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

Koji Yamaguchi,Yoshimasa Kanawaku,Youkichi Ohno,Hajime Miyaguchi

doi:10.1007/s11419-021-00611-9

Abstract

PurposeZolpidem (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).Methods7- 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.ResultsThe 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.ConclusionsThe presence of novel metabolites revealed new metabolic pathways, which involve formation of an epoxide on the pyridine ring as an intermediate.

Highlights

Hypnotics are used to treat insomnia, but they are sometimes involved in drug-facilitated crimes including sexual assaults, homicides, and robberies
In the past few decades, liquid chromatography–tandem mass spectrometry (LC–MS/MS) utilizing the triple quadrupole (QqQ) and quadrupole-time-of-flight (QqTOF) analyzers has become widely used in forensic toxicological analysis because of its excellent selectivity and sensitivity [4, 5]
We developed a synthetic method for ZOL metabolites and successfully synthesized M1–M4 [9]

Summary

Introduction

Hypnotics are used to treat insomnia, but they are sometimes involved in drug-facilitated crimes including sexual assaults, homicides, and robberies. In such cases, forensic toxicologists are asked to analyze the hypnotics and/or their metabolites in body fluids, such as blood, urine, and saliva, obtained from a victim. Drug metabolites can be found that are produced from only a small fraction of an administered drug By applying these technologies, novel metabolites not known at the time of launch can be discovered even for well-known hypnotics, such as Z-drugs (ZOL, and zopiclone) and benzodiazepines. We analyzed ZOL metabolites in human blood and urine using LC-QqQMS, and revealed the presence of an unknown metabolite, which is a hydroxyzolpidem with a hydroxy group on the pyridine ring. We searched for novel ZOL metabolites in postmortem urine using LC-QqQMS and LC-QqTOFMS

Materials and methods

Result

Discussion

Findings

Conclusions