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Purpose <p>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.</p> Methods <p>For sample preparation, 100&#xa0;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.</p> Results <p>The lower limits of quantification varied from 5 to 20&#xa0;ng/mL, with all substances demonstrating linearity up to 1000&#xa0;ng/mL. The method exhibited maximum precision values of 19.3%, while the bias ranged from −&#xa0;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%).</p> Conclusions <p>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.</p>

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

  • 期刊论文

  • 发表日期：

    2025年4月24日

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  • Letícia Birk,
  • Bruno Pereira dos Santos,
  • Daniela Souza Ossanes,
  • Patrícia de Souza Schwarz,
  • Mariana Lopes Mesquita,
  • Sarah Eller,
  • Tiago Franco de Oliveira

摘要

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.