A sensitive method for simultaneous screening exposure to 14 alkylating agents based on tripeptide adducts: an efficient approach for chemical weapon verification and chemical impurity profiling in plasma samples

For the implementation of the Chemical Weapons Convention, the Organisation for Prohibition of Chemical Weapons (OPCW) designates laboratories for the analysis of chemicals that can be used as chemical warfare agents (CWAs). In these laboratories, analytical methods for detecting CWAs have been developed for environmental and biomedical samples. Protein adducts from exposed biomedical samples are an important type of biomarker for verification of their abuse. Moreover, these adducts could also be applied in chemical impurity profiling studies of biomedical samples. Alkylating agents, as cytotoxicants, can react with Cys³⁴ in human serum albumin, and cysteine-proline-phenylalanine (CPF) tripeptide adducts generated from proteinase K digestion of modified albumin have been used as biomarkers for retrospective detection of exposure to sulfur mustards or nitrogen mustards. In this study, a sensitive screening method for detecting exposure to 14 alkylating agents in one analytical run was established through sample preparation and instrumental detection optimization. Ultrahigh-performance liquid chromatography–triple quadrupole mass spectrometry (UHPLC–TQ MS) operated in multiple reaction monitoring (MRM) mode and combined with an optimized chromatographic program was used for detecting the 14 corresponding CPF tripeptide adducts. The limits of detection (LODs) are in the range of 0.200–10.0 ng/mL exposure concentrations in human plasma. This method could also allow impurity profiling applicable for potential attribution of sulfur and nitrogen mustard in plasma through retrospective analysis of exposed biomedical samples with impurity compositions as low as 0.1%. This method has potential in clinical diagnosis, CWA verification and forensic identification applications.

Graphical Abstract