Introduction Forensic investigations involving either lethal intoxication or suicide, drug facilitated sexual assault, driving or workplace impairment, frequently require the analysis of fresh or post-mortem blood samples to check out a wide variety of pharmaceutical and illicit drugs, even after a single dose assumption. Sensitive and selective screening methods with rapid sample processing and analysis must be developed for the simultaneous monitoring of a large number of substances. Recent advancements in liquid chromatography and mass spectrometry expanded the multiresidue capability of LC-MS/MS protocols, with significant benefit for drugs screening. Our goal was to develop and validate a UHPLC-MS/MS method for the determination of many psychoactive drugs in blood samples, including the ones most frequently involved in forensic investigations. Methods Protein precipitation was performed by addiction of acetonitrile to the whole blood sample, followed by centrifugation. An LC-MS/MS method was developed using a Shimadzu Nexera UHPLC-system interfaced to an Applied Biosystem 5500TM triple-quadrupole mass spectrometer, operating in ESI-positive ion mode. LC separation was performed using a Agilent UHPLC column (XDB C-18, 100 x 2.1 mm i.d, 1.8 µm particle diameter). The data were acquired at unit mass resolution in the selected-reaction monitoring (SRM) mode. The method validation according to ISO 17025 and international guidelines is in progress. Results The developed method involved a simple and rapid sample pretreatment, followed by UHPLC-MS/MS analysis. The simultaneous screening, detection and approximate quantification of over 80 drugs (benzodiazepines, zolpidem, zopiclone and zaleplon, antiepileptics, antidepressants, barbiturates, opioid analgesics, antipsychotics, anesthetics and others) was achieved in the fast SRM mode using variable time-windows for increasing selectivity and detection capability at the same time. To maximize the fragment ion signals while maintaining comparable precursor ion abundance, different collision energies were optimizes for each compound. For all analytes, two SRM transitions were utilized for identification and quantification. According to international guidelines, linearity range, precision, accuracy and trueness, detection and quantification limits (LODs and LOQs), recovery, selectivity, specificity, carry-over and matrix effect phenomena were evaluated. The method was applied to the analysis of authentic blood (post-mortem) samples collected from victims of various crimes in routine casework. Furthermore, the present screening method can be easily expanded to encompass more drugs, either new or becoming important for criminal investigation. The method can also be modified and re-validated to analyze other biological fluids or solid materials.
Determination of over 80 pharmaceutical and illicit drugs in (post-mortem) blood by ultra-high performance liquid chromatography-tandem mass spectrometry
VINCENTI, Marco;GERACE, ENRICO;PIRRO, VALENTINA;A. Salomone
2012-01-01
Abstract
Introduction Forensic investigations involving either lethal intoxication or suicide, drug facilitated sexual assault, driving or workplace impairment, frequently require the analysis of fresh or post-mortem blood samples to check out a wide variety of pharmaceutical and illicit drugs, even after a single dose assumption. Sensitive and selective screening methods with rapid sample processing and analysis must be developed for the simultaneous monitoring of a large number of substances. Recent advancements in liquid chromatography and mass spectrometry expanded the multiresidue capability of LC-MS/MS protocols, with significant benefit for drugs screening. Our goal was to develop and validate a UHPLC-MS/MS method for the determination of many psychoactive drugs in blood samples, including the ones most frequently involved in forensic investigations. Methods Protein precipitation was performed by addiction of acetonitrile to the whole blood sample, followed by centrifugation. An LC-MS/MS method was developed using a Shimadzu Nexera UHPLC-system interfaced to an Applied Biosystem 5500TM triple-quadrupole mass spectrometer, operating in ESI-positive ion mode. LC separation was performed using a Agilent UHPLC column (XDB C-18, 100 x 2.1 mm i.d, 1.8 µm particle diameter). The data were acquired at unit mass resolution in the selected-reaction monitoring (SRM) mode. The method validation according to ISO 17025 and international guidelines is in progress. Results The developed method involved a simple and rapid sample pretreatment, followed by UHPLC-MS/MS analysis. The simultaneous screening, detection and approximate quantification of over 80 drugs (benzodiazepines, zolpidem, zopiclone and zaleplon, antiepileptics, antidepressants, barbiturates, opioid analgesics, antipsychotics, anesthetics and others) was achieved in the fast SRM mode using variable time-windows for increasing selectivity and detection capability at the same time. To maximize the fragment ion signals while maintaining comparable precursor ion abundance, different collision energies were optimizes for each compound. For all analytes, two SRM transitions were utilized for identification and quantification. According to international guidelines, linearity range, precision, accuracy and trueness, detection and quantification limits (LODs and LOQs), recovery, selectivity, specificity, carry-over and matrix effect phenomena were evaluated. The method was applied to the analysis of authentic blood (post-mortem) samples collected from victims of various crimes in routine casework. Furthermore, the present screening method can be easily expanded to encompass more drugs, either new or becoming important for criminal investigation. The method can also be modified and re-validated to analyze other biological fluids or solid materials.
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