User Benefits Application News # Type Name # Type Name 1 Target PFBA 1 EIS 13C4-PFBA 2 Target PFMPA 2 EIS 13C5-PFPeA 3 Target 3:3 FTCA 3 EIS 13C2-4:2 FTS 4 Target PFPeA 4 EIS 13C5-PFHxA 5 Target PFMBA 5 EIS 13C3-PFBS 6 Target 4:2 FTS 6 EIS 13C3-HFPO-DA 7 Target NFDHA 7 EIS 13C4-PFHpA 8 Target PFHxA 8 EIS 13C2-6:2FTS 9 Target PFBS 9 EIS 13C8-PFOA 10 Target HFPO-DA 10 EIS 13C3-PFHxS 11 Target 5:3 FTCA 11 EIS 13C9-PFNA 12 Target PFEESA 12 EIS 13C2-8:2FTS 13 Target PFHpA 13 EIS D3-NMeFOSAA 14 Target PFPeS 14 EIS 13C6-PFDA 15 Target ADONA 15 EIS D5-NEtFOSAA 16 Target 6:2 FTS 16 EIS 13C8-PFOS 17 Target PFOA 17 EIS 13C7-PFUnA 18 Target PFHxS 18 EIS 13C2-PFDoA 19 Target 7:3 FTCA 19 EIS 13C8-PFOSA 20 Target PFNA 20 EIS 13C2-PFTeDA 21 Target PFHpS 21 EIS D7-NMeFOSE 22 Target 8:2 FTS 22 EIS D3-NMeFOSA 23 Target NMeFOSAA 23 EIS D9-NEtFOSE 24 Target PFDA 24 EIS D5-EtFOSA 25 Target NEtFOSAA 1 NIS 13C3-PFBA 26 Target PFOS 2 NIS 13C2-PFHxA 27 Target PFUnA 3 NIS 13C4-PFOA 28 Target 9Cl-PF3ONS 4 NIS 18O2-PFHxS 29 Target PFNS 5 NIS 13C5-PFNA 30 Target PFDOA 6 NIS 13C2-PFDA 31 Target PFOSA 7 NIS 13C4-PFOS 32 Target PFDS 33 Target PFTrDA 34 Target 11Cl-PF3OUdS 35 Target PFTeDA 36 Target PFDOS 37 Target NMeFOSE 38 Target NMeFOSA 39 Target NEtFOSE 40 Target NEtFOSA Fig. 1 LCMS-8065XE. The combination of low-diffusion nebulizer nozzle and IonFocus TM technology achieve both high sensitivity and robustness.  Method Overview Table 1 EPA Method 1633A compound list This application describes the analysis of 40 native PFAS target compounds using 24 extracted internal standards (EIS), and 7 non-extracted internal standards (NIS). Stock standards were purchased from Wellington Laboratories (Ontario, Canada) as a series of native and mass-labelled PFAS mixtures in methanol (PFAC-MXF, PFAC-MXG, PFAC-MXH, PFAC-MXI, PFAC-MXJ, MPFAC-HIF-ES, and MPFAC-HIF-IS). Three spiking standards were prepared, containing the native targets, EIS and NIS compounds, by diluting the stock solutions in methanol. Calibration curves were generated by preparing methanol containing 4% water, 1% ammonium hydroxide, and 0.625% acetic acid. The stock standards were then diluted to yield concentration ranges of 2.5 to 62,400 ng/L for native PFAS targets, 1.0 to 20.0 μg/L for EIS, and 1.0 to 4.0 μg/L for NIS. 2.5 ng/L for native standards is equivalent to 0.00625 ng/g in tissue. Per- and polyfluoroalkyl substances (PFAS) are a diverse group of synthetic chemicals widely used in industry and consumer products. Owing to their persistence and potential health risks, PFAS have become a major environmental concern. The U.S. Environmental Protection Agency (EPA) has developed standardized analytical methods, including EPA Method 1633A, to monitor PFAS contamination in a wide range of environmental and biological matrices. This study evaluates the performance of LCMS-8065XE for PFAS analysis in accordance with EPA Method 1633A. By assessing calibration stability over the course of one week, we demonstrate the method’s suitability for routine PFAS monitoring. All aspects of the workflow including sample extraction, preparation, and analysis, were performed in compliance with EPA Method 1633A guidelines 1) . Our results highlight the system’s robustness and minimal downtime, underscoring its value for laboratories responsible for delivering rapid and reliable PFAS determinations.  Introduction Breakthrough Sensitivity and Robustness for PFAS Analysis for EPA Method 1633A Om Shrestha, Eishi Imoto, Toshiya Matsubara High Performance Liquid Chromatograph Mass Spectrometer Shimadzu Scientific Instruments, R&D center  LCMS-8065XE achieved up to 80 times lower limit of quantifications than LLOQ concentration in EPA Method 1633A.  The measurement time was 10 min while meeting the chromatographic requirements by EPA Method 1633A.  This method showed the excellent robustness across 7 days of over 900 continuous injections of the matrix sample. All standards were prepared for analysis in 200 μL silanized glass inserts in 1.5 mL amber silanized glass vials, sealed with PE/silicone blue screw caps. 2 grams of tissue were processed and extracted to a final volume for analysis of 5 mL.

Application News LCMS analyses were performed using a Shimadzu triple quadrupole mass spectrometer, LCMS-8065XE, coupled with a Shimadzu Nexera TM 40 series UHPLC. To minimize PFAS background contamination, a delay column was installed between the mixer and high-pressure valve. The LC and MS parameters are summarized in Table 2 and 3. Analyses included a calibration curve, instrument blank, a calibration verification (CV), method blanks, and spiked chicken tissue samples. A robustness test was conducted by monitoring calibration verification, method blanks, and spiked chicken tissue samples. Prior to each LC-MS/MS run, every vial was vortexed to resuspend PFAS compounds that may have adsorbed to the vial walls. This procedure helped improve the relative standard error (RSE), as PFAS compounds are knows to adsorb to glass surfaces.  Instrument and Operational Conditions System Nexera X3 Delay Column Shim-pack Scepter TM C18-120, 2.1 × 100 mm, 3 µm (P/N: 227-31014-05) Analytical Column Shim-pack Scepter C18-120, 2.1 × 50 mm, 1.9 µm (P/N: 227-31033-03) Flow rate 0.3 mL/min Mobile phase A 2mM Ammonium Acetate in Water Mobile phase B Acetonitrile Injection volume 5 µL standard injection Oven temperature 35 ˚C Measurement time 10 min Table 2 LC conditions Fig. 4 MS chromatogram of 40 PFAS and 3 cholic acids. Measuring time for 40 PFASs was 10 min. Samples were loaded onto WAX cartridges at a flow rate of 5 mL/min. The cartridges were rinsed with LC/MS grade water followed by 0.1 mol/L formic acid/methanol and then dried under vacuum for 15 seconds. Elution was performed by rinsing the inside of the tubes with 1% methanolic ammonium hydroxide and passing the eluate through the WAX cartridge. Acetic acid was added to each extract, which was then hand- shaken for up to five minutes and centrifuged for ten minutes. An aliquot of the supernatant was transferred to a 1 mL silanized amber glass vial and spiked with 0.5 µL MPFAC-HIF-IS this vial was vortexed prior to LC/MS analysis. Table 3. MS conditions System LCMS-8065XE Nebulizing gas 1.1 L/min Heating gas 15.0 L/min Drying gas 5.0 L/min Interface Temperature 225 °C DL Temperature 200 °C Heat Block Temperature 250 °C Interface voltage -0.5 kV Focus voltage 0 kV • Insertion of silanized glass wool to the WAX SPE cartridge • 15 mL 1% methanolic ammonium hydroxide • 5mL 0.3 M formic acid Condition • 5 mL/min Sample Loading • 5 mL Water (twice) • 5mL 1:1 0.1M formic acid/methanol Washing • 5mL 1% methanolic ammonium hydroxide Elution • 25 μL Acetic Acid • Handshake • Transfer to LC vial Cleanup Fig. 2 The appearance of EDGE PFAS (CEM) which automates the extraction of PFAS from field samples. 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 min -0.25 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 (x1,000,000) 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 min TDCA (RT: 3.6 min) PFOS (RT: 4.7 min) TCDCA (RT: 3.0 min) TUDCA (RT: 3.5 min) ΔRT > 1.0 min • Weigh 2 grams of tissue sample into Q-Cup • 0.05 M KOH in methanol, 10mL, 65 ˚C, 3 min Cycle 1 • Acetonitrile, 10mL, 65 ˚C, 3 min Cycle 2 • IPA, 10 mL Wash 1 • 0.05 M KOH in methanol, 10 mL, 65 ˚C, 0.5 min Wash 2 • 0.05 M KOH in methanol, 10 mL Wash 3 Automated extraction procedures by EDGE PFAS Clean-up procedures Fig. 3 Extraction and clean-up scheme  Sample Preparation and Extraction Automated extraction was performed using the EDGE PFAS system (see Fig. 2). Sample preparation procedures are described in Fig. 3. Approximately 2 grams of chicken tissue, as reference matrix, was weighed into a Q-Cup. Each sample was spiked with 25 μL of MPFAC-HIF-ES and 40 μL of native compounds. 10 mg of carbon was added to the combined extract and mixed by hand shaking for 5 min. Method Blanks (MB) were also prepared, spiked only with EIS. Samples were subsequently cleaned up by solid phase extraction (SPE). Silanized glass wool was added to each cartridge prior to extraction, and the cartridges were pre-conditioned with 1% methanolic ammonium hydroxide.

Application News  Calibration Curve Results The relative standard error (RSE) of all native target PFAS ranged from 3% to 17%, remaining below the maximum limit of 20% specified in the EPA Method 1633A. Table 4 summarizes the concentration ranges, RF RSE values and calibration correlations for 40 PFAS compounds. All calibration curves, blanks and MS chromatograms at the LLOQ are shown in Fig. 5. Each calibration curve included at least seven calibration standards within the linear quantitative range. The generated calibration curves exhibited linear regression with R 2 >0.995. All accuracy ranges at each calibration points were within 70-130%. # Name Calibration Range [ng/L] Equivalent LLOQ in tissue [ng/g] EPA LLOQ conc. [ng/L] RF RSE (curve) Linearity (R2) % Accuracy range 1 PFBA 10-10000 0.025 800 4 0.998 95-105 2 PFMPA 5-5000 0.0125 400 7 0.995 92-113 3 3:3 FTCA 62.4-62400 0.156 998 7 0.995 90-113 4 PFPeA 5-5000 0.0125 400 4 0.998 95-106 5 PFMBA 5-5000 0.0125 400 6 0.996 92-110 6 4:2 FTS 10-10000 0.025 800 7 0.995 88-106 7 NFDHA 5-5000 0.0125 400 7 0.995 89-111 8 PFHxA 2.5-2500 0.00625 200 6 0.996 94-114 9 PFBS 2.5-2500 0.00625 200 6 0.997 92-110 10 HFPO-DA 10-10000 0.025 800 4 0.998 93-106 11 5:3 FTCA 2.5-2500 0.00625 4992 5 0.997 92-106 12 PFEESA 5-5000 0.0125 400 3 0.999 95-104 13 PFHpA 2.5-2500 0.00625 200 5 0.997 92-109 14 PFPeS 2.5-2500 0.00625 200 6 0.996 92-108 15 ADONA 10-10000 0.025 800 5 0.998 91-105 16 6:2 FTS 2.5-2500 0.00625 800 7 0.995 94-111 17 PFOA 2.5-2500 0.00625 200 6 0.996 95-113 18 PFHxS 2.5-2500 0.00625 200 6 0.996 87-107 19 7:3 FTCA 25-25000 0.0625 4992 5 0.997 94-106 20 PFNA 2.5-2500 0.00625 200 6 0.996 94-109 21 PFHpS 2.5-2500 0.00625 200 7 0.995 92-111 22 8:2 FTS 2.5-2500 0.00625 800 17 0.995 85-116 23 NMeFOSAA 6.3-2500 0.01575 200 7 0.995 89-108 24 PFDA 2.5-2500 0.00625 200 6 0.996 92-109 25 NEtFOSAA 6.3-2500 0.01575 200 7 0.995 90-109 26 PFOS 6.3-2500 0.01575 200 6 0.997 94-110 27 PFUnA 2.5-2500 0.00625 200 4 0.998 94-105 28 9Cl-PF3ONS 10-10000 0.025 800 6 0.997 91-109 29 PFNS 2.5-2500 0.00625 200 7 0.995 90-110 30 PFDOA 2.5-2500 0.00625 200 5 0.997 94-109 31 PFOSA 2.5-2500 0.00625 200 5 0.997 95-111 32 PFDS 2.5-2500 0.00625 200 7 0.995 92-112 33 PFTrDA 2.5-2500 0.00625 200 5 0.997 90-106 34 11Cl-PF3OUdS 10-10000 0.025 800 4 0.998 94-106 35 PFTeDA 2.5-2500 0.00625 200 6 0.996 93-111 36 PFDOS 6.3-2500 0.01575 200 9 0.997 84-116 37 NMeFOSE 25-25000 0.0625 2000 6 0.996 93-113 38 NMeFOSA 2.5-2500 0.00625 200 5 0.997 92-111 39 NEtFOSE 25-25000 0.0625 2000 3 0.999 95-110 40 NEtFOSA 6.3-2500 0.01575 200 5 0.997 96-106 Table 4 Calibration ranges, RF RSE values and correlation R 2 and accuracy ranges for 40 PFAS  Chromatographic separation Cholic acids, such as taurodeoxycholic acid (TDCA), taurochenodeoxycholic acid (TCDCA), and tauroursodeoxycholic acid (TUDCA), can interfere with PFOS during the ionization process because their precursor and product ions are similar. These bile acids are present in tissue and wastewater samples. Therefore, one of the requirements of the LC method for EPA Method 1633A is to achieve a separation of at least 1 min between PFOS and these cholic acids. In this study, acetonitrile was used as mobile phase B to meet this requirement. Fig. 4 shows the chromatographic separation of forty PFAS listed in EPA Method 1633A. With acetonitrile, the cholic acids eluted much earlier than both branched and linear PFOS. The retention time difference was 1.5 min, which exceeded the required one-minute separation.

Application News RT (min) 3.0 3.2 RT (min) 3.0 3.2 RT (min) 3.2 3.4 RT (min) 3.6 3.8 RT (min) 3.8 4.0 RT (min) 4.0 4.2 RT (min) 4.2 4.3 4.4 4.5 RT (min) 4.5 4.6 4.7 4.8 RT (min) 4.6 4.8 RT (min) 5.0 5.1 5.2 5.3 RT (min) 5.2 5.4 Q 228.9500>85.1000 (-) RT (min) 3.0 3.2 Q 263.0000>219.0000 (-) RT (min) 3.2 3.4 Q 326.9500>307.1000 (-) RT (min) 3.6 3.8 Q 312.9500>269.1000 (-) RT (min) 3.8 4.0 Q 285.0000>169.0000 (-) RT (min) 4.0 4.2 Q 314.9500>135.1500 (-) RT (min) 4.2 4.3 4.4 4.5 Q 348.9500>80.0000 (-) RT (min) 4.5 4.6 4.7 4.8 Q 426.9500>406.9000 (-) RT (min) 4.6 4.8 Q 398.9500>80.0000 (-) RT (min) 5.0 5.1 5.2 5.3 Q 462.9500>419.1000 (-) RT (min) 5.2 5.4 Q 241.0000>177.1000 (-) RT (min) 3.0 3.2 RT (min) 2.8 3.0 RT (min) 3.4 3.6 RT (min) 3.8 4.0 RT (min) 3.9 4.0 4.1 4.2 RT (min) 4.0 4.2 RT (min) 4.4 4.6 RT (min) 4.6 4.8 RT (min) 4.8 5.0 RT (min) 5.0 5.2 Q 213.0000>169.0000 (-) RT (min) 2.8 3.0 Q 278.9500>85.0000 (-) RT (min) 3.4 3.6 Q 294.9500>200.9500 (-) RT (min) 3.8 4.0 Q 298.9500>79.9000 (-) RT (min) 3.9 4.0 4.1 4.2 Q 341.0000>237.2000 (-) RT (min) 4.0 4.2 Q 362.9500>319.1000 (-) RT (min) 4.4 4.6 Q 376.9500>251.0000 (-) RT (min) 4.6 4.8 Q 412.9500>169.0000 (-) RT (min) 4.8 5.0 Q 441.0000>317.1000 (-) RT (min) 5.0 5.2 Conc.Ratio 0 20 40 60 80 100 Area Ratio 0 2 4 6 7:3 FTCA y = 0.06809099x + 0.001706564 R² = 0.9969779 R = 0.9984878 Conc.Ratio 0 2 4 6 8 10 Area Ratio 0.0 0.5 1.0 1.5 PFOA y = 0.1605350x + 0.01299955 R² = 0.9960826 R = 0.9980394 Conc.Ratio 0 2 4 6 8 10 Area Ratio 0 5 10 15 ADONA y = 1.772719x + 0.002742974 R² = 0.9976114 R = 0.9988050 Conc.Ratio 0 2 4 6 8 10 Area Ratio 0 1 2 PFHpA y = 0.2664472x + 0.003087872 R² = 0.9971900 R = 0.9985940 Conc.Ratio 0 2 4 6 8 10 Area Ratio 0.0 2.5 5.0 7.5 5:3 FTCA y = 0.8369629x + 0.00006230707 R² = 0.9970049 R = 0.9985013 Conc.Ratio 0 2 4 6 8 10 Area Ratio 0 1 2 PFBS y = 0.2814981x - 0.00002885327 R² = 0.9966303 R = 0.9983137 Conc.Ratio 0 5 10 15 20 Area Ratio 0 1 2 NFDHA y = 0.1179902x + 0.002049916 R² = 0.9947646 R = 0.9973789 Conc.Ratio 0 2 4 6 8 10 Area Ratio 0.0 0.5 1.0 1.5 PFMBA y = 0.1798742x + 0.00001908034 R² = 0.9960060 R = 0.9980010 Conc.Ratio 0 20 40 60 80 100 120 Area Ratio 0.0 0.1 0.2 3:3 FTCA y = 0.001863205x + 0.0000007639463 R² = 0.9946779 R = 0.9973354 Conc.Ratio 0 2 4 6 8 10 Area Ratio 0 1 2 PFBA y = 0.2771505x + 0.001131668 R² = 0.9983958 R = 0.9991976 Conc.Ratio 0 5 10 15 20 Area Ratio 0 1 2 3 PFNA y = 0.1731396x + 0.002018995 R² = 0.9962865 R = 0.9981415 Conc.Ratio 0 2 4 6 8 10 Area Ratio 0.0 0.5 1.0 1.5 PFHxS y = 0.1808125x + 0.006932017 R² = 0.9956126 R = 0.9978039 Conc.Ratio 0 5 10 15 20 Area Ratio 0 5 10 15 6-2 FTS y = 0.8981036x + 0.01270995 R² = 0.9948639 R = 0.9974286 Conc.Ratio 0 2 4 6 8 10 Area Ratio 0 1 2 PFPeS y = 0.3088216x - 0.0005004488 R² = 0.9956084 R = 0.9978018 Conc.Ratio 0 5 10 15 20 Area Ratio 0 2 4 PFEESA y = 0.3000925x + 0.002783926 R² = 0.9988458 R = 0.9994227 Conc.Ratio 0 2 4 6 8 10 Area Ratio 0 1 2 HFPO-DA y = 0.2712730x + 0.0006879638 R² = 0.9979516 R = 0.9989753 Conc.Ratio 0 2 4 6 8 10 Area Ratio 0 1 2 PFHxA y = 0.2579368x + 0.003056870 R² = 0.9958522 R = 0.9979239 Conc.Ratio 0 5 10 15 20 Area Ratio 0 2 4 4-2 FTS y = 0.2468771x + 0.0006595192 R² = 0.9951936 R = 0.9975939 Conc.Ratio 0 2 4 6 8 10 Area Ratio 0 1 2 PFPeA y = 0.2344023x + 0.001442244 R² = 0.9981189 R = 0.9990590 Conc.Ratio 0 2 4 6 8 10 Area Ratio 0.0 0.5 1.0 PFMPA y = 0.1211639x + 0.0002593765 R² = 0.9953079 R = 0.9976512 PFBA PFMPA 3:3 FTCA PFPeA PFMBA 4:2 FTS NFDHA PFHxA PFBS HFPO-DA 5:3 FTCA PFEESA PFHpA ADONA PFPeS 6:2 FTS PFOA PFHxS 7:3 FTCA PFNA Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank 10 ng/L 62.4 ng/L 5 ng/L 5 ng/L 2.5 ng/L 2.5 ng/L 2.5 ng/L 10 ng/L 2.5 ng/L 25 ng/L Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank 5 ng/L 5 ng/L 10 ng/L 2.5 ng/L 10 ng/L 5 ng/L 2.5 ng/L 2.5 ng/L 2.5 ng/L 2.5 ng/L Fig. 5 Calibration curves and MS chromatograms of blank and LLOQ for 40 PFAS compounds

Application News RT (min) 5.4 5.6 RT (min) 5.6 5.8 6.0 RT (min) 5.8 6.0 RT (min) 6.0 6.2 6.4 RT (min) 6.0 6.2 RT (min) 6.0 6.2 RT (min) 6.4 6.6 6.8 RT (min) 6.6 6.8 RT (min) 5.4 5.6 5.8 RT (min) 5.6 5.8 RT (min) 6.4 6.6 6.8 RT (min) 6.0 6.2 RT (min) 6.0 6.2 RT (min) 5.8 6.0 RT (min) 5.8 6.0 RT (min) 5.6 5.8 RT (min) 5.4 5.6 RT (min) 6.4 6.6 6.8 RT (min) 6.2 6.4 RT (min) 5.6 5.8 Q 511.9500>219.1000 (-) RT (min) 6.4 6.6 6.8 Q 630.9000>451.0000 (-) RT (min) 6.0 6.2 Q 598.9000>79.9500 (-) RT (min) 6.0 6.2 Q 612.9500>569.0000 (-) RT (min) 5.8 6.0 Q 530.9000>351.1000 (-) RT (min) 5.8 6.0 Q 512.9500>469.1000 (-) RT (min) 5.6 5.8 Q 526.9500>506.8500 (-) RT (min) 5.4 5.6 Q 511.9500>219.1000 (-) RT (min) 6.4 6.6 6.8 Q 698.9000>79.9000 (-) RT (min) 6.2 6.4 Q 499.0000>80.0000 (-) RT (min) 5.6 5.8 Q 448.9500>80.0000 (-) RT (min) 5.4 5.6 Q 562.9500>518.9500 (-) RT (min) 5.6 5.8 6.0 Q 548.9500>99.0000 (-) RT (min) 5.8 6.0 Q 497.9500>78.0000 (-) RT (min) 6.0 6.2 6.4 Q 662.9500>618.8000 (-) RT (min) 6.0 6.2 Q 712.9500>668.8000 (-) RT (min) 6.0 6.2 Q 616.0000>59.0500 (-) RT (min) 6.4 6.6 6.8 Q 630.0000>59.0500 (-) RT (min) 6.6 6.8 Q 569.9500>418.9000 (-) RT (min) 5.4 5.6 5.8 Q 584.0000>419.0500 (-) RT (min) 5.6 5.8 Conc.Ratio 0 2 4 6 8 10 Area Ratio 0 1 2 PFHpS y = 0.3082597x + 0.0004445569 R² = 0.9947648 R = 0.9973790 Conc.Ratio 0 2 4 6 8 10 Area Ratio 0 1 2 NEtFOSE y = 0.2443665x + 0.0003468605 R² = 0.9987934 R = 0.9993965 Conc.Ratio 0 2 4 6 8 10 Area Ratio 0 1 2 NMeFOSE y = 0.2423128x + 0.001116607 R² = 0.9962365 R = 0.9981165 Conc.Ratio 0.0 0.5 1.0 1.5 2.0 2.5 Area Ratio 0 1 2 3 NEtFOSA y = 1.311801x + 0.0009666156 R² = 0.9967610 R = 0.9983792 Conc.Ratio 0 1 2 3 4 5 Area Ratio 0 1 2 NMeFOSA y = 0.5810172x + 0.001973844 R² = 0.9967759 R = 0.9983866 Conc.Ratio 0 2 4 6 8 10 Area Ratio 0.0 0.5 1.0 PFDOS y = 0.1341372x + 0.0001294327 R² = 0.9970636 R = 0.9985307 Conc.Ratio 0 5 10 15 20 Area Ratio 0 2 4 PFTeDA y = 0.2465830x + 0.001865513 R² = 0.9957001 R = 0.9978477 Conc.Ratio 0 2 4 6 8 10 Area Ratio 0 1 2 3 11Cl-PF3OUdS y = 0.2954397x + 0.0003174112 R² = 0.9983816 R = 0.9991905 Conc.Ratio 0 5 10 15 20 Area Ratio 0 2 4 PFTrDA y = 0.2157913x + 0.002375201 R² = 0.9968067 R = 0.9984021 Conc.Ratio 0 1 2 3 4 5 Area Ratio 0.0 0.2 0.4 0.6 PFDS y = 0.1362689x - 0.0003011733 R² = 0.9949140 R = 0.9974538 Conc.Ratio 0 1 2 3 4 5 Area Ratio 0 1 2 PFOSA y = 0.4625918x + 0.002946585 R² = 0.9969724 R = 0.9984851 Conc.Ratio 0 5 10 15 20 Area Ratio 0 2 4 PFDOA y = 0.2485202x + 0.002372830 R² = 0.9972547 R = 0.9986264 Conc.Ratio 0 1 2 3 4 5 Area Ratio 0.0 0.5 1.0 PFNS y = 0.2086781x - 0.0006097040 R² = 0.9947600 R = 0.9973766 Conc.Ratio 0 2 4 6 8 10 Area Ratio 0 2 4 9Cl-PF3ONS y = 0.4186749x + 0.0004884323 R² = 0.9965410 R = 0.9982690 Conc.Ratio 0 5 10 15 20 Area Ratio 0 2 4 PFUnA y = 0.2397342x + 0.001170976 R² = 0.9980286 R = 0.9990138 Conc.Ratio 0 2 4 6 8 10 Area Ratio 0.0 0.5 1.0 1.5 PFOS y = 0.1782836x + 0.01301463 R² = 0.9967206 R = 0.9983590 Conc.Ratio 0 1 2 3 4 5 Area Ratio 0.0 0.5 1.0 NEtFOSAA y = 0.2051660x - 0.000004308553 R² = 0.9947621 R = 0.9973776 Conc.Ratio 0 5 10 15 20 Area Ratio 0 2 4 PFDA y = 0.2487561x + 0.001585488 R² = 0.9963038 R = 0.9981502 Conc.Ratio 0 2 4 6 8 10 Area Ratio 0.0 0.5 1.0 NMeFOSAA y = 0.1071648x + 0.001790018 R² = 0.9947207 R = 0.9973568 Conc.Ratio 0 1 2 3 4 5 Area Ratio 0 1 2 8-2 FTS y = 0.5727685x + 0.001723162 R² = 0.9947000 R = 0.9973465 8:2 FTS NMeFOSAA NEtFOSAA PFUnA PFNS PFDS 11Cl-PF3OUdS PFOSA NMeFOSE PFHpS PFDOA PFTrDA PFTeDA PFDA PFOS 9Cl-PF3ONS PFDOS NMeFOSA NEtFOSA Fig. 5 (continued). Calibration curves and MS chromatograms of blank and LLOQ for 40 PFAS compounds Blank Blank Blank Blank Blank Blank Blank Blank Blank 6.3 ng/L 6.3 ng/L 2.5 ng/L 2.5 ng/L 2.5 ng/L 2.5 ng/L 2.5 ng/L 25 ng/L 25 ng/L Blank Blank Blank Blank Blank Blank Blank Blank Blank Blank 2.5 ng/L 2.5 ng/L 6.3 ng/L 10 ng/L 2.5 ng/L 2.5 ng/L 10 ng/L 6.3 ng/L 2.5 ng/L 6.3 ng/L Blank 2.5 ng/L NEtFOSE

Application News www.shimadzu.com/an/ Shimadzu Corporation © Shimadzu Corporation, 2025 For Research Use Only. Not for use in diagnostic procedures. This publication may contain references to products that are not available in your country. Please contact us to check the availability of these products in your country. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. See https://www.shimadzu.com/about/trademarks/index.html for details. Third party trademarks and trade names may be used in this publication to refer to either the entities or their products/services, whether or not they are used with trademark symbol “TM” or “”. Shimadzu disclaims any proprietary interest in trademarks and trade names other than its own. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its 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. First Edition: Aug. 2025 Revision A: Mar. 2026 01-01046A-EN IonFocus, Nexera and Shim-pack Scepter are trademarks of Shimadzu Corporation or its affiliated companies in Japan and/or other countries. Fig. 6 Accuracy plots for PFBS, HFPO-DA, PFOA, PFHxS, PFNA and PFOS in continuing calibration verification (CCV) samples. CCVs were injected after several chicken tissue samples during robustness test. CCV accuracies were within ±30% (gray line) from assigned concentration level over 900 injections.  Conclusion The LCMS-8065XE was able to detect concentrations up to 80 times lower than the LLOQ required in EPA Method 1633A, using a neat standard solution. Excellent linearity was achieved with the developed method, as indicated by RSE values below 20% and high R 2 values. This method demonstrates high throughput and robust instrument performance, maintaining accurate quantification in complex chicken tissue matrices without the need for maintenance or cleaning. The level 5 calibration standard was used as the continuing calibration verification (CCV). This concentration was four times lower than the required LLOQ specified in EPA Method 1633A. CCVs were analyzed according to EPA’s QA/QC requirements to monitor quantitative performance stability. Fig. 6 shows the accuracy of representative PFBS, HFPO-DA, PFOA, PFHxS, PFNA, and PFOS in CCV samples. A total of 930 injections of extracted tissue samples were performed. All CCVs (n=73), shown in Fig. 6, demonstrated accuracies within 70-130%. These results highlight the excellent robustness of the LCMS-8065XE for analysis in complex sample matrices.  Robustness test  Reference 1) Method 1633A, Analysis of Per- and Polyfluoroalkyl Substances (PFAS) in Aqueous, Solid, Biosolids, and Tissue Samples by LC- MS/MS  Acknowledgement The authors would like to thank CEM corporation for kindly providing the samples for their valuable technical support.

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