Institute of Toxicology Research, 45 Kasztanowa Street, Borowa 55093, Poland.
Wroclaw Medical University, Department of Forensic Medicine, 4 J. Mikulicza-Radeckiego Street, Wroclaw 50345, Poland.
J Pharm Biomed Anal. 2023 May 10;228:115318. doi: 10.1016/j.jpba.2023.115318. Epub 2023 Feb 27.
Barbiturates which are old pharmaceutical drugs are still widely used in medical treatment of epilepsy and for general anesthesia. To date, more than 2500 different barbituric acid analogs have been synthesized, and 50 of them were introduced into medical use over the last century. Due to their highly addictive properties, pharmaceuticals containing barbiturates are under strict control in many countries. However, by considering the worldwide problem with new psychoactive substances (NPS) the introduction of new designer barbiturate analogs into the dark market might serve a serious public health problem in the near future. For this reason there is an increasing need for application methods for barbiturates monitoring in biological samples. The UHPLC-QqQ-MS/MS method for determination of 15 barbiturates, phenytoin, methyprylon and glutethimide was developed and fully validated. The biological sample volume was reduced to only 50 µL. A simple LLE (pH 3 with ethyl acetate) was successfully applied. The lower LOQ was 10 ng/mL. The method enables differentiation of structural isomers: hexobarbital and cyclobarbital; as well as amobarbital and pentobarbital. Chromatographic separation was achieved with the use of the alkaline mobile phase (pH 9) and Acquity UPLC BEH C18 column. Furthermore, the novel fragmentation mechanism of barbiturates was proposed, which may have a great impact in identification of novel barbiturates analogs introduced to illegal marketplaces. The presented technique has a great potential to be applied in forensic, clinical and veterinary toxicological laboratories, as was evidenced by the positive results of international proficiency tests.
巴比妥类药物是一种古老的药物，目前仍广泛用于治疗癫痫和全身麻醉。迄今为止，已经合成了超过 2500 种不同的巴比妥酸类似物，其中 50 种在过去一个世纪被引入医学用途。由于其高度成瘾性，含有巴比妥类药物的药物在许多国家受到严格控制。然而，考虑到新精神活性物质（NPS）的全球问题，新的设计型巴比妥类类似物可能会在不久的将来成为一个严重的公共卫生问题。因此，在生物样本中监测巴比妥类药物的应用方法的需求不断增加。建立并完全验证了一种用于测定 15 种巴比妥类药物、苯妥英、甲丙氨酯和格鲁米特的 UHPLC-QqQ-MS/MS 方法。将生物样本体积减少至仅 50μL。成功应用了简单的 LLE（pH3 用乙酸乙酯）。最低定量限为 10ng/mL。该方法能够区分结构异构体：己巴比妥和环巴比妥；以及阿美巴比妥和戊巴比妥。使用碱性流动相（pH9）和 Acquity UPLC BEH C18 柱实现了色谱分离。此外，提出了巴比妥类药物的新的碎裂机制，这可能对鉴定引入非法市场的新型巴比妥类类似物产生重大影响。该技术具有在法医、临床和兽医毒理学实验室中应用的巨大潜力，国际能力验证的阳性结果证明了这一点。
