A02.11_PWS-Annex-2_EPA Method 537_EPA Doc 600 R-08 092.pdf

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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 Development, National Exposure

Research Laboratory

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

Research Laboratory

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

Program

NATIONAL EXPOSURE RESEARCH LABORATORY

OFFICE OF RESEARCH AND DEVELOPMENT

U. S. ENVIRONMENTAL PROTECTION AGENCY

CINCINNATI, OHIO 45268

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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 spectrometry (LC/MS/MS) method for the determination of selected perfluorinated alkyl acids (PFAAs) in drinking water.

Accuracy and precision data have been generated in reagent water, and finished ground and surface waters for the compounds listed in the table below.

Analyte

Acronym Chemical Abstract Services Registry Number (CASRN)

N-ethyl perfluorooctanesulfonamidoacetic acid NEtFOSAA ─

N-methyl perfluorooctanesulfonamidoacetic acid NMeFOSAA ─

Perfluorobutanesulfonic acid PFBS 375-73-5

Perfluorodecanoic acid PFDA 335-76-2

Perfluorododecanoic acid PFDoA 307-55-1

Perfluoroheptanoic acid PFHpA 375-85-9

Perfluorohexanesulfonic acid PFHxS 355-46-4

Perfluorohexanoic acid PFHxA 307-24-4

Perfluorononanoic acid PFNA 375-95-1

Perfluorooctanesulfonic acid PFOS 1763-23-1

Perfluorooctanoic acid PFOA 335-67-1

Perfluorotetradecanoic acid PFTA 376-06-7

Perfluorotridecanoic acid PFTrDA 72629-94-8

Perfluoroundecanoic acid PFUnA 2058-94-8

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

Data Quality Objectives (DQOs) that are developed based on the intended use of this method. The single laboratory lowest concentration MRL (LCMRL) is the lowest true concentration for which the future recovery is predicted to fall, with high confidence (99%), between 50 and 150% recovery. Single laboratory LCMRLs for analytes in this method range from 2.9-14 ng/L, and are listed in Table 5. The procedure used to determine the LCMRL is described elsewhere.1

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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 laboratory MRL for this method meets the requirements described in Section 9.2.5.

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

9.2.7). Detection limit is defined as the statistically calculated minimum concentration that can be measured with 99% confidence that the reported value is greater than zero.2 The DL is compound dependent and is dependent on extraction efficiency, sample matrix, fortification concentration, and instrument performance.

1.5 This method is intended for use by analysts skilled in solid phase extractions, the operation of LC/MS/MS instruments, and the interpretation of the associated data.

1.6 METHOD FLEXIBILITY – In recognition of technological advances in analytical systems and techniques, the laboratory is permitted to modify the evaporation technique, separation technique, LC column, mobile phase composition, LC conditions and MS and MS/MS conditions (Sect. 6.12, 9.1.1, 10.2, and 12.1). Changes may not be made to sample collection and preservation (Sect. 8), the sample extraction steps (Sect. 11), or to the quality control requirements (Sect. 9). Method modifications should be considered only to improve method performance. Modifications that are introduced in the interest of reducing cost or sample processing time, but result in poorer method performance, should not be used. Analytes must be adequately resolved chromatographically in order to permit the mass spectrometer to dwell on a minimum number of compounds eluting within a retention time window. Instrumental sensitivity (or signal-to-noise) will decrease if too many compounds are permitted to elute within a retention time window. In all cases where method modifications are proposed, the analyst must perform the procedures outlined in the initial demonstration of capability (IDC, Sect. 9.2), verify that all Quality Control (QC) acceptance criteria in this method (Sect. 9) are met, and that acceptable method performance can be verified in a real sample matrix (Sect. 9.3.6).

NOTE: The above method flexibility section is intended as an abbreviated summation of method flexibility. Sections 4-12 provide detailed information of specific portions of the method that may be modified. If there is any perceived conflict between the general method flexibility statement in Section 1.6 and specific information in Sections 4-12, Sections 4-12 supersede Section 1.6.

2. SUMMARY OF METHOD

2.1 A 250-mL water sample is fortified with surrogates and passed through a solid phase extraction (SPE) cartridge containing polystyrenedivinylbenzene (SDVB) to extract the method analytes and surrogates. The compounds are eluted from the solid phase with a small amount of methanol. The extract is concentrated to dryness with nitrogen in a heated water bath, and then adjusted to a 1-mL volume with 96:4% (vol/vol) methanol:water after adding the IS(s). A 10-µL injection is made into an LC equipped with a C18 column that is interfaced to an MS/MS. The analytes are separated and

537-4 identified by comparing the acquired mass spectra and retention times to reference spectra and retention times for calibration standards acquired under identical LC/MS/MS conditions. The concentration of each analyte is determined by using the internal standard technique. Surrogate analytes are added to all Field and QC Samples to monitor the extraction efficiency of the method analytes.

3. DEFINITIONS

3.1 ANALYSIS BATCH – A set of samples that is analyzed on the same instrument during a 24-hour period, including no more than 20 Field Samples, that begins and ends with the analysis of the appropriate Continuing Calibration Check (CCC) standards. Additional CCCs may be required depending on the length of the analysis batch and/or the number of Field Samples.

3.2 CALIBRATION STANDARD (CAL) – A solution prepared from the primary dilution standard solution and/or stock standard solution, internal standard(s), and the surrogate(s). The CAL solutions are used to calibrate the instrument response with respect to analyte concentration.

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

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

3.5 DETECTION LIMIT (DL) – The minimum concentration of an analyte that can be identified, measured, and reported with 99% confidence that the analyte concentration is greater than zero. This is a statistical determination of precision (Sect. 9.2.7), and accurate quantitation is not expected at this level.2

3.6 EXTRACTION BATCH – A set of up to 20 Field Samples (not including QC samples) extracted together by the same person(s) during a work day using the same lot of SPE devices, solvents, surrogate, internal standard and fortifying solutions.

Required QC samples include Laboratory Reagent Blank, Laboratory Fortified Blank, Laboratory Fortified Sample Matrix, and either a Field Duplicate or Laboratory Fortified Sample Matrix Duplicate.

3.7 FIELD DUPLICATES (FD1 and FD2) – Two separate samples collected at the same time and place under identical circumstances, and treated exactly the same throughout field and laboratory procedures. Analyses of FD1 and FD2 give a measure of the precision associated with sample collection, preservation, and storage, as well as laboratory procedures.

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3.8 FIELD REAGENT BLANK (FRB) – An aliquot of reagent water that is placed in a sample container in the laboratory and treated as a sample in all respects, including shipment to the sampling site, exposure to sampling site conditions, storage, preservation, and all analytical procedures. The purpose of the FRB is to determine if method analytes or other interferences are present in the field environment.

3.9 INTERNAL STANDARD (IS) – A pure chemical added to an extract or standard solution in a known amount(s) and used to measure the relative response of other method analytes and surrogates that are components of the same solution. The internal standard must be a chemical that is structurally similar to the method analytes, has no potential to be present in water samples, and is not a method analyte.

3.10 LABORATORY FORTIFIED BLANK (LFB) – A volume of reagent water or other blank matrix to which known quantities of the method analytes and all the preservation compounds are added in the laboratory. The LFB is analyzed exactly like a sample, and its purpose is to determine whether the methodology is in control, and whether the laboratory is capable of making accurate and precise measurements.

3.11 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) – A preserved field sample to which known quantities of the method analytes are added in the laboratory.

The LFSM is processed and analyzed exactly like a sample, and its purpose is to determine whether the sample matrix contributes bias to the analytical results. The background concentrations of the analytes in the sample matrix must be determined in a separate sample extraction and the measured values in the LFSM corrected for background concentrations.

3.12 LABORATORY FORTIFIED SAMPLE MATRIX DUPLICATE (LFSMD) – A

duplicate of the Field Sample used to prepare the LFSM. The LFSMD is fortified, extracted, and analyzed identically to the LFSM. The LFSMD is used instead of the Field Duplicate to assess method precision when the occurrence of method analytes is low.

3.13 LABORATORY REAGENT BLANK (LRB) – An aliquot of reagent water or other blank matrix that is treated exactly as a sample including exposure to all glassware, equipment, solvents and reagents, sample preservatives, internal standard, and surrogates that are used in the analysis batch. The LRB is used to determine if method analytes or other interferences are present in the laboratory environment, the reagents, or the apparatus.

3.14 LOWEST CONCENTRATION MINIMUM REPORTING LEVEL (LCMRL) – The single laboratory LCMRL is the lowest true concentration for which a future recovery is expected, with 99% confidence, to be between 50 and 150% recovery.1

3.15 MATERIAL SAFETY DATA SHEET (MSDS) – Written information provided by vendors concerning a chemical’s toxicity, health hazards, physical properties, fire, and reactivity data including storage, spill, and handling precautions.

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3.16 MINIMUM REPORTING LEVEL (MRL) – The minimum concentration that can be reported as a quantitated value for a method analyte in a sample following analysis.

This defined concentration can be no lower than the concentration of the lowest calibration standard for that analyte and can only be used if acceptable QC criteria for this standard are met. A procedure for verifying a laboratory’s MRL is provided in Section 9.2.5.

3.17 PRECURSOR ION – For the purpose of this method, the precursor ion is the deprotonated molecule ([M-H]-) of the method analyte. In MS/MS, the precursor ion is mass selected and fragmented by collisionally activated dissociation to produce distinctive product ions of smaller m/z.

3.18 PRIMARY DILUTION STANDARD (PDS) SOLUTION – A solution containing the analytes prepared in the laboratory from stock standard solutions and diluted as needed to prepare calibration solutions and other needed analyte solutions.

3.19 PRODUCT ION – For the purpose of this method, a product ion is one of the fragment ions produced in MS/MS by collisionally activated dissociation of the precursor ion.

3.20 QUALITY CONTROL SAMPLE (QCS) – A solution of method analytes of known concentrations that is obtained from a source external to the laboratory and different from the source of calibration standards. The second source SSS is used to fortify the QCS at a known concentration. The QCS is used to check calibration standard integrity.

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

3.22 SURROGATE ANALYTE (SUR) – A pure chemical which chemically resembles method analytes and is extremely unlikely to be found in any sample. This chemical is added to a sample aliquot in known amount(s) before processing and is measured with the same procedures used to measure other method analytes. The purpose of the SUR is to monitor method performance with each sample.

4. INTERFERENCES

4.1 All glassware must be meticulously cleaned. Wash glassware with detergent and tap water, rinse with tap water, followed by a reagent water rinse. Non-volumetric glassware can be heated in a muffle furnace at 400 °C for 2 h or solvent rinsed.

Volumetric glassware should be solvent rinsed and not be heated in an oven above 120 °C. Store clean glassware inverted or capped. Do not cover with aluminum foil because PFAAs can be potentially transferred from the aluminum foil to the glassware.

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NOTE: PFAA standards, extracts and samples should not come in contact with any glass containers or pipettes as these analytes can potentially adsorb to glass surfaces. PFAA analyte, IS and SUR standards commercially purchased in glass ampoules are acceptable; however, all subsequent transfers or dilutions performed by the analyst must be prepared and stored in polypropylene containers.

4.2 Method interferences may be caused by contaminants in solvents, reagents (including reagent water), sample bottles and caps, and other sample processing hardware that lead to discrete artifacts and/or elevated baselines in the chromatograms. The method analytes in this method can also be found in many common laboratory supplies and equipment, such as PTFE (polytetrafluoroethylene) products, LC solvent lines, methanol, aluminum foil, SPE sample transfer lines, etc.3 All items such as these must be routinely demonstrated to be free from interferences (less than 1/3 the MRL for each method analyte) under the conditions of the analysis by analyzing laboratory reagent blanks as described in Section 9.3.1. Subtracting blank values from sample results is not permitted.

4.3 Matrix interferences may be caused by contaminants that are co-extracted from the sample. The extent of matrix interferences will vary considerably from source to source, depending upon the nature of the water. Humic and/or fulvic material can be co-extracted during SPE and high levels can cause enhancement and/or suppression in the electrospray ionization source or low recoveries on the SPE sorbent.4-5 Total organic carbon (TOC) is a good indicator of humic content of the sample. Under the LC conditions used during method development, matrix effects due to total organic carbon (TOC) were not observed.

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

The potential exists for trace-level organic contaminants in these reagents. Interfer-ences from these sources should be monitored by analysis of laboratory reagent blanks (Sect. 9.3.1), particularly when new lots of reagents are acquired.

4.5 SPE cartridges can be a source of interferences. The analysis of field and laboratory reagent blanks can provide important information regarding the presence or absence of such interferences. Brands and lots of SPE devices should be tested to ensure that contamination does not preclude analyte identification and quantitation.

5. SAFETY

5.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely defined. Each chemical should be treated as a potential health hazard, and exposure to these chemicals should be minimized. Each laboratory is responsible for maintaining an awareness of OSHA regulations regarding safe handling of chemicals used in this method. A reference file of MSDSs should be made available to all personnel involved in the chemical analysis. Additional references to laboratory safety are available.6-8

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5.2 PFOA has been described as “likely to be carcinogenic to humans.”9 Pure standard materials and stock standard solutions of these method analytes should be handled with suitable protection to skin and eyes, and care should be taken not to breathe the vapors or ingest the materials.

6. EQUIPMENT AND SUPPLIES (Brand names and/or catalog numbers are included for illustration only, and do not imply endorsement of the product.) Due to potential adsorption of analytes onto glass, polypropylene containers were used for all standard, sample and extraction preparations. Other plastic materials (e.g., polyethylene) which meet the QC requirements of Section 9 may be substituted.

6.1 SAMPLE CONTAINERS – 250-mL polypropylene bottles fitted with polypropylene screw caps. Sample bottles must be discarded after use.

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

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

6.3 CENTRIFUGE TUBES – 15-mL conical polypropylene tubes with polypropylene screw caps for storing standard solutions and for collection of the extracts (Thomas Scientific Cat. No.: 2602A10 or equivalent).

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

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

NOTE: Polypropylene vials and caps are necessary to prevent contamination of the sample from PTFE coated septa. However, polypropylene caps do not reseal, so evaporation occurs after injection. Thus, multiple injections from the same vial are not possible.

6.5 POLYPROPYLENE GRADUATED CYLINDERS – Suggested sizes include 25, 50, 100 and 1000-mL cylinders.

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

1000-µL syringes.

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

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

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

6.9 SOLID PHASE EXTRACTION (SPE) APPARATUS FOR USING CARTRIDGES

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

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6.9.2 VACUUM EXTRACTION MANIFOLD – A manual vacuum manifold with Visiprep large volume sampler (Supelco Cat. No. 57030 and 57275 or equivalent) for cartridge extractions, or an automatic/robotic sample preparation system designed for use with SPE cartridges, may be used if all QC requirements discussed in Section 9 are met. Extraction and/or elution steps may not be changed or omitted to accommodate the use of an automated system. Care must be taken with automated SPE systems to ensure the PTFE commonly used in these systems does not contribute to unacceptable analyte concentrations in the LRB (Sect. 9.3.1).

6.9.3 SAMPLE DELIVERY SYSTEM – Use of a polypropylene transfer tube system, which transfers the sample directly from the sample container to the SPE cartridge, is recommended, but not mandatory. Standard extraction manifolds come equipped with PTFE transfer tube systems. These can be replaced with 1/8” O.D. x 1/16” I.D. polypropylene or polyethylene tubing (Hudson Extrusions LLDPE or equivalent) cut to an appropriate length to ensure no sample contamination from the sample transfer lines. Other types of non-PTFE tubing may be used provided it meets the LRB (Sect. 9.3.1) and LFB (Sect. 9.3.3) QC requirements. The PTFE transfer tubes may be used, but an LRB must be run on each PFTE transfer tube and the QC requirements in Section 9.2.1 must be met.

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

therefore, LRBs will need to be rotated among the ports and must meet the QC requirements of Sections 9.2.1 and 9.3.1.

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

6.11 LABORATORY OR ASPIRATOR VACUUM SYSTEM – Sufficient capacity to maintain a vacuum of approximately 10 to 15 inches of mercury for extraction cartridges.

6.12 LIQUID CHROMATOGRAPHY (LC)/TANDEM MASS SPECTROMETER

(MS/MS) WITH DATA SYSTEM

6.12.1 LC SYSTEM – Instrument capable of reproducibly injecting up to 10-µL aliquots, and performing binary linear gradients at a constant flow rate near the flow rate used for development of this method (0.3 mL/min). The LC must be capable of pumping the water/methanol mobile phase without the use of a degasser which pulls vacuum on the mobile phase bottle (other types of degassers are acceptable). Degassers which pull vacuum on the mobile phase bottle will volatilize the ammonium acetate mobile phase causing the analyte peaks to shift to earlier retention times over the course of the analysis batch. The usage of a column heater is optional.

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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 replaced with PEEK tubing and the PTFE solvent frits were replaced with stainless steel frits. It is not possible to remove all PFAA background contamination, but these measures help to minimize their background levels.

6.12.2 LC/TANDEM MASS SPECTROMETER – The LC/MS/MS must be capable of negative ion electrospray ionization (ESI) near the suggested LC flow rate of

0.3 mL/min. The system must be capable of performing MS/MS to produce unique product ions (Sect. 3.19) for the method analytes within specified retention time segments. A minimum of 10 scans across the chromatographic peak is required to ensure adequate precision. Data are demonstrated in Tables 5-9 using a triple quadrupole mass spectrometer (Waters Micromass Quattro Premier).

6.12.3 DATA SYSTEM – An interfaced data system is required to acquire, store, reduce, and output mass spectral data. The computer software should have the capability of processing stored LC/MS/MS data by recognizing an LC peak within any given retention time window. The software must allow integration of the ion abundance of any specific ion within specified time or scan number limits. The software must be able to calculate relative response factors, construct linear regressions or quadratic calibration curves, and calculate analyte concentrations.

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

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

Any column that provides adequate resolution, peak shape, capacity, accuracy, and precision (Sect. 9) may be used.

7. REAGENTS AND STANDARDS

7.1 GASES, REAGENTS, AND SOLVENTS – Reagent grade or better chemicals should be used. Unless otherwise indicated, it is intended that all reagents shall conform to the specifications of the Committee on Analytical Reagents of the American Chemical Society, where such specifications are available. Other grades may be used, provided it is first determined that the reagent is of sufficiently high purity to permit its use without lessening the quality of the determination.

7.1.1 REAGENT WATER – Purified water which does not contain any measurable quantities of any method analytes or interfering compounds greater than 1/3 the MRL for each method analyte of interest. Prior to daily use, at least 3 L of reagent water should be flushed from the purification system to rinse out any build-up of analytes in the system’s tubing.

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7.1.2 METHANOL (CH3OH, CAS#: 67-56-1) – High purity, demonstrated to be free of analytes and interferences (Tedia Absolv grade or equivalent).

7.1.3 AMMONIUM ACETATE (NH4C2H3O2, CAS#: 631-61-8) – High purity, demonstrated to be free of analytes and interferences (Sigma-Aldrich ACS grade or equivalent).

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

1.54 g ammonium acetate to 1 L of reagent water. This solution is prone to volatility losses and should be replaced at least every 48 hours.

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

Reagent grade. A premixed blend of Tris [Tris(hydroxymethyl)aminomethane] and Tris HCL [Tris(hydroxymethyl)aminomethane hydrochloride]. Alternatively, a mix of the two components with a weight ratio of 15.5/1 Tris HCL/Tris may be used. These blends are targeted to produce a pH near 7.0 at 25 °C in reagent water. Trizma functions as a buffer, and removes free chlorine in chlorinated finished waters (Sect. 8.1.2).

7.1.6 NITROGEN – Used for the following purposes:

7.1.6.1 Nitrogen aids in aerosol generation of the ESI liquid spray and is used as collision gas in some MS/MS instruments. The nitrogen used should meet or exceed instrument manufacturer’s specifications.

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

7.1.7 ARGON – Used as collision gas in MS/MS instruments. Argon should meet or exceed instrument manufacturer’s specifications. Nitrogen gas may be used as the collision gas provided sufficient sensitivity (product ion formation) is achieved.

7.2 STANDARD SOLUTIONS – When a compound purity is assayed to be 96% or greater, the weight can be used without correction to calculate the concentration of the stock standard. PFAA analyte, IS and SUR standards commercially purchased in glass ampoules are acceptable; however, all subsequent transfers or dilutions performed by the analyst must be prepared and stored in polypropylene containers. Solution concentrations listed in this section were used to develop this method and are included as an example. Alternate concentrations may be used as necessary depending on instrument sensitivity and the calibration range used. Standards for sample fortification generally should be prepared in the smallest volume that can be accurately measured to minimize the addition of excess organic solvent to aqueous samples.

Even though stability times for standard solutions are suggested in the following sections, laboratories should use standard QC practices to determine when their standards need to be replaced.

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

Primary dilution standards (Sect. 7.2.1.2, 7.2.2.2 and 7.2.3.2) were stored at room temperature to prevent adsorption of the method analytes onto the container surfaces that may occur when refrigerated. Storing the standards at room temperature will also minimize daily imprecision due to the potential of inadequate room temperature stabilization.

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

IS compounds listed in the table below. These isotopically labeled IS(s) were carefully chosen during method development because they encompass all the functional groups of the method analytes. Although alternate IS standards may be used provided they are isotopically labeled compounds with similar functional groups as the method analytes, the analyst must have documented reasons for using alternate IS(s). Alternate IS(s) must meet the QC requirements in Section 9.3.4. Note that different isotopic labels of the same IS(s) are acceptable (e.g., 13C2-PFOA and 13C4-PFOA) but will require modification of the MS/MS precursor and product ions.

Internal Standards Acronym

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

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

N-deuteriomethylperfluoro-1-octanesulfonamidoacetic acid d3-NMeFOSAA

7.2.1.1 IS STOCK STANDARD SOLUTIONS – These IS stocks can be obtained as individual certified stock standard solutions. During the development of this method, commercially obtained 50 µg/mL stock standard solutions in methanol were used (Wellington Labs, Perkin Elmer or equivalent).

Analysis of the IS(s) is less complicated if the IS(s) purchased contains only the linear isomer. IS stock standard solutions were stable for at least 6 months when stored at 4 °C.

7.2.1.2 INTERNAL STANDARD PRIMARY DILUTION (IS PDS) STANDARD

(1-4 ng/µL) – Prepare, or purchase commercially, the IS PDS at a suggested concentration of 1-4 ng/µL. If prepared from the individual stock standard solutions (Sect. 7.2.1.1), the table below can be used as a guideline for preparing the IS PDS. The IS PDS used in these studies was prepared in 96:4% (vol/vol) methanol:water. The IS PDS has been shown to be stable for at least two months when stored in polypropylene centrifuge tubes at room temperature. Use 10 µL of this 1-4 ng/µL solution to fortify the final 1-mL extracts (Sect. 11.5). This will yield a concentration of 10-40 pg/µL of each IS in the 1-mL extracts.

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IS

Conc. of IS Stock (µg/mL)

Vol. Of IS Stock

(µL)

Final Vol.

of IS PDS

(µL)

Final Conc.

of IS PDS (ng/µL)

13C-PFOA 1000 5.0 5000 1.0

13C-PFOS 50 300.0 5000 3.0

d3-NMeFOSAA 50 400.0 5000 4.0

7.2.2 SURROGATE (SUR) ANALYTE STANDARD SOLUTIONS – The three

SUR(s) for this method are listed in the table below. These isotopically labeled SUR standards were carefully chosen during method development because they encompass most of the functional groups, as well as the water solubility range of the method analytes. Although alternate SUR standards may be used provided they are isotopically labeled compounds with similar functional groups as the method analytes, the analyst must have documented reasons for using alternate SUR standards. The alternate SUR standards chosen must still span the water solubility range of the method analytes. In addition, alternate SUR standards must meet the QC requirements in Section 9.3.5.

Surrogates Acronym

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

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

N-deuterioethylperfluoro-1-octanesulfonamidoacetic acid d5-NEtFOSAA

7.2.2.1 SUR STOCK STANDARD SOLUTIONS – These SUR stocks can be obtained as individual certified stock standard solutions. During the development of this method, commercially obtained 50 µg/mL stock standard solutions in methanol containing 4% water (except for d5-NEtFOSAA which was purchased in pure methanol) were used (Wellington Labs or equivalent). Analysis of the SUR(s) is less complicated if the SUR(s) purchased contains only the linear isomer. SUR stock standard solutions were stable for at least 6 months when stored at 4 °C.

7.2.2.2 SURROGATE PRIMARY DILUTION STANDARD (SUR PDS)

(1-4 ng/µL) – Prepare, or purchase commercially, the SUR PDS at a suggested concentration of 1-4 ng/µL. If prepared from the individual stock standard solutions (Sect. 7.2.2.1), the table below can be used as a guideline for preparing the SUR PDS. The SUR PDS used in these studies was prepared in 96:4% (vol/vol) methanol:water. This solution is used to fortify all QC and Field Samples. The PDS has been shown to be stable for one year when stored in polypropylene centrifuge tubes at room temperature.

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SUR

Conc. Of SUR Stock

(µg/mL)

Vol. of SUR Stock

(µL)

Final Vol. of

SUR PDS

(µL)

Final Conc.

of SUR PDS

(ng/µL) 13C-PFHxA 50 100.0 5000 1.0

13C-PFDA 50 100.0 5000 1.0

d5-NEtFOSAA 50 400.0 5000 4.0

7.2.3 ANALYTE STANDARD SOLUTIONS – Analyte standards may be purchased commercially as ampoulized solutions or prepared from neat materials (see Table 3 for the analyte sources used during method development). If possible, purchase the method analytes as technical grade standards or neat materials.

Standards or neat materials that contain only the linear isomer can be substituted only if technical grade (linear and branched isomers) standards or neat material cannot be purchased. PFHxS and PFOS must be purchased as technical grade (containing branched and linear isomers), as well as NEtFOSAA and NMeFOSAA (if commercially available). PFHxS and PFOS are not available as the acids listed in Section 1.1, but rather as their corresponding salts, such as Na+ and K+ (see Table 3). These salts are acceptable starting materials for the stock standards provided the measured mass is corrected for the salt content according to the equation below. Prepare the Analyte Stock and Primary Dilutions Standards as described below.

salt acid saltacid MW

MW

MassMeasuredMass ×= where:

MWacid = the molecular weight of PFAA MWsalt = the molecular weight of purchased salt

7.2.3.1 ANALYTE STOCK STANDARD SOLUTION – If preparing from neat material, accurately weigh approximately 5 mg of pure material to the nearest 0.1 mg into a 4-mL or larger polypropylene bottle (Sect. 6.2). Add 1 mL of the solvent indicated in the table below. Repeat for each method analyte. These stock standards were stable for at least 6 months when stored at -15 °C. When using these stock standards to prepare a PDS, care must be taken to ensure that these standards are at room temperature and adequately vortexed.

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Analyte Analyte Stock Solvent PFHxA 96:4% (vol/vol) methanol:water PFHpA 96:4% (vol/vol) methanol:water PFOA 96:4% (vol/vol) methanol:water PFNA 96:4% (vol/vol) methanol:water PFDA 96:4% (vol/vol) methanol:water PFUnA 96:4% (vol/vol) methanol:water PFDoA 96:4% (vol/vol) methanol:water PFTrDA 100% ethyl acetate PFTA 100% ethyl acetate PFBS 100% methanol PFHxS 100% methanol PFOS 100% methanol NEtFOSAA 100% methanol NMeFOSAA 100% methanol

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

2.5 ng/µL) – The analyte PDS contains all the method analytes of interest at various concentrations in methanol containing 4% water. The ESI and MS/MS response varies by compound; therefore, a mix of concentrations may be needed in the analyte PDS. See Tables 5-9 in Section 17 for suggested concentrations for each analyte. During method development, the analyte PDS was prepared such that approximately the same instrument response was obtained for all the analytes. The analyte PDS is prepared by dilution of the combined Analyte Stock Standard Solutions and is used to prepare the CAL standards, and fortify the LFBs, the LFSMs, the LFSMDs and FDs with the method analytes. The analyte PDS has been shown to be stable for 6 months when stored at room temperature.

7.2.4 CALIBRATION STANDARDS (CAL) – At least five calibration concentrations are required to prepare the initial calibration curve spanning a 20-fold concentration range (Sect. 10.2). Larger concentration ranges will require more calibration points. Prepare the CAL standards over the concentration range of interest from dilutions of the analyte PDS in methanol containing 4% reagent water. The suggested analyte concentrations found in Tables 5-9 can be used as a starting point for determining the calibration range. The IS and SUR are added to the CAL standards at a constant concentration. During method development, the concentrations of the SUR(s) were 10-40 pg/µL in the standard (40-160 ng/L in the sample) and the IS(s) were 10-40 pg/µL. The lowest concentration CAL standard must be at or below the MRL, which may depend on system sensitivity.

The CAL standards may also be used as CCCs (Sect. 9.3.2). During method development, the CAL standards were shown to be stable for at least two weeks when stored at room temperature. Longer storage times are acceptable provided

537-16 appropriate QC measures are documented demonstrating the CAL standard stability.

8. SAMPLE COLLECTION, PRESERVATION, AND STORAGE

8.1 SAMPLE BOTTLE PREPARATION

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

8.1.2 The preservation reagent, listed in the table below, is added to each sample bottle as a solid prior to shipment to the field (or prior to sample collection).

Compound Amount Purpose

Trizma 5.0 g/L buffering reagent and removes free chlorine

8.2 SAMPLE COLLECTION

8.2.1 The sample handler must wash their hands before sampling and wear nitrile gloves while filling and sealing the sample bottles. PFAA contamination during sampling can occur from a number of common sources, such as food packaging and certain foods and beverages. Proper hand washing and wearing nitrile gloves will aid in minimizing this type of accidental contamination of the samples.

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

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

Samples do not need to be collected headspace free.

8.2.4 After collecting the sample, cap the bottle and agitate by hand until preservative is dissolved. Keep the sample sealed from time of collection until extraction.

8.3 FIELD REAGENT BLANKS (FRB)

8.3.1 A FRB must be handled along with each sample set. The sample set is composed of samples collected from the same sample site and at the same time. At the laboratory, fill the field blank sample bottle with reagent water and preservatives, seal, and ship to the sampling site along with the sample bottles. For each FRB shipped, an empty sample bottle (no preservatives) must also be shipped. At the sampling site, the sampler must open the shipped FRB and pour the preserved reagent water into the empty shipped sample bottle, seal and label this bottle as the FRB. The FRB is shipped back to the laboratory along with the samples and analyzed to ensure that PFAAs were not introduced into the sample during sample collection/handling.

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

8.3.3 The reagent water used for the FRBs must be initially analyzed for method analytes as a LRB and must meet the LRB criteria in Section 9.3.1 prior to use.

This requirement will ensure samples are not being discarded due to contaminated reagent water rather than contamination during sampling.

8.4 SAMPLE SHIPMENT AND STORAGE – Samples must be chilled during shipment and must not exceed 10 °C during the first 48 hours after collection. Sample temperature must be confirmed to be at or below 10 °C when the samples are received at the laboratory. Samples stored in the lab must be held at or below 6 °C until extraction, but should not be frozen.

NOTE: Samples that are significantly above 10° C, at the time of collection, may need to be iced or refrigerated for a period of time, in order to chill them prior to shipping. This will allow them to be shipped with sufficient ice to meet the above requirements.

8.5 SAMPLE AND EXTRACT HOLDING TIMES – Results of the sample storage stability study (Table 10) indicated that all compounds listed in this method have adequate stability for 14 days when collected, preserved, shipped and stored as described in Sections 8.1, 8.2, and 8.4. Therefore, water samples should be extracted as soon as possible but must be extracted within 14 days. Extracts must be stored at room temperature and analyzed within 28 days after extraction. The extract storage stability study data are presented in Table 11.

9. QUALITY CONTROL

9.1 QC requirements include the Initial Demonstration of Capability (IDC) and ongoing QC requirements that must be met when preparing and analyzing Field Samples. This section describes the QC parameters, their required frequencies, and the performance criteria that must be met in order to meet EPA quality objectives. The QC criteria discussed in the following sections are summarized in Tables 12 and 13. These QC requirements are considered the minimum acceptable QC criteria. Laboratories are encouraged to institute additional QC practices to meet their specific needs.

9.1.1 METHOD MODIFICATIONS – The analyst is permitted to modify LC columns, LC conditions, evaporation techniques, internal standards or surrogate standards, and MS and MS/MS conditions. Each time such method modifications are made, the analyst must repeat the procedures of the IDC. Modifications to LC conditions should still produce conditions such that co-elution of the method analytes is minimized to reduce the probability of suppression/enhancement effects.

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9.2 INITIAL DEMONSTRATION OF CAPABILITY – The IDC must be successfully performed prior to analyzing any Field Samples. Prior to conducting the IDC, the analyst must first generate an acceptable Initial Calibration following the procedure outlined in Section 10.2.

9.2.1 INITIAL DEMONSTRATION OF LOW SYSTEM BACKGROUND – Any time a new lot of SPE cartridges, solvents, centrifuge tubes, disposable pipets, and autosampler vials are used, it must be demonstrated that an LRB is reasonably free of contamination and that the criteria in Section 9.3.1 are met. If an automated extraction system is used, an LRB should be extracted on each port to ensure that all the valves and tubing are free from potential PFAA contamination.

9.2.2 INITIAL DEMONSTRATION OF PRECISION (IDP) – Prepare, extract, and analyze four to seven replicate LFBs fortified near the midrange of the initial calibration curve according to the procedure described in Section 11. Sample preservatives as described in Section 8.1.2 must be added to these samples. The relative standard deviation (RSD) of the results of the replicate analyses must be less than 20%.

9.2.3 INITIAL DEMONSTRATION OF ACCURACY (IDA) – Using the same set of replicate data generated for Section 9.2.2, calculate average recovery. The average recovery of the replicate values must be within ± 30% of the true value.

9.2.4 INITIAL DEMONSTRATION OF PEAK ASYMMETRY FACTOR – Peak

asymmetry factors must be calculated using the equation in Section 9.3.9 for the first two eluting peaks (if only two analytes are being analyzed, both must be evaluated) in a mid-level CAL standard. The peak asymmetry factors must fall in the range of 0.8 to 1.5. See guidance in Section 10.2.4.1 if the calculated peak asymmetry factors do not meet the criteria.

9.2.5 MINIMUM REPORTING LEVEL (MRL) CONFIRMATION – Establish a target concentration for the MRL based on the intended use of the method. The MRL may be established by a laboratory for their specific purpose or may be set by a regulatory agency. Establish an Initial Calibration following the procedure outlined in Section 10.2. The lowest CAL standard used to establish the Initial Calibration (as well as the low-level CCC, Section 10.3) must be at or below the concentration of the MRL. Establishing the MRL concentration too low may cause repeated failure of ongoing QC requirements. Confirm the MRL following the procedure outlined below.

9.2.5.1 Fortify, extract, and analyze seven replicate LFBs at the proposed MRL concentration. These LFBs must contain all method preservatives described in Section 8.1.2. Calculate the mean measured concentration (Mean) and standard deviation for these replicates. Determine the Half Range for the prediction interval of results (HRPIR) using the equation below

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HR sPIR = 3963.

where s = the standard deviation

3.963 = a constant value for seven replicates.1

9.2.5.2 Confirm that the upper and lower limits for the Prediction Interval of Result (PIR = Mean + HRPIR) meet the upper and lower recovery limits as shown below

The Upper PIR Limit must be ≤150% recovery.

150% %100 ≤× ononcentratiFortifiedC

HRMean PIR

The Lower PIR Limit must be ≥ 50% recovery.

50% %100 ≥×− ononcentratiFortifiedC

HRMean PIR

9.2.5.3 The MRL is validated if both the Upper and Lower PIR Limits meet the criteria described above (Sect. 9.2.5.2). If these criteria are not met, the MRL has been set too low and must be determined again at a higher concentration.

9.2.6 CALIBRATION CONFIRMATION – Analyze a QCS as described in Section

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

9.2.7 DETECTION LIMIT DETERMINATION (optional) – While DL determination is not a specific requirement of this method, it may be required by various regulatory bodies associated with compliance monitoring. It is the responsibility of the laboratory to determine if DL determination is required based upon the intended use of the data.

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

(i.e., both the sample extraction and the LC/MS/MS analyses should be done over at least three days). Prepare at least seven replicate LFBs at a concentration estimated to be near the DL. This concentration may be estimated by selecting a concentration at 2-5 times the noise level. The DLs in Table 5 were calculated from LFBs fortified at various concentrations as indicated in the table. The appropriate fortification concentrations will be dependent upon the sensitivity of the LC/MS/MS system used. All preservation reagents listed in Section 8.1.2 must also be added to these samples. Analyze the seven replicates through all steps of Section 11.

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NOTE: If an MRL confirmation data set meets these requirements, a DL may be calculated from the MRL confirmation data, and no additional analyses are necessary.

Calculate the DL using the following equation

)99.01 ,1( =−−×= αntsDL s = standard deviation of replicate analyses t (n-1, 1-α=0.99) = Student's t value for the 99% confidence level with n-1 degrees of freedom n = number of replicates.

NOTE: Do not subtract blank values when performing DL calculations. The DL is a statistical determination of precision only.2 If the DL replicates are fortified at a low enough concentration, it is likely that they will not meet the precision and accuracy criteria for CCCs. Therefore, no precision and accuracy criteria are specified.

9.2.7.2 If a laboratory is establishing their own MRL, the calculated DLs should not be used as the MRL for analytes that commonly occur as background contaminants. Method analytes that are seen in the background should be reported as present in Field Samples, only after careful evaluation of the background levels. It is recommended that a MRL be established at the mean LRB concentrations + 3σ or 3 times the mean LRB concentration, whichever is greater. This value should be calculated over a period of time, to reflect variability in the blank measurements. It is recommended that this value be used as an MRL in order to avoid reporting false positive results.

9.3 ONGOING QC REQUIREMENTS – This section summarizes the ongoing QC criteria that must be followed when processing and analyzing Field Samples.

9.3.1 LABORATORY REAGENT BLANK (LRB) – An LRB is required with each extraction batch (Sect. 3.6) to confirm that potential background contaminants are not interfering with the identification or quantitation of method analytes. If more than 20 Field Samples are included in a batch, analyze an LRB for every 20 samples. If the LRB produces a peak within the retention time window of any analyte that would prevent the determination of that analyte, determine the source of contamination and eliminate the interference before processing samples.

Background contamination must be reduced to an acceptable level before proceeding. Background from method analytes or other contaminants that inter-fere with the measurement of method analytes must be below 1/3 of the MRL.

Blank contamination is estimated by extrapolation, if the concentration is below the lowest CAL standard. This extrapolation procedure is not allowed for sample results as it may not meet data quality objectives. If the method analytes are

537-21 detected in the LRB at concentrations equal to or greater than this level, then all data for the problem analyte(s) must be considered invalid for all samples in the extraction batch. Because background contamination is a significant problem for several method analytes, it is highly recommended that the analyst maintain a historical record of LRB data.

9.3.2 CONTINUING CALIBRATION CHECK (CCC) – CCC Standards are analyzed at the beginning of each analysis batch, after every 10 Field Samples, and at the end of the analysis batch. See Section 10.3 for concentration requirements and acceptance criteria.

9.3.3 LABORATORY FORTIFIED BLANK (LFB) – An LFB is required with each extraction batch (Sect. 3.6). The fortified concentration of the LFB must be rotated between low, medium, and high concentrations from batch to batch. The low concentration LFB must be as near as practical to, but no more than two times, the MRL. Similarly, the high concentration LFB should be near the high end of the calibration range established during the initial calibration (Sect. 10.2).

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

Results of the medium and high-level LFB analyses must be 70-130% of the true value. If the LFB results do not meet these criteria for method analytes, then all data for the problem analyte(s) must be considered invalid for all samples in the extraction batch.

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

IS(s) in all injections during each analysis day. The IS responses (peak areas) in any chromatographic run must be within 70-140% of the response in the most recent CCC and must not deviate by more than 50% from the average area measured during initial analyte calibration. If the IS areas in a chromatographic run do not meet these criteria, inject a second aliquot of that extract aliquotted into a new capped autosampler vial. Random evaporation losses have been observed with the polypropylene caps causing high IS(s) areas.

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

9.3.4.2 If the reinjected extract fails again, the analyst should check the calibration by reanalyzing the most recently acceptable CAL standard. If the CAL standard fails the criteria of Section 10.3, recalibration is in order per Section 10.2. If the CAL standard is acceptable, extraction of the sample may need to be repeated provided the sample is still within the holding time.

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