537

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

METHOD 537. DETERMINATION OF SELECTED PERFLUORINAT ED 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 Deve lopment, National Exposure

Research Laboratory

P.E. Grimmett US EPA, Office of Research and Devel opment, 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 45268537

- 2 METHOD 537

DETERMINATION OF SELECTED PERFLUORINATED ALKYL ACID S 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 spe ctrometry (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 NEtFO SAA

─ N-methyl perfluorooctanesulfonamidoacetic acid NMeF OSAA

─

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 b ased 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 pred icted to fall, with high confidence (99%), between 50 and 150% recovery. Single labora tory 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. 1537

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1.3 Laboratories using this method will not be required to determine the LCMRL for this method, but will need to demonstrate that their lab oratory MRL for this method meets the requirements described in Section 9.2.5.

1.4 Determining the Detection Limit (DL) for analytes i n this method is optional (Sect. 9.2.7). Detection limit is defined as the statisti cally calculated minimum concentration

that can be measured with 99% confidence that the r eported value is greater than zero. 2
The DL is compound dependent and is dependent on ex traction 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 interpre tation of the associated data.

1.6 METHOD FLEXIBILITY – In recognition of technologica l 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 perform ance. Modifications that are introduced in the interest of reducing cost or samp le processing time, but result in poorer method performance, should not be used. Ana lytes must be adequately resolved chromatographically in order to permit the mass spectrometer to dwell on a minimum number of compounds eluting within a retent ion time window. Instrumental sensitivity (or signal-to-noise) will decrease if t oo 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 o utlined in the initial demonstration of capability (IDC, Sect. 9.2), verify that all Qua lity Control (QC) acceptance criteria in this method (Sect. 9) are met, and that acceptab le 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 detai led information of specific portions of the method that may be modified. If th ere is any perceived conflict between the general method flexibility sta tement in Section 1.6 and specific information in Sections 4-12, Sections 4-1 2 supersede Section 1.6.

2. SUMMARY OF METHOD

2.1 A 250-mL water sample is fortified with surrog ates and passed through a solid phase extraction (SPE) cartridge containing polystyrenedi vinylbenzene (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 co ncentrated 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 inj ection is made into an LC equipped

with a C 18 column that is interfaced to an MS/MS. The analyt es are separated and537

- 4 identified by comparing the acquired mass spectra a nd retention times to reference spectra and retention times for calibration standar ds acquired under identical

LC/MS/MS conditions. The concentration of each ana lyte is determined by using the internal standard technique. Surrogate analytes ar e 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 analy zed 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 Continuin g Calibration Check (CCC) standards. Additional CCCs may be required depend ing on the length of the analysis batch and/or the number of Field Samples.

3.2 CALIBRATION STANDARD (CAL) – A solution prepare d from the primary dilution standard solution and/or stock standard solution, i nternal standard(s), and the surrogate(s). The CAL solutions are used to calibr ate the instrument response with respect to analyte concentration.

3.3 COLLISIONALLY ACTIVATED DISSOCIATION (CAD) – Th e process of converting the precursor ion’s translational energy into internal energy by collisions with neutral gas molecules to bring about dissociat ion into product ions.

3.4 CONTINUING CALIBRATION CHECK (CCC) – A calibrat ion standard containing the method analytes, internal standard(s) and surro gate(s). The CCC is analyzed periodically to verify the accuracy of the existing calibration for those analytes.

3.5 DETECTION LIMIT (DL) – The minimum concentratio n of an analyte that can be identified, measured, and reported with 99% confide nce that the analyte concentration is greater than zero. This is a statistical determ ination 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 Samp les (not including QC samples) extracted together by the same person(s) d uring a work day using the same lot of SPE devices, solvents, surrogate, internal s tandard and fortifying solutions. Required QC samples include Laboratory Reagent Blan k, Laboratory Fortified Blank, Laboratory Fortified Sample Matrix, and either a Fi eld Duplicate or Laboratory Fortified Sample Matrix Duplicate.

3.7 FIELD DUPLICATES (FD1 and FD2) – Two separate s amples collected at the same time and place under identical circumstances, and t reated exactly the same throughout field and laboratory procedures. Analyses of FD1 a nd FD2 give a measure of the precision associated with sample collection, preser vation, and storage, as well as laboratory procedures.537

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3.8 FIELD REAGENT BLANK (FRB) – An aliquot of reage nt 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 p urpose 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 t he 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 si milar to the method analytes, has no potential to be present in water samples, and is no t a method analyte.

3.10 LABORATORY FORTIFIED BLANK (LFB) – A volume of reagent water or other blank matrix to which known quantities of the metho d 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 methodo logy is in control, and whether the laboratory is capable of making accurate and precis e measurements.

3.11 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) – A preserved field sample to which known quantities of the method anal ytes are added in the laboratory. The LFSM is processed and analyzed exactly like a s ample, and its purpose is to determine whether the sample matrix contributes bia s to the analytical results. The background concentrations of the analytes in the sa mple matrix must be determined in a separate sample extraction and the measured value s 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 L FSM. The LFSMD is fortified, extracted, and analyzed identically to the LFSM. T he 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 in cluding exposure to all glassware, equipment, solvents and reagents, sample preservati ves, internal standard, and surrogates that are used in the analysis batch. Th e 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 concentr ation 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 in formation provided by vendors concerning a chemical’s toxicity, health ha zards, physical properties, fire, and reactivity data including storage, spill, and handl ing precautions.537

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3.16 MINIMUM REPORTING LEVEL (MRL) – The minimum co ncentration that can be reported as a quantitated value for a method analyt e 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 i on is mass selected and fragmented by collisionally ac tivated dissociation to produce distinctive product ions of smaller m/z

.

3.18 PRIMARY DILUTION STANDARD (PDS) SOLUTION – A s olution containing the analytes prepared in the laboratory from stock stan dard solutions and diluted as needed to prepare calibration solutions and other needed a nalyte 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 d issociation of the precursor ion.

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

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

from the source of calibration standards. The seco nd source SSS is used to fortify the QCS at a known concentration. The QCS is used to c heck calibration standard integrity.

3.21 STOCK STANDARD SOLUTION (SSS) – A concentrated solution containing one or more method analytes prepared in the laboratory usi ng assayed reference materials or purchased from a reputable commercial source.

3.22 SURROGATE ANALYTE (SUR) – A pure chemical whic h chemically resembles method analytes and is extremely unlikely to be fou nd 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 an alytes. The purpose of the SUR is to monitor method performance with each sample.

4. INTERFERENCES

4.1 All glassware must be meticulously cleaned. Wa sh 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 n ot 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 t he aluminum foil to the glassware.537

- 7 NOTE: PFAA standards, extracts and samples should not c ome in contact with any glass containers or pipettes as these analytes can potentially adsorb to glass surfaces. PFAA analyte, IS and SUR standards comme rcially purchased in glass ampoules are acceptable; however, all subsequ ent transfers or dilutions performed by the analyst must be prepared and store d in polypropylene

containers.

4.2 Method interferences may be caused by contamina nts in solvents, reagents (including reagent water), sample bottles and caps, and other sample processing hardware that

lead to discrete artifacts and/or elevated baseline s in the chromatograms. The method analytes in this method can also be found in many c ommon laboratory supplies and equipment, such as PTFE (polytetrafluoroethylene) p roducts, 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 interfere nces (less than 1/3 the MRL for each method analyte) under the conditions of the an alysis 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 contamina nts that are co-extracted from the sample. The extent of matrix interferences will va ry considerably from source to source, depending upon the nature of the water. Hu mic and/or fulvic material can be co-extracted during SPE and high levels can cause e nhancement and/or suppression in the electrospray ionization source or low recoverie s on the SPE sorbent. 4-5 Total organic carbon (TOC) is a good indicator of humic c ontent of the sample. Under the LC conditions used during method development, matri x 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 contam inants in these reagents. Interferences from these sources should be monitored by ana lysis of laboratory reagent blanks (Sect. 9.3.1), particularly when new lots of reagen ts are acquired.

4.5 SPE cartridges can be a source of interferences . The analysis of field and laboratory reagent blanks can provide important information re garding the presence or absence of

such interferences. Brands and lots of SPE devices should be tested to ensure that contamination does not preclude analyte identificat ion 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. E ach laboratory is responsible for maintaining an awareness of OSHA regulations regard ing safe handling of chemicals

used in this method. A reference file of MSDSs sho uld be made available to all personnel involved in the chemical analysis. Addit ional references to laboratory safety are available. 6-8537

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

materials and stock standard solutions of these met hod 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 we re 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 bottle s fitted with polypropylene screw caps. Sample bottles must be discarded after use.

6.2 POLYPROPYLENE BOTTLES – 4-mL narrow-mouth poly propylene 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 c ollection of the extracts (Thomas Scientific Cat. No.: 2602A10 or equivalent).

6.4 AUTOSAMPLER VIALS – Polypropylene 0.3-mL autosample r 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 t o prevent contamination of the sample from PTFE coated septa. However, polypr opylene 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 s izes 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 USIN G CARTRIDGES

6.9.1 SPE CARTRIDGES – 0.5 g, 6-mL SPE cartridges c ontaining styrenedivinylbenzene (SDVB) sorbent phase (Varian Cat. No.: 1225 -5021 or equivalent).537

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6.9.2 VACUUM EXTRACTION MANIFOLD – A manual vacuum manifold with Visiprep  large volume sampler (Supelco Cat. No. 57030 and 5 7275 or equivalent) for cartridge extractions, or an automa tic/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 aut omated system. Care must be taken with automated SPE systems to ensure the P TFE commonly used in these systems does not contribute to unacceptable a nalyte concentrations in the LRB (Sect. 9.3.1).

6.9.3 SAMPLE DELIVERY SYSTEM – Use of a polypropyle ne transfer tube system, which transfers the sample directly from the sample container to the SPE cartridge, is recommended, but not mandatory. Stan dard extraction manifolds come equipped with PTFE transfer tube systems. The se can be replaced with 1/8” O.D. x 1/16” I.D. polypropylene or polyethylene tub ing (Hudson Extrusions LLDPE or equivalent) cut to an appropriate length t o ensure no sample contamination from the sample transfer lines. Othe r 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 l ines may not be feasible; therefore, LRBs will need to be rotated among the p orts and must meet the QC requirements of Sections 9.2.1 and 9.3.1.

6.10 EXTRACT CONCENTRATION SYSTEM – Extracts are co ncentrated by evaporation with nitrogen using a water bath set no higher than 65 °C (Meyer N-Evap, Model 111, Organomation Associates, Inc. or equival ent).

6.11 LABORATORY OR ASPIRATOR VACUUM SYSTEM – Suffic ient 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 inje cting up to 10- μ L aliquots, and performing binary linear gradients at a constan t 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 mo bile phase bottle will volatilize the ammonium acetate mobile phase causin g the analyte peaks to shift

to earlier retention times over the course of the a nalysis batch. The usage of a column heater is optional.537

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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 repla ced with

PEEK  tubing and the PTFE solvent frits were replaced wi th stainless steel frits. It is not possible to remov e all PFAA background contamination, but these measures help t o 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 performi ng MS/MS to produce unique product ions (Sect. 3.19) for the method ana lytes within specified retention time segments. A minimum of 10 scans across the ch romatographic peak is required to ensure adequate precision. Data are de monstrated in Tables 5-9 using a triple quadrupole mass spectrometer (Waters Micro mass Quattro Premier).

6.12.3 DATA SYSTEM – An interfaced data system is required to acquire, store, reduce, and output mass spectral data. The computer softwa re should have the capability of processing stored LC/MS/MS data by recognizing a n LC peak within any given

retention time window. The software must allow int egration of the ion abundance of any specific ion within specified time or scan n umber 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 C 18 column (2.1 x 150 mm) packed with

5 μ m d p C 18 solid phase particles (Waters #: 186001301 or equi valent) 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 o r better chemicals should be used. Unless otherwise indicated, it is intende d that all reagents shall conform to the specifications of the Committee on Analytical R eagents 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 suffi ciently 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 co mpounds 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 purificati on system to rinse out any build-up of analytes in the system’s tubing.537

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7.1.2 METHANOL (CH 3 OH, CAS#: 67-56-1) – High purity, demonstrated to b e free of analytes and interferences (Tedia Absolv  grade or equivalent).

7.1.3 AMMONIUM ACETATE (NH 4 C 2 H 3 O 2 , CAS#: 631-61-8) – High purity, demonstrated to be free of analytes and interferenc es (Sigma-Aldrich ACS grade or equivalent).

7.1.4 20 mM AMMONIUM ACETATE/REAGENT WATER – To pre pare 1 L, add 1.54 g ammonium acetate to 1 L of reagent water. T his solution is prone to volatility losses and should be replaced at least e very 48 hours.

7.1.5 TRIZMA  PRESET CRYSTALS, pH 7.0 (Sigma cat# T-7193 or equi valent) – Reagent grade. A premixed blend of Tris [Tris(hydr oxymethyl)aminomethane] and Tris HCL [Tris(hydroxymethyl)aminomethane hydro chloride]. 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 nea r 7.0 at 25 °C in reagent

water. Trizma  functions as a buffer, and removes free chlorine i n 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 nitro gen used should meet or exceed instrument manufacturer’s specifications.

7.1.6.2 Nitrogen is used to concentrate sample extr acts (Ultra High Purity or equivalent).

7.1.7 ARGON – Used as collision gas in MS/MS instru ments. Argon should meet or exceed instrument manufacturer’s specifications. N itrogen 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 tr ansfers or dilutions performed by the analyst must be prepared and stored in polyprop ylene containers. Solution

concentrations listed in this section were used to develop this method and are included as an example. Alternate concentrations may be use d as necessary depending on instrument sensitivity and the calibration range us ed. Standards for sample fortification generally should be prepared in the s mallest 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 pract ices 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 metho d analytes onto the container surfaces that may occur when refrigerated . Storing the standards at room temperature will also minimize daily imprecisi on 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 isot opically 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 compoun ds with similar functional groups as the method analytes, the analyst must hav e documented reasons for using alternate IS(s). Alternate IS(s) must meet t he QC requirements in Section 9.3.4. Note that different isotopic labels of the same IS(s) are acceptable (e.g., 13 C 2 -PFOA and 13 C 4 -PFOA) but will require modification of the MS/MS precursor and product ions.

Internal Standards Acronym

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

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

N-deuteriomethylperfluoro-1-octanesulfonamidoacetic acid d 3 -NMeFOSAA

7.2.1.1 IS STOCK STANDARD SOLUTIONS – These IS stoc ks can be obtained

as individual certified stock standard solutions. During the development of this method, commercially obtained 50 μg/mL stock s tandard solutions in methanol were used (Wellington Labs, Perkin Elmer o r equivalent). Analysis of the IS(s) is less complicated if the IS (s) purchased contains only

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

6 months when stored at 4 ° C.

7.2.1.2 INTERNAL STANDARD PRIMARY DILUTION (IS PDS) STANDAR D (1-4 ng/μL) – Prepare, or purchase commercially, th e IS PDS at a suggested concentration of 1-4 ng/μL. If prepared from the i ndividual stock standard solutions (Sect. 7.2.1.1), the table below can be u sed as a guideline for preparing the IS PDS. The IS PDS used in these stu dies was prepared in 96:4% (vol/vol) methanol:water. The IS PDS has bee n shown to be stable for at least two months when stored in polypropylen e centrifuge tubes at room temperature. Use 10 μL of this 1-4 ng/μL solu tion to fortify the final 1-mL extracts (Sect. 11.5). This will yield a conc entration 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)

13 C-PFOA 1000 5.0 5000 1.0

13 C-PFOS 50 300.0 5000 3.0

d 3 -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 belo w. These isotopically labeled SUR standards were carefully chosen during method d evelopment because they encompass most of the functional groups, as well as the water solubility range of the method analytes. Although alternate SUR standa rds may be used provided they are isotopically labeled compounds with simila r functional groups as the method analytes, the analyst must have documented r easons for using alternate

SUR standards. The alternate SUR standards chosen must still span the water solubility range of the method analytes. In additi on, alternate SUR standards must meet the QC requirements in Section 9.3.5.

Surrogates Acronym

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

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

N-deuterioethylperfluoro-1-octanesulfonamidoacetic acid d 5 -NEtFOSAA

7.2.2.1 SUR STOCK STANDARD SOLUTIONS – These SUR st ocks can be

obtained as individual certified stock standard sol utions. During the development of this method, commercially obtained 5 0 μg/mL stock standard solutions in methanol containing 4% water (except for

d 5 -NEtFOSAA which was purchased in pure methanol) wer e used (Wellington Labs or equivalent). Analysis of the S UR(s) is less complicated if the SUR(s) purchased contains only the linear is omer. SUR stock

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

7.2.2.2 SURROGATE PRIMARY DILUTION STANDARD (SUR PD S) (1-4 ng/μL) – Prepare, or purchase commercially, th e 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 the se studies was prepared in 96:4% (vol/vol) methanol:water. This so lution 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)

13 C-PFHxA 50 100.0 5000 1.0

13 C-PFDA 50 100.0 5000 1.0

d 5 -NEtFOSAA 50 400.0 5000 4.0

7.2.3 ANALYTE STANDARD SOLUTIONS – Analyte standar ds may be purchased commercially as ampoulized solutions or prepared fr om neat materials (see Table 3 for the analyte sources used during method development)

. If possible, purchase the method analytes as technical grade sta ndards or neat materials. Standards or neat materials that contain only the l inear isomer can be substituted only if technical grade (linear and branched isomer s) standards or neat material cannot be purchased. PFHxS and PFOS must be purcha sed as technical grade (containing branched and linear isomers), as well a s NEtFOSAA and NMeFOSAA (if commercially available)

. PFHxS and PFOS are not available as the acids listed in Section 1.1, but rather as thei r corresponding salts, such as Na +

and K + (see Table 3). These salts are acceptable startin g materials for the stock standards provided the measured mass is corrected f or the salt content according to the equation below. Prepare the Analyte Stock a nd Primary Dilutions

Standards as described below.

salt acid
salt acid MW MW Mass Measured Mass × =

where:

MW acid = the molecular weight of PFAA

MW salt = the molecular weight of purchased salt

7.2.3.1 ANALYTE STOCK STANDARD SOLUTION – If prepar ing from neat material, accurately weigh approximately 5 mg of pu re 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 P DS, care must be

taken to ensure that these standards are at room te mperature 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/ μ L) – The analyte PDS contains all the method analyt es of interest at various concentrations in methanol containing 4% wa ter. 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 i n Section 17 for suggested concentrations for each analyte. During method development, the analyte PDS was prepared such that approximately th e same instrument response was obtained for all the analytes. The an alyte PDS is prepared by dilution of the combined Analyte Stock Standard Sol utions and is used to prepare the CAL standards, and fortify the LFBs, th e 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 calibra tion concentrations are required to prepare the initial calibration cur ve spanning a 20-fold concentration range (Sect. 10.2). Larger concentra tion ranges will require more calibration points. Prepare the CAL standards over the concentration range of interest from dilutions of the analyte PDS in metha nol containing 4% reagent water. The suggested analyte concentrations found in Tables 5-9 can be used as a starting point for determining the calibration rang e. The IS and SUR are added to

the CAL standards at a constant concentration. Dur ing method development, the concentrations of the SUR(s) were 10-40 pg/ μ L in the standard (40-160 ng/L in the sample) and the IS(s) were 10-40 pg/ μ L. The lowest concentration CAL standard must be at or below the MRL, which may dep end 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 sta ble for at least two weeks when stored at room temperature. Longer storage ti mes are acceptable provided537

- 16 appropriate QC measures are documented demonstratin g 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 polypro pylene 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 sam pling 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 washi ng and wearing nitrile gloves will aid in minimizing this type of accidental cont amination of the samples.

8.2.2 Open the tap and allow the system to flush until th e water temperature has stabilized (approximately 3 to 5 min). Collect sam ples from the flowing system.

8.2.3 Fill sample bottles, taking care not to flush out t he sample preservation reagent. Samples do not need to be collected headspace free.

8.2.4 After collecting the sample, cap the bottle and agi tate by hand until preservative is dissolved. Keep the sample sealed from time of col lection 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 FR B 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 i nto 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 initial ly 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 samp ling.

8.4 SAMPLE SHIPMENT AND STORAGE – Samples must be chill ed 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 s ufficient ice to meet the above requirements.

8.5 SAMPLE AND EXTRACT HOLDING TIMES – Results of the s ample storage stability study (Table 10) indicated that all compo unds listed in this method have adequate stability for 14 days when collected, pres erved, 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 o f 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 qual ity 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 sta ndards 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 tha t 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 m ust be successfully performed prior to analyzing any Field Samples. Pr ior to conducting the IDC, the analyst must first generate an acceptable Initial C alibration following the procedure

outlined in Section 10.2.

9.2.1 INITIAL DEMONSTRATION OF LOW SYSTEM BACKGROUND – An y time a new lot of SPE cartridges, solvents, centrifuge t ubes, disposable pipets, and autosampler vials are used, it must be demonstrated that an LRB is reasonably

free of contamination and that the criteria in Sect ion 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 descri bed 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, calcula te average recovery. The average recovery of the replicate values must be wi thin ± 30% of the true value.

9.2.4 INITIAL DEMONSTRATION OF PEAK ASYMMETRY FACTOR – Pe ak asymmetry factors must be calculated using the equa tion in Section 9.3.9 for the first two eluting peaks (if only two analytes are b eing analyzed, both must be evaluated) in a mid-level CAL standard. The peak a symmetry factors must fall in the range of 0.8 to 1.5. See guidance in Section 1 0.2.4.1 if the calculated peak asymmetry factors do not meet the criteria.

9.2.5 MINIMUM REPORTING LEVEL (MRL) CONFIRMATION – Establ ish a target

concentration for the MRL based on the intended use of the method. The MRL may be established by a laboratory for their specif ic purpose or may be set by a regulatory agency. Establish an Initial Calibratio n 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 con centration 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 conc entration ( Mean ) and standard deviation for these replicates. Determine the Half Range for the prediction interval of results ( HR PIR ) using the equation below537

- 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 Pre diction Interval of Result ( PIR = Mean + HR PIR ) meet the upper and lower recovery limits as shown

below

The Upper PIR Limit must be ≤ 150% recovery.

150% % 100 ≤ × +

on oncentrati FortifiedC HR Mean PIR

The Lower PIR Limit must be ≥ 50% recovery.

50% % 100 ≥ × −

on oncentrati FortifiedC HR Mean PIR

9.2.5.3 The MRL is validated if both the Upper and Lower PI R 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 aga in at a higher

concentration.

9.2.6 CALIBRATION CONFIRMATION – Analyze a QCS as describ ed in Section 9.3.10 to confirm the accuracy of the standards/cal ibration curve.

9.2.7 DETECTION LIMIT DETERMINATION (optional)

– While DL determination is not a specific requirement of this method, it ma y be required by various regulatory bodies associated with compliance monito ring. 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 don e 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 co ncentration 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 v arious concentrations as indicated in the table. The appropriate fortifica tion concentrations will be dependent upon the sensitivity of the LC/MS/MS syst em 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 requi rements, a DL may be calculated from the MRL confirmation data, and no a dditional analyses

are necessary.

Calculate the DL using the following equation

) 99.0 1 ,1 ( =

− − × = α n t s DL

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 c alculations. The DL is a statistical determination of precision only. 2 If the DL replicates are fortified at a low enough concentration, it is like ly that they will not meet the precision and accuracy criteria for CCCs. Ther efore, no precision and accuracy criteria are specified.

9.2.7.2 If a laboratory is establishing their own M RL, 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 ca reful evaluation of the background levels. It is recommended that a MRL be established at the

mean LRB concentrations + 3 σ or 3 times the mean LRB concentration, whichever is greater. This value should be calcula ted 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 summariz es the ongoing QC criteria that must be followed when processing and analyzing Field Samples.

9.3.1 LABORATORY REAGENT BLANK (LRB) – An LRB is re quired with each extraction batch (Sect. 3.6) to confirm that potent ial background contaminants are not interfering with the identification or quantita tion of method analytes. If more than 20 Field Samples are included in a batch, anal yze an LRB for every 20 samples. If the LRB produces a peak within the ret ention time window of any analyte that would prevent the determination of tha t analyte, determine the source

of contamination and eliminate the interference bef ore processing samples. Background contamination must be reduced to an acce ptable level before proceeding. Background from method analytes or oth er contaminants that interfere with the measurement of method analytes must b e below 1/3 of the MRL. Blank contamination is estimated by extrapolation, if the concentration is below the lowest CAL standard. This extrapolation proced ure is not allowed for sample results as it may not meet data quality objectives. If the method analytes are537

- 21 detected in the LRB at concentrations equal to or g reater 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 t hat the analyst maintain a

historical record of LRB data.

9.3.2 CONTINUING CALIBRATION CHECK (CCC) – CCC Stan dards are analyzed at the beginning of each analysis batch, after ever y 10 Field Samples, and at the end of the analysis batch. See Section 10.3 for co ncentration requirements and acceptance criteria.

9.3.3 LABORATORY FORTIFIED BLANK (LFB) – An LFB is required with each extraction batch (Sect. 3.6). The fortified concen tration of the LFB must be rotated between low, medium, and high concentration s 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 50-15 0% of the true value. Results of the medium and high-level LFB analyses m ust be 70-130% of the true

value. If the LFB results do not meet these criter ia 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 mo nitor 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 t he response in the most recent CCC and must not deviate by more than 50% fr om the average area measured during initial analyte calibration. If th e IS areas in a chromatographic run do not meet these criteria, inject a second ali quot of that extract aliquotted into a new capped autosampler vial. Random evapora tion losses have been observed with the polypropylene caps causing high I S(s) areas.

9.3.4.1 If the reinjected aliquot produces an accep table 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 sta ndard. If the CAL standard fails the criteria of Section 10.3, recali bration is in order per Section 10.2. If the CAL standard is acceptable, e xtraction of the sample may need to be repeated provided the sample is stil l within the holding time. Otherwise, report results obtained from the reinjec ted extract, but annotate as suspect. Alternatively, collect a new sample an d 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 mean s of assessing method537

- 22 performance from extraction to final chromatographi c measurement. Calculate the recovery (%R) for the SUR using the following e quation

100 % ×

     

= B A R

where

A = calculated SUR concentration for the QC or Fiel d 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. C orrect the problem and reanalyze the extract.

9.3.5.2 If the extract reanalysis meets the SUR recovery cr iterion, report only data for the reanalyzed extract.

9.3.5.3 If the extract reanalysis fails the 70-130% recover y criterion, the analyst should check the calibration by injecting the last CAL standard that passed.

If the CAL standard fails the criteria of Section 1 0.3, recalibration is in order per Section 10.2. If the CAL standard is acc eptable, extraction of the sample should be repeated provided the sample is st ill within the holding time. If the re-extracted sample also fails the re covery criterion, report all data for that sample as suspect/SUR recovery to inf orm the data user that the results are suspect due to SUR recovery. Alternati vely, collect a new sample and re-analyze.

9.3.6 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) – Analysi s of an LFSM is required in each extraction batch and is us ed to determine that the sample matrix does not adversely affect method accu racy. Assessment of method precision is accomplished by analysis of a Field Du plicate (FD) (Sect. 9.3.7); however, infrequent occurrence of method analytes w ould 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, mu st 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 perfo rmance 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 ana lyzed. The LFSM is prepared by spiking a sample with an appropriate am ount of the Analyte537

- 23 PDS (Sect. 7.2.3.2). Select a spiking concentratio n that is greater than or equal to the matrix background concentration, if kn own. Use historical data and rotate through the low, mid and high concentrat ions when selecting a fortifying concentration.

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



## ( ) 100 % × −

= C B A 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 M RL (within a factor of 2-times the MRL concentration) where 50-150% recove ries are acceptable. If the accuracy of any analyte falls outside the de signated 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 SAMPL E MATRIX DUPLICATE (FD or LFSMD) – Within each extraction ba tch (not to exceed 20 Field Samples, Sect. 3.6), a minimum of one FD or L FSMD must be analyzed. Duplicates check the precision associated with samp le collection, preservation, storage, and laboratory procedures. If method anal ytes are not routinely observed in Field Samples, an LFSMD should be analyzed rathe r than an FD.

9.3.7.1 Calculate the relative percent difference ( RPD ) for duplicate measurements ( FD1 and FD2 ) using the equation



## ( ) 100

2/ 2 1 2 1

×
+ −

= 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 f actor 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 cont rol 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 inf orm 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, ca lculate the relative percent difference (RPD) for duplicate LFSMs (LFSM and LFSM D) using the equation



## ( ) 100 2/ ×
+ −

= LFSMD LFSM LFSMD LFSM RPD

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

above their native concentration. Greater variabil ity 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 s hould have RPDs that

are ≤ 50% for samples fortified at or above their native concentration. If the RPD of any analyte falls outside the designated ran ge, and the laboratory performance for that analyte is shown to be in cont rol 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 inf orm 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 inadve rtently introduced into the sample during sample collection/handling. Analysis of the FRB is required only if a Field Sample contains a method analyte or anal ytes at or above the MRL. The FRB is processed, extracted and analyzed in exa ctly the same manner as a Field Sample. If the method analyte(s) found in th e Field Sample is present in the FRB at a concentration greater than 1/3 the MRL, th en all samples collected with

that FRB are invalid and must be recollected and re analyzed.

9.3.9 PEAK ASYMMETRY FACTOR – A peak asymmetry fact or must be calculated using the equation below during the IDL and every t ime 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. M odifying 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 

### 10% 

## a b A b

a s =

where:

A

s = 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 lin e dropped perpendicularly from the peak apex

a = the width of the front half of the peak measured (a t 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 t he 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 sourc e of the CAL standards. If a second vendor is not available, then a different lo t 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 calculat ed amount for each analyte must be ± 30% of the expected value. If measured a nalyte 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 calibr ation is successful, a CCC is required at the beginning and end of each period in which an alyses 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 th e 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 μ g/mL of each analyte (prepared in the initial mobil e phase conditions) directly into the MS at the chosen LC m obile phase flow rate (approximately 0.3 mL/min). This tune can be done on a mix of the method analytes. The MS parameters (voltages, temperature s, gas flows, etc.) are537

- 26 varied until optimal analyte responses are determin ed. The method analytes may have different optima requiring some compromise between the optima.

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

10.2.1.3 Optimize the product ion (Sect. 3.19) for each analyte by infusing approximately 0.5-1.0 μ g/mL of each analyte (prepared in the initial mobil e phase conditions) directly into the MS at the chose n 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 g as pressure, collision energy, etc.) are varied until optimal analyte resp onses are determined. Typically, the carboxylic acids have very similar M S/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 res olution and peak shape. Suggested LC suggested conditions can be found in T able 1. The LC conditions listed in Table 1 may not be optimum for all LC sys tems and may need to be optimized by the analyst (See Sect. 10.2.4.1). Mod ifying the standard or extract composition to more aqueous content to prevent poor shape is not permitted.

Cautions: LC system components, as well as the m obile phase constituents, contain many of the method analytes in this method. Thus, these PFAAs will build up on the head of the LC column du ring mobile phase equilibration. To minimize the background PF AA peaks and to keep background levels constant, the time th e LC column sits at initial conditions must be kept constant an d as short as possible (while ensuring reproducible retention tim es). 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 compone nts such as wash syringes, sample needles or any other system c omponents before daily use.

Mobile phase modifiers other than 20 mM ammonium a cetate 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 ti mes shifted to shorter and shorter times as days progressed when m obile 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 PFT A, ensure that the LC conditions are adequate to prevent co-elution of PF TA and the mobile phase interferants shown in Figure 1. These interferants have the same precursor and products ions as PFTA, and under faster LC conditio ns may co-elute with PFTA. Divide the chromatogram into retention time windows each of which contains one537

- 27 or more chromatographic peaks. During MS/MS analys is, 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. T he product ions (also the quantitation ions) chosen during method development are in Table 4, although these will be instrument dependent. For maximum se nsitivity, small mass windows of ±0.5 daltons around the product ion mass were used for quantitation. If sufficient sensitivity exists to meet the MRL, w ider mass ranges may be used to

obtain more confirmation ions.

NOTE: As the NOTE in Section 10.2.4.1 indicates, P FOS has linear and branched isomers. There have been reports 10 that not all the products ions in the linear PFOS are produced in al l 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 th e m/z 499 → m/z 80 transition be used as the quantitation transitio n. Some MS/MS instruments, such as conventional ion tr aps, 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
reports 10 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, the re is not a single ion trap MS/MS transition that encompasses the line ar 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 w indow and that there are at least 10 scans across the peak for optimum precisio n.

10.2.4.1 If broad, split or fronting peaks are observed for the first two eluting chromatographic peaks (if only two analytes are bei ng analyzed, both must be evaluated), change the initial mobile phase cond itions to higher aqueous content until the peak asymmetry ratio for each pea k is 0.8 – 1.5. The peak asymmetry factor is calculated as described in Sect ion 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 ever y time a new calibration curve is generated. Modifying the standard or extr act composition to more aqueous content to prevent poor shape is not permit ted.

NOTE: PFHxS, PFOS, NMeFOSAA, and NEtFOSAA have mul tiple chromatographic peaks using the LC conditions in Ta ble 1 due to chromatographic resolution of the linear and bra nched isomers of these compounds. Most PFAAs are produce d by two different processes. One process gives rise to linear537

- 28 PFAAs only while the other process produces both li near and branched isomers. Thus, both branched and linear P FAAs can potentially be found in the environment. For t he aforementioned compounds that give rise to more tha n one peak, all the chromatographic peaks observed in the standard must be integrated and the areas totaled. Chromato graphic peaks in a sample must be integrated in the same wa y 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 bel ow the MRL, which may depend on system sensitivity. It is recommended th at at least four of the CAL standards are at a concentration greater than or eq ual to the MRL.

10.2.6 The LC/MS/MS system is calibrated using the IS tech nique. Use the LC/MS/MS data system software to generate a linear regressio n 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 z ero 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 u ndergo suppression in

the ESI source if the concentration of the co-eluti ng unlabeled method analyte(s) is too high. The analyte concentration at which suppression may occur can vary depending on the instrument, LC cond itions, ESI conditions, IS concentration, etc. To evaluate whether suppres sion is occurring during calibration, calculate the relative percent differe nce ( RPD ) between the high (H) and low (L) areas for each IS using the equatio n



## ( ) ( ) 100 2/ ×
+ −

= L H L H RPD

10.2.6.2 The RPD calculated above must be <20% for each IS d uring 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 quant itated using the initial calibration curve, each calibration point, except t he lowest point, for each analyte

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

should calculate to be within 50-150% of its true v alue. If these criteria cannot be met, the analyst will have difficulty meeting ongoi ng QC criteria. It is

recommended that corrective action is taken to rean alyze the CAL standards, restrict the range of calibration, or select an alt ernate 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 r eproducible537

- 29 retention times. If this is not done, the correct ions will not be monitored at the appropriate times. As a precautio nary 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 d aily calibration verification is as follows. Verify the initial cal ibration at the beginning and end of each group of analyses, and after every tenth sampl e during analyses. In this context, a “sample” is considered to be a Field Sample. LRB s, CCCs, LFBs, LFSMs, FDs FRBs and LFSMDs are not counted as samples. The be ginning CCC of each analysis

batch must be at or below the MRL in order to verif y 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 analy ze two CAL standards to meet this requirement. Alternatively, the analyte conce ntrations 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 concent ration CAL standard and analyze with the same conditions used during the initial calibra tion.

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

70-140% of the areas measured in the most recent co ntinuing 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 adjustmen ts 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 c hanges.

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 amo unt 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 exis t, then all data for the problem analyte must be considered invalid, and remedial ac tion should be taken (Sect. 10.3.4) which may require recalibration. An y Field or QC Samples that have been analyzed since the last acceptable calibr ation verification should be reanalyzed after adequate calibration has been rest ored, with the following exception. If the CCC fails because the calculated concentrati on is greater than 130% (150% for the low-level CCC) for a partic ular method analyte, and Field Sample extracts show no detection for tha t method analyte, nondetects may be reported without re-analysis.

10.3.4 REMEDIAL ACTION – Failure to meet CCC QC performanc e criteria may require remedial action. Major maintenance, such a s cleaning the electrospray537

- 30 probe, atmospheric pressure ionization source, clea ning the mass analyzer, replacing the LC column, etc., requires recalibrati on (Sect 10.2) and verification of sensitivity by analyzing a CCC at or below the M RL (Sect 10.3).

11. PROCEDURE

11.1 This procedure may be performed manually or in an a utomated mode using a robotic or automatic sample preparation device. The data p resented in Tables 5-11 demonstrate data collected by manual extraction. I f an automated system is used to prepare samples, follow the manufacturer's operatin g 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 acco mmodate 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 L RB requirements (Sect. 9.3.1).

11.2 Some of the PFAAs adsorb to surfaces, including pol ypropylene. Therefore, the aqueous sample bottles must be rinsed with the elution solvent (Sect 11.4.4) w hether 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 u se. 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, mus t contain the dechlorinating agent listed in Section 8.1.2. Dete rmine 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 volum e may NOT be transferred to a graduated cylinder for volume measurement. Th e LRB, LFB and FRB may be prepared by measuring 250 mL of reagent water wi th a polypropylene graduated cylinder or filling a 250-mL sample bottl e 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- μ L aliquot of the 1-4 ng/ μ L SUR PDS (Sect. 7.2.2.2) was added to 250 mL of sample for a final concentration of 40 ng/L for 13 C-PFHxA and 13 C-PFDA and 160 ng/L for d 5 -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 condit ioning 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 b elow the top edge of the packing. If the cartridge goes dry during the cond itioning 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 va cuum, and begin adding sample to the cartridge.

11.4.2 SAMPLE EXTRACTON – Adjust the vacuum so that the ap proximate 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 entir e sample has passed through the cartridge, rinse the sample bott les with two 7.5-mL aliquots of reagent water and draw each aliquot through the sam ple transfer tubes and the cartridges. Draw air or nitrogen through the cartr idge for 5 min at high vacuum (10-15 in. Hg).

NOTE: If empty plastic reservoirs are used in place of th e sample transfer tubes to pass the samples through the cartridges, t hese reservoirs

must be treated like the transfer tubes. After the entire sample has passed through the cartridge, the reservoirs must b e 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 inser t 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 th at the solvent exits the cartridge in a dropwise fashion. Repeat sample bot tle rinse and cartridge elution with a second 4-mL aliquot of methanol.

NOTE: If empty plastic reservoirs are used in plac e of the sample transfer tubes to pass the samples through the cartridges, t hese 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 th e reservoirs while eluting the cartridge to ensure that any method ana lytes on the

surface of the reservoirs are transferred to the ex tract.

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/methanol537

- 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 br ing the volume to 1 mL and vortex.
(10 μ L of the 1-4 ng/ μ L IS PDS for extract concentrations of 10-40 pg/ μ L 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 autosamp ler caps do not reseal after injection. Therefore, do not store the extracts in the autosampler vials as evaporation losses can occur o ccasionally 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 meas ure the volume of water required to fill the original sample bottle to the mark made pr ior 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 sub traction 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 b e 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 shou ld be optimized prior to the

initiation of the IDC.

11.7.2 Establish an appropriate retention time wind ow for each analyte. This should be

based on measurements of actual retention time vari ation for each method analyte in CAL standard solutions analyzed on the LC over t he 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 init ial calibration and completing the IDC can be used to calculate a suggested window size. However, the experience of the analyst should weigh heavily on t he 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 b y analyzing a CCC as described in Section 10.3. If establishing an initial calibrati on, 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 μ L was used in method development), under the same conditions used to ana lyze the CAL standards.537

- 33

11.7.5 At the conclusion of data acquisition, use t he 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 t he 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 particu larly useful given the limited ±0.5 dalton mass range around a single prod uct 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 origina l concentration. Re-inject the diluted extract. Incorporate the dilution factor i nto the final concentration calculations. Acceptable SUR performance (Sect. 9. 3.5.1) should be determined from the undiluted sample extract. The resulting d ata should be documented as a dilution, with an increased MRL.

12. DATA ANALYSIS AND CALCULATION

12.1. Complete chromatographic resolution is not necessar y for accurate and precise measurements of analyte concentrations using MS/MS. In validating this method, concentrations were calculated by measuring the pro duct ions listed in Table 4. Other ions may be selected at the discretion of the analy st.

12.2. Calculate analyte and SUR concentrations using the multipoint calibration established in Section 10.2. Do not use daily calibration veri fication data to quantitate analytes in samples. Adjust final analyte concentrations to re flect the actual sample volume

determined in Section 11.6.

12.3 Prior to reporting the data, the chromatogram shoul d 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 linea r and branch isomers of these compounds (Sect. 10.2.4.1). The areas of all the l inear and branched isomer peaks

observed in the CAL standards for each of these ana lytes must be summed and the concentrations reported as a total for each of thes e analytes.

12.5 Calculations must utilize all available digits of p recision, 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 illust rate 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 p resented for four water matrices: reagent water (Tables 6 and 7); chlorinated (finish ed) ground water (Table 8); high TOC chlorinated surface water (Table 9); and day ze ro of the aqueous holding time in chlorinated surface water (Table 10).

13.2 EVALUATION OF ADDITIONAL PERFLUORINATED ALKYL COMPO UNDS –

Nine additional compounds were evaluated for inclus ion in this method, but were not included for the following reasons. Due to vol atility, low recoveries following evaporation were obtained for PFOSA (perf luorooctane sulfonylamide), NEtFOSE (2-N-ethylperfluorooctane sulfonamide ethan ol), NMeFOSE (2-Nmethylperfluorooctane sulfonamide ethanol), and NEt FOSA (Nethylperfluorooctanesulfonamide). Poor chromatogra phic peak shape, which degraded with repeated injections, was obtained for PFOSAA (perfluorooctanesulfonamidoacetic acid). PFOSfn (p erfluorosulfinate) degraded in aqueous samples in under 24 hours. PFOSF (perfl uorooctane-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 PFB A (perfluorobutanoic acid) and PFPA (perfluoropentanoic acid).

13.3 SAMPLE STORAGE STABILITY STUDIES – An analyte stora ge stability study was

conducted by fortifying the analytes into chlorinat ed 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 chlorina ted surface water fortified with the method analytes. The precision and mean recover y (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 o f this method was demonstrated by multiple laboratories, with res ults similar to those reported in Section 17. The authors wish to acknowledge the wor k of the Suffolk County Water Authority Laboratory Hauppauge, NY and Lancaster La bs, a wholly owned subsidiary of Thermo Fisher Scientific, for their participatio n in the second laboratory

demonstration.

14. POLLUTION PREVENTION

14.1 This method utilizes SPE to extract analytes f rom water. It requires the use of very small volumes of organic solvent and very small qua ntities of pure analytes, thereby minimizing the potential hazards to both the analys t and the environment as compared537

- 35 to the use of large volumes of organic solvents in conventional liquid-liquid

extractions.

14.2 For information about pollution prevention tha t may be applicable to laboratory operations, consult “Less is Better: Laboratory Che mical Management for Waste

Reduction” available from the American Chemical Soc iety’s Department of Government Relations and Science Policy, 1155 16 th 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 me thod generate relatively small amounts of waste since only small amounts of reagents and s olvents are used. The matrices of concern are finished drinking water or source water . However, laboratory waste management practices must be conducted consistent w ith 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 regu lations, particularly the hazardous waste identification rules and land disposal restri ctions.

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 f or 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 Resear ch.” 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 Io nization/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-Wat er Samples by Solid-Phase Extraction and High-Performance Liquid Chromatograp hy 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, Jul y 1, 2001).

7. “Carcinogens-Working with Carcinogens,” Publication No. 77-206, Department of Health, Education, and Welfare, Public Health Service, Cent er for Disease Control, National Institute of Occupational Safety and Health, Atlanta, Georgia , August 1977.

8. "Safety in Academic Chemistry Laboratories,” Americ an Chemical Society Publication, Committee on Chemical Safety, 7th Edition. Informa tion 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 Poten tial Human Health Effects

Associated with PFOA and Its Salts.” Document avai lable at http://yosemite.epa.gov/sab/SABPRODUCT.NSF/A3C83648 E77252828525717F004B9099/ $File/sab_06_006.pdf (accessed August, 2008).

10. Langlois, I. and Oehme, M. “Structural Identificat ion of Isomers Present In Technical Perfluorooctane Sulfonate By Tandem Mass Spectromet ry.” Rapid Commun. Mass Spectrom.
2006, 20 , 844-850.537

- 37

17. TABLES, DIAGRAMS, FLOWCHARTS AND VALIDATION DA TA

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  dC 18 2.1 x 150 mm packed with 5.0 μ m C 18 stationary phase

Flow rate of 0.3 mL/min

10 μ L 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 ° C537

- 38

TABLE 3. METHOD ANALYTE SOURCE, RETENTION TIMES (RTs), AND

SUGGESTED IS REFERENCES

Analyte Method analyte

Source a 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

PFHxS b Fluka 5 14.48 2

PFOA Aldrich 6 16.73 1

PFNA Aldrich 8 18.76 1

PFOS b 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

13 C-PFHxA Wellington Labs 3 11.38 1

13 C-PFDA Wellington Labs 12 20.47 1

d 5 -NEtFOSAA Wellington Labs 16 21.99 3

13 C-PFOA– IS#1 PerkinElmer 7 16.73 -

13 C-PFOS– IS#2 Wellington Labs 10 18.71 -

d 3 -NMeFOSAA–IS#3 Wellington Labs 14 21.24 -

a The data presented in this method were obtained us ing analytes purchased from these vendors. Other vendors’ materials can be used prov ided 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 t he 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 CONDITIONS a

Segment b Analyte Precursor Ion c
(m/z) Product Ion c,d

( m/z ) Cone

Voltage (v) Collision

Energy e (v)

1 PFBS 299 80 40 25

2 PFHxA 313 269 15 10

3 PFHpA 363 319 12 10

3 PFHxS f 399 80 40 40

4 PFOA 413 369 15 10

4 PFNA 463 419 12 10

4 PFOS f

499 80 40 40

5 PFDA 513 469 15 10

5 NMeFOSAA f

570 419 25 20

5 NEtFOSAA f

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 13 C

- PFHxA 315 270 15 10

5 13 C

- PFDA 515 470 12 12

5 d 5 - NEtFOSAA 589 419 25 20

4 13 C

- PFOA 415 370 15 10

4 13 C

- PFOS 503 80 40 40

5 d 3 - 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 mult iple scan events occur.

c Precursor and product ions listed in this table a re nominal masses. During MS and MS/MS optimization, the analyst should determine the prec ursor and product ion masses to one decimal place by locating the apex of the mass spec tral peak place (e.g., m/z 498.9 → 79.9 for PFOS). These precursor and product ion masses (wit h 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 pea ks 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 DL b (ng/L) LCMRL c (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 seve n 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 REAGE NT

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

13 C

- PFHxA 40 89 6.1

13 C

- PFDA 40 102 7.3

d 5 - NEtFOSAA 160 100 4.9537

- 42

TABLE 7. PRECISION AND ACCURACY OF HIGH LEVEL FORTIFIED REAG ENT

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

13 C

- PFHxA 40 93 4.4

13 C

- PFDA 40 99 3.5

d 5 - NEtFOSAA 160 100 4.5537

- 43

TABLE 8. PRECISION AND ACCURACY IN FORTIFIED CHLORINATED GRO UND

WATER a (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 96 100 b 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

13 C

- PFHxA 40 98 2.0

13 C

- PFDA 40 98 2.1

d 5 - NEtFOSAA 160 97 5.0

a TOC = 0.96 mg/L and hardness = 342 mg/L.

b Recovery is corrected for a matrix PFOS concentrati on of 40 ng/L.537

- 44

TABLE 9. PRECISION AND ACCURACY IN FORTIFIED HIGH TOC CHLORINATED

SURFACE WATER a (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

13 C

- PFHxA 40 85 4.3

13 C

- PFDA 40 101 3.2

d 5 - NEtFOSAA 160 106 3.7

a TOC = 4.95 mg/L and hardness =137 mg/L.537-45TABLE 10. AQUEOUS SAMPLE HOLDING TIME DATA FOR SAMPLES FROM CHLORINATED SURFACEWATERa, FORTIFIED WITH METHOD ANALYTES AND PRESERVED ANDSTORED ACCORDINGTO SECTION 8 (n=5)FortifiedConc. (ng/L)Day 0Day 7Day 14AnalyteMean %Rec% RSDMean %Rec% RSDMean %Rec% RSDPFBS 91 83 8.5 87 3.3 90 12PFHxA 50 99 4.3 98 3.4 104 10PFHpA 41 105 1.1 97 4.2 102 7.0PFHxS 113 98 2.8 98 3.8 92 9.0PFOAb46 98 2.5 94 3.2 96 7.6PFNA 48 106 2.5 102 4.0 98 5.5PFOS 96 103 4.9 100 3.1 104 6.7PFDA 37 102 3.3 99 5.0 99 7.0NMeFOSAA 202 97 3.6 95 4.0 97 5.1NEtFOSAA 214 105 3.0 101 6.1 99 5.3PFUnA 54 104 3.0 97 4.8 95 10PFDoA 37 100 2.2 94 5.8 102 4.6PFTrDA 55 102 2.8 93 3.8 104 5.3PFTA 44 105 4.0 99 4.3 102 4.4aTOC = 1.8 mg/L and hardness =205 mg/L.bRecovery corrected for a native PFOA concentrationof 14 ng/L in the tap water used for this study.537-46TABLE 11. EXTRACT HOLDING TIME DATA FOR SAMPLES FROM CHLORINATED SURFACE WATER,FORTIFIED WITH METHOD ANALYTES AND PRESERVED AND STORED ACCORDING TOSECTION 8 (n=5)AnalyteFortified Conc.(ng/L)Day 0Day 7Day 14Day 22Day 28Mean%RecRSD%Mean%RecRSD%Mean%RecRSD%Mean%RecRSD%Mean%RecRSD%PFBS 91 83 8.5 89 10 91 8.392 11 90 7.5PFHxA 50 99 4.3 101 4.7103 5.7105 5.2102 4.1PFHpA 41 105 1.1 101 3.4109 3.5110 2.9108 1.7PFHxS 113 98 2.8 101 2.1107 3.5106 3.1104 2.5PFOAa46 98 2.5 100 1.4102 2.7101 3.4101 2.8PFNA 48 106 2.5 104 3.0102 3.5104 4.0107 3.1PFOS 96 103 4.9 100 1.6109 1.4103 3.6105 1.9PFDA 37 102 3.3 104 2.9100 3.3102 5.4105 2.9NMeFOSAA202 97 3.6 96 1.896 2.8102 3.499 3.9NEtFOSAA 214 105 3.0 100 2.599 3.4108 1.7105 3.7PFUnA 54 104 3.0 103 3.1100 4.4104 3.6104 4.8PFDoA 37 100 2.2 102 1.6101 3.0107 5.4110 4.6PFTrDA 55 102 2.8 95 3.897 2.5104 4.1104 2.3PFTA 44 105 4.0 100 2.693 3.4110 3.1110 2.7aRecovery corrected for a native PFOA concentrationof 14 ng/L in the tap water used for this study.537-47TABLE 12.INITIAL DEMONSTRATION OF CAPABILITY QUALITY CONTROLREQUIREMENTSMethodReferenceRequirementSpecification and FrequencyAcceptance CriteriaSect. 9.2.1Initial Demonstration ofLow System BackgroundAnalyze LRB prior to any other IDC steps.Demonstrate that all method analytes are below 1/3the MRLand that possible interferences from extraction media do notprevent the identification and quantification of methodanalytes.Sect. 9.2.2Initial Demonstration ofPrecision (IDP)Analyze four to seven replicate LFBs fortified nearthe midrange calibration concentration.%RSD must be <20%Sect. 9.2.3Initial Demonstration ofAccuracy (IDA)Calculate average recovery for replicates used inIDP.Mean recovery±30% of true valueSect. 9.2.4Initial Demonstration ofPeak Asymmetry FactorCalculate the peak asymmetry factor using theequation in Section 9.3.9 for the first two elutingchromatographic peaks in a mid-level CALstandard.Peak asymmetry factor of 0.8 - 1.5Sect. 9.2.5Minimum Reporting Limit(MRL) ConfirmationFortify, extract and analyze seven replicate LFBsat the proposed MRL concentration. Calculate theMean and the Half Range (HR). Confirm that theupper and lower limits for the Prediction Intervalof Result (Upper PIR, and Lower PIR, Sect.9.2.5.2) meet the recovery criteria.Upper PIR≤150%Lower PIR≥50%Sect. 9.2.6and 9.3.10Quality Control Sample(QCS)Analyze a standard from a second source, aspart of IDC.Results must be within 70-130% of true value.Sect. 9.2.7Detection Limit (DL)Determination (optional)Over a period of trhee days, prepare a minimum ofseven replicate LFBs fortified at a concentrationestimated to be near the DL. Analyze thereplicates through all steps of the analysis.Calculate the DL using the equation inSect. 9.2.7.1.Data from DL replicates are not requiredto meet methodprecision and accuracy criteria. If the DL replicates arefortified at a low enough concentration, it is likely that theywill not meet precision and accuracy criteria.NOTE:Table 12 is intended as an abbreviated summary ofQC requirements provided as a convenience to the method user. Because the information has beenabbreviated to fit the table format, there may be issues that need additional clarification, or areaswhere important additionalinformation from the method textis needed. In all cases, the full text of the QC in Section 9 supersedes any missing or conflicting information in this table.537-48TABLE 13.ONGOING QUALITY CONTROL REQUIREMENTS (SUMMARY)MethodReferenceRequirementSpecification and FrequencyAcceptance CriteriaSect. 8.1 -Sect. 8.5Sample Holding Time14 days with appropriate preservation and storage asdescribed in Sections 8.1-8.5.Sample results are valid only if samples are extracted within thesample holding time.Sect. 8.5 Extract Holding Time28 days when stored at room temperature inpolypropylene centrifuge tubes.Extract results are valid only if extracts are analyzed within theextract holding time.Sect. 9.3.1Laboratory Reagent Blank(LRB)Daily, or with each extraction batch of up to 20samples, whichever is more frequent.Demonstrate that all method analytes are below 1/3the MRL, andconfirm that possible interferences do not preventquantification ofmethod analytes. If targets exceed 1/3 the MRL orif interferencesare present, results for these subject analytes inthe extraction batchare invalid.Sect. 9.3.3Laboratory Fortified Blank(LFB)Analyze at least one LFB daily or one for eachextraction batch of up to 20 Field Samples. Rotatethe fortified concentrations between low, medium andhigh amounts.Results of LFB analyses must be 70-130% of the truevalue for eachmethod analyte for all fortified concentrations except the lowestCAL point. Results of the LFBs corresponding to the lowest CALpoint for each method analyte must be 50-150% of the true value.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 allstandards and sample extracts, including QC samples.Compare IS areas to the average IS area in the initialcalibration and to the most recent CCC.Peak area counts for all ISs in all injections mustbe within±50% ofthe average peak area calculated during the initialcalibration and70-140% from the most recent CCC. If ISs do not meet thiscriterion, corresponding target results are invalid.Sect. 9.3.5Surrogate Standards(SUR)Surrogate standards,13C-PFHxA,13C-PFDA, and d5-NEtFOSAA, are added to all CAL standards andsamples, including QC samples. Calculate SURrecoveries.SUR recoveries must be 70-130% of the true value.If a SUR failsthis criteria, report all results for sample as suspect/SUR recovery.537-49TABLE 13.(Continued)MethodReferenceRequirementSpecification and FrequencyAcceptance CriteriaSect. 9.3.6Laboratory FortifiedSample Matrix (LFSM)Analyze one LFSM per extraction batch (20samples or less) fortified with method analytes ataconcentration close to but greater than the nativeconcentration, if known. Calculate LFSMrecoveries.Recoveries at mid and high levels should be within70-130%and within 50-150% at the low-level fortified amount (nearthe MRL). If these criteria are not met, results are labeledsuspect due to matrix effects.Sect. 9.3.7Laboratory FortifiedSample Matrix Duplicate(LFSMD) orField Duplicates (FD)Extract and analyze at least one FD or LFSMDwith each extraction batch (20 samples or less). ALFSMD may be substituted for a FD when thefrequency of detects are low. Calculate RPDs.Method analyte RPDs for the LFMD or FD should be≤30% at mid and high levels of fortification and≤50% nearthe MRL. If these criteria are not met, results are labeledsuspect due to matrix effects.Sect. 9.3.8 Field Reagent Blank (FRB)Analysis of the FRB is required only if a FieldSample contains a method analyte or analytes at orabove the MRL. The FRB is processed, extractedand analyzed in exactly the same manner as aField Sample.If the method analyte(s) found in the Field Sampleis presentin the FRB at a concentration greater than 1/3 theMRL, thenall samples collected with that FRB are invalid andmust berecollected and reanalyzed.Sect. 9.3.9 Peak Asymmetry FactorCalculate the peak asymmetry factor for the firsttwo eluting chromatographic peaks in a mid-levelCAL standard every time a calibration curve isgenerated.Peak asymmetry factor of 0.8 - 1.5Sect. 9.3.10Quality Control Sample(QCS)Analyze at least quarterly or when preparing newstandards, as well as during the IDC.Results must be within 70-130% of true value.Sect. 10.2andSect. 9.3.2Initial CalibrationUse IS calibration technique to generate a first orsecond order calibration curve forced throughzero. Use at least five standard concentrations.Check the calibration curve as described inSect. 10.2.7.When each CAL standard is calculated as an unknownusingthe calibration curve, the analyte results should be 70-130%of the true value for all except the lowest standard, whichshould be 50-150% of the true value. Recalibrationisrecommended if these criteria are not met.Sect. 9.3.2and Sect.10.3Continuing CalibrationCheck (CCC)Verify initial calibration by analyzing a low level(at the MRL or below) CCC prior to analyzingsamples. CCCs are then injected after every 10samples and after the last sample, rotatingconcentrations to cover the calibrated range of theinstrument.Recovery for each analyte and SUR must be within 70-130%of the true value for all but the lowest level of calibration.Recovery for each analyte in the lowest CAL level CCCmust be within 50-150% of the true value and the SUR mustbe within 70-130% of the true value.NOTE:Table 13 is intended as an abbreviated summary ofQC requirements provided as a convenience to the method user. Because the information has beenabbreviated to fit the table format, there may be issues that need additional clarification, or areaswhere important additional information from the method textis needed. In all cases, the full text of the QC in Section 8-10 supersedes any missing or conflicting information in this table.537-50FIGURE 1. EXAMPLE CHROMATOGRAM FOR REAGENT WATER FORTIFIED WITH METHOD 537 ANALYTES ATCONCENTRATION LEVELS INDICATED IN TABLE 6. NUMBERED PEAKS ARE IDENTIFIED IN TABLE 3.aAbbreviated chromatographic conditions may lead toco-elution of PFTA and the potential interferantsshown here. SeeSection 10.2.3 for more information.10.0012.5015.0017.5020.0022.5025.000100Retention Time, min12,3456,78,9,1011,1213,1415,16,171819120PFHxSisomersPFOSisomersNEtFOSAAisomersNMeFOSAAisomersPFTApotentialinterferantsa