Bemethyl is an actoprotector, an antihypoxant, and a moderate psychostimulant. Even though the therapeutic effectiveness of bemethyl is well documented, there is a gap in knowledge regarding its metabolic products and their quantitative and qualitative characteristics. Since 2018, bemethyl is included to the Monitoring Program of the World Anti-Doping Agency, which highlights the challenge of identifying its urinary metabolites. The objective of the study was to investigate the biotransformation pathways of bemethyl using a combination of liquid chromatography-high-resolution mass spectrometry and in silico studies. Metabolites were analyzed in a 24 h rat urine collected after oral administration of bemethyl at a single dose of 330 mg/kg. The urine samples were prepared for analysis by a procedure developed in the present work and analyzed by high performance liquid chromatography–tandem mass spectrometry. For the first time, nine metabolites of bemethyl with six molecular formulas were identified in rat urine. The most abundant metabolite was a benzimidazole–acetylcysteine conjugate; this biotransformation pathway is associated with the detoxification of xenobiotics. The BioTransformer and GLORY computational tools were used to predict bemethyl metabolites in silico. The molecular docking of bemethyl and its derivatives to the binding site of glutathione S-transferase has revealed the mechanism of bemethyl conjugation with glutathione. The findings will help to understand the pharmacokinetics and pharmacodynamics of actoprotectors and to improve antihypoxant and adaptogenic therapy.

To date, chemical weapons in the Russian Federation have been completely destroyed. Former chemical weapons destruction facilities (CWDFs) are subject to re-profiling. The most important stage of this activity is to assess the safety of engineering elements of the facilities to find out whether they can be involved in national economy. The most toxic and persistent substance processed at the CWDFs was a V-series agent. The certified gas chromatographic methods previously used for control of residual amounts of this substance on the surfaces and in deep layers of materials were based on its conversion to acid fluoride on silver fluoride pads. The latter are no longer produced, and these methods are impossible to use. The use of liquid tandem chromatography-mass spectrometry (HPLC-MS/MS) provided a direct determination of VR in the different matrices at the level of the toxicity threshold. At the same time HPLC-MS/MS method allows to determine the toxic acid formed by the hydrolysis of the P–O bond in the VR molecule. Previously no methods for the determination of this acid were available. A unified procedure for the sensitive target determination of VR (O-isobutyl S- (2-diethylamino)ethyl methylphosphonothioate) and its most toxic hydrolysis product S-[(2-diethylamino)ethyl] methylphosphonothioic acid in surface wipes and in deep layers of various materials is proposed. The method involves the addition of an internal standard (paraoxon) to the sample, extraction with methanol, concentration of the extract, and analysis by HPLC-MS/MS in the multiple reaction monitoring (MRM) mode. The detection limits of the analytes are close to their toxic threshold levels. Special emphasis in the procedure is paid to the reliability of identification of the analytes, which is of key importance in international investigations under the Chemical Weapons Convention.

The review considers and summarizes separation and preconcentration methods used in the determination of toxicants of the 1st hazard class, namely, organophosphorus nerve agents (a group of Vx isomers, sarin, cyclosarin, soman, and tabun) and blister agents (sulfur mustard).

The capabilities and limitations of complex procedures for targeted metabolomic analysis using liquid chromatography in combination with tandem mass spectrometry (HPLC-MS/MS) are discussed. An HPLC-MS/MS procedure for the simultaneous determination of the concentrations of 15 biomarkers of the functional state of a human being in urine is presented. The target analytes are biogenic substances of various chemical natures, the basic concentrations of which in biomatrices can vary significantly, up to three orders of magnitude or more. Complex analysis is also difficult to perform due to significant differences in the hydrophilic and hydrophobic properties of the analytes. The testing of the procedure in a bioanalytical experiment made it possible to establish significant differences in the concentrations of a number of biomarkers in the urine of persons with different levels of physical fitness. With a high level of physical fitness, the concentrations of these compounds in urine have lower values in comparison with those in the control group.

Сформирован перечень веществ (маркеров) для ретроспективного установления факта загрязнения в прошлом различных материалов фосфорорганическими отравляющими веществами. К «долгоживущим» маркерам загрязнения относятся: O-изобутил-S-(2-диэтиламиноэтил) метилфосфонотиоат (VR) и 11 продуктов конверсии фосфороганических отравляющих веществ: диизопропилметилфосфонат, изопропил-изобутил метилфосфонат, изобутил-пинаколил метилфосфонат, дипинаколил метилфосфонат, диизобутил метилфосфонат, изобутил метилфосфоновая кислота, бис(2-диэтил-аминоэтил) дисульфид, S-2-(диэтиламиноэтил) метилфосфонотиоат, О-изопропил метилфосфоновая кислота, О-изобутил метилфосфоновая кислота, О-пинаколил метилфосфоновая кислота, метилфосфоновая кислота. Разработана методика обнаружения и идентификации перечисленных маркеров в измельченных твердых материалах методом высокоэффективной жидкостной хроматографии с тандемным масс-селективным детектированием. В случае обнаружения высокотоксичных веществ: VR и S-2-(диэтиламиноэтил) метилфосфонотиоата, предусмотрено их количественное определение по аттестованной методике ФР.1.31.2020.36539. A list of substances (markers) has been established for the retrospective determination of the fact of contamination of various materials with organophosphorus toxic substances. The "long-lived" markers of contamination include: O-isobutyl-S-(2-diethylaminoethyl) methylphosphonothioate (VR) and 11 products of the conversion of organophosphorus toxic substances: diisopropylmethylphosphonate (diPrMP), isopropyl-isobutyl methylphosphonate (iPr-iBMP), isobutyl-pinacolylmethylphosphonate (iBPMP), dipinacolylmethylphosphonate (dPMP), diisobutylmethylphosphonate (diBMP), isobutyl methylphosphonic acid (iBMPA), bis(2-diethyl-aminoethyl) disulfide (DEAEdiS); S-2-(diethylaminoethyl) methylphosphonothioate (DEMP); O-isopropyl methylphosphonic acid (iPrMPA), O-isobutyl methylphosphonic acid (iBMPA), O-pinacolylmethylphosphonic acid (PMPA), methylphosphonic acid (MPA). A method for detecting and identifying these markers in building materials by high-performance liquid chromatography with tandem mass-selective detection has been developed. In the case of detection of highly toxic VR and S-2-(diethylaminoethyl) methylphosphonothioate, their quantitative determination is provided according to the certified method of FR.1.31.2020.36539.

Wipe sampling is widely used for microbiological control purposes. Sanitary and chemical studies also include analysis of samples wiped from the work surfaces during routine and periodic working conditions safety inspections at chemical facilities. The analysis also allows assessing the toxicity and hazard of items/structures that could be in contact with highly toxic substances. This study aimed to investigate the capabilities and limitations of the surface wipe sample analysis method in control of residual contamination of equipment and building structures of a former chemical weapons destruction facilities (CWDF) with sulfur mustard and O-isobutyl-S(2-diethylaminoethyl) methylphosphonothioate (VR), as well as their degradation products. Gas chromatography with tandem mass spectrometry (GC-MS/MS) enabled identification of the sulfur mustard markers, high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) allowed identifying VR markers. An assessment of the matrix influence on the results of GC-MS/MS and HPLC-MS/MS analysis was carried out. The matrix effect was established to affect the results the most in case of HPLC-MS/MS analysis: for GC-MS/MS analysis of target substances, the matrix factor averaged at 60–80%, for HPLC-MS/ MS it was less than 40%. The average percent sulfur mustard recoveries from three types of surfaces (PVC tiles, laminate and metal plates) was 9 ± 2%, 0.13 ± 0.02% and 0.10 ± 0.03%; in case of VR, the recoveries was 2.7 ± 0.5%, 11.8 ± 0.3% and 0.8 ± 0.1%, respectively. The limits of detection for sulfur mustard by GC-MS/MS and VR by HPLC-MS/MS were established at 0.001 MPL and 0.02 MPL, respectively. The developed approaches were applied to the analysis of wipe samples from the surfaces of the equipment and engineering structures of the former CWDF.