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Stability of zopiclone in whole blood: Studies from a forensic perspective
Linköping University, Department of Medicine and Health Sciences, Clinical Pharmacology . Linköping University, Faculty of Health Sciences.
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: urn:nbn:se:liu:diva-58997ISBN: 978‐91‐7393‐339‐1 OAI: oai:DiVA.org:liu-58997DiVA: diva2:349342
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: 2010-09-07Bibliographically approved
List of papers
1. Stability tests of zopiclone in whole blood
Open this publication in new window or tab >>Stability tests of zopiclone in whole blood
2010 (English)In: Forensic Science International, ISSN 0379-0738, E-ISSN 1872-6283, Vol. 200, no 01-Mar, 130-135 p.Article in journal (Refereed) Published
Abstract [en]

Zopiclone is a common drug in forensic cases and it is frequently analyzed in biological materials using different analytical methods. Zopiclone is unstable in certain solvents and depending on storage conditions unstable in biological fluids; however its stability in human whole blood has not yet been established in detail. Therefore, the following investigation was performed to study the stability of zopiclone in both spiked and authentic human blood. First, spiked blood samples were stored at -20 degrees C, 5 degrees C and 20 degrees C and the degradation of zopiclone was investigated. Second, authentic and spiked blood samples were stored at 5 degrees C and differences in zopiclone stability were studied. Third, processed sample stability and effect of freeze/thaw cycles were evaluated. Analyses were performed by GC-NPD and zopiclone concentrations were measured at selected time intervals. The study showed that zopiclone degrades in human blood depending on time and temperature and may not be detected after long-term storage. 2-amino-5-chloropyridine was identified as the primary degradation product from zopiclone. At refrigerator temperature zopiclone was stable less than 1 month in both spiked and authentic human blood samples. The best storage condition was at -20 degrees C even at short storage times, as freeze-thaw had no influence on the results. In butyl acetate extracts, zopiclone was stable at least 2 days when kept in the autosampler at ambient temperature. We conclude that preanalytical factors have great impact on analytical results and should be addressed when interpreting whole blood zopiclone concentrations.

Place, publisher, year, edition, pages
Elsevier Science B.V., Amsterdam., 2010
Keyword
Degradation; Forensic toxicology; Stability; Storage; Zopiclone
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-58328 (URN)10.1016/j.forsciint.2010.04.001 (DOI)000279024800017 ()
Available from: 2010-08-13 Created: 2010-08-11 Last updated: 2014-04-08Bibliographically approved
2. Influence of pre-analytical conditions on the interpretation of zopiclone concentrations in whole blood
Open this publication in new window or tab >>Influence of pre-analytical conditions on the interpretation of zopiclone concentrations in whole blood
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
Keyword
Degradation; Forensic toxicology; Stability; Storage; Zopiclone
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:liu:diva-59046 (URN)10.1016/j.forsciint.2010.08.016 (DOI)000288851300028 ()20851542 (PubMedID)
Available from: 2010-09-07 Created: 2010-09-07 Last updated: 2015-01-13Bibliographically approved

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