Structural elucidation of in vitro metabolites of bavachinin in rat liver microsomes by LC-ESI-MSn and chemical synthesis

1.?Bavachinin isolated from Psoralea corylifolia has various activities, such as antimicrobial, antiallergic, antitumor and so on. Our previous study showed that natural bavachinin exhibits peroxisome proliferator-activated receptor γ-agonist activity.

2.?In vitro studies on bavachinin metabolism were conducted using rat liver microsomes incubated at 37?°C for 60?min.

3.?Structures of eight metabolites of the incubation mixtures were cautiously characterized using electrospray tandem mass spectra and three synthetic compounds. The results indicated that eight metabolites of bavachinin were biotransformed mainly through oxidation.

4.?The metabolic pathways of bavachinin were elucidated in vitro. These results contribute to the understanding of bavachinin’s in vivo metabolism.     

地非三唑在鼠肝微粒体中的体外代谢

目的　为了解地非三唑 (Dip)在不同预处理的鼠肝微粒体中主要受何种酶代谢影响 ,为其临床合理应用和进一步开发利用提供科学依据。方法　将Dip与 6种不同诱导剂〔苯巴比妥 (PB)、地塞米松(Dex)、β 萘黄酮 (BNF)、Dip、吡啶和空白对照〕诱导的鼠肝微粒体进行体外共孵育 ,用氯仿终止反应 ,以地西泮为内标 ,采用反相高效液相色谱 (RP HPLC)法测定孵育后剩余的Dip的含量。结果　BNF诱导的鼠肝微粒体对Dip代谢具有强烈的催化活性 ,Dip诱导的微粒体的催化能力次之 ,PB诱导组也有一定的催化能力 ,其他几种诱导剂诱导的微粒体对Dip代谢能力与对照组无明显差别。测得Dip在BNF诱导的鼠肝微粒体中的Km 为 (6 0 .5± 1.3) μmol·L- 1,vm 为 (5 .6± 0 .4 )mmol·g- 1·min- 1。结论　由BNF诱导的鼠肝微粒体 (主要为细胞色素P4 5 0 1A)和PB诱导的鼠肝微粒体 (主要为细胞色素P4 5 0 2B)在Dip的体外代谢中可能起主导作用 ;Dip诱导的鼠肝微粒体对其自身的代谢也起了重要作用。     

In vitro metabolic studies using homogenized horse liver in place of horse liver microsomes

The study of the metabolism of drugs, in particular steroids, by both in vitro and in vivo methods has been carried out in the authors' laboratory for many years. For in vitro metabolic studies, the microsomal fraction isolated from horse liver is often used. However, the process of isolating liver microsomes is cumbersome and tedious. In addition, centrifugation at high speeds (over 100 000 g) may lead to loss of enzymes involved in phase I metabolism, which may account for the difference often observed between in vivo and in vitro results. We have therefore investigated the feasibility of using homogenized horse liver instead of liver microsomes with the aim of saving preparation time and improving the correlation between in vitro and in vivo results. Indeed, the preparation of the homogenized horse liver was very simple, needing only to homogenize the required amount of liver. Even though no further purification steps were performed before the homogenized liver was used, the cleanliness of the extracts obtained, based on gas chromatography‐mass spectrometry (GC‐MS) analysis, was similar to that for liver microsomes. Herein, the results of the in vitro experiments carried out using homogenized horse liver for five anabolic steroids—turinabol, methenolone acetate, androst‐4‐ene‐3,6,17‐trione, testosterone, and epitestosterone—are discussed. In addition to the previously reported in vitro metabolites, some additional known in vivo metabolites in the equine could also be detected. As far as we know, this is the first report of the successful use of homogenized liver in the horse for carrying out in vitro metabolism experiments. Copyright © 2011 John Wiley & Sons, Ltd.     

Determination of amitriptyline and nortriptyline in human liver microsomes with reversed-phase HPLC in vitro

ＤｅｔｅｒｍｉｎａｔｉｏｎｏｆａｍｉｔｒｉｐｔｙｌｉｎｅａｎｄｎｏｒｔｒｉｐｔｙｌｉｎｅｉｎｈｕｍａｎｌｉｖｅｒｍｉｃｒｏｓｏｍｅｓｗｉｔｈｒｅｖｅｒｓｅｄｐｈａｓｅＨＰＬＣｉｎｖｉｔｒｏ１ＳＨＵＹａｎ，ＺＨＵＲｏｎｇＨｕａ２，ＸＵＺｈｅｎＨｕ...     

狼毒大戟中4种二萜类成分在大鼠肝微粒体中的代谢

目的 研究狼毒大戟中4种二萜类成分在大鼠肝微粒体中的代谢产物并探讨其代谢规律.方法 运用大鼠肝微粒体体外孵育法,将岩大戟内酯A、岩大戟内酯B、17-羟基岩大戟内酯A、17-羟基岩大戟内酯B加至由还原型烟酰胺腺嘌呤二核苷酸磷酸启动的大鼠肝微粒体孵育体系中,于37℃条件下孵育30 min,再用乙腈终止反应.以阴性组(先加乙...     

In vitro metabolism study of saikosaponin d and its derivatives in rat liver microsomes

1.?Saikosaponins, one of the representative bioactive ingredients in Radix Bupleuri, possess hepatoprotective, anti-inflammatory, antiviral, antitumor, and other pharmacological activities. Up to now, few studies focused on the further metabolism of saikosaponins and their secondary metabolites absorbed into the circulatory system.

2.?To understand the in vivo efficacy of saikosaponin d, the in vitro metabolism of saikosaponin d, and its two derivatives formed in the gastrointestinal tract, prosaikogenin G and saikogenin G was investigated in rat liver microsomes, respectively.

3.?Fifteen metabolites were detected using high-performance liquid chromatography hybrid ion trap and time-of-flight mass spectrometry and triple-quadrupole mass spectrometry, and the predominant metabolic reactions were hydroxylation, carboxylation and combinations of these steps on the aglycone moiety.

4.?The metabolic pathways of saikosaponin d, prosaikogenin G, and saikogenin G were proposed in vitro and the results contribute to the understanding of saikosaponins in vivo metabolism.     

2,5-Dihydroxy-4-methoxybenzophenone: a novel major in vitro metabolite of benzophenone-3 formed by rat and human liver microsomes

1.?When benzophenone-3 (2-hydroxy-4-methoxybenzophenone; BP-3) was incubated with liver microsomes of untreated rats in the presence of NADPH, the 5-hydroxylated metabolite, 2,5-dihydroxy-4-methoxybenzophenone (5-OH-BP-3), was formed as a major novel metabolite of BP-3. The 4-desmethylated metabolite, 2,4-dihydroxybenzophenone (2,4-diOH-BP), previously reported as the major in vivo metabolite of BP-3, was also detected. However, the amount of 5-OH-BP-3 formed in vitro was about the same as that of 2,4-diOH-BP.

2.?The oxidase activity affording 5-OH-BP-3 was inhibited by SKF 525-A and ketoconazole, and partly by quinidine and sulfaphenazole. The oxidase activity affording 2,4-diOH-BP was inhibited by SKF 525-A, ketoconazole and α-naphthoflavone, and partly by sulfaphenazole.

3.?The oxidase activity affording 5-OH-BP-3 was enhanced in liver microsomes of dexamethasone-, phenobarbital- and 3-methylcholanthrene-treated rats. The activity affording 2,4-diOH-BP was enhanced in liver microsomes of 3-methylcholanthrene- and phenobarbital-treated rats.

4.?When examined recombinant rat cytochrome P450 isoforms catalyzing the metabolism of BP-3, 5-hydroxylation was catalyzed by P450 3A2, 1A1, 2B1, 2C6 and 2D1, while 4-desmethylation was catalyzed by P450 2C6 and 1A1.     

反相高效液相色谱法测定体外人肝微粒体中氟西汀及其代谢产物去甲氟西汀

目的:建立同时测定体外肝微粒体中氟西汀及其代谢产物去甲氟西汀的反相高效液相色谱紫外检测法.方法:含微粒体蛋白和NADPH发生系统及氟西汀的孵育液加入冰乙腈酸终止反应后,再加入去甲替林作为内标并以 n-正己烷和乙腈的混合液进行萃取,然后以反相ODS柱分离,在226 nm处以紫外检测器进行检测.结果:孵育体系中没有明显的干扰峰出现,氟西汀和去甲氟西汀洗脱快,分离好,线性范围均为10-800μg/L,最低检测限均为5μg/L,相对回收率为94％-104％,变异系数少于9.1％.结论:本法快速,灵敏,回收率高,且萃取过程简单,可用于体外氟西汀的代谢研究.     

Identifying metabolites of diphenidol by liquid chromatography-quadrupole/orbitrap mass spectrometry using rat liver microsomes,human blood,and urine samples

In recent years, diphenidol [1,1-diphenyl-4-piperidino-1-butanol] has been one of the drugs that appears in suicide cases, but there are few research data on its metabolic pathways and main metabolites. Metabolite identification plays a key role in drug safety assessment and clinical application. In this study, in vivo and in vitro samples were analyzed with ultra-high-performance liquid chromatography-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry. Structural elucidation of the metabolites was performed by comparing their molecular weights and product ions with those of the parent drug. As a result, 10 Phase I metabolites and 5 glucuronated Phase II metabolites were found in a blood sample and a urine sample from authentic cases. Three other Phase I metabolites were identified in the rat liver microsomes incubation solution. The results showed that the main metabolic pathways of diphenidol in the human body include hydroxylation, oxidation, dehydration, N-dealkylation, methylation, and conjugation with glucuronic acid. This study preliminarily clarified the metabolic pathways and main metabolites of diphenidol. For the development of new methods for the identification of diphenidol consumption, we recommend using M2-2 as a marker of diphenidol entering the body. The results of this study provide a theoretical basis for the pharmacokinetics and forensic scientific research of diphenidol.     

Characterization of in vitro metabolites of luotonin A in human liver microsomes using electrospray/tandem mass spectrometry

1. The pharmacological activity of luotonin A varies, depending on the type of functional group and the site of derivatization. To understand the in vivo efficacy of luotonin A, the in vitro metabolism of luotonin A was investigated in human liver microsomes and recombinant cDNA-expressed cytochromeP450 (CYP).

2. Incubation of luotonin A with pooled human liver microsomes in the presence of NADPH-generating system resulted in the formation of four metabolites and the structures of each metabolite were tentatively characterized on the basis of electrospray tandem mass spectra.

3. The main metabolic pathway of luotonin A in human liver microsomes was hydroxylation, resulting in the generation of two mono-hydroxyl metabolites (M1 and M2) and two di-hydroxyl metabolites (M3 and M4). CYP1A2 was primarily involved in hydroxylation of the quinolone moiety (M1 andM3), while CYP3A4 was mainly responsible for hydroxylation of the quinazoline moiety of luotonin A (M2 and M4) in human liver microsomes.     

Nonspecific binding of drugs to human liver microsomes

AIMS: To characterize the nonspecific binding to human liver microsomes of drugs with varying physicochemical characteristics, and to develop a model for the effect of nonspecific binding on the in vitro kinetics of drug metabolism enzymes. METHODS: The extent of nonspecific binding to human liver microsomes of the acidic drugs caffeine, naproxen, tolbutamide and phenytoin, and of the basic drugs amiodarone, amitriptyline and nortriptyline was investigated. These drugs were chosen for study on the basis of their lipophilicity, charge, and extent of ionization at pH 7.4. The fraction of drug unbound in the microsomal mixture, fu(mic), was determined by equilibrium dialysis against 0.1 M phosphate buffer, pH 7.4. The data were fitted to a standard saturable binding model defined by the binding affinity KD, and the maximum binding capacity Bmax. The derived binding parameters, KD and Bmax, were used to simulate the effects of saturable nonspecific binding on in vitro enzyme kinetics. RESULTS: The acidic drugs caffeine, tolbutamide and naproxen did not bind appreciably to the microsomal membrane. Phenytoin, a lipophilic weak acid which is mainly unionized at pH 7. 4, was bound to a small extent (fu(mic) = 0.88) and the binding did not depend on drug concentration over the range used. The three weak bases amiodarone, amitriptyline and nortriptyline all bound extensively to the microsomal membrane. The binding was saturable for nortriptyline and amitriptyline. Bmax and KD values for nortriptyline at 1 mg ml-1 microsomal protein were 382 +/- 54 microM and 147 +/- 44 microM, respectively, and for amitriptyline were 375 +/- 23 microM and 178 +/- 33 microM, respectively. Bmax, but not KD, varied approximately proportionately with the microsome concentration. When KD is much less than the Km for a reaction, the apparent Km based on total drug can be corrected by multiplying by fu(mic). When the substrate concentration used in a kinetic study is similar to or greater than the KD (Km >/= KD), simulations predict complex effects on the reaction kinetics. When expressed in terms of total drug concentrations, sigmoidal reaction velocity vs substrate concentration plots and curved Eadie Hofstee plots are predicted. CONCLUSIONS: Nonspecific drug binding in microsomal incubation mixtures can be qualitatively predicted from the physicochemical characteristics of the drug substrate. The binding of lipophilic weak bases is saturable and can be described by a standard binding model. If the substrate concentrations used for in vitro kinetic studies are in the saturable binding range, complex effects are predicted on the reaction kinetics when expressed in terms of total (added) drug concentration. Sigmoidal reaction curves result which are similar to the Hill plots seen with cooperative substrate binding.     

山冈橐吾碱在雌性大鼠肝微粒体内的代谢(英文)

研究了山冈橐吾碱 (clivorine)在雌性大鼠肝微粒体内的代谢 .山冈橐吾碱在雌性大鼠肝微粒体内的主要代谢物为两个非吡咯代谢物M1和M2 .与雄性大鼠不同 ,生成肝毒性的吡咯代谢物为其次要的代谢途径 .文献报道山冈橐吾碱在雄性大鼠肝微粒体内的主要代谢方式是形成相应的吡咯代谢物 .这提示山冈橐吾碱在雌雄大鼠肝微粒体内的主要代谢方式不同 .CYP4 5 0特异性抑制剂黄胺苯吡唑(CYP2C) ,毛果芸香碱 (CYP2A1) ,二乙基二硫代氨基甲酸钠 (CYP2E1)和酮康唑 (CYP3A)对M1和M2的形成无明显的影响 .黄素单氧化酶的特异性抑制剂甲巯咪唑可以显著地抑制M2 的形成 ,但对M1的形成无明显的抑制作用 ,且M1在肝微粒体中的形成为NADPH非依赖性 ,上述结果提示参与M1和M2代谢的酶分别为肝微粒体中的水解酶和黄素单氧化酶 .另一方面 ,毛果芸香碱 ,黄胺苯吡唑和二乙基二硫代氨基甲酸钠对山冈橐吾碱的吡咯代谢物的形成无明显的影响 ,而CYP3A的特异性抑制剂酮康唑可以显著地抑制吡咯代谢物的生成 ,且山冈橐吾碱在重组的大鼠肝CYP2C12 ,CYP2E1温孵液中无代谢 ,而在重组的大鼠肝CYP3A1和CYP3A2的温孵液中山冈橐吾碱被代谢成相应的吡咯代谢物 .这提示CYP3A作为主要的CYP4 5 0酶参与了山冈橐吾碱的肝毒性吡咯代谢物的形成 .山冈橐吾碱?     

Tentative identification of in vitro metabolites of O-acetylpsilocin (psilacetin, 4-AcO-DMT) by UHPLC-Q-Orbitrap MS

4-Acetoxy-N,N-dimethyltryptamine (4-AcO-DMT, psilacetin, O-acetylpsilocin) is a synthetic tryptamine with psychedelic properties. Psilacetin may also act as precursor drug of psilocin, similar to psilocybin, but little is known about its metabolism. In this study, the phase I and phase II in vitro metabolism of 4-AcO-DMT was investigated with pooled human liver microsomes, and the reaction mixture was analyzed using liquid chromatography-quadrupole/electrostatic field orbitrap mass spectrometry. Fifteen metabolites were formed after incubation of pooled human liver microsomes with 4-AcO-DMT (12 phase I metabolites and 3 phase II metabolites). The proposed metabolite structures were based on accurate mass analysis and MS/MS fragmentation patterns. The biotransformations included hydrolysis, hydroxylation, N-demethylation, oxidation, and conjugation with glucuronic acid. The hydrolysis metabolite was the most abundant compound. For the development of new methods for the identification of 4-AcO-DMT consumption, the beta-hydroxylation metabolite of 4-AcO-DMT (M2-1) is recommended as a biomarker. The data reported in this work might be applicable to metabolic transformation of 4-AcO-DMT in vivo and also forensically helpful.     

The metabolism of valerylfentanyl using human liver microsomes and zebrafish larvae

Valerylfentanyl, a novel synthetic opioid less potent than fentanyl, has been reported in biological samples, but there are limited studies on its pharmacokinetic properties. The goal of this study was to elucidate the metabolism of valerylfentanyl using an in vitro human liver microsome (HLM) model compared with an in vivo zebrafish model. Nineteen metabolites were detected with N-dealkylation—valeryl norfentanyl and hydroxylation as the major metabolic pathways. The major metabolites in HLMs were also detected in 30 day postfertilization zebrafish. An authentic liver specimen that tested positive for valerylfentanyl, among other opioids and stimulants, revealed the presence of a metabolite that shared transitions and retention time as the hydroxylated metabolite of valerylfentanyl but could not be confirmed without an authentic standard. 4-Anilino-N-phenethylpiperidine (4-ANPP), a common metabolite to other fentanyl analogs, was also detected. In this study, we elucidated the metabolic pathway of valerylfentanyl, confirmed two metabolites using standards, and demonstrated that the zebrafish model produced similar metabolites to the HLM model for opioids.     

米氮平在鼠肝微粒体中的体外代谢研究

目的:通过不同诱导剂预处理鼠肝,研究米氮平在肝微粒体中的代谢主要受何种酶影响,同时研究米氮平对介导其自身代谢的P450酶亚型是否有影响,为米氮平的临床合理应用提供科学依据。方法:将米氮平与不同诱导剂诱导的鼠肝微粒体进行体外孵育代谢,以乙腈中止反应,样品用25%氨水碱化后以环己烷提取。用RPHPLC测定剩余的米氮平含量,流动相为甲醇水(含10mmol·L-1NH4AC,5mmol·L-1SDS,pH3.5)6238(VV),检测波长为307nm。结果:本文测定方法回收率高,日内和日间精密度均良好,符合生物样本检测要求。苯巴比妥诱导的鼠肝微粒体对米氮平的代谢具有明显的催化作用,利福平也有一定的催化能力,米氮平诱导的鼠肝微粒体与对照组对米氮平的代谢无明显差异。结论:由苯巴比妥诱导的P450酶亚型(主要为细胞色素P4503A4)和利福平诱导的P450酶亚型(主要为细胞色素P4502C92C19,同时也对P4503A4有一定的诱导作用)在米氮平的体外代谢中起着重要作用;而米氮平诱导组对于米氮平代谢无明显影响,预示米氮平对介导其自身代谢的P450酶亚型无明显的诱导或抑制作用。     

Metabolic profiling of deschloro-N-ethyl-ketamine and identification of new target metabolites in urine and hair using human liver microsomes and high-resolution accurate mass spectrometry

The aim of this study was to identify new markers of deschloro-N-ethyl-ketamine (O-PCE), a ketamine analogue that has been involved in acute intoxications with severe outcomes including death and whose metabolism has never been studied before. In vitro study after 2-h incubation with pooled human liver microsomes (HLMs) cross-checked by the analysis of urine and hair from a 43-year-old O-PCE user (male) were performed by liquid chromatography–high resolution mass spectrometry (LC-HRMS). Acquired data were processed by the Compound Discoverer® software, and a full metabolic profile of O-PCE was proposed. In total, 15 metabolites were identified, 10 were detected in vitro (HLMs) and confirmed in vivo (urine and/or hair), two were present only in HLMs, and the remaining three metabolites were identified only in biological specimens. While O-PCE was no longer detected in urine, nine metabolites were identified allowing to increase its detection window. In descending order of metabolites abundance, we suggest using 2-en-PCA-N-Glu (34%, first), M3 (16%, second), O-PCA-N-Glu (15.4%, third), OH-O-PCE (15%, fourth) and OH-PCE (11.9%, fifth) as target metabolites to increase the detection window of O-PCE in urine. In hair, nine metabolites were identified. OH-PCA was the major compound (78%) with a relevant metabolite to parent drug ratio (=6) showing its good integration into hair and making it the best marker for long-term monitoring of O-PCE exposure.     

Characterization of in vitro metabolites of cudratricusxanthone A in human liver microsomes

Cudratricusxanthone A (CTXA), isolated from the roots of Cudrania tricuspidata, exhibits several biological activities; however, metabolic biotransformation was not investigated. Therefore, metabolites of CTXA were investigated and the major metabolic enzymes engaged in human liver microsomes (HLMs) were characterized using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). CTXA was incubated with HLMs or human recombinant CYPs and UGTs, and analysed by an LC‐MS/MS equipped electrospray ionization (ESI) to qualify and quantify its metabolites. In total, eight metabolites were identified: M1–M4 were identified as mono‐hydroxylated metabolites during Phase I, and M5–M8 were identified as O‐glucuronidated metabolites during Phase II in HLMs. Moreover, these metabolite structures and a metabolic pathway were identified by elucidation of MSⁿ fragments and formation by human recombinant enzymes. M1 was formed by CYP2D6, and M2–M4 were generated by CYP1A2 and CYP3A4. M5–M8 were mainly formed by UGT1A1, respectively. While investigating the biotransformation of CTXA, eight metabolites of CTXA were identified by CYPs and UGTs; these data will be valuable for understanding the in vivo metabolism of CTXA. Copyright © 2015 John Wiley & Sons, Ltd.     

Antitumor activity of two gelsemine metabolites in rat liver microsomes

Gelsemine is one of the major alkaloids from Gelsemium elegans Benth., which has been used as an antitumor remedy in clinic. In this paper, metabolism of gelsemine has been investigated in vitro in phenobarbital-treated rat liver microsomes. The metabolites of gelsemine were separated and evaluated using the flash silica gel column, preparative HPLC, using NMR and MS methods. According to the spectral data, two metabolites, M1 and M2, were identified as 4-N-demethylgelsemine and 21-oxogelsemine, respectively. By the MTT method in vitro, the antitumor activities between gelsemine and its metabolites were compared in the HepG2 and HeLa cell lines. Moreover, the main metabolic pathway was further proposed.     

In vitro metabolism of the lignan (−)‐grandisin,an anticancer drug candidate,by human liver microsomes

(?)‐grandisin is a tetrahydrofuran lignan that displays important biological properties, such as trypanocidal, anti‐inflammatory, cytotoxic, and antitumor activities, suggesting its utility as a potential drug candidate. One important step in drug development is metabolic characterization and metabolite identification. To perform a biotransformation study of (?)‐grandisin and to determine its kinetic properties in humans, a high performance liquid chromatography (HPLC) method was developed and validated. After HPLC method validation, the kinetic properties of (?)‐grandisin were determined. (?)‐grandisin metabolism obeyed Michaelis‐Menten kinetics. The maximal reaction rate (V_max) was 3.96 ± 0.18 µmol/mg protein/h, and the Michaelis‐Menten constant (K_m) was 8.23 ± 0.99 μM. In addition, the structures of the metabolites derived from (?)‐grandisin were characterized via gas chromatography‐mass spectrometry (GC‐MS) and liquid chromatography‐mass spectrometry (LC‐MS) analysis. Four metabolites, 4‐O‐demethylgrandisin, 3‐O‐demethylgrandisin, 4,4′‐di‐O‐demethylgrandisin, and a metabolite that may correspond to either 3,4‐di‐O‐demethylgrandisin or 3,5‐di‐O‐demethylgrandisin, were detected. CYP2C9 isoform was the main responsible for the formation of the metabolites. These metabolites have not been previously described, demonstrating the necessity of assessing (?)‐grandisin metabolism using human‐derived materials. Copyright © 2015 John Wiley & Sons, Ltd.     

In vitro studies on flubromazolam metabolism and detection of its metabolites in authentic forensic samples

Flubromazolam is a triazole benzodiazepine with high potency and long‐lasting central nervous system depressant effects; however, limited data about its pharmacokinetics are available. Here, we report in vitro studies of the human flubromazolam metabolism analyzed by liquid chromatography high‐resolution mass spectrometry (LC‐HRMS). In vitro investigations were carried out in pooled human liver microsomes (pHLM) and recombinant cytochrome P450 (CYP)‐enzymes. To confirm those metabolites detected in vitro , authentic samples obtained from two forensic cases were also analyzed by LC‐HRMS. Additionally, determination of the unbound fraction of flubromazolam in pHLM and in plasma was performed by equilibrium dialysis with subsequent prediction of its hepatic clearance (CL_H ) using well‐stirred and parallel‐tube models. Additional findings obtained by routine screening methods of these forensic cases are also reported. Studies using incubations with nicotinamide adenine dinucleotide phosphate‐fortified pHLM with or without uridine 5'‐diphosphoglucuronic acid and incubations with CYP‐enzymes identified the main metabolic pathway of flubromazolam as hydroxylation on the α‐ and/or 4‐position mediated by CYP3A4 and CYP3A5, with subsequent glucuronidation of the hydroxylated metabolites as well as of the parent drug. Further, α‐hydroxy‐flubromazolam and its corresponding glucuronide were detected in vivo together with the N ‐glucuronide of flubromazolam. The predicted CL _H of flubromazolam using the well‐stirred and parallel‐tube models were 0.42 and 0.43 mL/min/kg, respectively. Based on the data presented here, flubromazolam is primarily metabolized by CYP3A4/5 with a high protein‐binding and a predicted low clearance. Analysis of authentic samples suggested that analytical targets for flubromazolam should be the compound itself and α‐hydroxy‐flubromazolam. Copyright © 2016 John Wiley & Sons, Ltd.