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A sensitive quantification approach for detection of HETE-CP adduct after benzyl chloroformate derivatization using ultra-high-pressure liquid chromatography tandem mass spectrometry

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Abstract

Sulfur mustard (HD) reacts with human serum albumin (HSA) at Cys34 and produces a long-term biomarker of HD exposure. Here, we present a novel, sensitive, and convenient method for quantification of HD exposure by detection of HD-HSA adducts using pronase digestion, benzyl chloroformate (Cbz-Cl) derivatization, and ultra-high-pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). The HSA in HD-exposed plasma in vitro was precipitated with acetone and digested (2 h, 50 °C) with pronase to form the alkylated dipeptide, S-hydroxyethylthioethyl-CysPro (HETE-CP). The HETE-CP adduct was derivatized with Cbz-Cl to generate N-carbobenzoxy HETE-CP (HETE-C(Cbz)P). The derivatized product was analyzed by UHPLC-MS/MS. HD surrogate, 2-chloroethyl ethyl sulfide (2-CEES), was introduced as a non-isotope internal standard (ISTD) instead of traditional d8-HD for quantification. The method was found to be linear between 1.00 and 200 ng/mL HD exposure (R2 > 0.998) with precision of ≤ 9.0% relative standard deviation (RSD) and accuracy ranged between 97.1 and 111%. The limit of detection (LOD) is 0.500 ng/mL (S/N~5), over 15 times lower than that of the previous method (7.95 ng/mL). Time-consuming affinity purification or solid phase extraction (SPE) is not needed in the experiment and the operation takes less than 5 h. This study provides a new strategy and useful tool for retrospective analysis of HD exposure by HETE-CP biomarker detection.

Flow diagram for quantification of sulfur mustard exposure by detection of HETE-CP dipeptide adduct after benzyl chloroformate derivatization using ultra-high-pressure liquid chromatography tandem mass spectrometry.

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Acknowledgments

The reference chemical of HETE-CP was synthetized by Dr. Dai Xiandong in Research Institute of Chemical Defence.

Funding

The work was financially supported by the State Key Laboratory of NBC Protection for Civilian (Grant No. SKLNBC201405).

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Authors and Affiliations

  1. State Key Laboratory of NBC Protection for Civilian, Beijing, 102205, China

    Bo Chen, Hui-Lan Yu, Shi-Lei Liu, Chang-Cai Liu, Long-Hui Liang, Xin-Hai Li, Xiao-Sen Li, Ji-Na Wu & Yang Yang

  2. Laboratory of Analytical Chemistry, Research Institute of Chemical Defence, Beijing, 102205, China

    Bo Chen, Hui-Lan Yu, Shi-Lei Liu, Chang-Cai Liu, Long-Hui Liang, Xin-Hai Li, Xiao-Sen Li, Ji-Na Wu & Yang Yang

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  1. Bo Chen
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Correspondence to Hui-Lan Yu or Shi-Lei Liu.

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Plasma samples were kindly provided by the 306th Hospital with consent of all individual volunteers, and approved by the Hospital’s Ethics Committee.

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Chen, B., Yu, HL., Liu, SL. et al. A sensitive quantification approach for detection of HETE-CP adduct after benzyl chloroformate derivatization using ultra-high-pressure liquid chromatography tandem mass spectrometry. Anal Bioanal Chem 411, 3405–3415 (2019). https://doi.org/10.1007/s00216-019-01820-3

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