A simple method for the determination of stimulant substances in postmortem blood: development, validation, and application in nearly 1000 forensic cases.

Purpose: Toxicological analyses of postmortem blood samples are essential to elucidate forensic cases involving toxic agents, such as illicit drugs. A simple method for determining stimulant substances in postmortem blood samples through protein precipitation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and applied in nearly 1000 samples from Brazilian forensic cases.

Methods: For sample preparation, 100 µL of postmortem blood was precipitated using acetonitrile. The supernatant was analyzed via LC-MS/MS system for sixteen substances, including amphetamine, benzoylecgonine, cocaethylene, cocaine, diethylpropion, dimethyltryptamine, ecgonine methyl ester (EME), ephedrine, fenproporex, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxyethylamphetamine, 3,4-methylenedioxymethamphetamine, methamphetamine, methylphenidate, phenylephrine, and sibutramine. The method was validated following the parameters established by the ANSI/ASB Standard 036 Guideline. After validation, a total of 971 postmortem blood samples were analyzed.

Results: The lower limits of quantification varied from 5 to 20 ng/mL, with all substances demonstrating linearity up to 1000 ng/mL. The method exhibited maximum precision values of 19.3%, while the bias ranged from - 15.4 to + 4.3%. A significant matrix effect was observed only for EME and phenylephrine. Approximately 20.1% of the analyzed samples tested positive for at least one substance, and 12 out of the 16 target analytes were detected. The most prevalent substances identified were benzoylecgonine (17.8%), ecgonine methyl ester (13.9%), and cocaine (13.0%).

Conclusions: A rapid and straightforward LC-MS/MS method for the quantitative analysis of drugs in postmortem blood was validated and successfully applied to nearly 1000 postmortem blood samples.