Forensic Science International Looking at flubromazolam metabolism from four different angles: Metabolite profiling in human liver microsomes, human hepatocytes, mice and authentic human urine samples with liquid chromatography high-resolution mass spectrometry  Ariane Wohlfarth, Svante Vikingsson, Markus Roman, Mikael Andersson, Fredrik C. Kugelberg, Henrik Green, Robert Kronstrand  Forensic Science International  Volume 274, Pages 55-63 (May 2017) DOI: 10.1016/j.forsciint.2016.10.021 Copyright © 2016 Elsevier Ireland Ltd Terms and Conditions

Fig. 1 Structures of flubromazolam and related benzodiazepines midazolam, alprazolam, triazolam, pyrazolam and flubromazepam. Forensic Science International 2017 274, 55-63DOI: (10.1016/j.forsciint.2016.10.021) Copyright © 2016 Elsevier Ireland Ltd Terms and Conditions

Fig. 2 Combined extracted ion chromatograms showing flubromazolam and metabolites in hydrolyzed (continuous line) and non-hydrolyzed (gray line) mouse urine and two representative human urine specimens. Arrows indicate the release of the phase I metabolite/parent from the phase II conjugates. Bold labeled metabolites represent major differences between mouse and human: whereas the mouse metabolism proceeds to dihydroxylated metabolites, the human metabolism rather conjugates monohydroxylated metabolites and the parent itself with glucuronic acid. Forensic Science International 2017 274, 55-63DOI: (10.1016/j.forsciint.2016.10.021) Copyright © 2016 Elsevier Ireland Ltd Terms and Conditions

Fig. 3 Product ion spectra and proposed structures of flubromazolam and metabolites. No product ion spectra were obtained for M1 and M4 due to low intensity. Forensic Science International 2017 274, 55-63DOI: (10.1016/j.forsciint.2016.10.021) Copyright © 2016 Elsevier Ireland Ltd Terms and Conditions

Fig. 4 Proposed structures and formulas, exact masses, mass measurement errors and electron configuration (EE=even-electron, OE=odd-electron configuration) for characteristic fragment ions present in the product ion spectrum of flubromazolam. The most intense ions are highlighted in bold. Forensic Science International 2017 274, 55-63DOI: (10.1016/j.forsciint.2016.10.021) Copyright © 2016 Elsevier Ireland Ltd Terms and Conditions

Fig. 5 Proposed structures and formulas, exact masses, mass measurement errors and electron configuration (EE=even-electron, OE=odd-electron configuration) for characteristic fragment ions present in the product ion spectrum of α-hydroxy-flubromazolam. The most intense ions are highlighted in bold. Structures for common fragment ions described for the analogous metabolites α-hydroxy-alprazolam and α-hydroxy-triazolam are shown at the right for purpose of comparison (see further explanation in the text). Forensic Science International 2017 274, 55-63DOI: (10.1016/j.forsciint.2016.10.021) Copyright © 2016 Elsevier Ireland Ltd Terms and Conditions

Fig. 6 Proposed structures and formulas, exact masses, mass measurement errors and electron configuration (EE=even-electron, OE=odd-electron configuration) for characteristic fragment ions present in the product ion spectrum of 4-hydroxy-flubromazolam. The most intense ions are highlighted in bold. Forensic Science International 2017 274, 55-63DOI: (10.1016/j.forsciint.2016.10.021) Copyright © 2016 Elsevier Ireland Ltd Terms and Conditions