Istituto Superiore di Sanità, Analytical Pharmacotoxicology, Viale Regina Elena, 299, 00161 Roma, Italy.
University of Murcia, Department of Legal and Forensic Medicine, Calle Campus Universitario, 11, 30100 Murcia, Spain.
J Anal Toxicol. 2021 Jan 21;44(9):985-992. doi: 10.1093/jat/bkaa020.
A procedure based on gas chromatography-mass spectrometry was developed for the analysis of benzodiazepines (nordiazepam, oxazepam, lormetazepam, lorazepam, clonazepam, bromazepam and alprazolam) in postmortem human ribs. Powdered bone samples, including marrow remains inside, with the internal standard diazepam-d5 were subjected to enzymatic hydrolysis with 100 μL of β-glucoronidase and were incubated in sodium hydroxide for 1 h in a 70°C oven. Samples underwent liquid phase extraction and ethyl acetate was used as eluent. Chromatography was performed on a fused silica capillary column and the selected-ion-monitoring mode was used for analytes determination. The method was validated in the range 0.1-0.5 ng/mg (depending on the benzodiazepine) to 100 ng/mg with average values of recovery, matrix effect and process efficiency ranged from 83.2 to 94.3%, from 97.3 to 102.1% and from 80.5 to 91.2%, respectively. The intra- and inter-day accuracy was <15%. The procedure was tested in rib specimens obtained during routine autopsies from 20 cases where these benzodiazepines were found in blood. Benzodiazepines were detected in the combined bone and marrow samples in 60% of cases. Lorazepam was detected in bone in the range of 0.3-0.7 ng/mg, nordiazepam at 1.3-4.2 ng/mg and oxazepam at 1.1-1.2 ng/mg. To our knowledge, this protocol for the simultaneous analysis of these benzodiazepines is the first performed and validated using human ribs.
建立了一种基于气相色谱-质谱法的程序，用于分析死后人体肋骨中的苯二氮䓬类药物（去甲安定、奥沙西泮、劳拉西泮、氯硝西泮、溴西泮和阿普唑仑）。将包括骨髓在内的粉末状骨样 品与内标地西泮-d5 一起用 100 μLβ-葡萄糖醛酸酶进行酶解，并在 70°C 烘箱中用氢氧化钠孵育 1 小时。样品进行液相萃取，并用乙酸乙酯作为洗脱剂。色谱在熔融石英毛细管柱上进行，采用选择离子监测模式进行分析物测定。该方法在 0.1-0.5ng/mg（取决于苯二氮䓬类药物）至 100ng/mg 的范围内进行验证，平均回收率、基质效应和过程效率分别为 83.2-94.3%、97.3-102.1%和 80.5-91.2%。日内和日间准确度<15%。该程序在 20 例常规尸检中获得的肋骨标本中进行了测试，这些标本中发现了这些苯二氮䓬类药物。在 60%的病例中，在骨和骨髓的组合样本中检测到了苯二氮䓬类药物。在骨中检测到劳拉西泮的浓度范围为 0.3-0.7ng/mg，去甲安定为 1.3-4.2ng/mg，奥沙西泮为 1.1-1.2ng/mg。据我们所知，该协议是首次使用人体肋骨进行的同时分析这些苯二氮䓬类药物的协议，并进行了验证。
