UHPLC‐MS/MS and UHPLC‐HRMS identification of zolpidem and zopiclone main urinary metabolites and method development for their toxicological determination

Abstract

Zolpidem and zopiclone (Z-compounds) are non-benzodiazepine hypnotics of new generation that can be used in drug-facilitated sexual assault (DFSA). Their determination in biological fluids, mainly urine, is of primary importance; nevertheless, although they are excreted almost entirely as metabolites, available methods deal mainly with the determination of the unmetabolized drug. This paper describes a method for the determination in urine of Z-compounds and their metabolites by ultra-high-pressure liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS) and UHPLC coupled with high resolution/high accuracy Orbitrap® mass spectrometry (UHPLC-HRMS). The metabolic profile was studied on real samples collected from subjects in therapy with zolpidem or zopiclone; the main urinary metabolites were identified and their MS behaviour studied by MS/MS and HRMS. Two carboxy- and three hydroxy- metabolites, that could be also detected by gas chromatography/mass spectrometry (GC-MS) as trimethylsylyl derivatives, have been identified for zolpidem. Also, at least one dihydroxilated metabolite was detected. As for zopiclone, the two main metabolites detected were N-demethyl and N-oxide zopiclone. For both substances, the unmetabolized compounds were excreted in low amounts in urine. In consideration of these data, a UHPLC-MS/MS method for the determination of Z-compounds and their main metabolites after isotopic dilution with deuterated analogues of zolpidem and zopiclone and direct injection of urine samples was set up. The proposed UHPLC-MS/MS method appears to be practically applicable for the analysis of urine samples in analytical and forensic toxicology cases, as well as in cases of suspected DFSA.

Zopiclone degradation in biological samples : Characteristics and consequences in forensic toxicology

“…Another study showed that ZOP metabolites are excreted mainly unconjugated [142]. Urine samples have also been processed without hydrolysis because of analyte instability during incubation [137].…”

“…Some assays (HPLC, LC-MS, LC-MS/MS) have also been developed to separate and determine ZOP [127,[137][138][139] and/or its metabolites (NDZOP and ZOPNO) in urine [140][141][142]. However, because of analytical detection or validation problems when ZOP was analysed in urine, the ZOP analyte was excluded from two of these methods [139,141].…”

“…However, because of analytical detection or validation problems when ZOP was analysed in urine, the ZOP analyte was excluded from two of these methods [139,141]. In another method the metabolites were identified but not quantitated [142]. A couple of methods have been developed to include also the analysis of ZOP metabolites NDZOP and/or ZOPNO in plasma.…”

“…Blood and urine are the commonest matrices for analysis of ZOP in forensic toxicology [87]. Stability tests of ZOP in stored urine samples was not investigated or reported in recently validated methods for forensic applications [137][138][139][140][141][142]. Because of the short half-life, ZOP is rapidly elimination from blood so urine may be a good choice of matrix, for example, in drug facilitated crimes when the victim might delay reporting the crime [153].…”

“…Stability tests of ZOP in stored urine samples have not been reported before [127,[137][138][139]142]. Storage stability tests in Paper III could be evaluated both from method validation and from the degradation and formation study.…”