Department of Experimental and Clinical Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, Center for Molecular Signaling (PZMS), Saarland University, Kirrberger Str. Geb. 46, 66421 Homburg, Germany.
Department of Applied Pharmacy, University of Applied Sciences Kaiserslautern, Campus Pirmasens, Carl-Schurz-Str. 10-16, 66953 Pirmasens, Germany.
J Anal Toxicol. 2020 Jul 31;44(6):549-558. doi: 10.1093/jat/bkaa019.
An increasing number of benzodiazepine-type compounds are appearing on the new psychoactive substances market. 8-Chloro-6-(2-fluorophenyl)-1-methyl-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepine (well known as flualprazolam) represents a potent 'designer benzodiazepine' that has been associated with sedation, loss of consciousness, memory loss and disinhibition. The aims of the present study were to tentatively identify flualprazolam metabolites using in vitro incubations with pooled human liver S9 fraction or HepaRG cells by means of liquid-chromatography-high resolution tandem mass spectrometry. Isozymes involved in phase I and II biotransformation were identified in vitro. Results were then confirmed using human biosamples of an 18-year old male who was admitted to the emergency department after suspected flualprazolam ingestion. Furthermore, the plasma concentration was determined using the standard addition method. Seven flualprazolam metabolites were tentatively identified. Several cytochrome P450 and UDP-glucuronosyltransferase isozymes, amongst them CYP3A4 and UGT1A4, were shown to be involved in flualprazolam biotransformation reactions, and an influence of polymorphisms as well as drug-drug or drug-food interactions cannot be excluded. Alpha-hydroxy flualprazolam glucuronide, 4-hydroxy flualprazolam glucuronide and the parent glucuronide were identified as most abundant signals in urine, far more abundant than the parent compound flualprazolam. These metabolites are thus recommended as urine-screening targets. If conjugate cleavage was performed during sample preparation, the corresponding phase I metabolites should be added as targets. Both hydroxy metabolites can also be recommended for blood screening. The flualprazolam plasma concentration determined in the intoxication case was as low as 8 μg/L underlining the need of analytical methods with sufficient sensitivity for blood-screening purposes.
越来越多的苯二氮䓬类化合物出现在新精神活性物质市场上。8-氯-6-(2-氟苯基)-1-甲基-4H-[1,2,4]三唑[4,3-a][1,4]苯并二氮䓬（俗称氟甲噻庚酮）是一种强效的“设计苯二氮䓬”，与镇静、意识丧失、记忆缺失和去抑制有关。本研究的目的是通过液相色谱-高分辨串联质谱法，用混合人肝 S9 级分或 HepaRG 细胞对氟甲噻庚酮进行体外孵育，初步鉴定氟甲噻庚酮的代谢物。鉴定了参与 I 相和 II 相生物转化的同工酶。然后使用一名 18 岁男性的人生物样本进行验证，该男性在疑似氟甲噻庚酮摄入后被送往急诊室。此外，使用标准添加法测定了血浆浓度。初步鉴定了 7 种氟甲噻庚酮代谢物。几种细胞色素 P450 和 UDP-葡萄糖醛酸转移酶同工酶，包括 CYP3A4 和 UGT1A4，被证明参与氟甲噻庚酮的生物转化反应，并且不能排除遗传多态性以及药物-药物或药物-食物相互作用的影响。α-羟基氟甲噻庚酮葡萄糖醛酸苷、4-羟基氟甲噻庚酮葡萄糖醛酸苷和母体葡萄糖醛酸苷被鉴定为尿液中最丰富的信号，远远超过母体化合物氟甲噻庚酮。因此，这些代谢物被推荐为尿液筛查靶标。如果在样品制备过程中进行了轭合物裂解，则应添加相应的 I 相代谢物作为靶标。两种羟基代谢物也可推荐用于血液筛查。在中毒案例中测定的氟甲噻庚酮血浆浓度低至 8μg/L，这表明需要具有足够灵敏度的分析方法用于血液筛查目的。
