Determination of over 80 pharmaceutical and illicit drugs in (post-mortem) blood by ultra-high performance liquid chromatography-tandem mass spectrometry

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 doses assumption. Sensitive and selective screening methods with shorter sample processing and analysis time must be developed for the simultaneous monitoring of a large number of different 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 in blood samples of many psychoactive drugs, including the ones mostly involved in forensic investigations. Methods Protein precipitation was performed by addiction of acetonitrile to the whole blood samples, 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, 100x2.1 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. Preliminary data The method developed involves a simple and rapid sample pretreatment, followed by UHPLC-MS/MS analysis. The simultaneously screening, detection and quantification of over 80 drugs (benzodiazepines, zolpidem, zopiclone, antiepileptics, antidepressants, barbiturates, opioid analgesics, antipsychotics, anesthetics and others) was achieved in the fast SRM mode using various time-windows for increasing selectivity and detection capability. To maximize the fragment ion signals while maintain comparable precursor ion abundance, different collision energies were optimizes for each compound. For all analytes, two-three 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 are 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 easily be 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. Novel aspect Development of a multiresidue, sensitive, and rapid UHPLC-MS/MS screening method for blood samples represents a powerful tool in forensic investigation.