A quantitative method to detect human exposure to sulfur and nitrogen mustards via protein adducts.

Affiliation

Emergency Response Branch, Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, 4770 Buford Highway, NE, Atlanta, GA 30341, United States. Electronic address: [Email]

Abstract

Sulfur and nitrogen mustards are internationally banned vesicants listed as Schedule 1 chemical agents in the Chemical Weapons Convention. These compounds are highly reactive electrophiles that form stable adducts to a variety of available amino acid residues on proteins upon exposure. We present a quantitative exposure assay that simultaneously measures agent specific protein adducts to cysteine for sulfur mustard (HD) and three nitrogen mustards (HN1, HN2, and HN3). Proteinase K was added to a serum or plasma sample to digest protein adducts and form the target analyte, the blister agent bound to the tripeptide cysteine-proline-phenylalanine (CPF). The mustard adducted-tripeptide was purified by solid phase extraction and analyzed using isotope dilution LC-MS/MS. Product ion structures were identified using high-resolution product ion scan data for HD-CPF, HN1-CPF, HN2-CPF, and HN3-CPF. Thorough matrix comparison, analyte recovery, ruggedness, and stability studies were incorporated during method validation to produce a robust method. The method demonstrated long term-stability, precision (RSD < 15%), and intra- and inter-day accuracies > 85% across the reportable range of 3.00-200 ng/mL for each analyte. Compared to previously published assays, this method quantitates both sulfur and nitrogen mustard exposure biomarkers, requires only 10 μL of sample volume, and can use either a liquid sample or dried sample spot.

Keywords

Cysteine adducts,LC-MS/MS,Nitrogen mustard,Protein biomarker,Quantitative method,Sulfur mustard,

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