Determination of benzodiazepines in blood and in dried blood spots collected from post‐mortem samples and evaluation of the stability over a three‐month period

• Published in: Drug testing and analysis 2019-09, Vol.11 (9), p.1403-1411
• Main Authors: Moretti, Matteo, Freni, Francesca, Tomaciello, Ilaria, Vignali, Claudia, Groppi, Angelo, Visonà, Silvia Damiana, Tajana, Luca, Osculati, Antonio Marco Maria, Morini, Luca
• Format: Article
• Language: English
• Subjects: Autopsy; benzodiazepines; Benzodiazepines - blood; Blood tests; Central Nervous System Depressants - blood; Chromatography, High Pressure Liquid - methods; dried blood spot; Dried Blood Spot Testing - methods; Drug testing; Forensic chemistry; Humans; LC–MS/MS; Limit of Detection; post‐mortem; stability; Substance Abuse Detection - methods; Tandem Mass Spectrometry - methods
• ISSN: 1942-7603; 1942-7611
• DOI: 10.1002/dta.2653

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 (