Simple implementation of muscle tissue into routine workflow of blood analysis in forensic cases - A validated method for quantification of 29 drugs in postmortem blood and muscle samples by UHPLC-MS/MS

Abstract

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.

Keywords: Automated SPE; Benzodiazepines; Muscle tissue; Postmortem toxicology; UHPLC–MS/MS; Whole blood.