Stability of zopiclone in whole blood : Studies from a forensic perspective

Abstract: Bio‐analytical results are influenced by in vivo factors like genetic, pharmacological and physiological conditions and in vitro factors like specimen composition, sample additives and storage conditions. The knowledge of stability of a drug and its major metabolites in biological matrices is very important in forensic cases for the interpretation of analytical results. Many drugs are unstable and undergo degradation during storage.Zopiclone is a short‐acting hypnotic drug, introduced as a treatment for insomnia in the 1980s. However, this drug is also subject to abuse and can be found in samples from drug‐impaired drivers, recreational drug users and forensic autopsy cases. Zopiclone is analyzed in biological materials using different analytical methods. It is unstable in certain solvents and depending on storage conditions unstable in biological fluids. The aim of this thesis was to investigate the stability of zopiclone in human whole blood and to compare stability between authentic and spiked samples. Interpretation of zopiclone concentrations in whole blood is important in forensic toxicology. The following investigations were performed to study the stability of zopiclone in both spiked and authentic human blood.First, different stability tests were performed. Spiked blood samples were stored at –20°C, 5°C and 20°C and the degradation of zopiclone was investigated in long‐ and short‐term stability. Authentic and spiked blood samples were stored at 5°C and differences in zopiclone stability were studied. Processed sample stability and effect of freeze/thaw cycles were also evaluated.Second, influence of pre‐analytical conditions on the interpretation of zopiclone concentrations in whole blood was investigated. Nine volunteers participated in the study. Whole blood was obtained before and after oral administration of 2 x 5 mg Imovane®. Aliquots of authentic and spiked blood were stored under different conditions and zopiclone stability was evaluated. In this study, the influence from physiological factors such as drug interactions, matrix composition and plasma protein levels were minimized.Analyses of zopiclone were performed by gas chromatography with nitrogen phosphorous detection and zopiclone concentrations were measured at selected time intervals. Degradation product of zopiclone was identified using liquid chromatography‐tandem mass spectrometry.The first study showed that zopiclone degrades in human blood depending on time and temperature and may not be detected after long‐term storage. The degradation product 2‐amino‐5‐chloropyridine was identified following zopiclone degradation. The best storage condition was at –20°C even for short storage times, because freeze‐thaw had no influence on the results. In butyl acetate extracts, zopiclone was stable for at least two days when kept in the autosampler. However, in blood samples stored at 20°C a rapid decrease in concentration, was noticed. This rapid degradation at ambient temperature can cause an underestimation of the true concentration and consequently flaw the interpretation.The second study showed no stability differences between authentic and spiked blood but confirmed the poor stability in whole blood at ambient temperature. The results showed that zopiclone was stable for less than 1 day at 20°C, less than 2 weeks at 5°C, but stable for 3 months at –20°C. This study, demonstrates the importance of controlling pre‐analytical conditions from sampling to analysis to avoid misinterpretation of toxicological results.