Qualitative analysis of 7- and 8-hydroxyzolpidem and discovery of novel zolpidem metabolites in postmortem urine using liquid chromatography–tandem mass spectrometry

Abstract

Purpose Zolpidem (ZOL) is a hypnotic sometimes used in drug-facilitated crimes. Understanding ZOL metabolism is important for proving ZOL intake. In this study, we synthesized standards of hydroxyzolpidems with a hydroxy group attached to the pyridine ring and analyzed them to prove their presence in postmortem urine. We also searched for novel ZOL metabolites in the urine sample using liquid chromatography–triple quadrupole mass spectrometry (LC-QqQMS) and liquid chromatography–quadrupole time-of-flight mass spectrometry (LC-QqTOFMS). Methods 7- and 8-Hydroxyzolpidem (7OHZ and 8OHZ, respectively) were synthesized and analyzed using LC-QqQMS. Retention times were compared between the synthetic standards and extracts of postmortem urine. To search for novel ZOL metabolites, first, the urine extract was analyzed with data-dependent acquisition, and the peaks showing the characteristic fragmentation pattern of ZOL were selected. Second, product ion spectra of these peaks at various collision energies were acquired and fragments that could be used for multiple reaction monitoring (MRM) were chosen. Finally, MRM parameters were optimized using the urine extract. These peaks were also analyzed using LC-QqTOFMS. Results The presence of 7OHZ and 8OHZ in urine was confirmed. The highest peak among hydroxyzolpidems was assigned to 7OHZ. The novel metabolites found were zolpidem dihydrodiol and its glucuronides, cysteine adducts of ZOL and dihydro(hydroxy)zolpidem, and glucuronides of hydroxyzolpidems. Conclusions The presence of novel metabolites revealed new metabolic pathways, which involve formation of an epoxide on the pyridine ring as an intermediate.

2-(4-Fluorophenyl)-1H-benzo[d]imidazole as a Promising Template for the Development of Metabolically Robust, α1β2γ2GABA-A Receptor-Positive Allosteric Modulators

“…First, a fluorine atom was placed as a substitute for the methyl group at the four position of the phenyl ring, which is the group most prone to metabolic degradation. 7 We envisioned that replacing this methyl group with incorporation of a fluorine atom might be harnessed to improve the metabolic stability of potential GABA-A receptor ligands. The value of this strategy steams out from our previous studies showing that single-site fluorination shows higher metabolic stability over compounds bearing two fluorine atoms attached at both sites of the aromatic system.…”

“…(8). The title compounds (7) and (8) were obtained as an inseparable mixture of regioisomers. The mixture of the title compounds was obtained using a mixture of ethyl 2-…”

“…The latter compound undergoes rapid biotransformation forming a reactive epoxide intermediate that induces glutathione deprivation and acute liver toxicity in patients. 7,19,20 In line with the literature data, 19 we observed that alpidem was heavily metabolized producing high content of metabolites (M1, M2, M3, M4, and M5), and solely 38.60% of the parent compound remained unmetabolized (Table 3), after 120 min of incubation with HLMs. Literature data suggest that the biotransformation pathway of alpidem occurs mainly within 7and 8-position of the imidazo[1,2-a]pyridine ring, producing an epoxide intermediate, which is further transformed into hydroxy counterparts or glutathione adducts (Figure 2).…”

“…Although these agents are excellent nanomolar binders of α1β2γ2GABA-A receptors, within the α1/γ2 interface, they are characterized by high metabolic vulnerability, which is suitable for rapid insomnia treatment, but would be challenging for long-term treatments in neurological patients. In the case of alpidem, the formation of the toxic epoxide metabolite that deprives the glutathione reservoir, , induces hepatotoxicity, and poses an additional hurdle (Figure ). This particular feature led to the final withdrawal of alpidem from the pharmaceutical marked .…”

“…Reported positive allosteric modulators (PAMs) of the GABA-A receptor based on the imidazo­[1,2- a ]­pyridine scaffold and their main metabolic sites, conferring high metabolic degradation . GSHglutathione.…”