Fatality involving ocfentanil documented by identification of metabolites

The use of new psychoactive substances (NPS) has rapidly increased over the last decade. In the last 4 years, producers increasingly appear to be targeting non‐controlled synthetic opioids, involving fentanyl derivatives such as ocfentanil (OcF). Identification of metabolites is of major importance in the context of NPS use, as it could improve the detection window in biological matrices in clinical and forensic intoxication cases. Hence, this work aims to report a fatality involving OcF documented by the identification of metabolites. A 30‐year‐old woman was found dead at home: an unidentified powder was found near her body and some injection sites were found at the autopsy. Toxicological analyses allowed to determine the presence of OcF in the powder, blood (3.7/3.9 μg/L, peripheral/cardiac) and in other post‐mortem samples. The most relevant potential CYP‐ and UGT‐dependent metabolites of OcF were investigated in vitro using human liver microsome incubation and liquid chromatography coupled with high resolution mass spectrometry, and subsequently confirmed in post‐mortem samples. Four OcF metabolites were produced in vitro (a mono‐hydroxylated OcF, O‐desmethylOcF, a hydroxylated desmethylOcF and a glucuronidated form of the O‐desmethylOcF), and all except the glucuronide were observed in blood and bile post‐mortem samples. Considering the relative intensity of the chromatographic peak areas, O‐desmethylOcF can be suggested to be an abundant metabolite of OcF. Nevertheless, the relevance of O‐desmethylOcF as being a complementary analytical target of OcF for OcF use detection needs further in vivo confirmation, especially through analysis of urines from users.     

Human urinary metabolic patterns of the designer benzodiazepines flubromazolam and pyrazolam studied by liquid chromatography–high resolution mass spectrometry

Over the past ~8 years, hundreds of unregulated new psychoactive substances (NPS) of various chemical categories have been introduced as recreational drugs through mainly open online trade. This study was performed to further investigate the human metabolic pattern of the NPS, or designer benzodiazepines flubromazolam and pyrazolam, and to propose analytical targets for urine drug testing of these substances. The urine samples originated from patient samples confirmed by liquid chromatography–high‐resolution tandem mass spectrometry (LC–HRMS/MS) analysis to contain flubromazolam or pyrazolam. The LC–HRMS/MS system consisted of a YMC‐UltraHT Hydrosphere C18 column (YMC, Dinslaken, Germany) coupled to a Thermo Scientific (Waltham, MA, USA) Q Exactive Orbitrap MS operating in positive electrospray mode. The samples were analyzed both with and without enzymatic hydrolysis using β‐glucuronidase. Besides the parent compounds, the main urinary excretion products were parent glucuronides, mono‐hydroxy metabolites, and mono‐hydroxy glucuronides. In samples prepared without hydrolysis, the most common flubromazolam metabolites were 1 of the mono‐hydroxy glucuronides and 1 of the parent glucuronides. For pyrazolam, a parent glucuronide was the most common metabolite. These 3 metabolites were detected in all samples and were considered the primary targets for urine drug testing and confirmation of intake. After enzymatic hydrolysis of the urine samples, a 2–19‐fold increase in the concentration of flubromazolam was found, highlighting the value of hydrolysis for this analyte. With hydrolysis, the flubromazolam hydroxy metabolites should be used as target metabolites.     

基于UHPLC-Q-Orbitrap HRMS和生物信息学探讨瓜蒌薤白汤治疗冠心病的潜在药效物质基础和作用机制

目的 系统分析并识别瓜蒌薤白汤主要化学成分和入血成分,预测其防治冠心病的药效物质基础和作用机制。方法 采用超高效液相色谱-四级杆/静电场轨道阱高分辨质谱（UHPLC-Q-Orbitrap HRMS）进行瓜蒌薤白汤体内外成分分析,结合色谱峰保留时间、精确相对分子质量、碎片离子、中性丢失等信息进行化合物指认。使用生物信息学方法,对潜在靶点进行GO和KEGG富集分析,借助Cytoscape软件构建瓜蒌薤白汤“活性成分-核心靶点-通路”网络图,对潜在活性成分和作用通路进行预测。利用AutoDock Tools 1.5.6软件对其活性成分与核心靶点进行分子对接验证。结果 瓜蒌薤白汤体外定性得到了68个化学成分,大鼠ig给药后在血清样本中检测到23个入血成分。其中芹菜素、木犀草素、酒渣碱、亚油酸等化合物为瓜蒌薤白汤发挥治疗冠心病药效的关键成分,前列腺素内过氧化物合成酶2（prostaglandin-endoperoxidesynthase 2,PTGS2）、肿瘤坏死因子（tumor necrosis factor,TNF）、基质金属蛋白酶-9（matrix metalloproteinase-9,MMP-9）、表皮生长因子受体（epidermal growth factor receptor,EGFR）、半胱氨酸蛋白水解酶3（cysteine-containing aspartate-specific protease 3,CASP3）等为瓜蒌薤白汤治疗冠心病的主要靶点,涉及到脂质与动脉粥样硬化、癌症的途径、TNF信号通路、NF⁃κB信号通路、MAPK信号通路。结论 初步鉴定了瓜蒌薤白汤防治冠心病的潜在药效成分并预测其作用靶点,为该方药效物质基础及作用机制的深入研究提供参考。     

奶制品中二恶英类化合物两种提取方法的比较

[目的]比较奶和奶制品中二恶英类化合物索氏抽提法和液液萃取法。[方法]采集市售4种纯牛奶和3种奶粉,分别通过索氏抽提和液液萃取,再用酸性硅胶处理,利用硅胶柱和铝柱净化,用高分辨气相色谱/高分辨质谱检测分析PCDDs/Fs和PCBs。[结果]索氏抽提方法对牛奶和奶制品中脂肪不能提取完全,得到的二恶英类化合物检测结果不能真实反映牛奶和奶制品中二恶英类化合物污染情况。液液萃取法能完全萃取牛奶和奶制品中脂肪以及溶于脂肪中的二恶英类化合物,得到的结果能真实反映牛奶和奶制品中二恶英类化合物污染情况。[结论]液液萃取法是适用于检测牛奶和牛奶制品中二恶英类化合物的检测方法。     

Wildtype motoneurons,ALS‐Linked SOD1 mutation and glutamate profoundly modify astrocyte metabolism and lactate shuttling

The selective degeneration of motoneuron that typifies amyotrophic lateral sclerosis (ALS) implicates non‐cell‐autonomous effects of astrocytes. However, mechanisms underlying astrocyte‐mediated neurotoxicity remain largely unknown. According to the determinant role of astrocyte metabolism in supporting neuronal function, we propose to explore the metabolic status of astrocytes exposed to ALS‐associated conditions. We found a significant metabolic dysregulation including purine, pyrimidine, lysine, and glycerophospholipid metabolism pathways in astrocytes expressing an ALS‐causing mutated superoxide dismutase‐1 (SOD1) when co‐cultured with motoneurons. SOD1 astrocytes exposed to glutamate revealed a significant modification of the astrocyte metabolic fingerprint. More importantly, we observed that SOD1 mutation and glutamate impact the cellular shuttling of lactate between astrocytes and motoneurons with a decreased in extra‐ and intra‐cellular lactate levels in astrocytes. Based on the emergent strategy of metabolomics, this work provides novel insight for understanding metabolic dysfunction of astrocytes in ALS conditions and opens the perspective of therapeutics targets through focusing on these metabolic pathways. GLIA 2017 GLIA 2017;65:592–605     

基于UPLC-Q-Orbitrap HRMS技术的参坤养血颗粒化学成分研究

目的为系统研究参坤养血颗粒的化学成分,采用超高效液相色谱-四级杆/静电场轨道阱高分辨质谱(UPLCQ-Orbitrap HRMS)法建立一种快速、准确识别中药中复杂化学成分的定性分析方法。方法通过UPLC-Q-Orbitrap HRMS捕捉化合物的精准相对分子质量以及多级碎片离子信息,同时将其与对照品的保留时间和质谱信息进行比对,并结合相关参考文献或Chemical Book等数据库信息最终实现对未知化合物的快速、准确定性。结果从参坤养血颗粒中共鉴定出51种化学成分,主要包括有机酸类、黄酮类、醌类、苷类和其他类。结论建立的方法可快速、准确、系统地识别参坤养血颗粒中多种化学成分,并为其质量控制、药效物质基础研究及进一步的临床应用提供扎实的理论依据和科学的研究思路。     

UHPLC-Q-Orbitrap HRMS快速鉴定羊红膻药材的化学成分

目的:应用超高液相色谱四级杆-静电场轨道肼高分辨质谱联用(UHPLC-Q-Orbitrap HRMS)技术,快速识别与鉴定羊红膻药材中主要化学成分。方法:采用Waters Acquity UPLC BEH C18色谱柱(2. 1 mm×50 mm,1. 7μm),流动相0. 1%甲酸水溶液(A)-乙腈(B)进行梯度洗脱。质谱采用ESI源,负离子一级全扫描-数据依赖性二级质谱扫描(MS2)模式采集数据。通过高分辨质谱数据解析,并结合对照品数据和相关文献对羊红膻药材中的化学成分进行识别和鉴定。结果:通过精确相对分子质量和二级碎片离子分析,并结合对照品确认、质谱裂解规律和相关文献检索,共鉴定29个化合物,包括绿原酸类化合物19个,黄酮类7个,小分子有机酸3个,其中除木犀草素葡萄糖醛酸苷和芹菜素葡萄糖醛酸苷外,其余27个化合物为首次从该药材中发现。结论:建立的UHPLC-Q-Orbitrap HRMS可快速鉴定羊红膻药材中的化学成分,研究结果为其药效物质基础、质量控制以及临床应用等研究提供了科学的依据。     

苗药水冬瓜叶化学成分的HPLC-HESI-HRMS分析

目的 采用HPLC-HESI-HRMS技术对苗药水冬瓜叶中的化学成分进行定性分析。方法 采用Thermo Fisher Hypersil GOLD aQ C₁₈色谱柱（100 mm×2.1 mm,1.9 μm）,以0.1%甲酸水溶液-乙腈（0.1%甲酸）为流动相梯度洗脱;质谱使用HESI离子源,正离子和负离子模式下采集数据。同时采用HPLC比较7-羟基香豆素、断氧化马钱苷、金丝桃苷、异槲皮苷对照品与样品中化合物17,20,24,25保留时间（t_R）差异性,比较薄层色谱荧光特征与比移值（Rf值）,并参考文献中有关7-羟基香豆素、断氧化马钱苷、金丝桃苷、异槲皮苷质谱裂解碎片特征综合进行验证。结果 通过高分辨质谱正、负离子质谱信息、数据库及相关文献数据对照,共推测出苗药水冬瓜叶中38个化合物,包括3个氨基酸类化合物,5个生物碱类化合物,10个有机酸类化合物,3个苯丙素类化合物,3个芳香含氧衍生物类化合物,3个萜类化合物,6个黄酮类化合物,1个酰胺类化合物,4个其他类化合物。验证结果表明,7-羟基香豆素、断氧化马钱苷、金丝桃苷、异槲皮苷对照品分别与化合物17,20,24,25的t_R相比差异均<0.1 min;在药材样品溶液、7-羟基香豆素、断氧化马钱苷、金丝桃苷、异槲皮苷对照品色谱相应的位置上分别显相同颜色的蓝色荧光斑点,Rf值一致;文献表明化合物17,20,24,25分别与7-羟基香豆素、断氧化马钱苷、金丝桃苷、异槲皮苷具有相似的质谱裂解碎片特征。结论 初步鉴定出化合物17,20,24,25分别为7-羟基香豆素、断氧化马钱苷、金丝桃苷、异槲皮苷。HPLC-HESI-HRMS对苗药水冬瓜叶中化学成分的初步推测具有一定的科学性,为阐明苗药水冬瓜叶药材的药效物质基础提供依据。     

Studies on the metabolism of the fentanyl‐derived designer drug butyrfentanyl in human in vitro liver preparations and authentic human samples using liquid chromatography‐high resolution mass spectrometry (LC‐HRMS)

Increasing numbers of new psychoactive substances (NPS) among them fentanyl derivatives has been reported by the European monitoring centre for drugs and drug addiction (EMCDDA). Butyrfentanyl is a new fentanyl derivative whose potency ratio was found to be seven compared to morphine and 0.13 compared to fentanyl. Several case reports on butyrfentanyl intoxications have been described. Little is known about its pharmacokinetic properties including its metabolism. However, knowledge of metabolism is essential for analytical detection in clinical and forensic toxicology. Therefore, in vitro and in vivo phase I and phase II metabolites of butyrfentanyl were elucidated combining liquid chromatography with a qTOF high resolution mass spectrometer. Human liver microsomes and recombinant cytochrome P450 enzymes (CYP) were used for in vitro assays. Authentic blood and urine samples from a fatal intoxication case were available for in vivo comparison. Butyrfentanyl was shown to undergo extensive metabolism. Six pathways could be postulated with hydroxylation and N ‐dealkylation being the major ones in vitro . In vivo , hydroxylation of the butanamide side chain followed by subsequent oxidation to the carboxylic acid represented the major metabolic step in the authentic case. Initial screening experiments with the most relevant CYPs indicated that mainly CYP2D6 and 3A4 were involved in the primary metabolic steps. Altered CYP2D6 and CYP3A4 status might cause a different metabolite pattern, making the inclusion of metabolites of different pathways recommendable when applying targeted screening procedures in clinical and forensic toxicology. Copyright © 2016 John Wiley & Sons, Ltd.