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Whole blood is most often the matrix of choice for postmortem analysis but it is not always available. In these cases, muscle tissue can be used as an alternative matrix. Therefore, an ultra-high-performance liquid chromatography-tandem mass spectrometry method for the quantification of 29 drugs and metabolites of toxicological interest in postmortem muscle tissue was developed and validated. Additionally, a validation of whole blood was carried out to compare the results from the two matrices. Solid-phase extraction was performed by an automated robotic system to minimize manual labour and risk of human errors, and increase robustness, sample throughput and sample traceability. The method was validated in terms of selectivity, matrix effect, extraction recovery, process efficiency, measuring range, lower limit of quantification, carry-over, stability, precision and accuracy. To correct for any inter-individual variability in matrix effects on analyte accuracy and precision, deuterated analogues of each analyte were used as internal standards. The lower limit of quantification in both blood and muscle homogenate ranged between 0.002 and 0.005 mg/kg, while the upper limit of quantification spanned from 0.20 to 1.0 mg/kg. Corrected with the 4-fold dilution factor, the corresponding concentrations in muscle tissue were 0.008-0.02 mg/kg at the lower limit of quantification and 0.80-4.0 mg/kg at the upper limit of quantification. The method showed acceptable precision and accuracy, with precision below 12% and accuracies ranging from 87% to 115% at up to 6 levels for all analytes in both matrices. In addition, comparison between calibration standards in spiked muscle homogenate and spiked blood showed that analyte concentrations in muscle samples could be quantified by using spiked blood samples as calibration standards with acceptable precision and accuracy when using deuterated analogues as internal standards. The investigation of matrix effects showed no great difference between blood and homogenates of non-decomposed and decomposed muscle tissue for most analytes. In the samples where high ion suppression or enhancement was observed, the results were corrected by the internal standards. Statistical comparison of quality control samples in blood and muscle tissue showed no obvious differences, and therefore muscle tissue was included in the routine method for analysis of blood samples and used in autopsy cases where no blood was available. By adding a semi-automated homogenization step before the remaining automated sample preparation, muscle tissue samples were easily incorporated into the workflow of the existing routine method. The present method has been successfully implemented in routine analysis of blood and muscle tissue since 2019. [ABSTRACT FROM AUTHOR]

In forensic investigations, such as drug-facilitated crimes, reference values are useful for interpretation of hair results. The aim of this study was to establish levels of zopiclone and two main metabolites, N-desmethylzopiclone and zopiclone N-oxide, in hair after the administration of a single dose of zopiclone, as very limited data are published. A controlled study was performed, where 16 volunteers consumed either 5 or 10mg zopiclone. Hair was sampled prior to consumption and 14, 30, 60, and 120 days after intake. The deposition of drug in hair segments of all sampling time points was followed in small hair segments of 5-mm, using a validated ultra-high performance liquid chromatography-tandem mass spectrometry method. In all participants, hair segments corresponding to the time of intake were positive for zopiclone, but also with lower concentrations in the neighbouring segments. The highest zopiclone concentrations were detected in samples collected 30 or 60 days after intake. For all sampling time points maximum values for the 5-mg dose ranged from 5.0-370pg/mg for zopiclone and 5.4 to 300pg/mg for N-desmethylzopiclone, where the maximum values for the 10-mg dose ranged from 17 to 590pg/mg for zopiclone and 25-410pg/mg for N-desmethylzopiclone for all sampling time points. No significant difference in concentrations was found between the two dosing groups for either zopiclone or N-desmethylzopiclone. Almost half of the participants showed lower levels 14 days after intake than in the later sampling time points. The metabolite to parent drug ratio of N-desmethylzopiclone to zopiclone varied from 0.6 to 3.4 (median=1.2) for the maximum levels of all sampling time points. N-desmethylzopiclone are suggested to serve as an additional marker to confirm the intake of zopiclone. Traces of zopiclone N-oxide were detected in hair from only eight participants. This study showed, that it was possible to follow zopiclone and N-desmethylzopiclone in hair for 4 months even though the drugs was divided into several segments in the latest collected hair samples, and no obvious wash-out effect between the sampling time points by e.g. personal hygiene could be discerned because the cumulated amount at each sampling time point was similar. We conclude that the analysis of short segments e.g. segments of 5-mm can help determine the time of a single intake of zopiclone and that obtaining a sample 1-2 months after a drug exposure provide the best conditions to detect and interpret the results. [ABSTRACT FROM AUTHOR]