合成大麻素MDMB-4en-PICA的体内和体外代谢研究

目的 随着新型合成大麻素的演变,其在体内代谢速度增快,对原药的检测难度增加,为确定是否吸食合成大麻素,其生物标志物可以作为强有力的证据,常见的代谢模型有体内代谢模型和体外代谢模型。方法 本研究采用液相色谱-高分辨质谱技术检测斑马鱼体内代谢模型和人肝微粒体体外代谢模型中MDMB-4en-PICA的代谢情况,并使用Trance Finder 4.1通用版进行数据采集和Compound Discoverer 2.1版本进行代谢物鉴定。结果斑马鱼模型代谢产生26种代谢物,人肝微粒体模型代谢产生17种代谢物,总共32种代谢物,其中包括23种Ⅰ相代谢物和9种Ⅱ相代谢物,涉及10种代谢途径。结论 结果显示,两种模型中二氢二醇代谢物（M16）的峰面积均最高,其次是羟基化代谢物（M22）,而且具有一定的特异性,可以推荐作为MDMB-4en-PICA潜在的生物标志物。     

合成大麻素类新精神活性物质的代谢物鉴定研究进展

合成大麻素类是目前涵盖物质种类最多、滥用最为严重的一类新精神活性物质,代谢物鉴定研究可以为合成大麻素滥用监测提供基础数据,是目前的研究热点。合成大麻素结构修饰的主要趋势是戊基吲哚或吲唑环戊基末端上的氟原子取代为氢原子,这大大提高了化合物的生物活性,涉及的主要代谢反应包括羟基化、氧化脱氟、酰胺水解、酯水解。液相色谱-高分辨质谱已成为代谢物结构鉴定的首选方法。本文基于合成大麻素的结构和分类,着重对代谢软件预测以及人肝细胞模型、人肝微粒体模型、大鼠体内模型、斑马鱼模型和真菌秀丽线虫模型在代谢物鉴定方面的研究进展进行综述。     

基于HPLC-TOF-MS代谢组学方法的溴鼠灵毒性作用评价

目的分析溴鼠灵中毒大鼠尿液的代谢特征,揭示溴鼠灵干预对大鼠毒性作用的分子机制。方法通过构建大鼠溴鼠灵中毒模型,采用高效液相色谱-飞行时间质谱(high performance liquid chromatographytime of flight mass spectrometry,HPLC-TOF-MS)获取大鼠尿液代谢轮廓,并用正交偏最小二乘法-判别分析(orthogonal partial least squares-discrimination analysis,OPLS-DA)进行多变量统计分析,找出与溴鼠灵毒性作用密切相关的差异代谢物。结果 OPLS-DA得分图显示给药前后不同时间的大鼠尿液样本代谢物轨迹在各时间段内相似度较好,呈现各自聚类现象。比较溴鼠灵给药前后大鼠尿液样本,筛选出22个与溴鼠灵毒性相关的差异代谢物。结论溴鼠灵主要通过干扰大鼠体内的三羧酸循环、糖酵解、鞘脂代谢和色氨酸代谢等代谢通路发挥毒性作用,且溴鼠灵毒性作用具有累积效应。基于尿液HPLC-TOF-MS代谢组学方法可为溴鼠灵毒性作用的分子机制研究提供新思路。     

体内咪达唑仑代谢研究

提取小鼠肝P450酶,建立咪达唑仑的体外代谢模型.确定了其在体内的主要代谢产物的结构;通过优化药物体外代谢的条件,获得了较高纯度的咪达唑仑代谢物(α-OH咪达唑仑)对照品.通过志愿者实验,考察了咪达唑仑在体内的代谢过程和咪达唑仑及其代谢物α-OH咪达唑仑在体内的消除过程.结果表明以代谢物α-0H咪达唑仑作为分析目标物,建立灵敏、快速的分析方法,可使体内咪达唑仑检测的检出时限从6h延长至48h.该研究结果可满足实际检案需要,并可为分析结果的评价提供依据.     

乌头生物碱及其代谢物在家兔死后体内分布研究

目的研究乌头生物碱及6种代谢物在中毒家兔死后体内的分布规律,为乌头生物碱中毒案件中检材的选取提供参考依据。方法将生川乌粉碎制成水煎剂,以其中乌头碱的LD50计算,给予家兔灌胃,待其死亡后立即解剖取材,采用高效液相色谱-串联质谱测定检材中乌头生物碱及6种代谢物的含量。结果乌头生物碱及6种代谢物在各脏器及体液中的死后分布情况为:(1)乌头碱:尿液外周血、心血、胃、肺、肾、心、肝、肌、脾;(2)新乌头碱:尿液外周血、心血、肺、胃、肾、肝、肌、脾、心;(3)次乌头碱:尿液外周血、心血、胃、肺、肝、脾、肾、肌、心;(4)苯甲酰乌头原碱:尿液外周血心血胃、脾、肾、肺、肌、心、肝;(5)苯甲酰新乌头原碱:尿液外周血心血胃、脾、肾、肺、肌、肝、心;(6)苯甲酰次乌头原碱:尿液外周血心血、胃、肾、脾、肺、肌、肝、心;(7)乌头原碱:尿液外周血、心血、肾、胃、心、脾、肝、肌、肺;(8)新乌头原碱:尿液外周血、心血、肾、胃、肝、心、肺、脾、肌;(9)次乌头原碱:尿液外周血、心血、肝、肾、脾、胃、心、肺、肌。结论乌头生物碱及6种代谢物分布以血液和尿液中为主,在脏器以胃、肺脏、肾脏和肝脏等含量较高。     

基于模式识别方法研究深静脉血栓形成大鼠尿液代谢物谱

目的采用代谢组学技术研究深静脉血栓形成(deep venous thrombosis,DVT)大鼠尿液中代谢物谱,筛选出可用于DVT诊断和法医学鉴定的小分子生物标志物。方法建立下腔静脉完全结扎DVT大鼠模型,分为模型组、假手术组和对照组,每组各10只。模型组和假手术组大鼠在建模后48 h于代谢笼中收集24 h尿液,同时对照组收集24 h尿液。核磁共振检测其代谢物谱,SIMCA-P 14.1软件进行模式识别,通过正交偏最小二乘法-判别分析(orthogonal PLS-DA,OPLS-DA)模型中的变量权重重要性排序(variable importance in projection,VIP)值结合Mann-Whitney U检验,寻找尿液中差异代谢物。结果模型组、假手术组和对照组大鼠尿液的代谢轮廓呈现显著性差异。通过偏最小二乘法-判别分析(partial least squares method-discriminant analysis,PLS-DA)模型可以有效判别模型组、假手术组与对照组。与对照组大鼠相比,DVT大鼠尿液中亮氨酸、谷氨酰胺、肌酸、肌酐和蔗糖水平上调,3-羟基丁酸、乳酸、丙酮、α-酮戊二酸、柠檬酸和马尿酸水平下调。结论 DVT大鼠尿液中的差异代谢物有望成为该疾病的候选生物标志物,该结果可为DVT的诊断、治疗和法医学鉴定提供研究基础。     

家兔死后体内2-氨基噻唑啉-4-羧酸分布研究

目的建立家兔氰化钾灌胃给药致死动物模型,研究氰化物代谢物2-氨基噻唑啉-4-羧酸(ATCA)在家兔体内的死后分布规律。方法雄性家兔7只(体重约2.0kg~2.5kg)经口灌胃2LD50(10mg/kg)氰化钾水溶液,观察家兔反应,待家兔呼吸、心跳和反射全部消失后立即对家兔进行解剖取心、肝、脾、肺、肾、脑、睾丸、胃壁、肌肉等组织检材以及心血、玻璃体液、尿液等体液检材置于-80℃冷冻保存待检。液相色谱-串联质谱联用法测定生物检材中氰化物代谢物ATCA的含量,对其在各个组织的分布进行比较并寻找规律。结果氰化钾灌胃后家兔出现呼吸频率加快,走路乏力,癫痫大发作样抽搐,后瞳孔散大,肌肉松弛,各种反射消失,似"电击样"死亡。死亡后测得心血中氰基(CN-)平均浓度为11.81μg/ml。死后0h氰化物代谢物ATCA在家兔体内的分布如下:脾>肺>肾>肝、脑>睾丸>心血>心、胃壁>玻璃体液>右下肢肌肉>尿。结论大剂量氰化物中毒致死后其代谢物ATCA在家兔体内分布不均匀,在脾中最高,尿中最低。在疑似氰化物中毒致死案件的法医学鉴定中,除采取心血外,还应全面正确采集分布量较高的脾、肺、肾和肝组织进行氰化物代谢物ATCA的定性定量分析。     

氯胺酮在家兔体内的毒物代谢动力学

目的研究氯胺酮及其代谢物去甲氯胺酮在家兔体内的毒物代谢动力学特征。方法家兔以氯胺酮0.15g/kg剂量灌胃,分别于给药前和给药后不同时间点收集血液和尿液,血清和尿液中氯胺酮及代谢物用GC-MS法定性、GC-NPD法定量检测,WinNorLin软件拟合房室模型并计算毒物代谢动力学参数。全程记录实验动物主要生命体征变化。结果氯胺酮和代谢物去甲氯胺酮在家兔体内的毒物代谢动力学过程均呈一级动力学特征,符合二室开放模型,氯胺酮毒物代谢动力学方程为ρt=121.760e-0.025t+0.980e-0.002t+4.579 e-0.021t,去甲氯胺酮毒物代谢动力学方程为ρt=640.919 e-0.03t+1.023 e-0.001t+9.784 e-0.031t。血液中氯胺酮质量浓度达峰时间为(40.950±12.098)min,血峰质量浓度为(9.015±1.344)μg/mL,消除半衰期为(430.370±28.436)min。给药后30～240 min内氯胺酮在血清和尿液中的质量浓度之间具有动态平衡的中度相关性。家兔给药后30min出现中毒症状,120min后渐恢复正常。结论建立的氯胺酮毒物代谢动力学方程和参数...     

兔体内三唑仑及其代谢物的检测

目的不同方法检测三唑仑及代谢物α-羟基三唑仑，为其及时、准确的检测提供科学证据和最优方法。方法用GC／NPD、GC／MS、MS／MS、GC／ECD对兔体内三唑仑及代谢物α-羟基三唑仑进行检测。结果GC／ECD法最易检测出三唑仑及其代谢物。结论兔胃及内容物、尿液中容易检测到三唑仑及其代谢物；运用酶水解后，检测率最高；GC／ECD检测三唑仑及其代谢物的灵敏度最高。     

氯硝安定在生物体内的代谢

目的推测氯硝安定在生物体内的主要代谢方式,检测其代谢产物。方法取5只Wistar大鼠,连续3d给每只大鼠灌胃氯硝安定（剂量分别为1、2、2mg）,收集灌胃后24h内尿液;给3只大鼠各灌胃2mg氯硝安定,2h后处死,取血液和肝组织等检材;收集3名口服5mg氯硝安定的志愿者24h内尿液。分别对不同检材进行处理后,气相色谱/质谱联用（GC/MS）检测。结果在大鼠阳性尿液和志愿者阳性尿液中,均检出7-乙酰氨基氯硝安定和7-氨基氯硝基安定;在大鼠血液中,主要检出氯硝安定和7-氨基氯硝基安定;在大鼠肝组织中,主要检出7-乙酰氨基氯硝安定,还有少量7-氨基氯硝安定和氯硝安定。结论氯硝安定进入生物体后,其7位硝基被还原为氨基,氨基接着被乙酰化,形成7-氨基氯硝基安定和7-乙酰氨基氯硝安定代谢物,其中7-乙酰氨基氯硝安定为主要代谢物。     

Metabolism and toxicological detection of the designer drug N-(1-phenylcyclohexyl)-3-methoxypropanamine (PCMPA) in rat urine using gas chromatography-mass spectrometry

Studies on the metabolism and the toxicological detection of the phencyclidine-derived designer drug N-(1-phenylcyclohexyl)-3-methoxypropanamine (PCMPA) in rat urine are described using gas chromatographic-mass spectrometric (GC-MS) techniques. Based on the identified metabolites, the following metabolic pathways could be postulated: N-dealkylation, O-demethylation partially followed by oxidation of the resulting alcohol to the corresponding carboxylic acid, hydroxylation of the cyclohexyl ring at different positions, and aromatic hydroxylation. The formed metabolites were identical to those of the homologue N-(1-phenylcyclohexyl)-3-ethoxypropanamine (PCEPA) with exception of the mono hydroxyl metabolites of PCEPA. All PCMPA metabolites were partially excreted in conjugated form. An intake of a common drug users' dose of PCMPA could be detected in rat urine by the authors' systematic toxicological analysis (STA) procedure using full-scan GC-MS after acid hydrolysis, liquid-liquid extraction and microwave-assisted acetylation. The STA should be suitable for proof of an intake of PCMPA also in human urine assuming similar metabolism.     

Characterization of In Vitro Metabolites of CP 47,497, a Synthetic Cannabinoid,in Human Liver Microsomes by LC‐MS/MS

CP 47,497, a potent cannabinoid receptor type 1 agonist, is the main active ingredient in the herbal mixture “Spice” sold in European countries. The illegal use of “Spice” for its psychoactive effects has become a social issue. In this study, the in vitro metabolism of CP 47,497 was investigated in human liver microsomes to characterize the metabolic fate of CP 47,497. CP 47,497 was incubated with human liver microsomes, and the reaction mixture was analyzed using liquid chromatography‐tandem mass spectrometry. A total of eight metabolites were detected in human liver microsomes and structurally characterized based on mass spectral data. The main metabolic pathways involved hydroxylations or oxygenations. The identified metabolites were mono‐oxygenated metabolites (M1 and M4), mono‐hydroxylated metabolites (M3, M5, M6, M7, and M8), and a di‐oxygenated metabolite (M2). The detection of these metabolites could confirm the presence of CP 47,497 in biological samples; therefore, collectively, they would be excellent indicators of “Spice” drug abuse.     

Assessment of UDP-glucuronosyltransferase catalyzed formation of ethyl glucuronide in human liver microsomes and recombinant UGTs

While ethanol is primarily metabolized to acetaldehyde and acetic acid via alcohol dehydrogenase, a minor but increasingly important pathway in the field of forensic science involves the conjugation of glucuronic acid to form an ethyl glucuronide (EtG) metabolite. The kinetics of ethyl glucuronide formation were examined in human liver microsomes (HLM) and recombinant UDP-glucuronosyltransferases (UGTs). The metabolite exhibited a relatively slow rate of formation in a human liver microsome mix of 75.4 pmol/(min/mg). Further investigation identified multiple UGT isoforms to be responsible for catalyzing the addition of glucuronic acid to ethanol, with UGT1A1 and 2B7 being the two most prevalent isoforms. Co-incubation with bilirubin or 3'-azido-3'-deoxythymidine (UGT1A1 and 2B7 inhibitors, respectively) inhibited the greatest amount of ethyl glucuronide formation, though other UGT inhibitors also showed some effect. Enzyme kinetics were performed in human liver microsomes and recombinant UGT enzymes. The apparent Km (Km app) and Vmax values were determined to be 0.17+/-0.08 mM and 75.98+/-5.63 pmol/(min/mg) (human liver microsomes), 0.03+/-0.01 mM and 25.22+/-3.45 pmol/(min/mg) (UGT1A1), and 0.11+/-0.04 mM and 52.03+/-9.8 pmol/(min/mg) (UGT2B7). Thus, it appears that multiple UGTs are responsible for the formation of ethyl glucuronide and that any functional differences in the enzymology underlying ethyl glucuronide formation would most likely be masked by a combination of other enzymatic pathways.     

尿中MDMA及其代谢物的GC和GC／MS分析

考察MDMA在人体内的代谢以及建立尿中MDMA和体内主要代谢物MDA的分析方法。尿样水解后经液-液提取处理,用GC／MS（EI、PCI）和GC／FID法分析。人摄入MDMA后尿中MDA和原体MDMA比约为0．10～0．14。GC／MS／SIM和GC／FID法的最低检出限为2ng／ml和50ng／ml,回收率大于85％,变异系数小于10％。该法简便快速、灵敏度高、结果可靠,可用于MDMA滥用者的尿样鉴定。MDA／MDMA浓度比可作为评判毒分结果的参考指标。     

Analysis of 4‐Bromo‐2,5‐DimethoxyphenethylamineAbuser's Urine: Identification and Quantitation of Urinary Metabolites

The metabolites of 4‐bromo‐2,5‐dimethoxyphenethylamine (2C‐B), a psychoactive drug with hallucinogenic activity, were investigated in a urine sample from a user of 2C‐B. The urine sample was deconjugated enzymatically and the metabolites were recovered by liquid–liquid extraction. The extract was analyzed by gas chromatography/mass spectrometry after derivatization, and the results were used to identify and quantitate the metabolites. 4‐Bromo‐2,5‐dimethoxyphenylacetic acid was the most abundant metabolite of 2C‐B in human urine and accounted for 73% of the total amount of detected metabolites, followed by 4‐bromo‐2‐hydroxy‐5‐methoxyphenylacetic acid (13%) and 4‐bromo‐2,5‐dimethoxyphenylethyl alcohol (4.5%). According to the literature, the main metabolites of 2C‐B in rat urine are N‐(4‐bromo‐2‐methoxy‐5‐hydroxyphenylethyl)acetamide and N‐(4‐bromo‐2‐hydroxy‐5‐methoxyphenylethyl)acetamide. However, these metabolites accounted for only a small proportion of the total amount of detected metabolites in human urine, which indicates that there are significant species‐specific differences in the metabolism of 2C‐B. 4‐Bromo‐2,5‐dimethoxyphenylacetic acid, which was the most abundant metabolite in human urine, is thought to be generated by deamination of 2C‐B by monoamine oxidase (MAO) followed by oxidation by aldehyde dehydrogenase. Our results suggest that MAO plays a crucial role in the metabolism of 2C‐B in humans.     

Determination of nimetazepam and 7-aminonimetazepam in human urine by using liquid chromatography–tandem mass spectrometry

We report determination of metabolites of popular drugs of abuse, including nimetazepam and nitrazepam, in urine by using liquid chromatography/mass spectrometry. Nimetazepam and its metabolites, 7-aminonimetazepam and nitrazepam, were extracted by solid-phase extraction using a DAU cartridge. An ammonium acetate buffer solution (pH 4) and a Luna polar-RP column were selected as the mobile and stationary phase, respectively, for liquid chromatography. Mass spectrometry was used for analysis and was optimized for operation in the positive mode for all analytes. The urine specimens were screened for the presence of nimetazepam and its metabolites nitrazepam and 7-aminonimetazepam at a concentration of 0.1 ng/mL. Presence of 7-aminonimetazepam in the urine was an indicator of the subject being a probable abuser of nimetazepam.     

CYP450在大鼠肝内氯胺酮N-去甲基代谢中的作用

目的考察细胞色素P450(CYP450)亚型酶在大鼠肝内氯胺酮N-去甲基代谢中的作用。方法选择CYP450亚型酶的专属性诱导剂β-萘黄酮、苯巴比妥钠、利福平、异烟肼和地塞米松,腹腔给药对实验大鼠肝微粒体进行诱导;考察经诱导后的各组肝微粒体氯胺酮N-去甲基代谢速率。选择CYP450专属性抑制剂α-萘黄酮、磺胺甲恶唑和奥美拉唑、硫酸奎宁和酮康唑,对大鼠空白肝微粒体进行体外抑制;考察各组氯胺酮N-去甲基代谢速率。上述结果分别与对照组进行比较。结果经苯巴比妥钠和地塞米松诱导,氯胺酮代谢速率有显著性变化(P<0.05～0.001),去甲氯胺酮的生成速率分别是对照组的2.3～4.6倍和1.4～1.9倍,其余诱导剂对氯胺酮代谢无影响。经酮康唑作用,氯胺酮的代谢产生显著抑制(P<0.001),N-去甲基活性为对照组的47.20%～28.97%,其余抑制剂对氯胺酮代谢无影响。结论CYP450亚型酶中CYP2B和CYP3A参与了氯胺酮N-去甲基代谢,氯胺酮可能在体内与上述酶底物发生相互作用。     

Analysis of dimethylamphetamine and its metabolites in human urine by liquid chromatography-electrospray ionization-mass spectrometry with direct sample injection

An analytical method to identify and determine dimethylamphetamine (DMA) and its metabolites in human urine was developed with liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) involving the direct injection of a urine sample. The urine samples were directly injected by using a gel permeation column, whose stationary phase was polyvinyl alcohol with a small amount of a carboxyl group, so DMA and its metabolites were analyzed rapidly and simply without pretreatment such as extraction, concentration and derivatization. DMA and its metabolites were identified in drug-free human urine spiked with 1 microg of DMA, dimethylamphetamine N-oxide (DMANO) and methamphetamine (MA), and 3 microg of amphetamine (AM) in 1 ml of urine under the full-scan mode. Under the selected ion monitoring (SIM) mode, the limits of detection (signal-to-noise ratio=5) for DMA, DMANO, MA and AM were 20, 20, 20 and 60 ng in 1 ml of urine, respectively. This method was applied to the identification and determination of DMA and its metabolites in urine samples of 10 DMA abusers. The concentrations of DMANO were higher than those of unchanged DMA in all urine samples; thus, DMANO is considered to be a useful metabolite as an indicator to prove DMA intake.     

气相色谱质谱联用法检测大鼠尿液中2C-B及其代谢物

目的研究4-溴-2,5-二甲氧基苯乙胺（2C—B）在大鼠体内的代谢物以及代谢途径。方法取Wistar大鼠3只,以2C-B灌胃,收集24h内尿液,用B葡萄糖醛酸酶水解,Oasis HLB柱固相提取,DB-35MS柱分析,气相色谱质谱联用检测。结果从大鼠尿液中检出6种2C-B的代谢产物,分别为：4-溴-2-羟基-5-甲氧基苯乙醇、4-溴-2,5-二甲氧基苯乙醇、4-溴-2-羟基-5-甲氧基苯乙酸、4-溴-2,5-二甲氧基苯乙酸、1-乙酰氨基-2-（4-溴-5-羟基-2-甲氧基苯）乙烷和1-乙酰氨基-2-（4-溴-2-羟基-5-甲氧基苯）乙烷。未检出2C—B原药。结论2C-B在大鼠尿液中主要以代谢物形式存在,其在大鼠体内至少仔在两种代谢途径：第一种是2C—B的2位和5位氧上去甲基后氨基被乙酰化;第二种是2C—B去氨基生成醛,接着被还原或氧化生成醇和羧酸。