Optimized Methods for Sensitive and Robust PFAS Analysis of Wastewater

User Benefits Application News ◆ Reproducible results can be achieved with the Shimadzu LCMS 8060NX for the analysis of wastewater according to EPA Method 1633. ◆ Achieve quantification 10x lower than the EPA’s Limit of Quantitation (LOQ). ◆ The excellent sensitivity achieved enables laboratories to reoptimize their sample preparation approach (i.e. reduce sample volume) while ensuring performance as required in EPA 1633 LCMS -8060NX High Performance Liquid Chromatograph Mass Spectrometer EPA Method 1633: Method Detection Limits of Perand Polyfluoroalkyl Substances (PFAS) in Aqueous Matrices using the Triple Quad LCMS-8060NX Megan Davis, Om Shrestha, Kathleen Lou, Ruth Marfil-Vega, Landon Wiest, Evelyn Wang Shimadzu Scientific Instruments, Inc. ■ Introduction This application note demonstrates that the LCMS8060NX meets and exceeds the method detection limits, required by the Environmental Protection Agency (EPA) in Method 1633 for aqueous matrices.1 All 40 Per- and Polyfluoroalkyl Substances (PFAS) compounds were successfully quantified at concentrations 10x lower than the Limit of Quantitation (LOQ). This improved sensitivity allows laboratories to minimize operational cost by decreasing the volume of sample that needs to be collected, shipped, and extracted. ■ Method Overview This application details the analysis of 40 native target PFAS compounds extracted from aqueous matrix along with 23 extracted internal standards (EIS), and 7 nonextracted internal standards (NIS). Stock standards were purchased from Wellington Laboratories as a series of native and mass-labelled PFAS mixtures in methanol (PFAC-MXF, PFAC-MXG, PFAC-MXH, PFAC-MXI, PFACMXJ, MPFAC-HIFES, and MPFAC-HIF-IS). Three spiking standards was made containing the native targets, EIS, and NIS compounds by diluting the stock solutions in methanol. The calibration curve was made by preparing methanol with 4% water, 1% ammonium hydroxide, and 0.625% acetic acid. The stock standards were then diluted to make a curve that ranged from 0.025 to 10.0 μg/L for PFBA, 1.0 to 20.0 μg/L for EIS, and 1.0 to 4.0 μg/L for NIS. All standards were prepared for analysis in 200 μL silanized glass inserts in 1.5 mL amber silanized glass vials a with PE/Silicone blue screw caps. Type Name Type Name Target PFBA Target NMeFOSE Target PFMPA Target NMeFOSA Target 3:3 FTCA Target NEtFOSE Target PFPeA Target NEtFOSA Target PFMBA EIS 13C4-PFBA Target 4-2 FTS EIS 13C5-PFPeA Target NFDHA EIS 13C2-4:2 FTS Target PFHxA EIS 13C5-PFHxA Target PFBS EIS 13C3-PFBS Target HFPO-DA EIS 13C3-HFPO-DA Target 5:3 FTCA EIS 13C4-PFHpA Target PFEESA EIS 13C2-6:2FTS Target PFHpA EIS 13C8-PFOA Target PFPeS EIS 13C3-PFHxS Target ADONA EIS 13C9-PFNA Target 6-2 FTS EIS 13C2-8:2FTS Target PFOA EIS D3-NMeFOSAA Target PFHxS EIS 13C6-PFDA Target 7:3 FTCA EIS D5-NEtFOSAA Target PFNA EIS 13C8-PFOS Target PFHpS EIS 13C7-PFUnA Target 8-2 FTS EIS 13C2-PFDoA Target NMeFOSAA EIS 13C8-PFOSA Target PFDA EIS 13C2-PFTeDA Target NEtFOSAA EIS D7-NMeFOSE Target PFOS EIS D3-NMeFOSA Target PFUnA EIS D9-NEtFOSE Target 9Cl-PF3ONS EIS D5-NEtFOSA Target PFNS NIS 13C3-PFBA Target PFDOA NIS 13C2-PFHxA Target PFOSA NIS 13C4-PFOA Target PFDS NIS 18O2-PFHxS Target PFTrDA NIS 13C5-PFNA Target 11Cl-PF3OUdS NIS 13C2-PFDA Target PFTeDA NIS 13C4-PFOS Target PFDOS Table 1: EPA Draft Method 1633 compound list ■ Sample Preparation and Extraction 500 mL of reagent water was spiked with 50 μL of EIS (800 μg/L 13C4-PFBA) and 200 μL of native compounds (2 μg/L PFBA). Method Blanks (MB) were also prepared and only spiked with EIS. Samples were extracted by solidphase extraction (SPE) using Biotage EVOLUTE® EXPRESS WAX 150-mg/6-mL cartridges. Silanized glass wool was added to each cartridge before extraction, and each was pre-conditioned with 1% methanolic ammonium hydroxide and 0.3 M formic acid. Samples were loaded onto the WAX cartridges at a rate of 5 mL/min. The cartridges were then rinsed with LCMS grade water and 0.1 M formic acid/methanol and were left to dry for 15 seconds by vacuum. Elution was then carried out by rinsing the sample bottles with 1% methanolic ammonium hydroxide and eluted onto the WAX cartridge. Acetic acid and carbon were added to each extracted sample, then shaken by hand for a maximum of five minutes and centrifuged for ten minutes. The extracted samples were then filtered using a NYLON Choice 25, 0.22 μm filter into a new collection tube containing 50 μL of NIS (400μg/L 13C3-PFBA) spiking solution. A portion was transferred to a 1 mL silanized amber glass vial and vortexed for LCMS analysis. Parameter Value LCMS Shimadzu LCMS-8060NX Analytical Column Shim-pack Scepter C18-120, 3.0 μm, 2.0 x 50mm Delay Column Shim-pack Scepter C18-120, 3.0 μm, 2.0 x 100mm Injection Volume 10 μL Pretreatment Mode Co-Injection Column Oven Temp. 40°C Mobile Phase A: 2 mM Ammonium Acetate in LCMS Grade Water B: Acetonitrile Flow Rate 0.4 mL/min Run Time 14 minutes Table 2: LCMS analysis method parameters Figure 1: Installation/placement of a delay column for PFAS Analysis. ■ Instrument and Operational Conditions The LCMS analysis was performed by using a Shimadzu triple quadrupole mass spectrometer LCMS-8060NX, coupled with a Shimadzu Nexera -40 Series UHPLC. To minimize PFAS background contamination, a delay column was installed between the mixer and high-pressure valve shown in Figure 1. The LCMS parameters are included in Table 2. Samples run for calculating MDLs, according to EPA Method 1633, occurred over a minimum of three days. Day 1 analyses included, a calibration curve, instrument blank, a calibration verification (CV), three method blanks, and three spiked water samples were analyzed. Day 2 consisted of analyzing the instrument blank, CV, three method blanks, and three spiked water samples. This was repeated on Day 3 with the instrument blank, CV, two method blanks, and two spiked water samples. Before each LC-MS/MS batch, every vial was vortexed to resuspend PFAS compounds that may have adsorbed to the walls of their respective vials. This helps to improve relative standard error (RSE), as PFAS compounds are known to adsorb to the walls of sample vials. Table 3: Retention time, calibration range, and resulted RSE for each target PFAS and EIS compound. Type Name Ret. Time CS1 (μg/L) CS9 (μg/L) RF RSE (curve) Type Name Ret. Time CS1 (μg/L) CS9 (μg/L) RF RSE (curve) Target PFBA 2.43 0.03 10.00 10.00 Target PFTrDA 9.10 0.01 2.50 7.00 Target PFMPA 2.73 0.01 5.00 9.00 Target 11Cl-PF3OUdS 9.23 0.03 10.00 10.00 Target 3:3 FTCA 2.82 0.03 12.50 11.00 Target PFTeDA 9.40 0.01 2.50 15.00 Target PFPeA 3.27 0.01 5.00 10.00 Target PFDOS 9.59 0.01 2.50 10.00 Target PFMBA 3.57 0.01 5.00 10.00 Target NMeFOSE 9.42 0.06 25.00 9.00 Target 4-2 FTS 3.87 0.03 10.00 10.00 Target NMeFOSA 9.50 0.01 2.50 19.00 Target NFDHA 4.09 0.01 5.00 10.00 Target NEtFOSE 9.60 0.06 25.00 9.00 Target PFHxA 4.19 0.01 2.50 17.00 Target NEtFOSA 9.67 0.01 2.50 10.00 Target PFBS 4.33 0.01 2.50 9.00 EIS 13C4-PFBA 2.43 8.00 8.00 1.00 Target HFPO-DA 4.57 0.03 10.00 17.00 EIS 13C5-PFPeA 3.27 4.00 4.00 4.00 Target 5:3 FTCA 4.56 0.16 62.50 9.00 EIS 13C2-4:2 FTS 3.87 4.00 4.00 4.00 Target PFEESA 4.84 0.01 5.00 3.00 EIS 13C5-PFHxA 4.19 2.00 2.00 1.00 Target PFHpA 5.13 0.01 2.50 9.00 EIS 13C3-PFBS 4.33 2.00 2.00 3.00 Target PFPeS 5.39 0.01 2.50 9.00 EIS 13C3-HFPO-DA 4.56 8.00 8.00 11.00 Target ADONA 5.45 0.03 10.00 9.00 EIS 13C4-PFHpA 5.13 2.00 2.00 3.00 Target 6-2 FTS 5.59 0.03 10.00 11.00 EIS 13C2-6:2FTS 5.60 4.00 4.00 5.00 Target PFOA 5.97 0.01 2.50 14.00 EIS 13C8-PFOA 5.97 2.00 2.00 1.00 Target PFHxS 6.32 0.01 2.50 11.00 EIS 13C3-PFHxS 6.33 2.00 2.00 1.00 Target 7:3 FTCA 6.23 0.16 62.50 11.00 EIS 13C9-PFNA 6.78 1.00 1.00 1.00 Target PFNA 6.78 0.01 2.50 10.00 EIS 13C2-8:2FTS 7.16 4.00 4.00 2.00 Target PFHpS 7.21 0.01 2.50 10.00 EIS D3-NMeFOSAA 7.47 2.00 2.00 1.00 Target 8-2 FTS 7.16 0.03 10.00 11.00 EIS 13C6-PFDA 7.57 1.00 1.00 1.00 Target NMeFOSAA 7.48 0.01 2.50 8.00 EIS D5-NEtFOSAA 7.78 4.00 4.00 2.00 Target PFDA 7.57 0.01 2.50 12.00 EIS 13C8-PFOS 8.03 2.00 2.00 2.00 Target NEtFOSAA 7.81 0.01 2.50 15.00 EIS 13C7-PFUnA 8.30 1.00 1.00 2.00 Target PFOS 8.03 0.01 2.50 9.00 EIS 13C2-PFDoA 8.77 1.00 1.00 3.00 Target PFUnA 8.30 0.01 2.50 13.00 EIS 13C8-PFOSA 8.72 2.00 2.00 3.00 Target 9Cl-PF3ONS 8.51 0.03 10.00 8.00 EIS 13C2-PFTeDA 9.39 1.00 1.00 4.00 Target PFNS 8.64 0.01 2.50 11.00 EIS D7-NMeFOSE 9.41 20.00 20.00 4.00 Target PFDOA 8.76 0.01 2.50 18.00 EIS D3-NMeFOSA 9.50 2.00 2.00 6.00 Target PFOSA 8.73 0.01 2.50 16.00 EIS D9-NEtFOSE 9.59 20.00 20.00 3.00 Target PFDS 9.00 0.01 2.50 9.00 EIS D5-NEtFOSA 9.67 2.00 2.00 2.00 ■ Calibration Curve Results Relative standard error (RSE) of all native target PFAS and EIS compounds ranged between 1% and 19% and were below the maximum level of 20% required in the EPA method. Table 3 shows the concentration range from CS1 to CS9 for each compound along with its retention time and RSE. The calibration curve for NMeFOSA can be seen in Figure 2. Each curve contained a minimum of 7 calibration standards within the linear quantitative range. Conc.Ratio 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Area Ratio 0.0 0.2 0.4 0.6 0.8 1.0 NMeFOSA RSE: 18.935798 Figure 2. Calibration curve for NMeFOSA ■ Method Detection Limit Calculations and Results The method detection limits for spiked samples (MDLs) were computed by taking the standard deviation of each compound’s concentration and multiplying it by the appropriate t-value (Equation 1). The method detection limit for the method blanks (MDLb) was computed if the compound was found to have a numerical result. If all seven samples did not give a numerical result, then it does not apply. If any of the method blanks gave a numerical result the MDLb is set to the highest recorded method blank. The MDLb was calculated using Equation 2. If the average concentration found was negative, then it was changed to zero) after multiplying the t-value and standard deviation of each compound. Equation 1: 𝑀𝐷𝐿s = 𝑡(𝑛−1,1−∝≡0.99)𝑆𝑠 Equation 2: 𝑀𝐷𝐿b = 𝑋ത + 𝑡(𝑛−1,1−∝≡0.99)𝑆𝑠 The greater value between the MDLs and MDLb for each compound becomes the initial MDL result.2 Out of the 40 compounds, 39 had higher MDLs values than their corresponding MDLb. PFHpA was the only compound for which the concentration quantified in the Method Blank (MB) was used to compute the max MDL value. This demonstrates that despite the high sensitivity achieved with this method, presence of PFAS in the MB was minimal and had negligible impact in the final MDLs. These results are all shown in Table 4 along with the values obtained by the EPA. 0 0.2 0.4 0.6 0.8 1 PFBA PFPeA PFHxA PFHpA PFOA PFNA PFDA PFUnA PFDOA PFTeDA PFTrDA PFBS PFPeS PFHxS PFHpS PFOS PFNS PFDS PFDOS MDL Range EPA reported: 0 - 1 ng/L Shimadzu: 0 - 0.4 ng/L EPA Reported Shimadzu Figure 3: MDLs reported in EPA 1633 and obtained with Shimadzu’s LCMS-8060NX of perfluoroalkyl carboxylic acids and sulfonic acids. Overall, the MDLs ranged from 0.10 ng/L for PFEESA to 1.48 ng/L for 5:3 FTCA and were up to 13.4x better than those reported in EPA Method 1633. Figures 3 and 4 compare the MDLs reported in EPA Method 1633 compared with those from Shimadzu’s LCMS-8060NX, based on the class of PFAS. For perfluoralkyl carboxylic and sulfonic acids (Figure 3), the highest MDL obtained with Shimadzu’s LCMS-8060NX was 0.34 ng/L (PFOA), 1.6x better than MDL reported in the method. The results from the other classes of PFAS included in EPA 1633 are shown in Figure 4. MDLs reported in EPA Method 1633 ranged from 0.32 ng/L (PFOSA) and 9.59 ng/L (5:3 FTCA); those obtained with Shimadzu’s LCMS-8060NX were between 0.1 ng/L (PFEESA) and 1.48 (5:3 FTCA). In addition to the improved sensitivity, which was up to 13.4x less as compared to results from the published EPA method, results presented less disparity in the concentrations determined from all PFAS classes targeted in the method. These results confirm concentrations of PFAS can be determined with 99% confidence at ppt levels and distinguishable from the method blank results. 0 2 4 6 8 10 HFPO-DA ADONA PFMPA PFMBA NFDHA 9Cl-PF3ONS 11Cl-PF3OUdS PFEESA 4-2 FTS 6-2 FTS PFOSA 8-2 FTS NMeFOSA NEtFOSA NMeFOSAA NEtFOSAA NMeFOSE NEtFOSE 3:3 FTCA 5:3 FTCA MDL Range 7:3 FTCA EPA reported: 0 - 10 ng/L Shimadzu: 0 - 2 ng/L EPA Reported Shimadzu Figure 4: MDLs reported in EPA 1633 and obtained with Shimadzu’s LCMS-8060NX of Per- and Polyfluoroether carboxylicacids, Ether sulfonic acids, Fluorotelomer sulfonic acids, Perfluorooctane sulfonamides, Perfluorooctane sulfonamidoacetic acids, Perfluorooctane sulfonamide ethanols, Fluorotelomer carboxylic acids. Table 4:Comparison of Method Detection Limits values obtained by this study and EPA Draft Method 1633 ■ References (1) Method 1633* Analysis of Per- and Polyfluoroalkyl Substances (PFAS) in Aqueous, Solid, Biosolids, and Tissue Samples by LC-MS/MS (2) Appendix B to Part 136, Title 40 -- Definition and Procedure for the Determination of the Method Detection Limit—Revision 2 ■ Conclusions • The Shimadzu LCMS-8060NX can detect 10x lower than EPA’s LOQ in a neat standard matrix and extracted aqueous matrix. • Low MDL values were determined using the Shimadzu LCMS-8060NX, confirming sufficient sensitivity and reproducibility to meet and exceed (up to 13.4x better) all EPA 1633 requirements. • Passing calibration curve linearity was obtained using this analysis method and consumables that were tested to ensure they did not interact with or contain any detectable PFAS constituents. Users must test every new lot number of consumables used n this analysis to ensure absence of detectable PFAS. ©Shimadzu Scientific Instruments, 2024 First Edition: March 2024 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. Established in 1975, Shimadzu Scientific Instruments (SSI), the American subsidiary of Shimadzu Corporation, provides a comprehensive range of analytical solutions to laboratories throughout North, Central, and parts of South America. SSI maintains a network of ten regional offices strategically located acrossthe United States, with experienced technicalspecialists,service and sales engineers situated throughout the country, as well as applicationslaboratories on both coasts. For information about Shimadzu Scientific Instruments and to contact your local office, please visit our Web site at www.ssi.shimadzu.com LCMS-8040 LCMS-8050 LCMS-8060NX LCMS-2020 Q-TOF LCMS-9030/9050 For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu.The information contained herein is provided to you “as is” without warranty of any kind including without limitation warranties as to is accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. LCMS-8045 LCMS-2050 Reference: SSI-LCMS-154 No. SSI-LCMS-150 This application news summarizes the performance of the Shimadzu LCMS-8060NX Liquid Chromatography Mass Spectrometer (LC/MS/MS) (Fig. 1) for all analytes listed in ASTM D8421. Results meet or exceed the requirements outlined in the method. The reporting range and the target analytes are listed in Table 1. The reporting limit (RL) for the test method is defined as an integer value that is equal to the concentration of the lowest calibration standard. Fig. 1 Shimadzu LCMS-8060NX Introduction and Background ASTM International published ASTM D84211 for the analysis of 44 per- and polyfluorinated alkyl substances and 24 labeled isotopes in non-potable water samples. This method extracts the substances in a 1+1 ratio of sample and methanol, filters and then measures the targeted compounds using external standard calibration liquid chromatography-tandem mass spectrometry (LC/MS/MS). The minimum reporting limit is 10 ng/L with an analytical range of 10 - 400 ng/L for most compounds. The method requires standard solutions be prepared by the laboratory from neat compounds. To save a laboratory’s time and effort from preparing stock standards individually, we optimized the method using commercially available native and labeled calibration standard mixes. Additionally, we optimized chromatography, achieving better peak shape for early-eluting compounds, such as PFBA and PFPrA. Table 1 Analyte List with D8421 Reporting Range Analyte Name Acronym CAS Number Range (ng/L) Perfluorotetradecanoic acid PFTreA 376-06-7 10-400 Perfluorotridecanoic acid PFTriA 72629-94-8 10-400 Perfluorododecanoic acid PFDoA 307-55-1 10-400 Perfluoroundecanoic acid PFUnA 2058-94-8 10-400 Perfluorodecanoic acid PFDA 335-76-2 10-400 Perfluorononanoic acid PFNA 375-95-1 10-400 Perfluorooctanoic acid PFOA 335-67-1 10-400 Perfluoroheptanoic acid PFHpA 375-85-9 10-400 Perfluorohexanoic acid PFHxA 307-24-4 10-400 Perfluoropentanoic acid PFPeA 2706-90-3 50-1000 Perfluorobutanoic acid PFBA 375-22-4 50-1000 Perfluorodecanesulfonic acid PFDS 335-77-3 10-400 User Benefits ◆ The LCMS-8060NX easily meets and exceeds method performance criteria of ASTM D8421 for 44 PFAS and 24 surrogates. ◆ Optimized chromatography and MS conditions for excellent peak shape for improved precision and accuracy. ◆ ASTM D8421 is a simple extraction procedure validated by ASTM for the analysis of PFAS in wastewater samples. Liquid Chromatography Mass Spectrometry ASTM D8421-22 Standard Test Method for Determination of Per- and Polyfluoroalkyl Substances (PFAS) in Aqueous Matrices by Co-solvation Followed by Analysis Using the Shimadzu LCMS-8060NX Nami Iwasa1 , Landon Wiest2 , William Lipps2 1 Shimadzu Corporation, 2 Shimadzu Scientific Instruments, Inc. Perfluorononanes`ulfonic acid PFNS 68259-12-1 10-400 Perfluorooctanesulfonic acid PFOS 1763-23-1 10-400 Perfluoroheptanesulfonic acid PFHpS 375-92-8 10-400 Perfluorohexanesulfonic acid PFHxS 355-46-4 10-400 Perfluoropentanesulfonic acid PFPeS 2706-91-4 10-400 Perfluorobutanesulfonic acid PFBS 375-73-5 10-400 Perfluorooctanesulfonamide PFOSA 754-91-6 10-400 8:2 Fluorotelomer sulfonic acid 8:2 FTS 39108-34-4 10-400 6:2 Fluorotelomer sulfonic acid 6:2 FTS 27619-97-2 10-400 4:2 Fluorotelomer sulfonic acid 4:2 FTS 757124-72-4 10-400 N-Ethylperfluorooctanesulfonamidoacetic acid NEtFOSAA 2991-50-6 10-400 N-Methylperfluorooctanesulfonamidoacetic acid NMeFOSAA 2355-31-9 10-400 Perfluorododecanesulfonic acid PFDoS 79780-39-5 10-400 N-Methylperfluorooctanesulfonamide NMeFOSA 31506-32-8 10-400 N-Ethylperfluorooctanesulfonamide NEtFOSA 4151-50-2 10-400 N-Methylperfluorooctanesulfonamidoethanol NMeFOSE 24448-09-7 10-400 N-Ethylperfluorooctanesulfonamidoethanol NEtFOSE 1691-99-2 10-400 Hexafluoropropylene oxide dimer acid HFPO-DA 13252-13-6 10-400 4,8-dioxa-3H-perfluorononanoic acid ADONA 919005-14-4 10-400 9-chlorohexadecafluoro-3-oxanonane-1-sulfonic acid 9Cl-PF3ONS 756426-58-1 10-400 11-chloroeicosafluoro-3-oxaundecane-1-sulfonic acid 11Cl-PF3OUdS 763051-92-9 10-400 Pentafluorpropanoic acid PFPrA 422-64-0 50-1000 Perfluoro-3,6-dioxaheptanoic acid NFDHA 151772-58-6 10-400 Perfluoro(2-ethoxyethane) sulfonic acid PFEESA 113507-82-7 10-400 Perfluoro-3-methoxypropanoic acid PFMPA 377-73-1 10-400 Perfluoro-4-methoxybutanoic acid PFMBA 863090-89-5 10-400 2H,2H,3H,3H-Perfluorohexanoic Acid 3:3 FTCA 356-02-05 10-400 2H,2H,3H,3H-Perfluorooctanoic Acid 5:3 FTCA 914637-49-3 10-400 2H,2H,3H,3H-Perfluorodecanoic acid 7:3 FTCA 812-70-4 10-400 2H-perfluoro-2-octenoic acid FHUEA 70887-88-6 10-400 2H-perfluoro-2-decenoic acid FOUEA 70887-84-2 10-400 Lithium Bis(trifluoromethane)sulfonimide * HQ-115 90076-65-6 10-400 Surrogates Perfluoro-n-[13C4] butanoic acid MPFBA NA 10-400 Perfluor0-n-[13C5] pentanoic acid M5PFPeA NA 10-400 Perfluoro-n-[1,2,3,4,6-13C5] hexanoic acid M5PFHxA NA 10-400 Perfluoro-n-[1,2,3,4-13C4] heptanoic acid M4PFHpA NA 10-400 Perfluoro-n-[13C8] octanoic acid M8PFOA NA 10-400 Perfluoro-n-[13C9] nonanoic acid M9PFNA NA 10-400 Perfluoro-n-[1,2,3,4,5,6-13C6] decanoic acid M6PFDA NA 10-400 Perfluoro-n-[1,2,3,4,5,6,7-13C7] undecanoic acid M7PFUnA NA 10-400 Perfluoro-n-[1,2-13C2] dodecanoic acid MPFDoA NA 10-400 Perfluoro-n-[1,2-13C2] tetradecanoic acid M2PFTreA NA 10-400 Perfluoro-1-[13C8] octanesulfonamide M8FOSA NA 10-400 N-methyl-d3-perfluoro-1-octanesulfonamidoacetic acid D3-N-MeFOSAA NA 10-400 N-ethyl-d5-perfluoro-1-octanesulfonamidoacetic acid D5-N-EtFOSAA NA 10-400 N-methyl-d3-perfluoro-1-octanesulfanamide d-N-MeFOSA NA 10-400 N-ethyl-d5-perfluoro-1-octanesulfanamide d-N-EtFOSA NA 10-400 2-(N-methyl-d3-perfluoro-1-octanesulfonamido) ethan-d4-ol d7-N-MeFOSE NA 10-400 2-(N-ethyl-d5-perfluoro-1-octanesulfonamido) ethan-d4-ol D9-N-EtFOSE NA 10-400 2,3,3,3-Tetrafluoro-2-(1,1,2,2,3,3,3-heptafluoropropoxy-13C3-propanoic acid MHFPO-DA NA 10-400 1H,1H,2H,2H-perfluoro-1-[1,2-13C2] hexane sulfonate M4:2FTS NA 10-400 1H,1H,2H,2H-perfluoro-1-[1,2-13C2]-octane sulfonate M6:2FTS NA 10-400 1H,1H,2H,2H-perfluoro-1-[1,2-13C2]-decane sulfonate M8:2FTS NA 10-400 Perfluoro-1-[13C8] octanesulfonate M8PFOS NA 10-400 Perfluoro-1-[2,3,4-13C3] butanesulfonate MPFBS NA 10-400 Perfluoro-1-[1,2,3-13C3] hexanesulfonate M3PFHxS NA 10-400 Compounds in red are not in the Method 1633 standards and need to be added separately The individual standard solution was prepared in 50:50 (vol: vol) methanol/water with 0.1% acetic acid to obtain final concentrations shown in Table 2. Materials and Methods Stock standard solutions containing native analytes and labeled isotopes (surrogates) were diluted from commercially available mixed stock standards (Wellington Method 1633 standard mixes) to be within the calibration range per analyte as shown in Table 1. Table 2 Concentrations of each Calibration Standard (CS) in ng/L These standards were not filtered. Calibration is performed using a 6 to 10-point curve, depending on the analyte. To obtain the calibration levels from the commercial stock solutions, 15 individual calibration standards were prepared and analyzed. Only the calibration points within the methodspecified range were used. The stock solutions were prepared and stored in PFAS-free polypropylene (PP) containers. Prior to the analysis, the solutions were shaken thoroughly, then transferred to a 2 mL PP LC vial and analyzed within 24 hours. If samples or standards are allowed to sit in the LC vials, some PFAS compounds may settle, rise, precipitate, or adsorb on the surface. To ensure a homogenous solution and optimum results, the solutions were vortexed prior to injection. 2.1 Sample Preparatio n The surrogate spiking mix is added to 5 mL of sample contained in a 15 mL polypropylene vial. Add 5 mL of methanol and mix by vortex for ~2 minutes. After mixing, add acetic acid and adjust the pH ~4. Transfer an aliquot to a LC vial and cap with a Shimadzu GLC PP vial with septum confirmed to not contain PFAS. Analyze per the conditions shown in Table 3. Concentrations obtained from the curve are multiplied by two to obtain the final concentration in the samples. Compounds CS1 CS2 CS3 CS4 CS5 CS6 CS7 CS8 CS9 CS10 CS11 CS12 CS13 CS14 CS15 Analyte All analytes unless otherwise noted 1 2.5 5 10 25 40 60 80 100 150 200 250 375 500 800 PFPeA 2 5 10 20 50 80 120 160 200 300 400 500 750 1000 1600 PFBA, 4:2-FTS, 6:2- FTS, 8:2-FTS 4 10 20 40 100 160 240 320 400 600 800 1000 1500 2000 3200 PFPrA, 5:3 FTCA, 7:3 FTCA 5 12.5 25 50 125 200 300 400 500 750 1000 1250 1875 2500 4000 NMeFOSE, NEtFOSE 10 25 50 100 250 400 600 800 1000 1500 2000 2500 3750 5000 8000 Surrogate 13C9-PFNA, 13C6- PFDA, 13C7-PFUnA, 13C2-PFDoA, 13C2- PFTreA 0.25 0.625 1.25 2.5 6.25 10 15 20 25 37.5 50 62.5 93.75 125 200 13C5-PFHxA, 13C4- PFHpA, 13C8-PFOA, 13C8-PFOSA, D3- NMeFOSA, D5- NEtFOSA, 13C8- PFOS, 13C3-PFBS, 13C3-PFHxS 0.5 1.25 2.5 5 12.5 20 30 40 50 75 100 125 187.5 250 400 13C5-PFPeA, 13C2- 4:2FTS,13C2-6:2FTS, 13C2-8:2FTS, D3- NMeFOSAA, D5- NEtFOSAA 1 2.5 5 10 25 40 60 80 100 150 200 250 375 500 800 13C4-PFBA, 13C3- HFPO-DA 2 5 10 20 50 80 120 160 200 300 400 500 750 1000 1600 D7-NMeFOSE, D9-NEtFOSE 5 12.5 25 50 125 200 300 400 500 750 1000 1250 1875 2500 4000 2.2 Analytical Conditions Table 3 Instrument Configuration and Analytical Conditions for ASTM D8421 PFAS using the Shimadzu LCMS-8060NX. Mobile Phase A: 2 mmol/L Ammonium Acetate in H2O/ Acetonitrile = 95/5 B: Acetonitrile Delay Column Shim-pack ScepterTM C18-120 2.1 mm x 100 mm, 3 µm (P/N: 227-31014-05) Analytical Column Shim-packTM GIST-HP C18 3.0 mm x 100 mm, 3 um (P/N: 227-30040-04) Gradient (%B) 10% (0 min) 22% (2.3-3.0 min) 45% (6.0 min) 80% (13.0 min) 95% (14.0-16 min ) 10% (16.01-20.0 min ) Interface IonFocus ESI Column Oven Temp. 40 oC Flow rate 0.6 mL/min Injection volume Multiple draw injection program Co-injection 25 µL Sample 25 µL 0.1% Acetic acid in H2O Interface Temp. 170 Probe position +3 mm Neblizer gas flow 3 L/min Heating gas flow 15 L/min Interface Voltage -0.5 kV (same value for all compounds DL Temp. 200 Heatblock Temp. 300 Drying gas flow 5 L/min Focus bias -2 kV (same value for all compounds Results and Discussion A single laboratory validation of this method for specificity, linearity, recovery, and precision in nine wastewater matrices according to ASTM D82722 was previously described.3 For this application news, a study was made to improve peak shape, particularly of early-eluting compounds, such as PFPrA and PFBA. This included evaluation of injection technique, columns, and flow rate. Co-injection of 25 µL sample with 25 µL 0.1% acetic acid in reagent water significantly improved the peak shapes of PFPrA, PFBA, and PFMPA (Fig. 2). A large diameter column with a long column length and large particle size, combined with a high flow rate, allowed greater axial diffusion, improving peak shape (Fig. 3). Finally, to better separate impurities from the mobile phase, a new delay column was chosen, and the gradient program was modified (Fig. 4). Upon optimization of chromatography and mass spectrometer conditions, calibration mixtures (Table 2) were prepared and used for subsequent analysis. Compound parameters, including quantitation ion, confirmation ion and collision energies, were optimized using LabSolutionsTM software. At least two MRM transitions, if available, were used. Fig. 2 Optimization of injection technique to improve peak shape Fig. 3 Example chromatograms for final column and flow rate with co-injection applied These compounds were chosen to illustrate because of their likelihood for regulation in wastewater. Additionally, calibration curves and midpoint chromatograms of PFPrA and NEtFOSE are shown in Figures 7 and 8. These compounds were chosen because they are the earliest and latest eluting compounds respectively. Linearity Study Calibration curves for each analyte were found by Shimadzu Lab Solutions Insight data processing software to have a % RSD of less than 30%, as required by ASTM D8421. Calibration data, MRM transitions for the quantitation and confirmation ions (when available), and retention times are shown in Table 4. Calibration curves along with a midpoint standard chromatogram of PFOA and PFOS are shown in Figures 5 and 6. Table 4 Summary of calibration data. Compound Quantitation Ion Confirmation Ion Retention Time (min) r 2 PFTreA 712.95>668.95 712.95>169.00 11.756 0.9962 PFTriA 662.95>618.95 662.95>169.00 11.026 0.9978 PFDoA 612.95>568.95 612.95>319.00 10.286 0.9987 PFUnA 562.95>518.95 562.95>269.00 9.543 0.9991 PFDA 512.95>468.95 512.95>219.00 8.813 0.9967 PFNA 462.95>418.95 462.95>219.00 8.11 0.9921 PFOA 412.95>369.00 412.95>169.00 7.451 0.9929 PFHpA 362.95>319.00 362.95>169.00 6.807 0.9989 PFHxA 312.95>269.00 312.95>119.00 6.028 0.9976 PFPeA 263.00>219.00 263.00>69.00 4.728 0.9996 PFBA 213.00>169.00 ---- 3.026 0.9985 PFDS 598.90>79.95 598.90>98.95 10.785 0.9971 PFNS 548.95>79.95 548.95>98.95 10.033 0.9975 PFOS 498.95>79.95 498.95>98.95 9.275 0.9951 PFHpS 448.95>79.95 448.95>98.95 8.522 0.9951 PFHxS 398.95>79.95 398.95>98.95 7.783 0.9917 PFPeS 348.95>79.95 348.95>98.95 7.059 0.9917 PFBS 298.95>79.95 298.95>98.95 6.17 0.9990 PFOSA 497.95>77.95 497.95>477.95 11.075 0.9979 8:2FTS 526.95>506.95 526.95>80.90 8.426 0.9976 6:2FTS 426.95>406.95 426.95>80.90 7.148 0.9960 4:2FTS 326.95>306.95 326.95>80.90 5.678 0.9970 Fig. 4 Final gradient with chromatogram of PFPrA, the earliest eluting peak NEtFOSAA 584.00>418.95 584.00>526.00 9.01 0.9950 NMeFOSAA 569.95>418.95 569.95>482.95 8.703 0.9929 PFDoS 698.90>79.95 698.90>98.95 12.228 0.9959 NMeFOSA 511.95>219.00 511.95>169.00 13.556 0.9956 NEtFOSA 526.00>219.00 526.00>169.00 14.149 0.9988 NMeFOSE 616.00>59.00 ---- 13.246 0.9996 NEtFOSE 630.00>59.00 ---- 13.853 0.9998 HFPO-DA 285.00>169.00 285.00>185.00 6.365 0.9971 ADONA 376.95>251.00 376.95>85.00 7.064 0.9980 9Cl-PF3ONS 530.90>350.95 532.90>352.95 9.809 0.9994 11Cl-PF3OUdS 630.90>450.95 632.90>452.95 11.308 0.9994 PFPrA 163.00>119.00 ---- 1.589 0.9996 NFDHA 294.95>201.00 294.95>85.00 5.937 0.9953 PFEESA 314.95>135.00 314.95>82.95 6.628 0.9978 PFMPA 228.95>85.00 ---- 3.656 0.9981 PFMBA 278.95>85.00 ---- 5.279 0.9979 3:3 FTCA 241.00>177.00 241.00>117.00 3.804 0.9717 5:3 FTCA 341.00>237.00 341.00>217.00 6.375 0.9945 7:3 FTCA 441.00>317.00 441.00>337.00 7.752 0.9964 FHUEA 357.00>293.00 ---- 6.472 0.9962 FOUEA 456.95>393.00 ---- 7.704 0.9973 HQ-115 279.90>146.95 279.90>210.90 7.259 0.9988 13C4-PFBA_Surr 217.00>172.00 ---- 3.023 0.9982 13C5-PFPeA_Surr 268.00>223.00 ---- 4.726 0.9976 13C5-PFHxA_Surr 318.00>273.00 318.00>120.00 6.026 0.9972 13C4-PFHpA_Surr 367.00>322.00 ---- 6.806 0.9994 13C8-PFOA_Surr 421.00>376.00 ---- 7.45 0.9959 13C9-PFNA_Surr 472.00>427.00 ---- 8.108 0.9947 13C6-PFDA_Surr 519.00>474.00 ---- 8.81 0.9989 13C7-PFUnA_Surr 570.00>525.00 ---- 9.541 0.9979 13C2-PFDoA_Surr 614.95>569.95 ---- 10.285 0.9968 13C2-PFTreA_Surr 714.95>669.95 ---- 11.755 0.9952 13C8-PFOSA_Surr 505.95>77.95 ---- 11.077 0.9983 D3-NMeFOSAA_Surr 573.00>418.95 ---- 8.697 0.9933 D5-NEtFOSAA_Surr 589.00>418.95 ---- 9 0.9976 D3-NMeFOSA_Surr 515.00>219.00 515.00>168.90 13.548 0.9993 D5-NEtFOSA_Surr 531.00>219.00 531.00>168.90 14.131 0.9983 D7-NMeFOSE_Surr 623.05>59.00 ---- 13.206 0.9957 D9-NEtFOSE_Surr 639.10>59.00 ---- 13.807 0.9994 13C3-HFPO-DA_Surr 287.00>169.00 284.90>185.00 6.363 0.9921 13C2-4:2FTS_Surr 329.00>308.95 329.00>80.90 5.678 0.9943 13C2-6:2FTS_Surr 428.95>408.95 428.95>80.90 7.147 0.9903 13C2-8:2FTS_Surr 528.95>508.95 528.95>80.90 8.425 0.9956 13C8-PFOS_Surr 506.95>79.95 506.95>98.95 9.274 0.9966 13C3-PFBS_Surr 301.95>79.95 301.95>98.95 6.17 0.9953 13C3-PFHxS_Surr 401.95>79.95 401.95>98.95 7.782 0.9921 Fig. 5 Calibration curve and midpoint chromatogram for PFOA Fig. 6 Calibration curve and midpoint chromatogram for PFOS Fig. 7 Calibration curve and midpoint chromatogram for PFPrA Fig. 8 Calibration curve and midpoint chromatogram for NEtFOSE These data are well within the 70 -130 % recovery and ≤ 30 %RSD limits of the method. Recovery and Repeatability Stu dy Recovery and repeatability (Table 5) were evaluated in reagent water and wastewater, each spiked four times at the concentration indicated. Recovery was calculated after subtracting the native PFAS found in the unspiked sample matrices. Table 5 Recovery and Repeatability in Reagent Water and Wastewater Compound Spike Concentration (ppt) Reagent Water % Recovery Reagent Water %RSD (n=4) Wastewater % Recovery Wastewater %RSD (n=4) PFTreA 160 110 3.76 119 2.71 PFTriA 160 109 2.08 79.9 4.82 PFDoA 160 104 4.33 107 4.6 PFUnA 160 113 5.53 105 2.49 PFDA 160 113 2.67 102 4.5 PFNA 160 113 6.71 107 2.34 PFOA 160 111 7.52 112 5.7 PFHpA 160 116 4.13 108 4.46 PFHxA 160 114 6.83 115 3.41 PFPeA 320 106 4.37 108 2.47 PFBA 640 107 0.55 108 2.06 PFDS 160 112 8.89 112 3.73 PFNS 160 113 3.72 116 5.58 PFOS 160 110 3.83 122 4.02 PFHpS 160 115 6.09 102 6.91 PFHxS 160 113 5.93 113 13.15 PFPeS 160 124 4.94 119 9.49 PFBS 160 109 4.59 114 5.12 PFOSA 160 101 2.44 100 4.59 8:2FTS 640 113 6.24 103 4.97 6:2FTS 640 119 3.2 107 3.3 4:2FTS 640 121 0.7 100 2.47 NEtFOSAA 160 113 7.5 89.4 10.15 NMeFOSAA 160 111 13.35 88.0 7.42 PFDoS 160 106 5.23 108 9.31 NMeFOSA 160 102 3.68 91.5 5.59 NEtFOSA 160 100 0.73 90.5 3.33 NMeFOSE 1600 97.2 0.34 93.6 0.94 NEtFOSE 1600 96.8 0.9 93.5 1.39 HFPO-DA 160 109 2.35 112 9.66 ADONA 160 110 1.01 104 4.22 9Cl-PF3ONS 160 111 2.01 111 1.92 11Cl-PF3OUdS 160 112 3.59 111 2.78 PFPrA 800 108 1.8 105 0.77 NFDHA 160 107 9.13 110 4.16 PFEESA 160 112 4.71 115 4.45 PFMPA 160 106 2.37 102 5.83 PFMBA 160 113 7.07 115 2.55 3:3 FTCA 160 87.1 20.57 94.8 14.52 5:3 FTCA 800 95.7 6.91 90.4 4.22 7:3 FTCA 800 92.5 2.56 88.8 2.73 FHUEA 160 99.1 3.91 95.3 2.16 FOUEA 160 102 4.2 97.2 3.5 HQ-115 160 112 2.56 111 0.78 Surrogates 13C4-PFBA_Surr 320 102 1.7 96.4 2.88 13C5-PFPeA_Surr 160 108 6.03 96.4 3.36 13C5-PFHxA_Surr 80 110 3.7 101 5.35 13C4-PFHpA_Surr 80 104 6.43 105 5.99 13C8-PFOA_Surr 80 107 11.32 100 10.19 13C9-PFNA_Surr 40 98.2 13.76 92.2 17.29 13C6-PFDA_Surr 40 106 5.74 96.5 8.59 13C7-PFUnA_Surr 40 98.6 6.01 90.8 6.68 13C2-PFDoA_Surr 40 96.1 4.31 94.9 5.43 13C2-PFTreA_Surr 40 98.9 10.44 119 9.46 13C8-PFOSA_Surr 80 92.2 2.22 91.4 6.54 D3-NMeFOSAA_Surr 160 98.4 3.95 90.6 6.56 D5-NEtFOSAA_Surr 160 95.0 3.55 81.3 3.26 D3-NMeFOSA_Surr 80 89.3 12.1 82.6 8.57 D5-NEtFOSA_Surr 80 90.5 8.83 84.2 8.94 D7-NMeFOSE_Surr 800 88.8 0.49 85.4 1.68 D9-NEtFOSE_Surr 800 89.6 0.97 85.9 1.49 13C3-HFPO-DA_Surr 320 103 3.19 101 8.25 13C2-4:2FTS_Surr 160 116 3.15 96.2 6.88 13C2-6:2FTS_Surr 160 119 2.25 97.3 5.65 13C2-8:2FTS_Surr 160 104 0.73 97.4 7.53 13C8-PFOS_Surr 80 103 9.45 105 9.61 13C3-PFBS_Surr 80 121 5.06 94.0 6.33 13C3-PFHxS_Surr 80 116 9.99 108 6.65 Conclusion This application news demonstrates the analysis of 44 PFAS and 24 surrogate compounds in non-potable water by ASTM D8421 using the Shimadzu LCMS-8060NX Liquid Chromatography Mass Spectrometer (LC/MS/MS). Chromatographic conditions were optimized to achieve excellent peak shape, even for the earliest eluting compounds, such as PFPrA and PFBA. The highly sensitive Shimadzu LCMS-8060NX easily exceeds method performance criteria of the ASTM method and provides testing laboratories with highly accurate and reliable, repeatable results for PFAS in wastewater samples. References 1. ASTM Test Method D8241 Determination of Per- and Polyfluoroalkyl Substances (PFAS) in Aqueous Matrices by Cosolvation followed by Liquid Chromatography Tandem Mass Spectrometry (LC/MS/MS) 2. ASTM Standard D8272-19 -Standard Guide for Development and Optimization of D19 Chemical Analysis Methods Intended for EPA Compliance Reporting / ASTM International / West Conshohocken / PA / 2020 / 10.1520/D8272-19/ https://www.astm.org/ 3. Lipps, W., ASTM D8421-22 Standard Test Method for Determination of Per- and Polyfluoroalkyl Substances (PFAS) in Aqueous Matrices by Co-solvation followed by Liquid Chromatography Tandem Mass Spectrometry (LC/MS/MS), Shimadzu Whitepaper, August 2023. Copyright © 2023 Shimadzu Corporation and/or its affiliates. All rights reserved. Shimadzu Corporation www.shimadzu.com/an/ Shimadzu Scientific Instruments, Inc. www.ssi.shimadzu.com Founded in 1875, Shimadzu Corpora�on, a leader in the development of advanced technologies, has a dis�nguished history of innova�on built on the founda�on of contribu�ng to society through science and technology. Established in 1975, Shimadzu Scien�fic Instruments (SSI), the American subsidiary of Shimadzu Corpora�on, provides a comprehensive range of analy�cal solu�ons to laboratories throughout North, Central, and parts of South America. 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