Rapid Extraction of Low Concentrations of PFAS in Food

User Benefits Application News ◆ Validated method for 30 PFAS in seafood (shrimp) meeting all criteria of AOAC SMPR 2023.003 ◆ High precision, excellent recovery, low Limit of Quantification (LOQ) ◆ Simple and rapid extraction using QuEChERS LCMS -8060NX High Performance Liquid Chromatograph Mass Spectrometer Nexera series High Performance Liquid Chromatograph Determination of 30 PFAS in Seafood by Liquid Chromatography Triple Quadrupole Mass Spectrometry (LC-MS/MS) ■ Introduction Per-and polyfluorinated substances (PFAS) are a diverse group of man-made chemicals used in numerous products since the 1950s. PFAS can enter the food supply by contact in environmentally contaminated areas, during food processing, or exposure to packaging. Because PFAS have been linked to serious health effects, accurate methodology is needed. In this application news, we describe a single laboratory validation study with a rapid extraction of low concentrations of 30 PFAS in shrimp using the QuEChERS technique followed by analysis using the Shimadzu Nexera Liquid Chromatograph coupled to a Shimadzu LCMS-8060NX triple quadrupole mass spectrometer (Figure 1). We optimized the chromatography and instrument operating parameters to achieve excellent peak shape, separation, and sensitivity. Sensitivity was improved for 4 PFAS, PFOA, PFHxS, PFNA, and PFOS. In this study, we spiked samples at three concentrations in triplicate. For greater accuracy, standards were matrixmatched and extracted and spikes were quantified using the isotope dilution technique. Recovery and precision were compared to the requirements of AOAC SMPR 2023.003. In addition, we determined the Limit of Quantitation (LOQ) as the lowest concentration meeting accuracy and precision, ion ratio, retention time, and signal-to-noise ratio criteria of the qualifier ion. All recovery, precision, and LOQ’s met the acceptance criteria of the SMPR. The target analytes, their acronym, chemical abstract number, and experimentally determined LOQ are shown in Table 1. 1 Shimadzu Scientific Instruments, Inc. William Lipps1 , Toshiya Matsubara1 , Dominika Gruszecka1 Figure 1: Nexera and LCMS -8060NX. The ion focus design improves signal intensity with higher gas flows and higher effective temperatures. Analyte name Acronym CAS No. LOQ (ppb) Perfluorobutanoic acid PFBA 375-22-4 0.055 Perfluoropentanoic acid PFPeA 2706-90-3 0.0055 Perfluorohexanoic acid PFHxA 307-24-4 0.0055 Perfluoroheptanoic acid PFHpA 375-85-9 0.055 Perfluorooctanoic acid PFOA 335-67-1 0.0055 Perfluorononanoic acid PFNA 375-95-1 0.0055 Perfluorodecanoic acid PFDA 335-76-2 0.0055 Perfluoroundecanoic acid PFUnA 2058-94-8 0.0055 Perfluorododecanoic acid PFDoA 307-55-1 0.055 Table 1: PFAS Analytes, Acronyms, CAS No. and Method LOQ ■ Sample Preparation and Analysis Conditions Samples were prepared by spiking prepared shrimp samples in triplicate at three different concentrations with 30 native PFAS (Table 1) and 16 isotopically labeled internal standards. Calibration curves for use in the quantitative analysis were prepared using shrimp test portions spiked with concentrations of 0.001, 0.01, 0.10, 1.0, and 10.0 ng/g. Quantitation was carried out on additional shrimp samples spiked in triplicate at 0.0055, 0.055, 0.55, and 5.5 ng/g. Since standards were extracted in a shrimp matrix and carried through the same procedure, the final concentration of each PFAS in the sample can be calculated directly from the curve. Frozen shrimp were thawed at room temperature and then crushed in a grinder with dry ice for 30 seconds at 4000 rpm. The ground sample was stored in a freezer overnight to remove all the dry ice. Ten-gram portions were weighed, and 10 mL of acetonitrile was added. The samples were vortexed for 1 minute and a QuEChERS packet was added. The sample was shaken for 1 minute and then centrifuged for 5 minutes at 4000 rpm. An aliquot of the acetonitrile layer was transferred to a tube and diluted 5 times with PFAS-free reagent water. The sample was then passed through a weak anion exchange (WAX) Solid Phase Extraction (SPE) cartridge and the PFAS were eluted with basic methanol. For greater sensitivity, 2 mL of the extract was concentrated in 400 µL of a methanol-water mixture. A volume suitable to obtain the required sensitivity of the extract was injected onto a UHPLC system (Shimadzu Nexera). Adequate separation of all compounds was achieved in nine minutes. (Figure 2 chromatogram shows the separation of all peaks). For this study, Shimadzu evaluated 1984 different instrument settings, and 6 different column and gradient combinations, to achieve excellent peak shape and resolution between peaks, as well as to maximize the signal-to-noise ratio of PFOA, PFHxS, PFNA, and PFOS. Mass spectrometry was performed on a Shimadzu LCMS8060NX with heated electrospray ionization operated in negative mode. Specific compound MRM transitions and associated internal standards are listed in Table 2. Chromatography was adjusted to provide sufficient separation of PFOA from potential cholic acid interferences, and to provide baseline resolution of branched and linear isomers (Figure 3). Perfluorotridecanoic acid PFTrDA 72629-94-8 0.055 Perfluorotetradecanoic acid PFTeDA 376-06-7 0.055 Perfluorobutanesulfonic acid PFBS 375-73-5 0.055 Perfluoropentansulfonic acid PFPeS 2706-91-4 0.055 Perfluorohexanesulfonic acid PFHxS 355-46-4 0.0055 Perfluoroheptanesulfonic acid PFHpS 375-92-8 0.0055 Perfluorooctanesulfonic acid PFOS 1763-23-1 0.055 Perfluorononanesulfonic acid PFNS 68259-12-1 0.0055 Perfluorodecanesulfonic acid PFDS 335-77-3 0.0055 Perfluoroundecanesulfonic acid PFUnDS 749786-16-1 0.0055 Perfluorododecanesulfonic acid PFDoS 79780-39-5 0.0055 Perfluorotridecanesulfonic acid PFTrDS 791563-89-8 0.055 Perfluorooctanesulfonamide PFOSA 754-91-6 0.055 9-Chlorohexadecafluoro-3-oxanonane-1-sulfonic acid 9Cl-PF3ONS 756426-58-1 0.0055 11-Chloroeicosafluoro-3-oxaundecane-1-sulfonic acid 11Cl-PF3OUdS 763051-92-9 0.0055 Hexafluoropropylene oxide dimer acid HFPO-DA 13252-13-6 0.055 4,8-Dioxa-3H-perfluorononanoic acid DONA 919005-14-4 0.0055 1H,1H, 2H, 2H-Perfluorohexane sulfonic acid 4:2 FTS 757124-72-4 0.055 1H,1H, 2H, 2H-Perfluorooctane sulfonic acid 6:2 FTS 27619-97-2 0.55 1H,1H, 2H, 2H-Perfluorodecane sulfonic acid 8:2 FTS 39108-34-4 0.0055 1H,1H, 2H, 2H-Perfluorododecane sulfonic acid 10:2 FTS 120226-60-0 0.0055 Analyte Quantitation Ion Qualifier Ion Internal Standard PFBA 212.9 > 168.8 13C4-PFBA PFPeA 262.9 > 218.8 13C5-PFPeA PFHxA 313 > 268.8 313 > 118.9 13C5-PFHxA PFHpA 362.9 > 318.9 362.9 > 168.8 13C4-PFHpA PFOA 412.9 > 368.9 412.9 > 168.8 13C8-PFOA PFNA 462.9 > 418.5 462.9 > 218.6 13C9-PFNA PFDA 512.9 > 468.95 512.9 > 268.55 13C6-PFDA PFUnA 562.9 > 518.95 562.9 > 269 13C7-PFUnA PFDoA 612.9 > 268.6 612.9 > 168.6 13C7-PFUnA PFTrDA 663 > 618.6 663 > 168.5 13C7-PFUnA PFTeDA 713 > 669 713 > 168.5 13C7-PFUnA PFBS 298.8 > 79.8 298.8 > 98.9 13C5-PFHxA PFPeS 348.8 > 79.8 348.8 > 98.9 13C4-PFHpA PFHxS 398.8 > 79.8 398.8 > 98.9 13C3-PFHxS PFHpS 448.8 > 79.8 448.8 > 99 13C6-PFDA PFOS 498.8 > 79.8 498.8 > 98.9 13C8-PFOS PFNS 548.9 > 79.8 548.8 > 99 13C7-PFUnA PFDS 598.8 > 99 598.8 > 79.8 13C7-PFUnA PFUnDS 649 > 99 649 > 80 13C8-PFOS PFDoS 699 > 80 699 > 99 13C2-PFDoA PFTrDS 749 > 80 749 > 279.6 13C7-PFUnA PFOSA 498 > 78 498 > 477.95 13C8-PFOSA 9Cl-PF3ONS 530.8 > 350.8 532.9 > 352.95 13C8-PFOS 11Cl-PF3OUdS 631 > 451 632.9 > 452.95 13C7-PFUnA HFPO-DA 284.8 > 168.8 284.8 > 118.9 13C3-HFPO-DA DONA 376.9 > 250.8 376.9 > 84.9 13C4-PFHpA 4:2 FTS 326.9 > 80.9 326.9 > 306.8 13C2-4:2 FTS 6:2 FTS 426.9 > 80.8 426.9 > 406.9 13C2-6:2FTS 8:2 FTS 526.9 > 506.45 526.9 > 80.55 13C3-PFHxS 10:2 FTS 626.9 > 606.7 626.9 > 80.9 13C7-PFUnA Figure 2: Chromatogram of 0.55 µg/kg PFAS in shrimp matrix with separation of all peaks in nine minutes 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 min 0 250000 500000 750000 1000000 1250000 1500000 1750000 2000000 2250000 2500000 2750000 3000000 3250000 Figure 3: Separation of PFOS from Cholic Acids and baseline resolution between PFOS branched and linear isomers Table 2: MRM Transitions and Internal Standard Associations ■ Quantitative Analysis Calibration standards were processed the same as samples. A linear model not forced through zero isotopic dilution calibration in matrix-matched standards provided the best fit and best recoveries of analytes. Residuals of each point in the curve were ±25% of the expected value. Calibration curves for PFOA, PFHxS, PFNA, and PFOS are shown in Figures 3 – 6 respectively. Branched and linear isomers of PFHXS and PFOS were integrated together. Exact labeled analogs were used as isotope dilution standards, except in a few cases where we found that 13C2 suffered matrix interferences. In these cases, another noninterfering isotope was chosen. The analytes tested and the associated calibration isotopes are shown in Table 3. During the single laboratory validation, the shrimp matrix contained an unknown interference coeluting with the 13C3 -PFBS isotope so 13C5 -PFHxA was used instead. Figure 3: PFOA Calibration Curve Figure 4: PFHxS Calibration Curve Figure 5: PFNA Calibration Curve Figure 6: PFOS Calibration Curve Conc. (ng/ kg) %Accuracy 0.001 101 0.01 92.8 0.1 94.8 1 99.6 10 112 Conc. (ng/ kg) %Accuracy 0.001 102 0.01 82.4 0.1 97.0 1 102 10 116 Conc. (ng/ kg) %Accuracy 0.001 99.9 0.01 100 0.1 101 1 108 10 90.3 Conc. (ng/ kg) %Accuracy 0.001 99.6 0.01 105 0.1 89.4 1 93.4 10 112 Blank matrixes and at least three different concentrations ranging from below the SMPR required LOQ to approximately 100 times the estimated LOQ were analyzed in triplicate. Recovery and repeatability for each analyte at each concentration are given in Table 3. The LOQ for each analyte was estimated by spiking at concentrations at, or below, the required LOQs listed in SMPR-2023_003. The spiked samples were analyzed in triplicate and the mean and repeatability standard deviation were calculated. Then, the standard deviation was divided by the mean and multiplied by 100% to calculate the repeatability percent relative standard deviation (RSD). The LOQs for all matrices and compounds were determined using an Excel worksheet that compared each of the requirements of the SMPR including retention time, recovery, repeatability, S/N > 3 for the qualifier ion and an ion ratio of ±30%. PFBA, PFPeA, and PFOSA LOQ were set at the minimum concentration, meeting recovery and repeatability requirements and a S/N > 10. The lowest concentration to meet all the requirements of the SMPR was set as the LOQ. Figure 7 shows examples of the LOQ spike for PFHxS, PFNA, PFOA, and PFOS and their corresponding internal standards. Analyte Spike conc. (ppb) Average conc. (ppb) Standard Deviation %RSD Average Recovery (%) PFBA Blank 0.007 0.055 0.056 0.98 0.98 100.3 0.55 0.518 0.32 0.34 94.1 5.5 5.478 0.35 0.35 99.6 PFPeA Blank ND 0.0055 0.005 3.52 3.81 92.6 0.055 0.054 2.17 2.23 97.6 0.55 0.517 0.67 0.71 94.1 5.5 5.751 0.26 0.25 104.6 PFHxA Blank 0.001 0.0055 0.005 9.08 9.50 95.6 0.055 0.057 1.48 1.43 103.7 0.55 0.538 0.66 0.67 97.8 5.5 5.871 0.15 0.14 106.8 PFHpA Blank 0.001 0.0055 0.005 3.90 4.06 96.1 0.055 0.051 0.75 0.82 92.0 0.55 0.518 0.26 0.28 94.2 5.5 6.161 1.25 1.12 112.0 PFOA Blank ND 0.0055 0.006 6.16 5.89 104.6 0.055 0.053 0.67 0.69 95.8 0.55 0.543 1.59 1.61 98.8 5.5 6.303 0.67 0.58 114.6 PFNA Blank 0.000 0.0055 0.006 7.32 6.77 108.1 0.055 0.056 0.68 0.67 102.1 0.55 0.583 0.55 0.52 106.0 5.5 5.340 2.53 2.61 97.1 PFDA Blank 0.000 0.0055 0.006 4.45 4.38 101.6 0.055 0.056 0.72 0.71 101.9 0.55 0.572 0.86 0.83 104.0 5.5 5.827 2.00 1.89 105.9 PFUnA Blank ND 0.0055 0.007 6.07 4.92 123.3 0.055 0.056 2.29 2.27 100.9 0.55 0.579 1.03 0.98 105.2 5.5 5.789 1.91 1.82 105.3 PFDoA Blank 0.001 0.055 0.054 6.52 6.67 97.7 0.55 0.528 3.51 3.66 96.0 5.5 5.863 3.10 2.91 106.6 PFTrDA Blank ND 0.055 0.063 2.31 2.01 115.0 0.55 0.586 3.89 3.65 106.6 5.5 6.215 2.56 2.26 113.0 PFTeDA Blank 0.001 0.055 0.055 5.22 5.23 99.7 0.55 0.555 4.27 4.23 100.8 5.5 6.313 3.03 2.64 114.8 PFBS Blank 0.001 0.055 0.051 5.55 5.93 93.6 0.55 0.527 2.83 2.95 95.8 5.5 5.940 2.31 2.14 108.0 PFPeS Blank 0.002 0.0055 0.004 15.88 20.67 76.8 0.055 0.055 3.92 3.89 100.7 0.55 0.598 3.29 3.02 108.8 5.5 5.713 0.71 0.68 103.9 Table 3: Recovery and repeatability for each analyte at each spike concentration PFHxS Blank ND 0.0055 0.004 14.59 17.62 82.8 0.055 0.052 2.32 2.45 94.9 0.55 0.535 1.06 1.09 97.2 5.5 6.070 0.25 0.23 110.4 PFHpS Blank 0.001 0.0055 0.006 20.96 18.27 114.8 0.055 0.057 9.51 9.14 104.0 0.55 0.590 8.60 8.02 107.2 5.5 6.223 4.30 3.80 113.1 PFOS Blank 0.001 0.055 0.053 5.12 5.28 96.9 0.55 0.517 1.77 1.88 94.0 5.5 5.700 1.03 0.99 103.6 PFNS Blank 0.001 0.0055 0.006 5.17 4.42 117.0 0.055 0.052 3.68 3.86 95.1 0.55 0.526 3.20 3.35 95.6 5.5 6.117 4.19 3.76 111.2 PFDS Blank 0.000 0.0055 0.006 9.46 8.11 116.6 0.055 0.056 0.87 0.85 102.6 0.55 0.569 4.82 4.67 103.4 5.5 6.148 4.56 4.08 111.8 PFUnDS Blank ND 0.0055 0.004 15.95 19.84 80.4 0.055 0.056 5.75 5.66 101.5 0.55 0.594 0.61 0.56 107.9 5.5 5.614 0.85 0.84 102.1 PFDoS Blank 0.000 0.0055 0.007 14.15 11.90 118.9 0.055 0.056 5.90 5.80 101.7 0.55 0.607 4.68 4.24 110.3 5.5 5.949 2.90 2.68 108.2 PFTrDS Blank 0.000 0.0055 0.005 8.10 7.94 102.0 0.055 0.057 4.16 4.03 103.2 0.55 0.581 5.25 4.97 105.7 5.5 6.228 3.96 3.50 113.2 PFOSA Blank 0.001 0.0055 0.006 9.45 8.91 106.1 0.055 0.055 3.80 3.81 99.8 0.55 0.556 0.78 0.77 101.1 5.5 5.973 1.75 1.61 108.6 9Cl -PF3ONS Blank 0.000 0.0055 0.004 7.22 8.57 84.3 0.055 0.054 1.65 1.67 98.6 0.55 0.570 0.78 0.75 103.6 5.5 5.896 2.62 2.44 107.2 11Cl -PF3OUdS Blank 0.001 0.0055 0.006 5.66 5.06 112.0 0.055 0.054 1.81 1.85 97.8 0.55 0.546 3.03 3.06 99.2 5.5 6.022 3.61 3.30 109.5 HFPO -DA Blank 0.002 0.0055 0.005 6.55 6.59 99.4 0.055 0.053 2.17 2.24 96.9 0.55 0.517 3.93 4.18 94.0 5.5 5.653 0.95 0.93 102.8 DONA Blank 0.000 0.0055 0.005 2.04 2.07 98.7 0.055 0.060 3.96 3.59 110.1 0.55 0.619 2.37 2.10 112.6 5.5 5.714 1.95 1.87 103.9 4:2 FTS Blank ND 0.055 0.052 1.92 2.05 93.6 0.55 0.535 0.35 0.36 97.2 5.5 5.663 0.80 0.78 103.0 6:2 FTS Blank 0.018 0.055 0.048 4.12 4.66 88.4 0.55 0.495 1.60 1.78 90.0 5.5 5.290 1.26 1.31 96.2 8:2 FTS Blank 0.000 0.0055 0.005 5.61 6.54 85.8 0.055 0.060 1.83 1.67 110.1 0.55 0.609 3.59 3.25 110.6 5.5 5.790 0.83 0.79 105.3 10:2 FTS Blank 0.000 0.0055 0.006 13.17 12.02 109.5 0.055 0.065 2.75 2.34 117.8 0.55 0.641 2.69 2.31 116.6 5.5 6.102 4.75 4.28 110.9 ND = average results less than zero Figure 7: LOQ Peaks with Internal Standards 4.25 4.50 4.75 5.00 0 5000 10000 15000 20000 25000 20:412.90>168.80(-) 20:412.90>368.90(-) 4.25 4.50 4.75 5.00 0 250000 500000 750000 19:421.00>375.90(-) 4.75 5.00 5.25 5.50 0 500 1000 1500 2000 22:398.80>98.90(-) 22:398.80>79.80(-) 4.75 5.00 5.25 5.50 0 25000 50000 75000 100000 125000 150000 21:402.00>79.80(-) 5.00 5.25 5.50 5.75 0 10000 20000 30000 40000 50000 60000 24:462.90>218.60(-) 24:462.90>418.50(-) 5.00 5.25 5.50 5.75 0 100000 200000 300000 400000 23:472.00>426.90(-) 5.75 6.00 6.25 6.50 0 2500 5000 7500 10000 30:498.80>98.90(-) 30:498.80>79.80(-) 5.75 6.00 6.25 6.50 0 25000 50000 75000 100000 125000 150000 31:507.00>79.80(-) PFOA PFHxS PFNA PFOS ■ Conclusion The Shimadzu LCMS-8060NX Triple Quadrupole Mass Spectrometer coupled with a Shimadzu Nexera Liquid Chromatograph was used in a single laboratory study to measure 30 PFAS compounds in a shrimp matrix and compared to criteria set by AOAC SMPR 2023.003. Chromatography conditions and the mass spectrometer were optimized to achieve excellent separation of all analytes, baseline resolution between linear and branched isomers, and a two-minute separation between PFOS and potentially interfering cholic acids. Precision and recovery and the experimentally determined LOQ are well within the requirements of the SMPR. ■ Reference 1) AOAC SMPR 2023.003 © Shimadzu Scientific Instruments, 2024 First Edition: May2024 SHIMADZU SCIENTIFIC INSTRUMENTS, INC. Applications Laboratory 7102 Riverwood Drive, Columbia, MD 21045 Phone: 800-477-1227 Fax: 410-381-1222 www.ssi.shimadzu.com Shimadzu Corporation www.shimadzu.com/an/ Founded in 1875, Shimadzu Corporation, a leader in the development of advanced technologies, has a distinguished history of innovation built on the foundation of contributing to society through science and technology. 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