Need to return call by / at/between and . Signature:

Project No.: 0172.09.005.412 Page 1 of 2

Project Name: 304m 2012 Annual Review CBI?

Yes

No

TELEPHONE CALL RECORD

Incoming

Outgoing

Date: 01/04/2012 Time:

1:00

am

pm

Company Name: U.S. EPA, OW EAD ERG Staff Name: Eleanor Codding, 

Elizabeth Sabol

Contact Name: Maria Gomez-Taylor

Lem Walker

William Swietlik

Contact Phone Number: 605-235-4801

General Subject: New Analytical Methods

TOPICS DISCUSSED AND ACTION TAKEN

Ellie Codding and Elizabeth Sabol, ERG and William Swietlik, EPA contacted Maria Gomez-Taylor and Lem

Walker, EPA (EAD Methods Staff) on January 4, 2012 to discuss the Clean Water Act’s (CWA) Update Rule 

for analytical methods. Ms. Gomez and Mr.Walker stated that the proposal for the methods update rule has been 

reviewed and that the final rule was expected to be published shortly. 

Currently, the Update Rule is updating EPA Methods 624 ad 625 to add additional organic compounds. 

Additionally, Methods 8270 and 8260 were updated to expand the pollutant covered. Ms. Gomez and 

Mr.Walker mentioned that the Office of Ground Water and Drinking Water is currently review biosolids to 

determine the industrial users that are generating these solids. 

Ms. Gomez and Mr.Walker discussed that methods are updated based on potentially water quality criteria 

(WQC) topics, biosolids surveys, fish studies, or identification of pollutants of concern. Currently, the data used 

to support the Update Rule is not publically available because the rule is not final.

Mr.Walker provided additional documentation after the call. One article discussed a draft procedure for the 

determination perfluorinated carboxylic acids and perfluorinated sulfonic acids in sewage sludge and biosolids. 

See the attached e-mails and documentation.
Need to return call by / at/between and . Signature:

Email Communication with Maria Gomez-Taylor and Lem Walker, EPA (EAD Methods Staff)

From: Lemuel Walker <walker.lemuel@epamail.epa.gov>

To: Swietlik, William

CC: Codding, Eleanor; Gomez-Taylor, Maria; Sabol, Elizabeth

Date: 1/4/2012 2:45 PM

Subject PPCP Methods with Analyte List

Hope you received the previous email!

Two EPA developed methods for PPCP with Contaminants of Emerging Concern

in EPA Methods 1694 and 1698

Link provided: http://water.epa.gov/scitech/methods/cwa/ppcp/index.cfm 

---------

Draft procedure for determination of perfluorinated carboxylic acids,

perfluorinated sulfonic acids, methyl and ethyl

perfluroro-octanesulfonamides, and (methyl and ethyl

sulfonamido)-ethanols in sewage sludge and biosolids.

(See attached file:

Draft-Procedure-for-Analysis-of-Perfluorinated-Carboxylic-Acids-and-Sulfonic-Acids-in-Sewage-Sludge-and-Biosolidsby-HPLC-MS-MS.pdf)

----------

The Nonylphenol method is an ASTM method and is subjected to copyright

usage.

Link: http://water.epa.gov/scitech/methods/cwa/other.cfm 

ASTM link:

http://www.astm.org/search/standards-search.html?query=nonyhenol&reskin=true#43781425 

===================================

Lemuel (Lem) Walker, Jr.

Clean Water Act ATP Coordinator

U.S. Environmental Protection Agency

Office of Science and Technology

Engineering and Analysis Division (EAD)

===================================

http://www.epa.gov/waterscience/methods 

Methods Questions? Write to OSTCWAMethods@epa.gov

Attachment: Draft-Procedure-for-Analysis-of-Perfluorinated-Carboxylic-Acids-and-Sulfonic-Acids-in-Sewage-Sludgeand-Biosolids-by-HPLC-MS-MS.pdf
United States Office of Water

Environmental Protection Agency (4303) December 2011

Draft Procedure for Analysis of 

Perfluorinated Carboxylic Acids 

and Sulfonic Acids in Sewage 

Sludge and Biosolids by 

HPLC/MS/MS

December 2011
U.S. Environmental Protection Agency

Office of Water (4303T)

1200 Pennsylvania Avenue, NW

Washington, DC 20460

EPA-821-R-11-007
Draft PFC Procedure i December 2011

Not approved for either general purpose or regulatory use

Introduction and Disclaimer

This document represents a draft procedure for the analysis of perfluorinated carboxylic acids and 

perfluorinated sulfonates in sewage sludge and biosolids, using high performance liquid chromatography 

(HPLC) combined with tandem mass spectrometry (MS/MS).

This draft procedure has been tested in one laboratory under contract to EPA’s Engineering and Analysis 

Division in the Office of Water, but it is incomplete and is undergoing continued development by EPA. 

Further method development work and validation must take place including: MDL studies, IPR studies, 

and generation of QC acceptance criteria. Following those development activities, the draft procedure 

should be validated in multiple laboratories.

However, EPA is publicly releasing this draft procedure because of interest in methods for perfluorinated 

compounds as “emerging contaminants.” 

This procedure does not determine fluorotelomer alcohols and related precursor compounds, which can 

degrade to form perfluorinated carboxylic acids and sulfonic acids, and thus may contribute to the overall 

concentrations of perfluorinated compounds in sewage sludge and biosolids.

This procedure should be restricted to use by analysts who are experienced in HPLC-MS/MS. 

Mention of trade names or commercial products does not constitute endorsement or recommendation for 

use. 

Acknowledgements

This draft procedure was written by staff in EPA’s Engineering and Analysis Division in the Office of 

Water, based on extraction, cleanup, and instrumental procedures developed at U.S. EPA’s National 

Exposure Research Laboratory in Athens, GA (References 1 and 2) and additional supplementary 

procedures from EPA Method 537 (Reference 3), developed by the National Exposure Research 

Laboratory in Cincinnati, OH.

Initial single-laboratory testing of the procedure was performed by AXYS Analytical Services Ltd., 

Sydney, British Columbia, Canada, under contract to EPA. Following that testing, a version of the 

procedure was subjected to a limited internal Agency peer review by two researchers from EPA’s

National Risk Management Research Laboratory in Cincinnati, and one researcher from EPA’s National 

Environmental Research Laboratory in Athens, GA.

EPA gratefully acknowledges the contributions of all of these organizations and individuals.

Contact Information

Questions concerning this draft procedure or its application should be addressed to:

Engineering and Analytical Support Branch

Engineering and Analysis Division (4303T) 

Office of Science and Technology, Office of Water

U.S. Environmental Protection Agency

1200 Pennsylvania Avenue NW

Washington, DC 20460

http://www.epa.gov/waterscience

ostcwamethods@epa.gov
Draft PFC Procedure ii December 2011

Not approved for either general purpose or regulatory use

Table of Contents

1.0 Scope and Application .....................................................................................................................1

2.0 Summary of Procedure ....................................................................................................................2

3.0 Definitions and Units of Measure ....................................................................................................2

4.0 Interferences and Contamination .....................................................................................................2

5.0 Safety ...............................................................................................................................................4

6.0 Equipment and Supplies ..................................................................................................................5

7.0 Reagents...........................................................................................................................................8

8.0 Standards.........................................................................................................................................9

9.0 Sample Collection, Preservation, Storage, and Holding Times.....................................................12

10.0 Quality Control (QC).....................................................................................................................12

11.0 Preparation, Extraction, and Cleanup of Field Samples and QC Samples.....................................15

12.0 LC/MS/MS Set Up and Calibration...............................................................................................19

13.0 Instrument Quality Control............................................................................................................21

14.0 Instrumental Analysis....................................................................................................................24

15.0 Qualitative Identification and Quantitation....................................................................................25

16.0 Method Performance......................................................................................................................28

17.0 Pollution Prevention and Waste Management...............................................................................28

18.0 References......................................................................................................................................28

19.0 Glossary .........................................................................................................................................29

20.0 Tables and Figures.........................................................................................................................32

Appendix 1..................................................................................................................................................46

Appendix 2..................................................................................................................................................50
Draft PFC Procedure 1 December 2011

Not approved for either general purpose or regulatory use

Draft Procedure for Analysis of Perfluorinated Carboxylic Acids and 

Sulfonic Acids in Sewage Sludge and Biosolids by HPLC/MS/MS

December 2011

1.0 Scope and Application

1.1 This draft procedure is intended for use by EPA in the development of standardized methods for 

the determination of perfluorinated carboxylic acids, perfluorinated sulfonic acids, methyl and 

ethyl perfluroro-octanesulfonamides, and (methyl and ethyl sulfonamido)-ethanols in sewage 

sludge and biosolids. The procedure employs high performance liquid chromatography (HPLC) 

combined with tandem mass spectrometry (MS/MS). The list of target analytes is presented in 

Table 1.

1.2 This procedure may be applied to various solids from sewage treatment plant operations,

including sewage sludge and biosolids. Sewage sludge is the solid, semisolid, or liquid organic 

material that results from the treatment of domestic wastewater by municipal wastewater 

treatment plants. Biosolids are defined in EPA regulations at 40 CFR Part 503 as sewage sludge 

that has had additional processing for land application. However, EPA often uses the terms 

sewage sludge and biosolids interchangeably, and may do so in this procedure as well.

1.3 This draft procedure is based on extraction, cleanup, and instrumental procedures developed at 

U.S. EPA’s National Exposure Research Laboratory in Athens, GA (References 1 and 2) and 

additional supplementary procedures from EPA Method 537 (Reference 3). The quality control 

protocols found in this draft procedure are based on existing EPA HPLC/MS/MS methods 

(Reference 4).

1.4 Early work on this procedure focused on analysis of the solid portion of sewage sludge samples 

and described discarding any supernatant aqueous liquid in the sample. Subsequent efforts 

suggest that the extraction procedures are capable of dealing with samples containing large 

amounts of water, which may better represent actual sewage sludges and biosolids from 

wastewater treatment operations. These capabilities will be the subject of further testing and 

refinement of this draft procedure by the EPA Office of Water’s Engineering and Analysis 

Division.

1.5 This draft procedure does not determine fluorotelomer alcohols and related precursor compounds,

which can degrade to form perfluorinated carboxylic acids and sulfonic acids, and thus may 

contribute to the overall concentrations of perfluorinated compounds in sewage sludge and 

biosolids.

1.6 This draft procedure should be restricted to use by analysts who are experienced in HPLC 

MS/MS.

1.7 The draft procedure has not been published in 40 CFR Part 136 and is not approved for either 

general purpose or regulatory use. This draft procedure has not been validated by EPA and is 

being used strictly for method development purposes. It is being publicly released because of 

interest in methods for perfluorinated compounds as “emerging contaminants.”
Draft PFC Procedure 2 December 2011

Not approved for either general purpose or regulatory use

2.0 Summary of Procedure

The general steps in this procedure are summarized in Secs. 2.1 to 2.3. A flow chart that 

summarizes the procedures for sample preparation, cleanup, and analysis is shown in Figure 1 at 

the end of the document.

2.1 Sample collection and digestion – Collect a sample of sewage sludge or biosolids sufficient to 

yield at least 0.5 g of wet solids. (Larger samples are recommended to ensure that they are more 

representative of the bulk source of the material.) Homogenize the sample and transfer a

subsample containing 0.5 g of wet solids to a centrifuge tube, spike the sample with the labeled 

compound spiking solution, and digest the sample for 30 min with 1M NaOH by heating and 

ultrasonic agitation, followed by overnight incubation. Samples are neutralized with HCl and 

extracted with solvent.

2.2 Solvent extraction and cleanup – Extract the digested sample twice by shaking and ultrasonic 

agitation, using 10 mL 50:50/ACN:MeOH (v/v). Dilute the sample extract, acidify it to pH 6.5, 

agitate it ultrasonically, and clean up the extract using a weak anion exchange (WAX) solid-phase 

extraction (SPE) cartridge. 

2.3 Analysis – Reconstitute the sample extract with 1 mL 0.3% NH4OH in methanol containing the 

labeled injection internal standards. Analyze a 15-μL aliquot on a dedicated HPLC/MS/MS 

equipped with a trapping column (if needed), using negative electrospray ionization (ESI-) mode. 

Sample concentrations are calculated using either isotope dilution quantitation for those analytes 

with exact labeled analogs in the labeled compounds spiking solution, or internal standard 

quantitation for those analytes without an exact labeled analog. The recoveries of the labeled 

analogs themselves are determined by internal standard quantitation and used as a quality control 

check on the overall analytical process.

3.0 Definitions and Units of Measure

Definitions of terms, acronyms, abbreviations, and units of measure are given in the glossary in 

Sec. 19 of this document.

4.0 Interferences and Contamination

4.1 Background levels of perfluorinated chemicals must be controlled during this analysis. To 

determine if background concentrations of perfluorinated compounds (PFCs) are under control, 

analysts must be able to see a significant difference between a blank and a low-level standard. 

This test is discussed in Sec. 12.5. If this cannot be achieved, it may be necessary to proof 

reagents and equipment to find the source of contamination or to modify the tubing on the 

LC/MS/MS system. Modifications to LC/MS/MS systems are discussed in Sec. 6.4.5.

4.2 Containers –Aqueous solutions of PFC compounds should be stable for at least 28 days when 

stored in glass or high density polyethylene (HDPE) containers. Storage of aqueous solutions in 

polypropylene containers has resulted in significant loss of certain perfluorocarboxylic acids (C11

and C12) after 7 days, although some researchers have suggested that stability of the 

perfluorocarboxylic acids may be improved through the inclusion of a substantial fraction of an 

organic solvent. PFC solutions in basic methanol have not been observed to degrade when in 

contact with any of these materials for 6 months. 
Draft PFC Procedure 3 December 2011

Not approved for either general purpose or regulatory use

4.3 All solvents and reagent water used in the analysis must be demonstrated to be free of PFC 

contamination and other interferences. A sample (equivalent in volume to the amount used in the 

procedure) from each lot number of solvent or water should be proofed before use. 

Concentrations of any detected target compounds are compared to the method detection limits 

(MDLs) and minimum levels (MLs) to determine whether they are acceptable for use. PFC-free 

reagent water is available (see Sec. 7.1.4), but a procedure for “polishing” deionized water is 

provided in Appendix 2. 

4.4 All materials used in the analysis, and the entire analytical process, must be demonstrated to be 

free of PFCs and other interferences by running reference matrix method blanks (Sec. 10.7) 

initially, and with each sample batch (samples started through the extraction process on a given 

12-hour shift, to a maximum of 20 samples).

4.5 False positives, false negatives, and co-extracted interferences – Interferences co-extracted from 

samples will vary considerably from source to source. Taurodeoxycholic Acid (TDCA) and some 

of its isomers, including tauroursodeoxycholic acid and taurochendeoxycholic acid, are known 

interferences which may overestimate or yield a false positive result for perfluoro-octanesulfonic 

acid (PFOS) (Ref. 5), while 5-pregnan-3,20-diol-3-sulfate and 34S-3-hydroxy-5-pregnan-20-one 

sulfate may interfere with perfluorohexanesulfonic acid (PFHxS) (Ref. 6). Protocols for ensuring 

chromatographic separation of PFOS from TDCA and for detecting interferences by monitoring 

secondary multiple reaction monitoring (MRM) transitions for both PFOS and PFHxS are 

discussed in Sec. 13.5.

4.6 Matrix Effects – Matrix effects can occur during LC/MS/MS analysis when the sample extract 

contains material that co-elutes with the target analytes causing enhancement or suppression of 

their MS/MS response, potentially impacting the reliability of data obtained for samples with 

complex matrices. This type of matrix effect manifests itself as either high or low labeled 

compound recovery (indicating that some of the labeled compounds or injection internal 

standards are either being enhanced or suppressed). 

If the labeled standard recoveries do not meet the acceptance criteria in Table 8, then the 

laboratory should determine whether a matrix effect is the cause. One diagnostic test is to dilute 

the sample extract and reanalyze it. Dilution decreases the amount of matrix entering the MS/MS 

system, diminishing its effect, and therefore dilution should produce a change in labeled standard 

recoveries as the effect of matrix is diminished. The “method of standard additions” also can be 

used to diagnose matrix effects, but because it introduces even more material into the ionization

chamber, it is unlikely to be useful in resolving those effects in specific samples.

When performing isotope dilution quantification, the labeled compound and its exact analogue 

enter the MS/MS system at the same time and are therefore similarly enhanced or suppressed. 

Therefore, the quantification of target compounds is not affected, even if the labeled standard 

recovery is outside of the specified range. For those target compounds whose concentration is 

determined by internal standard quantification (vs. isotope dilution), the matrix enhancement or 

suppression may have a different effect on the target compounds compared to the internal 

standard used for quantification and therefore, there can be an effect on quantified results. 

There are three possible remedies for this situation: the affected target compounds could be 

reported from the diluted analysis, the sample extract could be subjected to further cleanup 

(repeat the SPE cleanup on the finished extract), or the analysis could be repeated using a smaller 

sample size to reduce the matrix effects.
Draft PFC Procedure 4 December 2011

Not approved for either general purpose or regulatory use

5.0 Safety

5.1 The toxicity or carcinogenicity of each chemical used in this procedure has not been precisely 

determined; however, each compound should be treated as a potential health hazard. Pure 

standards of the compounds should be handled only by highly trained personnel thoroughly 

familiar with handling and cautionary procedures and the associated risks. It is recommended 

that the laboratory purchase dilute standard solutions of the analytes in this method. However, if 

primary solutions are prepared, they should be prepared in a hood, and a NIOSH/MESA approved 

toxic gas respirator may be necessary when high concentrations are handled.

5.2 This procedure does not address all safety issues associated with its use. The laboratory is 

responsible for maintaining a current awareness file of OSHA regulations regarding the safe 

handling of the chemicals specified in this procedure. A reference file of material safety data 

sheets (MSDSs) should also be made available to all personnel involved in these analyses. It is 

also suggested that the laboratory perform personal hygiene monitoring of each analyst who uses 

this procedure and that the results of this monitoring be made available to the analyst. Additional 

information on laboratory safety can be found in Refs. 7 to 9.

5.3 Spiking solutions or samples suspected to contain high concentrations of these compounds should 

be handled with care.

5.3.1 Facility – When finely divided samples (dusts, soils, dry chemicals) are handled, all 

operations (including removal of samples from sample containers, weighing, transferring, 

and mixing) should be performed in a glove box demonstrated to be leak tight or in a 

fume hood demonstrated to have adequate air flow. Gross losses to the laboratory 

ventilation system must not be allowed. Handling of the dilute solutions normally used 

in analytical and animal work presents no inhalation hazards except in the case of an 

accident.

5.3.2 Protective equipment – Disposable plastic gloves (Latex or non-Latex [such as nitrile]), 

apron or lab coat, safety glasses or mask, and a glove box or fume hood should be used. 

During analytical operations that may give rise to aerosols or dusts, personnel should 

wear respirators equipped with activated carbon filters. Eye protection (preferably full 

face shields) should be worn while working with exposed samples or pure analytical 

standards. Latex or non-Latex (such as nitrile) gloves are commonly used to reduce 

exposure of the hands. 

5.3.3 Training – Workers must be trained in the proper method of removing contaminated 

gloves and clothing without contacting the exterior surfaces.

5.3.4 Personal hygiene – Hands and forearms should be washed thoroughly after each 

operation involving high concentrations of the analytes of interest, and before breaks 

(coffee, lunch, and shift).

5.3.5 Confinement – Isolated work areas posted with signs, segregated glassware and tools, and 

plastic absorbent paper on bench tops will aid in confining contamination.

5.3.6 Waste handling – Good technique includes minimizing contaminated waste. Plastic bag

liners should be used in waste cans. Janitors and other personnel should be trained in the 

safe handling of waste. See Sec. 17 for additional information on waste handling and 

disposal.
Draft PFC Procedure 5 December 2011

Not approved for either general purpose or regulatory use

5.4 Sewage sludge and biosolids samples may contain high concentrations of biohazards, and must be 

handled with gloves and opened in a hood or biological safety cabinet to prevent exposure. 

Laboratory staff should know and observe the safety procedures required in a microbiology 

laboratory that handles pathogenic organisms when handling such samples.

6.0 Equipment and Supplies 

Note: Brand names, suppliers, and part numbers are cited for illustration purposes only. No 

endorsement is implied. Equivalent performance may be achieved using equipment and 

materials other than those specified here. Demonstration of equivalent performance that 

meets the requirements of this procedure is the responsibility of the laboratory.

6.1 Disposable lab equipment

6.1.1 Sample bottles and caps 

6.1.1.1 Sample collection bottle – High density polyethylene (HDPE) sample bottles 

with polypropylene lids, Nalgene, Part No.: 69032, Lima, Ohio, USA, or 

equivalent. 

6.1.1.2 Extract dilution bottle – HDPE bottles 125-mL Nalgene, Part No.: 332189-

0004, Lima, Ohio, USA, or equivalent. 

6.1.2 Centrifuge tubes

6.1.2.1 Polypropylene centrifuge tubes (15 mL) with polypropylene screw caps,

Corning®, Cat. No: 430766, Corning, NY, USA, or equivalent.

6.1.2.2 Glass centrifuge tubes (15 mL), Kimble Chase, Part No.: 7379015, Vineland, 

NJ, USA, or equivalent. 

6.1.3 Autosampler vials – Polypropylene 0.3-mL autosampler crimp-top vials (Canadian Life 

Sciences, Peterborough, ON, Canada, Cat. No.: 30300P-1232), or equivalent, with 

polypropylene snap caps (Canadian Life Sciences, Peterborough, ON, Canada. Cat. No.: 

30300P-1232), or equivalent. Polypropylene vials and caps are required, but do not 

adequately reseal after use. Therefore, due to potential evaporation, multiple injections 

from the same vial are not recommended.

6.1.4 Syringes – Polypropylene 5-mL NORM-JECT syringes with Luer-slip fitting (Fisher 

Scientific Cat. No. 1481728), or equivalent. 

6.1.5 Disposable pipets – Glass or polypropylene disposable pipets (Fisher Cat. No. 13-711-17)

or equivalent. 

6.1.6 Miscellaneous labware – Glass or polypropylene, as required.

6.1.7 pH paper – Whatman® Panpeha® pH indicator strips pH range 0 to 14 (Sigma-Aldrich, 

Cat. No.: Z134147), or equivalent. 

6.1.8 Syringe filters – Acrodisc 0.45-μm, 25-mm filters with nylon membranes (Ann Arbor, 

MI, USA, Part No.: 4614), or equivalent.
Draft PFC Procedure 6 December 2011

Not approved for either general purpose or regulatory use

6.2 Non-disposable lab equipment

6.2.1 Auto-pipettes – Gilson (Middleton, WI, USA) Microman® positive displacement pipettes,

volume 10-, 25-, 100-, and 1000-μL with disposable tips, or equivalent. 

6.2.2 Balances – An analytical balance capable of weighing 0.1 mg is used for sample weight 

measurements. An analytical balance capable of weighing 10 mg is used for measuring 

standards. 

6.2.3 Equipment for concentration of extracts – Extracts should be concentrated by evaporation 

with nitrogen using a water bath set at 40 oC (Meyer N-Evap, Model 111, Organomation 

Associates, Inc.) or equivalent.

6.2.4 Ultrasonic bath – Branson Model 5510 ultrasonic bath (Danbury, CT, USA) or 

equivalent. 

6.2.5 Shaker – Barnstead LabQuake Tube Shaker/Rotators (Waltham, MA, USA, Cat. No.: 

415110), or equivalent. 

6.2.6 Bench top Centrifuge – Thermo Scientific, Sorvall Legend RT+ (Waltham, MA, USA) or 

equivalent. 

6.2.7 Vortex mixer – Barnstead (Dubuque, IA, USA Thermolyne Model No: M16715) or 

equivalent. 

6.3 Solid-phase extraction 

6.3.1 Solid-phase extraction cartridge – Oasis weak anion exchange (WAX) extraction 

cartridge, 6-cc barrel size, 150-mg sorbent weight, 30-μm particle size (Waters Milford, 

MA, USA), or equivalent.

6.3.2 SPE extraction kit – Vacuum extraction manifold (Alltech/Grace Davison, Deerfield, IL, 

USA, Part No:. 210016) or equivalent, using 60-mL polypropylene reservoirs (Supelco/

Sigma Aldrich, Part No. 57022) or equivalent, with reservoir adapters (Supelco/Sigma 

Aldrich, Part No. 57020-U) or equivalent, and polypropylene needles (Alltech/Grace 

Davison, Deerfield, IL, USA, Part No. 210916) or equivalent. Either a manual or 

automatic vacuum manifold may be used for SPE extractions. 

Caution: Automated systems may contain parts made of polytetrafluoroethylene (PTFE). 

Before use, the system should be proofed to ensure it is free from 

contamination.

6.3.3 Laboratory vacuum system or aspirator vacuum system – Capable of maintaining 23 in. 

Hg, equipped with shutoff valve and vacuum gauge. 

6.4 LC/MS/MS system – The analytical instrumentation used should meet the following 

requirements:

6.4.1 HPLC system – The HPLC/MS/MS system used must have a high-pressure inlet, must 

have a post-column pump for admission of calibrant during mass spectrometry 

calibration and optimization, must be capable of multi-segment gradient separation, 

producing the separations for the analytical runs detailed in Table 2 under the instrument 
Draft PFC Procedure 7 December 2011

Not approved for either general purpose or regulatory use

conditions detailed in Table 5, and meeting other HPLC requirements in Sec. 12. This 

system must also be equipped with a 50-μL loop capable of using ‘partial loop with 

needle overfill’ mode.

6.4.2 Columns – Injections are introduced into a 50-μL loop using ‘partial loop with needle 

overfill’ mode connected to a C18 guard cartridge, followed by an analytical column (PN 

186000404, Waters Xterra MS C18 3.5 μm, 2.1 x 100 mm column) or equivalent. 

Alternative columns may be used as long as they provide comparable chromatography to 

that described in Table 2.

6.4.3 MS/MS system – The MS/MS system must be capable of negative electrospray ionization 

(ESI-) under the conditions in Table 5, producing unique product ions for analytes within 

specified retention time segments. A minimum of 10 scans across the chromatographic 

peak of the lowest concentration standard is required to ensure adequate precision 

(Waters Quattro Ultima tandem mass spectrometer) or equivalent. The system should be 

able to resolve native and labeled compounds with a mass difference of two.

6.4.4 Data system and other software – All system operations should be controlled by 

appropriate system software (Waters MassLynx 4.1 and QuanLynx 4.1), or equivalent. 

The software should be interfaced to the HPLC/MS/MS to control the LC gradient and 

other LC and MS/MS operating conditions, and to acquire, store, reduce and output 

HPLC/MS/MS data. The software must be able to identify a compound by retention time 

and precursor-product m/z, allow integration of an ion abundance of any specific ion 

within specified time or scan number limits, be able to quantify the compound using 

relative responses and response factors, or linear or quadratic multi-point weighted 

regressions, by isotope dilution and internal standard quantitation techniques. 

6.4.5 Elimination of PFC background – Background levels of perfluorinated chemicals must be 

controlled to ensure that they do not interfere with this analysis. To determine if 

background levels of PFCs interfere with this analysis, the background test in Sec. 12.5

must be conducted. 

If instrument background levels of PFCs are found (see diagnostic test in Sec. 12.5), they 

must be eliminated before analyses proceed. The following instrumentation 

modifications may be required: 

• substitution of tubing with PEEKTM tubing, 

• replacement of PTFE solvent frits with stainless steel frits, 

• inserting a trap column (PN WAT200650, Symmetry C18, 3.5 μm, 2.1 x 50 mm 

column) or equivalent, as part of the solvent manager at the most down-gradient 

point in the water-eluent line immediately before the solvent mixing cell, and

• injection of sufficient blanks to cleanse the system (3 to 5 blanks may be required).

Additionally, to minimize buildup of PFCs during mobile phase equilibration and keep 

background level constant, the time the system sits at initial conditions should be kept 

constant and as short as possible, but should also assure reproducible retention times in 

continuing calibration verifications. Prior to daily use, flush the LC column with elution 

solvents before initiating a sequence. It may also be necessary to flush other LC components 

such as syringes and other system components. 
Draft PFC Procedure 8 December 2011

Not approved for either general purpose or regulatory use

7.0 Reagents

7.1 Solvents - Each lot of solvent must be demonstrated to be free from contamination on a routine 

basis. 

7.1.1 Trace-grade methanol (BDH, VWR International, West Chester, PA, USA) or equivalent. 

7.1.2 HPLC-grade acetonitrile (VeX Chem, Aurora, ON, Canada) or equivalent. 

7.1.3 Plasma-grade reagent water (Fisher Chemicals) or equivalent, for use in the preparation 

of standards and samples.

7.1.4 HPLC-grade water (Fisher Chemicals) or equivalent, for use as the LC mobile phase.

7.2 Gases

7.2.1 Argon, used as the collision gas. Ultra high purity (Alphagaz 1, Air Liquide Canada, or 

Airgas, Radnor, PA) or equivalent. Argon gas used should meet or exceed instrument 

manufacturer’s specifications. Nitrogen may be used as the collision gas, provided that 

sufficient sensitivity can be achieved. 

7.2.2 Nitrogen, ultra high purity or from a nitrogen generator. Nitrogen may be used as a 

carrier gas and as a nebulizer gas in aerosol generation in ESI liquid spray (Alphagaz 1, 

Air Liquide Canada, or Airgas, Radnor, PA) or equivalent. Nitrogen also is used to 

concentrate sample extracts (Ultra High Purity) or equivalent. Nitrogen gas used should 

meet or exceed instrument manufacturer’s specifications.

7.3 Other reagents

7.3.1 Certified ACS grade sodium hydroxide (Fisher Chemical) or equivalent.

7.3.2 Ultra pure hydrochloric acid (Seastar Chemicals Inc., Sidney, BC, Canada) or equivalent. 

7.3.3 Glacial acetic acid, HPLC grade (Fisher Chemical, Fair Lawn, NJ, USA) or equivalent.

7.3.4 Ammonium hydroxide – (Fisher Chemicals, certified ACS+ grade, 30% in water, Fair 

Lawn, NJ, USA) is used as received.

7.3.5 Formic acid – (Alfa Aesar, greater than 96% purity, Ward Mill, MA, USA,) is used as 

received. 

7.3.6 Ammonium acetate, purity >98%. (Sigma Chemicals) or equivalent.

7.3.7 Reference matrices – Reference matrices must be free from contamination. It may be 

difficult to obtain a sewage sludge or biosolids sample that does not have detectable 

concentrations of one or more of the native PFCs covered by this procedure. Therefore, 

organic-component-rich, commercially available top soil may be used as a reference 

matrix if testing demonstrates that it does not contain the native PFCs. Other reference 

matrices will be evaluated. 
Draft PFC Procedure 9 December 2011

Not approved for either general purpose or regulatory use

7.4 Preparation of reagents 

7.4.1 Pretreatment reagent (1 M NaOH) – prepared by adding 40 g of NaOH to 1 L of reagent 

water.

7.4.2 Neutralization reagent (1M HCl) – prepared by dilution of 83.5 mL of concentrated 

hydrochloric acid (HCl) to 1 L with plasma-grade reagent water.

7.4.3 Extraction solvent (50:50/ACN:MeOH [v/v]) – prepared by mixing 500 mL of methanol 

(MeOH) and 500 mL of Acetonitrile. 

7.4.4 Acetic acid (3%, v/v) – prepared by dissolving 30 mL of glacial acetic acid in 1 L of 

reagent water.

7.4.5 SPE reagents

7.4.5.1 Basic methanol (0.3% NH4OH v/v in methanol) – prepared by mixing 30 mL

of ammonium hydroxide with 1 L of methanol.

7.4.5.2 Formic acid, 0.1 M – prepared by dissolving 4.8 g formic acid (96%) in 1 L of 

reagent water.

7.4.5.3 Methanol (20%): formic acid 0.1M, (80%) – prepared by mixing 200 mL of 

methanol with 800 mL of 0.1 M formic acid in reagent water. 

7.4.5.4 Aqueous ammonium hydroxide (0.3% v/v) – prepared by adding 1 mL, of 30% 

ammonium hydroxide to 99 mL of reagent water.

7.5 Preparation of LC mobile phases and wash solutions

7.5.1 Aqueous mobile phase – 12.1 mM ammonium acetate in 0.1% acetic acid (aqueous) is 

prepared by adding 4 g of ammonium acetate and 4 mL of acetic acid to 4 L of HPLCgrade water.

7.5.2 Organic mobile phase – 90% acetonitrile/10% HPLC water is prepared by adding 400 

mL of HPLC-grade water to 3600 mL of acetonitrile.

7.5.3. Seal wash solution – 10% acetonitrile/90% HPLC water is prepared by adding 400 mL of 

acetonitrile to 3600 mL HPLC-grade water.

7.5.4 Needle wash solution – methanol is used as received.

7.5.5 Purge solvent – HPLC-grade water is used as received, or aqueous mobile phase may be

used. 

8.0 Standards

8.1 Sources of standards

The standards used and suggested suppliers are listed in Table 9. Standards are used as received. 

If the stated chemical purity is 98 % or greater, the weight may be used without correction to 
Draft PFC Procedure 10 December 2011

Not approved for either general purpose or regulatory use

calculate the concentration of the standard. All solution concentrations and calculated results are 

reported in terms of the acid form. Where the obtained standards are salts of the analyte 

compounds, a salt-to-parent conversion factor must be applied to the concentration.

8.2 Validation

Before preparation of mixed standards, each individual standard is validated by analysis to 

confirm its identity and the absence of impurities. A combined working level solution containing 

native (unlabeled) standards, labeled standards, and labeled injection standards was prepared and 

analyzed to demonstrate accurate quantification against the calibration standards. A combined 

solution of labeled standards and labeled injection standards was prepared and analyzed to 

demonstrate cleanliness of these solutions. 

8.3 Stock solutions

Native (unlabeled) perfluoroheptanesulfonic acid (PFHpS) and all individual labeled compounds,

except perfluoro-n-[1,2-

13C9]undecanoic acid, were purchased as solutions in methanol. 

Individual native alky 1-octanesulfonamide and (1-octanesulfonamido)-ethanol compounds (NMe/Et-FOSA/Es) were purchased as solutions in 90% nonane/10% toluene. Individual natives 

for all compounds except PFHpS and perfluoro-n-[1,2-

13C9]undecanoic acid were prepared from 

solids. 

Individual standards stock solutions should be prepared in methanol, except perfluorotetradecanoic acid (PFTeDA), which is prepared in acetonitrile (ACN). See Appendix 1 for details on 

standards preparation. 

Mixed standards stocks and working level solutions should be prepared in 60:40 ACN:H2O or 

basic methanol in glass, Class A volumetric flasks. 60:40 ACN:H2O should be prepared from 

Optima grade ACN and PFC-free 18-MΩ water.

Mixed standards stocks and working level solutions for N-Me/Et-FOSA/Es should be prepared in 

methanol.

Prepare the following mixed stock standards. See Appendix 1 for preparation details.

8.3.1 Native stock mix (containing all native perfluorocarboxylic acids, perfluorosulfonic 

acids, and PFOSA)

8.3.2 Stock labeled standard mix

8.3.3 Stock labeled injection standard

8.4 Working-level native standards

8.4.1 Working-level native (unlabeled; authentic) compound spiking solution – This solution is 

spiked into calibration standards, IPR, OPR, and matrix spike samples. Prepare target 

analyte native compounds at the concentrations shown in Table 7 (and detailed in 

Appendix 1) in basic methanol. 40 μL of the native compound spiking solution is added 

to each OPR, IPR or matrix spike sample before digestion. See Sec. 11.3.5.
Draft PFC Procedure 11 December 2011

Not approved for either general purpose or regulatory use

8.4.2 Low-level native standard – This solution is spiked into calibration standards, IPR, OPR, 

MDL, and matrix spike samples. Prepare target analytes at the concentrations shown in 

Table 7 (and detailed in Appendix 1) in basic methanol.

8.4.3 Working-level native standard for N-Me/Et-FOSA/Es – This solution is spiked into 

calibration standards, IPR, OPR, MDL, and matrix spike samples. Prepare target analyte 

native compounds at the concentrations shown in Table 7 (and detailed in Appendix 1) in 

methanol containing 10% propan-2-ol. A 16-μL aliquot of this solution is added to each 

OPR, IPR, or matrix spike sample before digestion. See Sec. 11.3.5.

Note: Larger volumes of the solutions in Secs. 8.4.1 to 8.4.3 may be used, provided 

that the concentrations are adjusted accordingly.

8.5 Labeled internal standards 

Labeled internal standard solution (sometimes called the labeled compound spiking solution) –

This solution is spiked directly into samples prior to extraction. Labeled compounds are used to 

quantify unlabeled target compounds and perform recovery correction. Prepare the labeled 

compounds at the concentrations shown in Table 7 (and detailed in Appendix 1) in basic 

methanol. A 100-μL aliquot of this solution is added to each sample before extraction. See Sec.

11.3.6.

8.6 Labeled injection standards

Labeled injection standard solution – this solution contains the labeled compounds that are used 

to quantify all of the other labeled compounds in the labeled internal standard solution (Sec. 8.5),

and is added to the final sample extract prior to instrumental analysis. Prepare the labeled 

compounds used as injection standards at the concentrations shown in Table 7 (and detailed in 

Appendix 1) in basic methanol. A 12.5-μL aliquot of this solution is added to each extract in 

preparation for LC/MS/MS analysis. See Sec. 11.6.3.

Note: A larger volume of this solution may be used, provided that the concentration is adjusted 

accordingly.

8.7 Calibration standards 

Combine and dilute the solutions in Secs. 8.4, 8.5, and 8.6 in basic methanol to produce the 

calibration solutions at the levels shown in Table 6 or, if available, purchase prepared standards 

for calibration solutions. These solutions are used for initial calibration of the analytical system 

(Sec. 13.1). The CS-4 standard is used for ongoing calibration verification (Sec. 13.3). 

8.8 Storage

Standards are stored in glass in the dark at 4 °C. Longer-term storage stability is to be 

determined, but stability for 6 months has been observed. Place a mark on the vial or ampule at 

the level of the solution so that solvent loss by evaporation can be detected. Alternatively, weigh 

the vial or ampule before storage, record the mass, and reweigh the vial or ampule before the next 

use.
Draft PFC Procedure 12 December 2011

Not approved for either general purpose or regulatory use

8.9 Stability

Shelf life of purchased solutions in methanol is determined by the supplier. Stability of diluted 

solutions and working solutions remains to be determined.

Perfluorocarboxylic acid standards in methanol solution may undergo esterification to the methyl 

esters. Most purchased perfluorocarboxylic acid standard solutions were received in methanol 

containing 4 mole equivalents of NaOH. Basic methanol (Sec. 7.4.5.1), rather than straight 

methanol, is used for all standard dilutions to avoid this potential problem.

9.0 Sample Collection, Preservation, Storage, and Holding Times 

9.1 Sample collection – Collect samples in amber high density polyethylene (HDPE) containers with 

propylene caps/lids, following conventional sampling practices designed to obtain a sample that is 

representative of the material of interest. Lids and other materials containing PTFE must be 

avoided, due to possible leaching of fluorinated materials. 

9.2 Collect a sample of sewage sludge or biosolids sufficient to yield at least 0.5 g of wet solids for 

analysis, plus enough sample to allow the determination of % solids determination (Sec. 11.2) 

and to provide volume for QC samples (Sec. 10.5). Larger samples are recommended to ensure 

that they are more representative of the bulk source of the material. 

9.3 Holding times – EPA has not yet conducted a formal holding time study and will conduct one after

the procedure is finalized. Until that time, default holding times that begin at the time of sample 

collection are as follows: 

9.3.1 Begin sample extraction within 60 days of collection (to be validated).

9.3.2 Analyze extracts within 30 days of extraction (to be validated).

9.3.3 Store all samples and extracts at less than 4 oC in HDPE containers

10.0 Quality Control (QC) 

10.1 Each laboratory that uses this draft procedure is required to operate a formal quality assurance 

program. The minimum requirements of this program consist of initial and ongoing quality 

control samples. Initial quality control samples include: an initial precision and recovery (IPR) 

study described in Sec. 10.2, an MDL study described in Sec. 10.3, and a reporting limit sample

(RLS) described in Sec. 10.4. 

Ongoing quality control samples (Secs. 10.5 – 10.8) include calibration verification (CALVER) 

standards at the beginning of every shift, analysis of ongoing calibration verification standards

with every sample batch, and analysis of a method blank with every sample batch. 

Laboratory performance is compared to the draft performance criteria (Table 8) to determine if 

the results of analyses meet the performance characteristics of the procedure. (The performance 

criteria will be revised by EPA as the procedure is finalized.)

Additionally, the test for determination of PFC backgrounds in Sec. 12.5 must be performed on 

every instrument to determine the modifications to instrument plumbing that are required.
Draft PFC Procedure 13 December 2011

Not approved for either general purpose or regulatory use

10.2 Initial precision and recovery (IPR) – To establish the ability to generate acceptable precision and 

recovery in reference matrices, that the analytical system is performing properly, and that the 

laboratory may perform the procedure, the laboratory must perform the following operations. 

10.2.1 Spike four aliquots of the reference matrix (Sec. 7.3.7) with 40 μL of the working level 

native standard (Sec. 8.4.1), 16 μL of working-level native standard solution for NMe/Et-FOSA/Es (Sec. 8.4.3), and 100 μL of labeled internal standard solution (see Sec.

8.5), and analyze each aliquot according to the procedures in Secs. 11 through 14. All 

processing steps that are used for samples, including preparation, extraction, and cleanup 

(Sec. 11), must be included in this test. 

10.2.2 Using results of the set of four analyses, compute the average percent recovery (X) of 

each compound in each extract and the relative standard deviation (RSD) of the recovery 

for each compound, by isotope dilution for compounds with a labeled analog, and by 

injection internal standard for compounds without a labeled analog and for the labeled 

compounds.

10.2.3 For each native and labeled compound, compare RSD and X with the corresponding 

limits for initial precision and recovery in Table 8. If RSD and X for all compounds meet 

the acceptance criteria, then system performance is acceptable and analysis of blanks and 

samples may begin.

Note: EPA has not yet developed formal acceptance criteria for this procedure. 

Therefore, use the draft criteria in Table 8 as guidance. If more than one target 

compound fails the IPR recovery test, examine the system to determine the cause 

and repeat the test.

10.3 Method detection limit (MDL) study – Determine the MDL in accordance with the procedures 

described in 40 CFR Part 136, prior to the analysis of field samples.

10.4 Reporting limit sample (RLS) – Use 0.5 gram of the soil reference matrix (Sec. 7.3.7) to prepare 

the RLS. Spike this aliquot with 10 μL of the low-level native standard (Sec. 8.4.2) that is 

equivalent to the concentration in the lowest (CS-1) calibration standard. Analyze this sample as 

an unknown in the sample batch. Analyze the RLS immediately prior to analysis of the OPR, and 

samples from the same batch.

Note: The RLS is not required during routine sample analysis after method development has 

been completed and single-lab validation has occurred.

10.5 Matrix spike samples are used to assess performance of the procedure on the biosolids being 

analyzed. The laboratory must determine the recovery of both labeled and native compounds 

spiked into a biosolids matrix. By preparing two such samples, i.e., a matrix spike and a matrix

spike duplicate, the laboratory can also assess precision of the procedure in routine application.

10.5.1 Spike two 0.5-g aliquots of a sewage sludge or biosolids sample with 40 μL of the 

working-level native standard (Sec. 8.4.1), 16 μL of the working-level native standard 

solution for N-Me/Et-FOSA/Es (Sec. 8.4.3), and 100 μL of labeled internal standard 

solution (Sec. 8.5) and mix thoroughly. Analyze both matrix spike samples according to 

the procedures in Secs. 11 through 14.

10.5.2 Compute the recovery of the labeled compounds using the internal standard method 

(quantify using the injection internal standard). 
Draft PFC Procedure 14 December 2011

Not approved for either general purpose or regulatory use

10.5.3 It may be necessary to spike the native compounds at concentrations that will allow 

meaningful recovery data to be obtained, e.g., 3 to 5 times the background levels in the 

unspiked sample. Thus, it may be advisable to spike new aliquots of a sample that has 

already been analyzed. 

10.5.4 Report the background concentrations of native compounds, and recoveries and spiking 

concentrations for both the native and labeled compounds. Compare the recoveries to the 

draft acceptance criteria in Table 8.

10.5.5 Calculate and report the relative percent difference (RPD) of the recoveries of the native 

analytes in the matrix spike and matrix spike duplicate samples. Compare the RPDs to 

the draft acceptance criteria in Table 8.

10.6 Calibration verification 

Note: Initial calibration is discussed in Sec. 13.1 and must be performed prior to analysis of 

samples.

10.6.1 Frequency – Ongoing calibration verification consists of the analysis of bracketing 

calibration verification samples, one at the beginning of every 12-hour shift and another 

at the end of every batch or 12-hour shift, whichever is more frequent. 

10.6.2 In addition, a calibration verification sample is required during the middle of every 

sample batch larger than 10 samples. (Sample batches may be no larger than 20 samples.)

10.6.3 The standards used for calibration, calibration verification, and for initial and ongoing 

precision and recovery should be identical, so that the most precise results will be 

obtained. The CS-4 calibration standard is used for calibration verification samples.

10.6.4 Inject the VER (CS-4) standard (Table 6) using the analysis procedure in Sec. 14.

Note: The requirements in Sec. 15 must be met when analyzing ongoing calibration verification 

samples.

10.7 Method blank – A method blank is analyzed with each sample batch to demonstrate freedom 

from contamination. Use a 0.5-g aliquot of the reference matrix (Sec. 7.3.7) to prepare a method 

blank. Extract the sample following the procedure described in Sec. 11. The method blank 

should be analyzed immediately after the OPR sample. If native compounds will be carried from 

the OPR into the method blank, analyze one or more aliquots of solvent between the OPR and the 

method blank.

Note: If aliquots of solvent must be analyzed, record how many, and update the analytical 

sequence in Sec. 14 of this draft procedure to reflect the use of solvent blanks.

Alternatively, analyze the method blank before the OPR sample, and analyze one or more use 

solvent blanks after the OPR to prevent carryover from the OPR into the first sample.

10.8 Ongoing precision and recovery (OPR) sample – The laboratory must, on an ongoing basis, 

demonstrate through analysis of the ongoing precision and recovery standard that the analytical 

system is in control. Use a 0.5-gram aliquot of the soil reference matrix (Sec. 7.3.7) as the matrix 

for the OPR. Spike this sample with 40 μL of the working-level native standard (Sec. 8.4.1), 16 

uL of the working-level native standard for N-Me/Et-FOSA/Es (Sec. 8.4.3), and 100 μL of the 
Draft PFC Procedure 15 December 2011

Not approved for either general purpose or regulatory use

labeled internal standard solution (Sec. 8.5). The aliquot of native standard spiked is equivalent to 

the concentration in the CS-3 calibration standard. Extract the sample following the procedure 

described in Sec. 11. Analyze the extracts of the OPR aliquot prior to analysis of samples from 

the associated batch. Compare the results to the acceptance criteria in Table 8.

Note: EPA has not yet developed formal acceptance criteria for this procedure. Therefore, use 

the draft criteria in Table 8 as guidance. If more than one target compound fails the 

OPR test, examine the system to determine the cause and repeat the test.

11.0 Preparation, Extraction, and Cleanup of Field Samples and QC 

Samples

11.1 Sample preparation

Note: As noted in Sec. 1.4, early work on this procedure focused on analysis of the solid portion 

of sewage sludge samples and described discarding any supernatant aqueous liquid in 

the sample. EPA believes that this approach was intended to provide a consistent mass 

of solids from a bulk sample that could be used for testing the many extraction conditions 

studied by Yoo et al, 2009. At present, EPA believes that the extraction procedures are 

capable of dealing with samples containing large amounts of water, which may better 

represent actual sewage sludges and biosolids from wastewater treatment operations. 

Homogenize the entire sample in the original sample container, by shaking samples that are 

pourable liquids, or by stirring solids in their original container with a clean spatula, glass stirring 

rod, or other suitable implement. Once homogenized, remove an aliquot of the sample to 

determine the percent solids content of the sample using the procedure in Sec. 11.2. Remove a 

second aliquot of the sample for analysis, as described in Sec. 11.3.

11.2 Determination of solids content 

The percent solids of sewage sludge and biosolids will vary depending on the source of the sample and 

the treatment processes applied. The solids content of the bulk sample is determined from a subsample 

that is used only for the solids determination. Separate procedures are used for the solids determination, 

based on the nature of the sample, as described below.

11.2.1 Single-phase solid samples and multi-phase samples in which the main phase is not 

aqueous

11.2.1.1 Using a solvent-rinsed spatula, transfer a 1-gram subsample of the 

homogenized sample into tared weighing boat. Record weight of the 

subsample to three significant figures. (If there is not sufficient mass of the 

original sewage sludge and biosolid sample, a smaller subsample may be used 

for the solids determination.)

11.2.1.2 Dry the subsample for a minimum of 12 hours in a drying oven set at 110 ± 5 o 

C, and cool in a desiccator.
Draft PFC Procedure 16 December 2011

Not approved for either general purpose or regulatory use

11.2.1.3 Weigh the dried aliquot and calculate percent solids as follows:

11.2.2 Multi-phase biosolids samples consisting of mainly an aqueous phase

11.2.2.1 Dry a GF/A filter and weigh to three significant figures. Mix the bulk sample 

in the original container (e.g., cap the bottle and shake) and take a 10.0 ± 0.2 

mL aliquot. Filter that aliquot through the filter. Dry the filter in an oven for a 

minimum of 12 hours at 110 ± 5 oC and cool in a desiccator. 

11.2.2.2 Weigh the filter and calculate percent solids as follows:

11.3 Sample digestion

Each sample batch to be digested and extracted during the same 12-hour shift consists of a 

maximum of 20 field samples, plus one method blank, and one OPR sample. A reference matrix 

(e.g., reference soil) known to be free (below background levels) of the target analytes is used as 

the matrix for the method blank and OPR sample (Sec. 7.3.7). 

11.3.1 Place a 0.5-g (wet weight) subsample of thoroughly homogenized sample into a 15-mL 

polypropylene centrifuge tube. 

11.3.2 Place a 0.5-g (wet weight) aliquot of the reference soil into a 15-mL polypropylene 

centrifuge tube. This sample is used as the method blank. 

11.3.3 Place another 0.5-g (wet weight) aliquot of the reference soil into a 15-mL polypropylene 

centrifuge tube. This sample is used as the OPR sample.

11.3.4 If matrix spike samples are to be analyzed, prepare those aliquots as described in Sec. 

10.5 and spike them with the native analytes at 3 – 5 times the background 

concentrations.

11.3.5 Spike all of the QC samples (OPR, IPR, matrix spike samples) with 40 μL of the 

working-level native standard solution (Sec. 8.4.1) and 16 μL of the working-level native 

standard solution for N-Me/Et-FOSA/Es (Sec. 8.4.3).

Note: Larger volumes of these solutions may be used, provided that the concentrations 

are adjusted accordingly.

11.3.6 Spike all samples and QC samples with 100 μL of the labeled internal standard solution 

(Sec. 8.5). The volume spiked will yield a sample extract with labeled compounds 

present at a concentration equivalent to the native compounds in the mid-level calibration 

standard.

11.3.7 Add 0.5 mL of 1 M NaOH solution to each sample and QC sample. If a sample is very 

dry, add enough NaOH to wet and cover the sample. Record volume of NaOH used. 

100

10 g

Weight of sample aliquot after drying (g) - weight of filter (g) % Solids = x

100

Weight of sample aliquot before drying (g)

Weight of sample aliquot after drying (g) % Solids = x
Draft PFC Procedure 17 December 2011

Not approved for either general purpose or regulatory use

11.3.8 Sonicate the samples in a heated water bath at 40 oC for 30 min, followed by incubation 

overnight (12 h) at ambient temperature.

Note: Mixing the sample by sonication is described in Ref. 1. EPA has not tested 

whether or not this step is essential, but may do so in the future.

11.3.9 After the incubation period, neutralize the NaOH by adding an equivalent number of 

moles of the 1 M HCl solution (Sec. 7.4.2), allow the mixture to react for 1 min, and 

vortex.

Note: Some biosolids are treated with lime and will require additional HCl to 

neutralize them. If the samples are known or suspected to involve lime treatment, 

then use a clean glass stirring rod to remove a small amount of the supernatant 

from the incubated sample and test the pH with a wide-range pH paper. Do NOT 

dip the pH paper into the sample. If the pH of the incubated sample is not 7.0 ± 

0.5, add additional HCl, vortex, and retest the pH until the sample is neutral.

11.4 Sample extraction

11.4.1 Add 10 mL of 50:50/ACN:MeOH (v/v) to the sample in the centrifuge tube. 

11.4.2 Shake the mixture for 1 h on a tube shaker (Sec. 6.2.5) at a moderate speed.

11.4.3 Centrifuge the sample at a speed of 3000 x g for 20 min.

11.4.4 Decant the 10 mL supernatant into a 250-mL HDPE bottle containing 180 mL of plasmagrade reagent water (Sec. 7.1.3).

11.4.5 Repeat the extraction (Secs. 11.4.1 to 11.4.4) one more time and add the extract to the 

250-mL HDPE bottle.

11.4.6 After the second extraction, check that the pH of the diluted extract is 6.5 ± 0.5. If 

required, adjust the pH with 3% (v/v) acetic acid in reagent water or 0.3% (v/v) aqueous 

ammonium hydroxide (Sec. 7.4.5.4).

11.4.7 Sonicate the mixture for 30 min. The extract is ready for cleanup. 

11.5 Extract cleanup

All sample extracts are subjected to cleanup using an SPE cartridge, as described below.

11.5.1 Prepare the SPE extraction manifold, reservoirs, SPE adapters, and SPE needles. Use 

SPE cartridges that pass the SPE cartridge performance check described in Sec. 11.7.

11.5.2 Label each Oasis WAX SPE cartridge with the sample ID and place the cartridge on the 

extraction manifold.

11.5.3 Condition each cartridge with 5 mL of 0.3% NH4OH in methanol, followed by 5 mL of 

0.1M formic acid in reagent water. Discard eluants. 

11.5.4 Equilibrate the cartridge with 5 mL of plasma-grade reagent water (Sec. 7.1.3). Discard 

the eluant.
Draft PFC Procedure 18 December 2011

Not approved for either general purpose or regulatory use

11.5.5 Load the sample onto the cartridge drop-wise (~5 mL/min). 

11.5.6 Wash the sample with 5 mL of 20% MeOH in 80% 0.1M formic acid in reagent water, 

followed by 2 mL of 0.3% (v/v) NH4OH in reagent water. Discard the eluants. 

Note: Other wash solvents have been reported in the literature and may provide 

acceptable results. 

11.5.7 Dry the cartridge by pulling air under vacuum for 5 min.

11.5.8 Elute the cartridge into a 15-mL clean glass centrifuge tube with 4 mL 0.3% NH4OH 

(v/v) in MeOH.

11.6 Extract concentration 

11.6.1 Reduce the extract to about 50 μL using a gentle stream of nitrogen and a water bath set 

at 40 oC.

11.6.2 Reconstitute the extract with 938 μL of 0.3% NH4OH (v/v) in MeOH and vortex to mix. 

11.6.3 Spike the extract with 12.5 μL of the injection internal standard solution (Sec. 8.6), 

vortex to mix, and filter the sample extract through a syringe filter (Sec. 6.1.8) into a 

clean centrifuge tube. The final extract volume is 1.0 mL.

Note: Other volumes and solvents may be used in Secs. 11.6.1 to 11.6.3, provided that 

the mass of the injection internal standard is equal to the mass in the calibration 

standards and the final extract volume is the same for all sample extracts. 

However, avoid taking the sample extract to dryness in Sec. 11.6.1, because it 

may result in loss of short-chain analytes such as PFBA.

11.6.4 Vortex the extract and transfer 300 μL of the final extract to a polypropylene LC/MS/MS 

auto-sampler vial (Sec. 6.1.3) for analysis. Cap the centrifuge tube containing the 

remaining 700 μL and store at 4 oC for backup. Place a mark on the tube at the level of 

the solution so that solvent loss by evaporation can be detected. Alternatively, weigh the 

tube before storage, record the mass, and reweigh the tube before any subsequent analysis 

of the stored extract.

11.7 SPE cartridge performance check 

In order to be used for cleanup of sample extracts, the performance of the WAX SPE cartridges 

must be checked at least once for each manufacturer’s lot of cartridges. This performance check 

is accomplished by processing a spiked reagent water sample through the extraction procedure 

and analyzing the extract. Labeled compounds are not added to these check samples before 

extraction because the recovery correction inherent in isotope dilution will mask problems with 

the cartridges. Cartridge performance is acceptable if the recoveries of the native analytes are 

within the QC acceptance criteria for the OPR in Table 8. Perform this cartridge check as 

outlined below.

Note: This check is performed whenever a new lot number of cartridges is purchased.
Draft PFC Procedure 19 December 2011

Not approved for either general purpose or regulatory use

11.7.1 Spike 100 mL reagent water with 40 μL of the working-level native standard solution 

(Sec. 8.4.1) and 16 μL of the working-level native standard solution for N-Me/EtFOSA/Es (Sec. 8.4.3). Do NOT spike the labeled internal standard compounds. 

11.7.2 Process the solution through the SPE cleanup procedure in Sec. 11.5.

11.7.3 After cleanup, spike the solution with the labeled internal standard solution (Sec. 8.5) and 

complete the analysis as per Sec. 14.

11.7.4 Recovery of the native compounds must be within the QC acceptance criteria for the 

OPR in Table 8. If the compounds are not recovered in this range, adjust the elution 

volumes or reject the cartridge batch.

12.0 LC/MS/MS Set Up and Calibration

Samples are analyzed on a high performance liquid chromatograph coupled to a triple quadrupole mass 

spectrometer (LC/MS/MS), or equivalent, equipped with an electrospray ionization (ESI) source. The 

tandem MS systems operate at a nominal resolution of 1 amu. The LC/MS/MS is run in the negative ion 

electrospray (ESI-) mode using multiple reaction monitoring (MRM). Data acquisition and quantification 

are performed by recording the peak areas of the applicable parent ion /daughter ion transitions. The 

instrument manufacturer’s software is used to acquire data and calculate results using isotope dilution and 

internal standard quantitation (Sec. 15.2).

Once the mass spectrometer has been optimized and the LC/MS/MS operating conditions for the targeted 

compounds have been established, the same conditions must be used for the analysis of all standards, 

blanks, IPR and OPR standards, field samples, and QC samples.

12.1 Establishing LC/MS/MS Operating Conditions

Prior to any analyses, optimize the following instrumental conditions: mass calibration, MRM 

acquisition parameters, scans per peak, chromatographic resolution, retention time calibration, 

sensitivity and instrument background elimination. Example analyte-specific instrumental source 

parameters for PFCs analysis are found in Table 3, but actual tuning parameters are instrumentspecific and should be optimized according to manufacturer’s specifications. 

12.2 Mass calibration

The mass spectrometer system must undergo mass calibration according to manufacturer’s 

specifications to ensure accurate assignments of m/z values by the instrument. Mass calibration 

is performed at least annually, after performing major maintenance, or as required to maintain 

routine instrument sensitivity and stability performance. The reference calibrant used is a 50:50 

isopropanol:water (IPA:water) solution containing sodium cesium iodide (NaCsI), which is 

infused directly into the electrospray source during calibration. Mass calibration is performed at 

instrumental settings corresponding to a nominal unit mass resolution, but mass resolution is not 

directly measured.

In the absence of manufacturer-specific instructions and acceptance criteria, the following 

procedure may be used. 

12.2.1 Use a NaCsI calibration solution (in 50:50 IPA:water) containing 2 μg/μL sodium iodide 

and 50 ng/μL cesium iodide.
Draft PFC Procedure 20 December 2011

Not approved for either general purpose or regulatory use

12.2.2 Use a syringe pump to infuse the calibration solution as a stable aerosol spray at 10 

μL/min directly into the ESI-MS/MS source.

12.2.3 Scan the MS/MS over the mass range from 20 to 2000 Daltons. Adjust the source 

parameters to optimize peak intensity and shape across the mass range. The exact m/z 

values for NaCsI calibration are contained as a reference file on the instrument data 

system and are:

Calibration Masses (Daltons - m/z)

22.9898 922.3552 1971.6149

132.9054 1072.2494 2121.5091

172.8840 1222.1437 2271.4033

322.7782 1372.0379 2421.2976

472.6725 1521.9321 2571.1918

622.5667 1671.8264 2721.0861

772.4610 1821.7206 2870.9803

During the mass calibration, examine the instrument parameters to ensure detection of the 

specified ions.

Mass calibration is judged on the basis of the presence or absence of the exact calibration 

masses, e.g., a limit on the number of masses that are “missed.” Repeat the test if more 

than two masses are missed. 

12.3 MRM acquisition parameters 

During method setup, the mass spectrometer response must be separately optimized for each target 

compound, using a solution containing only the compound of interest. These parameters are then 

used for analysis of all standards and samples.

12.3.1 Using a post-column pump, infuse a solution mixture of methanol and mobile phase 

containing approximately 1 ppm of the compound of interest directly into the ESI-MS/MS 

source. This solution is prepared by adding mobile phase to a 1 ppm methanol solution 

of the compound being tested in the ratio of 1 part mobile phase/2 parts test solution.

12.3.2 For each compound, optimize the sensitivity to the specified parent-daughter transitions 

(see Table 3) by adjusting the collision energy and cone voltage. 

12.3.3 The optimum parameters are compound specific and a set of single settings cannot be 

used for all target compounds in the analysis. Use the optimized settings for the analysis 

of all standards and samples. 

12.4 Chromatographic separation

Establish liquid chromatography conditions suitable for the separation of the target compounds. 

To achieve the retention times in Table 2, the HPLC should be operated according to the 

parameters in Table 5. A C18 analytical column is used. The exact gradient is optimized for the 

chromatography system in use, but the conditions in Table 5 can be used as guidelines. The 

chromatographic separation should ensure that there is adequate resolution of target compounds 

from potentially interfering substances (Sec. 13.5). Note that the LC gradient selected will affect 

the chromatographic resolution of linear and branched PFC compounds. After the LC column 

conditions and gradient have been determined, they should be used for all analyses.
Draft PFC Procedure 21 December 2011

Not approved for either general purpose or regulatory use

12.5 Instrument background determination

To determine if background concentrations of PFCs significantly impact this analysis, a 40 part 

per trillion (ppt) standard containing all of the target analytes in 0.3% NH4OH (v/v) in methanol 

must be analyzed three times, with alternating instrument solvent (methanol) blank 

measurements. If the peak area from the standards is not greater than that of the solvent blank 

using a Student’s t-test (95% confidence), then it may be necessary to modify the “plumbing” of 

the analytical system as outlined in Sec. 6.4.5. This test should be performed prior to any 

analysis, at least annually, and after major instrument maintenance.

12.6 Establishing retention time windows

Analyze individual solutions of the each of the target compounds using the LC gradient and 

acquisition parameters determined above. Analyze a mixed solution of all target compounds to 

confirm their separation and identification. A total ion chromatogram (TIC) indicating the 

separation of the target analytes is shown in Figure 2, as an example.

12.7 Analytical data acquisition program

All of the information is now in place to finalize the analytical acquisition routine to be used for 

calibration of instrument response and analysis of all samples. The acquisition program will 

monitor each of the MRM ions listed in Table 3 at its optimum cone voltage and collision energy,

as determined in Sec. 12.3, and in the appropriate retention time window established in Table 2.

12.8 Instrument sensitivity

Prior to commencement of any analysis, and at least once every 24 hours during extended runs, 

determine that the instrument is meeting the sensitivity specifications. Analyze the lowest 

concentration calibration solution (CS-1) using the acquisition program described in Sec. 12.7. 

Ensure that all compounds are detected with S/N > 3. If sensitivity is inadequate, perform system 

cleaning and maintenance and repeat the test. If calibration verification can not be established 

after this maintenance, a new initial calibration (Sec. 13.2) is required. 

13.0 Instrument Quality Control 

The mass spectrometer is optimized and the LC/MS/MS operating conditions are established for the 

target compounds as described in Sec. 12. The same conditions must be used for the analysis of all 

standards, blanks, IPR and OPR standards, and samples.

13.1 Initial calibration

Initial calibration (ICAL) must be performed prior to analysis of any field sample or QC sample. 

Approaches to quantitation vary depending on the compound. Some compounds are quantified

using isotope dilution (for those target compounds with exact labeled analogs) and others are 

quantified using internal standard (those without an exact labeled analog). Additionally, labeled 

compounds are quantified using the internal standard approach. These two approaches are 

discussed in Secs. 13.2.1 and 13.2.2, respectively. The requirements for initial calibration are 

outlined in Sec. 13.2, and Table 8. After the initial calibration, analyze an initial calibration blank 

and an initial calibration verification (CALVER) standard.
Draft PFC Procedure 22 December 2011

Not approved for either general purpose or regulatory use

13.2 Calibration by isotope dilution and internal standard

Calibrations should be constructed using regressions of untransformed data and plotting 

normalized relative response versus the absolute concentration of analyte. Normalized relative 

response is defined as:

where:

NRR = Normalized relative response

An = Area of the peak for the native (unlabeled) analyte

Al = Area of the peak for the specified labeled compound

Cl = Concentration of the specified labeled compound (in pg/mL)

Efforts to date indicate that a linear equation may be used for all of the analytes except:

perfluorobutanesulfonic acid (PFBS), perfluoro-n-heptane sulfonic acid (PFHpS),

perfluorooctane sulfonamide (PFOSA), and those compounds containing more than 10 carbons 

(C11 – C14). For those analytes, the response is better fit (higher r

2

) with a quadratic equation. 

For either a linear regression or a quadratic fit, weight the equation using the inverse of the 

concentration (e.g., 1/x), and do not force the calibration through the origin.

13.2.1 Calibration by isotope dilution

Isotope dilution is used for calibration of each native compound for which an exact

labeled analog is available (Table 3). An 8-point initial calibration is prepared for each 

native compound. The calibration solutions are listed in Table 6.

13.2.1.1 To calibrate the analytical system by isotope dilution, inject the CS-1 through 

CS-8 calibration solutions (Sec. 8.7 and Table 6). Use an injection volume of 

15 μL.

13.2.1.2 For each compound determined by isotope dilution, compute its normalized 

relative response (NRR) over the calibration range. Determine the NRR of 

each compound using the area responses of the product m/z values specified in 

Table 3. Use the labeled compounds listed in the tables as the quantitation 

reference and the product m/z values of these labeled compounds for 

quantitation. Determine the calibration equation for each compound by 

regressing the NRR against the native compound concentration to produce a 

calibration weighted inversely proportional to concentration (i.e., a 1/x

weighted linear regression). Select the calibration equation (linear or 

quadratic) that provides the best fits to the data, as indicated by the coefficient 

of determination (r

2

). Examine the residuals of the regression (i.e., the distance 

between the observed response for each calibration standard and the response 

predicted by the regression) to determine if they are randomly distributed (i.e., 

the regression line passes between the calibration points, and not above or 

below all of the points). Whichever calibration is selected, r2 must be at least 

0.99. Use the resultant equation to calculate the concentration of analyte in

each sample or CALVER.

l

l

n x C

A

A NRR =
Draft PFC Procedure 23 December 2011

Not approved for either general purpose or regulatory use

13.2.1.3 Some of the labeled compounds may contain native analogs as impurities. If 

the labeled compounds are present at a constant concentration in calibration 

and other solutions (including sample extracts), a constant background 

response will be added to the response from analysis of calibration and other 

solutions. A calibration not forced through zero will accommodate any such 

residual.

13.2.2 Calibration by internal standard

Internal standard calibration is applied to the determination of the native compounds that 

do not have exact labeled analogs, and that are not being quantified by isotope dilution. 

Internal standard calibration is also used to quantify the labeled compounds themselves. 

The internal standard approach utilizes the injection internal standard (IIS) that is added 

to the extract after extraction and cleanup and prior to injection into the instrument as the 

quantitation reference. The reference compound for each native and/or labeled 

compound is listed in Table 3. For the labeled compounds, calibration is performed at a 

single concentration, using data from the 8 points in the calibration (all of which contain 

the labeled compounds at the same concentration).

13.2.2.1 To calibrate the system for native compounds for which isotope dilution is not 

being performed, use the data from the 8-point calibration and the labeled 

injection internal standard.

13.2.2.2 For each compound determined by internal standard quantitation, its NRR is 

computed over the calibration range. Determine the NRR of each compound 

using the area responses of the product m/z’s specified in Table 3. Use the 

labeled IIS compounds listed in the tables as the quantitation reference and the 

product m/z’s of these labeled compounds for quantitation. Determine the 

calibration equation for each compound by regressing the NRR against the 

native compound concentration to produce a calibration weighted inversely 

proportional to concentration (i.e., a 1/x weighted linear regression). Do not 

force through zero (Sec. 13.2.1.3). Select the calibration equation (linear or 

quadratic) that provides the best fits to the data, as indicated by the coefficient 

of determination (r2

). Examine the residuals of the regression (i.e., the distance 

between the observed response for each calibration standard and the response 

predicted by the regression) to determine if they are randomly distributed (i.e., 

the regression line passes between the calibration points, and not above or 

below all of the points). Whichever calibration is selected, r2 must be at least 

0.99. Use the resultant equation to calculate the concentration of analyte in 

each sample or CALVER.

13.2.2.3 For each labeled compound, regress its NRR against the concentration of the 

labeled compound using the labeled IIS as the quantitation reference as 

indicated in Table 3. The labeled compounds and the labeled injection internal 

standards are in each calibration solution at a constant concentration. The 

regression will simplify to a single-point calibration because the concentrations 

are constant.
Draft PFC Procedure 24 December 2011

Not approved for either general purpose or regulatory use

13.2.3 Quantification of the labeled injection internal standard concentration 

The injection internal standard (IIS) itself is quantified by external standard. Monitoring 

of the recovery of the IIS is used as a diagnostic test for matrix effects during the LC 

analysis (Sec. 4.6). 

13.3 Calibration verification 

Calibration verification (VER) requires the analysis of bracketing calibration verification samples, 

one at the beginning of every 12-hour shift, and another after the analysis of every 10 samples, or at 

the end of each 12-hour shift, whichever is more frequent. The standards used for calibration, 

calibration verification, and for initial and ongoing precision and recovery should be identical, so 

that the most precise results will be obtained. The CS-4 calibration standard is used for calibration 

verification samples. Inject the CS-4 calibration standard (Table 7) using the analysis procedure in 

Sec. 14. The requirements in Sec. 10.8, and Table 8 must be met when analyzing calibration 

verification samples. 

13.4 Quantitation of linear and branched isomers 

Some PFCs consist of linear and branched isomers, depending on manufacturing processes (Ref. 

5). There are reports that during MS/MS analysis, the linear and branched isomers ionize with 

different efficiencies, complicating the quantification of PFC compounds (Ref. 3). The LC 

analysis performed by this procedure results in partial chromatographic resolution of the isomers. 

Additionally, the composition of standards may differ by vendor, and differ from the distribution 

of the PFCs in environmental samples. Therefore, peak integration in samples should ensure that

the PFC target peaks include the linear and branched isomers as a single total response. 

13.5 Co-extracted interferences

Interferences co-extracted from samples will vary considerably from source to source. 

Taurodeoxychloic Acid (TDCA) is a known interference which may lead to an overestimate or 

yield a false positive result for PFOS (Ref. 5) while 5-pregnan-3,20-diol-3-sulfate and 34S-3-

hydroxy-5-pregnan-20-one sulfate may interfere with PFHxS (Ref. 6). The 499 > 80 transition is 

prominent in all TDCA isomers and in PFOS. However, the 499 > 99 transition for PFOS is not 

affected by the TDCA. In the absence of chromatographic separation of TDCA from PFOS, the 

499 > 80 transition will result in significant bias in PFOS concentrations. Therefore, both 

transitions must be monitored for PFOS and results must agree within 20%, to ensure accurate 

quantification of PFOS. Similarly, analysis for PFHxS can be biased by co-eluting interferences. 

In this case, the 399 > 80 and the 399 > 99 transitions may both be affected, and therefore, a third 

transition, 399 > 119, also must be monitored to demonstrate that there is not a bias from coeluting interferences. 

14.0 Instrumental Analysis

Once the operating conditions have been established and the instrument tuned (Sec. 12), inject a 

15-μL aliquot of sample extract into a 50-uL loop, using partial-loop-with-needle-overfill mode 

onto a trapping column (if needed). Start the gradient according to the parameters found in Table 

5. Start data collection 1 to 2 minutes prior to elution of the first analyte. Monitor the product 

m/z’s for each native and labeled analyte throughout its retention time window. Stop data 
Draft PFC Procedure 25 December 2011

Not approved for either general purpose or regulatory use

collection after elution of the last analyte in each group. Return the gradient to the initial mixture 

for analysis of the next sample extract or standard.

A typical instrument analysis sequence is as follows:

• 1-2 Instrument blanks (more may be included)

• 8 Initial calibration standards

• 2 Instrument blanks

• Opening calibration verification standard 

• OPR sample

• 1-2 Instrument blanks

• Method blank

• Up to 6 field samples

• Bracketing calibration verification standard (every 10th injection)

• Up to 10 field samples

• Closing calibration verification standard (every 10th injection, which may serve as the 

opening VER for the next cycle)

• OPR sample, etc.

15.0 Qualitative Identification and Quantitation

15.1 Qualitative identification – The following requirements must be met in all samples for a 

compound to be identified.

15.1.1 Signal to noise requirements – The LC peak representing the quantitation m/z of each 

native compound in the upper 7 ICAL standards must be present with a S/N of at least 10, 

and with a S/N of at least 3 for the lowest standard (CS-1). The LC peak representing each 

labeled compound quantitation m/z in the CALVER standard and in extracts from all other 

samples must be 10 or greater. If these requirements are not met in ICAL or CALVER 

samples, the LC/MS/MS system must be adjusted or recalibrated until these requirements 

can be met. If these requirements are not met in samples, the CALVER test should be 

repeated. 

15.1.2 Relative intensity – The ion intensity of each monitored ion transition in the ICAL should 

be recorded for use in qualitative identification for all other samples. The monitored ion

transitions are found in Table 3. These parameters may be instrument specific. If 

alternate transitions are monitored for diagnostic purposes (called confirmatory 

transitions), the following requirements should be met: 

1) The molecular ion shall preferably be the precursor of one of the selected diagnostic 

transitions (the molecular ion, characteristic adducts of the molecular ion, 

characteristic fragment ions and all their isotope ions),

2) Diagnostic transitions preferably should not originate from the same part of the 

molecule as that for the quantitation transition, and 

3) The signal-to-noise ratio for each diagnostic transition must be greater than or equal to 

3.
Draft PFC Procedure 26 December 2011

Not approved for either general purpose or regulatory use

Comparison of the ratio of the confirmation transition response to the quantitation 

transition response in a sample to the ratio in the most recent CALVER can assist in 

confirmation of the presence or absence of a target compound in samples. The relative 

ion ratio requirements are described below.

15.1.3 Relative ion intensity ratio requirement – Depending on the relative intensity of the 

confirmatory m/z to the quantitation m/z, the following requirements must be met to 

provide confirmation of the presence of the target analyte: 

Relative intensity of confirmation 

transition to quantitative transition in 

most recent CALVER

Agreement of ratio of confirmation transition to 

quantitative transition in sample relative to 

same ratio in most recent CALVER

>50 % ±20%

20 to 50 % ±25%

10 to 20 % ±30%

<10 % ±50%

Note: If the results for any of the qualitative identification criteria above are 

ambiguous, or if false negatives or false positives occur, the laboratory should 

consult EPA to determine corrective action and next steps during method 

development.

15.1.4 Retention time window requirement – There are two retention time requirements:

1) the retention times of the native and labeled compounds in the initial calibration must be 

stored in the system for verification of the retention time window requirement during 

analysis of all subsequent samples. Data acquired for all subsequent samples should be 

within the required retention time windows. If this is not the first time an initial 

calibration is being performed, retention times from this calibration should be checked 

against those from previous calibrations to determine if the separation of target analytes 

is being affected.

2) the relative retention times for native compounds and the labeled compound for which 

each native is being quantified must fall within a certain range of each other. Typical 

retention times, relative retention times and retention time windows are listed in Table 2.

15.1.5 Peak asymmetry factor – During initial calibration and calibration verification, the peak 

asymmetry factor must be calculated for all analytes using the following figure and 

equation:

As = b/a
Draft PFC Procedure 27 December 2011

Not approved for either general purpose or regulatory use

where:

As = peak asymmetry factor

b = width of the back half of the peak measured (at 10% peak height) from the trailing 

edge of the peak to a line dropped perpendicularly from the peak apex, and 

a = the width of the front half of the peak measured (at 10% peak height) from the 

leading edge of the peak to a line dropped perpendicularly from the apex. 

Calculated peak asymmetry factors for the first two eluting peaks in the CS-4 standard of 

the ICAL must fall within 0.8 to 1.5.

If this criterion cannot be achieved, the LC mobile phase conditions need to be modified. 

This criterion must be met each time a new calibration curve is generated.

15.2 Quantitation

15.2.1 Isotope dilution quantitation

Using the most recent multi-point calibration (Sec. 13.2.1), calculate native and labeled 

compound concentration in the extract. Do not use calibration verification data to 

quantify analytes.

15.2.2 Internal standard quantitation and labeled compound recovery 

Compute the concentration of each native compound in the extract that does not have an 

exact labeled analog and each labeled compound by internal standard, using the weighted 

regression established in Secs. 13.2.2.2 and 13.2.2.3, respectively.

Using the concentration in the extract determined above, compute the percent recovery of 

each labeled compound using the following equation:

15.2.3 The concentration of a native compound in the solid sample is computed using the 

concentration of the compound in the extract, and the wet weight of the solids, 

and the percent solids, as follows:

where:

Cex = Concentration of the compound in the extract in ng/mL

Vex = Extract volume in mL

Ws = Sample weight (wet weight) in kg

% solids = Percent solids determined in Sec. 11.2

If desired, divide the concentration by 1000 to convert ng/kg (ppt) to μg/kg (ppb).

15.3 Reporting results 

Unless otherwise specified, report results in ng/kg (parts-per-trillion) to three significant figures,

based on the dry weight of the sample. Also report the percent solids so that the result may be 

converted to wet-weight units by the end user.

W (%solids)

C V Concentration in solid sample (ng / kg)

s

ex ex =

x 100

Concentration spiked (ng/mL)

Concentration found (ng/mL) Recovery (%) =
Draft PFC Procedure 28 December 2011

Not approved for either general purpose or regulatory use

Results for a compound in a sample that has been diluted must be reported at the least dilute level 

at which the area at the quantitation m/z is within the calibration range. Results should be flagged 

to indicate that they are from a diluted analysis.

Project-specific reporting requirements may apply, including reporting results based on the 

volume of the original sample, e.g., ng/L or other weight/volume units

16.0 Method performance

This procedure is still under development. Preliminary method performance information can be found in 

Tables 4, 8, and 10. Additional performance data will be added by EPA as they are developed.

17.0 Pollution prevention and waste management 

17.1 The laboratory is responsible for complying with all federal, state, and local regulations governing 

waste management, particularly the hazardous waste identification rules and land disposal 

restrictions, and to protect the air, water, and land by minimizing and controlling all releases from 

fume hoods and bench operations. Compliance is also required with any sewage discharge 

permits and regulations. An overview of requirements can be found in Reference 12.

17.2 Samples at pH <2, or pH >12 are hazardous and must be neutralized before being poured down a 

drain, or must be handled as hazardous waste.

17.3 Standards should be prepared in volumes consistent with laboratory use to minimize the disposal 

of excess volumes of expired standards. For information about pollution prevention that may be 

applied to laboratories and research institutions, consult Less is Better: Laboratory Chemical 

Management for Waste Reduction, available from the American Chemical Society’s Department 

of Governmental Relations and Science Policy, 1155 16th Street NW, Washington DC 20036.

17.4 For further information on waste management, consult The Waste Management Manual for 

Laboratory Personnel and Less is Better: Laboratory Chemical Management for Waste 

Reduction, available from the American Chemical Society’s Department of Government 

Relations and Science Policy, 1155 16th Street NW, Washington, DC 20036. 

18.0 References

1. “Analysis of Perfluorinated Chemicals in Sludge: Method Development and Initial Results.” Yoo, 

Hoon, Washington, John W., Jenkins, Thomas M., Libelo, and E. Laurence. Journal of 

Chromatography A. Volume 1216, Issue 45, 6 November 2009, Pages 7831-7839. 

2. “Analysis of Perfluorinated Carboxylic Acids in Soils: Detection and Quantitation Issues at Low 

Concentrations.” Washington, John, W., Ellington, Jackson J., Jenkins, Thomas M., and John Evans.

J. Journal of Chromatography A. Volume 1154, Issues 1-2, 22 June 2007, Pages 111-120.

3. EPA Method 537. “Determination of Selected Perfluorinated Alkyl Acids in Drinking Water By 

Solids Phase Extraction and Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS),”

Revision 1.1, EPA Document Number EPA/600/R-08/092. September 2009.
Draft PFC Procedure 29 December 2011

Not approved for either general purpose or regulatory use

4. EPA Method 1694: Pharmaceuticals and Personal Care Products in Water, Soil, Sediment, and 

Biosolids by HPLC/MS/MS. EPA Document Number EPA-821-R-08-002. December 2007.

5. “An analytical method for the determination of perfluorinated compounds in whole blood using 

acetonitrile and solid phase extraction methods.” Leo W.Y., Yeung, Sachi Taniyasu, Kurunthachalam 

Kannan, Della Z.Y. Xu, Keerthi S. Guruge, Paul K.S. Lam, Nobuyoshi Yamashita. Journal of 

Chromatography A, 1216 (2009) 4950–4956. 

6. “Simultaneous characterization of perfluoroalkyl carboxylate, sulfonate and sulfonamide isomers by 

liquid chromatography – tandem mass spectrometry.” Benskin, J. P.; Bataineh, M.; and Martin, J. W. 

Anal. Chem. 2007, 79, 6455-6464.

7. “Working with Carcinogens,” Department of Health, Education, & Welfare, Public Health Service, 

Centers for Disease Control, NIOSH, Publication 77-206, September 1977, NTIS PB-277256. 

8. “OSHA Safety and Health Standards, General Industry,” OSHA 2206, 29 CFR 1910. 

9. “Safety in Academic Chemistry Laboratories,” ACS Committee on Chemical Safety, 1979.

10. “Structural Identification of Isomers Present In Technical Perfluorooctane Sulfonate By Tandem 

Mass Spectrometry.” Langlois, I. and Oehme, M. Rapid Commun. Mass Spectrom. 2006, 20, 844-

850.

11. “Commission Decision of 12 August 2002 implementing Council Directive 96/23/EC concerning the 

performance of analytical methods and the interpretation of results.”

http://eur-lex.europa.eu/LexUriServ/site/en/oj/2002/l_221/l_22120020817en00080036.pdf. 

12. “Environmental Management Guide for Small Laboratories” USEPA Office of the Administrator, 

Washington, DC, EPA 233-B-00-001, May 2000.

19.0 Glossary

These definitions and purposes are specific to this method but have been conformed to common usage to 

the extent possible.

Symbols

o C degrees Celsius

μL microliter

μm micrometer

< less than

> greater than

% percent

Abbreviations (in alphabetical order)

cm centimeter

ESI- Negative Electrospray Ionization

g gram

h hour

ID inside diameter
Draft PFC Procedure 30 December 2011

Not approved for either general purpose or regulatory use

in. inch

L liter

M molecular ion

m meter

mg milligram

min minute

mL milliliter

mm millimeter

m/z mass-to-charge ratio

N normal; gram molecular weight of solute divided by hydrogen equivalent of solute, per liter of 

solution

NRR Normalized relative response

OD outside diameter

pg picogram

ppb part-per-billion

ppm part-per-million

ppq part-per-quadrillion

ppt part-per-trillion

psig pounds-per-square inch gauge

v/v volume per unit volume

w/v weight per unit volume

Definitions and acronyms (in alphabetical order)

Analyte –A perfluorinated compound tested for by this method. The analytes are listed in Table 1.

Calibration standard (CAL) – A solution prepared from a secondary standard and/or stock solution and 

used to calibrate the response of the HPLC/MS/MS instrument. Referred to as CS-1, CS-2, CS-3, CS-4, 

CS-5, CS-6.

Calibration verification standard (CALVER) – A calibration standard close to the mid-point calibration 

standard that is used to verify calibration. 

HPLC – High performance liquid chromatograph or high performance liquid chromatography

ICAL – Initial calibration

Internal standard quantitation – A means of determining the concentration of (1) a naturally occurring 

(native) compound by reference to a compound other than its labeled analog and (2) a labeled compound 

by reference to another labeled compound.

IPR – Initial precision and recovery; four aliquots of a reference matrix spiked with the analytes of 

interest and labeled compounds and analyzed to establish the ability of the laboratory to generate 

acceptable precision and recovery. An IPR is performed prior to the first time this method is used and 

any time the method or instrumentation is modified.

Isotope dilution quantitation – A means of determining a naturally occurring (native) compound by 

reference to the same compound in which one or more atoms has been isotopically enriched. In this 

method, labeled compounds are enriched with deuterium to produce 2

H-labeled analogs or carbon-13 to 

produce 13C-labeled analogs. The labeled analogs are spiked into each sample to allow identification and 

correction of the concentration of the native compounds in the extraction, cleanup and the analytical 

process.
Draft PFC Procedure 31 December 2011

Not approved for either general purpose or regulatory use

Labeled compound, or labeled analog – A molecule in which one or more of the atoms is isotopically 

enriched, thereby increasing the mass of the molecule. As used in this procedure, all isotopic labels are 

stable (non-radioactive).

Labeled injection standard – A labeled compound used as a reference for quantitation of other labeled 

compounds and for quantitation of a native compound for which there is not an exact labeled analog. This 

compound is spiked into the sample extract prior to instrumental analysis. 

Labeled internal standard – A labeled compound used as a reference for quantitation of native 

compounds. This compound is spiked into the sample prior to extraction.

Method blank – An aliquot of a reference matrix that is treated exactly as a sample including exposure to 

all glassware, equipment, solvents, reagents, internal standards, and surrogates that are used with samples. 

The method blank is used to determine if analytes or interferences are present in the laboratory 

environment, the reagents, or the apparatus.

Method detection limit (MDL) – The minimum concentration of a substance that can be measured and 

reported with 99% confidence that the analyte concentration is greater than zero and is determined from 

analysis of a sample in a given matrix containing the analyte (see 40 CFR 136, appendix B).

Minimum level (ML) – The greater of a multiple of the MDL or the lowest calibration point (see 68 FR 

11790, March 12, 2003). 

MS – Mass spectrometer or mass spectrometry

Native compound – A molecule in which the atoms all have naturally occurring isotopic abundances

OPR – Ongoing precision and recovery standard (OPR); an aliquot of a reference matrix spiked with 

known quantities of analytes. Also known as a “laboratory control sample” (LCS). The OPR is analyzed 

exactly like a sample. Its purpose is to assure that the results produced by the laboratory remain within 

the limits specified in this method for precision and recovery.

Reagent water – water demonstrated to be free from the analytes of interest and potentially interfering 

substances at the method detection limit for the analyte.

Relative standard deviation (RSD) – The standard deviation times 100, divided by the mean. Also termed 

the “coefficient of variation.”

Relative percent difference (RPD) – The absolute difference between two values, divided by the mean of 

the two values. Used to compare results when there is no true value.

Signal-to-noise ratio (S/N) – The height of the signal as measured from the mean (average) of the noise to 

the peak maximum divided by the width of the noise.

SPE – Solid-phase extraction; an extraction technique in which an analyte is extracted from an aqueous 

solution by passage over or through a material capable of reversibly adsorbing the analyte. Also termed 

liquid-solid extraction. SPE is used in this procedure as a cleanup technique.
Draft PFC Procedure 32 December 2011

Not approved for either general purpose or regulatory use

20.0 Tables and figures

Table 1. Names and CAS Registry numbers for PFCs determined by isotope dilution and internal standard

Compound

CAS 

Number Formula Acronym

Native PFCs

Perfluoorobutanoic acid 375-22-4 CF3(CF2)2COOH PFBA

Perfluoropentanoic acid 2706-90-3 CF3(CF2)3COOH PFPA

Perfluoro-n-hexanoic acid 307-24-4 CF3(CF2)4COOH PFHxA (C6)

Perfluoro-n-heptanoic acid 375-85-9 CF3(CF2)5COOH PFHpA (C7)

Perfluoro-n-octanoic acid 335-67-1 CF3(CF2)6COOH PFOA (C8)

Perfluoro-n-nonanoic acid 375-95-1 CF3(CF2)7COOH PFNA (C9)

Perfluoro-n-decanoic acid 335-76-2 CF3(CF2)8COOH PFDA (C10)

Perfluoro-n-undecanoic acid 2058-94-8 CF3(CF2)9COOH PFUnDA (C11)

Perfluoro-n-dodecanoic acid 307-55-1 CF3(CF2)10COOH PFDoDA (C12)

Perfluoro-n-tridecanoic acid 72629-94-8 CF3(CF2)11COOH PFTriDA (C13)

Perfluoro-n-tetradecanoic acid 376-06-7 CF3(CF2)12COOH PFTeDA (C14)

Perfluorobutanesulfonic acid 375-73-5 CF3(CF2)3SO3H PFBS (S4)

Perfluorohexanesulfonic acid 355-46-4 CF3(CF2)5SO3H PFHxS (S6)

Perfluoro-n-heptanesulfonic acid 375-92-8 CF3(CF2)6SO3H PFHpS (S7)

Perfluorooctanesulfonic acid 1763-23-1 CF3(CF2)7SO3H PFOS (S8)

Perfluorooctane sulfonamide 754-91-6 CF3(CF2)6SO2NH2 PFOSA (S8)

N-methylperfluoro-1-octanesulfonamide 31506-32-8 CF3(CF2)7SO2N.H.CH3 N-MeFOSA

N-ethylperfluoro-1-octanesulfonamide 4151-50-2 CF3(CF2)7SO2N.H.C2H5 N-EtFOSA

2-(N-methylperfluoro-1-octanesulfonamido)-

ethanol 24448-09-7 CF3(CF2)7SO2N. CH3.C2H4OH N-MeFOSE

2-(N-ethylperfluoro-1-octanesulfonamido)-

ethanol 1691-99-2 CF3(CF2)7SO2N. C2H5.C2H4OH N-EtFOSE

Labeled Internal Standards

Perfluoro-n-[1,2,3,4-

13C4]butanoic acid 13CF3(13CF2)213COOH [13C4]PFBA-(MPFBA)

Perfluoro-n-[1,2-

13C2]hexanoic acid CF3(CF2)3(13CF2)13COOH [13C2]PFHxA-(MPFHxA)

Perfluoro-n-[1,2,3,4-

13C4]octanoic acid CF3(CF2)3(13CF2)313COOH [13C4]PFOA-(M4PFOA)

Perfluoro-n-[1,2,3,4,5-

13C5]nonanoic acid CF3(CF2)3(13CF2)413COOH [13C5]PFNA-(MPFNA)

Perfluoro-n-[1,2-

13C2]decanoic acid CF3(CF2)7(13CF2)13COOH [13C2]PFDA-(M2PFDA)

Perfluoro-n-[1,2,3,4,5,6,7,8,9-

13C9]decanoic 

acid CF3( 13CF2)8

13COOH [13C9]PFDA-(M9PFDA)

Perfluoro-n-[1,2-

13C2]undecanoic acid CF3(CF2)8(13CF2)13COOH 

[

13C2]PFUnDA-

(M2PFUnDA)

Perfluoro-n-[2,3,4,5,6,7,8,9,10-

13C9] undecanoic 

acid CF3(

13CF2)9COOH 

[

13C9]PFUnDA-

(M9PFUnDA)

Perfluoro-n-[1,2-

13C2]dodecanoic acid CF3(CF2)9(13CF2)13COOH [13C2]PFDoDA-MPFDoDA)

Perfluoro-1-[1,2-

18O2]-hexanesulfonic acid CF3(CF2)5SO18O2H [18O2]PFOS-(MPFHxS)

Perfluoro-n-[1,2,3,4-

13C4]-octanesulfonic acid CF3(CF2)3(13CF2)4SO3H [13C4]PFOS-(MPFOS)

2-(N-deuteriomethylperfluoro-1-octane

sulfonamido)-1,1,2,2-tetradeuterioethanol CF3(CF2)7SO2N. CD3.C2D4OH d7-N-MeFOSE

Injection Internal Standards 

Perfluoro-n-[1,2,3,4,5,6,7,8-

13C8]octanoic acid 13CF3(13CF2)613COOH [13C8]PFOA-(M8PFOA)

2H-Perfluoro-[1,2-

13C2]-2-decenoic acid CF3(CF2)6CF13CH13COOH [13C2]PFOUEA-MPFOUEA)
Draft PFC Procedure 33 December 2011

Not approved for either general purpose or regulatory use

Table 2. Typical retention times (RT), relative retention times (RRT), and RT windows

Compound

Mean RT in 

Calibration 

Standard(1)

Standard 

Deviation

of RT

Mean RT 

in 

Biosolids(2)

Standard 

Deviation 

of RT

RT 

Window

Mean RRT in 

a Calibration 

Standard(1)

Standard 

Deviation 

of RRT

Mean RRT

in 

Biosolids(2)

Standard 

deviation 

RRT

RRT

Window

Native PFCs

Perfluoorobutanoic acid 5.22 0.025 5.24 0.036 ± 0.3 1.000 0.0026 1.008 0.0116 ± 0.05

Perfluoropentanoic acid 6.36 0.016 6.37 0.016 ± 0.1 0.725 0.0018 0.723 0.0018 ± 0.05

Perfluoro-n-hexanoic acid 7.06 0.016 7.07 0.015 ± 0.1 1.001 0.0018 1.000 0.0000 ± 0.01

Perfluoro-n-heptanoic acid 7.81 0.014 7.82 0.020 ± 0.1 0.891 0.0015 0.887 0.0022 ± 0.05

Perfluoro-n-octanoic acid 8.78 0.026 8.80 0.016 ± 0.1 1.000 0.0000 1.000 0.0000 ± 0.01

Perfluoro-n-nonanoic acid 10.01 0.036 10.05 0.048 ± 0.5 1.001 0.0027 1.002 0.0039 ± 0.01

Perfluoro-n-decanoic acid 11.50 0.026 11.48 0.040 ± 0.5 1.002 0.0017 1.000 0.0000 ± 0.01

Perfluoro-n-undecanoic acid 13.16 0.030 13.02 0.113 ± 0.5 1.001 0.0013 1.001 0.0014 ± 0.01

Perfluoro-n-dodecanoic acid 15.12 0.032 14.99 0.168 ± 0.5 1.000 0.0009 1.001 0.0010 ± 0.01

Perfluoro-n-tridecanoic acid 17.48 0.038 17.46 0.073 ± 0.5 1.993 0.0043 1.981 0.0083 ± 0.05

Perfluoro-n-tetradecanoic acid 20.56 0.072 20.64 0.100 ± 0.5 2.344 0.0082 2.343 0.0114 ± 0.05

Perfluorobutanesulfonic acid 7.20 0.018 7.21 0.016 ± 0.1 0.821 0.0020 0.819 0.0018 ± 0.05

Perfluoro-n-hexane sulfonic acid 9.25 0.019 9.25 0.016 ± 0.1 1.001 0.0020 1.000 0.0000 ± 0.01

Perfluoro-n-heptane sulfonic acid 10.61 0.032 10.65 0.029 ± 0.5 1.210 0.0037 1.209 0.0032 ± 0.05

Perfluoro-n-octanesulfonic acid 12.20 0.047 12.15 0.073 ± 0.5 1.002 0.0017 1.001 0.0013 ± 0.01

Perfluorooctane sulfonamide 17.56 0.032 17.52 0.064 ± 0.5 2.002 0.0036 1.988 0.0073 ± 0.05

N-methylperfluoro-1-octanesulfonamide 23.15 0.063 23.07 0.096 ± 0.5 2.640 0.0072 2.619 0.0109 ± 0.05

N-ethylperfluoro-1-octanesulfonamide 24.91 0.039 24.95 0.031 ± 0.5 2.841 0.0045 2.832 0.0036 ± 0.05

2-(N-methylperfluoro-1-octane

sulfonamido)-ethanol 21.53 0.057 21.36 0.127 ± 0.5 1.009 0.0024 1.005 0.0022 ± 0.01

2-(N-ethylperfluoro-1-octane sulfonamido)-

ethanol 24.27 0.034 24.22 0.070 ± 0.5 2.767 0.0039 2.749 0.0080 ± 0.05

Mass-labeled PFCs

Perfluoro-n-[1,2,3,4-

13C4]butanoic acid 5.22 0.028 5.20 0.084 ±0.3 0.595 0.0032 0.590 0.0096 ± 0.05

Perfluoro-n-[1,2-

13C2]hexanoic acid 7.05 0.011 7.07 0.015 ± 0.1 0.804 0.0012 0.802 0.0018 ± 0.05

Perfluoro-n-[1,2,3,4-

13C4]octanoic acid 8.78 0.026 8.80 0.016 ± 0.1 1.001 0.0029 0.999 0.0019 ± 0.01

Perfluoro-n-[1,2,3,4,5-

13C5]nonanoic acid 9.99 0.038 10.03 0.036 ± 0.1 1.139 0.0043 1.138 0.0041 ± 0.05

Perfluoro-n-[1,2-

13C2]decanoic acid 11.49 0.033 11.48 0.040 ± 0.5 1.310 0.0037 1.303 0.0045 ± 0.05

Perfluoro-n-[1,2,3,4,5,6,7,8,9-

13C9]decanoic 

acid 11.48 0.034 11.48 0.040 ± 0.5 1.309 0.0039 1.303 0.0045 ± 0.05

Perfluoro-n-[1,2-

13C2]undecanoic acid 13.15 0.029 13.06 0.069 ± 0.5 1.499 0.0033 1.482 0.0079 ± 0.05
Draft PFC Procedure 34 December 2011

Not approved for either general purpose or regulatory use

Table 2. Typical retention times (RT), relative retention times (RRT), and RT windows

Compound

Mean RT in 

Calibration 

Standard(1)

Standard 

Deviation

of RT

Mean RT 

in 

Biosolids(2)

Standard 

Deviation 

of RT

RT 

Window

Mean RRT in 

a Calibration 

Standard(1)

Standard 

Deviation 

of RRT

Mean RRT

in 

Biosolids(2)

Standard 

deviation 

RRT

RRT

Window

Perfluoro-n-[2,3,4,5,6,7,8,9,10- 13C9]undecanoic acid 13.15 0.031 13.01 0.124 ± 0.5 1.499 0.0035 1.477 0.0141 ± 0.05

Perfluoro-n-[1,2-

13C2]dodecanoic acid 15.11 0.025 14.98 0.162 ± 0.5 1.723 0.0029 1.700 0.0184 ± 0.05

Perfluoro-1-[1,2-

18O2]-hexanesulfonic acid 9.24 0.021 9.25 0.016 ± 0.1 1.054 0.0024 1.050 0.0019 ± 0.05

Perfluoro-n-[1,2,3,4-

13C4]-octanesulfonate 12.18 0.032 12.14 0.083 ± 0.5 1.389 0.0036 1.378 0.0095 ± 0.05

2-(N-deuteriomethylperfluoro-1-octane

sulfonamido)-1,1,2,2-tetradeuterioethanol 21.34 0.071 21.26 0.094 ± 0.5 2.433 0.0081 2.414 0.0106 ± 0.05

Injection Internal Standards (compound added after extraction, but prior to injection)

Perfluoro-n-[1,2,3,4,5,6,7,8-

13C8]octanoic 

acid 8.78 0.026 8.80 0.016 ± 0.1 -- -- -- -- --

2H-Perfluoro-[1,2-

13C2]-2-decenoic acid 9.64 0.037 9.65 0.016 ± 0.1 -- -- -- -- --
Draft PFC Procedure 35 December 2011

Not approved for either general purpose or regulatory use

Table 3. Analytes, ions, quantification references, and instrument conditions

Compound

Precursor

Ion (m/z)

Quant 

Ion 

(m/z)

Precursor 

formula

Primary Quant 

Ion formula

Cone 

Voltage 

(V)

Collision 

(eV)

2nd Qual 

Ion Mass 

(m/z)

2nd Qual Ion 

formula

Collision 

(eV)

Quant 

by: Quantitation Reference

Native PFCs

Perfluoorobutanoic acid 213 169 [CF3(CF2)2CO2]

- [CF3(CF2)2]- 27 8 ID 13CF3(13CF2)213COOH

Perfluoropentanoic acid 263 219 [CF3(CF2)3CO2]

- [CF3(CF2)3]- 27 8 IS 13CF3(13CF2)613COOH

Perfluoro-n-hexanoic acid 313 269 [CF3(CF2)4CO2]

- [CF3(CF2)4]- 27 20 119 [CF3CF2]- 8 ID CF3(CF2)3(13CF2)13COOH

Perfluoro-n-heptanoic acid 363 319 [CF3(CF2)5CO2]

- [CF3(CF2)5]- 27 12 169 [CF3(CF2)2]- 8 IS 13CF3(13CF2)613COOH

Perfluoro-n-octanoic acid 413 369 [CF3(CF2)6CO2]

- [CF3(CF2)6]- 19 12 169 [CF3(CF2)2]- 12 ID CF3(CF2)3(

13CF2)3 13COOH

Perfluoro-n-nonanoic acid 463 419 [CF3(CF2)7CO2]

- [CF3(CF2)7]- 20 13 219 [CF3(CF2)3]- 12 ID CF3(CF2)3(

13CF2)4 13COOH

Perfluoro-n-decanoic acid 513 469 [CF3(CF2)8CO2]

- [CF3(CF2)8]- 21 11 219 [CF3(CF2)3]- 13 ID CF3( 13CF2)813COOH

Perfluoro-n-undecanoic 

acid 563 519 [CF3(CF2)9CO2]

- [CF3(CF2)9]- 21 15 269 [CF3(CF2)4]- 12 ID CF3( 13CF2)9COOH 

Perfluoro-n-dodecanoic 

acid 613 569 [CF3(CF2)10CO2]

- [CF3(CF2)10]- 22 15 319 [CF3(CF2)5]- 12 ID CF3(CF2)9( 13CF2)13COOH

Perfluoro-n-tridecanoic 

acid 663 619 [CF3(CF2)11CO2]

- [CF3(CF2)11]- 20 17 319 [CF3(CF2)5]- 13 IS 13CF3(13CF2)6

13COOH

Perfluoro-n-tetradecanoic 

acid 713 669 [CF3(CF2)12CO2]

- [CF3(CF2)12]- 27 21 319 [CF3(CF2)5]- 11 IS 13CF3(13CF2)6

13COOH

Perfluorobutanesulfonic 

acid 299 80 [CF3(CF2)3SO3]

- [SO3]- 70 40 99 [FSO3]- 35 IS 13CF3(13CF2)6

13COOH

Perfluoro-n-hexane 

sulfonic acid 1 399 80 [CF3(CF2)5SO3]

- [SO3]- 30 45 99 [FSO3]- 40 ID CF3(CF2)5S(18O)2OH

Perfluoro-n-heptane 

sulfonic acid 449 80 [CF3(CF2)6SO3]

- [SO3]- 50 39 99 [FSO3]- 38 IS 13CF3(13CF2)6

13COOH

Perfluoro-n-octanesulfonic 

acid 499 80 [CF3(CF2)7SO3]

- [SO3]- 80 45 99 [FSO3]- 40 ID CF3(CF2)3(13CF2)4SO3H

Perfluorooctane 

sulfonamide 498 78 [CF3(CF2)7SO2NH]- [SO2N]- 80 40 478 [(CF2)8SO2N]- 16 IS 13CF3(

13CF2)6

13COOH

N-methylperfluoro-1-

octanesulfonamide 512 169 [CF3(CF2)7SO2 N(CH3) ]

- [CF3(CF2)2]

- 27 45 IS 13CF3(13CF2)6

13COOH

N-ethylperfluoro-1-

octanesulfonamide 526 169 [CF3(CF2)7SO2 N(C2H5) ]

- [CF3(CF2)2]

- 27 45 IS 13CF3(13CF2)6

13COOH

2-(N-methylperfluoro-1-

octanesulfonamido)-ethanol 616 59

[CF3(CF2)7SO2N(

CH3)C2H4OH·CH

3CO2]

-

[CH3CO2]

- 27 45 ID CF3(CF2)7SO2N

(CD3)C2D4OH·CH3COOH

2-(N-ethylperfluoro-1-

octanesulfonamido)-ethanol 630 59

[CF3(CF2)7SO2N(

C2H5)C2H4OH·C

H3CO2]

-

[CH3CO2]

- 27 45 IS 13CF3(13CF2)6

13COOH
Draft PFC Procedure 36 December 2011

Not approved for either general purpose or regulatory use

Table 3. Analytes, ions, quantification references, and instrument conditions

Compound

Precursor

Ion (m/z)

Quant 

Ion 

(m/z)

Precursor 

formula

Primary Quant 

Ion formula

Cone 

Voltage 

(V)

Collision 

(eV)

2nd Qual 

Ion Mass 

(m/z)

2nd Qual Ion 

formula

Collision 

(eV)

Quant 

by: Quantitation Reference

Mass-labeled PFCs

Perfluoro-n-[1,2,3,4- 13C4]butanoic acid 217 172 [

13CF3(13CF2)2 13CO2]

- [

13CF3(13CF2)2]- 27 8 IS 13CF3(13CF2)6

13COOH

Perfluoro-n-[1,2- 13C2]hexanoic acid 315 270 [CF3(CF2)3(

13CF2) 13CO2]

-

[CF3(CF2)3(

13CF

2) ]- 27 8 IS 13CF3(

13CF2)6

13COOH

Perfluoro-n-[1,2,3,4-

13

C4]octanoic acid 417 372 [CF3(CF2)3(

13CF2)313CO2]-

[CF3(CF2)3

(

13CF2)3]- 21 12 IS 13CF3(

13CF2)6

13COOH

Perfluoro-n-[1,2,3,4,5-

13

C5]nonanoic acid 468 423 [CF3(CF2)3(

13CF2)413CO2]-

[CF3(CF2)3(

13CF

2)4]

- 20 12 IS 13CF3(

13CF2)6

13COOH

Perfluoro-n-[1,2- 13C2]decanoic acid 515 470 [CF3(CF2)7(

13CF2) 13CO2]

-

[CF3(CF2)2(

13CF

2) ]- 21 12 IS 13CF3(

13CF2)6

13COOH

Perfluoro-n-

[1,2,3,4,5,6,7,8,9- 13C9]decanoic acid

522 477 [CF3( 13CF2)8 13CO2]

- [CF3( 13CF2)8]

- 20 12 IS 13CF3(13CF2)6

13COOH

Perfluoro-n-[1,2- 13C2]undecanoic acid 565 520 [CF3(CF2)8(

13CF2) 13CO2]

-

[CF3(CF2)8(

13CF

2) ]- 20 12 IS 13CF3(

13CF2)6

13COOH

Perfluoro-n-

[2,3,4,5,6,7,8,9,10- 13C9]undecanoic acid572 528 [CF3( 13CF2)9CO2]

- [CF3( 13CF2)9]

- 20 12 IS 13CF3(13CF2)6

13COOH

Perfluoro-n-[1,2- 13C2]dodecanoic acid 615 570 [CF3(CF2)9

( 13CF2)

13CO2]-

[CF3(CF2)9

( 13CF2) ]- 22 12 IS 13CF3(

13CF2)6

13COOH

Perfluoro-1-[1,2-

18 O2]-

hexanesulfonic acid 403 84 [CF3(CF2)5S(18O)2O]- [S(18O)2O]- 30 45 103 [FS(18O)2 O]- 45 IS 13CF3(

13CF2)6

13COOH

Perfluoro-n-[1,2,3,4-

13 C4]-

octanesulfonate 503 80 [CF3(CF2)3(

13CF2)4SO3]- [SO3]

- 40 45 99 [FSO3]- 40 IS 13CF3(13CF2)6

13COOH

2-(Ndeuteriomethylperfluoro-1-

octanesulfonamido)-

1,1,2,2-tetradeuterioethanol

623 59

[CF3(CF2)7SO2N

(CD3)C2D4OH·C

H3CO2]

-

[CH3CO2]

- 27 45 IS 13CF3(13CF2)6

13COOH

Injection Internal Standards (compound added after extraction, but prior to injection)

Perfluoro-n-

[1,2,3,4,5,6,7,8-

13

C8]octanoic acid

421 376 [

13CF3( 13CF2)6 13CO2]

- [

13CF3( 3

CF2)6]

- 21 12 ES

2H-Perfluoro-[1,2-

13C2]-2-

decenoic acid 459 394 [CF3(CF2)6CF13CH13CO2]

-

[CF3(CF2)3

(

13CF2)3]- 21 11 ES

1 This analyte has a third ion that can be used for qualitative identification. The m/z of that ion is 119, the formula is CF3CF2-, and the collision energy is 30 eV.
Draft PFC Procedure 37 December 2011

Not approved for either general purpose or regulatory use

Table 4. Method detection limits, minimum levels of quantitation, and provisional health advisory

Compound MDL (ng/g) ML (ng/g)

OW Provisional Health 

Advisory

Perfluoorobutanoic acid 0.125 0.250

Perfluoropentanoic acid 0.185 0.250

Perfluoro-n-hexanoic acid 0.136 0.250

Perfluoro-n-heptanoic acid 0.054 0.250

Perfluoro-n-octanoic acid 0.085 0.250 0.4 ppb

Perfluoro-n-nonanoic acid 0.127 0.250

Perfluoro-n-decanoic acid 0.084 0.250

Perfluoro-n-undecanoic acid 0.080 0.250

Perfluoro-n-dodecanoic acid 0.067 0.250

Perfluoro-n-tridecanoic acid 0.067 0.250

Perfluoro-n-tetradecanoic acid 0.028 0.250

Perfluorobutanesulfonic acid 0.115 0.500

Perfluorohexanesulfonic acid 0.268 0.500

Perfluoroheptanesulfonic acid 0.291 0.500

Perfluorooctanesulfonic acid 0.208 0.500 0.2 ppb

Perfluorooctane sulfonamide 0.040 0.250

N-methylperfluoro-1-octanesulfonamide 3.65 10.0

N-ethylperfluoro-1-octanesulfonamide 3.05 10.0

2-(N-methylperfluoro-1-octanesulfonamido)-ethanol 7.65 5.00

2-(N-ethylperfluoro-1-octanesulfonamido)-ethanol 1.88 5.00
Draft PFC Procedure 38 December 2011

Not approved for either general purpose or regulatory use

Table 5. Instrument conditions

Instruments Waters Acquity high performance liquid chromatography (HPLC) or equivalent

Waters Quattro Ultima tandem mass spectrometer or equivalent

Trapping cartridge NA

HPLC Column Waters Xtera C18MS analytical column, 100 mm length, 2.1 mm ID, 3.5 μm 

particle size, or equivalent

Ionization Negative Ion Electrospray

Acquisition MRM mode, unit resolution

Injection Volume 15 μL

Final Extract Volume 1 mL

HPLC Gradient Program HPLC Flow 

rate 

Gradient 

Curve

HPLC conditions

Time 

(min) Flow mixture (mL/min)

0.00 A=15% 

B=85% 0.150 1 Column Temp (°C) 40

1.00 A=15% 

B=85% 0.150 1 Max Pressure (bar) 345

5.00 A=50% 

B=50% 0.200 4

MS Conditions

20.00 A=65% 

B=35% 0.200 4

23.00 A=100% 

B=0% 0.200 4

26.00 A=100% 

B=0% 0.200 4

26.50 A=15% 

B=85% 0.200 2 Source Temp (°C) 120

30.00 A=15% 

B=85% 0.200 2 Desolvation Temp (oC) 325

32.00 A=15% 

B=85% 0.150 2 Capillary voltage (kV) 3.50

Solvent A = 90% ACN:10% water (organic phase) 

Solvent B = 12.1 mM ammonium acetate and 0.1% acetic acid in water (aqueous phase)
Draft PFC Procedure 39 December 2011

Not approved for either general purpose or regulatory use

Table 6. Concentrations of calibration standards (CS) in pg/mL

CS-1 CS-2 CS-3 CS-4 (VER) CS-5 CS-6 CS-7 CS-8

Native Analytes

Perfluoorobutanoic acid 125 312.5 1,250 5,000 25,000 50,000 125,000 312,500

Perfluoropentanoic acid 125 312.5 1,250 5,000 25,000 50,000 125,000 312,500

Perfluoro-n-hexanoic acid 125 312.5 1,250 5,000 25,000 50,000 125,000 312,500

Perfluoro-n-heptanoic acid 125 312.5 1,250 5,000 25,000 50,000 125,000 312,500

Perfluoro-n-octanoic acid 125 312.5 1,250 5,000 25,000 50,000 125,000 312,500

Perfluoro-n-nonanoic acid 125 312.5 1,250 5,000 25,000 50,000 125,000 312,500

Perfluoro-n-decanoic acid 125 312.5 1,250 5,000 25,000 50,000 125,000 312,500

Perfluoro-n-undecanoic acid 125 312.5 1,250 5,000 25,000 50,000 125,000 312,500

Perfluoro-n-dodecanoic acid 125 312.5 1,250 5,000 25,000 50,000 125,000 312,500

Perfluoro-n-tridecanoic acid 125 312.5 1,250 5,000 25,000 50,000 125,000 312,500

Perfluoro-n-tetradecanoic acid 125 312.5 1,250 5,000 25,000 50,000 125,000 312,500

Perfluorobutanesulfonic acid 250 625 2,500 10,000 50,000 100,000 250,000 625,000

Perfluorohexanesulfonic acid 250 625 2,500 10,000 50,000 100,000 250,000 625,000

Perfluoro-n-heptanesulfonic acid 250 625 2,500 10,000 50,000 100,000 250,000 625,000

Perfluorooctanesulfonic acid 250 625 2,500 10,000 50,000 100,000 250,000 625,000

Perfluorooctane sulfonamide 125 312.5 1,250 5,000 25,000 50,000 125,000 312,500

N-methylperfluoro-1-octanesulfonamide 5,000 10,000 20,000 40,000 80,000 160,000 320,000 --

N-ethylperfluoro-1-octanesulfonamide 5,000 10,000 20,000 40,000 80,000 160,000 320,000 --

2-(N-methylperfluoro-1-octanesulfonamido)-ethanol 2,500 5,000 10,000 20,000 40,000 80,000 160,000 --

2-(N-ethylperfluoro-1-octanesulfonamido)-ethanol 2,500 5,000 10,000 20,000 40,000 80,000 160,000 --

Labeled Internal Standards

Perfluoro-n-[1,2,3,4-

13C4]butanoic acid 3,000 3,000 3,000 3,000 3,000 3,000 3,000 3,000

Perfluoro-n-[1,2-

13C2]hexanoic acid 3,000 3,000 3,000 3,000 3,000 3,000 3,000 3,000

Perfluoro-n-[1,2,3,4-

13C4]octanoic acid 3,000 3,000 3,000 3,000 3,000 3,000 3,000 3,000

Perfluoro-n-[1,2,3,4,5-

13C5]nonanoic acid 3,000 3,000 3,000 3,000 3,000 3,000 3,000 3,000

Perfluoro-n-[1,2-

13C2]decanoic acid 3,000 3,000 3,000 3,000 3,000 3,000 3,000 3,000

Perfluoro-n-[1,2,3,4,5,6,7,8,9-

13C9]decanoic acid 3,000 3,000 3,000 3,000 3,000 3,000 3,000 3,000

Perfluoro-n-[1,2-

13C2]undecanoic acid 3,000 3,000 3,000 3,000 3,000 3,000 3,000 3,000

Perfluoro-n-[2,3,4,5,6,7,8,9,10-

13C9]undecanoic acid 3,000 3,000 3,000 3,000 3,000 3,000 3,000 3,000

Perfluoro-n-[1,2-

13C2]dodecanoic acid 3,000 3,000 3,000 3,000 3,000 3,000 3,000 3,000

Perfluoro-1-[1,2-

18O2]-hexanesulfonic acid 4,270 4,270 4,270 4,270 4,270 4,270 4,270 4,270

Perfluoro-n-[1,2,3,4-

13C4]-octanesulfonic acid 4,310 4,310 4,310 4,310 4,310 4,310 4,310 4,310

2-(N-deuteriomethylperfluoro-1-octanesulfonamido)-1,1,2,2-

tetradeuterioethanol 20,000 20,000 20,000 20,000 20,000 20,000 20,000

Internal Injection Standards

Perfluoro-n-[1,2,3,4,5,6,7,8-

13C8]octanoic acid 3,000 3,000 3,000 3,000 3,000 3,000 3,000 3,000

2H-Perfluoro-[1,2-

13C2]-2-decenoic acid 2,500 2,500 2,500 2,500 2,500 2,500 2,500 2,500 1Calibration standards are stored in ~ 92% MeOH, up to 2.75% Nonane, 2% Propan-2-ol, 1%MeCN, 1% H2O, up to 0.3% Toluene, 0.3% NH4OH solution.
Draft PFC Procedure 40 December 2011

Not approved for either general purpose or regulatory use

Table 7. Detailed concentrations of actual working level standards (pg/mL)

Working-level Standards (pg/mL)

Native standards Working level Low level

Perfluoorobutanoic acid 125,000 12,500

Perfluoropentanoic acid 125,000 12,500

Perfluoro-n-hexanoic acid 125,000 12,500

Perfluoro-n-heptanoic acid 125,000 12,500

Perfluoro-n-octanoic acid 125,000 12,500

Perfluoro-n-nonanoic acid 125,000 12,500

Perfluoro-n-decanoic acid 125,000 12,500

Perfluoro-n-undecanoic acid 125,000 12,500

Perfluoro-n-dodecanoic acid 125,000 12,500

Perfluoro-n-tridecanoic acid 125,000 12,500

Perfluoro-n-tetradecanoic acid 125,000 12,500

Perfluorobutanesulfonic acid 250,000 25,000

Perfluorohexanesulfonic acid 250,000 25,000

Perfluoro-n-heptanesulfonic acid 250,000 25,000

Perfluorooctanesulfonic acid 250,000 25,000

Perfluorooctane sulfonamide 125,000 12,500

N-Me/Et-FOSA/Es Natives (NAT2)

N-methylperfluoro-1-octanesulfonamide 2,500,000

N-ethylperfluoro-1-octanesulfonamide 2,500,000

2-(N-methylperfluoro-1-octanesulfonamido)-ethanol 1,250,000

2-(N-ethylperfluoro-1-octanesulfonamido)-ethanol 1,250,000

Labeled Internal Standard Solution

Mass Labeled PFCs

Perfluoro-n-[1,2,3,4-

13C4]butanoic acid 30,000

Perfluoro-n-[1,2-

13C2]hexanoic acid 30,000

Perfluoro-n-[1,2,3,4-

13 C4]octanoic acid 30,000

Perfluoro-n-[1,2,3,4,5-

13 C5]nonanoic acid 30,000

Perfluoro-n-[1,2-

13C2]decanoic acid 30,000

Perfluoro-n-[1,2-

13C9]decanoic acid 30,000

Perfluoro-n-[1,2-

13C2]undecanoic acid 30,000

Perfluoro-n-[1,2-

13C9]undecanoic acid 30,000

Perfluoro-n-[1,2-

13C2]dodecanoic acid 30,000

Perfluoro-1-[1,2-

18 O2]-hexanesulfonic acid 42,700

Perfluoro-n-[1,2,3,4-

13 C4]-octanesulfonic acid 43,100

2-(N-deuteriomethylperfluoro-1-octanesulfonamido)-1,1,2,2-tetradeuterioethanol 200,000

Labeled Injection Standards

Perfluoro-n-[1,2,3,4,5,6,7,8-

13 C8]octanoic acid 12,000

2H-Perfluoro-[1,2-

13 C2]-2-decenoic acid 10,000
Draft PFC Procedure 41 December 2011

Not approved for either general purpose or regulatory use

Table 8. QC acceptance criteria for analytes, labeled compounds and internal standards in VER, IPR, OPR, matrix spikes, and samples

Compound Quant

ICAL and CALVER 

Recovery (%) IPR Recovery (%)

OPR 

Recovery (%)

RLS Recovery 

(%)

Recovery in Matrix 

Spikes and Samples

Low High RSD Low High RSD Low High Low High Low High RPD

Native PFCs

Perfluoorobutanoic acid ID 70 130 20 70 130 20 70 130 50 200 50 150 30

Perfluoropentanoic acid IS 70 130 20 70 130 20 70 130 50 200 50 150 30

Perfluoro-n-hexanoic acid ID 70 130 20 70 130 20 70 130 50 150 50 150 30

Perfluoro-n-heptanoic acid IS 70 130 20 70 130 20 70 130 50 150 50 150 30

Perfluoro-n-octanoic acid ID 70 130 20 70 130 20 70 130 50 150 50 150 30

Perfluoro-n-nonanoic acid ID 70 130 20 50 150 30 50 150 50 150 50 150 30

Perfluoro-n-decanoic acid ID 70 130 20 70 130 20 70 130 50 150 50 150 30

Perfluoro-n-undecanoic acid ID 70 130 20 70 130 20 70 130 50 150 50 150 30

Perfluoro-n-dodecanoic acid ID 70 130 20 70 130 20 70 130 50 150 50 150 30

Perfluoro-n-tridecanoic acid IS 70 130 20 20 130 30 20 130 50 150 50 150 30

Perfluoro-n-tetradecanoic acid IS 70 130 20 20 130 30 20 130 50 150 50 150 30

Perfluorobutanesulfonic acid IS 70 130 20 60 130 20 60 130 50 150 50 150 30

Perfluorohexanesulfonic acid IS 70 130 20 70 130 20 70 130 50 150 50 150 30

Perfluoroheptanesulfonic acid ID 70 130 20 70 130 20 70 130 50 150 50 150 30

Perfluorooctanesulfonic acid ID 70 130 20 70 130 20 70 130 50 150 50 150 30

Perfluorooctane sulfonamide IS 70 130 20 20 130 30 20 130 50 150 50 150 30

N-methylperfluoro-1-octanesulfonamide IS 70 130 20 5 130 20 5 130 10 150 5 150 40

N-ethylperfluoro-1-octanesulfonamide IS 70 130 20 5 130 40 5 130 10 150 5 150 40

2-(N-methylperfluoro-1-octanesulfonamido)-ethanol ID 70 130 20 40 130 30 40 130 50 150 50 150 30

2-(N-ethylperfluoro-1-octanesulfonamido)-ethanol IS 70 130 20 10 130 40 10 130 25 150 10 150 40

Labeled Internal Standards: Compounds added before extraction

Perfluoro-n-[1,2,3,4-

13C4]butanoic acid IS 70 130 20 60 130 20 60 130 60 130 60 130 20

Perfluoro-n-[1,2-

13C2]hexanoic acid IS 70 130 20 70 130 20 70 130 70 130 70 130 20

Perfluoro-n-[1,2,3,4-

13C4]octanoic acid IS 70 130 20 60 130 20 60 130 60 130 60 130 20

Perfluoro-n-[1,2,3,4,5-

13C5]nonanoic acid IS 70 130 20 50 130 25 50 130 50 130 50 130 25

Perfluoro-n-[1,2,3,4,5,6,7,8,9-

13C9]decanoic acid IS 70 130 20 60 130 20 60 130 60 130 60 130 20

Perfluoro-n-[1,2,3,4,5,6,7,8,9-

13C9]undecanoic acid IS 70 130 20 60 130 20 60 130 60 130 60 130 20

Perfluoro-n-[1,2-

13C2]dodecanoic acid IS 70 130 20 30 130 20 30 130 30 130 30 130 20

Perfluoro-n-[1,2,3,4-

13C4]-octanesulfonic acid IS 70 130 20 60 130 20 60 130 60 130 60 130 20

Perfluoro-1-[

18O2]-hexanesulfonic acid IS 70 130 20 60 130 20 60 130 60 130 60 130 20

2-(N-deuteriomethylperfluoro-1-octane sulfonamido)-

1,1,2,2-tetradeuterioethanol IS 70 130 20 5 130 70 5 130 5 130 5 130 70

Injection Internal Standard: Compound added after extraction, but prior to injection

Perfluoro-n-[1,2,3,4,5,6,7,8-

13C8]octanoic acid ES 80 120 10 80 120 10 80 120 80 120 80 120 10

Quantitation methods: ID = Isotope dilution IS = Internal standard ES = External standard
Draft PFC Procedure 42 December 2011

Not approved for either general purpose or regulatory use

Table 9. Sources used for standards, materials, and equipment during method development1

Native Compounds Vendor Part Number Shelf Life

Perfluoorobutanoic acid Sigma-Aldrich 164194 To be determined

Perfluoropentanoic acid Sigma-Aldrich 77285 To be determined

Perfluoro-n-hexanoic acid Sigma-Aldrich 29226 To be determined

Perfluoro-n-heptanoic acid Sigma-Aldrich 34,204-1 To be determined

Perfluoro-n-octanoic acid Sigma-Aldrich 17,146-8 To be determined

Perfluoro-n-nonanoic acid Sigma-Aldrich 77284 To be determined

Perfluoro-n-decanoic acid Sigma-Aldrich 17,774-1 To be determined

Perfluoro-n-undecanoic acid Sigma-Aldrich 446777 To be determined

Perfluoro-n-dodecanoic acid Sigma-Aldrich 40,644-9 To be determined

Perfluoro-n-tridecanoic acid Sigma-Aldrich 654973 To be determined

Perfluoro-n-tetradecanoic acid Sigma-Aldrich 446785 To be determined

Perfluorobutanesulfonic acid (Tetrabutylammonium salt) Sigma-Aldrich 86909 To be determined

Perfluorohexanesulfonic acid (Potassium salt) Sigma-Aldrich 50929 To be determined

Perfluoro-n-heptanesulfonic acid (Sodium salt) Wellington Labs L-PFHpS To be determined

Perfluorooctanesulfonic acid (Potassium salt) Sigma-Aldrich 77282 To be determined

Perfluorooctane sulfonamide Chiron 2043.8 To be determined

N-methylperfluoro-1-octanesulfonamide Wellington Labs N-MeFOSA To be determined

N-ethylperfluoro-1-octanesulfonamide Wellington Labs N-EtFOSA To be determined

2-(N-methylperfluoro-1-octanesulfonamido)-ethanol Wellington Labs N-MeFOSE To be determined

2-(N-ethylperfluoro-1-octanesulfonamido)-ethanol Wellington Labs N-EtFOSE To be determined

Labeled Internal Standards

Perfluoro-n-[1,2,3,4-

13C4]butanoic acid Wellington Labs MPFBA To be determined

Perfluoro-n-[1,2-

13C2]hexanoic acid Wellington Labs MPFHxA To be determined

Perfluoro-n-[1,2,3,4-

13C4]octanoic acid Wellington Labs MPFOA To be determined

Perfluoro-n-[1,2,3,4,5-

13C5]nonanoic acid Wellington Labs MPFNA To be determined

Perfluoro-n-[1,2-

13C2]decanoic acid Wellington Labs MPFDA To be determined

Perfluoro-n-[1,2,3,4,5,6,7,8,9-

13C9]decanoic acid CIL CLM-8172-S To be determined

Perfluoro-n-[1,2-

13C2]undecanoic acid Wellington Labs MPFUdA To be determined

Perfluoro-n-[2,3,4,5,6,7,8,9,10-

13C9]undecanoic acid CIL CLM-8240 To be determined

Perfluoro-n-[1,2-

13C2]dodecanoic acid Wellington Labs MPFDoA To be determined

Perfluoro-1-[1,2-

18O2]-hexanesulfonic acid Wellington Labs MPFHxS To be determined

Perfluoro-n-[1,2,3,4-

13C4]-octanesulfonic acid Wellington Labs MPFOS To be determined

2-(N-deuteriomethylperfluoro-1-octanesulfonamido)-1,1,2,2-

tetradeuterioethanol Wellington Labs d7-N-MeFOSE-M To be determined

Injection Internal Standards

Perfluoro-n-[1,2,3,4,5,6,7,8-

13 C8]octanoic acid CIL CLM-8005-S To be determined

2H-Perfluoro-[1,2-

13C2]-2-decenoic acid Wellington Labs MFOUEA To be determined

1 Provided for informational purposes only. Part numbers subject to change. Other suppliers may have equivalent materials.
Draft PFC Procedure 43 December 2011

Not approved for either general purpose or regulatory use

Table 10. Performance data from single-laboratory validation

Compound Transition

Solid IPR, n=4 Sludge, n=6

Mean

% Rec

Standard

Deviation %RSD

Mean

% Rec

Standard

Deviation %RSD

Native PFCs

Perfluorobutanoic acid Primary 105 1.61 1.54 95.8 9.55 9.97

Perfluoropentanoic acid Primary 114 4.46 3.81 56.4 31.4 55.6

Perfluoro-n-hexanoic acid Primary 116 2.28 1.98 115 9.83 8.52

Perfluoro-n-heptanoic acid Primary 95.6 3.25 3.40 91.1 16.3 17.9

Perfluoro-n-octanoic acid Primary 108 5.59 5.20 111 7.57 6.83

Perfluoro-n-octanoic acid Secondary 109 8.65 7.96 119 14.9 12.6

Perfluoro-n-nonanoic acid Primary 102 13.1 12.7 97.3 16.8 17.2

Perfluoro-n-decanoic acid Primary 101 2.37 2.34 111 8.41 7.59

Perfluoro-n-undecanoic acid Primary 101 2.20 2.18 104 7.35 7.09

Perfluoro-n-dodecanoic acid Primary 106 2.64 2.48 109 6.50 5.96

Perfluoro-n-tridecanoic acid Primary 41.3 2.41 5.83 126 29.1 23.1

Perfluoro-n-tetradecanoic acid Primary 48.6 4.52 9.30 131 16.1 12.2

Perfluorobutanesulfonic acid Primary 84.0 5.09 6.07 69.0 17.1 24.7

Perfluorobutanesulfonic acid Secondary 86.3 5.55 6.43 69.9 18.8 26.9

Perfluoro-n-hexane sulfonate Primary 101 2.18 2.16 105 11.0 10.5

Perfluoro-n-hexane sulfonate Secondary 105 4.18 4.00 107 11.5 10.8

Perfluoro-n-heptane sulfonate Primary 83.7 14.6 17.4 90.2 7.72 8.57

Perfluoro-n-heptane sulfonate Secondary 93.5 4.95 5.30 92.7 5.28 5.70

Perfluoro-n-octanesulfonate Primary 98.1 5.64 5.75 97.3 8.36 8.59

Perfluoro-n-octanesulfonate Secondary 102 4.30 4.21 99.6 7.32 7.35

Perfluorooctane sulfonamide Primary 39.5 3.69 9.33 50.7 3.63 7.15

N-methylperfluoro-1-octane sulfonamide Primary 9.33 0.542 5.81 48.8 13.3 27.2

N-ethylperfluoro-1-octanesulfonamide Primary 10.1 0.939 9.31 14.6 3.75 25.7

2-(N-methylperfluoro-1-octane sulfonamido)-

ethanol Primary 87.6 10.6 12.1 96.9 8.20 8.46

2-(N-ethylperfluoro-1-octane sulfonamido)-

ethanol Primary 21.8 1.37 6.27 50.3 13.6 27.0

Labeled Internal Standards

Perfluoro-n-[1,2,3,4-

13C4]butanoic acid Primary 95.2 6.84 7.19 81.6 37.5 45.9

Perfluoro-n-[1,2-

13C2]hexanoic acid Primary 98.9 2.55 2.58 72.0 18.2 25.3

Perfluoro-n-[1,2,3,4-

13C4]octanoic acid Primary 90.4 6.62 7.32 96.1 5.03 5.23

Perfluoro-n-[1,2,3,4,5-

13C5] nonanoic acid Primary 85.6 8.35 9.75 92.5 8.80 9.52

Perfluoro-n-[1,2,3,4,5,6,7,8,9-

13C9] decanoic 

acid Primary 85.2 4.61 5.41 107 11.1 10.4

Perfluoro-n-[2,3,4,5,6,7,8,9,10-

13C9]

undecanoic acid Primary 76.7 0.853 1.11 80.3 28.9 36.0

Perfluoro-n-[1,2-

13C2]dodecanoic acid Primary 53.5 3.50 6.54 109 13.1 12.0

Perfluoro-n-[1,2,3,4-

13C4]-octane sulfonic 

acid Primary 76.9 4.82 6.27 78.8 7.97 10.1

Perfluoro-1-[1,2-

18O2]-hexane sulfonic acid Primary 76.8 3.33 4.33 79.4 7.73 9.74

2-(N-deuteriomethylperfluoro-1-octanesulfon

amido)-1,1,2,2-tetradeuterioethanol Primary 18.3 3.32 18.2 45.5 13.0 28.6

Injection Internal Standards

Perfluoro-n-[1,2,3,4,5,6,7,8-

13C8] octanoic 

acid Primary 108 11.0 10.2 84.5 7.42 8.79
Draft PFC Procedure 44 December 2011

Not approved for either general purpose or regulatory use

Figure 1. Flow chart for determination of PFCs by LC/MS/MS

11.3.1 Place 0.5 g of wet 

sludge in a 16-mL 

polypropylene centrifuge 

tube

11.3.5. Spike samples with 

labeled compounds

11.3.6-11.3.7

Digestion with 0.5 mL 1M 

NaOH (1/2 hr sonication 

with heat) overnight 

incubation.

11.3.8 Neutralize with HCl

11.4.1. Add 10 mL 

50:50/ACN:MeOH (v/v)

11.4.2 Shake moderately 

for 1 h

11.4.3 Centrifuge at 3000 x 

g for 20 min

11.4.4. Decant 10 mL 

supernatant and collect in 

250-mL HDPE bottle 

containing 180 mL of 

reagent water

Cleanup

11.4.6

Adjust pH of diluted extract 

to 6.5 ± 0.5 with 3% acetic 

acid or 0.3% NH4OH, as 

needed

11.4.7. Sonication 

(30 min)

11.5. WAX Cartridge Cleanup

1) Condition SPE cartridge 

with 5 mL 0.3% NH4OH in 

MeOH and 5 mL 0.1 M 

formic acid

2) Equilibrate with 5mL 

reagent water

3) Load sample drop-wise (~5 

mL/min)

4) Wash with 5 mL 20% 

MeOH in 80% 0.1 M 

formic acid

5) Dry SPE for 5 min under 

vacuum

6) Elute with 4 mL 0.3% 

NH4OH in MeOH 

11.6.1 Reduce to near 

dryness (~50 uL) under N2 

and water bath at 40°C

Instrumental

11.6.2. Reconstitute 

with 938 uL of 0.3% 

NH4OH in MeOH and 

vortex mix

11.6.3 Filter extract 

using 0.45-μm nylon 

membranes filter

14.0. LC/MS/MS 

analysis of 20 μL of 

extract 

Determine % Solids

(Sec. 11.2)

11.4.5 Repeated once

Digestion and 

Extraction

11.4.5. Collect 20 mL total 

in 250-mL HDPE bottle 

containing 180 mL reagent 

water 

11.6.3 Spike 12.5 μL 

of internal standard 

and vortex mix

15.0. Quantify target 

PFCs
Draft PFC Procedure 45 December 2011

Not approved for either general purpose or regulatory use

Figure 2. Example Chromatogram for CS-4 Calibration Standard

Time

6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

%

0

N-EtFOSA

N-EtFOSE

N-MeFOSA

N-MeFOSE

PFTeDA

PFOSA and PFTriDA

PFOSA

PFDoDA

PFOS

PFDA

PFUnDA

PFHpS

PFNA

PFHxS

PFOA

PFHpA

PFBS and PFHxA

PFPA

PFBA
Draft PFC Procedure 46 December 2011

Not approved for either general purpose or regulatory use

APPENDIX 1

Details on Preparation of Standards
Draft PFC Procedure 47 December 2011

Not approved for either general purpose or regulatory use

Appendix 1. Details on Preparation of Standards

Individual PFCs are used to make standard solutions. From solids – native PFBS, PFHxS, and PFOS were 

obtained as sodium, potassium or tetrabutylammonium salts – all others were acids.

[

13C9]PFUnDA was obtained as a solid.

Native PFHpS and labeled compounds except [13C9]PFUnDA, were obtained as 50μg/mL solutions in 

methanol; 1.2 mL ampule. The PFHpS was a solution of its sodium salt.

Native N-Me/Et-FOSA/Es were obtained as 50μg/mL solutions in 90% Nonane/ 10% Toluene.

1. Use Fisher brand, Plasma grade water (has 18 MΩ specification and a bottle was proofed for PFC 

compounds of interest).

2. Prepare 60/40 (v/v) acetonitrile/ plasma grade water mix and 99% methanol / 0.7% water / 0.3% 

NH4OH (basic methanol) mixes and store in dedicated 3X methanol-washed 1L HDPE solvent 

bottles.

3. Prepare stock solutions of individual native compounds in methanol at ~ 20-60 mg/mL by weighing 

20 – 60 mg into a 10 mL glass grade A volumetric flask using a 4 place balance, then diluting to mark 

with methanol. For carboxylic acids, 4 mole equivalents of NaOH in methanol was added to reduce 

esterification in accordance with purchased standards from Wellington. The same procedure was used 

for [13C9]PFUnDA except 2 mg in a 5 mL flask was used. PFTeDA is much less soluble than the 

other compounds so a solution is prepared by weighing 10 mg into a 50 mL volumetric flask and 

diluting with acetonitrile without NaOH added.

Note that PFTeDA, as well as being difficult to dissolve may also be much more likely to stick to 

glass, so the flask used to prepare it should receive more extensive cleaning after use than other 

glassware. Sonication for 20 minutes in basic Methanol followed by regular cleaning is 

recommended. Similar precautions for PFTriDA are also recommended.

4. Clean glass syringes by rinsing with 20X Toluene, 20X Hexane then sonicating for 20 minutes in 

80:20 Toluene/Acetone then rinsing with 3X basic Methanol , 3X Toluene, 3X Hexane.

Clean class A volumetric flasks rinsing with 3X Toluene, 3X Hexane then sonicating for 20 minutes 

in 80:20 Toluene/Acetone, then rinsing with 3X Toluene, 3X Hexane, 3X Dichloromethane, then a 

final rinse before use with the dilution solvent being used for the standard.

Prepare Stock Mass-Labeled internal standard mix – does not include injection internal standard 

compounds.

Calculate the volumes required of each labeled standard to give a concentration of 600 ng/mL for 

each carboxylic acid and 900 ng/mL for each sulfonic acid.

Using a cleaned glass syringe that is reserved for mass labeled standards, transfer and combine the 

required measured quantities of each labeled standard into a cleaned volumetric flask. Dilute to the 

mark with the prepared 60:40 acetonitrile/water mix.

Prepare Labeled Internal Standard Solution (LINT)

4.1 Objective: prepare a solution of 30-45 pg/μL for the labeled carboxylic and sulfonic 

acids, plus 200 pg/μL of d7-N-MeFOSE, in basic methanol.

4.2 Using cleaned syringes and volumetric flasks transfer a portion of the Stock MassLabeled internal standard mix so that it will be diluted 20X, plus a portion of the 50 

μg/mL d7-N-MeFOSE so that it will be diluted 200X and dilute to mark with the prepared 

basic methanol mix.
Draft PFC Procedure 48 December 2011

Not approved for either general purpose or regulatory use

Prepare Stock Mass-Labeled Injection Internal Standard.

(Can be used as Injection Internal Standard or to prepare a more diluted INJ.)

Calculate the volumes required of each labeled standard to give a concentration of 240 ng/mL 13C8-

Perfluorooctanoic acid and 200 ng/mL 2

H-Perfluoro-[1,2-

13C2]-2-decenoic acid.

Using a cleaned glass syringe that is reserved for mass labeled standards, transfer and combine the 

required measured quantities of each labeled standard into the cleaned volumetric flask. Dilute to the 

mark with the prepared 60:40 acetonitrile/water mix.

Prepare Mass-Labeled injection standard

4.3 Objective: prepare a solution of 10-12 ng/mL in basic methanol.

4.4 Using cleaned syringes and volumetric flasks transfer a portion of the Stock MassLabeled internal standard mix and dilute to mark with the prepared basic methanol mix so 

that it is diluted 20X.

Prepare Intermediate Stock Mix of native PF Carboxylic and Sulfonic acids

Calculate the volumes required of each native stock standard to give a concentration of 50 μg/mL for 

each carboxylic acid and 100 μg/mL for each sulfonic acid.

Using a cleaned glass syringe that is reserved for native standards, transfer and combine the required 

measured quantities of each individual native standard into the cleaned volumetric flask. 

Dilute to the mark with the prepared 60:40 acetonitrile/water mix.

5. Prepare Full Native Stock Mix containing all native PF Carboxylic and Sulfonic acids

Calculate the volumes required of the intermediate Stock Mix standard and native PFHpS solution to 

give a concentration of 2.5 μg/mL for each carboxylic acid and 5 μg/mL for each sulfonic acid.

Using a cleaned glass syringe that is reserved for native standards, transfer and combine the required 

measured quantities of each standard into the cleaned volumetric flask. 

Dilute to the mark with the prepared basic methanol mix.

Prepare working level native standard of PF Carboxylic and Sulfonic acids

5.1. Objective: prepare a solution of 125-250 ng/mL in basic methanol.

5.2. Using appropriate cleaned syringes and volumetric flasks, transfer a portion of the Full 

Native Stock Mix and dilute to mark with the prepared basic methanol mix so that it is 

diluted 20X.

Prepare working-level native standard of N-Me/Et-FOSA/Es

5.3. Objective: prepare a solution of 1250 ng/mL of the FOSEs and 2500 ng/mL of the 

FOSAs in methanol, plus 10% propan-2-ol which is required to allow full mixing of the

nonane and methanol.

5.4. Using appropriate cleaned syringes and volumetric flasks, transfer and combine the 

required portion of each individual 50 μg/mL standard, add propan-2-ol so that it is 10% 

of the flask volume and dilute to mark with methanol.

Prepare low level native standard for low level spiking and Calibration standard preparation

5.5. Objective: prepare a solution of 12.5-25 ng/mL in basic methanol.

5.6. Using appropriate cleaned syringes and volumetric flasks, transfer a portion of the Full 

Native Stock Mix and dilute to mark with the prepared basic methanol mix so that it is 

diluted 200X.
Draft PFC Procedure 49 December 2011

Not approved for either general purpose or regulatory use

6. Prepare Calibration Standards (see Table 6 in the body of the procedure)

Label baked disposable 12mL glass vials.

Using disposable tip pipettors, transfer a 200μL portion of the Labeled Internal Standard Solution

and 25 μL of the Stock Mass-Labeled injection internal standard to each vial.

Using disposable tip pipettors, transfer portion of the full Native Stock Mix, working level native 

standard or low level native standard to give levels of native compounds matching those shown in 

Table 7.

Using disposable tip pipettors, add basic methanol to dilute each Calibration Standard to 2.0 mL. Cap 

and mix thoroughly.

For Matrix Matched Calibration standards, only 1 mL of each CAL was prepared, so volumes of 

standards used were halved and 0.5 mL of soil extract (containing the equivalent of 0.5 g of clean soil 

in 0.5 mL basic Methanol) was added before diluting each Calibration Standard to 1.0 mL with basic 

methanol.

Table of Calibration Standards

STD 

ID

Internal 

Standard 

Solution

(LINT) 

μL

Stock MassLabeled injection 

internal standard 

mix (INJ) μL

Low level 

native 

standard 

μL

Working 

level native 

standard 

(NAT)

μL

Full 

Native 

Stock 

Mix μL

N-Me/EtFOSA/Es

native standard 

(NAT2)

μL

Final 

Volume

μL

CS-1 200 25 20 4 2000

CS-2 200 25 50 8 2000

CS-3 200 25 20 16 2000

CS-4 200 25 80 32 2000

CS-5 200 25 20 64 2000

CS-6 200 25 40 128 2000

CS-7 200 25 100 256 2000

CS-8 200 25 250 2000
Draft PFC Procedure 50 December 2011

Not approved for either general purpose or regulatory use

APPENDIX 2

Procedure for Polishing Deionized Water
Draft PFC Procedure 51 December 2011

Not approved for either general purpose or regulatory use

Appendix 2. Procedure for Polishing Deionized Water

Laboratory produced de-ionized water can be purified to produce PFC-free water for use in this analysis. 

Polished water is used for preparation of strong needle wash solutions, seal wash solutions, 60:40 

ACN:H2O standards solutions LC aqueous mobile phase and during the extraction and cleanup procedure.

1.0 Equipment for polishing water

1.1 Cartridge for polishing 18MΩ water – Waters Oasis 35cc (6g) HLB Extraction Cartridge.

1.2 Glassware for polishing 18MΩ water – Fisher Scientific, 2000 mL Kimax Brand Volumetric 

Flask (or equivalent), Fisher Scientific: 2000 mL Pyrex Filtering Flask (or equivalent) and Fisher 

Scientific Glass Magnetic Stir Bar 1.5” x 3/8” (or equivalent). All glassware should be washed 3 

times using PFC free methanol. 

2.0 Procedure for Polishing Water

2.1 Use glassware dedicated to water polishing (See Sec. 1.2 for specified glassware).

2.2 Pass 2L of 18MΩ (nanopure or equivalent) water through a 60 cc “Oasis HLB” cartridge. This 

cartridge should be used no more than 3 times.

Polished nanopure water should be stored in dedicated 1-L HDPE containers.

Figure 1. Setup for Water Polishing