Chinese Journal of Chromatography

Chinese Journal of Chromatography ›› 2026, Vol. 44 ›› Issue (7): 764-776.DOI: 10.3724/SP.J.1123.2025.07008

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Determination of 29 per- and polyfluoroalkyl substances and 22 organophosphate esters and diester metabolites in human serum by high-throughput solid-phase extraction- ultra performance liquid chromatography- high resolution mass spectrometry

ZHAO Shiqi1,3, DING Hao4, ZHANG Xuwenqi4, HOU Minmin1,3,*(), ZHOU Tingting4, SHI Yali1,2,3, CAI Yaqi1,2,3   

  1. 1.Zhejiang Key Laboratory of Environment & Health of New Pollutants，School of Environment，Hangzhou Institute for Advanced Study，University of Chinese Academy of Sciences，Hangzhou 310024，China
    2.State Key Laboratory of Environment Chemistry and Ecotoxicology，Research Center for Eco-Environmental Sciences，Chinese Academy of Sciences，Beijing 100085，China
    3.University of Chinese Academy of Sciences，Beijing 100049，China
    4.Zhejiang Key Laboratory of Environment and Health of New Pollutants，Ecological and Environmental Science and Research Institute of Zhejiang Province，Hangzhou 310007，China
  • Received:2025-07-23 Online:2026-07-08 Published:2026-07-09
  • Supported by:
    National Natural Science Foundation of China(22320102005)

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Abstract:

Monitoring pollutants in human blood is a crucial basis for assessing human exposure levels and health risks. Per- and polyfluoroalkyl substances （PFAS）， organophosphate esters （OPEs）， and their diester metabolites （di-OPEs） are widespread environmental co-contaminants with significant toxic effects， making it crucial to monitor their internal human exposure levels. However， existing studies have predominantly investigated these substances in isolation， lacking comprehensive research that simultaneously quantifies PFAS， OPEs， and di-OPEs in human serum. Based on the pretreatment method of 96-well solid phase extraction columns， this study compared three extraction columns and optimized the pretreatment steps to establish an ultra performance liquid chromatography-high resolution mass spectrometry （UPLC-HRMS） method for 29 PFAS， 17 OPEs， and 5 di-OPEs in human serum samples. Phree PLR 96-Wellplate was used as a cleanup plate. 300 µL of 1% formic acid in acetonitrile， 100 µL of the serum sample， and internal standards were added to the cleanup device in sequence. After standing for 5 minutes， the 96-well plate positive pressure device was used to press the mixture into the 96-well collection plate. Finally， the sample was eluted with 100 µL of 1% formic acid in acetonitrile. The eluate was collected and concentrated for the detection of PFAS， OPEs， and di-OPEs by UPLC-HRMS. PFAS and OPEs were detected using an Acclaim RSLC 120 C18 column， while di-OPEs were detected using an Acquity UPLC BEH C18 column. Both were subjected to gradient elution with methanol and 5 mmol/L ammonium acetate in water as the mobile phases. Sample ionization was performed using a heated electrospray ionization source （H-ESI）. PFAS and di-OPEs were analyzed in negative ion mode， while OPEs were analyzed in positive ion mode. Data acquisition was conducted in full-scan/data-dependent tandem mass spectrometry （Full MS/ddMS2） mode. Quantification was achieved using the internal standard calibration method to ensure measurement accuracy. The results showed that under the optimized conditions， the target compounds had good linear relationships in the range of 0.05–50 ng/mL （R2 > 0.99）， and the method detection limits （MDLs） of 29 PFAS， 17 OPEs and 5 di-OPEs were 0.000 120–0.274 ng/mL， 0.011 0–0.250 ng/mL， and 0.012 0–0.220 ng/mL， respectively， and the spiked recoveries were between 45.9% and 147.8%. The relative standard deviations （RSDs） were 1.2%–29.0%. Most PFAS and di-OPEs had matrix enhancement effects， and most OPEs had matrix inhibition effects. Among them， hexafluoropropylene oxide dimer acid （GenX） （196.5%） and trimethylphenyl phosphate （TMPP） （54.6%） had significant matrix enhancement and inhibition effects， respectively， which could be corrected with appropriate internal standards. The recoveries of these two substances after correction were 127.3% and 78.7%， respectively， which met the analysis requirements. The proposed approach offers significant practical benefits， combining straightforward operation with shortened extraction time and enhanced throughput， which was validated through analysis of 15 human serum samples collected in Jinan in 2024. The total contents of 29 PFAS were 6.71–379 ng/mL， with a median value of 22.9 ng/mL. Eight PFAS were detected with a detection frequency of 100.0%， with median contents of perfluorooctanoic acid （PFOA） and perfluorooctane sulfonate （PFOS） reaching 8.96 ng/mL and 4.07 ng/mL， respectively. The total contents of 17 OPEs were 0.015 0–10.5 ng/mL， with a median value of 2.81 ng/mL. The most frequently detected OPEs， with detection frequencies exceeding 60.0%， were triethyl phosphate （TEP）， tri-n-butyl phosphate （TnBP）， and triphenyl phosphate （TPHP）. The total contents of 5 di-OPEs were <MDL–0.443 ng/mL， with a median value of 0.015 0 ng/mL. Therefore， the combined exposure to these pollutants in human blood and its potential health risks demand serious attention.

Key words: per- and polyfluoroalkyl substances （PFAS）, organophosphate esters （OPEs）, 96-wellplate, ultra performance liquid chromatography-high resolution mass spectrometry （UPLC-HRMS）, serum

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