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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., (© 2019 John Wiley & Sons, Ltd.)

The study aims the development and validation of a LC-MS/MS method for the identification and quantification of benzodiazepines and zolpidem in nails as alternative keratinized matrix to hair in long-term monitoring of anxiolytic and hypnotic drugs. Both fingernail and toenail samples (1-2 mm) were collected by clipping the excess overhang of the nail from volunteers and from postmortem cases. They were washed twice with organic solvents, dried under nitrogen stream, pulverized, immersed in a methanol solution (internal standard: diazepam-D5) and sonicated up to two hours. The solution was then direct injected in the LC-MS/MS system. Mass spectrometry was set in MRM mode, selecting two transitions for each substance. 32 analytes among benzodiazepines, metabolites and hypnotics were included in the list. The method fulfilled the internationally required criteria for validation. Limits of detection ranged from 0.03 pg/mg (zolpidem) to 13.1 pg/mg (bromazepam). 9 subjects under therapy were positive at 7 different benzodiazepines and/or metabolites (lorazepam, desalkylflurazepam, bromazepam, diazepam, alprazolam, lormetazepam and prazepam), while 5 molecules were measured in 4 postmortem cases (diazepam, desmethyldiazepam, delorazepam, 7-aminoclonazepam and zolpidem). In vitro experiments on eight authentic samples suggested that benzodiazepines in nails are influenced by the prolonged exposure to chlorinated water., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Introduction: An in vitro study on authentic positive samples was carried out, aiming the evaluation of the effect of chlorinated water on benzodiazepines in hair. Two subjects exposed to chlorinated water for several consecutive times were also investigated., Method: Seven hair samples collected from autopsy cases, previously tested positive for benzodiazepines, were washed with dichloromethane and methanol. They were longitudinally divided in six aliquots of about 30mg. An aliquot was processed without treatment while other five ones were soaked in chlorinated water (0.1% sodium dichloroisocyanurate and 0,1M sulfuric acid at pH 5.5) for 4, 20, 24 and 30h respectively. Hair samples were then processed following a fully validated and previously published method. Briefly hair samples were sonicated in 600 microliters methanol containing halazepam (IS) up to two hours. Ten microliters were injected in a liquid chromatographic tandem mass spectrometric (LC-MS/MS) system. Analytes were eluted from a C18 reversed-phased column. Two transitions on multiple reaction monitoring and positive ionization mode were monitored for each compound., Result and Discussions: Six compounds among benzodiazepines and metabolites were identified and quantified in the seven hair samples: diazepam (575pg/mg), desmethyldiazepam (562pg/mg), chlordesmethyldiazepam (173pg/mg), desalkylflurazepam (320pg/mg), clonazepam (three cases-195, 119 and 111pg/mg respectively), lormetazepam (two cases-182 and 416pg/mg respectively). Traces of 7-aminoclonazepam were identified into 2 samples. Stability of benzodiazepines in water was evaluated by soaking an aliquot of hair for up to 30h in deionized water. No significant degradation was observed. Samples soaked in chlorinated water showed considerable decreasing from the initial concentration even after the 4-h treatment: the fastest degradation was provided by clonazepam that showed a 61% loss. The greatest loss was measured for diazepam (86% loss after 30-h soaking)., Conclusion: To the best of our knowledge this is the first in vitro study that evaluated benzodiazepines stability in hair after prolonged exposure to chlorinated water. The results showed that the longer the exposure the higher the degradation. Prolonged exposure to chlorinated water and sunlight must be always taken into account as possible causes of false negative results., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Introduction: The use of a LC-MS/MS system for benzodiazepines detection remarkably increased the analytical sensitivity of these drugs in biological matrices, in particular in non-conventional ones such as hair. Since the amount of hair sample available for the analysis is frequently limited and, moreover, it needs to be checked for many other drugs and compounds of forensic interest, it is important to develop a sample preparation procedure able to detect either benzodiazepines and as many as possible other substances. The aim of this study was to compare the sensitivity of two different hair sample preparation procedures for benzodiazepines detection in hair., Methods: About 20mg hair, previously washed with organic solvent and cut into small pieces, were ultrasonicated with a phosphate buffer (pH 8.4) up to 1h and then extracted with dichloromethane/diethyl ether. The organic solvent was then dried under nitrogen flow and samples were reconstituted with 60μl methanol. Finally a 5μl aliquot was injected in the LC-MS/MS system. The second procedure consisted of an ultrasonication of hair samples in 700μl of methanol. Samples were then directly analyzed. Both the methods were fully validated., Results: Thirty-five compounds among benzodiazepines and their metabolites were screened using both the procedures. The methods fulfilled all the validation parameters and were applied on either spiked blank hair and real positive samples. While phosphate extraction allowed to reach a LOQ for almost all the substances ranging from 0.1 to 5pg/mg, thus guaranteeing to evaluate even a single dose administration (as confirmed by real positive cases) the sensitivity of the methanol extraction showed a LOQ ranging from 1 to 20pg/mg, still enough to assess a therapeutic use of almost all the benzodiazepines; yet the methanolic incubation allows a simple and rapid analytical procedure due to the direct injection of the extraction solvent., Conclusion: Even though a methanol extraction procedure for benzodiazepines determination is useful for forensic toxicological purposes also when a wider range of substances is needed and in case of a small amount of hair available, it is advisable to prefer a phosphate extraction when detection of a single dose administration is required., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)