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Quantitative Analysis of Zopiclone, N-desmethylzopiclone, Zopiclone N-oxide and 2-Amino-5-chloropyridine in Urine Using LC-MS-MS
Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences. Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, Linköping, Sweden.
Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences. Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, Linköping, Sweden.
Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences. Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, Linköping, Sweden.
Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences. Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, Linköping, Sweden.
2014 (English)In: Journal of Analytical Toxicology, ISSN 0146-4760, E-ISSN 1945-2403, Vol. 38, no 6, 327-334 p.Article in journal (Refereed) Published
Abstract [en]

A simple LC-MS/MS method was validated to allow determination of zopiclone (ZOP), Ndesmethylzopiclone (NDZOP), zopiclone N-oxide (ZOPNO) and 2-amino-5 chloropyridine (ACP) in urine at concentrations up to 3000 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. Additionally, 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.

Place, publisher, year, edition, pages
Oxford University Press (OUP): Policy F , 2014. Vol. 38, no 6, 327-334 p.
Keyword [en]
Degradation; Forensic toxicology; Zopiclone; LC-MS-MS
National Category
Clinical Medicine Pharmacology and Toxicology
Identifiers
URN: urn:nbn:se:liu:diva-105819DOI: 10.1093/jat/bku042ISI: 000340069000004OAI: oai:DiVA.org:liu-105819DiVA: diva2:710924
Available from: 2014-04-08 Created: 2014-04-08 Last updated: 2017-12-05Bibliographically approved
In thesis
1. Zopiclone degradation in biological samples: Characteristics and consequences in forensic toxicology
Open this publication in new window or tab >>Zopiclone degradation in biological samples: Characteristics and consequences in forensic toxicology
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Bio-analytical results are influenced by in vivo factors such as genetics, pharmacological and physiological conditions and in vitro factors such as specimen composition, sample additives and storage conditions. Zopiclone (ZOP) is a short-acting hypnotic drug (Imovane®) used for treatment of insomnia. ZOP is metabolized by three major pathways; oxidation to the active zopiclone N-oxide (ZOPNO), demethylation to the inactive N-desmethylzopiclone (NDZOP) and oxidative decarboxylation to other inactive metabolites. ZOP is increasingly being encountered in forensic cases and is a common finding in samples from drug-impaired drivers, users of illicit recreational drugs, victims of drug facilitated sexual assaults and forensic autopsy cases. ZOP is a notoriously unstable analyte in biological matrices and analytical results depend on pre-analytical factors, such as storage time and temperature. The overall aim of this thesis was to investigate the stability of ZOP and the factors of importance for degradation during storage in biological samples and to identify consequences for interpretation of results in forensic toxicology.

In paper I, different stability tests in spiked samples were performed including short-term, longterm, freeze-thaw and processed stability. Analyses of ZOP were performed by gas chromatography with nitrogen phosphorous detection and ZOP concentrations were measured at selected time intervals. The degradation product 2-amino-5-chloropyridine (ACP) was identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The stability investigations showed a very poor short-term storage stability of ZOP.

Therefore, in paper II, the influence of pre-analytical conditions was further investigated in dosed subjects. Whole blood from volunteers was obtained before and after oral administration of Imovane®. In this study, the influence from physiological factors such as drug interactions, matrix composition and plasma protein levels were minimized. The results showed that ZOP was stable in whole blood for only one day at room temperature, one week in a refrigerator and at least three months frozen in authentic as well as in spiked whole blood. The rapid degradation of ZOP at ambient temperature can cause an underestimation of the true concentration and consequently flaw the interpretation. However, by also analyzing the degradation product ACP the original concentration of ZOP may be estimated.

In papers III and IV, two LC-MS-MS methods were validated for the quantitation of ACP, ZOP and NDZOP in blood and ACP, ZOP, NDZOP and ZOPNO in urine. These methods were used in a controlled pharmacokinetic study where whole blood and urine were obtained after oral administration of Imovane®. Samples of blood and urine were aliquoted, analyzed and stored under different conditions and the formation of ACP was monitored. Additionally, at each studied time point the pH of the blood and urine samples was measured using i-STAT® system. The results showed that ACP was formed in equimolar amounts to the degradation of ZOP and its metabolites.

In urine samples, the formation of ACP occurred at elevated pH or temperature and mirrored the degradation of ZOP, NDZOP and ZOPNO. The high concentrations of metabolites, which also degraded to ACP, made it impossible to estimate the original ZOP concentration.

The results from analysis of blood samples containing ACP were also used to develop mathematical models to estimate the original ZOP concentration. Both models showed strong correlation to the original ZOP concentration (r=0.960 and r=0.955) with p<0.01. This study showed that the equimolar degradation of ZOP and NDZOP to ACP could be used to estimate the original concentration of ZOP in blood samples.

Absence of ACP in the blood or urine samples analysed strongly suggests that degradation has not occurred and that the measured concentration of ZOP is reliable. For proper interpretation in forensic cases, it is strongly recommended that ZOP and its metabolites as well as ACP are included in the analysis.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2014. 68 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1395
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-105821 (URN)10.3384/diss.diva-105821 (DOI)978-91-7519-397-7 (ISBN)
Public defence
2014-04-24, Eken, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 13:00 (Swedish)
Opponent
Supervisors
Available from: 2014-04-08 Created: 2014-04-08 Last updated: 2014-04-08Bibliographically approved

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Nilsson, GunnelKugelberg, FredrikAhlner, JohanKronstrand, Robert

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