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. 2019 Sep;11(9):1403-1411.
doi: 10.1002/dta.2653. Epub 2019 Jun 20.

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

Matteo Moretti  1 , Francesca Freni  1 , Ilaria Tomaciello  1 , Claudia Vignali  1 , Angelo Groppi  1 , Silvia Damiana Visonà  1 , Luca Tajana  1 , Antonio Marco Maria Osculati  1 , Luca Morini  1
Affiliations

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

Matteo Moretti et al. Drug Test Anal. 2019 Sep.

Abstract
in English, Italian

We successfully developed and validated an LC-MS/MS method for the identification of 27 and quantification of 9 benzodiazepines and metabolites in whole blood and DBSs. The results provided a good qualitative and quantitative correlation between DBSs stored at room temperature and whole blood stored at -20°C. A good stability for a three-month period was observed for most of the compounds detected in real post-mortem samples.

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.

Keywords: LC-MS/MS; benzodiazepines; dried blood spot; post-mortem; stability.

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References

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