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Validation of an LC-MS/MS method for the determination of zopiclone, N-desmethylzopiclone and 2-amino-5-chloropyridine in whole blood and its application to estimate the original zopiclone concentration in stored specimens

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Abstract

2-Amino-5-chloropyridine (ACP) is a degradation product of zopiclone (ZOP) and its two main metabolites N-desmethylzopiclone (NDZOP) and zopiclone N-oxide (ZOPNO). ACP may be formed when specimens are stored. ZOP instability in blood makes interpretation of concentrations difficult especially in cases of prolonged sample storage. This study investigated how ACP could be used to estimate the original concentration of ZOP in authentic samples. For that purpose, an analytical liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantitation of ACP, ZOP, and NDZOP in blood was developed and validated. Due to poor extraction recovery, ZOPNO was not included in the analytical method. The method was then applied to investigate ACP formation, ZOP and NDZOP degradation in stored ZOP post-dosed authentic whole blood and two mathematical models were used to calculate the original concentration of ZOP. During storage, ACP was formed in amounts equimolar to the ZOP and NDZOP degradation. Results from samples in which ACP had been formed were used to test two models to estimate the original ZOP concentration. The correlation tests of the models showed strong correlations to the original ZOP concentration (r = 0.960 and r = 0.955) with p < 0.01 and explained more than 90 % of the ZOP concentration. This study showed that the equimolar degradation of ZOP and NDZOP to ACP could be used to estimate the original concentration of ZOP.

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Acknowledgments

The authors would like to thank the National Board of Forensic Medicine and the Centre of Forensic Science Linköping University for providing resources for this study.

Conflict of interest

There were no external sources of funding for this project. None of the authors consider that any conflicts of interest exist.

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Authors and Affiliations

  1. Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, Artillerigatan 12, 587 58, Linköping, Sweden

    Gunnel H. Nilsson, Fredrik C. Kugelberg, Johan Ahlner & Robert Kronstrand

  2. Department of Medical and Health Sciences, Faculty of Health Sciences, Linköping University, 581 85, Linköping, Sweden

    Gunnel H. Nilsson, Fredrik C. Kugelberg, Johan Ahlner & Robert Kronstrand

Authors
  1. Gunnel H. Nilsson
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  2. Fredrik C. Kugelberg
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  3. Johan Ahlner
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  4. Robert Kronstrand
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Correspondence to Gunnel H. Nilsson.

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Nilsson, G.H., Kugelberg, F.C., Ahlner, J. et al. Validation of an LC-MS/MS method for the determination of zopiclone, N-desmethylzopiclone and 2-amino-5-chloropyridine in whole blood and its application to estimate the original zopiclone concentration in stored specimens. Int J Legal Med 129, 269–277 (2015). https://doi.org/10.1007/s00414-014-1049-2

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  • DOI: https://doi.org/10.1007/s00414-014-1049-2

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