Challenges in the indirect quantitation of acyl‐glucuronide metabolites of a cardiovascular drug from complex biological mixtures in the absence of reference standards

This paper describes the quantitation of acyl‐glucuronide metabolites (M26 and M5) of a cardiovascular‐drug (torcetrapib) from monkey urine, in the absence of their reference standards. LC/MS/MS assays for M1 and M4 (aglycones of M26 and M5, respectively) were characterized from normal and base‐treated urine, as their respective reference standards were available. The in vivo study samples containing M26 and M5 were treated with 1 n sodium hydroxide to hydrolyze them to their respective aglycones. The study samples were assayed for M1 and M4 before and after alkaline hydrolysis and the difference in the concentrations provided an estimate of the urinary levels of M26 and M5. Prior to the main sample analysis, conditions for alkaline hydrolysis of the glucuronides were optimized by incubating pooled study samples. During incubations, a prolonged increase in M4 levels over time was observed, which is inconsistent with the base‐hydrolysis of an acyl‐glucuronide (expected to hydrolyze rapidly). Possible interference of the metabolite M9 (an ether‐glucuronide metabolite isobaric to M4) was investigated to explain this observation using chromatographic and wet‐chemistry approaches. The strategies adopted herein established that the LC/MS/MS assay and our approach were reliable. The metabolite exposure was then correlated to toxicological observations to gain initial insights into the physiological role of these metabolites. Copyright © 2009 John Wiley & Sons, Ltd.     

绿茶多酚对自由基诱导的红细胞膜过氧化的抑制作用

采用水溶性偶氮引发剂2,2'-偶氮二(2-脒基丙烷)二盐酸盐(AAPH)在37引发入血红细胞膜的过氧化,通过测定氧气吸收及维生素E的消耗研究了过氧化过程的动力学,并对从绿茶中提取的主要多酚类化合物的抗氧化活性做了定量研究。使用的绿茶多酚有:(-)-表儿茶素(EC),(-)-表儿茶素(EGC),(-)-表儿茶素酸酯(ECG)和(-)-表儿茶素培酸酯(EGCG)。结果表明,这些绿茶多酚能够显著缩短过氧化反应的动力学链长,有效地抑制红细胞膜的过氧化。抗氧化活性顺序为:EC〉GCG〉EGCG〉EGC。     

Identification of metabolites of novel Anti‐MRSA fluoroquinolone WCK 771 in mouse,rat, rabbit,dog, monkey and human urine using liquid chromatography tandem mass spectrometry

WCK 771 is an l ‐arginine salt of levonadifloxacin (LND) being developed in intravenous dosage form and has recently completed a phase III trial in India. The pharmacokinetics of WCK 771, a novel anti‐MRSA fluoroquinolone, were examined in mice, rats, rabbits, dogs, monkeys and humans after systemic administration during pre‐clinical and clinical investigations. Urine and serum were evaluated for identification of metabolites. It was observed that LND mainly follows phase II biotransformation pathways. All of the species showed a different array of metabolites. In mice, rabbit and dog, the drug was mainly excreted in the form of O‐glucuronide (M7) and acyl glucuronide (M8) conjugates, whereas in rat and human major metabolite was sulfate conjugate (M6). Monkeys exhibited equal distribution of sulfate (M6) and glucuronide conjugates (M7, M8). In addition to these three major phase II metabolites; five phase I oxidative metabolites (M1, M2, M3, M4 and M5) were identified using liquid chromatography tandem mass spectrometry. Out of these eight metabolites M2, M3, M5, M7 and M8 are reported for the first time.     

Urinary metabolic profile of 19-norsteroids in humans: glucuronide and sulphate conjugates after oral administration of 19-nor-4-androstenediol

19-Nor-4-androstenediol (NOL) is a prohormone of nandrolone (ND). Both substances are included in the WADA List of Prohibited Classes of Substances and their administration is determined by the presence of 19-norandrosterone (NA) with the urinary threshold concentration of 2 ng mL(-1). Routine analytical procedures allow the determination of NA excreted free and conjugated with glucuronic acid, but amounts of ND and NOL metabolites are also excreted in the sulphate fraction. The aim of this study is to determine the urinary metabolic profile after oral administration of a nutritional supplement containing NOL. Urine samples were collected up to 96 h following supplement administration and were extracted to obtain separately three metabolic fractions: free, glucuronide and sulphate. Extraction with tert-butyl methyl ether was performed after the hydrolysis steps and trimethylsilyl derivatives were analyzed by gas chromatography/mass spectrometry (GC/MS). After oral administration of NOL, the main metabolites detected were NA and noretiocholanolone (NE) in the glucuronide and sulphate fractions. The relative abundances of each metabolite in each fraction fluctuate with time; a few hours after administration the main metabolite was NA glucuronide whereas in the last sample (4 days after administration) the main metabolite was the NA sulphate and the second was the NE glucuronide. During the studied period almost half of the dose was excreted and the main metabolites were still found in urine after 96 h. Norepiandrosterone and norepietiocholanolone were also detected only in the sulphate fraction. Our results suggest that sulphate metabolites should be taken into consideration in order to increase the retrospectivity in the detection of 19-norsteroids after oral administration. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Characterization of metabolites of a NK1 receptor antagonist, CJ-11,972, in human liver microsomes and recombinant human CYP isoforms by liquid chromatography/tandem mass spectrometry

The in vitro metabolism of CJ-11,972, (2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-yl)-(5-tert-butyl-2-methoxybenzyl)amine, an NK1 receptor antagonist, was studied in human liver microsomes and recombinant human CYP isoforms. Liquid chromatography/mass spectrometry (LC/MS) and tandem mass spectrometry (LC/MS/MS) coupled to radioactive detection were used to detect and identify the metabolites. CJ-11,972 was extensively metabolized in human liver microsomes and recombinant human CYP 3A4/3A5 isoforms. A total of fourteen metabolites were identified by a combination of various MS techniques. The major metabolic pathways were due to oxidation of the tert-butyl moiety to form an alcohol (M6) and/or O-demethylation of the anisole moiety. The alcohol metabolite M6 was further oxidized to the corresponding aldehyde (M7) and carboxylic acid (M4). Two unusual metabolites (M13, M17), formed by C-demethylation of the tert-butyl group, were identified as 2-{3-[(2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-ylamino)methyl]-4-methoxyphenyl}propan-2-ol and (2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-yl)-(5-isopropenyl-2-methoxybenzyl)amine. A plausible mechanism for C-demethylation may involve oxidation of M6 to form an aldehyde metabolite (M7), followed by cytochrome P450-mediated deformylation leaving an unstable carbon-centered radical, which would quickly form either the alcohol metabolite M13 and the olefin metabolite M17.     

Metabolism of eupatilin in rats using liquid chromatography/electrospray mass spectrometry

Eupatilin (5,7-dihydroxy-3',4',6-trimethoxy flavone) is an active ingredient of an ethanol extract of Artemisia asiatica (DA-9601) that is used in the treatment of gastritis. In vitro and in vivo metabolism of eupatilin in the rats has been studied by LC-electrospray mass spectrometry. Rat liver microsomal incubation of eupatilin in the presence of NADPH and UDPGA resulted in the formation of four metabolites (M1-M4). M1, M2, M3 and M4 were tentatively identified as 3'- or 4'-O-demethyl-eupatilin glucuronide, eupatilin glucuronide, 6-O-demethyleupatilin and 3'- or 4'-O-demethyl-eupatilin, respectively. Those metabolites from in vitro study were also characterized in bile, plasma or urine samples after an intravenous administration of eupatilin to rats. In rat bile, plasma and urine samples, eupatilin glucuronide (M2) was a major metabolite, whereas M3, M4 and M4 glucuronide (M1) were the minor metabolites.     

茶中茶多酚的高效液相色谱法分离分析

用改进的Ａｇａｒｗａｌ方法萃取不同种类茶叶和茶饮料中的茶多酚,建立了用高效液相色谱（ＨＰＬＣ）法对茶多酚进行分离分析方法。ＨＰＬＣ可有效分离ＧＴＰｓ主要组成成分ＥＣ、ＥＧＣ、ＥＣＧ和ＥＧＣＧ并精确定量,相对标准偏差小于５％。茶叶加工过程对ＧＴＰｓ含量有很大影响,绿茶总ＧＴＰｓ含量在６￣１５ｇ／１００ｇ干茶叶、乌龙茶总ＧＴＰｓ含量在５￣７ｇ／１００ｇ干茶叶,红茶总ＧＴＰｓ含量低于２ｇ／１００ｇ干茶叶     

New clostebol metabolites in human urine by liquid chromatography time‐of‐flight tandem mass spectrometry and their application for doping control

In this study, clostebol metabolic profiles were investigated carefully. Clostebol was administered to one healthy male volunteer. Urinary extracts were analyzed by liquid chromatography quadrupole time‐of‐flight mass spectrometry (MS) using full scan and targeted MS/MS techniques with accurate mass measurement for the first time. Liquid–liquid extraction and direct injection were applied to processing urine samples. Chromatographic peaks for potential metabolites were found by using the theoretical [M–H]^? as target ion in full scan experiment, and their actual deprotonated ions were analyzed in targeted MS/MS mode. Fourteen metabolites were found for clostebol, and nine unreported metabolites (two free ones and seven sulfate conjugates) were identified by MS, and their potential structures were proposed based on fragmentation and metabolism pathways. Four glucuronide conjugates were also first reported. All the metabolites were evaluated in terms of how long they could be detected and S1 (4ξ‐chloro‐5ξ‐androst‐3ξ‐ol‐17‐one‐3ξ‐sulfate) was considered to be the long‐term metabolite for clostebol misuse detected up to 25 days by liquid–liquid extraction and 14 days by direct injection analysis after oral administration. Five conjugated metabolites (M2, M5, S2, S6 and S7) could also be the alternative biomarkers for clostebol misuse. Copyright © 2015 John Wiley & Sons, Ltd.     

Targeted metabolic profiling of phenolics in urine and plasma after regular consumption of cocoa by liquid chromatography–tandem mass spectrometry

The biological properties of cocoa (Theobroma cacao L.) polyphenols are strictly dependent on their bioavailability. A long-term cocoa feeding trial was performed with subjects at high risk for cardiovascular disease. Subjects (n = 42) received two sachets of 20 g of cocoa powder/day with 250 mL of skimmed milk each, or only 500 mL/day of skimmed milk, both for two 4-week periods. The phenolic metabolic profile including phase II conjugated metabolites and phenolic acids derived from the intestinal microbiota was determined by LC-MS/MS in both 24-h urine and fasting plasma. The analysis of 24-h urine revealed significant increases of phase II metabolites, including glucuronides and sulfate conjugates of (−)-epicatechin, O-methyl-epicatechin, 5-(3′,4′-dihydroxyphenyl)-γ-valerolactone and 5-(3′-methoxy-4′-hydroxyphenyl)-γ-valerolactone, after regular cocoa intake. In the case of plasma, only glucuronide conjugates of dihydroxyphenylvalerolactones increased. Regular consumption of cocoa also resulted in a significant increase in the urinary excretion of colonic microbial-derived phenolic metabolites, including vanillic, 3,4-dihydroxyphenylacetic and 3-hydroxyphenylacetic acids, and particularly 5-(3′,4′-dihydroxyphenyl)-γ-valerolactone, whereas only the two latter metabolites showed a significant increase in fasting plasma. The results found herein indicate that 5-(3′,4′-dihydroxyphenyl)-γ-valerolactone and hydroxyphenylacetic acids could be good biomarkers of the regular consumption of cocoa and therefore, of flavanol-rich foods.     

Use of on-line hydrogen/deuterium exchange to facilitate metabolite identification

Biotransformation studies performed on an investigational compound (I, represented by R1-CH(NH(2))-CO-N(R2)-CH(2)-S-R3) led to the identification of five metabolites (M1-M5). Based on LC/MS (liquid chromatography/mass spectrometry) analysis which included the use of H(2)O and D(2)O in the mobile phases, they were identified as the sulfoxide (M1), sulfone (M2), carbamoyl glucuronide (M3), N-glucuronide (M4), and N-glucoside (M5) metabolites, respectively. The structure of M3, a less commonly seen carbamoyl glucuronide metabolite, was established using on-line H/D (hydrogen/deuterium) exchange experiments conducted by LC/MS. H/D exchange experiments were also used to distinguish the S-oxidation structures of M1 and M2 from hydroxylation. Herein, the application of deuterium oxide as the LC/MS mobile phase for structural elucidation of drug metabolites in biological matrices is demonstrated.     

A metabonomic investigation of the biochemical effects of mercuric chloride in the rat using 1H NMR and HPLC-TOF/MS: time dependent changes in the urinary profile of endogenous metabolites as a result of nephrotoxicity

The effects of the administration of a single dose of the model nephrotoxin mercuric chloride (2.0 mg kg(-1), subcutaneous) to male Wistar-derived rats on the urinary metabolite profiles of a range of endogenous metabolites has been investigated using (1)H NMR and HPLC-MS. Urine samples were collected daily for 9 days from both dosed and control animals. Analysis of these samples revealed marked changes in the pattern of endogenous metabolites as a result of HgCl(2) toxicity. Peak disturbances in the urinary metabolite profiles were observed (using both NMR and HPLC-MS) at 3 days post dose. Thereafter the urinary metabolite profile gradually returned to a more normal composition. Markers of toxicity identified by (1)H NMR spectroscopy were raised concentrations of lactate, alanine, acetate, succinate, trimethylamine (TMA), and glucose. Reductions in the urinary excretion of citrate and alpha-ketoglutarate were also seen. Markers identified by HPLC-MS, in positive ion mode, were kynurenic acid, xanthurenic acid, pantothenic acid and 7-methylguanine which decreased after dosing. In addition an ion at m/z 188, probably 3-amino-2-naphthoic acid, was observed to increase after dosing. As well as these identified compounds other ions at m/z 297 and 267 decreased after dosing. In negative ion mode a range of sulfated compounds were observed, including phenol sulfate and benzene diol sulfate, which decreased after dosing. As well as the sulfated components an unidentified glucuronide at m/z 326 was also observed to decrease after dosing. The results of this study demonstrate the complementary nature of the NMR and MS-based techniques for metabonomic analysis.     

Chemical constituents of two sages with free radical scavenging activity

From the aerial parts of Salvia trichoclada Bentham and S. verticillata L. one new and two known phenolic acids, 3-(3',4'-dihydroxyphenyl)-2-hydroxymethyl propionic acid (1), 3-(3',4'-dihydroxyphenyl) lactic acid (2), and rosmarinic acid (3); two flavonoids, apigenin 4'-methyl ether 7-O-glucuronide (4), and luteolin 7-O-beta glucuronide (5); two lupan type triterpene aglycones, lupeol (6), and 30-hydroxylup-20 (29)-en-3-on (7); an oleanane-type triterpene acid, oleanolic acid (8); and an ursan-type triterpene acid, ursolic acid (9) were isolated. The structures of the compounds were elucidated by spectroscopic analysis. Different extracts of the plants were examined for their free radical scavenging activities by DPPH (2,2-Diphenyl-1-picrylhydrazyl) assay. Some of the polar extracts showed high free radical scavenging activity.     

Comparison of sulfo‐conjugated and gluco‐conjugated urinary metabolites for detection of methenolone misuse in doping control by LC‐HRMS,GC‐MS and GC‐HRMS

Methenolone (17β‐hydroxy‐1‐methyl‐5α‐androst‐1‐en‐3‐one) misuse in doping control is commonly detected by monitoring the parent molecule and its metabolite (1‐methylene‐5α‐androstan‐3α‐ol‐17‐one) excreted conjugated with glucuronic acid using gas chromatography‐mass spectrometry (GC‐MS) and liquid chromatography mass spectrometry (LC‐MS) for the parent molecule, after hydrolysis with β‐glucuronidase. The aim of the present study was the evaluation of the sulfate fraction of methenolone metabolism by LC‐high resolution (HR)MS and the estimation of the long‐term detectability of its sulfate metabolites analyzed by liquid chromatography tandem mass spectrometry (LC‐HRMSMS) compared with the current practice for the detection of methenolone misuse used by the anti‐doping laboratories. Methenolone was administered to two healthy male volunteers, and urine samples were collected up to 12 and 26 days, respectively. Ethyl acetate extraction at weak alkaline pH was performed and then the sulfate conjugates were analyzed by LC‐HRMS using electrospray ionization in negative mode searching for [M‐H]^? ions corresponding to potential sulfate structures (comprising structure alterations such as hydroxylations, oxidations, reductions and combinations of them). Eight sulfate metabolites were finally detected, but four of them were considered important as the most abundant and long term detectable. LC clean up followed by solvolysis and GC/MS analysis of trimethylsilylated (TMS) derivatives reveal that the sulfate analogs of methenolone as well as of 1‐methylene‐5α‐androstan‐3α‐ol‐17‐one, 3z‐hydroxy‐1β‐methyl‐5α‐androstan‐17‐one and 16β‐hydroxy‐1‐methyl‐5α‐androst‐1‐ene‐3,17‐dione were the major metabolites in the sulfate fraction. The results of the present study also document for the first time the methenolone sulfate as well as the 3z‐hydroxy‐1β‐methyl‐5α‐androstan‐17‐one sulfate as metabolites of methenolone in human urine. The time window for the detectability of methenolone sulfate metabolites by LC‐HRMS is comparable with that of their hydrolyzed glucuronide analogs analyzed by GC‐MS. The results of the study demonstrate the importance of sulfation as a phase II metabolic pathway for methenolone metabolism, proposing four metabolites as significant components of the sulfate fraction. Copyright © 2015 John Wiley & Sons, Ltd.     

Determination of the site of glucuronidation in an N-(3,5-dichlorophenyl)succinimide metabolite by electrospray ionization tandem mass spectrometry following derivatization to picolinyl esters

Derivatization using 3-pyridylcarbinol coupled with liquid chromatography electrospray ionization tandem mass spectrometry (LC/MS/MS) was used to characterize a novel Phase II metabolite of the nephrotoxic agricultural fungicide, N-(3,5-dichlorophenyl)succinimide (NDPS). A glucuronide conjugate of N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA) was identified in the urine from a rat dosed with [14C]NDPS. However, 2-NDHSA contains an aliphatic hydroxyl group and a carboxylic acid group, both of which are potential sites for glucuronidation. Mass spectrometry alone was unable to distinguish between these possibilities. Since the position of glucuronidation may be important in the mechanism of NDPS-induced nephrotoxicity, chemical derivatization in conjunction with mass spectrometry was used to characterize the glucuronide. The 2-NDHSA glucuronide conjugate was isolated from rat urine, derivatized with 3-pyridylcarbinol, and the derivatized metabolite was then analyzed by LC/MS/MS. Two known NDPS metabolites, 2-NDHSA and N-(3,5-dichlorophenyl)succinamic acid (NDPSA), were also isolated from rat urine and derivatized similarly. 3-Pyridinylcarbinol reacted rapidly with the carboxylic acid groups and formation of the picolinyl esters increased the ionization potential under positive ion conditions. The urinary glucuronide of 2-NDHSA was identified as an alcohol-linked glucuronide by examination of the molecular ions and the collision-induced dissociation (CID) product ion spectra of the derivatized products. When used in combination with mass spectrometry, derivatization of carboxylic acids with 3-pyridylcarbinol provided useful mass fragmentations and is a rapid way to obtain structural information about the position of glucuronidation of NDPS metabolites.     

Structural identification of mouse urinary metabolites of pterostilbene using liquid chromatography/tandem mass spectrometry

Pterostilbene, the dimethoxy derivative of resveratrol, has drawn much attention recently due to its potential beneficial health effects. The metabolic fate of pterostilbene, however, is not well understood. In the present study, we identified nine novel mouse urinary pterostilbene metabolites, pterostilbene glucuronide, pterostilbene sulfate, mono‐demethylated pterostilbene glucuronide, mono‐demethylated pterostilbene sulfate, mono‐hydroxylated pterostilbene, mono‐hydroxylated pterostilbene glucuronide, mono‐hydroxylated pterostilbene sulfate, and mono‐hydroxylated pterostilbene glucuronide sulfate, using liquid chromatography/atmospheric pressure chemical ionization and electrospray ionization tandem mass spectrometry. The structures of these metabolites were confirmed by analyzing the MSⁿ (n = 1–3) spectra. To our knowledge, this is the first report of the identification of urinary metabolites of pterostilbene in mice. Copyright © 2010 John Wiley & Sons, Ltd.     

Liquid chromatography/electrospray tandem mass spectrometry characterization of styrene metabolism in man and in rat

Liquid chromatography with electrospray tandem mass spectrometry was used to characterize the metabolism of styrene in man and in rat. To improve identification and characterization of minor styrene metabolites, rats were co-exposed to styrene and styrene-d(8). In addition to the main styrene metabolites, mandelic acid and phenylglyoxylic acid, and specific mercapturic acids, phenylhydroxyethylmercapturic acids (PHEMAs), other minor metabolites, including phenylglycine, N-acetyl-S-(phenacyl)cysteine, 4-vinylphenol and styreneglycol conjugates (glucuronides and sulfates) were identified and determined both in human and rat urine. Phenylglycine and N-acetyl-S-(phenacyl)cysteine have been hypothesized to occur, but never detected in human or rat urine after styrene exposure. 4-Vinylphenol and styrene glycol had already been recognized as styrene metabolites, but never determined as intact glucuronide and sulfate conjugates. Failure to identify 1- and 2-phenylethanol conjugates suggests that phenylethanol might be an intermediate metabolite, but it is not a conjugated catabolite. A method for the simultaneous determination of mandelic acid, phenylglyoxylic acid, phenyglycine and the four PHEMA diastereoisomers has been developed and validated. For those glucuronide and sulfate conjugates whose standards are not commercially available, a method for semiquantitative analysis, based on the use of structurally similar compounds as standards, has been developed. This approach was found to be valid for the determination of 4-vinylphenol glucuronide and 4-vinylphenol sulfate.     

New potential biomarkers for mesterolone misuse in human urine by liquid chromatography quadrupole time‐of‐flight mass spectrometry

In this paper, mesterolone metabolic profiles were investigated carefully. Mesterolone was administered to one healthy male volunteer. Urinary extracts were analyzed by liquid chromatography quadruple time‐of‐flight mass spectrometry (LC‐QTOFMS) for the first time. Liquid–liquid extraction was applied to processing urine samples, and dilute‐shoot analyses of intact metabolites were also presented. In LC‐QTOFMS analysis, chromatographic peaks for potential metabolites were hunt down by using the theoretical [M–H]^? as target ions in full scan experiment, and their actual deprotonated ions were analyzed in targeted MS/MS mode. Ten metabolites including seven new sulfate and three glucuronide conjugates were found for mesterolone. Because of no useful fragment ion for structural elucidation, gas chromatography–mass spectrometry instrumentation was employed to obtain structural details of the trimethylsilylated phase I metabolite released after solvolysis. Thus, their potential structures were proposed particularly by a combined MS approach. All the metabolites were also evaluated in terms of how long they could be detected, and S1 (1α‐methyl‐5α‐androst‐3‐one‐17β‐sulfate) together with S2 (1α‐methyl‐5α‐androst‐17‐one‐3β‐sulfate) was detected up to 9 days after oral administration, which could be the new potential biomarkers for mesterolone misuse. Copyright © 2015 John Wiley & Sons, Ltd.     

Structural identification of mouse fecal metabolites of theaflavin 3,3'-digallate using liquid chromatography tandem mass spectrometry

Black tea consumption has been associated with many health benefits including the prevention of cancer and heart disease. Theaflavins are the major bioactive polyphenols present in black tea. Unfortunately, limited information is available on their biotransformation. In the present study, we investigated the metabolic fate of theaflavin 3,3'-digallate (TFDG), one of the most abundant and bioactive theaflavins, in mouse fecal samples using liquid chromatography/electrospray ionization tandem mass spectrometry by analyzing the MS(n) (n=1-3) spectra. Four metabolites theaflavin, theaflavin 3-gallate, theaflavin 3'-gallate, and gallic acid were identified as the major mouse fecal metabolites of TFDG. Glucuronidated and sulfated, instead of methylated metabolites of theaflavin 3-gallate, theaflavin 3'-gallate, and TFDG were detected and identified as the minor mouse fecal metabolites of TFDG. Our results indicate that TFDG can be degraded in mice. Further studies on the formation of those metabolites in TFDG-treated mice in germ-free conditions are warranted. To our knowledge, this is the first report on the biotransformation of TFDG in mice.     

In vitro metabolism of β‐lapachone (ARQ 501) in mammalian hepatocytes and cultured human cells

ARQ 501 (3,4‐dihydro‐2,2‐dimethyl‐2H‐naphthol[1,2‐b]pyran‐5,6‐dione, β‐lapachone) is an anticancer agent, currently in multiple phase II clinical trials as monotherapy and in combination with other cytotoxic drugs. This study focuses on in vitro metabolism in cryopreserved hepatocytes from mice, rats, dogs and humans using [¹⁴C]‐labeled ARQ 501. Metabolite profiles were characterized using liquid chromatography/mass spectrometry combined with an accurate radioactivity counter. Ion trap mass spectrometry was employed for further structural elucidation. A total of twelve metabolites were detected in the mammalian hepatocytes studied; all of which but one were generated from phase II conjugation reactions. Ten of the observed metabolites were produced by conjugations occurring at the reduced ortho‐quinone carbonyl groups of ARQ 501. The metabolite profiles revealed that glucuronidation was the major biotransformation pathway in mouse and human hepatocytes. Monosulfation was the major pathway in dog, while, in rat, it appears glucuronidation and sulfation pathways contributed equally. Three major metabolites were found in rats: monoglucuronide M1, monosulfate M6, and glucuronide‐sulfate M9. Two types of diconjugation metabolites were formed by attachment of the second glycone to an adjacent hydroxyl or to an existing glycone. Of the diconjugation metabolites, glucosylsulfate M10, diglucuronide M5, and glucuronide‐glucoside M11 represent rarely observed phase II metabolites in mammals. The only unconjugated metabolite was generated through hydrolysis and was observed in rat, dog and human hepatocytes. ARQ 501 appeared less stable in human hepatocytes than in those of other species. To further elucidate the metabolism of ARQ 501 in extrahepatic sites, its metabolism in human kidney, lung and intestine cells was also studied, and only monoglucuronide M1 was observed in all the cell types examined. Copyright © 2008 John Wiley & Sons, Ltd.