The aims of this study were (1) to identify and quantify cocaine (COC), benzoylecgonine (BE), ecgonine methyl ester (EME), and cocaethylene (CE) in DBS; (2) to compare dried blood spots (DBSs) analytical results with the routine blood analyses; (3) to monitor analytes stability on DBS within a 3-month period. Eighty-five μL of blood from postmortem cases were put on a card for DBS analysis and kept in the dark, at room temperature. Samples were extracted through solid-phase extraction (SPE) cartridges and injected in the liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. The analytical procedure is simple, sensitive, and specific. Limits of detection (LODs) and quantification (LOQs) were calculated at 1.0 and 5.0 ng/mL(g) for COC and CE, and at 0.5 and 2 ng/mL for EME and BE, respectively. Validation parameters fulfilled all the acceptance criteria. Fifty-five postmortem cases were evaluated. Eighteen cases were positive for COC (44-2456 ng/mL) and BE (228-4700 ng/mL), 12 for EME (92-1500 ng/mL), and 11 cases for CE (11-273 ng/mL). Stability was evaluated on 8 cases collected in the period January 2017-January 2018. For each case, 5 DBSs were collected at T0. Four DBSs were analyzed within the 4 following weeks and 1 sample was analyzed after 3 months. The concentrations on DBSs, stored at room temperature, always matched the ones obtained on blood samples kept at -20°C (<20% variation, both at T0 and after 3 months). BE and COC concentrations remained stable after a 3-month storage, EME concentrations slightly increased after 3 weeks in the 2 analyzed samples, while CE provided a less homogeneous stability depending on the sample.

The aims of this study were (a) to develop a liquid chromatography−tandem mass spectrometry (LC–MS/MS) method for the identification of 27 benzodiazepines and metabolites in dried blood spots (DBSs) and in whole blood; (b) to compare the diagnostic reliability of DBSs with blood analyses; and c) to monitor analytes stability on DBSs within a three‐month period. Aliquots of 85 μL of blood from post‐mortem cases were pipetted on cards for DBS analysis. We also collected a tube of blood and stored it at − 20°C. The cards were allowed to dry at room temperature. For each case, DBSs were analyzed immediately (T0), within the following 3 weeks (T1, T2, T3) and after 3 months (T4). The method was applied to 60 post‐mortem cases. A screening procedure was applied to all 27 molecules, while the method was fully validated for the 9 molecules detected in real samples. Limits of detection (LODs) and limits of quantification (LOQs) were in the range 0.1–50.0 ng/mL and 5.0–100.0 ng/mL, respectively. Nine analytes were detected and quantitated in 14 cases: diazepam (n = 6, 58–162 ng/mL), desmethyldiazepam (n = 6, 99‐ > 500 ng/mL), 7‐aminoclonazepam (n = 5, 43‐ > 500 ng/mL), alprazolam (n = 2, 15 and 69 ng/mL), chlordesmethyldiazepam (n = 1, 55 ng/mL), desalkylflurazepam (n = 1, 270 ng/mL), flurazepam (n = 1, 50 ng/mL), medazepam (n = 1, 155 ng/mL), and midazolam (n = 1, 227 ng/mL). The concentrations on DBSs were in good agreement with those obtained on conventional blood samples (<20% variation). Except for midazolam (degraded in 1 week), desalkylflurazepam and medazepam (decreased more than 50% after three months), a good stability for a three‐month period was observed for most of the compounds detected in real post‐mortem samples.