METHOD 533: DETERMINATION OF PER- AND

POLYFLUOROALKYL SUBSTANCES IN DRINKING WATER BY

ISOTOPE DILUTION ANION EXCHANGE SOLID PHASE

EXTRACTION AND LIQUID CHROMATOGRAPHY/TANDEM

MASS SPECTROMETRY

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Technical questions concerning this analytical method should be addressed to:

Steven C. Wendelken, Ph.D.

U.S. EPA, Office of Ground Water and Drinking Water, Standards and Risk Management Division, Technical Support Center, 26 W. Martin Luther King Dr. Cincinnati, OH 45268 Phone: (513) 569-7491 wendelken.steve@epa.gov

Questions concerning this document or policy should be addressed to: safewater@epa.gov

Office of Water (MS-140) EPA Document No. 815-B-19-020 EPA contract EP-C-17-014 November 2019

Authors Laura Rosenblum, Ph.D., APTIM (Cincinnati, OH) Contractor’s role did not include establishing Agency policy.

Steven C. Wendelken, Ph.D., U.S. EPA (Cincinnati, OH)

Acknowledgements Alan Zaffiro, APTIM (Cincinnati, OH)

The following organizations completed a validation study in their laboratories using this method, provided valuable feedback on the method procedures and reviewed the draft method manuscript:

Babcock Laboratories, Inc. (Riverside, CA) Eurofins Eaton Analytical, LLC (South Bend, IN) Eurofins TestAmerica (Sacramento, CA) Merit Laboratories, Inc. (East Lansing, MI) Shimadzu Scientific Instruments (Columbia, MD) Thermo Fisher Scientific (Sunnyvale, CA) Weck Laboratories, Inc. (City of Industry, CA) Vogon Laboratory Services Ltd. (Cochrane, Alberta, Canada)

Disclaimer This analytical method may support a variety of monitoring applications, which include the analysis of multiple short-chain per- and polyfluoroalkyl substances (PFAS) that cannot be measured by Method

537.1. This publication meets an agency commitment identified within the 2019 EPA PFAS Action Plan.

Publication of the method, in and of itself, does not establish a requirement, although the use of this method may be specified by the EPA or a state through independent actions. Terms such as “must” or “required,” as used in this document, refer to procedures that are to be followed to conform with the method. References to specific brands and catalog numbers are included only as examples and do not imply endorsement of the products. Such reference does not preclude the use of equivalent products from other vendors or suppliers.

mailto:wendelken.steve@epa.gov mailto:wendelken.steve@epa.gov mailto:safewater@epa.gov https://www.epa.gov/pfas/epas-pfas-action-plan

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Table of Contents 1 Scope and Application

2 Method Summary

3 Definitions

4 Interferences

5 Safety

6 Equipment and Supplies

7 Reagents and Standards

8 Sample Collection, Preservation, and Storage

9 Quality Control

10 Calibration and Standardization

11 Procedure

12 Data Analysis and Calculations

13 Method Performance

14 Pollution Prevention

15 Waste Management

16 References

17 Tables, Figures and Method Performance Data

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Tables Table 1. HPLC Method Conditions

Table 2. ESI-MS Method Conditions

Table 3. Isotopically Labeled Isotope Performance Standards and Retention Times

Table 4. Isotope Dilution Analogues: RTs and Suggested Isotope Performance Standard References

Table 5. Method Analytes, Retention Times and Suggested Isotope Dilution Analogue References

Table 6. MS/MS Method Conditions

Table 7. LCMRL Results

Table 8. Precision and Accuracy Data for Reagent Water

Table 9. P&A in Reagent Water: Isotope Dilution Analogue Recovery Data

Table 10. Precision and Accuracy Data for Finished Ground Water

Table 11. P&A in Finished Ground Water: Isotope Dilution Analogue Recovery Data

Table 12. Precision and Accuracy Data for a Surface Water Matrix

Table 13. P&A in Surface Water Matrix: Isotope Dilution Analogue Recovery Data

Table 14. Aqueous Sample Holding Time Data

Table 15. Extract Holding Time Data

Table 16. Initial Demonstration of Capability (IDC) Quality Control Requirements

Table 17. Ongoing Quality Control Requirements

Figures Figure 1. Example Chromatogram for Reagent Water Fortified with Method Analytes at 80 ng/L

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1 Scope and Application

This is a solid phase extraction (SPE) liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of select per- and polyfluoroalkyl substances (PFAS) in drinking water.

Method 533 requires the use of MS/MS in Multiple Reaction Monitoring (MRM) mode to enhance selectivity. Accuracy and precision data have been generated in reagent water and drinking water for the compounds included in the Analyte List.

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

Analyte List Analytea Abbreviation CASRN 11-Chloroeicosafluoro-3-oxaundecane-1-sulfonic acid 11Cl-PF3OUdS 763051-92-9 9-Chlorohexadecafluoro-3-oxanonane-1-sulfonic acd 9Cl-PF3ONS 756426-58-1 4,8-Dioxa-3H-perfluorononanoic acid ADONA 919005-14-4 Hexafluoropropylene oxide dimer acid HFPO-DA 13252-13-6 Nonafluoro-3,6-dioxaheptanoic acid NFDHA 151772-58-6 Perfluorobutanoic acid PFBA 375-22-4 Perfluorobutanesulfonic acid PFBS 375-73-5 1H,1H, 2H, 2H-Perfluorodecane sulfonic acid 8:2FTS 39108-34-4 Perfluorodecanoic acid PFDA 335-76-2 Perfluorododecanoic acid PFDoA 307-55-1 Perfluoro(2-ethoxyethane)sulfonic acid PFEESA 113507-82-7 Perfluoroheptanesulfonic acid PFHpS 375-92-8 Perfluoroheptanoic acid PFHpA 375-85-9 1H,1H, 2H, 2H-Perfluorohexane sulfonic acid 4:2FTS 757124-72-4 Perfluorohexanesulfonic acid PFHxS 355-46-4 Perfluorohexanoic acid PFHxA 307-24-4 Perfluoro-3-methoxypropanoic acid PFMPA 377-73-1 Perfluoro-4-methoxybutanoic acid PFMBA 863090-89-5 Perfluorononanoic acid PFNA 375-95-1 1H,1H, 2H, 2H-Perfluorooctane sulfonic acid 6:2FTS 27619-97-2 Perfluorooctanesulfonic acid PFOS 1763-23-1 Perfluorooctanoic acid PFOA 335-67-1 Perfluoropentanoic acid PFPeA 2706-90-3 Perfluoropentanesulfonic acid PFPeS 2706-91-4 Perfluoroundecanoic acid PFUnA 2058-94-8

a. Some PFAS are commercially available as ammonium, sodium, and potassium salts. This method measures all forms of the analytes as anions while the identity of the counterion is inconsequential. Analytes may be purchased as acids or as any of the corresponding salts.

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1.1 Detection of PFAS Isomers

Both branched and linear PFAS isomers may be found in the environment. This method includes procedures for summing the contribution of multiple isomers to the final reported concentration. In those cases where standard materials containing multiple isomers are commercially available, laboratories should obtain such standards for the method analytes.

1.2 Lowest Concentration Minimum Reporting Limits

The lowest concentration minimum reporting level (LCMRL) is the lowest concentration for which the future recovery is predicted to fall between 50 and 150% with high confidence (99%). Single-laboratory LCMRLs determined for the method analytes during method development are reported in Table 7. It should be noted that most of the LCMRL values determined during the second laboratory evaluation were lower than the values listed in Table 7. The values that a laboratory can obtain are dependent on the design and capability of the instrumentation used. The procedure used to determine the LCMRL is described elsewhere.1,2 Laboratories using this method are not required to determine LCMRLs, but they must demonstrate that they are able to meet the minimum reporting level (MRL) (Sect. 3.15) for each analyte per the procedure described in Section 9.1.4.

1.3 Method Flexibility

The laboratory may select LC columns, LC conditions, and MS conditions different from those used to develop the method. At a minimum, the isotope dilution standards and the isotope performance standards specified in the method must be used, if available. The laboratory may select the aqueous sample volume within the range of 100–250 mL that meets their objectives. During method development, 250 mL aqueous samples were extracted using a 500 mg solid phase extraction (SPE) sorbent bed volume. The ratio of sorbent mass to aqueous sample volume may not be decreased. If a laboratory uses 100 mL aqueous samples, the sorbent mass must be at least 200 mg. Changes may not be made to sample preservation, the quality control (QC) requirements, or the extraction procedure.

The chromatographic separation should minimize the number of compounds eluting within a retention window to obtain a sufficient number of scans across each peak. Instrumental sensitivity (or signal-to-noise) will decrease if too many compounds are permitted to elute within a retention time window.

Method modifications should be considered only to improve method performance. In all cases where method modifications are proposed, the analyst must perform the procedures outlined in the Initial Demonstration of Capability (IDC, Sect. 9.1), verify that all QC acceptance criteria in this method (Sect. 9.2) are met, and verify method performance in a representative sample matrix (Sect. 9.3.2).

2 Method Summary A 100–250 mL sample is fortified with isotopically labeled analogues of the method analytes that function as isotope dilution standards. The sample is passed through an SPE cartridge containing polystyrene divinylbenzene with a positively charged diamino ligand to extract the method analytes and isotope dilution analogues. The cartridge is rinsed with sequential washes of aqueous ammonium acetate followed by methanol, then the compounds are eluted from the solid phase sorbent with methanol containing ammonium hydroxide. The extract is concentrated to dryness with nitrogen in a heated water bath. The extract volume is adjusted to 1.0 mL with 20% water in methanol (v/v), and three isotopically labeled isotope performance standards are added. Extracts are analyzed by LC-MS/MS

533-3 in the MRM detection mode. The concentration of each analyte is calculated using the isotope dilution technique. For QC purposes, the percent recoveries of the isotope dilution analogues are calculated using the integrated peak areas of isotope performance standards, which are added to the final extract and function as traditional internal standards, exclusively applied to the isotope dilution analogues.

3 Definitions

3.1 Analysis Batch

A set of samples that are analyzed on the same instrument during a 24-hour period 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 the number of field samples.

3.2 Calibration Standard

A solution of the method analytes, isotope dilution analogues, and isotope performance standards prepared from the Primary Dilution Standards and stock standards. The calibration standards are used to calibrate the instrument response with respect to analyte concentration.

3.3 Continuing Calibration Check (CCC)

A calibration standard that is analyzed periodically to verify the accuracy of the existing calibration.

3.4 Extraction Batch

A set of up to 20 field samples (not including QC samples) extracted together using the same lot of solid phase extraction devices, solvents, and fortifying solutions.

3.5 Field Duplicates (FD)

Separate samples collected at the same time and sampling location, shipped and stored under identical conditions. Method precision, including the contribution from sample collection procedures, is estimated from the analysis of Field Duplicates. Field Duplicates are used to prepare Laboratory Fortified Sample Matrix and Laboratory Fortified Sample Matrix Duplicate QC samples. For the purposes of this method, Field Duplicates are collected to support potential repeat analyses (if the original field sample is lost or if there are QC failures associated with the analysis of the original field sample).

3.6 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, and all analytical procedures. The purpose of the FRB is to determine if method analytes or other interferences are introduced into the sample from shipping, storage, and the field environment.

3.7 Isotope Dilution Analogues

Isotopically labeled analogues of the method analytes that are added to the sample prior to extraction in a known amount. Note: Not all target PFAS currently have an isotopically labelled analogue. In these cases, an alternate isotopically labelled analogue is used as recommended in Table 5.

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3.8 Isotope Dilution Technique

An analytical technique for measuring analyte concentration using the ratio of the peak area of the native analyte to that of an isotopically labeled analogue, added to the original sample in a known amount and carried through the entire analytical procedure.

3.9 Isotope Performance Standards

Quality control compounds that are added to all standard solutions and extracts in a known amount and used to measure the relative response of the isotopically labelled analogues that are components of the same solution. For this method, the isotope performance standards are three isotopically labeled analogues of the method analytes. The isotope performance standards are indicators of instrument performance and are used to calculate the recovery of the isotope dilution analogues through the extraction procedure. In this method, the isotope performance standards are not used in the calculation of the recovery of the native analytes.

3.10 Laboratory Fortified Blank (LFB)

An aliquot of reagent water to which known quantities of the method analytes and isotope dilution analogues are added. The results of the LFB verify method performance in the absence of sample matrix.

3.11 Laboratory Fortified Sample Matrix (LFSM)

An aliquot of a field sample to which known quantities of the method analytes and isotope dilution analogues are added. The purpose of the LSFM is to determine whether the sample matrix contributes bias to the analytical results. Separate field samples are required for preparing fortified matrix so that sampling error is included in the accuracy estimate.

3.12 Laboratory Fortified Sample Matrix Duplicate (LFSMD)

A Field Duplicate of the sample used to prepare the LFSM that is fortified and analyzed identically to the LFSM. The LFSMD is used instead of the Field Duplicate to assess method precision when the method analytes are rarely found at concentrations greater than the MRL.

3.13 Laboratory Reagent Blank (LRB)

An aliquot of reagent water fortified with the isotope dilution analogues and processed identically to a field sample. An LRB is included in each Extraction Batch to determine if the method analytes or other interferences are introduced from the laboratory environment, the reagents, glassware, or extraction apparatus.

3.14 Lowest Concentration Minimum Reporting Level (LCMRL)

The single-laboratory LCMRL is the lowest spiking concentration such that the probability of spike recovery in the 50% to 150% range is at least 99%.1,2

3.15 Minimum Reporting Level (MRL)

The minimum concentration that may be reported by a laboratory as a quantified value for a method analyte. For each method analyte, the concentration of the lowest calibration standard must be at or

533-5 below the MRL and the laboratory must demonstrate its ability to meet the MRL per the criteria defined in Section 9.1.4.

3.16 Precursor Ion

The gas-phase species corresponding to the method analyte that is produced in the electrospray ionization interface. During tandem mass spectrometry, or MS/MS, the precursor ion is mass selected and fragmented by collision-activated dissociation to produce distinctive product ions of smaller mass to charge (m/z) ratio. For this method, the precursor ion is usually the deprotonated molecule ([M – H]–) of the method analyte, except for HFPO-DA. For this analyte, the precursor ion is formed by decarboxylation of HFPO-DA.

3.17 Primary Dilution Standard (PDS)

A solution that contains method analytes (or QC analytes) prepared from stock standards. PDS solutions are used to fortify QC samples and diluted to prepare calibration standards.

3.18 Product Ion

One of the fragment ions that is produced in MS/MS by collision-activated dissociation of the precursor ion.

3.19 Quality Control Standard (QCS)

A calibration standard prepared independently from the primary calibration solutions. For this method, the QCS is a repeat of the entire dilution scheme starting with the same stock materials (neat compounds or purchased stock solutions) used to prepare the primary calibration solutions.

Independent sources and separate lots of the starting materials are not required, provided the laboratory has obtained the purest form of the starting materials commercially available. The purpose of the QCS is to verify the integrity of the primary calibration standards.

3.20 Quantitative Standard

A quantitative standard of assayed concentration and purity traceable to a Certificate of Analysis.

3.21 Stock Standard Solution

A concentrated standard that is prepared in the laboratory using assayed reference materials or that is purchased from a commercial source with a Certificate of Analysis.

3.22 Technical-Grade Standard

As defined for this method, a technical-grade standard includes a mixture of the branched and linear isomers of a method analyte. For the purposes of this method, technical-grade standards are used to identify retention times of branched and linear isomers of method analytes.

4 Interferences

4.1 Labware, Reagents and Equipment

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 or

533-6 elevated baselines in the chromatograms. The 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, deactivated syringes, SPE sample transfer lines, etc.3 Laboratories must demonstrate that these items are not contributing to interference by analyzing LRBs as described in Section 9.2.1.

4.2 Sample Contact with Glass

Aqueous samples should not come in contact with any glass containers or pipettes as PFAS analytes can potentially adsorb to glass surfaces. Standards dissolved in organic solvent may be purchased in glass ampoules. These standards in organic solvent are acceptable and subsequent transfers may be performed using glass syringes and pipets. Following extraction, the eluate must be collected in a polypropylene tube prior to concentration to dryness. Concentration to dryness in glass tubes may cause poor recovery.

4.3 Matrix Interferences

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 fulvic material may be co-extracted during SPE and high levels may cause enhancement or suppression in the electrospray ionization source.4 Inorganic salts may cause low recoveries during the anion-exchange SPE procedure.

4.3.1 Co-extracted Organic Material

Under the LC conditions used during method development, matrix effects due to co-extracted organic material enhanced the ionization of 4:2 FTS appreciably. Total organic carbon (TOC) is a good indicator of humic content of the sample.

4.3.2 Inorganic Salts

The authors confirmed acceptable method performance for matrix ion concentrations up to 250 mg/L chloride, 250 mg/L sulfate, and 340 mg/L hardness measured as CaCO3. Acceptable performance was defined as recovery of the isotope dilution analogues between 50–200%.

4.3.3 Ammonium Acetate

Relatively large quantities of ammonium acetate are used as a preservative. The potential exists for trace-level organic contaminants in this reagent. Interferences from this source should be monitored by analysis of LRBs, particularly when new lots of this reagent are acquired.

4.3.4 SPE Cartridges

Solid phase extraction cartridges may be a source of interferences. The analysis of LRBs provides important information regarding the presence or absence of such interferences. Each brand and lot of SPE devices must be monitored to ensure that contamination does not preclude analyte identification and quantitation. SPE cartridges should be sealed while in storage to prevent ambient contamination of the SPE sorbent.

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4.4 Bias Caused by Isotopically Labeled Standards

During method development, no isotopically labeled standard solution yielded any signal that gave the same mass and retention time as any native analyte. However, due to isotopic impurity, the 13C3-PFBA isotope performance standard contained a small amount of 13C4-PFBA, slightly contributing to the signal of the isotope dilution analogue. Further, due to natural abundance of 34S, the native telomer sulfonates produced a small contribution to the 13C2 labeled telomer sulfonate isotope dilution analogues. The effects on quantitation are insignificant. However, these cases are described below in Sections 4.4.2 and

4.4.3 to alert the user that these situations could occur.

4.4.1 Method Analytes

At the concentrations used to collect method performance data, the authors could not detect any contribution from the isotope dilution analogues or isotope performance standards to the corresponding native analyte response. However, the user should evaluate each source of isotopically labeled analogues and isotope performance standards to verify that they do not contain any native analyte at concentrations greater than 1/3 of the MRL.

4.4.2 Isotopic purity of 13C3-PFBA

In this method, 13C3-PFBA is used as an isotope performance standard and 13C4-PFBA is used as an isotope dilution analogue. Both share the same product ion, m/z 172. Ten nanograms per liter of 13C4- PFBA is added to the sample prior to extraction (10 ng/mL extract concentration assuming 100% recovery), and 10 ng/mL of 13C3-PFBA is added to the final extract. Because the natural abundance of 13C is 1.1%, there is a 1.1% contribution to the 13C4-PFBA area from the lone, unlabeled 12C atom in 13C3- PFBA. The authors confirmed this contribution empirically. Users of this method may consider this bias to the area of the PFBA isotope dilution analogue insignificant.

4.4.3 Isotopic purity of 13C4-PFBA

A trace amount of 13C3-PFBA was detected in the 13C4-PFBA. The contribution was no greater than 1%.

The contribution of the isotope performance standard to the isotope dilution analogue is insignificant.

4.4.4 Telomer Sulfonates

Each of the three telomer sulfonates in the analyte list (4:2FTS, 6:2FTS, and 8:2FTS) are referenced to their 13C2 isotope dilution analogue. The mass difference between the telomer sulfonates and the isotope dilution analogues is 2 mass units. The single sulfur atom in each of the unlabeled molecules has a naturally occurring M+2 isotope (34S) at 4.25%. Thus, the precursor ions of the 13C2 isotopically labeled analogues and the naturally occuring 34S analogues present in the native analytes have the same nominal masses. The product ions of the telomer sulfonate isotope dilution analogues listed in Table 6 would contain a small contribution from the 34S analogue of the native telomer sulfonates. At the concentrations used in this study, the contribution of the 34S analogue to the isotope dilution analogue was not greater than 2.7%. Alternate product ions may be used if there is sufficient abundance.

5 Safety 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

533-8 safe handling of chemicals used in this method. A reference file of safety data sheets should be made available to all personnel involved in the chemical analysis.

6 Equipment and Supplies References to specific brands and catalog numbers are included as examples only and do not imply endorsement of the products. Such reference does not preclude the use of equivalent products from other vendors or suppliers. Due to potential adsorption of analytes onto glass, polypropylene containers were used for sample preparation and extraction steps. Other plastic materials (e.g., polyethylene) that meet the QC requirements of Section 9 may be substituted.

6.1 Sample Containers

Polypropylene bottles with polypropylene screw caps (for example, 250 mL bottles, Fisher Scientific, Cat.

No. 02-896-D or equivalent).

6.2 Polypropylene Vials

These vials are used to store stock standards and PDS solutions (4 mL, VWR Cat. No. 16066-960 or equivalent).

6.3 Centrifuge Tubes

Conical polypropylene centrifuge tubes (15 mL) with polypropylene screw caps for storing standard solutions and for collection of the eluate during the extraction procedure (Thomas Scientific Cat. No.

2602A10 or equivalent).

6.4 Autosampler Vials

Polypropylene autosampler vials (ThermoFisher, Cat. No. C4000-14) with polypropylene caps (ThermoFisher, Cat. No. C5000-50 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, creating the potential for evaporation to occur after injection. Multiple injections from the same vial are not permissible unless the cap is replaced immediately after injection.

6.5 Micro Syringes

Suggested sizes include 10, 25, 50, 100, 250, 500 and 1000 µL.

6.6 Pipets

Polypropylene or glass pipets may be used for methanolic solutions.

6.7 Analytical Balance

Capable of weighing to the nearest 0.0001 g.

6.8 Solid Phase Extraction (SPE) Apparatus

6.8.1 SPE Cartridges

SPE cartridges containing weak anion exchange, mixed-mode polymeric sorbent (polymeric backbone and a diamino ligand), particle size approximately 33 µm. The SPE sorbent must have a pKa above 8 so that it remains positively charged during extraction. SPE cartridges containing 500 mg sorbent

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(Phenomenex Cat. No. 8B-S038-HCH) were used during method development. Use of 200 mg cartridges is acceptable for the extraction of 100 mL samples.

6.8.2 Vacuum Extraction Manifold

Equipped with flow and vacuum control [Supelco Cat. No. 57030-U, UCT Cat. No. VMF016GL (the latter requires UCT Cat. No. VMF02116 control valves), or equivalent systems]. Automated devices designed for use with SPE cartridges may be used; however, all extraction and elution steps must be the same as in the manual procedure. Care must be taken with automated SPE systems to ensure that Teflon tubing and other PTFE components commonly used in these systems, do not contribute to unacceptable analyte concentrations in LRBs.

6.8.3 Sample Delivery System

Use of large volume sampling lines, constructed with polyethylene tubing, are recommended, but not mandatory. Large volume sample transfer lines, constructed with PTFE tubing, are commercially available for standard extraction manifolds (Supelco Cat. No. 57275 or equivalent). The PTFE tubing can be replaced with 1/8” o.d. x 1/16” i.d. polyethylene tubing [Freelin-Wade (McMinnville, Oregon) LLDPE or equivalent] cut to an appropriate length. This prevents potential contamination from PTFE transfer lines. Other types of non-PTFE tubing may be used provided it meets the LRB and LFB QC requirements.

PTFE tubing may be used, but an LRB must be run on each individual transfer line 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, acceptable performance for the LRB must be met for each port during the IDC (Sect 9.1.1). LRBs must be rotated among the ports during routine analyses thereafter. Plastic reservoirs are difficult to rinse during elution and their use may lead to lower recovery.

6.9 Extract Concentration System

Extracts are concentrated by evaporation with high-purity nitrogen using a water bath set no higher than 60 °C [N-Evap, Model 11155, Organomation Associates (Berlin, MA), Inc., or equivalent].

6.10 Laboratory Vacuum System

Sufficient capacity to maintain a vacuum of approximately 15 to 20 inches of mercury for extraction cartridges.

6.11 pH Meter

Used to verify the pH of the phosphate buffer and to measure the pH of the aqueous sample prior to anion exchange SPE.

6.12 LC-MS/MS System

6.12.1 LC System

The LC system must provide consistent sample injection volumes and be capable of performing binary linear gradients at a constant flow rate. On some LC systems, PFAS may build up in PTFE transfer lines when the system is idle for more than one day. To prevent long delays in purging high levels of PFAS from the LC solvent lines, it may be useful to replace PTFE tubing with PEEKTM tubing and the PTFE solvent frits with stainless steel frits. These modifications were not used on the LC system used for method development. However, a delay column, HLB Direct Connect 2.1 x 30 mm (Waters 186005231), 533-10 was placed in the mobile phase flow path immediately before the injection valve. This direct connect column may have reduced the co-elution of PFAS originating from sources prior to the sample loop from the PFAS injected in the sample. It may not be possible to remove all PFAS background contamination.

6.12.2 Analytical Column

C18 liquid chromatography column (2 x 50 mm) packed with 3 µm C18 solid phase particles (Phenomenex Part Number 00B-4439-B0 or equivalent).

6.12.3 Electrospray Ionization Tandem Mass Spectrometer (ESI-MS/MS)

The mass spectrometer must be capable of electrospray ionization in the negative ion mode. The system must be capable of performing MS/MS to produce unique product ions for the method analytes within specified retention time segments. A minimum of 10 scans across the chromatographic peak is needed to ensure adequate precision. Some ESI-MS/MS instruments may not be suitable for PFAS analysis. See the procedures in Section 10.1.2.1 to ensure that the selected MS/MS platform is capable of monitoring all the required MS/MS transitions for the method analytes.

6.12.4 MS/MS Data System

An interfaced data system is required to acquire, store, and output MS data. The computer software must have the capability of processing stored data by recognizing a chromatographic peak within a given retention time window. The software must allow integration of the abundance of any specific ion between specified time or scan number limits. The software must be able to construct a linear regression or quadratic regression calibration curve and calculate analyte concentrations using the internal standard technique.

7 Reagents and Standards Reagent grade or better chemicals must be used. Unless otherwise indicated, all reagents must conform to the specifications of the Committee on Analytical Reagents of the American Chemical Society (ACS), where such specifications are available. Other grades may be used if the reagent is demonstrated to be free of analytes and interferences and all requirements of the IDC are met when using these reagents.

7.1 Reagent Water

Purified water which does not contain any measurable quantities of any method analytes or interfering compounds greater than one-third of the MRL for each method analyte. It may be necessary to flush the water purification unit to rinse out any build-up of PFAS in the system prior to collection of reagent water.

7.2 Methanol

CH3OH, CASRN 67-56-1, LC grade (Fisher Scientific, Cat. No. A456 or equivalent).

7.3 Ammonium Acetate

NH4C2H3O2, CASRN 631-61-8, HPLC grade, molecular weight equals 77.08 g/mole.

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7.3.1 20 mM Ammonium Acetate

Chromatographic mobile phase. To prepare 1 L, add 1.54 g ammonium acetate to 1 L of reagent water.

This solution is volatile and must be replaced at least once per week. More frequent replacement may be necessary if unexplained losses in sensitivity or retention time shifts are encountered.

7.3.2 1 g/L Ammonium Acetate

Used to rinse SPE cartridges after loading the aqueous sample and prior to the methanol rinse. Prepare in reagent water.

7.4 Concentrated Ammonium Hydroxide Reagent

NH4OH, CASRN 1336-21-6, approximately 56.6% in water as ammonium hydroxide (w/w), approximately 28% in water as ammonia, approximately 14.5 N (Fisher Scientific, Cat. No. A669, Certified ACS Plus grade, or equivalent).

7.5 Solution of Ammonium Hydroxide in Methanol

Used for elution of SPE cartridges. Dilute 2 mL of concentrated ammonium hydroxide (56.6% w/w) in 100 mL methanol. This solution should be made fresh on the day of extraction.

7.6 Sodium Phosphate Dibasic (Na2HPO4)

Used for creating the aqueous buffer for conditioning the SPE cartridges. Dibasic sodium phosphate may be purchased in either the anhydrous or any hydrated form. The formula weight will vary based on degree of hydration.

7.7 Sodium Phosphate Monobasic (NaH2PO4)

Used for creating the aqueous buffer for conditioning the SPE cartridges. Monobasic sodium phosphate may be purchased in either the anhydrous or any hydrated form. The formula weight will vary based on degree of hydration.

7.8 0.1 M Phosphate Buffer pH 7.0

Mix 500 mL of 0.1 M dibasic sodium phosphate with approximately 275 mL of 0.1 M monobasic sodium phosphate. Verify that the solution pH is approximately 7.0.

7.9 Nitrogen

7.9.1 Nitrogen Nebulizer Gas

Nitrogen used as a nebulizer gas in the ESI interface and as collision gas in some MS/MS platforms should meet or exceed the instrument manufacturer’s specifications.

7.9.2 Nitrogen used for Concentrating Extracts

Ultra-high-purity-grade nitrogen should be used to concentrate sample extracts.

7.10 Argon

Used as collision gas in MS/MS instruments. Argon should meet or exceed instrument manufacturer’s specifications. Nitrogen may be used as the collision gas if recommended by the instrument manufacturer.

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7.11 Sodium Hydroxide

May be purchased as pellets or as aqueous solution of known concentration. Added to methanolic solutions of PFAS to prevent esterification.

7.12 Acetic Acid (glacial)

May be necessary to adjust pH of aqueous samples. The pH of the aqueous sample containing 1 g/L ammonium acetate must be between 6 and 8.

7.13 Standard Solutions

7.13.1 Stability of Methanolic Solutions

Fluorinated carboxylic acids will esterify in anhydrous acidic methanol. To prevent esterification, standards must be stored under basic conditions. If base is not already present, this may be accomplished by the addition of sodium hydroxide (approximately 4 mole equivalents) when standards are diluted in methanol. When calculating molarity for solutions containing multiple PFAS, the molecular weight can be estimated as 250 atomic mass units (amu). It is necessary to include sodium hydroxide in solutions of both isotopically labeled and native analytes. The amount of sodium hydroxide needed may be calculated using the following equation:

𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 (𝑔𝑔) × 160( 𝑔𝑔

𝑚𝑚𝑚𝑚𝑚𝑚)

250 ( 𝑔𝑔 𝑚𝑚𝑚𝑚𝑚𝑚)

= 𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀 𝑜𝑜𝑜𝑜 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅 (𝑔𝑔)

7.13.2 Preparation of Standards

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. Sorption of PFAS analytes in methanol solution to glass surfaces after prolonged storage has not been evaluated. PFAS analyte and isotopically labeled analogues commercially purchased in glass ampoules are acceptable; however, all subsequent transfers or dilutions performed by the analyst must be stored in polypropylene containers.

Solution concentrations listed in this section were used to develop this method and are included as examples. 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. Laboratories should use standard QC practices to determine when standards need to be replaced. The analyte supplier’s guidelines may be helpful when making this determination.

7.14 Storage Temperatures for Standards Solutions

Store stock standards at less than 4 °C unless the vendor recommends otherwise. The Primary Dilution Standards may be stored at any temperature, but cold storage is recommended to prevent solvent evaporation. During method development, the PDS was stored at –20 °C and no change in analyte concentrations was observed over a period of 6 months.

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7.15 Isotope Performance Standards

This method requires three isotope performance standards listed in the table below. These isotopically labeled compounds were chosen during method development to include the analogues of three method analytes: two carboxylates with different chain lengths and a sulfonate.

Obtain the isotope performance standards as certified standard solutions, if available, or as the neat compounds. During method development, the isotope performance standards were obtained from Wellington Laboratories (Guelph, ON, Canada) as certified stocks in basic methanol. Note that Chemical Abstracts Registry Numbers are not currently available for these compounds. The concentrations of the stocks supplied by Wellington are listed in the table below.

Isotope Performance Standards Abbreviation

Wellington Stock, µg/mL

PDS, ng/µL

Perfluoro-n-[2,3,4-13C3]butanoic acid 13C3-PFBA 50 1.0 Perfluoro-[1,2-13C2]octanoic acid 13C2-PFOA 50 1.0 Sodium perfluoro-1-[1,2,3,4-13C4]octanesulfonate 13C4-PFOS 50a 3.0

a. 47.8 µg/mL as the anion.

All the isotope performance standards listed in this section must be used, if available. Additional isotope performance standards may be used provided they are isotopically labeled analytes or labeled analytes with similar functional groups as the method analytes. Linear isomers are recommended to simplify peak integration. Method modification QC requirements must be met (Sect. 9.3) whenever additional isotope performance standards are used.

7.15.1 Isotope Performance Standard PDS

Prepare the isotope performance standard PDS in methanol and add sodium hydroxide if not already present to prevent esterification as described in Section 7.13.1. The PDS concentrations used to develop the method are listed in the table above (Sect. 7.15). During collection of method performance data, the final extracts were fortified with 10 µL of the PDS to yield a concentration of 10 ng/mL for 13C3-PFBA and 13C2-PFOA, and 30 ng/mL for 13C4-PFOS (28.7 ng/mL as the anion).

7.16 Isotope Dilution Analogues

Obtain the isotopically labeled analogues listed in the table in this section as individual certified standard solutions or as certified standard mixes. All listed isotope dilution analogues must be used, if available. Linear isomers are recommended to simplify peak integration. During method development, the isotope dilution analogues were obtained from Wellington Laboratories (Guelph, ON, Canada) as certified stocks in basic methanol. These analogues were chosen during method development because they encompass most of the functional groups, as well as the molecular weight range of the method analytes. Note that Chemical Abstracts Registry Numbers are not currently available for these isotopically labeled analogues.

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Isotope Dilution Standards

Abbreviation

PDS,

ng/µLa Perfluoro-n-[1,2,3,4-13C4]butanoic acid 13C4-PFBA 0.50 Perfluoro-n-[1,2,3,4,5-13C5]pentanoic acid 13C5-PFPeA 0.50 Sodium perfluoro-1-[2,3,4-13C3]butanesulfonate 13C3-PFBS 0.50 Sodium 1H,1H,2H,2H-perfluoro-1-[1,2-13C2]hexane sulfonate 13C2-4:2FTS 2.0 Perfluoro-n-[1,2,3,4,6-13C5]hexanoic acid 13C5-PFHxA 0.50 2,3,3,3-Tetrafluoro-2-(1,1,2,2,3,3,3-heptafluoropropoxy-13C3-propanoic acid 13C3-HFPO-DA 0.50 Perfluoro-n-[1,2,3,4-13C4]heptanoic acid 13C4-PFHpA 0.50 Sodium perfluoro-1-[1,2,3-13C3]hexanesulfonate 13C3-PFHxS 0.50 Sodium 1H,1H,2H,2H-perfluoro-1-[1,2-13C2]-octane sulfonate 13C2-6:2FTS 2.0 Perfluoro-n-[13C8]octanoic acid 13C8-PFOA 0.50 Perfluoro-n-[13C9]nonanoic acid 13C9-PFNA 0.50 Sodium perfluoro-[13C8]octanesulfonate 13C8-PFOS 0.50 Sodium 1H,1H,2H,2H-perfluoro-1-[1,2-13C2]-decane sulfonate 13C2-8:2FTS 2.0 Perfluoro-n-[1,2,3,4,5,6-13C6]decanoic acid 13C6-PFDA 0.50 Perfluoro-n-[1,2,3,4,5,6,7-13C7]undecanoic acid 13C7-PFUnA 0.50 Perfluoro-n-[1,2-13C2]dodecanoic acid 13C2-PFDoA 0.50

a. Concentrations used during method development.

As additional isotopically labelled PFAS analogues become commercially available they may be integrated into the method provided they have similar functional groups as the method analytes or are isotopically labeled analogues of the method analytes. Method modification QC requirements must be met (Sect. 9.3) whenever new analogues are proposed.

7.16.1 Isotope Dilution Analogue PDS

Prepare the isotope dilution analogue PDS in methanol and add sodium hydroxide if not already present to prevent esterification as described in Section 7.13.1. The PDS concentrations used during method development are listed in the table above. Method performance data were collected using 20 µL of this PDS to yield concentrations of 40–160 ng/L in the 250 mL aqueous samples. Note that the concentrations of sulfonates in the isotope dilution analogue PDS is based on the weight of the salt. It is not necessary to account for difference in the formula weight of the salt compared to the free acid for sample quantitation.

7.17 Analyte Standard Materials

Analyte standards may be purchased as certified standard solutions or prepared from neat materials of assayed purity. If available, the method analytes should be purchased as technical-grade (as defined in Sect. 3.22) to ensure that linear and branched isomers are represented. Standards or neat materials that contain only the linear isomer can be substituted if technical-grade analytes are not available as quantitative standards.

During method development, analyte standards were obtained from AccuStandard, Inc. (New Haven, CT), Absolute Standards (Hamden, CT), Wellington Laboratories (Guelph, Ontario, Canada), Santa Cruz Biotechnology (Dallas, TX), and Synquest Laboratories, Inc. (Alachua, FL). Stock standards are made by dilution in methanol containing 4 mole equivalents of sodium hydroxide as described in Section 7.13.1

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7.17.1 PFOA

A quantitative standard for PFOA is currently available only for the linear isomer; however, a technical-grade standard (Sect. 3.22) is available for PFOA that contains the linear and branched isomers (Wellington Labs, Cat. No. T-PFOA, or equivalent). This product or a similar technical-grade PFOA standard must be used to identify the retention times of the branched and linear PFOA isomers.

However, the linear-only PFOA standard must be used for quantitation until a quantitative PFOA standard containing the branched and linear isomers becomes commercially available.

7.17.2 PFHxS and PFOS

Technical grade, quantitative PFHxS and PFOS standards containing branched and linear isomers must be used when available.

7.17.3 Correction for Analytes Obtained in the Salt Form

This method measures all forms of the analytes as anions while the identity of the counterion is inconsequential. Analytes may be commercially available as neat materials or as certified stock standards as their corresponding ammonium, sodium, or potassium salts. These salts are acceptable standards provided the measured mass, or concentration, is corrected for the salt content. The equation for this correction is provided below.

𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚(𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓) = 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚(𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓) ×

𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀

𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀

7.17.4 Analyte PDS

The analyte PDS is used to prepare the calibration standards and to fortify the LFBs, LFSMs and LFSMDs with the method analytes. Prepare the analyte PDS by combining and diluting the analyte stock standards in 100% methanol and add sodium hydroxide if not already present to prevent esterification as described in Section 7.13.1. Select nominal analyte concentrations for the PDS such that between 5 and 100 µL of the PDS is used to fortify samples and prepare standard solutions. More than one PDS concentration may be necessary to meet this requirement. During method development, the analyte PDS was prepared at an identical concentration for all analytes, 0.5 ng/µL. The user may modify the concentrations of the individual analytes based on the confirmed MRLs and the desired monitoring range. If the PDS is stored cold, warm the vials to room temperature and vortex prior to use.

7.17.5 Calibration Standards

Prepare a series of calibration standards of at least five levels by diluting the analyte PDS into methanol containing 20% reagent water. The lowest calibration standard must be at or below the MRL for each analyte. The calibration standards may also be used as Continuing Calibration Checks (CCCs). Using the PDS solutions, add a constant amount of the isotope performance standards and the isotope dilution analogues to each calibration standard. The concentration of the isotope dilution analogues should match the concentration of the analogues in sample extracts, assuming 100% recovery through the extraction process. During method development, the concentrations of the isotope dilution analogues were 40 ng/mL extract concentration (160 ng/L in the aqueous sample) for 4:2FTS, 6:2FTS and 8:2FTS, and 10 ng/mL (40 ng/L) for all others. The analyte calibration ranged from approximately 0.50 ng/mL to 25 ng/mL extract concentration.

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8 Sample Collection, Preservation, and Storage

8.1 Sample Bottles

Samples must be collected in plastic bottles: polypropylene bottles fitted with polypropylene screw-caps, or polyethylene bottles with polypropylene screw caps. Discard sample bottles after a single use.

The bottle volume should approximate the volume of the sample. Subsampling from a single bottle is not permitted except as described in Section 12.5.

8.2 Sample Preservation

Based on sample volume, add ammonium acetate to each sample bottle as a solid (prior to shipment to the field or immediately prior to sample collection) to achieve a 1g/L concentration of ammonium acetate. Ammonium acetate will sequester free chlorine to form chloramine.

8.3 Sample Collection

8.3.1 Precautions against Contamination

Workers must wash their hands before sampling and wear nitrile gloves while filling and sealing the sample bottles. Users should seek to minimize accidental contamination of the samples.

8.3.2 Collection Procedure

Open the tap and allow the system to flush until the water temperature has stabilized. Collect samples from the flowing system. Samples do not need to be collected headspace free. After collecting the sample, cap the bottle and agitate by hand until the preservative is dissolved. Keep the sample sealed from time of collection until extraction.

8.4 Field Reagent Blanks (FRB)

Each sample set must include an FRB. A sample set is defined as samples collected from the same site and at the same time. The same lot of preservative must be used for the FRBs as for the field samples.

8.4.1 Analysis of Reagent Water used for FRBs

Reagent water used for the FRBs must be analyzed prior to shipment to ensure the water has minimal residual PFAS. Extract an LRB prepared with reagent water using the same lot of sample bottles destined for shipment to the sampling site and ensure that analyte concentrations are less than one-third the MRL, as described in Section 9.2.1. This will ensure that any significant contamination detected in the FRBs originated from exposure in the field.

8.4.2 Field Reagent Blank Procedure

In the laboratory, fill the FRB sample bottle with the analyzed reagent water (Sect. 8.4.1), then seal and ship to the sampling site with the sample bottles. For each FRB shipped, a second FRB sample bottle containing only preservative must also be shipped. At the sampling site, open the FRB bottle and pour the reagent water into the second sample bottle containing preservative; seal and label this bottle as the FRB with the date, time and location of the site.

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8.5 Sample Shipment and Storage

Samples must be shipped on ice. Samples are valid if any ice remains in the cooler when it is received at the laboratory or bottles are received within 2 days of collection and below 10 0C. Once at the laboratory, samples must be stored at or below 6 °C until extraction. Samples must not be frozen.

8.6 Sample and Extract Holding Times

Analyze samples as soon as possible. Samples must be extracted within 28 days of collection. Extracts are generally stored at room temperature and must be analyzed within 28 days after extraction.

9 Quality Control QC procedures include the IDC and ongoing QC requirements. This section describes each QC parameter, its required frequency, and the performance criteria that must be met in order to satisfy method objectives. The QC criteria discussed in the following sections are summarized in Table 16 and Table 17. These QC requirements are considered the minimum for an acceptable QC program.

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

9.1 Initial Demonstration of Capability

The IDC must be successfully performed prior to analyzing field samples. The IDC must be repeated if changes are made to analytical parameters not previously validated during the IDC. This may include, for example, changing the sample volume, selecting alternate quantitation ions, extending the calibration range, adding additional isotope performance standards, or adding additional isotope dilution analogues. Prior to conducting the IDC, the analyst must meet the calibration requirements outlined in Section 10. The same calibration range used during the IDC must be used for the analysis of field samples.

9.1.1 Demonstration of Low System Background

Analyze an LRB immediately after injecting the highest calibration standard in the selected calibration range. Confirm that the blank is free from contamination as defined in Section 9.2.1. If an automated extraction system is used, an LRB must be extracted on each port to fulfil this requirement.

9.1.2 Demonstration of Precision

Prepare, extract, and analyze seven replicate LFBs in a valid Extraction Batch (seven LFBs and an LRB).

Fortify…