Postmortem blood screening

Mass spectrometry data was collated retrospectively to the sample analyses, after study population inclusion. Standardised procedures for mass spectrometry analyses were carried out in accordance with previously described.⁴⁶ Briefly, data have been derived from femoral blood samples that were collected from all postmortem cases and analyzed using an ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry system (UHPLC-QTOF; Agilent 1290 Infinity LC with Agilent 6540 QTOF; Agilent Technologies Sweden AB, Sweden). The method used is routinely performed at the National Board of Forensic Medicine (Linköping, Sweden), and is an accredited method for the targeted analysis of medicine and drugs via UHPLC-QTOF in positive ionisation mode, however, data recorded and saved from the entire analytical run. Femoral whole blood samples were prepared by protein precipitation (acetonitrile:ethanol, 90:10), including the addition of three internal standards (amphetamine-d₈, diazepam-d₅, and mianserin-d₃). Samples were then injected into the UHPLC-QTOF system. The chromatography separation is performed via a gradient elution on a C18 reverse-phase column (150 mm × 2.1 mm, 1.8 μM; Waters Acquity HSS T2 column, Waters Sverige AB, Sweden). MS data were collected in positive ionisation mode and the total acquisition time for each sample was 12 min. Quality control samples, blank drug-free bovine whole blood containing the three internal standards, were included in every analytical run at the beginning and end. Mass spectra were pre-processed using XCMS and CAMERA packages in R (v.4.1.2) for peak list generation and peak annotation, according to parameters detailed in full previously.¹⁶