Unraveling the metabolic transformation of tetrazepam to diazepam with mass spectrometric methods
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Abstract
The metabolic transformation pathways of the 1,4-benzodiazepine tetrazepam (C16H17ClN2O, average mass: 288.772) were studied with capillary LC-QqTOF-MS and -MS/MS by analyzing human plasma and urine samples collected from healthy volunteers. Each volunteer took 50 mg of tetrazepam, given in the form of one tablet of Myolastan (Sanofi-Synthelabo, Vienna, Austria). Accurate molecular mass measurements in full-scan mode (scan range: 50–700) were used to survey the collected samples for putative metabolic transformation products. Full-scan fragment ion mass spectra were collected in subsequent LC/MS/MS experiments. Each spectrum was matched to a spectral library containing 3759 MS/MS-spectra of 402 compounds, including eighteen different benzodiazepines, to prove the structural relatedness of a tentative metabolite to tetrazepam. This “similarity search” approach provided a rapid and powerful tool to exclude non-drug-related species, even without any knowledge of the fragmentation chemistry. Interpretation of tandem mass spectrometric data was only required in order to elucidate the site of transformation. Using this strategy, 11 major classes of tetrazepam metabolites were identified. Possible metabolic routes from tetrazepam to diazepam (C16H13ClN2O, average mass: 284.740) via repeated hydroxylation and dehydration of the cylohexenyl moiety were discovered. No evidence for extensive hydroxylation of tetrazepam at position 3 of the diazepine ring was found. In contrast to what is commonly believed, this distinct transformation reaction may be of only minor importance. Furthermore, the occurrence of demethylation, hydration, and glucuronidation reactions was proven.
Principle workflow applied for the identification of tetrazepam metabolites
Keywords
Mass spectrometry Liquid chromatography Electrospray ionization Tetrazepam Metabolism Library searchNotes
Acknowledgments
The authors wish to thank Applied Biosystems for their generous provision of the mass spectrometer and the associated equipment. Furthermore, financial support from the Austrian Research Promotion Agency (FFG, Österreichisches Sicherheitsforschungs-Förderprogramm “KIRAS—eine Initiative des Bundesministeriums für Verkehr, Innovation, Technologie (BMVIT), Projekt 813786”) is acknowledged.
References
- 1.Keane PE, Simiand J, Morre M, Biziere K (1988) J Pharmacol Exp Ther 245:692Google Scholar
- 2.Keane PE, Bachy A, Morre M, Biziere K (1988) J Pharmacol Exp Ther 245:699Google Scholar
- 3.Baumgartner MG, Cautreels W, Langenbahn H (1984) Arzneimittelforschung 34:724Google Scholar
- 4.Pavlic M, Libiseller K, Grubwieser P, Schubert H, Rabl W (2007) Int J Legal Med 121:169CrossRefGoogle Scholar
- 5.Laloup M, del Mar Ramirez Fernandez M, Wood M, Maes V, De Boeck G, Vanbeckevoort Y, Samyn N (2007) Anal Bioanal Chem 388:1545CrossRefGoogle Scholar
- 6.Kostiainen R, Kotiaho T, Kuuranne T, Auriola S (2003) J Mass Spectrom 38:357CrossRefGoogle Scholar
- 7.Ma S, Chowdhury SK, Alton KB (2006) Curr Drug Metab 7:503CrossRefGoogle Scholar
- 8.Prakash C, Shaffer CL, Nedderman A (2007) Mass Spectrom Rev 26:340CrossRefGoogle Scholar
- 9.Oberacher H, Krajete A, Parson W, Huber CG (2000) J Chromatogr A 893:23CrossRefGoogle Scholar
- 10.Pavlic M, Libiseller K, Oberacher H (2006) Anal Bioanal Chem 386:69CrossRefGoogle Scholar
- 11.Smyth WF, McClean S, Ramachandran VN (2000) Rapid Commun Mass Spectrom 14:2061CrossRefGoogle Scholar
- 12.Risoli A, Cheng JB, Verkerk UH, Zhao J, Ragno G, Hopkinson AC, Siu KW (2007) Rapid Commun Mass Spectrom 21:2273CrossRefGoogle Scholar
- 13.Staak M, Sticht G, Kaferstein H, Norpoth T (1984) Beitr Gerichtl Med 42:75Google Scholar
- 14.Staak M, Sticht G, Saternus KS, Kaferstein H (1982) Beitr Gerichtl Med 40:323Google Scholar
- 15.Maurer H, Pfleger K (1987) J Chromatogr 422:85CrossRefGoogle Scholar