Micro-QuEChERS extraction method optimization for quantification of designer benzodiazepines in forensic postmortem blood samples

The aim of this study was to develop and validate a quantitative method for the analysis of designer benzodiazepines in postmortem blood samples using micro-QuEChERS extraction and liquid chromatography tandem mass spectrometry (LC-MS/MS). A comprehensive optimization of the method was performed using a multivariate statistical approach, incorporating validation criteria in line with established practices for method validation in forensic toxicology. The method showed linearity between 1 and 200 ng/mL (r2>0.990), with good imprecision (<9.8%) and inaccuracy (<11.1%) evaluated at three different quality control concentrations. Matrix effects and recovery rates were found to be better than 58% and 77.5%, respectively. No carryover or interferences were detected during the analysis. The method was effectively utilized on two real forensic postmortem blood samples, both of which tested positive for bromazolam, showing concentrations of 31 ng/mL and 40 ng/mL. The micro-QuEChERS extraction method demonstrated satisfactory analytical performance and is an environmentally sustainable option, minimizing the use of solvents and reagents, with potential for application in both clinical and forensic analyses, aligning with green analytical toxicology principles.