Russian Journal of Forensic MedicineRussian Journal of Forensic Medicine2411-87292409-4161Eco-Vector70510.17816/fm705Research ArticleDetection of clobazam and its metabolites in urine during poisoning using HPLC-QqQ-MS/MSVolkovaAlla A.<p>Cand. Sci. (Pharm.)</p>himija@rc-sme.ruhttps://orcid.org/0000-0002-9882-2330KalekinRoman A.<p>Dr. Sci. (Pharm.)</p>himija@rc-sme.ruhttps://orcid.org/0000-0002-4989-3511OrlovaAlevtina M.<p>Cand. Sci. (Pharm.)</p>himija@rc-sme.ruhttps://orcid.org/0000-0002-5419-1418Russian Centre of Forensic Medical ExpertisePeoples› Friendship University of Russia171220228447551203202213102022Copyright © 2022, Eco-Vector2022<p><strong><em>BACKGROUND</em></strong><em>: </em>Determining the incidence of acute and lethal poisoning with clobazam remains an urgent task of analytical toxicology. The medicinal substance clobazam belongs to the group of benzodiazepines, which is included in the list of psychoactive substances whose turnover is limited because of its high toxicity profile in overdose and abuse.</p>
<p><strong><em>AIM</em></strong><em>: </em>To propose a simple, reliable, and sensitive technique for the identification of clobazam and its metabolite in the urine by the modern high-performance liquid chromatography-tandem mass spectrometry (HPLC-QqQ-MS/MS).</p>
<p><strong><em>MATERIALS AND METHODS</em></strong><em>:</em> We have described a simple and sensitive HPLC-QqQ-MS/MS method for the qualitative determination of clobazam in the urine.</p>
<p><strong><em>RESULTS</em></strong><em>: </em>The retention time in the selected chromatography conditions for clobazam was 5.17 min, and its metabolite (norclobazam) in urine was 4.56 min. The base peaks for clobazam and norclobazam were 259 m/z and 245 m/z, respectively.</p>
<p><strong><em>CONCLUSION</em></strong><em>: </em>For the first time, this study provides a validated method of chemical-toxicological examination for poisoning with clobazam by HPLC-MS/MS, tested both on a model mixture and real biological matrix of the patients urine after taking clobazam. This technique can be a confirmatory research method in addition to the clinical picture in forensic medical examination.</p>clobazamhigh-performance liquid chromatography with tandem mass spectrometric detectionQqQ mass analyzerHPLC-QqQ-MS/MSdetectionurine sample preparationклобазамвысокоэффективная жидкостная хроматография с тандемным масс-спектрометрическим детектированиеммасс-анализатор QqQВЭЖХ-QqQ-МС/МСдетекцияподготовка образца мочи氯巴占高效液相色谱串联质谱检测QqQ质量分析仪HPLC-QqQ-MS/MS检测尿样制备[Volkova AA, Orlova AM, Kalekin RA, Nevmyatova SR. Analysis of the possibility of conducting a forensic chemical study in case of Clobazam poisoning. Forensic Medical examination. 2022;65(1):35–40. (In Russ). doi: 10.17116/sudmed20226501135][Markin PA, Moskaleva NE, Appolonova SA, et al. Development of a thin-layer chromatography method for simultaneous determination of Clobazam and zaleplon in a mixture. In: Topical issues of forensic medicine and law. Collection of scientific and practical articles. Kazan; 2021. Р. 158–161. (In Russ).][Orlova AM, Kalekin RA, Volkova AA, et al. Detection of Clobazam in urine by thin-layer chromatography. Bulletin Voronezh State University. 2021;(3):106–113. (In Russ).][Gauthier AC, Mattson RH. Clobazam: A safe. Efficacious and newly rediscovered therapeutic for epilepsy. CNS Neurosci Ther. 2015;21(7):543–548. doi: 10.1111/cns.12399][Arya R, Giridharan N, Anand V, Garg SK. Clobazam monotherapy for focal or generalized seizures. Cochrane Database Syst Rev. 2018;2018(7):CD009258. doi: 10.1002/14651858.CD009258.pub3][Tolbert D, Larsen FA. A comprehensive overview of the clinical pharmacokinetics of Clobazam. J Clin Pharmacol. 2019;59(1):7–19. doi: 10.1002/jcph.1313][Kheireldin R. A case report of Clobazam toxicity related to cannabidiol and Clobazam drug-drug interaction. Translation University Toledo J Med Sci. 2019;(6):35–36. doi: 10.46570/utjms.vol6-2019-339][Brigo F, Lattanzi S. Anticonvulsant agents: Benzodiazepines (Clobazam. Clonazepam. Diazepam. Lorazepam. Midazolam). In book: Riederer P., Laux G., Nagatsu T., et al., editors. NeuroPsychopharmacotherapy. Springer Nature Switzerland AG; 2020. Р. 1–8. doi: 10.1007/978-3-319-56015-1_440-1][Kheireldin R. A case report and literature review of Clobazam toxicity related to cbd and Clobazam drug-drug interaction. J Clin Med Res. 2019. doi: 10.37191/Mapsci-2582-4333-1(2)-012][Aung T. Rare but life-threatening aspiration pneumonia related to initiation of Clobazam therapy. Epilepsy Behavior Rep. 2020;14:100406. doi: 10.1016/j.ebr.2020.100406][Huddart R, Leeder JS, Altman RB, Klein TE. Pharm GKB summary: Clobazam pathway. Pharmacokinetics. Pharmacogenet Genomics. 2018;28(4):110–115. doi: 10.1097/FPC.0000000000000327][Hammer H, Ebert B, Jensen HS, Jensen AA. Functional characterization of the 1.5-benzodiazepine Clobazam and its major active metabolite N-desmethylclobazam at human GABAA receptors expressed in xenopus laevis oocytes. PLoS One. 2015;10(3):e0120239. doi: 10.1371/journal.pone.0120239][Souri E, Farahani AD, Ahmadkhaniha R, Amini M. A stability indicating HPLC method for the determination of Clobazam and its basic degradation product characterization. Daru. 2014;22(1):49. doi: 10.1186/2008-2231-22-49][Jensen HS, Nichol K, Lee D, Ebert B. Clobazam and its active metabolite N-desmethylclobazam display significantly greater affinities for α2- versus α1-GABAA-receptor complexes. PLoS One. 2014;9(2):e88456. doi: 10.1371/journal.pone.0088456][Bajaja AO, Ly D, Johnson-Davisab KL. Retrospective analysis of metabolite patterns of Clobazam and N-desmethylclobazam in human plasma by LC-MS/MS. J Mass Spectrom Adv Clin Lab. 2022;(24):100–106. doi: 10.1016/j.jmsacl.2022.04.005][Laloup M, Fernandez MR, De Boeck G, et al. Validation of a liquid chromatography-tandem mass spectrometry method for the simultaneous determination of 26 benzodiazepines and metabolites, zolpidem and zopiclone, in blood, urine, and hair. J Anal Toxicol. 2005;29(7):616–626. doi: 10.1093/jat/29.7.616]