C18, 2.1 x 150 mm, 3.5 μm 

Column temperature: 30 oC 

Column flow rate: 0.200 mL/min

Autosampler temperature: 10 OC 

Injection volume: 10 μL

Gradient: 

Time

(min)

%5 mM 

ammonium 

acetate in 10% 

MeOH/90% 

reagent water

%MeOH

 

0.00 90 10 

0.50 90 10 

0.51 50 50 

8.00 25 75 

8.01 0 100 

10.00 0 100 

14.00 90 10 

24.00 End Run 

ESI negative

HPLC

Column: Waters Xterra® MS C18, 2.1 x 150 mm, 3.5 μm 

Column temperature: 30 oC 

Column flow rate: 0.200 mL/min

Autosampler temperature: 10 OC 

Injection volume: 50 μL 

Gradient: 

Time

(min)

%5 mM 

ammonium 

acetate in 10% 

MeOH/90% 

reagent water

%MeOH

 

0.00 90 10 

0.50 90 10 

0.51 40 60 

8.00 0 100 

11.00 0 100 

15.00 90 10 

25.00 End Run 
 

A World of SolutionsTM 7 

INSTRUMENTAL METHOD

MS CONDITIONS

MS Parameter HPLC-MS/MS

Polarity Positive ion electrospray 

Capillary Voltage, kV 2.50 

Source Temperature, oC 120 

N2 Desolvation Temperature, oC 400 

N2 Desolvation Gas Flow, L/hr 900 

Cone Gas Flow, L/hr 50 

Extractor Lens, V 2.00 

RF Lens, V 0.2 

MS Parameter HPLC-MS/MS

Polarity Negative ion electrospray

Capillary Voltage, kV 2.50 

Source Temperature, oC 120 

N2 Desolvation Temperature, oC 400 

N2 Desolvation Gas Flow, L/hr 900 

Cone Gas Flow, L/hr 50 

Extractor Lens, V 1.00 

RF Lens, V 0.1 
 

A World of SolutionsTM 8 

ESI POSTIVE CHROMATOGRAM 
 ESI NEGATIVE CHROMATOGRAMS

A World of SolutionsTM 9 
A World of SolutionsTM 10

 LCMRL

Analyte LCMRL (ng/L)a

Carbamazepine 2.4 

Diazepam 0.27 

Diclofenac 1.1 

Enalapril 0.60 

Fluoxetine 0.98 

Gemfibrozil 1.4 

Naproxen 4.5 

Phenytoin 1.4 

Sulfamethoxazole 0.28 

Triclosan 3.4 

Trimethoprim 4.1 

Erythromycin 5.0 
PRECISION AND ACCURACY

A World of SolutionsTM 11

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Precision and accuracy measured in different matrixes (e.g. 

reagent water, high TOC tap water, hard tap water)

Measured at two concentrations

– Low and Mid/High of calibration range

Accuracy

– Low: 50–150% recovery

– Mid/High: 70–130% recovery

Precision

– Low: ≤30% RSD

– Mid/High: ≤20% RSD
 

A World of SolutionsTM 12



PRECISION AND ACCURACY

Reagent water

– 9.4 g/L potassium citrate, 350 mg/L EDTA, and 100 mg/L ascorbic acid

Analyte

Low Concentration High Concentration

Fortified 

Concentration 

(ng/L)

Avg. %Recovery %RSD

Fortified 

Concentration 

(ng/L)

Avg. 

%Recovery %RSD

Carbamazepine 4.94 87.9 4.0 31.3 91.5 0.88 

Diazepam 0.340 72.9 7.8 20.0 92.1 3.4 

Diclofenac 2.04 97.2 4.2 102 95.1 1.2 

Enalapril 1.22 105 6.0 40.4 89.7 1.6 

Fluoxetine 1.24 70.3 8.2 104 84.6 2.3 

Gemfibrozil 3.00 92.7 1.4 100 95.6 2.1 

Naproxen 9.20 101 4.0 100 98.5 1.2 

Phenytoin 2.42 76.0 4.5 30.7 90.2 4.1 

Sulfamethoxazole 0.410 92.2 8.0 29.4 90.9 0.51 

Triclosan 7.00 91.5 5.0 311 99.7 1.5 

Trimethoprim 8.80 90.5 4.2 30.4 91.1 2.4 

Erythromycin 5.60 80.4 6.2 40.0 83.9 1.7 

Diclofenac-d4 100 94.0 2.4 100 93.9 1.8 

13C3-Trimethoprim 20.0 86.6 2.3 20.0 87.3 3.3 
 

A World of SolutionsTM 13

PRECISION AND ACCURACY

 Ground source tap water

– 9.4 g/L potassium citrate, 350 mg/L EDTA, and 100 mg/L ascorbic acid

– Free chlorine: 0.76 mg/L

– Total chlorine: 0.97 mg/L

– Hardness: 331 mg/L

– TOC: 0.75 ppm

Analyte

Low Concentration High Concentration

Fortified 

Concentration 

(ng/L)

Avg. %Recovery %RSD

Fortified 

Concentration 

(ng/L)

Avg. 

%Recovery %RSD

Carbamazepine 4.94 93.0 2.5 31.3 95.3 1.2 

Diazepam 0.340 73.5 13 20.0 97.7 1.8 

Diclofenac 2.04 104 7.6 102 97.4 1.4 

Enalapril 1.22 94.3 2.9 40.4 94.4 1.1 

Fluoxetine 1.24 86.1 6.4 104 94.9 2.4 

Gemfibrozil 3.00 92.7 4.1 100 96.4 2.0 

Naproxen 9.20 89.0 3.8 100 104 2.1 

Phenytoin 2.42 83.0 5.9 30.7 80.2 2.1 

Sulfamethoxazole 0.410 87.8 12 29.4 93.8 1.3 

Triclosan 7.00 88.5 10 311 96.7 1.0 

Trimethoprim 8.80 92.4 5.2 30.4 94.3 1.2 

Erythromycin 5.60 69.8 7.3 40.0 78.7 5.5 

Diclofenac-d4 100 98.9 1.5 100 88.9 2.2 

13C3-Trimethoprim 20.0 98.4 1.8 20.0 97.7 1.8 
 

A World of SolutionsTM 14

PRECISION AND ACCURACY

 Surface source tap water

– 9.4 g/L potassium citrate, 350 mg/L EDTA, and 100 mg/L ascorbic acid

– Free chlorine: 1.16 mg/L

– Total chlorine: 1.65 mg/L

– Hardness: 124 mg/L

– TOC: 4.23 ppm

Analyte

Low Concentration High Concentration

Fortified 

Concentration 

(ng/L)

Avg. %Recovery %RSD

Fortified 

Concentration 

(ng/L)

Avg. 

%Recovery %RSD

Carbamazepine 4.94 83.7 2.7 31.3 91.1 2.8 

Diazepam 0.340 54.7 27 20.0 95.7 0.92 

Diclofenac 2.04 71.8 5.6 102 73.6 1.4 

Enalapril 1.22 99.5 8.8 40.4 94.1 0.42 

Fluoxetine 1.24 96.6 4.3 104 105 1.8 

Gemfibrozil 3.00 104 3.3 100 111 0.91 

Naproxen 9.20 92.7 4.8 100 98.6 1.9 

Phenytoin 2.42 95.4 6.6 30.7 95.4 1.6 

Sulfamethoxazole 0.410 92.2 28 29.4 93.3 0.96 

Triclosan 7.00 93.5 12 311 99.2 0.94 

Trimethoprim 8.80 69.6 5.2 30.4 72.5 2.1 

Erythromycin 5.60 85.0 6.3 40.0 87.2 2.0 

Diclofenac-d4 100 72.3 0.92 100 75.3 1.7 

13C3-Trimethoprim 20.0 80.2 1.6 20.0 81.4 1.3 
 

A World of SolutionsTM 15

HOLDING TIME - SAMPLE

 Surface source tap water

Analyte

Fortified 

Conc. 

(ng/L)

Day 0 Day 7 Day 14 Day 21 Day 28

Avg. 

%Rec %RSD

% 

Change 

from 

Day 0

%RSD

% 

Change 

from 

Day 0

%RSD

% 

Change 

from 

Day 0

%RSD

% 

Change 

from 

Day 0

%RSD

Carbamazepine 7.82 106 6.1 -1.3 6.6 -2.2 3.0 -6.7 3.4 -8.4 0.46 

Diazepam 5.00 114 1.1 -8.9 1.5 -6.7 1.8 -6.7 1.8 -11 1.7 

Diclofenac 25.6 105 0.84 -c -c 4.3 1.8 3.3 0.92 -13 1.2 

Enalapril 10.1 90.8 2.3 0.29 2.9 -2.6 2.8 -1.3 0.78 1.1 2.2 

Fluoxetine 26.0 113 2.4 0.56 7.8 15 41 -6.6 3.6 -4.1 1.9 

Gemfibrozil 25.0 104 1.2 7.3 2.3 3.3 2.1 4.1 1.1 -5.3 2.5 

Naproxen 25.0 106 1.2 3.4 2.0 2.5 2.7 0.39 2.7 0.50 2.2 

Phenytoin 7.68 94.8 11 -c -c -30 3.4 -15 9.2 -18 2.8 

Sulfamethoxazole 7.34 93.0 3.3 -1.2 5.5 -2.7 0.53 -7.8 0.56 -6.1 2.6 

Triclosan 77.8 103 2.8 3.1 3.5 5.1 2.2 1.5 1.3 -1.1 0.76 

Trimethoprim 7.60 74.8 2.0 1.7 4.1 -10 3.0 -10 4.5 -16 3.4 

Erythromycin 10.0 102 1.9 -1.3 22 -6.2 4.5 -0.62 1.5 -3.7 2.8 

Surrogate

Fortified 

Conc. 

(ng/L)

Avg. 

%Rec %RSD Avg. %Rec %RSD Avg. %Rec %RSD Avg. %Rec %RSD Avg. %Rec %RSD

Diclofenac-d4 100 85.5 1.2 103 5.1 97.8 1.7 99.7 0.55 82.2 0.68 

13C3-Trimethoprim 20.0 79.4 0.84 78.9 0.84 74.7 0.43 76.7 2.4 79.1 1.9 
 HOLDING TIME - EXTRACT

 Surface source tap water

A World of SolutionsTM 16

Analyte

Fortified 

Conc. 

(ng/L)

Day 0 Day 7 Day 14 Day 21 Day 28

Avg. 

%Rec %RSD

% 

Change 

from 

Day 0

%RSD

% 

Change 

from 

Day 0

%RSD

% 

Change 

from 

Day 0

%RSD

% 

Change 

from 

Day 0

%RSD

Carbamazepine 7.82 106 6.1 -10 3.8 -9.4 3.5 -12 2.3 -14 3.5 

Diazepam 5.00 114 1.1 -14 2.5 -12 4.1 -11 2.4 -13 3.0 

Diclofenac 25.6 105 0.84 -6.4 1.1 -6.4 3.2 -0.74 1.0 -17 1.1 

Enalapril 10.1 90.8 2.3 4.4 2.2 0.69 0.50 2.4 0.79 4.3 1.5 

Fluoxetine 26.0 113 2.4 13 2.2 2.6 4.6 8.8 4.9 18 6.0 

Gemfibrozil 25.0 104 1.2 -2.5 1.8 -3.3 1.7 3.0 0.41 -11 0.63 

Naproxen 25.0 106 1.2 -0.48 1.3 3.6 3.1 4.0 1.8 3.2 2.8 

Phenytoin 7.68 94.8 11 - - -35 7.4 -20 4.3 -16 13 

Sulfamethoxazole 7.34 93.0 3.3 -12 4.0 -9.6 1.2 -7.6 2.2 -8.2 1.6 

Triclosan 77.8 103 2.8 -1.0 1.8 -0.10 1.1 1.5 1.6 -4.1 2.7 

Trimethoprim 7.60 74.8 2.0 -3.1 3.0 -12 3.1 -9.7 0.41 -11 3.4 

Erythromycin 10.0 102 1.9 -24 6.6 -14 4.5 -12 1.8 -16 4.8 

Diclofenac-d4 100 85.5 1.2 -0.94 2.4 -1.8 5.2 10 5.1 -11 4.2 

13C3-Trimethoprim 20.0 79.4 0.84 11 3.7 -1.9 4.5 1.6 2.8 1.2 2.0 
 

A World of SolutionsTM 17











CONCLUSIONS

Various pharmaceuticals/personal care products (PPCPs) analyzed in 

both ESI positive and ESI negative modes

Erythromycin measured as erythromycin–H2O 

– Likely transforms prior to analysis

Extraction necessary to reach desired concentration levels

– HLB cartridges found to provide best overall extraction recovery for both 

positive and negative analytes

All analytes meet precision and accuracy QC requirements in three 

different matrixes

Sample and extract hold times change less than 20% after 28 days
 

A World of SolutionsTM

ANALYSIS OF CYLINDROSPERMOPSIN 

AND ANATOXIN-A BY LC-MS/MS

CB&I

William A. Adams, Ph.D.
CC L3 ALGAL TOXIN TARGET ANALYTES

A World of SolutionsTM 1 

Anatoxin-a 

Cylindrospermopsin









Cylindrospermopsin and anatoxin-a 

– Toxins produced by blue-green algae

Water soluble, polar molecules

Analysis by LC-MS/MS using a high 

aqueous composition eluent

– Cylindrospermopsin: 416.2 > 194.0 m/z

– Anatoxin-a: 165.8 > 148.8 m/z

Direct injection
SE LECTION OF INTERNAL STANDARDS

A World of SolutionsTM 2 







Uracil L-phenylalanine

Isotopically labeled standards not available for 

cylindrospermopsin or anatoxin-a 

Cylindrospermopsin is a uracil derivative

– Deuterated uracil readily available and elutes near 

retention time of cylindrospermopsin (Uracil-d5) 

Anatoxin is isobaric and structurally similar to L-phenylalanine

– Deuterated phenylalanine is readily available and appears 

to track well with anatoxin-a (L-phenylalanine-d4) 
 

A World of SolutionsTM 3 

CHROMATOGRAPHIC CONDITIONS

INSTRUMENTAL METHOD

HPLC

Column: Waters XSelect® HSS T3, 2.1 x 150 mm, 3.5 μm 

Column temperature: 30 oC 

Column flow rate: 0.200 mL/min

Autosampler temperature: 10 oC 

Injection volume: 50 μL 

Gradient: 

Time

(min)

%100 mM acetic 

acid in reagent 

water

%MeOH

 

0.00 100 0 

0.50 90 10 

8.50 100 0 

13.50 End Run 

MS CONDITIONS

MS Parameter HPLC-MS/MS

Polarity Positive ion electrospray 

Capillary Voltage, kV 2.50 

Source Temperature, oC 120 

N2 Desolvation Temperature, oC 400 

N2 Desolvation Gas Flow, L/hr 900 

Cone Gas Flow, L/hr 50 

Extractor Lens, V 2.00 

RF Lens, V 0.2 
 

A World of SolutionsTM 4 







Preservation

Store samples in refrigerator

100 mg/L ascorbic acid

– Reduces free chlorine present in tap water samples

– Easy to handle

– Solid can be added to bottles before sampling

1000 mg/L sodium bisulfate

– Acts as a microbial inhibitor

– pH less than 3

– Solid can be added to bottles before sampling
CHR OMATOGRAM – 5 ng/mL ANALYTES

A World of SolutionsTM 5 

Uracil-d4

L-phenylalanine-d5

CYN

ANA
 

A World of SolutionsTM 6 







MRL TARGETS

HRL values FROM CCL3

– Cylindrospermopsin – 0.21 μg/L

– Anatoxin-a – 3.5 μg/L

Calibration range

– Cylindrospermopsin – 0.050 to 10.0 μg/L

– Anatoxin-a – 0.050 to 10.0 μg/L

MRL confirmed at 0.100 μg/L for both analytes
 

A World of SolutionsTM 7 

LCMRL
 

A World of SolutionsTM 8 









PRECISION AND ACCURACY

Precision and accuracy measured in different matrixes (e.g. 

reagent water, high TOC tap water, hard tap water)

Measured at two concentrations

– Low and Mid/High of calibration range

Accuracy

– Low: 50–150% recovery

– Mid/High: 70–130% recovery

Precision

– Low: ≤30% RSD

– Mid/High: ≤20% RSD
 

A World of SolutionsTM 9 



PRECISION AND ACCURACY

Reagent water

– 1000 mg/L sodium bisulfate and 100 mg/L ascorbic acid

Reagent Water Low

Compound Samples (ug/L) Mean

(ug/L) SD

Unfortified 

blank

(ug/L)

Theoretical 

spike

(ug/L)

%RSD %Rec ESI (+) 1 2 3 4 5 6 7 

cylindrospermopsin 0.117 0.128 0.117 0.103 0.100 0.094 0.102 0.109 0.012 0.000 0.100 11.136 108.7 

anatoxin-a 0.108 0.116 0.108 0.106 0.102 0.127 0.111 0.111 0.008 0.000 0.100 7.391 111.1

Reagent Water High

Compound Samples (ug/L) Mean

(ug/L) SD

Unfortified 

blank

(ug/L)

Theoretical 

spike

(ug/L)

%RSD %Rec ESI (+) 1 2 3 4 5 6 7 

cylindrospermopsin 2.837 2.609 2.834 2.767 2.614 2.681 2.664 2.715 0.097 0.000 2.500 3.588 108.6 

anatoxin-a 2.757 2.689 2.810 2.580 2.688 2.798 2.709 2.719 0.079 0.000 2.500 2.898 108.7 
 

A World of SolutionsTM 10



PRECISION AND ACCURACY

Ground source tap water

– 1000 mg/L sodium bisulfate and 100 mg/L ascorbic acid

– Free chlorine: 0.69 mg/L

– Total chlorine: 0.97 mg/L

– Hardness: 325 mg/L

– Conductivity: 798 μS

– pH: 7.76 

Ground Water Low

Compound Samples (ug/L) Mean

(ug/L) SD

Unfortified 

blank

(ug/L)

Theoretical 

spike

(ug/L)

%RSD %Rec ESI (+) 1 2 3 4 5 6 7 

cylindrospermopsin 0.137 0.132 0.120 0.128 0.119 0.122 0.125 0.126 0.007 0.000 0.100 5.247 126.1 

anatoxin-a 0.102 0.080 0.087 0.099 0.092 0.096 0.093 0.093 0.007 0.000 0.100 8.016 92.7 

Ground Water High

Compound Samples (ug/L) Mean

(ug/L) SD

Unfortified 

blank

(ug/L)

Theoretical 

spike

(ug/L)

%RSD %Rec ESI (+) 1 2 3 4 5 6 7 

cylindrospermopsin 2.406 2.516 2.413 2.435 2.380 2.487 2.508 2.449 0.054 0.000 2.500 2.206 98.0 

anatoxin-a 2.316 2.394 2.370 2.324 2.382 2.419 2.424 2.376 0.043 0.000 2.500 1.790 95.0 
 

A World of SolutionsTM 11



PRECISION AND ACCURACY

Surface source tap water

– 1000 mg/L sodium bisulfate and 100 mg/L ascorbic acid

– Free chlorine: 0.64 mg/L

– Total chlorine: 1.14 mg/L

– Hardness: 142 mg/L

– Conductivity: 344 μS

– pH: 7.20 

– TOC 3.04 mg/L

Surface Water Low

Compound Samples (ug/L) Mean

(ug/L) SD

Unfortified 

blank

(ug/L)

Theoretical 

spike

(ug/L)

%RSD %Rec ESI (+) 1 2 3 4 5 6 7 

cylindrospermopsin 0.139 0.124 0.111 0.112 0.119 0.109 0.104 0.117 0.012 0.000 0.100 10.070 116.9 

anatoxin-a 0.106 0.091 0.096 0.101 0.101 0.093 0.090 0.097 0.006 0.000 0.100 6.178 96.9 

Surface Water High

Compound Samples (ug/L) Mean

(ug/L) SD

Unfortified 

blank

(ug/L)

Theoretical 

spike

(ug/L)

%RSD %Rec ESI (+) 1 2 3 4 5 6 7 

cylindrospermopsin 2.762 2.768 2.699 2.714 2.715 2.649 2.679 2.712 0.043 0.000 2.500 1.570 108.5 

anatoxin-a 2.450 2.506 2.383 2.585 2.484 2.565 2.548 2.503 0.071 0.000 2.500 2.833 100.1 
 

A World of SolutionsTM 12











CONCLUSIONS

Cylindrospermopsin and anatoxin-a show good response with method

Labeled analogs of target analytes not available

– Internal standards chosen based on tracking ability and response

– Uracil-d4 has shown some inconsistency in dirtier matrixes and may be 

removed

Precision and accuracy are within the acceptable range in three different 

matrixes

MRL is confirmed below HRL of both analytes and LCMRL gives 

reasonable results

Method development will continue with storage/stability study and 

second laboratory demonstration
 

A World of SolutionsTM

Update to Method 539 

CB&I

Steven D. Winslow
 

A World of SolutionsTM 1 





Update to Method 539

EPA Method 539: LC-MSMS analysis , multiple reaction 

monitoring (MRM) , SPE extraction

Original Target Analyte List

Method 539 CASRN

4-Androstene-3,17-dione 63-05-8 

Equilin 474-86-2 

17β-Estradiol 50-28-2 

Estriol 50-27-1 

Estrone 53-16-7 

17α-Ethynylestradiol 57-63-6 

Testosterone 58-22-0 
 

A World of SolutionsTM

β-estradiol 

2 

Estriol 

Estrone 

Ethynylestradiol 

Androstenedione 

Equilin Testosterone 

Update to Method 539

 EPA METHOD 539 TARGET ANALYTES
 

A World of SolutionsTM 3 



Update to Method 539



Proposed additional hormones to Method 539

Five compounds, introduced late in Method 539 

development, were initially rejected from method

COMPOUNDS CAS# ISSUES HRL 

(ug/L)

alpha- Estradiol 57-91-0 M539: enhancement issue 0.35 

Equilenin 517-09-9 M539: recovery in finished ground water failed QC 0.35 

Mestranol 72-33-3 M539: did not work with ESI 0.28 

Norethindrone 68-22-4 M539: ESI+ internal standard did not track 0.04 

Progesterone 57-83-0 M539: ESI+ internal standard did not track

Nominated for CCL4

---

Internal standards now available

– Norethindrone-2,2,4,6,6,10-d6

– Progesterone-2,3,4-13C3 
 

A World of SolutionsTM 4 

Equilenin 

α-Estradiol Norethindrone 

Mestranol 

Update to Method 539

 Structures of new hormones

Progesterone 
 

A World of SolutionsTM 5 



Update to Method 539

Also add bisphenol A to method Bisphenol A 

– Nominated for CCL4 

– Deuterated bisphenol A used as surrogate M539

• Extraction not an issue

– But, bisphenol A contamination from instrument 

and solvent at least 8 ppb 

– Quantitation level can be no lower than 3 times 

LRB level

• MRL would be above 24 ppb
 

A World of SolutionsTM 6 

Update to Method 539





Bisphenol A 

Instrument manufacturer suggests use of an 

“isolator” column for bisphenol A

– Contamination temporarily retained in short 

column placed between solvent mixer & pumps

– Contamination separated on chromatogram by 

later elution

Would bisphenol A require too many 

precautions for a “hormone” method? Separate 

method?
 

A World of SolutionsTM 7 



Update to Method 539

Proposed target analyte list for Method 539 revision 

with additional five hormones & bisphenol A (in bold)

CAS#

Androstenedione 63-05-8

Bisphenol A 80-05-7

Equilenin 517-09-9

Equilin 474-86-2

α-Estradiol 57-91-0

β-Estradiol 50-28-2

Estriol 50-27-1

Estrone 53-16-7

α-Ethynylestradiol 57-63-6

Mestranol 72-33-3

Norethindrone 68-22-4

Progesterone 57-83-0

Testosterone 58-22-0
 

A World of SolutionsTM 8 









Update to Method 539

Initial chromatographic challenges

– alpha- & beta-estradiol must be well separated, 

because precursor and product ions are same

– Must use high water content (90%) in initial segment 

of gradient mobile phase program to focus bisphenol 

A contamination 

Develop calibration range

– Test for reproducibility

– Estimate lowest calibration level

Extract spiked samples with C18 disks

– If needed, use Oasis HLB disks

Check for contamination or interference in Laboratory 

Reagent Blanks (LRBs) that contain preservation 

reagents, sodium omadine & sodium thiosulfate
 

A World of SolutionsTM 9 

 First 6 Norethindrone 299.52 > 108.88

Androstenedione 287.53 > 96.92

Equilin 267.46 > 143.08

Equilinen 265.44 > 221.32

Bisphenol A 227.45 >212.28

Estriol 287.48 > 171.18

Update to Method 539
 

A World of SolutionsTM

 Last 7 Mestranol 311.53 > 121.10

Progesterone 315.55 > 96.85

Testosterone 289.58 > 96.89

alpha-ethynylestradiol 295.48 > 145.09

beta-estradiol alpha-estradiol 271.48 > 145.08

Estrone 269.48> 145.11

10

Update to Method 539
 

A World of SolutionsTM 11



Update to Method 539

Collect data for demonstration of capability

– Determine Lowest Concentration Minimum Reporting 

Level (LCMRL)

– Run Precision & Accuracy studies at a low and a mid 

level in reagent water, surface water and ground water

– Conduct holding time study for samples with 

preservatives

• Holding time study for extracts

– Conduct Multi-Laboratory Validation 

• At least two other labs

• Use data to calculate MRL for method
Microbiology Methods Overview

Sandhya Parshionikar, Ph.D.

Office of Ground Water and Drinking Water

Standards and Risk Management Division

Technical Support Center

May 15, 2013

5/10/2013 U.S. Environmental Protection Agency 1 
Candidate Contaminant List 3

Microbial Contaminant Name Information 

Adenovirus Virus -respiratory and gastrointestinal illness

Caliciviruses Virus (includes Norovirus) Virus - gastrointestinal illness

Campylobacter jejuni Bacterium - gastrointestinal illness

Enterovirus Group of viruses - mild respiratory illness

Escherichia coli (0157) Toxin-producing bacterium - gastrointestinal illness and 

kidney failure

Helicobacter pylori Bacterium - ulcers and cancer

Hepatitis A virus Virus - liver disease and jaundice

Legionella pneumophila Bacterium -lung diseases when inhaled

Mycobacterium avium Bacterium - lung infection in those with underlying lung 

disease, and disseminated infection in the severely 

immuno compromised

Naegleria fowleri Protozoan parasite -warm surface and ground water 

causing primary amebic meningoencephalitis

Salmonella enterica Bacterium -gastrointestinal illness

Shigella sonnei Bacterium -gastrointestinal illness and bloody diarrhea

5/10/2013 U.S. Environmental Protection Agency 2 
Challenges to Microbial monitoring under UCMR

• Pathogen presence in biofilms 

– Biofilm sampling presents a challenge for UCMR

• Could be present in low concentrations

– Need to sample large volumes of water 

• Presents logistical challenges for UCMR

• Pathogen presence in unconventional locations

– Hospital and nursing home hot water tanks and storage tanks

• Public health concern but challenging to monitor

• Some pathogens hard to grow in culture

• Risk assessment with PCR data is a challenge

5/10/2013 U.S. Environmental Protection Agency 3 
Method Development Rationale

• Depends on which pathogen to target for UCMR 

monitoring

– What is the likelihood of a pathogen being found in finished drinking 

water?

– Which pathogen has a validated method ready?

– Which public health issue to address during UCMR monitoring?

• Outbreaks 

• Other public health concerns

– Under what conditions do we monitor?

• Frequency

• Volume of water 

• Seasonality 

• Type of public water system

5/10/2013 U.S. Environmental Protection Agency 4 
Current Method Development Efforts

• H.pylori

• E.coli O157:H7

• Adenovirus

• Mycobacterium

5/10/2013 U.S. Environmental Protection Agency 5 
Development of Methods to 

 detect H. pylori and E. coli 

O157:H7 in Water

CCL Methods Meeting/Webinar

Keya Sen, 

Technical Support Center, SRMD, 

OGWDW, OW US EPA

May 15, 2013

5/10/2013 1 U.S. Environmental Protection Agency
Facts about H. pylori

• H. pylori a gram negative bacteria

– Causes chronic active and persistent gastritis 

– Stomach and duodenal ulcers and in few of these cases, 

gastric cancer

• Approximately 25 Million Americans suffer from the disease

– Each year 500,000 to 850,000 new cases

• Culturable from stomach but unculturable from water

– necessitates the use of PCR based methods

5/10/2013 U.S. Environmental Protection Agency 2 
H. Pylori Method Development

• Analyzes 1 liter of sample collected

• Detects a segment of the gene ureA, 

conserved in all H. pylori, by qPCR

• Uses an internal control which is a process 

control and is added to every sample- a 

Bioball

– Confirms whether a sample is a true negative

5/10/2013 U.S. Environmental Protection Agency 3 
Detection of H. pylori from Water

Collect 1 liter of drinking water. Spike with 10-

1000 cells of H. pylori. Add 1 Bioball

Filter through 47mM, 0.4 micron

5/10/2013 U.S. Environmental Protection Agency 4 

Extract filter with lysis solution

Remove solution to centrifuge tube. 

Extract DNA into 33 μl water. Use 11 μl (x3) for qPCR 
Inter-laboratory validation of 

qPCR method for H. pylori detection

• Five volunteer laboratories selected

• Three drinking water samples (1L) tested

• One sterile reagent grade water (1L)

• 2 levels of spike used

– 15 or 100 cells of H. pylori

• BioBall-KS 10 containing 30 cells of E. coli used as 

matrix spike (process control)

5/10/2013 U.S. Environmental Protection Agency 5 
Initial and Ongoing Precision and Recovery (IPR 

and OPR) Acceptance Criteria1

Performance test* E. coli KS10 BioBallTM 

acceptance criteria

Initial precision and recovery (IPR)

• Mean percent recovery 25% - 379% (8-114 gene copies)2

• Precision (as maximum relative 

standard deviation) 107%

Ongoing precision and recovery (OPR) as 

percent recovery

Detect - 482% (145 gene copies)

1IPR requires the analysis of 4 reagent-grade water samples; OPR requires the analysis of a 

single reagent water sample

2One gene copy can be considered to be equivalent to one cell since each E. coli KS10 cell 

has the target cloned into a single copy plasmid.

5/10/2013 U.S. Environmental Protection Agency 6 
E. Coli O157:H7 Method 

Development

5/10/2013 U.S. Environmental Protection Agency 7 
Facts about E. coli O157:H7 

• Gram negative bacteria that lives in the guts of ruminants

• Causes severe illness 

– bloody diarrhea and vomiting

– About 10 % develop hemolytic uremic syndrome (HUS) that can 

result in non-functioning kidneys

• About 95,400 cases each year from E. coli O157:H7 in the US

• Bacteria can be cultured from water but can be difficult to identify

– Needs specialized approach

5/10/2013 U.S. Environmental Protection Agency 8 
Traditional Methods for Detection of 

E. coli O157:H7 

5/10/2013 U.S. Environmental Protection Agency 9 

Filter Water

• 0.1L-1L

Enrich filter in broth

• Tryptic Soy or Buffered Peptone Water

Immunomagnetic separation 

• Specific Antibodies

Grow in CT-SMAC or Rainbow 

Agar with antibiotics 

• select colonies with characteristic 

morphology

Verify by PCR/qPCR • Primers and Probes to virulence factors

• Primers and Probes to Structural genes
Issues With Detection of E. coli O157:H7 by 

Traditional Methods

5/10/2013 U.S. Environmental Protection Agency 10

False Positives

obtained

• Closely related pathogenic E. coli serotypes 

having similar phenotypic and genotypic 

properties

• Growth of O157:H7 suppressed

Takes 3-5 days

Labor 

intensive
•

•

•

•

•

Rapid method 

Goals for EPA Method for E. coli O157:H7

Detect in source and drinking

water within a day 

5/10/2013 U.S. Environmental Protection Agency 11

Highly 

Sensitive

Able to detect 1-5 cells

Highly specific

Able to detect 

viable cells 

No false positives

Appropriate QC reagents

Allow better risk assessment
Strategy

• Add an enrichment medium directly to water sample

• Provide conditions for stressed cells (starvation, 

chlorination induced), if present , to recover and grow

• Perform qPCR before and after enrichment without 

isolation of E. coli cells

– Change in Ct value from pre to post enrichment will indicate 

viable cells

– A decrease of 3.3 Ct value corresponds to 1 log increase in 

gene copies, if the amplification efficiency is 100%

5/10/2013 U.S. Environmental Protection Agency 12
Development of E. coli O157: H7 

qPCR assays

• Searched literature for primers and probes 

• Tested primers and probes with an ATCC strain

• Tested 4 commercially available kits with an ATCC strain

• Obtained 61 strains from USDA, FDA and in-house 

collection 

– Tested kits and probes

5/10/2013 U.S. Environmental Protection Agency 13
Selection of Targets/kits

• ABI MicroSeq kit produced no false positives

– Targets two non-coding regions on the E. coli genome

– Has an internal control for qPCR

• Additional triplex qPCR reaction optimized

– Targets genes stx1, stx2, eae

– Developed MGB probes 

• Additional monoplex assay that targets rfbE gene optimized for 

confirmation 

– rfbE specific to O157 serotype

– Developed MGB probes 

(Environmental Science and Technology (2011): 45, 2250-2256

Sen, K1. Sinclair1, J, Boczek, L2, Rice, E.G3.)

5/10/2013 U.S. Environmental Protection Agency 14
Demonstration of Viability

• Bacterial Cells starved for 12- 14 days

• Dilutions made, 4-400 cells

• Spiked water samples with different dilutions

• Added 6X Presence-Absence broth to sample 

 -1 mL aliquot removed and stored at – 200 C (pre-enrichment).

• Allowed to grow:35 0C for 18-23 hrs

– 1 mL aliquot removed

• DNA extracted from both aliquots

– Centrifuge to pellet cells

– Add 100 μL of PrepMan buffer; Heat at 99 0 C 

• Perform 3 qPCR assays with 3 μL extract.

• Examine whether there is a shift in Ct values from pre to post 

enrichment samples

5/10/2013 U.S. Environmental Protection 

Agency 15
5/10/2013 U.S. Environmental Protection Agency 16

Stx1

Stx2 eae 

18 hr 

0 hr 

Demonstration of Viability

 Detects less than 10 

 cells 

Examines only 30 μL of 

sample
Addition of Process Control

• Bioball-KS10 used for H. pylori considered good for 

the purpose since they are E. coli cells but not 

expected to occur naturally

• Optimized the number of cells to add

• BTF-Biomerieux packaged 5000 cells into Bioball.

• Bioball added at the time of extraction

• Detected in a separate Monoplex assay, 

– Used same primers and probe used in H. pylori assay

5/10/2013 U.S. Environmental Protection Agency 17
5/10/2013 U.S. Environmental Protection Agency 18

S

S

ID

Collection to Detection

Collect 1L water

Add 200 mLPresence /Absence broth 

Remove 1 mL. Store at -20 0C 

Grow 1199 mL x 18-23 hrs at 35 0C till yellow. 

Remove 2 x 1 mL

Add 1 Bioball-5000 to all three tubes

Centrifuge all tubes

Remove supernatant 

Suspend Pellet in PrepMan Buffer

 Heat to 950 C x 10 mins 

Centrifuge tubes x 2 mins

Use 3 μL of supernatant from each tube in 3 qPCR assays

rfbE ABI MicroSeq ( Triplex 1) 

tx1, Stx2, eae (Triplex 2)

PC (Monoplex)
Testing Method With Field Samples

• Six Surface water tested

– Four had pathogenic E. coli 

– Only one sample had E. coli O157:H7

5/10/2013 U.S. Environmental Protection Agency 19
Inter-laboratory validation of 

qPCR method for detection

• Five volunteer laboratories selected

• Three drinking (ground) water samples (1L) tested by 

all 5 labs

• One sterile reagent grade water (1L) by all 5 labs

• One spike level used <10 cells

5/10/2013 U.S. Environmental Protection Agency 20
Initial and Ongoing Demonstration of Capability (IDC 

and ODC) Acceptance Criteria

5/10/2013 U.S. Environmental Protection Agency 21

Performance Test Acceptance Criteria

Initial Demonstration of Capability ( IDC) (as Ct difference between pre and post enrichment 

samples). IPR requires the analysis of 4 reagent-grade water samples

Target Ct difference Precision (as standard deviation 

of Ct difference) 

MicroSEQ FAM 15.86- 25.21 1.79

MicroSEQ VIC 17.51-23.90 1.49

eae 15.11- 22.85 2.46

stx1 16.42-25.02 2.38

stx2 10.40-29.01 3.85

Ongoing Demonstration of Capability ( IDC) (as Ct difference between pre and post enrichment 

samples). OPR requires the analysis of a single reagent water sample

Target Ct difference

MicroSEQ FAM 15.74-25.33

MicroSEQ VIC 17.44-23.97

eae 13.40-24.56

stx1 14.95-26.49

stx2 10.55-28.86
Conclusions and Next Steps

• Both methods have been developed and 

validated

• Currently being processed for EPA publication

5/10/2013 U.S. Environmental Protection Agency 22
Office of Research and Development

National Exposure Research Laboratory, Cincinnati, Ohio May 2013

G. Shay Fout, Jennifer L. Cashdollar, and Nichole 

E. Brinkman

U.S. Environmental Protection Agency, 

Cincinnati, OH

Adenovirus Methods: Method 1615 

Modification and Small Volume 

Approach
1 

Office of Research and Development

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Presentation Outline

•

•

•

Method 1615







Rationale for the development and enhancement of 

Method 1615

Method overview

Water Cluster modifications

 Method performance

Small Volume Approaches

 Method overview and performance

Future development
2 

•

•

•

•





EPA Method 1615 Rationale

Office of Research and Development

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Groundwater-Borne Disease Outbreaks in the USA

From 1971 to 2008 there were nearly 846 outbreaks associated with an 

infectious agent in drinking water; about 50% of the outbreaks were 

attributable to groundwater 

During the same period, 8% of the outbreaks were caused by viruses and 

another 43% had a likely viral cause

Numerous studies have found human enteric viruses in about 25% of wells 

studied

Two studies focused on small community systems that use untreated 

groundwater

A study of communities in Southeast Michigan found virus presence in 

24% of the wells (Francy et al., 2004)

A study of communities in Wisconsin found virus in 95% of the wells

(Borchardt et al. 2012)
3 

Virus Types, Frequencies, and Concentrations 

in Tap Water

Virus

Type

Number

qPCR

Positive 

Samples

Virus Concentration

Genomic copies/L

Number

Culture 

Positive 

Mean Maximum Samples

Adenovirus 157 (13%) 0.07 9.5 40/157 (25%)

Enterovirus 109 (9%) 0.8 851.1 31/109 (28%)

GI Norovirus 51 (4%) 0.6 115.7 

GII Norovirus 0 (0%) 0 0 

Hepatitis A 10 (1%) 0.006 4.1 

Rotavirus 1 (0.1%) 2 x 10-5 0.03 

All Viruses 287 (24%) 1.5 853.6 

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N = 1,204 samples from 14 Communities; Infectivity was measured on qPCR 

positive samples only; Reference: Borchardt et al. 2012. 
4 

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EPA Method 1615 Rationale

• The Wisconsin study demonstrated that 22% of the AGI in the study 

communities was from virus-contaminated tap water (Borchardt et 

al. 2012. Environmental Health Perspectives 120:1272-1279)

• For children < 5 yrs in the spring of 2006, the fraction of AGI from 

norovirus in drinking water was 63%! 
5 

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EPA Method 1615 Rationale

 Virus in U.S. Source Waters

 Percent Cell Culture Positive

 Samples Plant Intakes

Region

East 28 85 

South 22 86

Midwest 21 90

West 20 83

Totals 24 87

Average titer all samples: 1.5 MPN/100 L (range: 0-1974) 
6 

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EPA Method 1615 Enhancement

Adenovirus in Wastewater

Adenovirus Genomic Copies/Liter

Raw/Primary Effluent 2,000 – 800,000,000 

Tertiary Effluent 930 – 8,900,000 

References:

Bofill-Mas et al., 2006

Katayama et al., 2008

Dong et al., 2009

Kuo et al., 2010

Hewitt et al., 2011

Sidhu et al., 2013

Brinkman et al., submitted
7 

EPA Method 1615 Overview

Filtration/Sample Concentration

Samples: Controls: 

NanoCeram filters (1800 L of groundwater) PT and PE: Filters containing unknown amounts of virus (10 L spiked)

 QC: Filters containing 0 or 500 MPN of virus (10 L spiked)

 Matrix: Filters containing 1,000 MPN of virus (1790 + 10 L spiked)

Elution

2 × 500 mL 1.5% beef extract, 0.05 M glycine, pH 9.0

Organic flocculation

pH 3.5 for 30 min

Resuspend floc in 30 mL 0.15 M sodium phosphate, pH 9.0 

Subsample 1: approx. 9 mL

Cell Culture

Quantal assay

MPN/L

Subsample 3

Approx. 12 

mL

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Archive

Subsample 2: approx. 9 mL

Tertiary Concentration

Vivaspin 20, 30 kDa MWCO 

RNA Extraction

QIAamp DNA Blood Mini Kit

RT-qPCR

Genomic copies/L

References:

Fout et al. 2012. EPA/600/R-10/181, January 2012 revision

Cashdollar et al. 2012. Applied Environmental Microbiology 79:215-223
8 

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Total Culturable Virus Assay

BGM cells showing early cytopathic 

effect from poliovirus

QUANTITATION OF TOTAL CULTURABLE VIRUS DATA SHEET

Sample Number: Sample 1

Sample

Number 

Replicates 

Inoculated

Number 

with CPE (1) MPN/mL(2)

95% Confidence 

Limits/mL

Lower Upper 

Undiluted 10 6

1.1 0.4 2.2 1:5 Dilution 0

1:25 Dilution 0

1:125 Dilution 0

D

M S = M m L

L = (1.1 MPN/mL * 8.33 mL)/1800 L = 0.02 MPN/L 
9 

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Additional Concentration for the Molecular Assay

Tertiary Concentration with Vivaspin 

Concentrators 

• Fill Vivaspin 20 unit with PBS, 0.2% BSA

• Soak overnight at 4 °C. Discard PBS

• Rinse with sterile water. Discard water

• Add an amount of Subsample 2 = S

• Centrifuge at 3,000 – 6,000 x g to a volume 

less than 0.4 mL

• Add 1 mL of 0.15 M sodium phosphate, pH 

7-7.5

• Centrifuge at 3,000 – 6,000 x g to a volume 

less than 0.4 mL

• Add 1 mL of 0.15 M sodium phosphate, pH 

7-7.5

• Centrifuge at 3,000 – 6,000 x g to a volume 

less than 0.4 mL

• Transfer sample to a 1.5 mL microcentrifuge 

tube

• Bring to a volume of 400 ± 2 μL 
10

Tertiary Concentration

Recovery of Poliovirus and Murine 

Norovirus Using Vivaspin 20

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Poliovirus Murine

Norovirus

30,000 

MWCO

80.9% 45.6%

100,000

MWCO

55.1% 26.1%
11

Subsample 2

Reverse Transcription – Quantitative PCR

Volume = S

Tertiary Concentration

Nucleic Acid Extraction

0.2 mL

Volume = 0.4 mL

Volume = 100 μL 

RNA 

6.7 μL per replicate

16.5 μL RT Master Mix 1

RT1 RT2 RT3

Step 10.4.9 99°C 99°C 99°C 

16.8 μL RT Master Mix 2

Step 10.4.12 42°C 42°C 42°C 

qPCR qPCR qPCR 

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12

Reverse Transcription

Reverse Transcription 

• Prepare master mixes

• Add 16.5 μL RT Mix 1

• Add 6.7 μL of sample

• Add 6.7 μL PCR-grade 

water as no template 

controls

• Heat at 99 °C for 4

min

• Quench on ice

• Add 16.8 μL RT Mix 2

• Run at 25 °C for 15 

min, 42 °C for 60 

min, 99 °C for 5 min, 

hold of 4 °C 

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Ingredient Volume per reaction (1) Final concentration Volume per Master Mix (2)

RT Master Mix 1

Random primer (Item 7.5.11) 0.8 10 ng/μL 84.0

Hepatitis G Armored RNA (3) (Item 

7.5.12) 1.0 105.0

PCR grade water (Item 7.5.2) 14.7 1543.5

Total 16.5 1732.5

RT Master Mix 2

10X PCR Buffer II (Item 7.5.13) 4.0 10 mM tris, pH 8.3, 50 mM KCL 420.0

25-mM MgCl2 (Item 7.5.13) 4.8 3 mM 504.0

10-mM dNTPs (Item 7.5.14) 3.2 0.8 mM 336.0

100-mM DTT (Item 7.5.15) 4.0 10 mM 420.0

RNase Inhibitor (Item 7.5.10) 0.5 0.5 units/μL 52.5

SuperScript II RT (Item 7.5.16) 0.3 1.6 units/μL 31.5

Total 16.8 1764.0

(1) The volumes given are for 40-μL RT assays.

(2) Reagent amounts sufficient for a 96-well PCR plate are given. The volumes shown were calculated by multiplying the volume per 

reaction amount by the number of assays to be performed, plus an additional 9 assays to account for losses during transfer of the master 

mix to plates (Item 6.6.15) using items 6.6.9 and 6.6.12. The amount of additional assays to add can be reduced if experience shows 

that lower amounts are adequate.

(3) Hepatitis G Armored RNA is supplied as an untitered stock. The amount to use must be determined for each lot, as described in Step 

13.6.1.
13

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Quantitative PCR

Ingredient Volume per reaction 

(μL) (1)

Final concentration Volume per Master Mix 

(μL) (2)

2X LightCycler 480 Probes Master Mix

(Item 7.4.17) (3) 10.0 Proprietary 1050.0

ROX reference dye (Item 7.4.18) (4) 0.4 0.5 mM 42.0

PCR grade water (Item 7.4.2) 1.0 105.0

10 μM EntF 0.6 300 nM 63.0

10 μM EntR 1.8 900 nM 189.0

10 μM EntP 0.2 100 nM 21.0

Total 14.0 1470.0

(1) The volumes given are for using 6 μL of cDNA from Step 12.5.3 in a qPCR assay using a total qPCR volume of 

20 μL.

(2) Reagent amounts sufficient for a 96-well PCR plate are given. The volumes shown were calculated by 

multiplying the volume per reaction amount by the number of assays to be performed, plus an additional 9 assays 

to account for losses during transfer of the master mix to tubes or plates. The amount of additional assays to add 

can be reduced if experience shows that lower amounts are adequate.

(3) 10X PCR Buffer II , 25-mM MgCl2 , and AmpliTaq Gold can be substituted for the LightCycler 480 Probe Master 

Mix. 

(4) This reagent is necessary for use with Applied Biosystems and similar instruments. It should be substituted with 

PCR grade water for use with the LightCycler and similar instruments.
14

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Quantitative PCR

Standard Curves Using Armored RNA® EPA-1615

• All results must be based upon standard curves run in duplicate on 

every RT-qPCR plate

• Standard curves are prepared at concentrations of 2.5 x 108 to 2.5 x 

103, giving 502,500 to 5 genomic copies per standard

• Acceptable standard curves must have a R2 value of > 0.97 and an 

efficiency of 80-110%

• R2 and slope are calculated by the thermocycler

• Efficiency is calculated by the thermocycler or as shown in the 

example below 

• For example, a curve having a slope of -3.29 has a % efficiency of 

100 x (10-1/-3.29

 -1) = 101.3%
15

NorGIA:

y = -3.2032x + 41.422

R2 = 0.9931

Nor GIB:

y = -3.3328x + 41.144

20.00 R2 = 0.9969

25.00

30.00

35.00

40.00

45.00

0 2 4 6

Ct Value

Log Copies/Reaction

NorGIA

NorGIB

Armored RNA® EPA-1615 Norovirus GI Standard Curve

Target

NorGIA

NorGIB

Efficiency 

 

 105.4% 

 99.7% 

Theoretical Detection

 Limit (# Particles)

 3 

 2 

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16

Mean Recovery (%) of Spiked Poliovirus and Murine Norovirus 

from Ground, Surface, and Reagent-Grade Water Samples

Assay 

Groundwater 

 n=7 

Reagent-Grade Water 

(High Titerb) 

n=6 

Reagent-Grade Water 

(Low Titerc

) 

n=6 

Recovery CVa Recovery CVa Recovery CVa

Total Culturable 

Virus Assay

58 79 42 34 122 96

Poliovirus RTqPCR 20 64 48 36 39 29

Murine 

Norovirus RTqPCR 

30 75 0.6 100 8 83

a

CV is % coefficient of variation 

bHigh titer spike at 1000 MPN of poliovirus and 1000 PFU of murine norovirus per 10 L sample c

Low titer spike at 300 MPN of poliovirus and 300 PFU of murine norovirus per 10 L sample 

Reference: Cashdollar et al., 2012

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16
17

Office of Research and Development

National Exposure Research Laboratory, Cincinnati, OH

Issues with EPA 1615

• Filter clogging in turbid waters

• An assay for adenoviruses was not included in the method

• Poor virus recovery from turbid waters

• Turbid waters have RT-qPCR inhibitors
18

Subobjective 1.A.i. Prefilter and electropositive filter options: Enhance 

ability to collect required sample volume when testing turbid waters

Subobjective 1.A.ii. Ultrafiltration versus NanoCeram – Determine the 

relative effectiveness of using ultrafiltration versus the NanoCeram 

electropositive filter for concentration of secondary and tertiary effluent

Subobjective 1.A.iii. Celite versus NanoCeram – Determine the relative 

effectiveness of the NanoCeram versus the small volume method

Subobjective 1.A.iv. Addition of Sea Salts – Determine whether the 

addition of sea salts can enhance recovery of norovirus recovery

Subobjective 1.A.v. Sonification, Dispersants, and Surfactants – Determine 

if sonication or treatment with dispersants and surfactants will improve 

recovery as measured by plaque assay and RT-qPCR

Project P1.5.3A.D.1: Improvements, Field Evaluation, 

and Commercialization of EPA Method 1615

Water Cluster Modifications

Office of Research and Development

National Exposure Research Laboratory, Cincinnati, OH
Subobjective 1.B. Dilutions for Samples with High Virus Levels – quantify 

the loss of precision resulting from the use of 10-fold rather than 5-fold 

dilutions when assaying samples with high virus titer

Subobjective 1.C. Tertiary Concentration - Large Volume Extraction Option 

– Examine if virus recoveries can be improved by replacing the tertiary 

concentration followed by a small volume RNA extraction approach with a 

large volume RNA extraction

Subobjective 1.D. Inhibitor Removal - Proprietary versus EPA Options: 

improve ability to remove RT-qPCR inhibitors from turbid waters

Subobjective 2.A. and 2.B. Add an adenovirus culture and molecular assay 

to 1615

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Water Cluster Modifications
20

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Water Cluster Modifications

Objective 3. Develop a RT-qPCR Commercial Kit and compare it to our 

current approach

Objective 4. Develop an Infectious Virus RT-qPCR assay using PMA

Objective 5. Measurement of Virus Occurrence

• Measure virus in primary, secondary, and tertiary effluents at AAA 

Wastewater Services, Hamilton Water Reclamation Facility (biological with 

chlorine treatment), Sycamore Wastewater Treatment Plant (nutrient removal 

plant), Twin Creek Preserve, and Upper Mill Creek Regional Water 

Reclamation Facility (biological with UV)

• Compare virus occurrence with indicator occurrence
21

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Addition of Adenovirus to Method 1615 

Adenovirus Culture Assay using A549 cells

Process Control % Recovery

 Negative - 

 Positive 1 18

 Positive 2 32

 Positive 3 28

 Positive 4 12

 Positive 5 7 

 Positive 6 3 

 Positive 7 12

 Positive 8 96

 Positive 9 29

Mean Recovery 26

CV 108
22

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Addition of Adenovirus to Method 1615 

AdV PCR

AdV PCR

AdV PCR
23

Office of Research and Development

National Exposure Research Laboratory, Cincinnati, OH

Adenovirus PCR

Prepare Standard Curves for Adenovirus using 

quantified Adenovirus 5 from OD260, Inc.

y = -3.363x + 36.003

R2 = 0.9951

Efficiency = 98.3%

0

5

10

15

20

25

30

35

40

0 1 2 3 4

Standard Curve for Adenovirus
24

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Small Volume Method

Celite Method

1. Add 1.5g of celite to the eluent 

2. Drop the pH to 4 and mix for 10-30 minutes

3. Collect celite on a sterile pre-filter 

4. Elute virus from celite with 40-80 ml of 0.15M sodium phosphate 

5. Adjust pH to 7-7.5. Filter sterilize. Freeze for analysis 

Recoveries range from 60-90% for enteroviruses and 65-69% for adenovirus
25

Office of Research and Development

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Future Development

• As part of the Water Cluster Project

Improve virus detection for turbid waters

Test robustness of EPA Method 1615 for measuring virus 

occurrence at wastewater “End-of-Pipe” settings and in 

recreational settings

• Test whether the small volume method is scalable to 10 L of 

secondary or tertiary effluent
Questions? 

Although this work was reviewed by EPA and approved for publication, it may not necessarily reflect official 

Agency policy. Mention of trade names or commercial products does not constitute endorsement or 

recommendation for use. 
Office of Research and Development

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Photo image area measures 2” H x 6.93” W and can be masked by a 

collage strip of one, two or three images.

The photo image area is located 3.19” from left and 3.81” from top of page. 

Each image used in collage should be reduced or cropped to a maximum of 

2” high, stroked with a 1.5 pt white frame and positioned edge-to-edge with 

accompanying images.

May 15, 2013

Detection Methods for Mycobacteria

Stacy Pfaller

Ohio River and Downtown Cincinnati, OH

Disclaimer: This presentation does not necessarily reflect official U.S. EPA policy
Office of Research and Development

National Exposure Research Laboratory, Microbial and Chemical Exposure and Assessment Research Division 1 

Background 













Waterborne illness caused by nontuberculous mycobacteria (NTM) costs 

the US nearly $500 M in hospitalizations per year (aCollier et al, 2012). 

Pulmonary NTM infections account for almost half of all NTM 

hospitalizations in the US, and are typically caused by Mycobacterium 

avium (MA) and M. intracellulare (MI) 

In addition to pulmonary infections, cause skin, soft tissue, lymph node, 

systemic infections, among others

Primary source of human exposure: WATERa

CCL’s 1 and 2: Mycobacterium avium Complex (MAC)

CCL 3: M. avium

 4 subspecies: M. avium subsp. hominissuis

 

 

 

 

M. avium subsp. avium

M. avium subsp. silvaticum

M. avium subsp. paratuberculosis

 aCollier et al (2012) Epidemiology and Infection, 140: 2003-2013
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Culture and Molecular Methods

Culture Method

 Standard Methods for the Examination of Water and Wastewatera

section 9260M

 Modifications described in Covert et al (1999) Appl Environ Microbiol 

65:2492-2496

Quantitative PCR (qPCR)

 Beumer et al, (2010) Appl Environ Microbiol 76:7367-7370 and Figure 

S1, Supplemental Material http://aem.asm.org/content/76/21/7367/suppl/DC1

 Primers and probe for M. avium and M. intracellulare have been 

submitted for publication

aEaton, A. D., L. S. Clesceri, E. W. Rice, and A. E. Greenberg (ed.). 2005. Standard methods for 

the examination of water and wastewater, 21st ed. American Public Health Association, 

Washington, DC.
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Sample Collection









Sample collection is identical for both culture and qPCR

Bulk Water: collected in 1L sterile polypropylene bottles 

according to sections 9060A and B of Standard Methods 

for the Examination of Water and Wastewatera, except 

NO preservative (Na2S2O3).

Biofilm: collected by swabbing surface of tap, pipe, 

tubing, etc. 

Samples transported back to lab on ice, stored at 4 °C 

until processing, within 48 hours.

 aEaton, A. D., L. S. Clesceri, E. W. Rice, and A. E. Greenberg (ed.). 

 2005. Standard methods for the examination of water and wastewater, 

 21st ed. American Public Health Association, Washington, DC. 
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Culture method













1L water split into 2 x 500 ml aliquots or some 

volume of biofilm slurry (1 – 2 ml typically)

cetyl pyridinium chloride (CPC) is added to a final 

concentration of 0.04% for reduction of 

background organisms

Samples shaken and incubated at room 

temperature for 30 min

Samples filtered through 0.45 um pore-size, 47 

mm black-grid, cellulose ester filter by vacuum 

filtration, washing the filter with sterile deionized 

water.

filter aseptically transferred to Middlebrook 7H10 

agar containing 500 mg L-1 cycloheximide

Plates are incubated a minimum 8 weeks at 37 

°C and inspected weekly for growth
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QC samples for Culture method

Sterile medium negative control

• Performed when medium is made, in advance of samples arriving

• Incubation of un-inoculated medium to ensure sterility

Method blank negative control

• Sterile deionized water filtered processed at the same time in the 

same way as unknowns

No CPC control, if possible

5 
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Benefits of Culture Method







Many NTM can grow on medium

Live only detection

Obtain a culture collection for future characterization

•Genotype

•Virulence genes

Drawbacks of Culture Method 



•



Method has not been characterized for specificity or sensitivity

medium is not selective for mycobacteria

CPC disinfection may reduce recovery of target by 70%

(Personal communication: Terry Covert)





•





Every colony is an unknown in need of identification

Only a subset of colonies can be chosen for identification

method is not quantitative

Months to years before results are obtained

Performs poorly on biofilm
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











8 

I/MAP-specific 

ta Analysis

te Quantification

qPCR Method and/or 

r Biofilm

MA/M assay

Da

Absolu

Bulk wate

Water/biofilm slurry is vacuum filtered through 47.0 mm, 

0.45 um polycarbonate membrane (no CPC treatment 

necessary)

Membrane rolled and placed in 2.0ml tube containing 

0.3g glass beads and buffer

Microorganisms trapped on membrane lysed physically 

by bead beating

DNA from crude lysate extracted using WaterMaster kit 

reagents from (EpiCenter Biotechnologies, Madison, WI)

DNA resuspended in sterile, molecular biology-grade 

water

Three replicate qPCR reactions analyzed/ DNA extract

• Two replicates must be positive for a sample to be 

considered positive
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Absolute Quantification from Master Standard Curves









Generated from six independents series of 10-fold 

serial dilutions of purified genomic DNA from 

ATCC Type strains of MA, MI, and MAP

Each dilution series contains eight standards, 

ranging in concentration from 106 target copies to 

1 copy, run in triplicate = 18 measurements/ 

standard

CT measurements plotted against log target 

number and analyzed by linear regression to 

generate line equation 

Target number in unknown sample estimated from 

line equation

9 
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QC Samples for qPCR



•



•



•



•

Method Blank (negative control)

Sterile molecular biology grade water or sterile swab filtered and 

processed at the same time in the same way as unknowns

Standards (positive control)

Purified genomic DNA from target, serially diluted

No Template Control (negative control)

Sterile molecular biology grade water added to qPCR reaction instead of 

DNA extract

Internal positive Control (IC) 

Commercially available kit (TaqMan Exogenous Internal Positive Control 

Kit, Life Technologies, Carlsbad, CA)

10
Office of Research and Development

National Exposure Research Laboratory, Microbial and Chemical Exposure and Assessment Research Division 11

Characteristics of qPCR assays for drinking water and biofilm

qPCR assay Target 

(copies/

genome)

E¶

Amplification

Efficiency

LOD‡ 

Targets/qPCR

reaction

LOQ† 

Targets/qPCR

reaction

Specificity£ Sensitivity §

Drinking 

Water

Sensitivity§

Biofilm 

M. avium 16S rDNA

(1)

1.92 10 10 100% 100 cells/L 1000 

cells/swab

M. avium subsp.

paratuberculosis

IS900

(14-20)

2.01 1.8 1.8 100% 100 cells/L Not 

determined

Beumer et al, 

2010, Appl

Environ Microbiol

Target 

251

(1)

Not 

determined

Not 

determined

Not 

determined

95% Not 

determined

Not 

determined

M. intracellulare 16S rDNA

(1) 

1.91 10 10 100% 10 cells/L 1000 

cells/swab

¶Amplification Efficiency = 10(1/-slope). If E = 2, the reaction is 100% efficient.

‡LOD = Limit of detection = lowest copy number/assay giving CT < 40 in 6/6 independent assays.

†LOQ = Limit of quantification = lowest copy number/assay yielding a coefficient of variation < 25%.

£Specificity = Number of target testing positive/total number targets tested x 100.

§Sensitivity = lowest copy number detected when spiking serial dilutions of known cell quantities

into actual tap water samples, processed as described in Beumer et al, 2010,

Appl Environ Microbiol, 76:7367-70
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National Exposure Research Laboratory, Microbial and Chemical Exposure and Assessment Research Division 12

Benefits of qPCR Method







Assays are specific for MA and MI (CCL)

No CPC treatment

Time to results = 3 days

Drawbacks of qPCR Method 





Assays are specific for MA , MI, and MAP only

Cannot distinguish between live and dead organisms though studies

 have demonstrated that DNA contained within chlorine disinfected

 cells does not typically persist in water with a chlorine residual

Page et al, 2010, Appl Environ Microbiol, 29:2946-2954

Sen et al, 2010, Current Microbiol, 62:727-732
Office of Research and Development

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National Occurrence Study using Culture







Covert et al (1999) Appl Environ Microbiol, 65:2492-2496

139 samples

– Drinking water (ground and surface water sources)

– Bottled water

– Cistern

– Ice 

– Reservoir

Results

– 1/139 samples positive for MA (drinking water from surface water 

source)

– 5/139 samples positive for MI (drinking water from surface water 

source)

13
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Occurrence Revisited

MJ Study: National Survey of MA and MI Occurrence using 

Culture and qPCR, 2009-2010





40 sites across the US

68 taps (1 to 2 taps/site)

 ground + no treatment = 2

 ground + chlorine = 6

 ground + chloramine = 1

 surface + no treatment = 0

 surface + chlorine = 15

 surface + chloramine = 12

 mixed + chlorine = 2

 mixed + chloramine = 2

Samples collected 3 times in 2009, 

once in 2010

14

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qPCR and Culture Results by Site

15

Number of Sites

N = 40

qPCR Culture

Number of Sites

N = 40

9 19 5 

7 

+ MI Only + MA Only

+ MA & MI

- MA & MI

6 6 8 

19

+ MI Only + MA Only

+ MA & MI

- MA & MI
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qPCR and Culture Results by Tap

16

Number of Taps

N = 68

qPCR Culture

Number of Taps

N = 68

15 23 9 

21

+ MI Only + MA Only

+ MA & MI

- MA & MI

7 5 11

45

+ MI Only + MA Only

+ MA & MI

- MA & MI
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Persistence of MA and MI at taps

17

Number Times Positive Number Times Positive Number Taps Positive

0

10

20

30

40

50

60

MA

MI 

Culture

0 Times 1 Time 2 Times 3 Times 4 Times 0 Times 1 Time 2 Times 3 Times 4 Times

qPCR

Number Taps Positive

0

10

20

30

40

MA

MI
Office of Research and Development

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Agreement Between Culture and qPCR

Target No. Samples Positive

Both Methods

Culture Positive

Only

qPCR Positive 

Only

M. avium 12 16 37

M. intracellulare 11 4 67

18
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PHASE II Study Leads Susan Glassmeyer(USEPA), Ed Furlong (USGS), 

and Dana Kolpin(USGS): Occurrence of Contaminants of Emerging 

Concern at Drinking Water Treatment Plants (DWTPs)

19

EPA Region Number of 

DWTPs

1 1 

2 1 

3 4 

4 5 

5 3 

6 5 

7 1 

8 2 

9 2 

10 1 
Office of Research and Development

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



20

DWTPs positive by qPCR

Twelve DWTPs were 

positive for MA, MI, 

and/or MAP by qPCR 

Estimated concentrations 

of MA, MI, and MAP are 

higher in untreated 

source water than 

finished treated water

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Estimated Targets L-1

1e+0

1e+1

1e+2

1e+3

1e+4

1e+5

1e+6

MA

MI

MAP

UNTREATED

Drinking Water Treatment Plant

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Estimated Targets L-1

1e+0

1e+1

1e+2

1e+3

1e+4

1e+5

MA

MI 

MAP

TREATED
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Description of qPCR Positive Samples

target N untreated untreated N treated treated

geometric mean 

(target copies/L)

maximum

(target copies/L)

geometric mean 

(target copies/L)

maximum

(target copies/L)

M. avium 4 900 5200 2 240 300

M. intracellulare 3 1600 5300 3 40 50

M. avium subsp. 

paratuberculosis 0 3 20 300

21
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Culture: NTM isolates from treated water at DWTPs

No NTM isolated from untreated 

source water samples. 

• All samples lost prior to 8 week 

minimum incubation period for 

MAC 

 

Thirty one isolates obtained from 

treated finished water samples 

from 6 DWTPs. 

• None are MAC though 71% (22/31) 

have been putatively identified as 

other clinically relevant NTM 

species by 16S sequencing

22

6 (27%)

8 (36%)

5 (23%)

M. septicum

M. mucogenicum

M. phocaicum

M. triplex

M. fortuitum

M. lentiflavum

1 (5%)

1 

(5%)

1 (5%)
Office of Research and Development

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Method for Mycobacterium Detection for UCMR?







Both, if possible, as both provide important information

qPCR performs well regardless of sample matrix (water 

or biofilm)

Culture does not perform well on microbiologically 

complex samples (contaminated water and biofilm) but 

does perform well on samples where microbiological 

water quality is good (i.g. treated water before 

distribution)

23
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National Exposure Research Laboratory, Microbial and Chemical Exposure and Assessment Research Division

Acknowledgments 

•

•

•

•

Dawn King (NERL)

•

•

•

•

•

Amy Beumer (NERL)

Eunice Chern (NERL)

Terry Covert (NERL)

Maura Donohue (NERL)

Jatin Mistry (EPA Region 6)

Susan Glassmeyer (NERL)

Ed Furlong (USGS)

Dana Kolpin (USGS) 

24