Quantitative analysis of zopiclone, N-desmethylzopiclone, zopiclone N-oxide and 2-amino-5-chloropyridine in urine using LC-MS-MS.

A simple liquid chromatography-tandem mass spectrometry method was validated to allow determination of zopiclone (ZOP), N-desmethylzopiclone (NDZOP), zopiclone N-oxide (ZOPNO) and 2-amino-5-chloropyridine (ACP) in urine at concentrations up to 3,000 ng/mL within 3.5 min. This method was used for quantitative analysis of the analytes in authentic urine samples obtained 10 h after oral administration of zopiclone (Imovane(®)) and in aliquots of the same urine samples after different storage conditions. In addition, pH of each studied urine sample was measured over time. The results showed that formation of ACP occurred at elevated pH and/or temperature by degradation of ZOP, NDZOP and ZOPNO. This method was also applied to samples obtained from two female victims of drug-facilitated assault. One sample had been exposed to long-term storage conditions at different temperatures and at pH >8.2, which resulted in high concentrations of ACP. The other sample, which was exposed to pH <6.5, showed no formation of ACP. ACP is formed both from ZOP and from its metabolites NDZOP and ZOPNO depending on the pH of the urine, time of storage and/or the temperature conditions. For correct interpretation in forensic cases, ZOP, its major metabolites and ACP should be analyzed. When ACP is identified in urine, the concentrations of ZOP, NDZOP and ZOPNO should be interpreted with great caution.