537-1 

EPA Document #: EPA/600/R-08/092 

METHOD 537. DETERMINATION OF SELECTED PERFLUORINATED ALKYL 

ACIDS IN DRINKING WATER BY SOLID PHASE EXTRACTION 

AND LIQUID CHROMATOGRAPHY/TANDEM MASS 

SPECTROMETRY (LC/MS/MS)

Version 1.1 

September 2009 

J.A. Shoemaker US EPA, Office of Research and Development, National Exposure 

Research Laboratory

P.E. Grimmett US EPA, Office of Research and Development, National Exposure 

Research Laboratory

B.K. Boutin The National Council on Aging, Senior Environmental Employment 

Program 

 

NATIONAL EXPOSURE RESEARCH LABORATORY 

OFFICE OF RESEARCH AND DEVELOPMENT 

U. S. ENVIRONMENTAL PROTECTION AGENCY 

CINCINNATI, OHIO 45268
 

537-2 

METHOD 537 

DETERMINATION OF SELECTED PERFLUORINATED ALKYL ACIDS IN 

DRINKING WATER BY SOLID PHASE EXTRACTION AND LIQUID

CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY (LC/MS/MS) 

1. SCOPE AND APPLICATION

1.1 This is a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for 

the determination of selected perfluorinated alkyl acids (PFAAs) in drinking water. 

Accuracy and precision data have been generated in reagent water, and finished 

ground and surface waters for the compounds listed in the table below. 

Analyte Acronym

Chemical Abstract Services 

Registry Number (CASRN)

N-ethyl perfluorooctanesulfonamidoacetic acid NEtFOSAA ─

N-methyl perfluorooctanesulfonamidoacetic acid NMeFOSAA ─

Perfluorobutanesulfonic acid PFBS 375-73-5 

Perfluorodecanoic acid PFDA 335-76-2 

Perfluorododecanoic acid PFDoA 307-55-1 

Perfluoroheptanoic acid PFHpA 375-85-9 

Perfluorohexanesulfonic acid PFHxS 355-46-4 

Perfluorohexanoic acid PFHxA 307-24-4 

Perfluorononanoic acid PFNA 375-95-1 

Perfluorooctanesulfonic acid PFOS 1763-23-1 

Perfluorooctanoic acid PFOA 335-67-1 

Perfluorotetradecanoic acid PFTA 376-06-7 

Perfluorotridecanoic acid PFTrDA 72629-94-8 

Perfluoroundecanoic acid PFUnA 2058-94-8 

1.2 The Minimum Reporting Level (MRL) is the lowest analyte concentration that meets 

Data Quality Objectives (DQOs) that are developed based on the intended use of this 

method. The single laboratory lowest concentration MRL (LCMRL) is the lowest true 

concentration for which the future recovery is predicted to fall, with high confidence 

(99%), between 50 and 150% recovery. Single laboratory LCMRLs for analytes in 

this method range from 2.9-14 ng/L, and are listed in Table 5. The procedure used to 

determine the LCMRL is described elsewhere.1
 

537-3 

1.3 Laboratories using this method will not be required to determine the LCMRL for this 

method, but will need to demonstrate that their laboratory MRL for this method meets 

the requirements described in Section 9.2.5. 

1.4 Determining the Detection Limit (DL) for analytes in this method is optional (Sect. 

9.2.7). Detection limit is defined as the statistically calculated minimum concentration 

that can be measured with 99% confidence that the reported value is greater than zero.2 

The DL is compound dependent and is dependent on extraction efficiency, sample 

matrix, fortification concentration, and instrument performance. 

1.5 This method is intended for use by analysts skilled in solid phase extractions, the 

operation of LC/MS/MS instruments, and the interpretation of the associated data. 

1.6 METHOD FLEXIBILITY – In recognition of technological advances in analytical 

systems and techniques, the laboratory is permitted to modify the evaporation 

technique, separation technique, LC column, mobile phase composition, LC conditions 

and MS and MS/MS conditions (Sect. 6.12, 9.1.1, 10.2, and 12.1). Changes may not 

be made to sample collection and preservation (Sect. 8), the sample extraction steps 

(Sect. 11), or to the quality control requirements (Sect. 9). Method modifications 

should be considered only to improve method performance. Modifications that are 

introduced in the interest of reducing cost or sample processing time, but result in 

poorer method performance, should not be used. Analytes must be adequately 

resolved chromatographically in order to permit the mass spectrometer to dwell on a 

minimum number of compounds eluting within a retention time window. Instrumental 

sensitivity (or signal-to-noise) will decrease if too many compounds are permitted to 

elute within a retention time window. In all cases where method modifications are 

proposed, the analyst must perform the procedures outlined in the initial demonstration 

of capability (IDC, Sect. 9.2), verify that all Quality Control (QC) acceptance criteria 

in this method (Sect. 9) are met, and that acceptable method performance can be 

verified in a real sample matrix (Sect. 9.3.6). 

 

NOTE: The above method flexibility section is intended as an abbreviated summation 

of method flexibility. Sections 4-12 provide detailed information of specific 

portions of the method that may be modified. If there is any perceived 

conflict between the general method flexibility statement in Section 1.6 and 

specific information in Sections 4-12, Sections 4-12 supersede Section 1.6. 

2. SUMMARY OF METHOD

2.1 A 250-mL water sample is fortified with surrogates and passed through a solid phase 

extraction (SPE) cartridge containing polystyrenedivinylbenzene (SDVB) to extract 

the method analytes and surrogates. The compounds are eluted from the solid phase 

with a small amount of methanol. The extract is concentrated to dryness with nitrogen 

in a heated water bath, and then adjusted to a 1-mL volume with 96:4% (vol/vol) 

methanol:water after adding the IS(s). A 10-μL injection is made into an LC equipped 

with a C18 column that is interfaced to an MS/MS. The analytes are separated and 
 

537-4 

identified by comparing the acquired mass spectra and retention times to reference 

spectra and retention times for calibration standards acquired under identical 

LC/MS/MS conditions. The concentration of each analyte is determined by using the 

internal standard technique. Surrogate analytes are added to all Field and QC Samples 

to monitor the extraction efficiency of the method analytes. 

3. DEFINITIONS

 

3.1 ANALYSIS BATCH – A set of samples that is analyzed on the same instrument 

during a 24-hour period, including no more than 20 Field Samples, that begins and 

ends with the analysis of the appropriate Continuing Calibration Check (CCC) 

standards. Additional CCCs may be required depending on the length of the analysis 

batch and/or the number of Field Samples. 

3.2 CALIBRATION STANDARD (CAL) – A solution prepared from the primary dilution 

standard solution and/or stock standard solution, internal standard(s), and the 

surrogate(s). The CAL solutions are used to calibrate the instrument response with 

respect to analyte concentration. 

3.3 COLLISIONALLY ACTIVATED DISSOCIATION (CAD) – The process of 

converting the precursor ion’s translational energy into internal energy by collisions 

with neutral gas molecules to bring about dissociation into product ions. 

3.4 CONTINUING CALIBRATION CHECK (CCC) – A calibration standard containing 

the method analytes, internal standard(s) and surrogate(s). The CCC is analyzed 

periodically to verify the accuracy of the existing calibration for those analytes. 

3.5 DETECTION LIMIT (DL) – The minimum concentration of an analyte that can be 

identified, measured, and reported with 99% confidence that the analyte concentration 

is greater than zero. This is a statistical determination of precision (Sect. 9.2.7), and 

accurate quantitation is not expected at this level.

2

3.6 EXTRACTION BATCH – A set of up to 20 Field Samples (not including QC 

samples) extracted together by the same person(s) during a work day using the same 

lot of SPE devices, solvents, surrogate, internal standard and fortifying solutions. 

Required QC samples include Laboratory Reagent Blank, Laboratory Fortified Blank, 

Laboratory Fortified Sample Matrix, and either a Field Duplicate or Laboratory 

Fortified Sample Matrix Duplicate. 

3.7 FIELD DUPLICATES (FD1 and FD2) – Two separate samples collected at the same 

time and place under identical circumstances, and treated exactly the same throughout 

field and laboratory procedures. Analyses of FD1 and FD2 give a measure of the 

precision associated with sample collection, preservation, and storage, as well as 

laboratory procedures. 
 

537-5 

3.8 FIELD REAGENT BLANK (FRB) – An aliquot of reagent water that is placed in a 

sample container in the laboratory and treated as a sample in all respects, including 

shipment to the sampling site, exposure to sampling site conditions, storage, 

preservation, and all analytical procedures. The purpose of the FRB is to determine if 

method analytes or other interferences are present in the field environment. 

3.9 INTERNAL STANDARD (IS) – A pure chemical added to an extract or standard 

solution in a known amount(s) and used to measure the relative response of other 

method analytes and surrogates that are components of the same solution. The internal 

standard must be a chemical that is structurally similar to the method analytes, has no 

potential to be present in water samples, and is not a method analyte. 

3.10 LABORATORY FORTIFIED BLANK (LFB) – A volume of reagent water or other 

blank matrix to which known quantities of the method analytes and all the preservation 

compounds are added in the laboratory. The LFB is analyzed exactly like a sample, 

and its purpose is to determine whether the methodology is in control, and whether the 

laboratory is capable of making accurate and precise measurements. 

 

3.11 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) – A preserved field 

sample to which known quantities of the method analytes are added in the laboratory. 

The LFSM is processed and analyzed exactly like a sample, and its purpose is to 

determine whether the sample matrix contributes bias to the analytical results. The 

background concentrations of the analytes in the sample matrix must be determined in 

a separate sample extraction and the measured values in the LFSM corrected for 

background concentrations. 

3.12 LABORATORY FORTIFIED SAMPLE MATRIX DUPLICATE (LFSMD) – A 

duplicate of the Field Sample used to prepare the LFSM. The LFSMD is fortified, 

extracted, and analyzed identically to the LFSM. The LFSMD is used instead of the 

Field Duplicate to assess method precision when the occurrence of method analytes is 

low. 

3.13 LABORATORY REAGENT BLANK (LRB) – An aliquot of reagent water or other 

blank matrix that is treated exactly as a sample including exposure to all glassware, 

equipment, solvents and reagents, sample preservatives, internal standard, and 

surrogates that are used in the analysis batch. The LRB is used to determine if method 

analytes or other interferences are present in the laboratory environment, the reagents, 

or the apparatus. 

3.14 LOWEST CONCENTRATION MINIMUM REPORTING LEVEL (LCMRL) – The 

single laboratory LCMRL is the lowest true concentration for which a future recovery 

is expected, with 99% confidence, to be between 50 and 150% recovery.1

3.15 MATERIAL SAFETY DATA SHEET (MSDS) – Written information provided by 

vendors concerning a chemical’s toxicity, health hazards, physical properties, fire, and 

reactivity data including storage, spill, and handling precautions. 
 

537-6 

3.16 MINIMUM REPORTING LEVEL (MRL) – The minimum concentration that can be 

reported as a quantitated value for a method analyte in a sample following analysis. 

This defined concentration can be no lower than the concentration of the lowest 

calibration standard for that analyte and can only be used if acceptable QC criteria for 

this standard are met. A procedure for verifying a laboratory’s MRL is provided in 

Section 9.2.5. 

3.17 PRECURSOR ION – For the purpose of this method, the precursor ion is the 

deprotonated molecule ([M-H]-) of the method analyte. In MS/MS, the precursor ion 

is mass selected and fragmented by collisionally activated dissociation to produce 

distinctive product ions of smaller m/z. 

3.18 PRIMARY DILUTION STANDARD (PDS) SOLUTION – A solution containing the 

analytes prepared in the laboratory from stock standard solutions and diluted as needed 

to prepare calibration solutions and other needed analyte solutions. 

3.19 PRODUCT ION – For the purpose of this method, a product ion is one of the fragment 

ions produced in MS/MS by collisionally activated dissociation of the precursor ion. 

3.20 QUALITY CONTROL SAMPLE (QCS) – A solution of method analytes of known 

concentrations that is obtained from a source external to the laboratory and different 

from the source of calibration standards. The second source SSS is used to fortify the 

QCS at a known concentration. The QCS is used to check calibration standard 

integrity. 

3.21 STOCK STANDARD SOLUTION (SSS) – A concentrated solution containing one or 

more method analytes prepared in the laboratory using assayed reference materials or 

purchased from a reputable commercial source. 

3.22 SURROGATE ANALYTE (SUR) – A pure chemical which chemically resembles 

method analytes and is extremely unlikely to be found in any sample. This chemical is 

added to a sample aliquot in known amount(s) before processing and is measured with 

the same procedures used to measure other method analytes. The purpose of the SUR 

is to monitor method performance with each sample. 

4. INTERFERENCES

4.1 All glassware must be meticulously cleaned. Wash glassware with detergent and tap 

water, rinse with tap water, followed by a reagent water rinse. Non-volumetric 

glassware can be heated in a muffle furnace at 400 °C for 2 h or solvent rinsed. 

Volumetric glassware should be solvent rinsed and not be heated in an oven above 

120 °C. Store clean glassware inverted or capped. Do not cover with aluminum foil 

because PFAAs can be potentially transferred from the aluminum foil to the glassware.
 

537-7 

NOTE: PFAA standards, extracts and samples should not come in contact with any 

glass containers or pipettes as these analytes can potentially adsorb to glass 

surfaces. PFAA analyte, IS and SUR standards commercially purchased in 

glass ampoules are acceptable; however, all subsequent transfers or dilutions 

performed by the analyst must be prepared and stored in polypropylene 

containers. 

4.2 Method interferences may be caused by contaminants in solvents, reagents (including 

reagent water), sample bottles and caps, and other sample processing hardware that 

lead to discrete artifacts and/or elevated baselines in the chromatograms. The method 

analytes in this method can also be found in many common laboratory supplies and 

equipment, such as PTFE (polytetrafluoroethylene) products, LC solvent lines, 

methanol, aluminum foil, SPE sample transfer lines, etc.3 All items such as these must 

be routinely demonstrated to be free from interferences (less than 1/3 the MRL for 

each method analyte) under the conditions of the analysis by analyzing laboratory 

reagent blanks as described in Section 9.3.1. Subtracting blank values from sample 

results is not permitted.

4.3 Matrix interferences may be caused by contaminants that are co-extracted from the 

sample. The extent of matrix interferences will vary considerably from source to 

source, depending upon the nature of the water. Humic and/or fulvic material can be 

co-extracted during SPE and high levels can cause enhancement and/or suppression in 

the electrospray ionization source or low recoveries on the SPE sorbent.4-5 Total 

organic carbon (TOC) is a good indicator of humic content of the sample. Under the 

LC conditions used during method development, matrix effects due to total organic 

carbon (TOC) were not observed. 

4.4 Relatively large quantities of the preservative (Sect. 8.1.2) are added to sample bottles. 

The potential exists for trace-level organic contaminants in these reagents. Interferences from these sources should be monitored by analysis of laboratory reagent blanks 

(Sect. 9.3.1), particularly when new lots of reagents are acquired. 

4.5 SPE cartridges can be a source of interferences. The analysis of field and laboratory 

reagent blanks can provide important information regarding the presence or absence of 

such interferences. Brands and lots of SPE devices should be tested to ensure that 

contamination does not preclude analyte identification and quantitation. 

5. SAFETY

5.1 The toxicity or carcinogenicity of each reagent used in this method has not been 

precisely defined. Each chemical should be treated as a potential health hazard, and 

exposure to these chemicals should be minimized. Each laboratory is responsible for 

maintaining an awareness of OSHA regulations regarding safe handling of chemicals 

used in this method. A reference file of MSDSs should be made available to all 

personnel involved in the chemical analysis. Additional references to laboratory 

safety are available.6-8
 

537-8 

5.2 PFOA has been described as “likely to be carcinogenic to humans.”9 Pure standard 

materials and stock standard solutions of these method analytes should be handled 

with suitable protection to skin and eyes, and care should be taken not to breathe the 

vapors or ingest the materials. 

6. EQUIPMENT AND SUPPLIES (Brand names and/or catalog numbers are included for 

illustration only, and do not imply endorsement of the product.) Due to potential adsorption 

of analytes onto glass, polypropylene containers were used for all standard, sample and 

extraction preparations. Other plastic materials (e.g., polyethylene) which meet the QC 

requirements of Section 9 may be substituted. 

6.1 SAMPLE CONTAINERS – 250-mL polypropylene bottles fitted with polypropylene 

screw caps. Sample bottles must be discarded after use. 

6.2 POLYPROPYLENE BOTTLES – 4-mL narrow-mouth polypropylene bottles (VWR 

Cat. No.: 16066-960 or equivalent). 

6.3 CENTRIFUGE TUBES – 15-mL conical polypropylene tubes with polypropylene 

screw caps for storing standard solutions and for collection of the extracts (Thomas 

Scientific Cat. No.: 2602A10 or equivalent). 

6.4 AUTOSAMPLER VIALS – Polypropylene 0.3-mL autosampler vials (SUN SRi Cat. 

No.: 501-354) with polypropylene caps (SUN SRi Cat. No.: 501-357 or equivalent). 

 

NOTE: Polypropylene vials and caps are necessary to prevent contamination of 

the sample from PTFE coated septa. However, polypropylene caps do 

not reseal, so evaporation occurs after injection. Thus, multiple 

injections from the same vial are not possible. 

6.5 POLYPROPYLENE GRADUATED CYLINDERS – Suggested sizes include 25, 50, 

100 and 1000-mL cylinders. 

6.6 MICRO SYRINGES – Suggested sizes include 5, 10, 25, 50, 100, 250, 500 and 

1000-μL syringes. 

6.7 PLASTIC PIPETS – Polypropylene or polyethylene disposable pipets (Fisher Cat. 

No.: 13-711-7 or equivalent). 

6.8 ANALYTICAL BALANCE – Capable of weighing to the nearest 0.0001 g. 

6.9 SOLID PHASE EXTRACTION (SPE) APPARATUS FOR USING CARTRIDGES

 

6.9.1 SPE CARTRIDGES – 0.5 g, 6-mL SPE cartridges containing styrenedivinylbenzene (SDVB) sorbent phase (Varian Cat. No.: 1225-5021 or equivalent). 
 

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6.9.2 VACUUM EXTRACTION MANIFOLD – A manual vacuum manifold with 

Visiprep large volume sampler (Supelco Cat. No. 57030 and 57275 or 

equivalent) for cartridge extractions, or an automatic/robotic sample preparation 

system designed for use with SPE cartridges, may be used if all QC requirements 

discussed in Section 9 are met. Extraction and/or elution steps may not be 

changed or omitted to accommodate the use of an automated system. Care must 

be taken with automated SPE systems to ensure the PTFE commonly used in 

these systems does not contribute to unacceptable analyte concentrations in the 

LRB (Sect. 9.3.1). 

6.9.3 SAMPLE DELIVERY SYSTEM – Use of a polypropylene transfer tube system, 

which transfers the sample directly from the sample container to the SPE 

cartridge, is recommended, but not mandatory. Standard extraction manifolds 

come equipped with PTFE transfer tube systems. These can be replaced with 1/8” 

O.D. x 1/16” I.D. polypropylene or polyethylene tubing (Hudson Extrusions 

LLDPE or equivalent) cut to an appropriate length to ensure no sample 

contamination from the sample transfer lines. Other types of non-PTFE tubing 

may be used provided it meets the LRB (Sect. 9.3.1) and LFB (Sect. 9.3.3) QC 

requirements. The PTFE transfer tubes may be used, but an LRB must be run on 

each PFTE transfer tube and the QC requirements in Section 9.2.1 must be met. 

In the case of automated SPE, the removal of PTFE lines may not be feasible; 

therefore, LRBs will need to be rotated among the ports and must meet the QC 

requirements of Sections 9.2.1 and 9.3.1. 

6.10 EXTRACT CONCENTRATION SYSTEM – Extracts are concentrated by 

evaporation with nitrogen using a water bath set no higher than 65 °C (Meyer N-Evap, 

Model 111, Organomation Associates, Inc. or equivalent). 

6.11 LABORATORY OR ASPIRATOR VACUUM SYSTEM – Sufficient capacity to 

maintain a vacuum of approximately 10 to 15 inches of mercury for extraction 

cartridges. 

6.12 LIQUID CHROMATOGRAPHY (LC)/TANDEM MASS SPECTROMETER

(MS/MS) WITH DATA SYSTEM 

6.12.1 LC SYSTEM – Instrument capable of reproducibly injecting up to 10-μL aliquots, 

and performing binary linear gradients at a constant flow rate near the flow rate 

used for development of this method (0.3 mL/min). The LC must be capable of 

pumping the water/methanol mobile phase without the use of a degasser which 

pulls vacuum on the mobile phase bottle (other types of degassers are 

acceptable). Degassers which pull vacuum on the mobile phase bottle will 

volatilize the ammonium acetate mobile phase causing the analyte peaks to shift 

to earlier retention times over the course of the analysis batch. The usage of a 

column heater is optional.
 

537-10 

 NOTE: During the course of method development, it was discovered that 

while idle for more than one day, PFAAs built up in the PTFE 

solvent transfer lines. To prevent long delays in purging high levels 

of PFAAs from the LC solvent lines, they were replaced with 

PEEK tubing and the PTFE solvent frits were replaced with 

stainless steel frits. It is not possible to remove all PFAA 

background contamination, but these measures help to minimize 

their background levels. 

6.12.2 LC/TANDEM MASS SPECTROMETER – The LC/MS/MS must be capable of 

negative ion electrospray ionization (ESI) near the suggested LC flow rate of 

0.3 mL/min. The system must be capable of performing MS/MS to produce 

unique product ions (Sect. 3.19) for the method analytes within specified retention 

time segments. A minimum of 10 scans across the chromatographic peak is 

required to ensure adequate precision. Data are demonstrated in Tables 5-9 using 

a triple quadrupole mass spectrometer (Waters Micromass Quattro Premier). 

6.12.3 DATA SYSTEM – An interfaced data system is required to acquire, store, reduce, 

and output mass spectral data. The computer software should have the capability 

of processing stored LC/MS/MS data by recognizing an LC peak within any given 

retention time window. The software must allow integration of the ion abundance 

of any specific ion within specified time or scan number limits. The software 

must be able to calculate relative response factors, construct linear regressions or 

quadratic calibration curves, and calculate analyte concentrations. 

6.12.4 ANALYTICAL COLUMN – An LC C18 column (2.1 x 150 mm) packed with 

5 μm dp C18 solid phase particles (Waters #: 186001301 or equivalent) was used. 

Any column that provides adequate resolution, peak shape, capacity, accuracy, 

and precision (Sect. 9) may be used. 

7. REAGENTS AND STANDARDS

7.1 GASES, REAGENTS, AND SOLVENTS – Reagent grade or better chemicals should 

be used. Unless otherwise indicated, it is intended that all reagents shall conform to 

the specifications of the Committee on Analytical Reagents of the American Chemical 

Society, where such specifications are available. Other grades may be used, provided 

it is first determined that the reagent is of sufficiently high purity to permit its use 

without lessening the quality of the determination. 

7.1.1 REAGENT WATER – Purified water which does not contain any measurable 

quantities of any method analytes or interfering compounds greater than 1/3 the 

MRL for each method analyte of interest. Prior to daily use, at least 3 L of 

reagent water should be flushed from the purification system to rinse out any 

build-up of analytes in the system’s tubing. 
 

537-11 

7.1.2 METHANOL (CH3OH, CAS#: 67-56-1) – High purity, demonstrated to be free of 

analytes and interferences (Tedia Absolv grade or equivalent). 

7.1.3 AMMONIUM ACETATE (NH4C2H3O2, CAS#: 631-61-8) – High purity, 

demonstrated to be free of analytes and interferences (Sigma-Aldrich ACS grade 

or equivalent). 

 

7.1.4 20 mM AMMONIUM ACETATE/REAGENT WATER – To prepare 1 L, add 

1.54 g ammonium acetate to 1 L of reagent water. This solution is prone to 

volatility losses and should be replaced at least every 48 hours. 

7.1.5 TRIZMA

 PRESET CRYSTALS, pH 7.0 (Sigma cat# T-7193 or equivalent) – 

Reagent grade. A premixed blend of Tris [Tris(hydroxymethyl)aminomethane] 

and Tris HCL [Tris(hydroxymethyl)aminomethane hydrochloride]. Alternatively, 

a mix of the two components with a weight ratio of 15.5/1 Tris HCL/Tris may be 

used. These blends are targeted to produce a pH near 7.0 at 25 °C in reagent 

water. Trizma

 functions as a buffer, and removes free chlorine in chlorinated 

finished waters (Sect. 8.1.2).

7.1.6 NITROGEN – Used for the following purposes: 

7.1.6.1 Nitrogen aids in aerosol generation of the ESI liquid spray and is used as 

collision gas in some MS/MS instruments. The nitrogen used should meet 

or exceed instrument manufacturer’s specifications.

7.1.6.2 Nitrogen is used to concentrate sample extracts (Ultra High Purity or 

equivalent). 

7.1.7 ARGON – Used as collision gas in MS/MS instruments. Argon should meet or 

exceed instrument manufacturer’s specifications. Nitrogen gas may be used as 

the collision gas provided sufficient sensitivity (product ion formation) is 

achieved. 

7.2 STANDARD SOLUTIONS – When a compound purity is assayed to be 96% or 

greater, the weight can be used without correction to calculate the concentration of the 

stock standard. PFAA analyte, IS and SUR standards commercially purchased in glass 

ampoules are acceptable; however, all subsequent transfers or dilutions performed by 

the analyst must be prepared and stored in polypropylene containers. Solution 

concentrations listed in this section were used to develop this method and are included 

as an example. Alternate concentrations may be used as necessary depending on 

instrument sensitivity and the calibration range used. Standards for sample 

fortification generally should be prepared in the smallest volume that can be accurately 

measured to minimize the addition of excess organic solvent to aqueous samples. 

Even though stability times for standard solutions are suggested in the following 

sections, laboratories should use standard QC practices to determine when their 

standards need to be replaced. 
 

537-12 

NOTE: Stock standards (Sect. 7.2.1.1, 7.2.2.1 and 7.2.3.1) were stored at ≤4 °C. 

Primary dilution standards (Sect. 7.2.1.2, 7.2.2.2 and 7.2.3.2) were stored at 

room temperature to prevent adsorption of the method analytes onto the 

container surfaces that may occur when refrigerated. Storing the standards at 

room temperature will also minimize daily imprecision due to the potential of 

inadequate room temperature stabilization. 

7.2.1 INTERNAL (IS) STOCK STANDARD SOLUTIONS – This method uses three 

IS compounds listed in the table below. These isotopically labeled IS(s) were 

carefully chosen during method development because they encompass all the 

functional groups of the method analytes. Although alternate IS standards may be 

used provided they are isotopically labeled compounds with similar functional 

groups as the method analytes, the analyst must have documented reasons for 

using alternate IS(s). Alternate IS(s) must meet the QC requirements in 

Section 9.3.4. Note that different isotopic labels of the same IS(s) are acceptable 

(e.g., 13C2-PFOA and 13C4-PFOA) but will require modification of the MS/MS 

precursor and product ions. 

Internal Standards Acronym 

Perfluoro-[1,2-13C2]octanoic acid 13C-PFOA 

Sodium perfluoro-1-[1,2,3,4-13C4]octanesulfonate 13C-PFOS 

N-deuteriomethylperfluoro-1-octanesulfonamidoacetic acid d3-NMeFOSAA 

7.2.1.1 IS STOCK STANDARD SOLUTIONS – These IS stocks can be obtained 

as individual certified stock standard solutions. During the development of 

this method, commercially obtained 50 μg/mL stock standard solutions in 

methanol were used (Wellington Labs, Perkin Elmer or equivalent). 

Analysis of the IS(s) is less complicated if the IS(s) purchased contains only 

the linear isomer. IS stock standard solutions were stable for at least 

6 months when stored at 4 °C. 

7.2.1.2 INTERNAL STANDARD PRIMARY DILUTION (IS PDS) STANDARD 

(1-4 ng/μL) – Prepare, or purchase commercially, the IS PDS at a suggested 

concentration of 1-4 ng/μL. If prepared from the individual stock standard 

solutions (Sect. 7.2.1.1), the table below can be used as a guideline for 

preparing the IS PDS. The IS PDS used in these studies was prepared in 

96:4% (vol/vol) methanol:water. The IS PDS has been shown to be stable 

for at least two months when stored in polypropylene centrifuge tubes at 

room temperature. Use 10 μL of this 1-4 ng/μL solution to fortify the final 

1-mL extracts (Sect. 11.5). This will yield a concentration of 10-40 pg/μL 

of each IS in the 1-mL extracts. 
 

537-13 

IS 

Conc. of 

IS Stock 

(μg/mL) 

Vol. Of 

IS Stock 

(μL) 

Final Vol. 

of IS PDS 

(μL) 

Final Conc. 

of IS PDS 

(ng/μL) 

13C-PFOA 1000 5.0 5000 1.0 

13C-PFOS 50 300.0 5000 3.0 

d3-NMeFOSAA 50 400.0 5000 4.0 

7.2.2 SURROGATE (SUR) ANALYTE STANDARD SOLUTIONS – The three 

SUR(s) for this method are listed in the table below. These isotopically labeled 

SUR standards were carefully chosen during method development because they 

encompass most of the functional groups, as well as the water solubility range of 

the method analytes. Although alternate SUR standards may be used provided 

they are isotopically labeled compounds with similar functional groups as the 

method analytes, the analyst must have documented reasons for using alternate 

SUR standards. The alternate SUR standards chosen must still span the water 

solubility range of the method analytes. In addition, alternate SUR standards 

must meet the QC requirements in Section 9.3.5. 

Surrogates Acronym 

Perfluoro-n-[1,2-13C2]hexanoic acid 13C-PFHxA 

Perfluoro-n-[1,2-13C2]decanoic acid 13C-PFDA 

N-deuterioethylperfluoro-1-octanesulfonamidoacetic acid d5-NEtFOSAA 

7.2.2.1 SUR STOCK STANDARD SOLUTIONS – These SUR stocks can be 

obtained as individual certified stock standard solutions. During the 

development of this method, commercially obtained 50 μg/mL stock 

standard solutions in methanol containing 4% water (except for 

d5-NEtFOSAA which was purchased in pure methanol) were used 

(Wellington Labs or equivalent). Analysis of the SUR(s) is less complicated 

if the SUR(s) purchased contains only the linear isomer. SUR stock 

standard solutions were stable for at least 6 months when stored at 4 °C. 

7.2.2.2 SURROGATE PRIMARY DILUTION STANDARD (SUR PDS) 

(1-4 ng/μL) – Prepare, or purchase commercially, the SUR PDS at a 

suggested concentration of 1-4 ng/μL. If prepared from the individual stock 

standard solutions (Sect. 7.2.2.1), the table below can be used as a guideline 

for preparing the SUR PDS. The SUR PDS used in these studies was 

prepared in 96:4% (vol/vol) methanol:water. This solution is used to fortify 

all QC and Field Samples. The PDS has been shown to be stable for one 

year when stored in polypropylene centrifuge tubes at room temperature. 
 

537-14 

SUR 

Conc. Of 

SUR Stock 

(μg/mL) 

Vol. of 

SUR Stock 

(μL) 

Final Vol. of 

SUR PDS 

(μL) 

Final Conc. 

of SUR PDS 

(ng/μL) 

13C-PFHxA 50 100.0 5000 1.0 

13C-PFDA 50 100.0 5000 1.0 

d5-NEtFOSAA 50 400.0 5000 4.0 

 7.2.3 ANALYTE STANDARD SOLUTIONS – Analyte standards may be purchased 

commercially as ampoulized solutions or prepared from neat materials (see 

Table 3 for the analyte sources used during method development). If possible, 

purchase the method analytes as technical grade standards or neat materials. 

Standards or neat materials that contain only the linear isomer can be substituted 

only if technical grade (linear and branched isomers) standards or neat material 

cannot be purchased. PFHxS and PFOS must be purchased as technical grade 

(containing branched and linear isomers), as well as NEtFOSAA and 

NMeFOSAA (if commercially available). PFHxS and PFOS are not available as 

the acids listed in Section 1.1, but rather as their corresponding salts, such as Na+

and K+ (see Table 3). These salts are acceptable starting materials for the stock 

standards provided the measured mass is corrected for the salt content according 

to the equation below. Prepare the Analyte Stock and Primary Dilutions 

Standards as described below. 

 

salt

acid

acid salt MW

MW Mass = Measured Mass ×

where: 

 MWacid = the molecular weight of PFAA 

 MWsalt = the molecular weight of purchased salt 

 

7.2.3.1 ANALYTE STOCK STANDARD SOLUTION – If preparing from neat 

material, accurately weigh approximately 5 mg of pure material to the 

nearest 0.1 mg into a 4-mL or larger polypropylene bottle (Sect. 6.2). Add 

1 mL of the solvent indicated in the table below. Repeat for each method 

analyte. These stock standards were stable for at least 6 months when stored 

at -15 °C. When using these stock standards to prepare a PDS, care must be 

taken to ensure that these standards are at room temperature and adequately 

vortexed.
 

537-15 

Analyte Analyte Stock Solvent 

PFHxA 96:4% (vol/vol) methanol:water

PFHpA 96:4% (vol/vol) methanol:water

PFOA 96:4% (vol/vol) methanol:water

PFNA 96:4% (vol/vol) methanol:water

PFDA 96:4% (vol/vol) methanol:water

PFUnA 96:4% (vol/vol) methanol:water

PFDoA 96:4% (vol/vol) methanol:water

PFTrDA 100% ethyl acetate 

PFTA 100% ethyl acetate 

PFBS 100% methanol 

PFHxS 100% methanol 

PFOS 100% methanol 

NEtFOSAA 100% methanol 

NMeFOSAA 100% methanol 

7.2.3.2 ANALYTE PRIMARY DILUTION STANDARD (PDS) SOLUTION (0.5-

2.5 ng/μL) – The analyte PDS contains all the method analytes of interest at 

various concentrations in methanol containing 4% water. The ESI and 

MS/MS response varies by compound; therefore, a mix of concentrations 

may be needed in the analyte PDS. See Tables 5-9 in Section 17 for 

suggested concentrations for each analyte. During method development, the 

analyte PDS was prepared such that approximately the same instrument 

response was obtained for all the analytes. The analyte PDS is prepared by 

dilution of the combined Analyte Stock Standard Solutions and is used to 

prepare the CAL standards, and fortify the LFBs, the LFSMs, the LFSMDs 

and FDs with the method analytes. The analyte PDS has been shown to be 

stable for 6 months when stored at room temperature. 

 

7.2.4 CALIBRATION STANDARDS (CAL) – At least five calibration concentrations 

are required to prepare the initial calibration curve spanning a 20-fold 

concentration range (Sect. 10.2). Larger concentration ranges will require more 

calibration points. Prepare the CAL standards over the concentration range of 

interest from dilutions of the analyte PDS in methanol containing 4% reagent 

water. The suggested analyte concentrations found in Tables 5-9 can be used as a 

starting point for determining the calibration range. The IS and SUR are added to 

the CAL standards at a constant concentration. During method development, the 

concentrations of the SUR(s) were 10-40 pg/μL in the standard (40-160 ng/L in 

the sample) and the IS(s) were 10-40 pg/μL. The lowest concentration CAL 

standard must be at or below the MRL, which may depend on system sensitivity. 

The CAL standards may also be used as CCCs (Sect. 9.3.2). During method 

development, the CAL standards were shown to be stable for at least two weeks 

when stored at room temperature. Longer storage times are acceptable provided 
 

537-16 

appropriate QC measures are documented demonstrating the CAL standard 

stability. 

8. SAMPLE COLLECTION, PRESERVATION, AND STORAGE

8.1 SAMPLE BOTTLE PREPARATION 

8.1.1 Samples must be collected in a 250-mL polypropylene bottle fitted with a 

polypropylene screw-cap. 

8.1.2 The preservation reagent, listed in the table below, is added to each sample bottle 

as a solid prior to shipment to the field (or prior to sample collection). 

 

Compound Amount Purpose

Trizma 5.0 g/L buffering reagent and removes free chlorine

 

8.2 SAMPLE COLLECTION 

8.2.1 The sample handler must wash their hands before sampling and wear nitrile 

gloves while filling and sealing the sample bottles. PFAA contamination during 

sampling can occur from a number of common sources, such as food packaging 

and certain foods and beverages. Proper hand washing and wearing nitrile gloves 

will aid in minimizing this type of accidental contamination of the samples. 

8.2.2 Open the tap and allow the system to flush until the water temperature has 

stabilized (approximately 3 to 5 min). Collect samples from the flowing system. 

8.2.3 Fill sample bottles, taking care not to flush out the sample preservation reagent. 

Samples do not need to be collected headspace free.

8.2.4 After collecting the sample, cap the bottle and agitate by hand until preservative is 

dissolved. Keep the sample sealed from time of collection until extraction. 

8.3 FIELD REAGENT BLANKS (FRB) 

8.3.1 A FRB must be handled along with each sample set. The sample set is composed 

of samples collected from the same sample site and at the same time. At the 

laboratory, fill the field blank sample bottle with reagent water and preservatives, 

seal, and ship to the sampling site along with the sample bottles. For each FRB 

shipped, an empty sample bottle (no preservatives) must also be shipped. At the 

sampling site, the sampler must open the shipped FRB and pour the preserved 

reagent water into the empty shipped sample bottle, seal and label this bottle as 

the FRB. The FRB is shipped back to the laboratory along with the samples and 

analyzed to ensure that PFAAs were not introduced into the sample during sample 

collection/handling. 
 

537-17 

8.3.2 The same batch of preservative must be used for the FRBs as for the field 

samples. 

8.3.3 The reagent water used for the FRBs must be initially analyzed for method 

analytes as a LRB and must meet the LRB criteria in Section 9.3.1 prior to use. 

This requirement will ensure samples are not being discarded due to contaminated 

reagent water rather than contamination during sampling. 

8.4 SAMPLE SHIPMENT AND STORAGE – Samples must be chilled during shipment 

and must not exceed 10 °C during the first 48 hours after collection. Sample 

temperature must be confirmed to be at or below 10 °C when the samples are received 

at the laboratory. Samples stored in the lab must be held at or below 6 °C until 

extraction, but should not be frozen. 

NOTE: Samples that are significantly above 10° C, at the time of collection, may need 

to be iced or refrigerated for a period of time, in order to chill them prior to 

shipping. This will allow them to be shipped with sufficient ice to meet the 

above requirements. 

8.5 SAMPLE AND EXTRACT HOLDING TIMES – Results of the sample storage 

stability study (Table 10) indicated that all compounds listed in this method have 

adequate stability for 14 days when collected, preserved, shipped and stored as 

described in Sections 8.1, 8.2, and 8.4. Therefore, water samples should be extracted 

as soon as possible but must be extracted within 14 days. Extracts must be stored at 

room temperature and analyzed within 28 days after extraction. The extract storage 

stability study data are presented in Table 11. 

9. QUALITY CONTROL

9.1 QC requirements include the Initial Demonstration of Capability (IDC) and ongoing 

QC requirements that must be met when preparing and analyzing Field Samples. This 

section describes the QC parameters, their required frequencies, and the performance 

criteria that must be met in order to meet EPA quality objectives. The QC criteria 

discussed in the following sections are summarized in Tables 12 and 13. These QC 

requirements are considered the minimum acceptable QC criteria. Laboratories are 

encouraged to institute additional QC practices to meet their specific needs. 

9.1.1 METHOD MODIFICATIONS – The analyst is permitted to modify LC columns, 

LC conditions, evaporation techniques, internal standards or surrogate standards, 

and MS and MS/MS conditions. Each time such method modifications are made, 

the analyst must repeat the procedures of the IDC. Modifications to LC 

conditions should still produce conditions such that co-elution of the method 

analytes is minimized to reduce the probability of suppression/enhancement 

effects. 
 

537-18 

9.2 INITIAL DEMONSTRATION OF CAPABILITY – The IDC must be successfully 

performed prior to analyzing any Field Samples. Prior to conducting the IDC, the 

analyst must first generate an acceptable Initial Calibration following the procedure 

outlined in Section 10.2. 

9.2.1 INITIAL DEMONSTRATION OF LOW SYSTEM BACKGROUND – Any time 

a new lot of SPE cartridges, solvents, centrifuge tubes, disposable pipets, and 

autosampler vials are used, it must be demonstrated that an LRB is reasonably 

free of contamination and that the criteria in Section 9.3.1 are met. If an 

automated extraction system is used, an LRB should be extracted on each port to 

ensure that all the valves and tubing are free from potential PFAA contamination. 

9.2.2 INITIAL DEMONSTRATION OF PRECISION (IDP) – Prepare, extract, and 

analyze four to seven replicate LFBs fortified near the midrange of the initial 

calibration curve according to the procedure described in Section 11. Sample 

preservatives as described in Section 8.1.2 must be added to these samples. The 

relative standard deviation (RSD) of the results of the replicate analyses must be 

less than 20%. 

9.2.3 INITIAL DEMONSTRATION OF ACCURACY (IDA) – Using the same set of 

replicate data generated for Section 9.2.2, calculate average recovery. The 

average recovery of the replicate values must be within ± 30% of the true value. 

9.2.4 INITIAL DEMONSTRATION OF PEAK ASYMMETRY FACTOR – Peak 

asymmetry factors must be calculated using the equation in Section 9.3.9 for the 

first two eluting peaks (if only two analytes are being analyzed, both must be 

evaluated) in a mid-level CAL standard. The peak asymmetry factors must fall in 

the range of 0.8 to 1.5. See guidance in Section 10.2.4.1 if the calculated peak 

asymmetry factors do not meet the criteria. 

9.2.5 MINIMUM REPORTING LEVEL (MRL) CONFIRMATION – Establish a target 

concentration for the MRL based on the intended use of the method. The MRL 

may be established by a laboratory for their specific purpose or may be set by a 

regulatory agency. Establish an Initial Calibration following the procedure 

outlined in Section 10.2. The lowest CAL standard used to establish the Initial 

Calibration (as well as the low-level CCC, Section 10.3) must be at or below the 

concentration of the MRL. Establishing the MRL concentration too low may 

cause repeated failure of ongoing QC requirements. Confirm the MRL following 

the procedure outlined below. 

9.2.5.1 Fortify, extract, and analyze seven replicate LFBs at the proposed MRL 

concentration. These LFBs must contain all method preservatives described 

in Section 8.1.2. Calculate the mean measured concentration (Mean) and 

standard deviation for these replicates. Determine the Half Range for the 

prediction interval of results (HRPIR) using the equation below 
 

537-19 

HR s PIR = 3963 .

where 

 s = the standard deviation 

 3.963 = a constant value for seven replicates.1 

9.2.5.2 Confirm that the upper and lower limits for the Prediction Interval of Result 

(PIR = Mean + HRPIR) meet the upper and lower recovery limits as shown

below 

 The Upper PIR Limit must be ≤150% recovery. 

×100% ≤150% +

FortifiedConcentration

Mean HRPIR

 The Lower PIR Limit must be ≥ 50% recovery. 

×100% ≥ 50% −

FortifiedConcentration

Mean HRPIR

9.2.5.3 The MRL is validated if both the Upper and Lower PIR Limits meet the 

criteria described above (Sect. 9.2.5.2). If these criteria are not met, the 

MRL has been set too low and must be determined again at a higher 

concentration. 

9.2.6 CALIBRATION CONFIRMATION – Analyze a QCS as described in Section 

9.3.10 to confirm the accuracy of the standards/calibration curve. 

9.2.7 DETECTION LIMIT DETERMINATION (optional) – While DL determination 

is not a specific requirement of this method, it may be required by various 

regulatory bodies associated with compliance monitoring. It is the responsibility 

of the laboratory to determine if DL determination is required based upon the 

intended use of the data. 

9.2.7.1 Replicate analyses for this procedure should be done over at least three days 

(i.e., both the sample extraction and the LC/MS/MS analyses should be done 

over at least three days). Prepare at least seven replicate LFBs at a 

concentration estimated to be near the DL. This concentration may be 

estimated by selecting a concentration at 2-5 times the noise level. The DLs 

in Table 5 were calculated from LFBs fortified at various concentrations as 

indicated in the table. The appropriate fortification concentrations will be 

dependent upon the sensitivity of the LC/MS/MS system used. All 

preservation reagents listed in Section 8.1.2 must also be added to these 

samples. Analyze the seven replicates through all steps of Section 11. 
 

537-20 

NOTE: If an MRL confirmation data set meets these requirements, a DL may be 

calculated from the MRL confirmation data, and no additional analyses 

are necessary. 

Calculate the DL using the following equation 

 

 ( − 1 ,1 − = 99.0 )= ×

n α

DL s t

 

 where 

 s = standard deviation of replicate analyses 

 t (n-1, 1-α=0.99) = Student's t value for the 99% confidence 

level with n-1 degrees of freedom 

n = number of replicates. 

NOTE: Do not subtract blank values when performing DL calculations. The DL 

is a statistical determination of precision only.2 If the DL replicates are 

fortified at a low enough concentration, it is likely that they will not meet 

the precision and accuracy criteria for CCCs. Therefore, no precision 

and accuracy criteria are specified. 

9.2.7.2 If a laboratory is establishing their own MRL, the calculated DLs should not 

be used as the MRL for analytes that commonly occur as background 

contaminants. Method analytes that are seen in the background should be 

reported as present in Field Samples, only after careful evaluation of the 

background levels. It is recommended that a MRL be established at the 

mean LRB concentrations + 3σ or 3 times the mean LRB concentration, 

whichever is greater. This value should be calculated over a period of time, 

to reflect variability in the blank measurements. It is recommended that this 

value be used as an MRL in order to avoid reporting false positive results. 

9.3 ONGOING QC REQUIREMENTS – This section summarizes the ongoing QC 

criteria that must be followed when processing and analyzing Field Samples. 

9.3.1 LABORATORY REAGENT BLANK (LRB) – An LRB is required with each 

extraction batch (Sect. 3.6) to confirm that potential background contaminants are 

not interfering with the identification or quantitation of method analytes. If more 

than 20 Field Samples are included in a batch, analyze an LRB for every 20 

samples. If the LRB produces a peak within the retention time window of any 

analyte that would prevent the determination of that analyte, determine the source 

of contamination and eliminate the interference before processing samples. 

Background contamination must be reduced to an acceptable level before 

proceeding. Background from method analytes or other contaminants that interfere with the measurement of method analytes must be below 1/3 of the MRL. 

Blank contamination is estimated by extrapolation, if the concentration is below 

the lowest CAL standard. This extrapolation procedure is not allowed for sample 

results as it may not meet data quality objectives. If the method analytes are 
 

537-21 

detected in the LRB at concentrations equal to or greater than this level, then all 

data for the problem analyte(s) must be considered invalid for all samples in the 

extraction batch. Because background contamination is a significant problem for 

several method analytes, it is highly recommended that the analyst maintain a 

historical record of LRB data. 

9.3.2 CONTINUING CALIBRATION CHECK (CCC) – CCC Standards are analyzed 

at the beginning of each analysis batch, after every 10 Field Samples, and at the 

end of the analysis batch. See Section 10.3 for concentration requirements and 

acceptance criteria. 

9.3.3 LABORATORY FORTIFIED BLANK (LFB) – An LFB is required with each 

extraction batch (Sect. 3.6). The fortified concentration of the LFB must be 

rotated between low, medium, and high concentrations from batch to batch. The 

low concentration LFB must be as near as practical to, but no more than two 

times, the MRL. Similarly, the high concentration LFB should be near the high 

end of the calibration range established during the initial calibration (Sect. 10.2). 

Results of the low-level LFB analyses must be 50-150% of the true value. 

Results of the medium and high-level LFB analyses must be 70-130% of the true 

value. If the LFB results do not meet these criteria for method analytes, then all 

data for the problem analyte(s) must be considered invalid for all samples in the 

extraction batch. 

9.3.4 INTERNAL STANDARDS (IS) – The analyst must monitor the peak areas of the 

IS(s) in all injections during each analysis day. The IS responses (peak areas) in 

any chromatographic run must be within 70-140% of the response in the most 

recent CCC and must not deviate by more than 50% from the average area 

measured during initial analyte calibration. If the IS areas in a chromatographic 

run do not meet these criteria, inject a second aliquot of that extract aliquotted 

into a new capped autosampler vial. Random evaporation losses have been 

observed with the polypropylene caps causing high IS(s) areas. 

 

9.3.4.1 If the reinjected aliquot produces an acceptable IS response, report results 

for that aliquot. 

9.3.4.2 If the reinjected extract fails again, the analyst should check the calibration 

by reanalyzing the most recently acceptable CAL standard. If the CAL 

standard fails the criteria of Section 10.3, recalibration is in order per 

Section 10.2. If the CAL standard is acceptable, extraction of the sample 

may need to be repeated provided the sample is still within the holding time. 

Otherwise, report results obtained from the reinjected extract, but annotate 

as suspect. Alternatively, collect a new sample and re-analyze. 

9.3.5 SURROGATE RECOVERY – The SUR standard is fortified into all samples, 

CCCs, LRBs, LFBs, LFSMs, LFSMDs, FD, and FRB prior to extraction. It is 

also added to the CAL standards. The SUR is a means of assessing method 
 

537-22 

performance from extraction to final chromatographic measurement. Calculate 

the recovery (%R) for the SUR using the following equation 

%  ×100 









=

B

A

R

where 

A = calculated SUR concentration for the QC or Field Sample 

 B = fortified concentration of the SUR. 

9.3.5.1 SUR recovery must be in the range of 70-130%. When SUR recovery from 

a sample, blank, or CCC is less than 70% or greater than 130%, check 1) 

calculations to locate possible errors, 2) standard solutions for degradation, 

3) contamination, and 4) instrument performance. Correct the problem and 

reanalyze the extract. 

9.3.5.2 If the extract reanalysis meets the SUR recovery criterion, report only data 

for the reanalyzed extract. 

9.3.5.3 If the extract reanalysis fails the 70-130% recovery criterion, the analyst 

should check the calibration by injecting the last CAL standard that passed. 

If the CAL standard fails the criteria of Section 10.3, recalibration is in 

order per Section 10.2. If the CAL standard is acceptable, extraction of the 

sample should be repeated provided the sample is still within the holding 

time. If the re-extracted sample also fails the recovery criterion, report all 

data for that sample as suspect/SUR recovery to inform the data user that the 

results are suspect due to SUR recovery. Alternatively, collect a new 

sample and re-analyze. 

9.3.6 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) – Analysis of an 

LFSM is required in each extraction batch and is used to determine that the 

sample matrix does not adversely affect method accuracy. Assessment of method 

precision is accomplished by analysis of a Field Duplicate (FD) (Sect. 9.3.7); 

however, infrequent occurrence of method analytes would hinder this assessment. 

If the occurrence of method analytes in the samples is infrequent, or if historical 

trends are unavailable, a second LFSM, or LFSMD, must be prepared, extracted, 

and analyzed from a duplicate of the Field Sample. Extraction batches that 

contain LFSMDs will not require the extraction of a FD. If a variety of different 

sample matrices are analyzed regularly, for example, drinking water from 

groundwater and surface water sources, method performance should be 

established for each. Over time, LFSM data should be documented by the 

laboratory for all routine sample sources. 

9.3.6.1 Within each extraction batch (Sect. 3.6), a minimum of one Field Sample is 

fortified as an LFSM for every 20 Field Samples analyzed. The LFSM is 

prepared by spiking a sample with an appropriate amount of the Analyte 
 

537-23 

PDS (Sect. 7.2.3.2). Select a spiking concentration that is greater than or 

equal to the matrix background concentration, if known. Use historical data 

and rotate through the low, mid and high concentrations when selecting a 

fortifying concentration. 

9.3.6.2 Calculate the percent recovery (%R) for each analyte using the equation 

( ) % ×100 −

=

C

A B

R

 where 

 A = measured concentration in the fortified sample 

 B = measured concentration in the unfortified sample 

 C = fortification concentration. 

9.3.6.3 Analyte recoveries may exhibit matrix bias. For samples fortified at or 

above their native concentration, recoveries should range between 70-130%, 

except for low-level fortification near or at the MRL (within a factor of 

2-times the MRL concentration) where 50-150% recoveries are acceptable. 

If the accuracy of any analyte falls outside the designated range, and the 

laboratory performance for that analyte is shown to be in control in the 

CCCs, the recovery is judged to be matrix biased. The result for that analyte 

in the unfortified sample is labeled suspect/matrix to inform the data user 

that the results are suspect due to matrix effects.

9.3.7 FIELD DUPLICATE OR LABORATORY FORTIFIED SAMPLE MATRIX 

DUPLICATE (FD or LFSMD) – Within each extraction batch (not to exceed 20 

Field Samples, Sect. 3.6), a minimum of one FD or LFSMD must be analyzed. 

Duplicates check the precision associated with sample collection, preservation, 

storage, and laboratory procedures. If method analytes are not routinely observed 

in Field Samples, an LFSMD should be analyzed rather than an FD. 

9.3.7.1 Calculate the relative percent difference (RPD) for duplicate measurements 

(FD1 and FD2) using the equation 

( ) 100

1 2 2/

1 2

×

+

−

=

FD FD

FD FD

RPD

9.3.7.2 RPDs for FDs should be ≤30%. Greater variability may be observed when 

FDs have analyte concentrations that are within a factor of 2 of the MRL. 

At these concentrations, FDs should have RPDs that are ≤50%. If the RPD 

of any analyte falls outside the designated range, and the laboratory 

performance for that analyte is shown to be in control in the CCC, the 

recovery is judged to be matrix biased. The result for that analyte in the 

unfortified sample is labeled suspect/matrix to inform the data user that the 

results are suspect due to matrix effects. 
 

537-24 

9.3.7.3 If an LFSMD is analyzed instead of a FD, calculate the relative percent 

difference (RPD) for duplicate LFSMs (LFSM and LFSMD) using the 

equation 

( ) 100

2/

×

+

−

=

LFSM LFSMD

LFSM LFSMD

RPD

9.3.7.4 RPDs for duplicate LFSMs should be ≤30% for samples fortified at or 

above their native concentration. Greater variability may be observed when 

LFSMs are fortified at analyte concentrations that are within a factor of 2 of 

the MRL. LFSMs fortified at these concentrations should have RPDs that 

are ≤50% for samples fortified at or above their native concentration. If the 

RPD of any analyte falls outside the designated range, and the laboratory 

performance for that analyte is shown to be in control in the CCC, the 

recovery is judged to be matrix biased. The result for that analyte in the 

unfortified sample is labeled suspect/matrix to inform the data user that the 

results are suspect due to matrix effects. 

9.3.8 FIELD REAGENT BLANK (FRB) – The purpose of the FRB is to ensure that 

PFAAs measured in the Field Samples were not inadvertently introduced into the 

sample during sample collection/handling. Analysis of the FRB is required only 

if a Field Sample contains a method analyte or analytes at or above the MRL. 

The FRB is processed, extracted and analyzed in exactly the same manner as a 

Field Sample. If the method analyte(s) found in the Field Sample is present in the 

FRB at a concentration greater than 1/3 the MRL, then all samples collected with 

that FRB are invalid and must be recollected and reanalyzed. 

9.3.9 PEAK ASYMMETRY FACTOR – A peak asymmetry factor must be calculated 

using the equation below during the IDL and every time a calibration curve is 

generated. The peak asymmetry factor for the first two eluting peaks in a midlevel CAL standard (if only two analytes are being analyzed, both must be 

evaluated) must fall in the range of 0.8 to 1.5. Modifying the standard or extract 

composition to more aqueous content to prevent poor shape is not permitted. See 

guidance in Section 10.2.4.1 if the calculated peak asymmetry factors do not meet 

the criteria. 
 

537-25 

a b

10%

 A

b

a

s =

 

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 

 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. 

9.3.10 QUALITY CONTROL SAMPLES (QCS) – As part of the IDC (Sect. 9.2), each 

time a new Analyte PDS (Sect. 7.2.3.2) is prepared, and at least quarterly, analyze 

a QCS sample from a source different from the source of the CAL standards. If a 

second vendor is not available, then a different lot of the standard should be used. 

The QCS should be prepared and analyzed just like a CCC. Acceptance criteria 

for the QCS are identical to the CCCs; the calculated amount for each analyte 

must be ± 30% of the expected value. If measured analyte concentrations are not 

of acceptable accuracy, check the entire analytical procedure to locate and correct 

the problem. 

10. CALIBRATION AND STANDARDIZATION

10.1 Demonstration and documentation of acceptable initial calibration is required before 

any samples are analyzed. After the initial calibration is successful, a CCC is required 

at the beginning and end of each period in which analyses are performed, and after 

every tenth Field Sample. 

10.2 INITIAL CALIBRATION 

10.2.1 ESI-MS/MS TUNE 

10.2.1.1 Calibrate the mass scale of the MS with the calibration compounds and 

procedures prescribed by the manufacturer. 

10.2.1.2 Optimize the [M-H]-

 for each method analyte by infusing approximately 

0.5-1.0 μg/mL of each analyte (prepared in the initial mobile phase 

conditions) directly into the MS at the chosen LC mobile phase flow rate 

(approximately 0.3 mL/min). This tune can be done on a mix of the method 

analytes. The MS parameters (voltages, temperatures, gas flows, etc.) are 
 

537-26 

varied until optimal analyte responses are determined. The method analytes 

may have different optima requiring some compromise between the optima. 

See Table 2 for ESI-MS conditions used in method development. 

10.2.1.3 Optimize the product ion (Sect. 3.19) for each analyte by infusing 

approximately 0.5-1.0 μg/mL of each analyte (prepared in the initial mobile 

phase conditions) directly into the MS at the chosen LC mobile phase flow 

rate (approximately 0.3 mL/min). This tune can be done on a mix of the 

method analytes. The MS/MS parameters (collision gas pressure, collision 

energy, etc.) are varied until optimal analyte responses are determined. 

Typically, the carboxylic acids have very similar MS/MS conditions and the 

sulfonic acids have similar MS/MS conditions. See Table 4 for MS/MS 

conditions used in method development. 

10.2.2 Establish LC operating parameters that optimize resolution and peak shape. 

Suggested LC suggested conditions can be found in Table 1. The LC conditions 

listed in Table 1 may not be optimum for all LC systems and may need to be 

optimized by the analyst (See Sect. 10.2.4.1). Modifying the standard or extract 

composition to more aqueous content to prevent poor shape is not permitted. 

 Cautions: LC system components, as well as the mobile phase constituents, 

contain many of the method analytes in this method. Thus, these 

PFAAs will build up on the head of the LC column during mobile 

phase equilibration. To minimize the background PFAA peaks 

and to keep background levels constant, the time the LC column 

sits at initial conditions must be kept constant and as short as 

possible (while ensuring reproducible retention times). In 

addition, prior to daily use, flush the column with 100% methanol 

for at least 20 min before initiating a sequence. It may be 

necessary on some systems to flush other LC components such as 

wash syringes, sample needles or any other system components 

before daily use. 

 Mobile phase modifiers other than 20 mM ammonium acetate may 

be used at the discretion of the analyst, provided that the retention 

time stability criteria in Sect. 11.7.2 can be met over a period of 

two weeks. During method development, retention times shifted to 

shorter and shorter times as days progressed when mobile phases 

with less then 20 mM ammonium acetate were used. 

10.2.3 Inject a mid-level CAL standard under LC/MS conditions to obtain the retention 

times of each method analyte. If analyzing for PFTA, ensure that the LC 

conditions are adequate to prevent co-elution of PFTA and the mobile phase 

interferants shown in Figure 1. These interferants have the same precursor and 

products ions as PFTA, and under faster LC conditions may co-elute with PFTA. 

Divide the chromatogram into retention time windows each of which contains one 
 

537-27 

or more chromatographic peaks. During MS/MS analysis, fragment a small 

number of selected precursor ions ([M-H]-; Sect. 3.17) for the analytes in each 

window and choose the most abundant product ion. The product ions (also the 

quantitation ions) chosen during method development are in Table 4, although 

these will be instrument dependent. For maximum sensitivity, small mass 

windows of ±0.5 daltons around the product ion mass were used for quantitation. 

If sufficient sensitivity exists to meet the MRL, wider mass ranges may be used to 

obtain more confirmation ions. 

 

NOTE: As the NOTE in Section 10.2.4.1 indicates, PFOS has linear and 

branched isomers. There have been reports10 that not all the 

products ions in the linear PFOS are produced in all the branched 

PFOS isomers. (This phenomenon probably exists for PFHxS and 

PFBS also, although it has not been studied to date.) Thus, in an 

attempt to reduce PFOS bias, it is required that the 

m/z 499 → m/z 80 transition be used as the quantitation transition. 

Some MS/MS instruments, such as conventional ion traps, may not 

be able to scan a product ion with such a wide mass difference from 

the precursor ion; therefore, they may not be used for this method if 

PFOS, PFBS, or PFHxS analysis is to be conducted. Literature 

reports10 indicate for the most abundant PFOS isomer, which is the 

linear isomer, that all the products ions obtained on an ion trap have 

less than 10% relative abundance. In addition, there is not a single 

ion trap MS/MS transition that encompasses the linear isomer and 

the majority of the branch isomers; thus, the bias would be 

unacceptably high. 

10.2.4 Inject a mid-level CAL standard under optimized LC/MS/MS conditions to ensure 

that each method analyte is observed in its MS/MS window and that there are at 

least 10 scans across the peak for optimum precision. 

10.2.4.1 If broad, split or fronting peaks are observed for the first two eluting 

chromatographic peaks (if only two analytes are being analyzed, both must 

be evaluated), change the initial mobile phase conditions to higher aqueous 

content until the peak asymmetry ratio for each peak is 0.8 – 1.5. The peak 

asymmetry factor is calculated as described in Section 9.3.9 on a mid-level 

CAL standard. The peak asymmetry factor must meet the above criteria for 

the first two eluting peaks during the IDL and every time a new calibration 

curve is generated. Modifying the standard or extract composition to more 

aqueous content to prevent poor shape is not permitted.

NOTE: PFHxS, PFOS, NMeFOSAA, and NEtFOSAA have multiple 

chromatographic peaks using the LC conditions in Table 1 due 

to chromatographic resolution of the linear and branched 

isomers of these compounds. Most PFAAs are produced by 

two different processes. One process gives rise to linear 
 

537-28 

PFAAs only while the other process produces both linear and 

branched isomers. Thus, both branched and linear PFAAs 

can potentially be found in the environment. For the 

aforementioned compounds that give rise to more than one 

peak, all the chromatographic peaks observed in the standard 

must be integrated and the areas totaled. Chromatographic 

peaks in a sample must be integrated in the same way as the 

CAL standard. 

10.2.5 Prepare a set of at least five CAL standards as described in Section 7.2.4. The 

lowest concentration CAL standard must be at or below the MRL, which may 

depend on system sensitivity. It is recommended that at least four of the CAL 

standards are at a concentration greater than or equal to the MRL. 

10.2.6 The LC/MS/MS system is calibrated using the IS technique. Use the LC/MS/MS 

data system software to generate a linear regression or quadratic calibration curve 

for each of the analytes. This curve must always be forced through zero and may 

be concentration weighted, if necessary. Forcing zero allows for a better estimate 

of the background levels of method analytes. 

10.2.6.1 The isotopically labeled IS(s) in this method may undergo suppression in 

the ESI source if the concentration of the co-eluting unlabeled method 

analyte(s) is too high. The analyte concentration at which suppression may 

occur can vary depending on the instrument, LC conditions, ESI conditions, 

IS concentration, etc. To evaluate whether suppression is occurring during 

calibration, calculate the relative percent difference (RPD) between the high 

(H) and low (L) areas for each IS using the equation 

( )

( ) 100

2/

×

+

−

=

H L

H L

RPD

10.2.6.2 The RPD calculated above must be <20% for each IS during calibration. If 

the calculated RPD is >20% for any IS, the analyst must recalibrate at lower 

analyte concentrations until the IS RPDs are <20%. 

10.2.7 CALIBRATION ACCEPTANCE CRITERIA – When quantitated using the initial 

calibration curve, each calibration point, except the lowest point, for each analyte 

should calculate to be within 70-130% of its true value. The lowest CAL point 

should calculate to be within 50-150% of its true value. If these criteria cannot be 

met, the analyst will have difficulty meeting ongoing QC criteria. It is 

recommended that corrective action is taken to reanalyze the CAL standards, 

restrict the range of calibration, or select an alternate method of calibration 

(forcing the curve through zero is still required). 

CAUTION: When acquiring MS/MS data, LC operating conditions must be 

carefully reproduced for each analysis to provide reproducible 
 

537-29 

retention times. If this is not done, the correct ions will not be 

monitored at the appropriate times. As a precautionary measure, the 

chromatographic peaks in each window must not elute too close to 

the edge of the segment time window. 

10.3 CONTINUING CALIBRATION CHECK (CCC) – Minimum daily calibration 

verification is as follows. Verify the initial calibration at the beginning and end of 

each group of analyses, and after every tenth sample during analyses. In this context, 

a “sample” is considered to be a Field Sample. LRBs, CCCs, LFBs, LFSMs, FDs 

FRBs and LFSMDs are not counted as samples. The beginning CCC of each analysis 

batch must be at or below the MRL in order to verify instrument sensitivity prior to 

any analyses. If standards have been prepared such that all low CAL points are not in 

the same CAL solution, it may be necessary to analyze two CAL standards to meet 

this requirement. Alternatively, the analyte concentrations in the analyte PDS may be 

customized to meet this criteria. Subsequent CCCs should alternate between a 

medium and high concentration CAL standard. 

10.3.1 Inject an aliquot of the appropriate concentration CAL standard and analyze with 

the same conditions used during the initial calibration. 

10.3.2 Determine that the absolute areas of the quantitation ions of the IS(s) are within 

70-140% of the areas measured in the most recent continuing calibration check, 

and within 50-150% from the average areas measured during initial calibration. If 

any of the IS areas has changed by more than these amounts, adjustments must be 

made to restore system sensitivity. These adjustments may include cleaning of the 

MS ion source, or other maintenance as indicated in Section 10.3.4. Major 

instrument maintenance requires recalibration (Sect 10.2) and verification of 

sensitivity by analyzing a CCC at or below the MRL (Sect 10.3). Control charts 

are useful aids in documenting system sensitivity changes. 

10.3.3 Calculate the concentration of each analyte and SUR in the CCC. The calculated 

amount for each analyte and SUR for medium and high level CCCs must be 

within ± 30% of the true value. The calculated amount for the lowest calibration 

point for each analyte must be within ± 50% and the SUR must be within ± 30% 

of the true value. If these conditions do not exist, then all data for the problem 

analyte must be considered invalid, and remedial action should be taken 

(Sect. 10.3.4) which may require recalibration. Any Field or QC Samples that 

have been analyzed since the last acceptable calibration verification should be 

reanalyzed after adequate calibration has been restored, with the following 

exception. If the CCC fails because the calculated concentration is greater 

than 130% (150% for the low-level CCC) for a particular method analyte, 

and Field Sample extracts show no detection for that method analyte, nondetects may be reported without re-analysis.

10.3.4 REMEDIAL ACTION – Failure to meet CCC QC performance criteria may 

require remedial action. Major maintenance, such as cleaning the electrospray 
 

537-30 

probe, atmospheric pressure ionization source, cleaning the mass analyzer, 

replacing the LC column, etc., requires recalibration (Sect 10.2) and verification 

of sensitivity by analyzing a CCC at or below the MRL (Sect 10.3). 

 11. PROCEDURE

11.1 This procedure may be performed manually or in an automated mode using a robotic 

or automatic sample preparation device. The data presented in Tables 5-11 

demonstrate data collected by manual extraction. If an automated system is used to 

prepare samples, follow the manufacturer's operating instructions, but all extraction 

and elution steps must be the same as in the manual procedure. Extraction and/or 

elution steps may not be changed or omitted to accommodate the use of an automated 

system. If an automated system is used, the LRBs should be rotated among the ports 

to ensure that all the valves and tubing meet the LRB requirements (Sect. 9.3.1). 

11.2 Some of the PFAAs adsorb to surfaces, including polypropylene. Therefore, the 

aqueous sample bottles must be rinsed with the elution solvent (Sect 11.4.4) whether 

extractions are performed manually or by automation. The bottle rinse is passed 

through the cartridge to elute the method analytes and is then collected (Sect. 11.4.4). 

NOTE: The SPE cartridges and sample bottles described in this section are designed 

as single use items and should be discarded after use. They may not be 

refurbished for reuse in subsequent analyses. 

11.3 SAMPLE PREPARATION 

11.3.1 Samples are preserved, collected and stored as presented in Section 8. All Field 

and QC Samples, including the LRB, LFB and FRB, must contain the 

dechlorinating agent listed in Section 8.1.2. Determine sample volume. An 

indirect measurement may be done in one of two ways: by marking the level of 

the sample on the bottle or by weighing the sample and bottle to the nearest 10 g. 

After extraction, proceed to Section 11.6 for final volume determination. Some of 

the PFAAs adsorb to surfaces, thus the sample volume may NOT be transferred 

to a graduated cylinder for volume measurement. The LRB, LFB and FRB may 

be prepared by measuring 250 mL of reagent water with a polypropylene 

graduated cylinder or filling a 250-mL sample bottle to near the top. 

11.3.2 Add an aliquot of the SUR PDS (Sect. 7.2.2.2) to each sample, cap and invert to 

mix. During method development, a 10-μL aliquot of the 1-4 ng/μL SUR PDS 

(Sect. 7.2.2.2) was added to 250 mL of sample for a final concentration of 

40 ng/L for 13C-PFHxA and 13C-PFDA and 160 ng/L for d5-NEtFOSAA. 

11.3.3 In addition to the SUR(s) and dechlorination agent, if the sample is an LFB, FD, 

LFSM, or LFSMD, add the necessary amount of analyte PDS (Sect. 7.2.3.2). Cap 

and invert each sample to mix. 
 

537-31 

11.4 CARTRIDGE SPE PROCEDURE 

 

11.4.1 CARTRIDGE CLEAN-UP AND CONDITIONING – DO NOT allow cartridge 

packing material to go dry during any of the conditioning steps. Rinse each 

cartridge with 15 mL of methanol. Next, rinse each cartridge with 18 mL of 

reagent water, without allowing the water to drop below the top edge of the 

packing. If the cartridge goes dry during the conditioning phase, the conditioning 

must be started over. Add 4-5 mL of reagent water to each cartridge, attach the 

sample transfer tubes (Sect. 6.9.3), turn on the vacuum, and begin adding sample 

to the cartridge. 

11.4.2 SAMPLE EXTRACTON – Adjust the vacuum so that the approximate flow rate 

is 10-15 mL/min. Do not allow the cartridge to go dry before all the sample has 

passed through. 

11.4.3 SAMPLE BOTTLE AND CARTRIDGE RINSE – After the entire sample has 

passed through the cartridge, rinse the sample bottles with two 7.5-mL aliquots of 

reagent water and draw each aliquot through the sample transfer tubes and the 

cartridges. Draw air or nitrogen through the cartridge for 5 min at high vacuum 

(10-15 in. Hg). 

NOTE: If empty plastic reservoirs are used in place of the sample transfer 

tubes to pass the samples through the cartridges, these reservoirs 

must be treated like the transfer tubes. After the entire sample has 

passed through the cartridge, the reservoirs must be rinsed to waste 

with reagent water. 

11.4.4 SAMPLE BOTTLE AND CARTRIDGE ELUTION – Turn off and release the 

vacuum. Lift the extraction manifold top and insert a rack with collection tubes 

into the extraction tank to collect the extracts as they are eluted from the 

cartridges. Rinse the sample bottles with 4 mL of methanol and elute the analytes 

from the cartridges by pulling the 4 mL of methanol through the sample transfer 

tubes and the cartridges. Use a low vacuum such that the solvent exits the 

cartridge in a dropwise fashion. Repeat sample bottle rinse and cartridge elution 

with a second 4-mL aliquot of methanol. 

NOTE: If empty plastic reservoirs are used in place of the sample transfer 

tubes to pass the samples through the cartridges, these reservoirs 

must be treated like the transfer tubes. After the reservoirs have 

been rinsed in Section 11.4.3, the elution solvent used to rinse the 

sample bottles must be swirled down the sides of the reservoirs while 

eluting the cartridge to ensure that any method analytes on the 

surface of the reservoirs are transferred to the extract. 

11.5 EXTRACT CONCENTRATION – Concentrate the extract to dryness under a gentle 

stream of nitrogen in a heated water bath (60-65 °C) to remove all the water/methanol 
 

537-32 

mix. Add the appropriate amount of 96:4% (vol/vol) methanol:water solution and the 

IS PDS (Sect. 7.2.1.2) to the collection vial to bring the volume to 1 mL and vortex. 

(10 μL of the 1-4 ng/μL IS PDS for extract concentrations of 10-40 pg/μL were used 

for method development). Transfer a small aliquot with a plastic pipet (Sect. 6.7) to a 

polypropylene autosampler vial. 

NOTE: It is recommend that the entire 1-mL aliquot not be transferred to the 

autosampler vial because the polypropylene autosampler caps do not 

reseal after injection. Therefore, do not store the extracts in the 

autosampler vials as evaporation losses can occur occasionally in these 

autosampler vials. Extracts can be stored in 15-mL centrifuge tubes 

(Sect. 6.3). 

11.6 SAMPLE VOLUME DETERMINATION – If the level of the sample was marked on 

the sample bottle, use a graduated cylinder to measure the volume of water required to 

fill the original sample bottle to the mark made prior to extraction. Determine to the 

nearest 10 mL. If using weight to determine volume, weigh the empty bottle to the 

nearest 10 g and determine the sample weight by subtraction of the empty bottle 

weight from the original sample weight (Sect. 11.3.1). Assume a sample density of 

1.0 g/mL. In either case, the sample volume will be used in the final calculations of 

the analyte concentration (Sect. 12.2). 

11.7 EXTRACT ANALYSIS 

11.7.1 Establish operating conditions equivalent to those summarized in Tables 1-4 of 

Section 17. Instrument conditions and columns should be optimized prior to the 

initiation of the IDC. 

11.7.2 Establish an appropriate retention time window for each analyte. This should be 

based on measurements of actual retention time variation for each method analyte 

in CAL standard solutions analyzed on the LC over the course of time. A value 

of plus or minus three times the standard deviation of the retention time obtained 

for each method analyte while establishing the initial calibration and completing 

the IDC can be used to calculate a suggested window size. However, the 

experience of the analyst should weigh heavily on the determination of the 

appropriate retention window size. 

11.7.3 Calibrate the system by either the analysis of a calibration curve (Sect. 10.2) or by 

confirming the initial calibration is still valid by analyzing a CCC as described in 

Section 10.3. If establishing an initial calibration, complete the IDC as described 

in Section 9.2. 

11.7.4 Begin analyzing Field Samples, including QC samples, at their appropriate 

frequency by injecting the same size aliquots (10 μL was used in method 

development), under the same conditions used to analyze the CAL standards. 
 

537-33 

11.7.5 At the conclusion of data acquisition, use the same software that was used in the 

calibration procedure to identify peaks of interest in predetermined retention time 

windows. Use the data system software to examine the ion abundances of the 

peaks in the chromatogram. Identify an analyte by comparison of its retention 

time with that of the corresponding method analyte peak in a reference standard. 

Comparison of the MS/MS mass spectra is not particularly useful given the 

limited ±0.5 dalton mass range around a single product ion for each method 

analyte. 

11.7.6 The analyst must not extrapolate beyond the established calibration range. If an 

analyte peak area exceeds the range of the initial calibration curve, the extract 

may be diluted with 96%:4% (vol/vol) methanol:water solution and the 

appropriate amount of IS added to match the original concentration. Re-inject the 

diluted extract. Incorporate the dilution factor into the final concentration 

calculations. Acceptable SUR performance (Sect. 9.3.5.1) should be determined 

from the undiluted sample extract. The resulting data should be documented as a 

dilution, with an increased MRL. 

12. DATA ANALYSIS AND CALCULATION

12.1. Complete chromatographic resolution is not necessary for accurate and precise 

measurements of analyte concentrations using MS/MS. In validating this method, 

concentrations were calculated by measuring the product ions listed in Table 4. Other 

ions may be selected at the discretion of the analyst. 

12.2. Calculate analyte and SUR concentrations using the multipoint calibration established 

in Section 10.2. Do not use daily calibration verification data to quantitate analytes in 

samples. Adjust final analyte concentrations to reflect the actual sample volume 

determined in Section 11.6. 

 

12.3 Prior to reporting the data, the chromatogram should be reviewed for any incorrect 

peak identification or poor integration. 

12.4 PFHxS, PFOS, NMeFOSAA, and NEtFOSAA have multiple chromatographic peaks 

using the LC conditions in Table 1 due to the linear and branch isomers of these 

compounds (Sect. 10.2.4.1). The areas of all the linear and branched isomer peaks 

observed in the CAL standards for each of these analytes must be summed and the 

concentrations reported as a total for each of these analytes. 

12.5 Calculations must utilize all available digits of precision, but final reported 

concentrations should be rounded to an appropriate number of significant figures (one 

digit of uncertainty), typically two, and not more than three significant figures. 

 

NOTE: Some data in Section 17 of this method are reported with more than two 

significant figures. This is done to better illustrate the method performance. 
 

537-34 

13. METHOD PERFORMANCE

13.1 PRECISION, ACCURACY, AND MINIMUM REPORTING LEVELS – Tables for 

these data are presented in Section 17. LCMRLs and DLs for each method analyte are 

presented in Table 5. Precision and accuracy are presented for four water matrices: 

reagent water (Tables 6 and 7); chlorinated (finished) ground water (Table 8); high 

TOC chlorinated surface water (Table 9); and day zero of the aqueous holding time in 

chlorinated surface water (Table 10). 

13.2 EVALUATION OF ADDITIONAL PERFLUORINATED ALKYL COMPOUNDS – 

Nine additional compounds were evaluated for inclusion in this method, but were 

not included for the following reasons. Due to volatility, low recoveries 

following evaporation were obtained for PFOSA (perfluorooctane sulfonylamide), 

NEtFOSE (2-N-ethylperfluorooctane sulfonamide ethanol), NMeFOSE (2-Nmethylperfluorooctane sulfonamide ethanol), and NEtFOSA (Nethylperfluorooctanesulfonamide). Poor chromatographic peak shape, which 

degraded with repeated injections, was obtained for PFOSAA 

(perfluorooctanesulfonamidoacetic acid). PFOSfn (perfluorosulfinate) degraded 

in aqueous samples in under 24 hours. PFOSF (perfluorooctane-1-

sulfonylfluoride) had the same LC retention time as PFOS and appeared to 

undergo conversion in the ESI source to PFOS under method conditions. Finally, 

acceptable SPE recoveries were not obtained for PFBA (perfluorobutanoic acid) 

and PFPA (perfluoropentanoic acid). 

13.3 SAMPLE STORAGE STABILITY STUDIES – An analyte storage stability study was 

conducted by fortifying the analytes into chlorinated surface water samples that 

were collected, preserved, and stored as described in Section 8. The precision and 

mean recovery (n=5) of analyses, conducted on Days 0, 7, and 14 are presented in 

Table 10. 

13.4 EXTRACT STORAGE STABILITY STUDIES – Extract storage stability studies 

were conducted on extracts obtained from a chlorinated surface water fortified with 

the method analytes. The precision and mean recovery (n=5) of injections conducted 

on Days 0, 7, 14, 22, and 28 are reported in Table 11. 

13.5 SECOND LABORATORY DEMONSTRATION – The performance of this method 

was demonstrated by multiple laboratories, with results similar to those reported in 

Section 17. The authors wish to acknowledge the work of the Suffolk County Water 

Authority Laboratory Hauppauge, NY and Lancaster Labs, a wholly owned subsidiary 

of Thermo Fisher Scientific, for their participation in the second laboratory 

demonstration. 

14. POLLUTION PREVENTION

14.1 This method utilizes SPE to extract analytes from water. It requires the use of very 

small volumes of organic solvent and very small quantities of pure analytes, thereby 

minimizing the potential hazards to both the analyst and the environment as compared 
 

537-35 

to the use of large volumes of organic solvents in conventional liquid-liquid 

extractions. 

14.2 For information about pollution prevention that may be applicable to laboratory 

operations, consult “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 N.W., Washington, D.C., 

20036 or on-line at http://membership.acs.org/c/ccs/pub_9.htm (accessed August 

2008). 

15. WASTE MANAGEMENT

15.1 The analytical procedures described in this method generate relatively small amounts 

of waste since only small amounts of reagents and solvents are used. The matrices of 

concern are finished drinking water or source water. However, laboratory waste 

management practices must be conducted consistent with all applicable rules and 

regulations, and that laboratories protect the air, water, and land by minimizing and 

controlling all releases from fume hoods and bench operations. Also, compliance is 

required with any sewage discharge permits and regulations, particularly the hazardous 

waste identification rules and land disposal restrictions. 

16. REFERENCES

1. Winslow, S.D., Pepich, B.V., Martin, J.J., Hallberg, G.R., Munch, D.J., Frebis, C.P., 

Hedrick, E.J., Krop, R.A. “Statistical Procedures for Determination and Verification of 

Minimum Reporting Levels for Drinking water Methods.” Environ. Sci. & Technol. 2004,

40, 281-288. 

2. Glaser, J.A., D.L. Foerst, G.D. McKee, S.A. Quave, W.L. Budde, "Trace Analyses for 

Wastewaters.” Environ. Sci. Technol. 1981, 15, 1426-1435. 

3. Martin, J.W., Kannan, K., Berger, U., De Voogt, P., Field, J., Franklin, J, Giesy, J.P., Harner, 

T., Muir, D.C., Scott, B., Kaiser, M., Järnberg, U., Jones, K.C., Mabury, S.A., Schroeder, H., 

Simcik, M., Sottani, C., van Bavel, B., Kärrman, A., Lindström, G., van Leeuwen, S. 

“Analytical Challenges Hamper Perfluoroalkyl Research.” Environ Sci Technol. 2004, 38, 

248A-255A. 

4. Leenheer, J.A., Rostad, C.E., Gates, P.M., Furlong, E.T., Ferrer, I. “Molecular Resolution 

and Fragmentation of Fulvic Acid by Electrospray Ionization/Multistage Tandem Mass 

Spectrometry.” Anal. Chem. 2001, 73, 1461-1471. 

5. Cahill, J.D., Furlong E.T., Burkhardt, M.R., Kolpin, D., Anderson, L.G. “Determination of 

Pharmaceutical Compounds in Surface- and Ground-Water Samples by Solid-Phase 

Extraction and High-Performance Liquid Chromatography Electrospray Ionization Mass 

Spectrometry.” J. Chromatogr. A 2004, 1041, 171-180. 
 

537-36 

6. “OSHA Safety and Health Standards, General Industry," (29CRF1910). Occupational Safety 

and Health Administration, OSHA 2206, (Revised, July 1, 2001). 

7. “Carcinogens-Working with Carcinogens,” Publication No. 77-206, Department of Health, 

Education, and Welfare, Public Health Service, Center for Disease Control, National Institute 

of Occupational Safety and Health, Atlanta, Georgia, August 1977. 

8. "Safety in Academic Chemistry Laboratories,” American Chemical Society Publication, 

Committee on Chemical Safety, 7th Edition. Information on obtaining a copy is available at 

http://membership.acs.org/C/CCS/pub_3.htm (accessed August, 2008). Also available by 

request at OSS@acs.org. 

9. “SAB Review of EPA’s Draft Risk Assessment of Potential Human Health Effects 

Associated with PFOA and Its Salts.” Document available at 

http://yosemite.epa.gov/sab/SABPRODUCT.NSF/A3C83648E77252828525717F004B9099/

$File/sab_06_006.pdf (accessed August, 2008). 

10. Langlois, I. and Oehme, M. “Structural Identification of Isomers Present In Technical 

Perfluorooctane Sulfonate By Tandem Mass Spectrometry.” Rapid Commun. Mass Spectrom.

2006, 20, 844-850. 
 

537-37 

17. TABLES, DIAGRAMS, FLOWCHARTS AND VALIDATION DATA

TABLE 1. LC METHOD CONDITIONS 

Time (min) % 20 mM ammonium acetate % Methanol

Initial 60.0 40.0 

1.0 60.0 40.0 

25.0 10.0 90.0 

32.0 10.0 90.0 

32.1 60.0 40.0 

37.0 60.0 40.0 

Waters Atlantis dC18 2.1 x 150 mm packed with 5.0 μm C18 stationary phase 

Flow rate of 0.3 mL/min 

10 μL injection 

TABLE 2. ESI-MS METHOD CONDITIONS 

ESI Conditions 

Polarity Negative ion 

Capillary needle voltage -3 kV 

Cone gas flow 98 L/hr 

Nitrogen desolvation gas 1100 L/hr 

Desolvation gas temp. 350 °C
 

537-38 

TABLE 3. METHOD ANALYTE SOURCE, RETENTION TIMES (RTs), AND 

SUGGESTED IS REFERENCES

Analyte 

Method analyte 

Sourcea

Peak # 

(Fig. 1) 

RT 

(min) IS# Ref 

PFBS Aldrich 1 8.48 2 

PFHxA Fluka 2 11.38 1 

PFHpA Aldrich 4 14.33 1 

PFHxSb

Fluka 5 14.48 2 

PFOA Aldrich 6 16.73 1 

PFNA Aldrich 8 18.76 1 

PFOSb

Fluka 9 18.72 2 

PFDA Aldrich 11 20.47 1 

NMeFOSAA c 13 21.28 3 

NEtFOSAA c 15 22.01 3 

PFUnA Aldrich 17 21.95 1 

PFDoA Aldrich 18 23.20 1 

PFTrDA Exfluor 19 24.32 1 

PFTA Aldrich 20 25.27 1 

13C-PFHxA Wellington Labs 3 11.38 1 

13C-PFDA Wellington Labs 12 20.47 1 

d5-NEtFOSAA Wellington Labs 16 21.99 3 

13C-PFOA– IS#1 PerkinElmer 7 16.73 - 

13C-PFOS– IS#2 Wellington Labs 10 18.71 - 

d3-NMeFOSAA–IS#3 Wellington Labs 14 21.24 - 

a

 The data presented in this method were obtained using analytes purchased from these 

vendors. Other vendors’ materials can be used provided the QC requirements in 

Section 9 can be met. 

b

 PFHxS and PFOS neat materials were purchased as K+ salts. The CASRN of the 

PFOS potassium salt is 2795-39-3 and the CASRN of the PFHxS potassium salt is 

3871-99-6. 

c

 These analytes were not commercially available at the time of method development 

as technical grade mixtures containing both linear and branched isomers.
 

537-39 

TABLE 4. MS/MS METHOD CONDITIONSa

Segmentb Analyte 

Precursor Ion c

(m/z)

Product Ionc,d

(m/z)

Cone 

Voltage (v)

Collision 

Energye (v)

1 PFBS 299 80 40 25

2 PFHxA 313 269 15 10

3 PFHpA 363 319 12 10

3 PFHxSf

399 80 40 40

4 PFOA 413 369 15 10

4 PFNA 463 419 12 10

4 PFOSf

 499 80 40 40

5 PFDA 513 469 15 10

5 NMeFOSAAf

 570 419 25 20

5 NEtFOSAAf

 584 419 25 20

5 PFUnA 563 519 15 10

5 PFDoA 613 569 15 10

6 PFTrDA 663 619 15 10

6 PFTA 713 669 15 10

2

13C-PFHxA 315 270 15 10 

5

13C-PFDA 515 470 12 12 

5 d5-NEtFOSAA 589 419 25 20 

4

13C-PFOA 415 370 15 10

4

13C-PFOS 503 80 40 40

5 d3-NMeFOSAA 573 419 25 20

a

 An LC/MS/MS chromatogram of the analytes is shown in Figure 1. 

b Segments are time durations in which single or multiple scan events occur. 

c

 Precursor and product ions listed in this table are nominal masses. During MS and MS/MS 

optimization, the analyst should determine the precursor and product ion masses to one 

decimal place by locating the apex of the mass spectral peak place (e.g., m/z 498.9→79.9 for 

PFOS). These precursor and product ion masses (with one decimal place) should be used in 

the MS/MS method for all analyses. 

d

 Ions used for quantitation purposes. 

e

 Argon used as collision gas at a flow rate of 0.3 mL/min. 

f

 Analyte has multiple resolved chromatographic peaks due to linear and branched isomers. All 

peaks summed for quantitation purposes. 
 

537-40 

TABLE 5. DLs AND LCMRLs IN REAGENT WATER 

Analyte Fortified Conc. (ng/L)a DLb (ng/L) LCMRLc (ng/L) 

PFBS 9.1 3.1 3.7 

PFHxA 5.0 1.6 2.9 

PFHpA 4.1 0.5 3.8 

PFHxS 11 2.0 8.0 

PFOA 4.6 1.7 5.1 

PFNA 4.8 0.7 5.5 

PFOS 9.6 1.4 6.5 

PFDA 3.7 0.7 3.8 

NMeFOSAA 20 6.5 14 

NEtFOSAA 21 4.2 14 

PFUnA 5.4 2.8 6.9 

PFDoA 3.7 1.1 3.5 

PFTrDA 5.5 2.2 3.8 

PFTA 4.4 1.7 4.7 

a

 Spiking concentration used to determine DL. 

b

 Detection limits were determined by analyzing seven replicates over three days according 

to Section 9.2.7. 

c

 LCMRLs were calculated according to the procedure in reference 1. 
 

537-41 

TABLE 6. PRECISION AND ACCURACY OF LOW LEVEL FORTIFIED REAGENT 

WATER (n=7) 

Analyte Fortified Conc. (ng/L) Mean % Recovery % RSD 

PFBS 9.1 93 12 

PFHxA 5.0 108 9.3 

PFHpA 8.3 111 6.2 

PFHxS 11 116 4.9 

PFOA 9.1 110 6.5 

PFNA 9.6 121 7.5 

PFOS 9.6 116 4.1 

PFDA 7.3 114 4.1 

NMeFOSAA 20 109 9.3 

NEtFOSAA 21 113 5.5 

PFUnA 11 120 5.4 

PFDoA 7.3 116 4.6 

PFTrDA 5.5 118 11 

PFTA 8.7 117 2.4 

13C-PFHxA 40 89 6.1 

13C-PFDA 40 102 7.3 

d5-NEtFOSAA 160 100 4.9 
 

537-42 

TABLE 7. PRECISION AND ACCURACY OF HIGH LEVEL FORTIFIED REAGENT 

WATER (n=7) 

Analyte Fortified Conc. (ng/L) Mean % Recovery % RSD 

PFBS 91 92 5.9 

PFHxA 50 103 4.6 

PFHpA 41 106 3.4 

PFHxS 113 101 2.5 

PFOA 46 104 3.6 

PFNA 48 109 3.5 

PFOS 96 101 3.8 

PFDA 37 106 4.8 

NMeFOSAA 202 100 3.7 

NEtFOSAA 214 105 5.3 

PFUnA 54 107 3.3 

PFDoA 37 107 3.9 

PFTrDA 55 107 1.9 

PFTA 44 111 2.7 

13C-PFHxA 40 93 4.4 

13C-PFDA 40 99 3.5 

d5-NEtFOSAA 160 100 4.5 
 

537-43 

TABLE 8. PRECISION AND ACCURACY IN FORTIFIED CHLORINATED GROUND 

WATERa

 (n=7)

Analyte Fortified Conc. (ng/L) Mean % Recovery % RSD 

PFBS 91 101 3.9 

PFHxA 50 112 0.9 

PFHpA 41 105 1.8 

PFHxS 113 103 2.2 

PFOA 46 107 2.2 

PFNA 48 103 2.4 

PFOS 96 100b

2.2 

PFDA 37 101 2.6 

NMeFOSAA 202 96 2.6 

NEtFOSAA 214 100 2.7 

PFUnA 54 99 2.6 

PFDoA 37 99 2.3 

PFTrDA 55 96 3.4 

PFTA 44 97 4.2 

13C-PFHxA 40 98 2.0 

13C-PFDA 40 98 2.1 

d5-NEtFOSAA 160 97 5.0 

a

 TOC = 0.96 mg/L and hardness = 342 mg/L. 

b Recovery is corrected for a matrix PFOS concentration of 40 ng/L. 
 

537-44 

TABLE 9. PRECISION AND ACCURACY IN FORTIFIED HIGH TOC CHLORINATED 

SURFACE WATERa (n=7)

Analyte Fortified Conc. (ng/L) Mean % Recovery % RSD 

PFBS 91 85 4.3 

PFHxA 50 101 3.2 

PFHpA 41 106 3.7 

PFHxS 113 101 4.9 

PFOA 46 110 3.1 

PFNA 48 112 3.9 

PFOS 96 104 4.0 

PFDA 37 109 4.3 

NMeFOSAA 202 95 4.0 

NEtFOSAA 214 100 3.4 

PFUnA 54 106 3.7 

PFDoA 37 104 4.2 

PFTrDA 55 102 4.7 

PFTA 44 105 3.5 

13C-PFHxA 40 85 4.3 

13C-PFDA 40 101 3.2 

d5-NEtFOSAA 160 106 3.7 

a

 TOC = 4.95 mg/L and hardness =137 mg/L. 
537-45 

TABLE 10. AQUEOUS SAMPLE HOLDING TIME DATA FOR SAMPLES FROM CHLORINAT

WATERa, FORTIFIED WITH METHOD ANALYTES AND PRESERVED AND STOR

TO SECTION 8 (n=5) 

Fortified 

Conc. (ng/L) 

Day 0 Day 7 Da

Analyte Mean %Rec % RSD Mean %Rec % RSD Mean %Rec 

PFBS 91 83 8.5 87 3.3 90 PFHxA 50 99 4.3 98 3.4 104 PFHpA 41 105 1.1 97 4.2 102 PFHxS 113 98 2.8 98 3.8 92 PFOAb 46 98 2.5 94 3.2 96 PFNA 48 106 2.5 102 4.0 98 PFOS 96 103 4.9 100 3.1 104 PFDA 37 102 3.3 99 5.0 99 NMeFOSAA 202 97 3.6 95 4.0 97 NEtFOSAA 214 105 3.0 101 6.1 99 PFUnA 54 104 3.0 97 4.8 95 PFDoA 37 100 2.2 94 5.8 102 PFTrDA 55 102 2.8 93 3.8 104 PFTA 44 105 4.0 99 4.3 102 

 a TOC = 1.8 mg/L and hardness =205 mg/L. 

b Recovery corrected for a native PFOA concentration of 14 ng/L in the tap water used for this study.
537-46 

TABLE 11. EXTRACT HOLDING TIME DATA FOR SAMPLES FROM CHLORINATED SURFA

FORTIFIED WITH METHOD ANALYTES AND PRESERVED AND STORED ACCOSECTION 8 (n=5)

Analyte 

Fortified Conc.

 (ng/L) 

Day 0 Day 7 Day 14 Day 22 

Mean

%Rec

RSD

% 

Mean

%Rec

RSD 

% 

Mean

%Rec

 RSD

% 

Mean

%Rec

 RSD 

% 

Me

%R

PFBS 91 83 8.5 89 10 91 8.3 92 11 9PFHxA 50 99 4.3 101 4.7 103 5.7 105 5.2 10PFHpA 41 105 1.1 101 3.4 109 3.5 110 2.9 10PFHxS 113 98 2.8 101 2.1 107 3.5 106 3.1 10PFOAa 46 98 2.5 100 1.4 102 2.7 101 3.4 10PFNA 48 106 2.5 104 3.0 102 3.5 104 4.0 10PFOS 96 103 4.9 100 1.6 109 1.4 103 3.6 10PFDA 37 102 3.3 104 2.9 100 3.3 102 5.4 10NMeFOSAA 202 97 3.6 96 1.8 96 2.8 102 3.4 9NEtFOSAA 214 105 3.0 100 2.5 99 3.4 108 1.7 10PFUnA 54 104 3.0 103 3.1 100 4.4 104 3.6 10PFDoA 37 100 2.2 102 1.6 101 3.0 107 5.4 11PFTrDA 55 102 2.8 95 3.8 97 2.5 104 4.1 10PFTA 44 105 4.0 100 2.6 93 3.4 110 3.1 11

a Recovery corrected for a native PFOA concentration of 14 ng/L in the tap water used for this study.
537-47 

TABLE 12. INITIAL DEMONSTRATION OF CAPABILITY QUALITY CONTROL REQUIREMENTS 

Method Reference Requirement Specification and Frequency Acceptance Criteria 

Sect. 9.2.1 Initial Demonstration of Low System Background Analyze LRB prior to any other IDC steps. 

Demonstrate that all method analyt

and that possible interferences from 

prevent the identification and qu

analytes. 

Sect. 9.2.2 Initial Demonstration of Precision (IDP) Analyze four to seven replicate LFBs fortified near the midrange calibration concentration. %RSD must be Sect. 9.2.3 Initial Demonstration of Accuracy (IDA) Calculate average recovery for replicates used in IDP. Mean recovery ± 30% Sect. 9.2.4 Initial Demonstration of Peak Asymmetry Factor 

Calculate the peak asymmetry factor using the 

equation in Section 9.3.9 for the first two eluting

chromatographic peaks in a mid-level CAL 

standard. 

Peak asymmetry facto

Sect. 9.2.5 Minimum Reporting Limit (MRL) Confirmation 

Fortify, extract and analyze seven replicate LFBs 

at the proposed MRL concentration. Calculate the 

Mean and the Half Range (HR). Confirm that the 

upper and lower limits for the Prediction Interval 

of Result (Upper PIR, and Lower PIR, Sect. 

9.2.5.2) meet the recovery criteria. 

Upper PIR ≤ 1

Lower PIR ≥ 

Sect. 9.2.6 

and 9.3.10 

Quality Control Sample 

(QCS) 

Analyze a standard from a second source, as 

part of IDC. Results must be within 70-1

Sect. 9.2.7 Detection Limit (DL) Determination (optional) 

Over a period of trhee days, prepare a minimum of 

seven replicate LFBs fortified at a concentration 

estimated to be near the DL. Analyze the 

replicates through all steps of the analysis. 

Calculate the DL using the equation in 

Sect. 9.2.7.1. 

Data from DL replicates are not r

precision and accuracy criteria. 

fortified at a low enough concentra

will not meet precision and 

NOTE: Table 12 is intended as an abbreviated summary of QC requirements provided as a convenience to the method user. Because the 

abbreviated to fit the table format, there may be issues that need additional clarification, or areas where important additional infor

is needed. In all cases, the full text of the QC in Section 9 supersedes any missing or conflicting information in this table.
537-48 

TABLE 13. ONGOING QUALITY CONTROL REQUIREMENTS (SUMMARY) 

Method Reference Requirement Specification and Frequency Acceptance Criteria 

Sect. 8.1 - 

Sect. 8.5 Sample Holding Time 14 days with appropriate preservation and storage as described in Sections 8.1-8.5. Sample results are valid only if sam

sample holdi

Sect. 8.5 Extract Holding Time 28 days when stored at room temperature in polypropylene centrifuge tubes. Extract results are valid only if ex

extract holdi

Sect. 9.3.1 Laboratory Reagent Blank (LRB) Daily, or with each extraction batch of up to 20 samples, whichever is more frequent. 

Demonstrate that all method analyt

confirm that possible interferences d

method analytes. If targets exceed 

are present, results for these subject 

are inva

Sect. 9.3.3 Laboratory Fortified Blank (LFB) 

Analyze at least one LFB daily or one for each 

extraction batch of up to 20 Field Samples. Rotate

the fortified concentrations between low, medium and 

high amounts. 

Results of LFB analyses must be 70-

method analyte for all fortified con

CAL point. Results of the LFBs co

point for each method analyte must 

Sect. 9.3.4 Internal Standard (IS) 

Internal standards, 13C-PFOA (IS#1), 13C-PFOS 

(IS#2), and d3-NMeFOSAA (IS#3), are added to all 

standards and sample extracts, including QC samples. 

Compare IS areas to the average IS area in the initial 

calibration and to the most recent CCC. 

Peak area counts for all ISs in all inje

the average peak area calculated du

70-140% from the most recent C

criterion, corresponding tar

Sect. 9.3.5 Surrogate Standards (SUR) 

Surrogate standards, 13C-PFHxA, 13C-PFDA, and d5- NEtFOSAA, are added to all CAL standards and 

samples, including QC samples. Calculate SUR 

recoveries. 

SUR recoveries must be 70-130% o

this criteria, report all results for sam
537-49 

TABLE 13. (Continued) 

Method Reference Requirement Specification and Frequency Acceptance Criteria 

Sect. 9.3.6 Laboratory Fortified Sample Matrix (LFSM) 

Analyze one LFSM per extraction batch (20 

samples or less) fortified with method analytes at a 

concentration close to but greater than the native 

concentration, if known. Calculate LFSM 

recoveries. 

Recoveries at mid and high levels 

and within 50-150% at the low-le

the MRL). If these criteria are n

suspect due to ma

Sect. 9.3.7 

Laboratory Fortified 

Sample Matrix Duplicate 

(LFSMD) or 

Field Duplicates (FD) 

Extract and analyze at least one FD or LFSMD 

with each extraction batch (20 samples or less). A

LFSMD may be substituted for a FD when the 

frequency of detects are low. Calculate RPDs. 

Method analyte RPDs for the 

≤30% at mid and high levels of f

the MRL. If these criteria are n

suspect due to ma

Sect. 9.3.8 Field Reagent Blank (FRB) 

Analysis of the FRB is required only if a Field 

Sample contains a method analyte or analytes at or 

above the MRL. The FRB is processed, extracted 

and analyzed in exactly the same manner as a 

Field Sample. 

 If the method analyte(s) found in 

in the FRB at a concentration grea

all samples collected with that FR

recollected and r

Sect. 9.3.9 Peak Asymmetry Factor 

Calculate the peak asymmetry factor for the first 

two eluting chromatographic peaks in a mid-level 

CAL standard every time a calibration curve is 

generated. 

Peak asymmetry fact

Sect. 9.3.10 Quality Control Sample (QCS) Analyze at least quarterly or when preparing new standards, as well as during the IDC. Results must be within 70-

Sect. 10.2 

and 

Sect. 9.3.2 

Initial Calibration 

Use IS calibration technique to generate a first or

second order calibration curve forced through 

zero. Use at least five standard concentrations. 

Check the calibration curve as described in 

Sect. 10.2.7. 

When each CAL standard is calcu

the calibration curve, the analyte 

of the true value for all except th

should be 50-150% of the true 

recommended if these cr

Sect. 9.3.2 

and Sect. 

10.3 

Continuing Calibration 

Check (CCC) 

Verify initial calibration by analyzing a low level

(at the MRL or below) CCC prior to analyzing 

samples. CCCs are then injected after every 10 

samples and after the last sample, rotating 

concentrations to cover the calibrated range of the

instrument. 

Recovery for each analyte and SU

of the true value for all but the lo

Recovery for each analyte in th

must be within 50-150% of the tru

be within 70-130% of 

NOTE: Table 13 is intended as an abbreviated summary of QC requirements provided as a convenience to the method user. Because the 

abbreviated to fit the table format, there may be issues that need additional clarification, or areas where important additional infor

is needed. In all cases, the full text of the QC in Section 8-10 supersedes any missing or conflicting information in this table. 
537-50 

FIGURE 1. EXAMPLE CHROMATOGRAM FOR REAGENT WATER FORTIFIED WITH METHOD 5

CONCENTRATION LEVELS INDICATED IN TABLE 6. NUMBERED PEAKS ARE IDEN

a Abbreviated chromatographic conditions may lead to co-elution of PFTA and the potential interferants 

Section 10.2.3 for more information. 

10.00 12.50 15.00 17.50 20.00 22.50 25.00

0

100

Retention Time, min

1

2,3

4

5

6,7

8,9,10 

11,12

13,14

15,16,17

18

19

1

20

PFHxS isomers 

PFOS isomers 

NEtFOSAA 

isomers 

NMeFOSAA 

isomers 

 

 in