Characterization of in vitro metabolites of rutaecarpine in rat liver microsomes using liquid chromatography/tandem mass spectrometry

Following incubation of rutaecarpine, a new cyclooxygenase-2 inhibitor, with rat liver microsomes, the structures of the metabolites were characterized by liquid chromatography with tandem mass spectrometry. Nine metabolites corresponding to mono- or dihydroxylated rutaecarpine were formed. Characteristic product ions for the identification of rutaecarpine metabolites were observed at m/z 136, 158 and 286. The loss of water led to the fragment ion at m/z 286, indicating the hydroxylation of the aliphatic ring. The fragment ion at m/z 136 indicated the hydroxylated form of the phenyl group of the quinazolinone moiety, while that at m/z 158 indicated the hydroxylated form of the aromatic ring of the indole moiety.     

Characterization of in vitro metabolites of irisflorentin by rat liver microsomes using high‐performance liquid chromatography coupled with tandem mass spectrometry

Belamcanda chinensis has been extensively used as antibechic, expectorant and anti‐inflammatory agent in traditional medicine. Irisflorentin is one of the major active ingredients. However, little is known about the metabolism of irisflorentin so far. In this work, rat liver microsomes (RLMs) were used to investigate the metabolism of this compound for the first time. Seven metabolites were detected. Five of them were identified as 6,7‐dihydroxy‐5,3′,4′,5′‐tetramethoxy isoflavone (M1), irigenin (M2), 5,7,4′‐trihydroxy‐6,3′,5′‐trimethoxy isoflavone (M3), 6,7,4′‐trihydroxy‐5,3′,5′‐trimethoxy isoflavone (M4) and 6,7,5′‐trihydroxy‐5,3′,4′‐trimethoxy isoflavone (M5) by means of NMR and/or HPLC‐ESI‐MS. The structures of M6 and M7 were not elucidated because they produced no MS signals. The predominant metabolite M1 was noted to be a new compound. Interestingly, it was found to possess anticancer activity much higher than the parent compound. The enzymatic kinetic parameters of M1 revealed a sigmoidal profile, with V_max = 12.02 μm /mg protein/min, K_m = 37.24 μm , CL_int = 0.32 μL/mg protein/min and h = 1.48, indicating the positive cooperation. For the first time in this work, a new metabolite of irisflorentin was found to demonstrate a much higher biological activity than its parent compound, suggesting a new avenue for the development of drugs from B. chinensis, which was also applicable for other herbal plants. Copyright © 2016 John Wiley & Sons, Ltd.     

Characterization of the metabolites of rosmarinic acid in human liver microsomes using liquid chromatography combined with electrospray ionization tandem mass spectrometry

Rosmarinic acid (RA) is a phenolic acid originally isolated from the herb medicine Rosmarinus officinalis. The purpose of this study was to identify the metabolites of RA. RA was incubated with human liver microsomes in the presence of β-nicotinamide adenine dinucleotide phosphate tetrasodium salt and/or uridine diphosphate glucuronic acid using glutathione (GSH) as a trapping agent. After 60-min incubation, the samples were analyzed using high-resolution liquid chromatography tandem mass spectrometry. Under the current conditions, 14 metabolites were detected and identified. Our data revealed that RA was metabolized through the following pathways: the first pathway is the oxidation of catechol to form ortho-quinone intermediates, which react with GSH to form mono-GSH adducts (M1, M2, and M3) and bis-GSH adducts (M4 and M5); the second pathway is conjugation with glucuronide to yield acylglucuronide (M7), which further reacts with GSH to form RA-S-acyl-GSH adduct (M9); the third pathway is hydroxylation to form M10, M11, and M12, which further react with GSH to form mono-GSH adducts (M13 and M14); the fourth pathway is conjugation with GSH through Michael addition (M6); the fifth pathway is conjugation with glucuronidation, forming M8, which is the major metabolic pathway of RA.     

In vitro metabolites characterization of ponatinib in human liver microsomes using ultra-high performance liquid chromatography combined with Q-Exactive-Orbitrap tandem mass spectrometry

Ponatinib is an oral drug for the treatment of chronic myeloid leukemia and acute lymphoblastic leukemia, which has been reported to increase the risk of hepatotoxicity. The aim of this study was to characterize the metabolites of ponatinib in human liver microsomes as well as its reactive metabolites. Ponatinib was incubated with human liver microsomes in the presence of NADPH and trapping agents (glutathione or potassium cyanide). The metabolites were characterized by liquid chromatography in combination with Q-Exactive-Orbitrap-MS. Under the current conditions, six metabolites were detected and structurally identified on the basis of their accurate masses, fragmentation patterns, and retention times. M3 (N-demethylation) was unambiguously identified by matching its retention time and fragment ions with those of its reference standard. N-demethylation and oxygenation were proved to be the predominant metabolic pathways of ponatinib. In addition, two reactive metabolites (cyano adducts) were detected in human liver microsomes in the presence of potassium cyanide and NADPH, suggesting that ponatinib underwent CYP450-mediated metabolic activation, which could be one of the causative mechanisms for its hepatotoxicity. The current study provides new information regarding the metabolic profiles of ponatinib and would be helpful in understanding the effectiveness and toxicity of ponatinib, especially the mechanism of hepatotoxicity.     

Identification of primary and sequential bioactivation pathways of carbamazepine in human liver microsomes using liquid chromatography/tandem mass spectrometry

Carbamazepine (CBZ)-induced idiosyncratic toxicities are commonly believed to be related to the formation of reactive metabolites. CBZ is metabolized primarily into carbamazepine-10,11-epoxide (CBZE), 2-hydroxycarbamazepine (2-OHCBZ) and 3-hydroxycarbamazepine (3-OHCBZ), in human liver microsomes (HLM). Over the past two decades, the 2,3-arene oxidation has been commonly assumed to be the major bioactivation pathway of CBZ. Recently, CBZE has been also confirmed to be chemically reactive. In order to identify other possible primary and sequential CBZ bioactivation pathways, individual HLM incubations of CBZ, CBZE, 2-OHCBZ and 3-OHCBZ were conducted in the presence of glutathione (GSH). In the CBZ incubation, a variety of GSH adducts were formed via individual or combined pathways of 10,11-epoxidation, arene oxidation and iminoquinone formation. In the CBZE incubation, the only detected GSH adducts were CBZE-SG1 and CBZE-SG2, which represented the two most abundant conjugates observed in the CBZ incubation. In the incubation of either 2-OHCBZ or 3-OHCBZ, a number of sequential GSH adducts were observed. However, none of the 2-OHCBZ-derived GSH adducts were detected in the CBZ incubation. Meanwhile, several GSH adducts were only observed in the CBZ incubation. In conclusion, CBZ can be bioactivated in HLM via 10,11-epoxidation, 2,3-arene oxidation, and several other pathways. In addition, the sequential bioactivation of 3-OHCBZ appeared to play a more important role than that of either CBZE or 2-OHCBZ in the overall bioactivation of CBZ in HLM. The identification of several new bioactivation pathways of CBZ in HLM demonstrates that possible CBZ bioactivation can be more complex than previously thought.     

Characterization of metabolites of Celecoxib in rabbits by liquid chromatography/tandem mass spectrometry

The metabolism of the anti-inflammatory drug Celecoxib in rabbits was characterized using liquid chromatography (LC)/tandem mass spectrometry (MS/MS) with precursor ion and constant neutral loss scans followed by product ion scans. After separation by on-line liquid chromatography, the crude urine samples and plasma and fecal extracts were analyzed with turbo-ionspray ionization in negative ion mode using a precursor ion scan of m/z 69 (CF(3)) and a neutral loss scan of 176 (dehydroglucuronic acid). The subsequent product ion scans of the [M - H] ions of these metabolites yielded the identification of three phase I and four phase II metabolites. The phase I metabolites had hydroxylations at the methyl group or on the phenyl ring of Celecoxib, and the subsequent oxidation product of the hydroxymethyl metabolite formed the carboxylic acid metabolite. The phase II metabolites included four positional isomers of acyl glucuronide conjugates of the carboxylic acid metabolite. These positional isomers were caused by the alkaline pH of the rabbit urine and were not found in rabbit plasma. The chemical structures of the metabolites were characterized by interpretation of their product ion spectra and comparison of their LC retention times and the product ion spectra with those of the authentic synthesized standards.     

Characterization of limonin glucoside metabolites from human prostate cell culture medium using high-performance liquid chromatography/electrospray ionization mass spectrometry and tandem mass spectrometry

The metabolism of limonin 17-beta-D-glucopyranoside (LG) by non-cancerous (RWPE-1) and cancerous (PC-3) human prostate epithelial cells was investigated using high-performance liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) with in-source fragmentation and tandem mass spectrometry (MS/MS). During positive ion LC/ESI-MS, LG formed an abundant sodiated species ([M+Na]+) while the protonated molecule was barely observable. [M+Na]+ further fragmented into the less abundant [LARL+H]+ and a predominantly protonated aglycone molecule (limonin) due to in-source fragmentation. The major metabolite, limonin A-ring lactone (LARL), formed an abundant protonated molecule that was fragmented into a protonated molecule of limonin by loss of one molecule of water. In MS/MS by collisionally activated dissociation (CAD), LG produced the sodiated aglycone, [aglycone+Na]+, while LARL fragmented into [M+H]+ of limonin and fragment ions resulted by further loss of water, carbon monoxide and carbon dioxide, indicating the presence of oxygenated-ring structures. The limits of detection of LG were 0.4 and 20 fmol in selected-ion monitoring (SIM) and selected-reaction monitoring (SRM) detection, respectively.     

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.     

Characterization of metabolites of tanshinone IIA in rats by liquid chromatography/tandem mass spectrometry

The metabolism of tanshinone IIA was studied in rats after a single-dose intravenous administration. In the present study, 12 metabolites of tanshinone IIA were identified in rat bile, urine and feces with two LC gradients using LC-MS/MS. Seven phase I metabolites and five phase II metabolites of tanshinone IIA were characterized and their molecular structures proposed on the basis of the characteristics of their precursor ions, product ions and chromatographic retention time. The seven phase I metabolites were formed, through two main metabolic routes, which were hydroxylation and dehydrogenation metabolism. M1, M4, M5 and M6 were supposedly tanshinone IIB, hydroxytanshinone IIA, przewaquinone A and dehydrotanshinone IIA, respectively, by comparing their HPLC retention times and mass spectral patterns with those of the standard compounds. The five phase II metabolites identified in this research were all glucuronide conjugates, all of which showed a neutral loss of 176 Da. M9 and M12 were more abundant than other identified metabolites in the bile, which was the main excretion path of tanshinone IIA and the metabolites. M12 was the main metabolite of tanshinone IIA. M9 and M12 were proposed to be the glucuronide conjugates of two different semiquinones and these semiquinones were the hydrogenation products of dehydrotanshinone IIA and tanshinone IIA, respectively. This hydrogenized reaction may be catalyzed by the NAD(P)H: quinone acceptor oxidoreductase (NQO). The biotransformation pathways of tanshinone IIA were proposed on the basis of this research.     

Identification of seven metabolites of oxyresveratrol in rat urine and bile using liquid chromatography/tandem mass spectrometry

Oxyresveratrol (trans‐2,4,3′,5′‐tetrahydroxystilbene) is a major compound isolated from Smilax china, a Chinese herbal medicine. The rat urine and bile samples were pretreated by solid‐phase extraction method after oral administration at a dose of 100 mg/kg of oxyresveratrol. Seven metabolites were identified by LC‐MS/MS method with electrospray ionization in negative ion mode. The results indicated that main metabolites of oxyresveratrol were monoglucuronided and monosulfated oxyresveratrol. Based on the results, the metabolic pathway of oxyresveratrol in rat urine and bile was proposed. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous determination of MK-0767 and seven metabolites in rat urine using liquid chromatography/tandem mass spectrometry

MK-0767, 5-[2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl)phenyl]methyl]benzamide (I, Table 1), is a dual peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonist previously studied for the treatment of type 2 diabetes and dyslipidemia. To support further toxicological studies in one of the animal species used in chronic testing of I, a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the simultaneous quantification of I and seven metabolites in rat urine was developed and validated. In this method, urine samples were diluted with acetonitrile/methanol (50:50, v/v) and injected directly onto the column of an LC system. Detection was achieved by MS/MS using a turbo ion spray probe monitoring precursor --> product ion combinations in selected reaction monitoring (SRM) mode. The linear range for I and three metabolites was 0.8-800 ng/mL, and 8-8000 ng/mL for four other metabolites found to be present in urine at higher concentrations than I. Intra-day and inter-day variation using this method were < or = 13.0%. The method exhibited good linearity, reproducibility, specificity and sufficient sensitivity when used for the analysis of rat urine samples. Concentrations of I and its major metabolites in rat urine were determined in samples collected between 0-24 h after dosing on the last day of administration of nine daily oral doses to three male (1000 mg/kg/day) and three female (300 mg/kg/day) Sprague-Dawley rats. The urinary concentrations of I and its metabolites were similar in male and female rats. The average concentrations of I were 0.51 and 0.33 microg/mL in male and female rats, respectively. Concentrations of four of the seven metabolites quantified were 6- to 45-fold higher than those of I. The most abundant metabolite, with concentrations of 24.2 and 13.3 microg/mL in male and female rat urine, respectively, was a methyl sulfoxide derivative formed by oxidative cleavage of the thiazolidinedione ring, followed by S-methylation and oxidation of the sulfide intermediate.     

Quantitative analysis of acetaminophen and its six metabolites in rat plasma using liquid chromatography/tandem mass spectrometry

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug. It is mainly metabolized by phase 1 and 2 reactions in the liver, and thus it could be involved in many drug–drug interactions. Therefore, the study of APAP metabolism is important in toxicological and pharmacokinetic studies. The objective of this study was to develop a rapid and sensitive method for the determination of APAP and its six metabolites in rat plasma for the pharmacokinetic studies. APAP and its metabolites were separated through a Capcell Pak MGII C₁₈ column and quantitated with a 16 min run in a triple‐quadruple mass spectrometer. The mobile phases were composed of 0.1% formic acid in either 95% water or 95% acetonitrile and analysis was performed twice in positive and negative modes. Validations such as accuracy, precision, recovery, matrix effect and stability were found to be within acceptance criteria of validation guidelines, indicating that the assay was applicable to the determination of the plasma concentrations of drug and its six metabolites. In conclusion, we developed an LC‐MS/MS method for the quantitative analysis of APAP and its six metabolites in rat plasma, and this method appears to be useful for pharmacokinetic/toxicokinetic studies of APAP and its metabolites in rats. Copyright © 2012 John Wiley & Sons, Ltd.     

Determination of nandrolone metabolites in human urine: comparison between liquid chromatography/tandem mass spectrometry and gas chromatography/mass spectrometry

Nandrolone (19‐nortestosterone) is an androgenic anabolic steroid illegally used as a growth‐promoting agent in animal breeding and as a performance enhancer in athletics. Therefore, its use was officially banned in 1974 by the Medical Commission of the International Olympic Committee (IOC). Following nandrolone administration, the main metabolites in humans are 19‐norandrosterone, 19‐norethiocolanolone and 19‐norepiandrosterone, and their presence in urine is the basis of detecting its abuse. The present work was undertaken to determine, in human urine, nandrolone metabolites (phase I and phase II) by developing and comparing multiresidue liquid chromatography/tandem mass spectrometry (LC/MS/MS) and gas chromatography/mass spectrometry (GC/MS) methods. A double extraction by solid‐phase extraction (SPE) was necessary for the complete elimination of the interfering compounds. The proposed methods were also tested on a real positive sample, and they allow us to determine the conjugated/free fractions ratio reducing the risk of false positive or misleading results and they should allow laboratories involved in doping control analysis to monitor the illegal use of steroids. The advantages of LC/MS/MS over GC/MS (which is the technique mainly used) include the elimination of the hydrolysis and derivatization steps: it is known that during enzymatic hydrolysis several steroids can be converted into related compounds and deconjugation is not always 100% effective. The validation parameters for the two methods were similar (limit of quantification (LOQ) <1 ng/mL and percentage coefficient of variance (CV%) <16.4), and both were able to confirm unambiguously all the analytes, thus confirming the validity of both techniques. Copyright © 2010 John Wiley & Sons, Ltd.     

Quantification of cyclophosphamide and its metabolites in urine using liquid chromatography/tandem mass spectrometry

A reliable and easy to use liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the simultaneous quantification of urinary concentrations of cyclophosphamide (CP) and its main metabolites excreted in urine, i.e. N-dechloroethylcyclophosphamide (DCL-CP), 4-ketocyclophosphamide (4KetoCP), and carboxyphosphamide (CarboxyCP). Sample preparation consisted of dilution of urine with an aqueous solution of the internal standard D(4)-CP and methanol, and centrifugation. LC/MS/MS detection was performed using a triple-quadrupole mass spectrometer working in selected reaction monitoring mode. All analytes were quantified in a single run within 11.5 min. The limits of detection were 5 ng/mL for CP and 4KetoCP, 1 ng/mL for DCL-CP, and 30 ng/mL for CarboxyCP. Quantification ranges were adjusted to the expected concentrations in 24-h urine collections of patients treated with a polychemotherapy regimen (3-175 microg/mL for CP, 0.5-27 microg/mL for 4KetoCP and 0.17-9 microg/mL for CarboxyCP and DCL-CP, respectively). The method was validated according to international guidelines of the ICH and the FDA.     

Quantification of rat brain neurotransmitters and metabolites using liquid chromatography/electrospray tandem mass spectrometry and comparison with liquid chromatography/electrochemical detection

Analytical methodology based on solid-phase extraction, polar reversed-phase liquid chromatography, and electrospray tandem mass spectrometry (LC/MS/MS) with isotope dilution was developed and validated for quantifying the neurotransmitters, dopamine and serotonin, and their major metabolites in brain tissue. Limits of detection (0.1-20 pg/mg tissue) were sufficient for analysis of multiple neurotransmitters in rat brain regions, including parietal cortex, hypothalamus, pituitary, substantia nigra, and striatum. Method performance was compared with contemporaneous measurements using a well-established procedure based on ion-pairing reversed-phase liquid chromatography and amperometric detection. The principal advantages of the LC/MS/MS method include a more robust sample purification procedure, an optimized chromatographic separation, and the qualitative and quantitative assurance that comes from coeluting isotopically labeled internal standards; however, sensitivity did not consistently improve upon that provided by amperometric detection. This methodology may be particularly useful for applications in which simultaneous determinations are required for drugs and their affected neurotransmitters in specific brain regions.     

Characterization of explosives by liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry using electrospray ionization and parent-ion scanning techniques

Analytical techniques for the detection of small amounts of explosives (in the picogram range) are now involved in various application. Some of them concern soil, water and air monitoring in order to face environmental problems related to improper handling procedures either in stocking or in wasting of the explosive products. Other areas are strictly related to forensic analysis of samples coming either from explosion areas where the matrix is various (metal, glass, wood, scraps), or from explosives transportation related to international terrorism. Generally speaking, for these applications the bulk of the matrix seriously interferes in the detection of the explosive analyte, which is usually present at trace levels. Unfortunately, despite some improvements, analytical techniques developed up today in this domain are still faced to two main constraints: the introduction of new products with unanticipated chemico-physical properties and the requirement of a routine and fast analytical method which can handle any matrix with a minimal clean-up and performing a sensitivity compatible either with the ever-decreasing demanded detection limit and with the ever-decreasing available specimen amount. These requirements can be fulfilled now by the new LC-MS and LC-MSMS techniques: mass spectrometry (MS) is likely an universal detector but even specific, especially when implemented in tandem MS (MSMS); LC is by far the most suitable technique to handle such a kind of compounds. Moreover, of a particular concern are some explosives which are reported to be thermally stable but difficult to dissolve. Some of the experiments on characterization of explosives [Octagen (HMX), Ethyleneglycol dinitrate (EGDN), Exogen (RDX), Propanetriol trinitrate (NG), Trinitrotoluene (TNT), N-Methyl-N-tetranitrobenzenamine (TETRYL), Dintrotoluene (DNT), Bis-(nitrooxy-methyl) propanediol dinitrate (PETN), Hexanitrostilbene (HNS), Triazido-trinitrobenzene (TNTAB), Tetranitro-acridone (TENAC), Hexa-nitrodiphenylamine (HEXYL), Nitroguanidine (NQ)] by LC-MS and LC-MSMS with the API-IonSpray source and using the Parent-Scan technique are presented.     

Characterization of in vitro metabolites of trimethoprim and diaveridine in pig liver microsomes by liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry

Trimethoprim (TMP) and diaveridine (DVD) are used in combination with sulfonamides and sulfaquinoxlaine as an effective antibacterial agent and antiprotozoal agent, respectively, in humans and animals. To gain a better understanding of the metabolism of TMP and DVD in the food-producing animals, the metabolites incubated with liver microsomes of pigs were analyzed for the first time with high-performance liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry. Seven TMP-related and six DVD-related metabolites were characterized based on the accurate MS2 spectra and known structure of the parent drug, respectively. The metabolites of TMP were identified as two O-demethylation metabolites, a di-O-demethylation metabolite, two N-oxides metabolites, a hydroxylated metabolite on the methylene carbon and a hydroxylated metabolite on the methyl group. DVD was also biotransformed to two O-demethylation metabolites, a di-O-demethylation metabolite, an N-oxide metabolite, a hydroxylation metabolite on the methylene carbon and a hydroxylation metabolite followed by O-demethylation. The results indicate that the two compounds have similar biotransformation pathways in pigs. O-Demethylation was the major metabolic route of TMP and DVD in the pig liver microsomes. The proposed metabolic pathways of TMP and DVD in liver microsomes will provide a basis for further studies of the in vivo metabolism of the two drugs in food-producing animals.