Development and validation of an analytical method for quantitation of PFAS constituents in rat plasma, urine, and liver by UPLC-MS/MS

Abstract

Aqueous film-forming foams (AFFF) containing per- and polyfluoroalkyl substances (PFAS) used during firefighting training exercises have been released into the environment and have contaminated drinking water supplies. In support of studies investigating bioaccumulation of PFAS following exposure of rats to AFFF formulations, an analytical method was developed and validated to quantitate major PFAS present in AFFF formulations. The PFAS investigated were 6:2 fluorotelomer sulfonic acid (FTS), N,N-dimethyl-3-((perfluorohexyl)ethylsulfonyl)amino-propanamine N-oxide (FTNO), 6:2 fluorotelomer sulfonamide betaine (FtSaB), 6:2 fluorotelomer sulfinyl amido sulfonic acid (FtSiAoS), and 6:2 fluorotelomer thioether amido sulfonic acid (FtTAoS). The method was validated in rat plasma for FTS, FTNOand FtSaB. Since standards were not available for FtSiAoS and FtTAoS, they were quantitated using FTS calibration curves. Standards were prepared by fortifying
100 μL plasma with FTS/FTNO/FtSaB and internal standard (FTS-13C,d4). Samples were extracted with 300 μL methanol, frozen at -80°C to separate phospholipids, and analyzed by UPLC-MS/MS in positive ion mode for the first 6 min (FTNO and FtSaB), then switched to negative ion mode for the remaining 12 min (FTS, FtSiAoS,
and FtTAoS). The method was linear over the range 0.25-100 ng/mL, accurate (mean RE≤±12.0%), and precise (RSD≤8.0%). Mean recoveries were ≥81%; the limits of detection were 0.0507, 0.0354, and 0.118 ng/mL for FTS, FTNO, and FtSaB, respectively. Since standards of FtSiAoS and FtTAoS were not available, matrix QC standards prepared using AFFF formulation containing all five components were used to assess precision. Intra- and inter-day RSD values were ≤13.2% for all components, except inter-day precision for FtTAoS, which showed day-to-day variation in chromatography. The method was evaluated in male Hsd:Sprague Dawley®SD® rat plasma, urine, and liver homogenate (mean %RE≤±18.7 and
%RSD≤7.2 for FTS, FTNO, and FtSaB). Stability of FTS, FTNO, and FtSaB in extracted samples was demonstrated for 5 d at ambient and refrigerated temperatures, as well as in plasma, urine, and liver homogenate stored at -80°C for 63 d (86.5-116% of day 0 concentrations), except for FTNO in liver homogenate (59.8-62.6% of day 0). These data demonstrate that this simple method is suitable for determination of PFAS in rat matrices following exposure of rats to AFFF, but the method could be improved with use of standards for FtSiAoS and FtTAoS.