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Integration of Ultrashort-Chain Compounds into the Biomonitoring of Per- and Polyfluorinated Substances in Human Plasma and Serum

Author(s): Shin-Hsin Liang, Justin A. Steimling
Restek Corporation

Published By: Journal of Chromatography Open

Issue: Volume 5

Year of Publication: 2024


Abstract: Ultrashort-chain (USC) per- and polyfluoroalkyl substances (PFAS) are small and very polar compounds with carbon chain lengths of shorter than C4. Their ubiquitous and high levels of occurrence in environmental aquatic systems have raised significant concern in conjunction with long-chain PFAS contamination. Measuring USC PFAS in blood can not only monitor human exposure but also serves as a valuable tool for studying the potential risks associated with USC PFAS exposure. The high polarity of USC PFAS poses a challenge to current analytical practices based on the reversed-phase liquid chromatography, primarily due to insufficient chromatographic retention. In this study, a simple and reliable workflow was developed for the simultaneous analysis of C1 to C10 perfluoroalkyl carboxylic and sulfonic acids, along with four alternative PFAS, in human plasma and serum. The chromatographic analysis was conducted using a polar-embedded reversed-phase LC column. Fetal bovine serum (FBS) was chosen for method validation due to its absence of all analytes, except for trifluoroacetic acid (TFA). Subsequently, a TFA isotope, 13C-TFA, was employed as a surrogate to assess the method accuracy for TFA in FBS. A single-step sample preparation procedure, conducted by mixing 100 µL of FBS with 200 µL of methanol, was demonstrated to be effective for the accurate and precise analysis of fortified FBS samples. Ten isotopes of C3 to C10 PFAS, serving as extracted internal standards, were added to the samples at 1 ppb to validate the accuracy of the entire workflow. Calibration standards were prepared in reverse osmosis water due to its cleanliness for all analytes. Phosphate-buffered saline was incorporated into the calibration standard solution to achieve similar chromatographic performance between standard and sample solutions. The calibration ranges varied among different analytes, spanning from 0.05 – 40 ppb, 0.1 – 40 ppb, 0.25 – 40 ppb, and 0.5 – 40 ppb. Method accuracy and precision were evaluated at three fortification levels, ranging from 0.4 to 30 ppb. All analytes and extracted internal standards exhibited recovery values within 20 % of the nominal concentration across all fortification levels. Satisfactory method precision was demonstrated with%RSD values <12 %. The validated method was applied to measure PFAS in NIST 1950 and 1957 standard reference human plasma and serum, affirming the established workflow is suitable for accurate quantification of C1 to C10 PFAS in human plasma and serum matrices.