Characterization and tentative identification of new flunitrazepam metabolites in authentic human urine specimens using liquid chromatography‐Q exactive‐HF hybrid quadrupole‐Orbitrap‐mass spectrometry (LC‐QE‐HF‐MS)

Abstract

Flunitrazepam (FNZ) is a potent hypnotic, sedative, and amnestic drug used to treat severe insomnia. In our recent study, FNZ metabolic profiles were investigated carefully. Six authentic human urine samples were purified using solid phase extraction (SPE) without enzymatic hydrolysis, and urine extracts were then analyzed by liquid chromatography-Q exactive-HF hybrid quadrupole-Orbitrap-mass spectrometry (LC-QE-HF-MS), using the full scan positive ion mode and targeted MS/MS (ddms2) technique to make accurate mass measurements. There were 25 metabolites, including 13 phase I and 12 phase II metabolites, which were detected and tentatively identified by LC-QE-HF-MS. In addition, nine previously unreported phase II glucuronide conjugates and four phase I metabolites are reported here for the first time. Eight metabolic pathways, including N-reduction and O-reduction, Nglucuronidation, O-glucuronidation, mono-hydroxylation and di-hydroxylation, demethylation, acetylation, and combinations, were implicated in this work, and 2-O-reduction together with dihydroxylation were two novel metabolic pathways for FNZ that were identified tentatively. Although 7-amino FNZ is widely considered to be the primary metabolite, a previously unreported metabolites (M12) can also serve as a potential biomarker for FNZ misuse. KEYWORDS chemical structural identification, flunitrazepam, human urine, liquid chromatography-Q exactive-HF hybrid quadrupole-Orbitrap-mass spectrometer, metabolic profiles

Detection of Flunitrazepam in Biological Fluids and Beverages:A Review

“…They found that after oral administration of FLU, about 90 % of the metabolites were excreted in urine and 10 % in feces. [23] Therefore, in handling cases related to FLU, whole blood, plasma and urine samples should be collected as early as possible for qualitative and quantitative detection of FLU and its metabolites, 7AF.…”

“…Qin and coworkers developed a liquid chromatography-Q Extractive-HF Hybrid Quadrupole-Orbitrapmass spectrometry (LC-QE-HF-MS) detection system. [23] This MS with high resolution and equipped with a heated-EIS detects peaks of potential metabolites for direct analysis of intact metabolites in full-scan and data-dependent scan (DDMS2) mode with the theoretical [M + H] + as the target ion. A total of 25 metabolites were detected, 12 of which have been reported and 13 unreported, but only 3 metabolites belonged to the FLU type and the others to the 7AF type.…”