Method for Analysis of PFAS in Pesticide Products Containing Non-ionic Surfactants and Non-volatile Oils Scope of Method and Application This method is for the analysis of poly- and per-fluorinated alkyl substances (PFAS) in pesticide formulations containing non-ionic surfactants and oil. It is based on a QuEChERS (Quick. Easy, Cheap, Effective, Rugged and Safe) extraction approach, followed by Solid Phase Extraction (SPE) cleanup to remove excess oily substances, and analysis using Liquid Chromatography- Tandem Mass Spectrometry (LC-MS/MS). This method is not applicable if formulations contain ionic surfactants (such as sodium lauryl sulfate, quaternary ammonium compounds, etc.) or only organic solvents/liquids (petroleum distillates, mineral oil, etc.). A different method Analysis of PFAS in Oily Matrix (epa.gov) can be used for pesticide products formulated in organic solvents/oils. Note: Due to the wide occurrence of PFAS in the environment, it is highly recommended to verify that all supplies and equipment are free of PFAS above the limit of detection. Certain PFAS compounds have been found in SPE cartridges, SPE manifold, and filters during the method development. 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. EPA has validated this method through the Analytical Chemistry Branch (ACB) of the Biological and Economic Analysis Division, Office of Pesticide Programs. Sample Preparation Solvents: • Milli-Q water • Ethyl acetate • Hexane • Methanol Materials: • QuEChERS salt mix (6 g MgSOVl .5 g NaCl) • Ammonium acetate • Solid Phase Extraction cartridge -Florisil 1 g/6 mL column • Polypropylene test tubes 15 and 50 mL Solutions: • Mobile phase A: Aqueous 20 mM ammonium acetate • Methanol/water (99/1, v/v) • Hexanes/ethyl acetate (9/1, v/v) ------- Standards: Equipment: Extraction Standard: Mixture of isotopically labeled PFAS standards, different from injection standards Injection Standard: Mixture of isotopically labeled PFAS standards, different from Extraction standards Native PFAS standard: Mixture of all the target PFAS compounds. Geno/Grinder or equivalent Centrifuge N-Evap or equivalent Sonicator Liquid chromatography/tandem mass spectrometry (LC-MS/MS) Extraction Procedure: 1. Weigh approximately 4 grams of pesticide products into 50 mL polypropylene centrifuge tubes. 2. For the procedural blank, transfer approximately 4 grams of Milli-Q water into a 50 mL tube. 3. For blank spikes and matrix spikes, weigh approximately 4 grams of Milli-Q water and pesticide product, respectively, into 50 mL tubes. 4. Add appropriate amount of "Extraction Standard" into each sample. 5. Add appropriate amount of spiking solution containing PFAS to spike samples. 6. Mix by vortexing or shaking and then let samples equilibrate after addition of PFAS standards for 15 minutes. 7. Add 5 mL of Milli-Q water and 25 mL of ethyl acetate to each sample. 8. Shake each sample on Geno/Grinder for 20 minutes at 1000 rpm. 9. Add QuEChERS salt mix (6 g MgS04/l .5 g NaCl) to each sample, shaking by hand to break all salt clumps. 10. Shake all samples on Geno/Grinder for 20 minutes at 1000 rpm, followed by centrifugation for 10 minutes at 4000 rpm. 11. Transfer 20 mL of organic supernatant to a new 50 mL centrifuge tube and concentrate to dryness under N2 flow at 50°C-60°C. Note: Some oil may remain after concentration depending on product formulation. 12. Add 20 mL of hexane/ethyl acetate (9/1, v/v) to the dried extracts and sonicate for 30 minutes, followed by a round of brief hand-shaking and then centrifugation at 4000 rpm for 10 minutes. 13. For solid precipitates: Decant entire supernatant into a new 50 mL tube. 14. For biphasic layers: Carefully transfer 20 mL of organic supernatant to a new 50 mL tube. 15. Concentrate samples as much as possible as in Step 11. Then combine with 5 mL of hexane/ethyl acetate (9/1, v/v) and proceed to SPE cleanup. 16. Attach Florisil SPEs to manifold and condition with 10 mL of methanol, followed by 10 mL of hexane/ethyl acetate (9/1, v/v). ------- 17. Load sample onto SPE, and wash with 10 mL of hexane/ethyl acetate (9/1, v/v). Do not let the column ran dry. 18. Place collection tubes under the manifold and elute samples with 10 mL of methanol. 19. For all samples: Add appropriate amounts of "Injection Standard" mixture to all solutions. 20. Concentrate all samples to dryness. Reconstitute with 1 mL of methanol/water (99/1, v/v). Note: If precipitate is visible in tube, centrifuge the tubes. 21. Transfer the solutions to LC vials for instrument analysis with LC-MS/MS. Sample Analysis and Procedure Calibration: • Prepare a calibration curve of at least 5 levels in the range of 0.02 - 20 ng/mL of "Native" compounds. • Each calibration point should also have "Extraction Standards" and "Injection Standards" at, for example, 0.50 ng/mL. Data Analysis Note: • Quantitation calculations are based on the response ratio of "Native PFAS" signal to "Extraction Standard" signal. • Matrix effects can be assessed by comparing responses of "Injection Standards" in samples and calibration sets. LC-MS/MS Specifications/Parameters Equipment: Mobile Phase A: Mobile Phase B: Flow Rate: Solvent Gradient: Total Run Time: Agilent 6470 LC-MS/MS or Equivalent Aqueous 20 mM Ammonium Acetate Methanol 0.400 mL/min 70% Mobile Phase A to 5% Mobile Phase A in 13 min. 26 minutes + 5 minutes Post Time Equilibration MS Operation Mode: Electrospray Negative Ionization (ESI) mode List of Analyzed PFAS Compounds Acronym Chemical Name Limits of Quantitation (ppb) Comments PFBA Perfluoro-n-butanoic acid 0.40 High background PFPeA Perfluoro-n-pentanoic acid 0.40 High background PFHxA Perfluoro-n-hexanoic acid 0.40 PFHpA Perfluoro-n-heptanoic acid 0.40 PFOA Perfluoro-n-octanoic acid 0.40 PFNA Perfluoro-n-nonanoic acid 0.40 PFDA Perfluoro-n-decanoic acid 0.40 PFUdA Perfluoro-n-undecanoic acid 0.40 PFDoA Perfluoro-n-dodecanoic acid 0.40 PFTrDA Perfluoro-n-tridecanoic acid 0.40 PFTeDA Perfluoro-n-tetradecanoic acid 0.40 ------- Acronym Chemical Name Limits of Quantitation (ppb) Comments PFHxDA Perfluoro-n-hexadecanoic acid 0.40 PFODA Perfluoro-n-octadecanoic acid 2.00 Low recovery PFPeS Perfluoro-1 -pentanesulfonate, Potassium Salt 0.40 PFHxS Perfluoro-1-hexanesulfonate, Sodium Salt 0.40 PFHpS Perfluoro-1-heptanesulfonate, Sodium Salt 0.40 PFOS Perfluoro-1-octanesulfonate, Sodium Salt 0.40 PFNS Perfluoro-1-nonanesulfonate, Sodium Salt 0.40 PFDS Perfluoro-1-decanesulfonate, Sodium Salt 0.40 PFDoS Perfluoro-1 -dodecanesulfonate, Sodium Salt 0.40 FOSAA Perfluoro -1 -octane sulfonamidioacetic acid 2.00 N-MeFOSAA N-methylperfluoro-1 - octanesulfonamidoacetic acid 0.40 N-EtFOSAA N-ethylperfluoro-1 -octanesulfonamidoacetic acid 2.00 11C1- PF30UDS 11 -chloroeicosafluoro-3 -oxaundecane-1 - sulfonate, Potassium Salt 0.40 9-CL-PF30NS 9-chlorohexadecafluoro-3 -oxanonane-1 - sulfonate, Potassium Salt 0.40 4:2 FTS 1H, 1H, 2H, 2H-perfluorohexanesulfonate, Sodium Salt 2.0 6:2 FTS 1H, 1H, 2H, 2H-perfluorooctanesulfonate, Sodium Salt 2.0 High background 8:2 FTS 1H, 1H, 2H, 2H-perfluorodecanesulfonate, Sodium Salt 0.40 ADONA Dodecafluoro -3H-4,8 -dioxanonanoate, Sodium Salt 0.40 Note: PFBA, PF] >eA, and 6:2 FTS have high background leve s in this procedure. PFO 3 A have low recovery by this extraction procedure. ------- |