The Latest Analytical Techniques for PFAS Monitoring

Untargeted PFAS identification and targeted PFAS library screening workflows for groundwater analysis using a QTOF mass spectrometer Ethan Hain, Om Shrestha, Kathleen Luo, Christopher Gilles, Evelyn Wang, Xiaomeng Xia, Robert English, Tiffany Liden Shimadzu Scientific Instruments, 7102 Riverwood Dr., Columbia, MD 21046 U.S.A. WP 304 Table of LC and LCMS parameters Introduction Per- and poly-fluoroalkylated substances (PFAS) have garnered increasing regulatory and public health interest as a result of the widespread occurrence and validated toxicity of these emerging contaminants. Although there are several specific PFAS that are currently being regulated on a federal and state level, there are potentially thousands of different PFAS that may exist in the environment. The toxicity associated with each of these individual PFAS will drive regulations; therefore, the identification of novel PFAS is a critical primary step in documenting the need for individual PFAS regulations. Once novel PFAS are characterized, these analytes of interest can be quickly identified in environmental matrices (e.g., groundwater) through targeted screening for easier monitoring. Structures of PFAS studied. A neat standard and spiked groundwater sample containing the 40 PFAS from Environmental Protection Agency (EPA) Method 1633 were chromatographically separated using a Shim-pack Scepter C18-120 column (2.1 × 100 mm; 3 μm) with a Shim-pack Scepter C18-120 delay column (2.1 × 50 mm; 3 μm) and mobile phases of 5 mM ammonium acetate in water and methanol (no additives) at a flow rate of 0.25 mL/min. Data were acquired on a Shimadzu LCMS-9030 quadrupole time-of-flight mass spectrometer using a negative mode MS scan ranging from 40-950 m/z, targeted MS/MS scans based off of target analyte m/z, and data-independent acquisition (DIA) MS/MS scans of a variable precursor isolation width and collision energy spread of 5-55 V. LabSolutions and LabSolutions Insight Explore software were used to obtain the data and perform data analyses. Methods Conclusions The 40 PFAS from EPAM1633 were used to generate a library from a single mixture, and then were confirmed to function for targeted library screening and untargeted identification in groundwater using the Insight Explore Assign function. Disclaimer: All content contained herein resulted solely from Shimadzu, and no conflict of interest exists. The products and applications are intended for Research Use Only (RUO). Not for use in diagnostic procedures. Nexera LC LCMS-9030 Flow Rate: 0.25 mL/min Nebulizing Gas: 3 L/min Oven Temp.: 45 °C Drying Gas: 5 L/min Injection Vol.: 1 µL Heating Gas: 15 L/min Mobile Phase: A: 5 mM Ammonium acetate in water B: Methanol Desolvation Temp.: 160 °C Delay Column: Shim-pack Scepter C18-120 (2.1 × 50 mm; 3 μm) DL Temp.: 150 °C Analytical Column: Shim-pack Scepter C18-120 (2.1 × 100 mm; 3 μm) Heat Block Temp.: 250 °C Time Course: 0 min %B = 5; 1 min %B = 40; 8 min %B = 95; 8.1 min %B = 100; 13 min %B = 100; 13.1 min %B = 5; 18 min %B = 5 Interface Temp: 100 °C Probe Position: +1 mm Interface: ESI Name Formula Theoretical m/z Acquired m/z Mass Error (ppm) Found RT RT Diff. Lib. SI 11Cl-PF3OUdS C10HClF20O4S 630.8892 630.8876 -2.504 8.726 0.04 100 3:3 FTCA C6H5F7O2 241.0105 241.0095 -4.025 5.143 0.02 94 4:2 FTS C6H5F9O3S 326.9743 326.9732 -3.272 5.909 0.03 94 5:3 FTCA C8H5F11O2 341.0041 341.0034 -2.023 6.847 0.03 97 6:2 FTS C8H5F13O3S 426.9679 426.9670 -2.084 7.288 0.05 100 7:3 FTCA C10H5F15O2 440.9977 440.9967 -2.449 7.966 0.04 98 8:2 FTS C10H5F17O3S 526.9615 526.9604 -2.220 8.235 0.03 99 9Cl-PF3ONS C8HClF16O4S 530.8956 530.8945 -1.997 8.011 0.04 94 ADONA C7H2F12O4 376.9689 376.9680 -2.334 6.779 0.03 100 FOSA C8H2F17NO2S 497.9462 497.9450 -2.510 8.638 0.02 100 HFPO-DA C6HF11O3 284.9779 284.9772 -2.421 6.221 0.04 97 NEtFOSA C10H6F17NO2S 525.9775 525.9761 -2.662 9.564 0.01 100 NEtFOSAA C12H8F17NO4S 583.9830 583.9816 -2.363 8.600 0.03 93 NEtFOSE C12H10F17NO3S 630.0249 630.0233 -2.492 9.533 0.01 100 NFDHA C5HF9O4 294.9658 294.9646 -4.272 5.852 0.03 97 NMeFOSA C9H4F17NO2S 511.9619 511.9608 -2.070 9.358 0.01 100 NMeFOSAA C11H6F17NO4S 569.9673 569.9660 -2.281 8.426 0.04 95 NMeFOSE C11H8F17NO3S 616.0092 616.0079 -2.143 9.332 0.01 100 PFBA C4HF7O2 212.9792 212.9785 -3.146 4.038 0.02 93 PFBS C4HF9O3S 298.9430 298.9424 -2.107 5.236 0.02 100 PFDA C10HF19O2 512.9600 512.9585 -3.100 8.220 0.04 89 PFDoA C12HF23O2 612.9537 612.9520 -2.773 8.909 0.05 90 PFDoS C12HF25O3S 698.9174 698.9158 -2.332 9.149 0.05 100 PFDS C10HF21O3S 598.9238 598.9225 -2.204 8.558 0.04 100 PFEESA C4HF9O4S 314.9379 314.9372 -2.350 5.602 0.03 99 PFHpA C7HF13O2 362.9696 362.9688 -2.369 6.704 0.04 92 PFHpS C7HF15O3S 448.9334 448.9325 -2.027 7.289 0.03 100 PFHxA C6HF11O2 312.9728 312.9720 -2.460 5.993 0.04 93 PFHxS C6HF13O3S 398.9366 398.9360 -1.404 6.707 0.03 100 PFMBA C5HF9O3 278.9709 278.9701 -2.904 5.400 0.03 94 PFMPA C4HF7O3 228.9741 228.9735 -2.489 4.456 0.02 100 PFNA C9HF17O2 462.9632 462.9620 -2.592 7.798 0.04 88 PFNS C9HF19O3S 548.9270 548.9256 -2.587 8.200 0.04 100 PFOA C8HF15O2 412.9664 412.9657 -1.768 7.291 0.04 97 PFOS C8HF17O3S 498.9302 498.9290 -2.465 7.779 0.04 100 PFPeA C5HF9O2 262.9760 262.9751 -3.422 5.112 0.03 92 PFPeS C5HF11O3S 348.9398 348.9388 -2.780 6.045 0.03 100 PFTeDA C14HF27O2 712.9473 712.9452 -2.875 9.433 0.06 98 PFTrDA C13HF25O2 662.9505 662.9491 -2.006 9.188 0.05 97 PFUnA C11HF21O2 562.9568 562.9553 -2.665 8.593 0.04 98 EPAM1633PFAS Library EPAM1633 PFAS spiked into groundwater (15.625-390 ppb) and identified by DIA. EPAM1633 PFAS were identified by targeted library screening and untargeted analysis. Sample information such as filenames, sample names, sample type, and flags are shown in the sample list. Compound details of the highlighted compound, including chromatogram, theoretical (top) and actual (bottom) MS and MS/MS spectrum and structure. The Assign function can be used to search ChemSpider or PubChem based off the acquired mass or a formula. Acquired MS/MS spectra are compared and fragments are assigned with relevant structures and formula. The compound table shows the name, formula, theoretical and acquired precursor m/z, mass error, many more compound specific parameters. Structure of the highlighted library compound. MS/MS spectrum of the highlighted library compound. Library information including CAS #, formula, RT, and CE spread. Analysis of Per- and Poly-fluoroalkylated Substances (PFAS) Specified in EPA Method 1633 Using Triple Quadrupole LC-MS/MS Om Shrestha, Ethan Hain, Kathleen Luo, Christopher Gilles, Evelyn Wang, Xiaomeng Xia, Robert English, Tiffany Liden , Samantha Olendorff Shimadzu Scientific Instruments, 7102 Riverwood Dr., Columbia, MD 21046 U.S.A. Disclaimer: All content contained herein resulted solely from Shimadzu, and no conflict of interest exists. The products and applications in this presentation are intended for Research Use Only (RUO). Not for use in diagnostic procedures. 2. Introduction The growing importance of per and poly-fluoroalkylated substances (PFAS) as a global public health threat is driving regulatory action. The EPA has developed methods for the measurement of PFAS in various matrices, such as Draft Method 1633 (EPA1633), which describes the analysis of forty PFAS in wastewater, solids, biosolids, and tissue samples. If wastewater becomes regulated, treatment utilities will be required to adopt methods such as EPA1633 which have been validated for analysis of PFAS in complex wastewater matrices. EPA1633 is currently in the third draft, which lists low detection and reporting limits for wastewater that may result in EPA establishing permit limits that will challenge existing water systems. Laboratories will need to measure at these low method reporting limits, and perhaps even lower to provide utilities a higher confidence in the results at these concentrations. 1. Overview A triple quadrupole mass spectrometer was used to quantify per- and polyfluoroalkylated substances (PFAS) to meet the requirements set by the EPA 1633 draft method. 4. Results LabSolutions Insight software was used to efficiently review and analyze the data (see Figure 3).The signalto-noise ratio for all PFAS at LOQ concentrations were above 10. The calibration range has the linearly of 0.99 or greater with %RSE of <20%. The IDL was calculated using 10 replicates of spiked ultrapure water samples. The theoretical MDL was calculated based on an assumption of 100% extraction efficiency and a concentration factor of 100X; these conditions may vary with laboratory environment, sample matrix, and analyst extraction proficiency. The Shimadzu LCMS-8060NX was not only able to meet EPA 1633 draft but was able to detect much lower concentrations. ThP 073 3. Method The instrument was calibrated according to the method using the Calibration Standards listed in Table 2 (Wellington PFAC) and verified to meet the performance criteria of EPA1633. The forty PFAS listed in EPA1633 were chromatographically separated on a Shim-pack Scepter C18 column (50 x 2.1 mm, 3 μm) by gradient elution using 2 mM ammonium acetate in water and acetonitrile (no additives) as the mobile phases at a flow rate of 0.4 mL/min (Figure 1). A Shimadzu GIST C18 column (50 × 3 mm, 5 µm) was used as a delay column to reduce the system PFAS interferences. Multiple reaction monitoring (MRM) analysis was performed on a Shimadzu LCMS-8060NX triple quadrupole mass spectrometer with neat standards of the forty PFAS listed in EPAM1633 ranging from 0.3 ppt to 625 ppb. LabSolutions and LabSolutions Insight software were used to obtain the data and perform data analyses. Acronym Precursor m/Z Ref.(1) m/z Ref.(2) m/z Ret. Time %RSE LOQ (ng/mL) EPA LOQ (ng/mL) IDL (ng/mL) n=10 Theoretic al MDL (ng/L) EPA Pooled MDL (ng/L) PFBA 212.98 168.90 N/A 2.16 11 0.0250 0.80 0.004 0.041 0.800 PFMPA 228.97 85.00 N/A 2.42 10 0.0125 0.40 0.053 0.526 0.540 3:3 FTCA 241.01 177.00 117.00 2.53 11 0.1248 1.00 0.053 0.526 2.540 PFPeA 262.98 219.00 N/A 2.91 9 0.0125 0.40 0.003 0.028 0.530 PFMBA 278.97 85.00 N/A 3.19 10 0.0250 0.40 0.003 0.025 0.530 4:2 FTS 326.97 307.00 80.90 3.50 10 0.1000 0.80 0.027 0.267 1.740 NFDHA 294.97 201.15 85.00 3.70 11 0.0063 0.40 0.005 0.049 1.920 PFHxA 312.97 269.00 119.10 3.80 11 0.0125 0.20 0.004 0.037 0.480 PFBS 298.94 80.10 99.10 3.88 12 0.0125 0.20 0.009 0.092 0.370 HFPO-DA 328.97 169.00 118.90 4.12 12 0.0125 0.80 0.007 0.067 1.540 5:3 FTCA 341.00 237.10 217.10 4.20 12 0.0780 5.00 0.055 0.552 9.920 PFEESA 314.94 134.85 N/A 4.34 10 0.0063 0.40 0.001 0.007 0.790 PFHpA 362.97 319.00 169.00 4.67 10 0.0125 0.20 0.002 0.023 0.390 PFPeS 348.94 80.00 99.00 4.87 14 0.0125 0.20 0.010 0.098 0.530 ADONA 376.97 250.90 84.80 4.98 12 0.0063 0.80 0.002 0.019 1.470 6:2 FTS 426.97 407.00 81.00 5.14 9 0.0250 0.80 0.013 0.132 2.520 PFHxS 398.94 80.10 99.10 5.47 11 0.0125 0.20 0.008 0.079 0.560 PFOA 412.97 369.00 169.00 5.48 11 0.0250 0.20 0.008 0.076 0.550 7:3 FTCA 441.00 317.00 337.00 5.83 11 0.0790 5.00 0.066 0.657 9.140 PFNA 462.96 418.90 219.00 6.25 11 0.0125 0.20 0.004 0.036 0.460 PFHpS 448.93 80.00 99.00 6.61 11 0.0125 0.20 0.010 0.095 0.870 8:2 FTS 526.96 506.95 81.05 6.63 12 0.1000 0.80 0.036 0.358 2.580 NMeFOSAA 569.97 483.00 419.00 6.95 15 0.0250 0.20 0.019 0.186 1.040 PFDA 512.96 469.00 269.05 7.00 16 0.0250 0.20 0.038 0.379 0.530 NEtFOSAA 583.98 418.95 482.95 7.25 19 0.0500 0.20 0.050 0.501 0.800 PFOS 498.93 80.00 99.05 7.40 10 0.0250 0.20 0.015 0.147 0.640 PFUnA 564.97 518.95 269.00 7.72 14 0.0250 0.20 0.007 0.069 0.440 9Cl-PF3ONS 530.90 351.00 83.10 7.96 10 0.0063 0.80 0.002 0.021 1.420 PFNS 548.93 80.05 99.00 8.15 11 0.0125 0.20 0.009 0.092 0.490 PFDoA 612.95 569.00 433.10 8.36 11 0.0125 0.20 0.007 0.074 0.370 PFOSA 497.95 78.05 168.90 8.53 15 0.0031 0.20 0.004 0.040 0.320 PFDS 598.92 80.05 98.95 8.63 11 0.0125 0.20 0.005 0.052 0.900 PFTrDA 662.95 619.00 168.95 8.74 10 0.0125 0.20 0.003 0.032 0.460 11Cl-PF3OUdS 630.89 451.00 452.95 8.87 12 0.0063 0.80 0.004 0.041 1.780 PFTeDA 712.95 668.90 169.00 9.02 11 0.0125 0.20 0.004 0.041 0.510 PFDoS 698.92 80.00 98.95 9.19 13 0.0125 0.20 0.006 0.060 0.640 NMeFOSE 555.99 58.95 N/A 9.26 12 0.0156 2.00 0.010 0.101 3.930 NMeFOSA 511.96 169.05 219.10 9.36 19 0.0250 0.20 0.032 0.317 0.410 NEtFOSE 570.00 59.05 N/A 9.45 10 0.0156 2.00 0.006 0.063 5.130 The signal-to-noise ratio (SNR) for CS1 was well above 10 for all analytes, easily meeting the method’s sensitivity requirements. To determine how much lower in concentration each of the instruments can detect, we serially diluted and analyzed CS1. PFAS were detected at or below the limits of quantitation (LOQ) established in EPA1633 in neat standards These data are shown in Table 2. Silanized glass vials and silicone/polyethylene polymer caps were used to hold PFAS standards which significantly reduced PFAS interferences compared to other materials. Shimadzu LabSolutions Insight LCMS software was used to quickly process the data and determine that the sensitivity would meet the performance criteria of EPA1633 (e.g., confirm the relative standard error and relative standard deviation were less than 20%). 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min 0 500000 1000000 1500000 2000000 2500000 3000000 LCMS-8060NX Parameters LC Parameters Ion source ESI Ion Focus Column Shimadzu Scepter C18, 2.1 × 50 mm, 3 µm Nebulizing gas 3.0 L/min Flow rate 0.4 mL/min Heating gas 15.0 L/min Mobile phase A 2mM Ammonium Acetate in Water Drying gas 5.0 L/min Mobile phase B Acetonitrile Interface Temperature 190 °C Injection volume 1 µL DL Temperature 200°C Column oven temperature 40 °C Heat Block Temperature 300°C Diluent Methanol with 4% water, 1% ammonium hydroxide and 0.625% acetic acid Table 1. Summary of LCMS method parameters Q 212.9000>168.9000 (-) 4.63e5 RT (min) 2.0 2.2 2.4 0.0e0 1.0e5 2.0e5 3.0e5 4.0e5 Conc.Ratio 0 100 200 Area Ratio 0 10 20 30 y = 0.1300997x + 0.007354381 R² = 0.9911610 R = 0.9955707 Q 241.0000>177.0000 (-) 2.40e5 RT (min) 2.2 2.4 2.6 0.0e0 5.0e4 1.0e5 1.5e5 2.0e5 Conc.Ratio 0 200 Area Ratio 0 1 2 3:3 FTCA y = 0.009562491x + 0.0001144136 R² = 0.9932052 R = 0.9965968 Q 327.0000>307.0000 (-) 7.11e4 RT (min) 3.2 3.4 3.6 0.0e0 2.0e4 4.0e4 6.0e4 Conc.Ratio 0 10 20 Area Ratio 0 2 4 4-2 FTS y = 0.2886572x + 0.01306672 R² = 0.9905024 R = 0.9952399 Q 285.0000>169.0000 (-) 2.99e6 RT (min) 3.8 4.0 4.2 0.0e0 1.0e6 2.0e6 Conc.Ratio 0 100 200 Area Ratio 0 10 20 30 40 HFPO-DA y = 0.1888084x + 0.001658244 R² = 0.9907273 R = 0.9953528 Q 412.9000>169.0000 (-) 4.76e5 RT (min) 5.2 5.4 0.0e0 1.0e5 2.0e5 3.0e5 4.0e5 Conc.Ratio 0 25 50 Area Ratio 0 5 10 PFOA y = 0.2150613x + 0.008414333 R² = 0.9915872 R = 0.9957847 Q 498.9500>80.0000 (-) 1.04e5 RT (min) 7.0 7.2 7.4 7.6 7.8 0.0e0 2.5e4 5.0e4 7.5e4 1.0e5 Conc.Ratio 0 25 50 Area Ratio 0 20 40 PFOS y = 0.8785163x + 0.02294125 R² = 0.9975630 R = 0.9987807 Q 398.9500>80.1000 (-) 8.19e4 RT (min) 5.0 5.2 5.4 5.6 5.8 0.0e0 2.0e4 4.0e4 6.0e4 8.0e4 Conc.Ratio 0 25 50 Area Ratio 0 5 10 15 20 PFHxS y = 0.3711037x + 0.001718047 R² = 0.9913654 R = 0.9956733 Q 298.9000>80.1000 (-) 6.16e5 RT (min) 3.6 3.8 4.0 0.0e0 2.0e5 4.0e5 6.0e5 Conc. Ratio 0 25 50 Area Ratio 0 10 20 30 PFBS y = 0.5829727x - 0.0001142251 R² = 0.9923528 R = 0.9961690 PFBA 3:3 FTCA 4-2 FTS HFPO-DA PFOA PFOS PFHxS PFBS 5. Conclusions The Shimadzu LCMS-8060NX was easily able to meet the LOQ requirements set by the EPA 1633 3rd draft method and Shimadzu LabSolutions Insight was used to efficiently process the acquired data. This processing software calculated %RSD, SNR, and %RSE as required by EPA 1633. Figure 1. Chromatograms of 1633 PFAS analytes with non- and extracted internal standards Figure 2. Examples of PFAS chromatograms and respective calibration curves Table 2. Summary of LCMS acquisition parameters and performance for EPAM1633 standards. Figure 3. Screenshot of EPAM1633 data in LabSolutions Insight software. Reference United States Environmental Protection Agency, Office of Water. 3rd Draft Method 1633 Analysis of Per- and Polyfluoroalkyl Substances (PFAS) in Aqueous, Solid, Biosolids, and Tissue Samples by LC/MS/MS, 2022. https://nepis.epa.gov/Exe/ZyPURL.cgi?Dockey=P101687F.txt (accessed June 1, 2023) Analysis of Per- and Poly-fluoroalkylated Substances (PFAS) Specified in EPA Method 1633 Using Triple Quadrupole LC-MS/MS Om Shrestha, Ethan Hain, Kathleen Luo, Christopher Gilles, Evelyn Wang, Xiaomeng Xia, Robert English, Tiffany Liden , Samantha Olendorff Shimadzu Scientific Instruments, 7102 Riverwood Dr., Columbia, MD 21046 U.S.A. Disclaimer: All content contained herein resulted solely from Shimadzu, and no conflict of interest exists. The products and applications in this presentation are intended for Research Use Only (RUO). Not for use in diagnostic procedures. 2. Introduction The growing importance of per and poly-fluoroalkylated substances (PFAS) as a global public health threat is driving regulatory action. The EPA has developed methods for the measurement of PFAS in various matrices, such as Draft Method 1633 (EPA1633), which describes the analysis of forty PFAS in wastewater, solids, biosolids, and tissue samples. If wastewater becomes regulated, treatment utilities will be required to adopt methods such as EPA1633 which have been validated for analysis of PFAS in complex wastewater matrices. EPA1633 is currently in the third draft, which lists low detection and reporting limits for wastewater that may result in EPA establishing permit limits that will challenge existing water systems. Laboratories will need to measure at these low method reporting limits, and perhaps even lower to provide utilities a higher confidence in the results at these concentrations. 1. Overview A triple quadrupole mass spectrometer was used to quantify per- and polyfluoroalkylated substances (PFAS) to meet the requirements set by the EPA 1633 draft method. 4. Results LabSolutions Insight software was used to efficiently review and analyze the data (see Figure 3).The signalto-noise ratio for all PFAS at LOQ concentrations were above 10. The calibration range has the linearly of 0.99 or greater with %RSE of <20%. The IDL was calculated using 10 replicates of spiked ultrapure water samples. The theoretical MDL was calculated based on an assumption of 100% extraction efficiency and a concentration factor of 100X; these conditions may vary with laboratory environment, sample matrix, and analyst extraction proficiency. The Shimadzu LCMS-8060NX was not only able to meet EPA 1633 draft but was able to detect much lower concentrations. ThP 073 3. Method The instrument was calibrated according to the method using the Calibration Standards listed in Table 2 (Wellington PFAC) and verified to meet the performance criteria of EPA1633. The forty PFAS listed in EPA1633 were chromatographically separated on a Shim-pack Scepter C18 column (50 x 2.1 mm, 3 μm) by gradient elution using 2 mM ammonium acetate in water and acetonitrile (no additives) as the mobile phases at a flow rate of 0.4 mL/min (Figure 1). A Shimadzu GIST C18 column (50 × 3 mm, 5 µm) was used as a delay column to reduce the system PFAS interferences. Multiple reaction monitoring (MRM) analysis was performed on a Shimadzu LCMS-8060NX triple quadrupole mass spectrometer with neat standards of the forty PFAS listed in EPAM1633 ranging from 0.3 ppt to 625 ppb. LabSolutions and LabSolutions Insight software were used to obtain the data and perform data analyses. Acronym Precursor m/Z Ref.(1) m/z Ref.(2) m/z Ret. Time %RSE LOQ (ng/mL) EPA LOQ (ng/mL) IDL (ng/mL) n=10 Theoretic al MDL (ng/L) EPA Pooled MDL (ng/L) PFBA 212.98 168.90 N/A 2.16 11 0.0250 0.80 0.004 0.041 0.800 PFMPA 228.97 85.00 N/A 2.42 10 0.0125 0.40 0.053 0.526 0.540 3:3 FTCA 241.01 177.00 117.00 2.53 11 0.1248 1.00 0.053 0.526 2.540 PFPeA 262.98 219.00 N/A 2.91 9 0.0125 0.40 0.003 0.028 0.530 PFMBA 278.97 85.00 N/A 3.19 10 0.0250 0.40 0.003 0.025 0.530 4:2 FTS 326.97 307.00 80.90 3.50 10 0.1000 0.80 0.027 0.267 1.740 NFDHA 294.97 201.15 85.00 3.70 11 0.0063 0.40 0.005 0.049 1.920 PFHxA 312.97 269.00 119.10 3.80 11 0.0125 0.20 0.004 0.037 0.480 PFBS 298.94 80.10 99.10 3.88 12 0.0125 0.20 0.009 0.092 0.370 HFPO-DA 328.97 169.00 118.90 4.12 12 0.0125 0.80 0.007 0.067 1.540 5:3 FTCA 341.00 237.10 217.10 4.20 12 0.0780 5.00 0.055 0.552 9.920 PFEESA 314.94 134.85 N/A 4.34 10 0.0063 0.40 0.001 0.007 0.790 PFHpA 362.97 319.00 169.00 4.67 10 0.0125 0.20 0.002 0.023 0.390 PFPeS 348.94 80.00 99.00 4.87 14 0.0125 0.20 0.010 0.098 0.530 ADONA 376.97 250.90 84.80 4.98 12 0.0063 0.80 0.002 0.019 1.470 6:2 FTS 426.97 407.00 81.00 5.14 9 0.0250 0.80 0.013 0.132 2.520 PFHxS 398.94 80.10 99.10 5.47 11 0.0125 0.20 0.008 0.079 0.560 PFOA 412.97 369.00 169.00 5.48 11 0.0250 0.20 0.008 0.076 0.550 7:3 FTCA 441.00 317.00 337.00 5.83 11 0.0790 5.00 0.066 0.657 9.140 PFNA 462.96 418.90 219.00 6.25 11 0.0125 0.20 0.004 0.036 0.460 PFHpS 448.93 80.00 99.00 6.61 11 0.0125 0.20 0.010 0.095 0.870 8:2 FTS 526.96 506.95 81.05 6.63 12 0.1000 0.80 0.036 0.358 2.580 NMeFOSAA 569.97 483.00 419.00 6.95 15 0.0250 0.20 0.019 0.186 1.040 PFDA 512.96 469.00 269.05 7.00 16 0.0250 0.20 0.038 0.379 0.530 NEtFOSAA 583.98 418.95 482.95 7.25 19 0.0500 0.20 0.050 0.501 0.800 PFOS 498.93 80.00 99.05 7.40 10 0.0250 0.20 0.015 0.147 0.640 PFUnA 564.97 518.95 269.00 7.72 14 0.0250 0.20 0.007 0.069 0.440 9Cl-PF3ONS 530.90 351.00 83.10 7.96 10 0.0063 0.80 0.002 0.021 1.420 PFNS 548.93 80.05 99.00 8.15 11 0.0125 0.20 0.009 0.092 0.490 PFDoA 612.95 569.00 433.10 8.36 11 0.0125 0.20 0.007 0.074 0.370 PFOSA 497.95 78.05 168.90 8.53 15 0.0031 0.20 0.004 0.040 0.320 PFDS 598.92 80.05 98.95 8.63 11 0.0125 0.20 0.005 0.052 0.900 PFTrDA 662.95 619.00 168.95 8.74 10 0.0125 0.20 0.003 0.032 0.460 11Cl-PF3OUdS 630.89 451.00 452.95 8.87 12 0.0063 0.80 0.004 0.041 1.780 PFTeDA 712.95 668.90 169.00 9.02 11 0.0125 0.20 0.004 0.041 0.510 PFDoS 698.92 80.00 98.95 9.19 13 0.0125 0.20 0.006 0.060 0.640 NMeFOSE 555.99 58.95 N/A 9.26 12 0.0156 2.00 0.010 0.101 3.930 NMeFOSA 511.96 169.05 219.10 9.36 19 0.0250 0.20 0.032 0.317 0.410 NEtFOSE 570.00 59.05 N/A 9.45 10 0.0156 2.00 0.006 0.063 5.130 The signal-to-noise ratio (SNR) for CS1 was well above 10 for all analytes, easily meeting the method’s sensitivity requirements. To determine how much lower in concentration each of the instruments can detect, we serially diluted and analyzed CS1. PFAS were detected at or below the limits of quantitation (LOQ) established in EPA1633 in neat standards These data are shown in Table 2. Silanized glass vials and silicone/polyethylene polymer caps were used to hold PFAS standards which significantly reduced PFAS interferences compared to other materials. Shimadzu LabSolutions Insight LCMS software was used to quickly process the data and determine that the sensitivity would meet the performance criteria of EPA1633 (e.g., confirm the relative standard error and relative standard deviation were less than 20%). 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min 0 500000 1000000 1500000 2000000 2500000 3000000 LCMS-8060NX Parameters LC Parameters Ion source ESI Ion Focus Column Shimadzu Scepter C18, 2.1 × 50 mm, 3 µm Nebulizing gas 3.0 L/min Flow rate 0.4 mL/min Heating gas 15.0 L/min Mobile phase A 2mM Ammonium Acetate in Water Drying gas 5.0 L/min Mobile phase B Acetonitrile Interface Temperature 190 °C Injection volume 1 µL DL Temperature 200°C Column oven temperature 40 °C Heat Block Temperature 300°C Diluent Methanol with 4% water, 1% ammonium hydroxide and 0.625% acetic acid Table 1. Summary of LCMS method parameters Q 212.9000>168.9000 (-) 4.63e5 RT (min) 2.0 2.2 2.4 0.0e0 1.0e5 2.0e5 3.0e5 4.0e5 Conc.Ratio 0 100 200 Area Ratio 0 10 20 30 y = 0.1300997x + 0.007354381 R² = 0.9911610 R = 0.9955707 Q 241.0000>177.0000 (-) 2.40e5 RT (min) 2.2 2.4 2.6 0.0e0 5.0e4 1.0e5 1.5e5 2.0e5 Conc.Ratio 0 200 Area Ratio 0 1 2 3:3 FTCA y = 0.009562491x + 0.0001144136 R² = 0.9932052 R = 0.9965968 Q 327.0000>307.0000 (-) 7.11e4 RT (min) 3.2 3.4 3.6 0.0e0 2.0e4 4.0e4 6.0e4 Conc.Ratio 0 10 20 Area Ratio 0 2 4 4-2 FTS y = 0.2886572x + 0.01306672 R² = 0.9905024 R = 0.9952399 Q 285.0000>169.0000 (-) 2.99e6 RT (min) 3.8 4.0 4.2 0.0e0 1.0e6 2.0e6 Conc.Ratio 0 100 200 Area Ratio 0 10 20 30 40 HFPO-DA y = 0.1888084x + 0.001658244 R² = 0.9907273 R = 0.9953528 Q 412.9000>169.0000 (-) 4.76e5 RT (min) 5.2 5.4 0.0e0 1.0e5 2.0e5 3.0e5 4.0e5 Conc.Ratio 0 25 50 Area Ratio 0 5 10 PFOA y = 0.2150613x + 0.008414333 R² = 0.9915872 R = 0.9957847 Q 498.9500>80.0000 (-) 1.04e5 RT (min) 7.0 7.2 7.4 7.6 7.8 0.0e0 2.5e4 5.0e4 7.5e4 1.0e5 Conc.Ratio 0 25 50 Area Ratio 0 20 40 PFOS y = 0.8785163x + 0.02294125 R² = 0.9975630 R = 0.9987807 Q 398.9500>80.1000 (-) 8.19e4 RT (min) 5.0 5.2 5.4 5.6 5.8 0.0e0 2.0e4 4.0e4 6.0e4 8.0e4 Conc.Ratio 0 25 50 Area Ratio 0 5 10 15 20 PFHxS y = 0.3711037x + 0.001718047 R² = 0.9913654 R = 0.9956733 Q 298.9000>80.1000 (-) 6.16e5 RT (min) 3.6 3.8 4.0 0.0e0 2.0e5 4.0e5 6.0e5 Conc. Ratio 0 25 50 Area Ratio 0 10 20 30 PFBS y = 0.5829727x - 0.0001142251 R² = 0.9923528 R = 0.9961690 PFBA 3:3 FTCA 4-2 FTS HFPO-DA PFOA PFOS PFHxS PFBS 5. Conclusions The Shimadzu LCMS-8060NX was easily able to meet the LOQ requirements set by the EPA 1633 3rd draft method and Shimadzu LabSolutions Insight was used to efficiently process the acquired data. This processing software calculated %RSD, SNR, and %RSE as required by EPA 1633. Figure 1. Chromatograms of 1633 PFAS analytes with non- and extracted internal standards Figure 2. Examples of PFAS chromatograms and respective calibration curves Table 2. Summary of LCMS acquisition parameters and performance for EPAM1633 standards. Figure 3. Screenshot of EPAM1633 data in LabSolutions Insight software. Reference United States Environmental Protection Agency, Office of Water. 3rd Draft Method 1633 Analysis of Per- and Polyfluoroalkyl Substances (PFAS) in Aqueous, Solid, Biosolids, and Tissue Samples by LC/MS/MS, 2022. https://nepis.epa.gov/Exe/ZyPURL.cgi?Dockey=P101687F.txt (accessed June 1, 2023)