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Influence of pre-analytical conditions on the interpretation of zopiclone concentrations in whole blood
Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. 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, Clinical Pharmacology. 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, Clinical Pharmacology. 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.
2011 (English)In: Forensic Science International, ISSN 0379-0738, E-ISSN 1872-6283, Vol. 207, no 1-3, 35-39 p.Article in journal (Refereed) Published
Abstract [en]

Zopiclone is a short-acting hypnotic drug used for treatment of insomnia and its stability has been described in some detail. However, data especially on short-term pre-analytical stability is missing. This study investigated zopiclone stability differences between spiked and authentic whole blood from subjects dosed with zopiclone. In this way influence from physiological factors such as drug interactions, matrix composition and plasma protein levels were minimized. Nine volunteers participated in the study. Whole blood was obtained before and after oral administration of 10 mg Imovane®. Aliquots of 1 g of authentic and spiked blood were after initial measuring, stored at 20°C during 5 days, 5°C or -20°C during 3 months, and zopiclone was measured by gas chromatography with nitrogen phosphorus detection. The results showed no stability differences between authentic and spiked blood but confirmed the very short stability in whole blood at ambient temperature. In summary, the stability was 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.

Place, publisher, year, edition, pages
Elsevier , 2011. Vol. 207, no 1-3, 35-39 p.
Keyword [en]
Degradation; Forensic toxicology; Stability; Storage; Zopiclone
National Category
Pharmacology and Toxicology
Identifiers
URN: urn:nbn:se:liu:diva-59046DOI: 10.1016/j.forsciint.2010.08.016ISI: 000288851300028PubMedID: 20851542OAI: oai:DiVA.org:liu-59046DiVA: diva2:349538
Available from: 2010-09-07 Created: 2010-09-07 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Stability of zopiclone in whole blood: Studies from a forensic perspective
Open this publication in new window or tab >>Stability of zopiclone in whole blood: Studies from a forensic perspective
2010 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2010. 58 p.
Series
Linköping Studies in Health Sciences. Thesis, ISSN 1100-6013 ; 113
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-58997 (URN)9789173933391 (ISBN)
Presentation
2010-10-01, Eken, Campus Valla, Linköping University, Linköping, 13:00 (English)
Supervisors
Available from: 2010-09-07 Created: 2010-09-06 Last updated: 2017-12-14Bibliographically approved
2. 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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