Structural identification and comparative assessment of the content of BZ incapacitant metabolite in rat urine and blood plasma

Highlights
- use of non-lethal chemical agents by Ukrainian armed and terrorist formations has been documented in the Special Military Operation zone, constituting a violation of the Chemical Weapons Convention;
- a chromatographic-mass spectrometric method has been developed to confirm exposure to BZ and its analogs through identification of their metabolites in urine and blood plasma.
Relevance. Since 2022, two instances of EA-3167 detection have been recorded in the SMO zone - a structural and functional analog of the incapacitating agent BZ listed in Schedule 2 of the CWC. Cases of intoxication among Russian military personnel have been reported. Despite BZ being known for decades, information about its metabolism and potential exposure markers in open sources remains extremely limited and insufficient.
Purpose of the study is to detect and structurally identify BZ metabolites in rat urine and blood plasma; to assess the relative content of BZ and its metabolites.
Materials and Methods. High-resolution liquid and gas chromatography-mass spectrometry. Evaluation of relative metabolite content was based on peak intensity (area) measurements under HPLC-HRMS conditions. Characteristics of metabolites specific to BZ exposure were determined.
Results. Over 40 BZ metabolites formed through oxidative and (partially) hydrolytic modifications.
Conclusion. For of the parent molecule were tentatively identified in rat urine and plasma. Structures of Phase I oxidized metabolites contained up to four additional oxygen atoms incorporated as hydroxyl groups on phenyl residues and N-oxide. Phase I metabolites undergo intensive conjugation forming O-methylated and glucuronidated forms, and their combinations. Levels of several metabolites in urine (including glucuronide of dihydroxylated methylated metabolite) increased up to 24 hours post-exposure. Unchanged BZ was present in rat urine only in minor concentrations.forensic confirmation of BZ exposure using chromatography-mass spectrometry, monohydroxylated, N-oxidized, and dihydroxylated methylated forms are most suitable. When significant time has passed after exposure, detection of glucuronides of monohydroxylated and dihydroxylated methylated metabolites is recommended.
Practical significance of the work. The developed approach allows for the objective confirmation of poisoning by the incapacitating agent BZ and its analogues through the detection of stable metabolites in biological samples. The results are of high practical importance for chemical-analytical support in monitoring compliance with the Chemical Weapons Convention and for forensic medical examination.

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