Metabolic profiles of ribociclib in rat and human liver microsomes using liquid chromatography combined with electrospray ionization high-resolution mass spectrometry

Ribociclib is a highly specific CDK4/6 inhibitor. Determination of the metabolism of ribociclib is required during the drug development stage. In this study, metabolic profiles of ribociclib were investigated using rat and human liver microsomes. Metabolites were structurally identified by liquid chromatography electrospray ionization high-resolution mass spectrometry operated in positive-ion mode. The metabolites were characterized by accurate masses, MS² spectra and retention times. With rat and human liver microsomes, a total of 10 metabolites were detected and further identified. No human-specific metabolites were detected. The metabolic pathways of ribociclib were oxygenation, demethylation and dealkylation. Most importantly, two glutathione (GSH) adducts were identified in human liver microsomes fortified with GSH. The formation of the GSH adducts was hypothesized to be through the oxidation of electron-rich 1,4-benzenediamine to a 1,4-diiminoquinone intermediate, which is highly reactive and can be trapped by GSH to form stable metabolites. The current study provides an overview of the metabolic profiles of ribociclib in vitro, which will be of great help in understanding the efficacy and toxicity of this drug.     

In vivo and in vitro metabolism of aspirin eugenol ester in dog by liquid chromatography tandem mass spectrometry

Aspirin eugenol ester (AEE) is a promising drug candidate for treatment of inflammation, pain and fever and prevention of cardiovascular diseases with fewer side effects than its precursor, aspirin. Investigation into its metabolic process in target animal species will help to illustrate its mechanism of action and to establish its residual mark compound to formulate its dosage. Six beagle dogs were orally given a dose of 20 mg kg^?1 of AEE and one dog was used to prepare blank liver microsomes. Their liver microsomes were prepared for in vitro study and their plasma and urine were collected for in vivo metabolic analysis using liquid chromatography tandem mass spectrometry. In this study we identified 10 metabolites, M1, M2, M3, M4, M5 in phase I and M6, M7, M8, M9, M10 in phase II. Based on the metabolites of AEE, the pathways of AEE metabolism in dog were demonstrated. Copyright © 2014 John Wiley & Sons, Ltd.     

Characterization of the metabolite of AdipoRon in rat and human liver microsomes by ultra‐high‐performance liquid chromatography combined with Q‐Exactive Orbitrap tandem mass spectrometry

AdipoRon is an orally active adiponectin receptor agonist. The aim of this study was to characterize the metabolites of AdipoRon in rat and human liver microsomes using ultra‐high performance liquid chromatography combined with Q‐Exactive Orbitrap tandem mass spectrometry (UPLC‐Q‐Exactive‐Orbitrap‐MS) together with data processing techniques including extracted ion chromatograms and a mass defect filter. AdipoRon (10 μm ) was incubated with liver microsomes in the presence of NADPH and this resulted in a total of 11 metabolites being detected. The identities of these metabolites were characterized by comparing their accurate masses and fragment ions as well as their retention times with those of AdipoRon using MetWorks software. Metabolites M1–M3, M6, and M8–M11 were identified for the first time. Metabolite M4, the major metabolite both in rat and human liver microsomes, was further confirmed using the reference standard. Our results revealed that the metabolic pathways of AdipoRon in liver microsomes were N‐dealkylation (M2), hydroxylation (M, M5–M9), carbonyl reduction (M4) and the formation of amide (M10 and M11). Our results provide valuable information about the in vitro metabolism of AdipoRon, which would be helpful for us to understand the mechanism of the elimination of AdipoRon and, in turn, its effectiveness and toxicity.     

In vitro metabolism of corydaline in human liver microsomes and hepatocytes using liquid chromatography-ion trap mass spectrometry

Corydaline is a pharmacologically active isoquinoline alkaloid isolated from Corydalis tubers. It exhibits the antiacetylcholinesterase, antiallergic, antinociceptive, and gastric emptying activities. The purposes of this study were to establish in vitro metabolic pathways of corydaline in human liver microsomes and hepatocytes by identification of their metabolites using liquid chromatography-ion trap mass spectrometry. Human liver microsomal incubation of corydaline in the presence of an NADPH-generating system resulted in the formation of nine metabolites, namely, four O-desmethylcorydaline [M1 (yuanhunine), M2 (9-O-desmethylcorydaline), M3 (isocorybulbine), and M4 (corybulbine)], three di-O-desmethylcorydaline [M5 (9,10-di-O-desmethylcorydaline), M6 (2,10-di-O-desmethylcorydaline), and M7 (3,10-di-O-desmethylcorydaline)], M8 (hydroxyyuanhunine), and M9 (hydroxycorydaline). Incubation of corydaline in human hepatocytes produced four metabolites including M1, M5, M6, and M9. O-Demethylation and hydroxylation were the major metabolic pathways for the metabolism of corydaline in human liver microsomes and hepatocytes.     

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.     

Characterization of xylazine metabolism in rat liver microsomes using liquid chromatography–hybrid triple quadrupole–linear ion trap–mass spectrometry

ABSTRACT: Xylazine is an α₂‐adrenoceptor agonist and it is widely used in veterinary anesthesia in combination with ketamine. There is limited information on the metabolism of xylazine. A quantitative method for the determination of xylazine by HPLC‐ESI/MS/MS was developed. The method consisted of a protein precipitation extraction followed by analysis using liquid chromatography electrospray tandem mass spectrometry. The chromatographic separation was achieved using a Thermo Betasil Phenyl 100 × 2 mm column combined with an isocratic mobile phase composed of acetonitrile, methanol, water and formic acid (60:20:20:0.4) at a flow rate of 300 μL/min. The mass spectrometer was operating in selected reaction monitoring mode and the analytical range was set at 0.05–50 μm . The precision (%CV) and accuracy (%NOM) observed were 2.3–7.2 and 88.2–96.4%. In vitro metabolism studies were performed in rat liver microsomes and results showed moderate cytochrome P450 affinity (K_m = 10.1 μm ) and a low metabolic stability of xylazine with a half‐life of 4.1 min in rat liver microsomes. Five phase 1 metabolites were observed. The main metabolite observed was an oxidation of the thiazine moiety at m/z 235 and, to a lesser extent, we observed the formation of N‐(2,6‐dimethylphenyl)thiourea at m/z 181 and three distinctive hydroxylated metabolites at m/z 237. Further experiments with ketamine and ketoconazole strongly supported that the metabolism of xylazine to its main metabolite is mediated by CYP3A in rat liver microsomes. Copyright © 2013 John Wiley & Sons, Ltd.     

Characterization of in vitro metabolites of deoxypodophyllotoxin in human and rat liver microsomes using liquid chromatography/tandem mass spectrometry

The in vitro metabolism of deoxypodophyllotoxin (DPT), a medicinal herbal product isolated from Anthriscus sylvestris (Apiaceae), was investigated in rats and human microsomes and human recombinant cDNA-expressed CYPs. The incubation of DPT with pooled human microsomes in the presence of NADPH generated five metabolites while its incubation with dexamethasone (Dex)-induced rat liver resulted in seven metabolites (M1-M7) with major metabolic reactions including mono-hydroxylation, O-demethylation and demethylenation. Reasonable structures of the seven metabolites of DPT could be proposed, based on the electrospray tandem mass spectra. Chemical inhibition by ketoconazole and metabolism studies with human recombinant cDNA-expressed CYPs indicated that CYP 3A4 and 2C19 are the major CYP isozymes in the metabolism of DPT in human liver microsomes.     

Rapid structural characterization of in vivo and in vitro metabolites of tinoridine using UHPLC–QTOF–MS/MS and in silico toxicological screening of its metabolites

Tinoridine is a nonsteroidal anti‐inflammatory drug and also has potent radical scavenger and antiperoxidative activity. However, metabolism of tinoridine has not been thoroughly investigated. To identify in vivo metabolites, the drug was administered to Sprague–Dawley rats (n = 5) at a dose of 20 mg kg^?1, and blood, urine and feces were collected at different time points up to 24 h. In vitro metabolism was delved by incubating the drug with rat liver microsomes and human liver microsomes. The metabolites were enriched by optimized sample preparation involving protein precipitation using acetonitrile, followed by solid‐phase extraction. Data processes were carried out using multiple mass defects filters to eliminate false‐positive ions. A total of 11 metabolites have been identified in urine samples including hydroxyl, dealkylated, acetylated and glucuronide metabolites; among them, some were also observed in plasma and feces samples. Only two major metabolites were formed using liver microsomal incubations. These metabolites were also observed in vivo. All the 11 metabolites, which are hitherto unknown and novel, were characterized by using ultrahigh‐performance liquid chromatography–quadrupole time‐of‐flight tandem mass spectrometry in combination with accurate mass measurements. Finally, in silico toxicological screening of all metabolites was evaluated, and two metabolites were proposed to show a certain degree of lung or liver toxicity. Copyright © 2015 John Wiley & Sons, Ltd.     

Studies on metabolites and metabolic pathways of bulleyaconitine A in rat liver microsomes using LC‐MSn combined with specific inhibitors

Bulleyaconitine A (BLA) from Aconitum bulleyanum plants is usually used as anti‐inflammatory drug in some Asian countries. It has a variety of bioactivities, and at the same time some toxicities. Since the bioactivities and toxicities of BLA are closely related to its metabolism, the metabolites and the metabolic pathways of BLA in rat liver microsomes were investigated by HPLC–MSⁿ. In this research, the 12 metabolites of BLA were identified according to the results of HPLC‐MSⁿ data and the relevant literature. The results showed that there are multiple metabolites of BLA in rat liver microsomes, including demethylation, deacetylation, dehydrogenation deacetylation and hydroxylation. The major metabolic pathways of BLA in rat liver microsomes were clarified by HPLC‐MS combined with specific inhibitors of CYP450 isoforms. As a result, CYP3A and 2C were found to be the principal CYP isoforms contributing to the metabolism of BLA. Moreover, CYP2D6 and 2E1 are also more important CYP isoforms for the metabolism of BLA. While CYP1A2 only affected the formation rate of M11, its effect on the metabolism of BLA is very small. Copyright © 2014 John Wiley & Sons, Ltd.     

Structural elucidation of metabolites of ginkgolic acid in rat liver microsomes by ultra‐performance liquid chromatography/electrospray ionization tandem mass spectrometry and hydrogen/deuterium exchange

Ginkgolic acids have been shown to possess allergenic as well as genotoxic and cytotoxic properties. The question arises whether the metabolism of ginkgolic acids in the liver could decrease or increase their toxicity. In this study, the in vitro metabolism of ginkgolic acid (15:1, GA), one component of ginkgo acids, was investigated as a model compound in Sprague‐Dawley rat liver microsomes. The metabolites were analyzed by ultra‐performance liquid chromatography coupled with photodiode array detector/negative‐ion electrospray ionization tandem mass spectrometry (UPLC‐PDA/ESI‐MS/MS) and hydrogen/deuterium (H/D) exchange. The result showed that the benzene ring remained unchanged and the oxidations occurred at the side alkyl chain in rat liver microsomes. At least eight metabolites were found. Among them, six phase I metabolites were tentatively identified. This study might be useful for the investigation of toxicological mechanism of ginkgolic acids. Copyright © 2009 John Wiley & Sons, Ltd.     

Investigation of the metabolic biotransformation of substance P in liver microsomes by liquid chromatography quadrupole ion trap mass spectrometry

Substance P (SP) belongs to the tachykinin family and plays an essential role in pain transmission and in neurogenic inflammation. It can be detected in the central and peripheral nervous systems. The objectives of this study were to establish SP metabolic stability in liver microsomes in three species (rat, mouse and human), and identify and characterize SP metabolites by LC‐MS/MS. Endogenous peptide metabolism is not well documented and this is particularly true for neuropeptides participating in neurogenic inflammation. In vitro, T_1/2 results in pooled liver microsomes were 9.2, 5.6 and 18.6 min for rat, mouse and human liver microsomes, respectively. Five major SP metabolites were identified and quantified, including C‐terminal SP fragments SP_3–11, SP_5–11, SP_6–11, SP_8–11 as well as N‐terminal fragment SP_1–7. The results suggest significant differences between species in SP metabolite concentrations. Consequently, the metabolic profile of each species is distinctive and may have a significant impact on biomolecular mechanisms involved in specific pathophysiological changes. Copyright © 2012 John Wiley & Sons, Ltd.     

Metabolism studies on prim‐O‐glucosylcimifugin and cimifugin in human liver microsomes by ultra‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry

Prim‐O‐glucosylcimifugin (PGCN) and cimifugin (CN) are major constituents of Radix Saposhnikoviae that have antipyretic, analgesic and anti‐inflammatory pharmacological activities. However, there were few reports with respect to the metabolism of PGCN and CN in vitro. In this paper, we describe a strategy using ultra‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry (UPLC‐Q‐TOF‐MS) for fast analysis of the metabolic profile of PGCN and CN in human liver microsomes. In total, five phase I metabolites of PGCN, seven phase I metabolites and two phase II metabolites of CN were identified in the incubation of human liver microsomes. The results revealed that the main phase I metabolic pathways of PGCN were hydroxylation and hydrolysis reactions. The phase I metabolic pathways of CN were found to be hydroxylation, demethylation and dehydrogenation. Meanwhile, the results indicated that O‐glucuronidation was the major metabolic pathway of CN in phase II metabolism. The specific UDP‐glucuronosyltransferase (UGT) enzymes responsible for CN glucuronidation metabolites were identified using recombinant UGT enzymes. The results indicated that UGT1A1, UGT1A9, UGT2B4 and UGT2B7 might play major roles in the glucuronidation of CN. Overall, this study may be useful for the investigation of metabolic mechanism of PGCN and CN, and it can provide reference and evidence for further pharmacodynamic experiments. Copyright © 2016 John Wiley & Sons, Ltd.     

In Vitro and In Vivo Primary Metabolic Characterization of F18, a Novel Histone Deacetylase-6 (HDAC6) Inhibitor,Using UHPLC–QqQ–MS/MS and Q-TOF–MS Methods

F18, N-hydroxy-4-(2-methoxy-5-(methyl (2-methylquinazolin-4-yl) amino) phenoxy) butanamide, is a novel selective HDAC6 inhibitor with good antitumor activity. In the early drug development, drug-metabolism studies are a crucial and indispensable part. In this study, we proposed to evaluate the in vitro primary metabolism of F18 in phase Ι in liver microsomes from human, rat, dog, monkey and mouse and investigate the metabolite profile both in vitro and in vivo using LC–MS/MS methods. F18 showed high metabolic stability in human, rat, dog, monkey and mouse liver microsomes over 120 min, with t _1/2 >8 h in human, rat, and dog, and t _1/2 <3.5 h in monkey, with almost no clearance in mouse. Human cytochrome P450 (P450) phenotyping showed that F18 was predominantly metabolized by CYP2C9, CYP2E1, CYP2D6 and CYP3A4. The investigation of the effect of F18 on CYP enzymes in HLM demonstrated that this compound did not significantly inhibit CYP 1A2 (IC₅₀ >100 μM), was a moderate inhibitor of CYP3A4 (IC₅₀ = 1.63 μM) and had negligible effects on CYP3A1/2 activity in rats. The results will be valuable in understanding drug–drug interactions (DDI) when F18 is co-administered with other drugs. The metabolites of F18 were investigated in rat plasma, urine, feces and different liver microsomes in NADPH samples, yielding at least 11 metabolites in these biological samples. The prominent metabolic pathways were de-methylation, de-amination, de-oxidation and O-glucuronidation. In summary, this work provides the first clues regarding F18 metabolism, providing important information for comprehensive understanding of F18 metabolites.

     

Metabolism of olaquindox in rat liver microsomes: structural elucidation of metabolites by high-performance liquid chromatography combined with ion trap/time-of-flight mass spectrometry

Olaquindox (N-(2-hydroxyethyl)-3-methyl-2-quinoxalincarboxamide-1,4-dioxide) is a growth-promoting feed additive for food-producing animals. Its toxicity is closely related to the metabolism. The complete metabolic pathways of olaquindox are not revealed. To improve studies of the metabolism and toxicity of olaquindox, its biotransformation in rat liver microsomes and the structure of its metabolites using high-performance liquid chromatography combined with ion trap/time-of-flight mass spectrometry (LC/MS-ITTOF) were investigated. When olaquindox was incubated with an NADPH-generating system and rat liver microsomes, ten metabolites (M1-M10) were detected. The structures of these metabolites were identified from mass spectra and comparison of their changes in their accurate molecular masses and fragment ions with those of the parent drug. With the high resolution and good mass accuracy achieved by this technique, the elemental compositions of the metabolites and their fragment ions were exactly determined. The results indicate that the N --> O group reduction is the main metabolic pathway of olaquindox metabolism in rat liver microsomes, because abundant 1-desolaquindox (M2), 4-desolaquindox (M1) and bisdesoxyolaquindox (M9) were produced during the incubation step. Seven other minor metabolites were revealed which were considered to be hydroxylation metabolites, based on the position of the quinoxaline ring or 3-methyl group and a carboxylic acid derivative on the side chain at position 2 of the quinoxaline ring. Among the identified metabolites, five new hydroxylated metabolites (M3-M7) were found for the first time in rat liver microsomes. This work will conduce to complete clarification of olaquindox metabolism, and improve the in vivo metabolism of olaquindox in food animals.     

In vivo and in vitro metabolite profiling of nirmatrelvir using LC–Q-ToF–MS/MS along with the in silico approaches for prediction of metabolites and their toxicity

Nirmatrelvir (NRV), a 3C-like protease or M^pro inhibitor of SARS-CoV-2, is used for the treatment of COVID-19 in adult and paediatric patients. The present study was accomplished to investigate the comprehensive metabolic fate of NRV using in vitro and in vivo models. The in vitro models used for the study were microsomes (human liver microsomes, rat liver microsomes, mouse liver microsomes) and S9 fractions (human liver S9 fractions and rat liver S9 fractions) with the appropriate cofactors, whereas Sprague–Dawley rats were used as the in vivo models. Nirmatrelvir was administered orally to Sprague–Dawley rats, which was followed by the collection of urine, faeces and blood at pre-determined time intervals. Protein precipitation was used as the sample preparation method for all the samples. The samples were then analysed by liquid chromatography–quadrupole time-of-flight tandem mass spectrometry (LC-Q-ToF-MS/MS) using an Acquity BEH C18 column with 0.1% formic acid and acetonitrile as the mobile phase. Four metabolites were found to be novel, which were formed via amide hydrolysis, oxidation and hydroxylation. Furthermore, an in silico analysis was performed using Meteor Nexus software to predict the probable metabolic changes of NRV. The toxicity and mutagenicity of NRV and its metabolites were also determined using DEREK Nexus and SARAH Nexus.     

Metabolite identification of the antimalarial naphthoquine using liquid chromatography–tandem high‐resolution mass spectrometry in combination with multiple data‐mining tools

Naphthoquine (NQ) is one of important partner drugs of artemisinin‐based combination therapy (ACT), which is recommended for the treatment of uncomplicated Plasmodium falciparum. NQ shows a high cure rate after a single oral administration. It is absorbed quickly (time to peak concentration 2–4 h) and has a long elimination half‐life (255 h). However, the metabolism of NQ has not been clarified. In this work, the metabolite profiling of NQ was studied in six liver microsomal incubates (human, cynomolgus monkey, beagle dog, mini pig, rat and CD1 mouse), seven recombinant CYP enzymes (1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4) and rat (plasma, urine, bile and feces) using liquid chromatography tandem high‐resolution LTQ‐Orbitrap mass spectrometry (HRMSⁿ) in conjunction with online hydrogen/deuterium exchange. The biological samples were pretreated by protein precipitation and solid‐phase extraction. For data processing, multiple data‐mining tools were applied in tandem, i.e. background subtraction and followed by mass defect filter. NQ metabolites were characterized by accurate MS/MS fragmentation characteristics, the hydrogen/deuterium exchange data and cLogP simulation. As a result, five phase I metabolites (M1–M5) of NQ were characterized for the first time. Two metabolic pathways were involved: hydroxylation and N‐oxidation. This study demonstrates that LC‐HRMSⁿ in combination with multiple data‐mining tools in tandem can be a valuable analytical strategy for rapid metabolite profiling of drugs.     

Metabolite identification of the antimalarial piperaquine in vivo using liquid chromatography–high‐resolution mass spectrometry in combination with multiple data‐mining tools in tandem

Artemisinin‐based combination therapy is widely used for the treatment of uncomplicated Plasmodium falciparum malaria, and piperaquine (PQ) is one of important partner drugs. The pharmacokinetics of PQ is characterized by a low clearance and a large volume of distribution; however, metabolism of PQ has not been thoroughly investigated. In this work, the metabolite profiling of PQ in human and rat was studied using liquid chromatography tandem high‐resolution LTQ‐Orbitrap mass spectrometry (HRMS). The biological samples were pretreated by solid‐phase extraction. Data processes were carried out using multiple data‐mining techniques in tandem, i.e., isotope pattern filter followed by mass defect filter. A total of six metabolites (M1–M6) were identified for PQ in human (plasma and urine) and rat (plasma, urine and bile). Three reported metabolites were also found in this study, which included N‐oxidation (M1, M2) and carboxylic products (M3). The subsequent N‐oxidation of M3 resulted in a new metabolite M4 detected in urine and bile samples. A new metabolic pathway N‐dealkylation was found for PQ in human and rat, leading to two new metabolites (M5 and M6). This study demonstrated that LC‐HRMSⁿ in combination with multiple data‐mining techniques in tandem can be a valuable analytical strategy for rapid metabolite profiling of drugs. Copyright © 2016 John Wiley & Sons, Ltd.     

Characterization of in vitro metabolism of capsazepine,a vanilloid transient receptor potential channel antagonist,by liquid chromatography quadrupole ion trap mass spectrometry

Capsazepine is an antagonist of the transient receptor potential channel vanilloid 1 (TRPV1), which is known to play an important role in the regulation of pain and inflammation. A selective and sensitive quantitative method for the determination of capsazepine by HPLC‐ESI/MS/MS was developed. The method consisted of a protein precipitation extraction followed by analysis using liquid chromatography electrospray quadrupole ion trap mass spectrometry. The chromatographic separation was achieved using a 100 × 2 mm C₁₈ Waters Symmetry column combined with a gradient mobile phase composed of acetonitrile and 0.1% formic acid aqueous solution at a flow rate of 220 µL/min. The mass spectrometer was operating in full‐scan MS/MS mode using two‐segment analysis. An analytical range of 10–5000 ng/mL was used in the calibration curve constructed in rat plasma. The inter‐batch precision and accuracy observed were 10.1, 6.4 and 6.1% and 100.8, 98.5 and 106.2% at 50, 500 and 5 000 ng/mL, respectively. An in vitro metabolic stability using rat, dog or mouse liver microsomes was performed to determine the intrinsic clearance of capsazepine. The results suggest a very rapid degradation with T_1/2 ranging from 2.6 to 4.3 min and a high clearance, suggesting that drug bioavailability is considerably reduced following extravascular administrations, consequently affecting drug response. Three metabolites were identified by HPLC‐MS/MS. S‐hydroxylation (M + 16), oxidative desulfuration (M − 16) and desulfuration (M − 32) metabolites of capsazepine were observed following exposure to rat, dog and mouse microsomes. Copyright © 2010 John Wiley & Sons, Ltd.     

Metabolism of cyadox in rat,chicken and pig liver microsomes and identification of metabolites by accurate mass measurements using electrospray ionization hybrid ion trap/time‐of‐flight mass spectrometry

Cyadox (CYX), (2‐formylquinoxaline)‐N¹,N⁴‐dioxide cyanoacetylhydrazone, is a growth promoter, which is more efficient and less toxic to animals. Few studies have been performed to reveal the metabolism of CYX in animals till now. In this study, the metabolic fate of CYX in the liver microsomes of animal was investigated firstly using high‐performance liquid chromatography combined with hybrid ion trap/time‐of‐flight mass spectrometry. CYX was incubated with rat, chicken and pig liver microsomes in the presence of a NADPH‐generating system. Multiple scans of metabolites in MS and MS² modes and accurate mass measurements were performed simultaneously through data‐dependent acquisition. Most measured mass errors were less than 10 ppm for both protonated molecules and fragment ions using external mass calibration. The structures of metabolites and their fragment ions were easily and reliably characterized based on the accurate MS² spectra and known structure of CYX. The relative biotransformation of CYX into characterized metabolites was estimated based on the UV absorption and the assumption that all metabolites had the same extinction coefficient as the parent compound at 305 nm. Totally, seven metabolites were identified as three reduced metabolites (cyadox 1‐monoxide (Cy1), cyadox 4‐monoxide (Cy2) and bisdesoxycyadox (Cy4)), three hydrolysis metabolites of the amide bond (N‐decyanoacetyl cyadox (Cy5), N‐decyanoacetyl cyadox 1‐monoxide (Cy6) and N‐decyanoacetyl bisdesoxycyadox (Cy7)) and a hydroxylation metabolite of Cy1 (Cy3). Cy1–Cy6 could be detected in rat, chicken and pig liver microsomes while metabolite Cy7 could only be observed in pig. The amounts of the metabolites in three species are different. For the formations of Cy1 and Cy3, the rank order was rat～chicken > pig. For Cy4 and Cy5, the order was pig > rat > chicken. Cy1 and Cy4 have been previously reported, whereas the other five metabolites were novel. The N→O group reduction and hydroxylation were the main metabolic pathways for CYX in the three species. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification and characterization of in vitro and in vivo fidarestat metabolites: Toxicity and efficacy evaluation of metabolites

The progression of diabetic complications can be prevented by inhibition of aldose reductase and fidarestat considered to be highly potent. To date, metabolites of the fidarestat, toxicity, and efficacy are unknown. Therefore, the present study on characterization of hitherto unknown in vitro and in vivo metabolites of fidarestat using liquid chromatography–electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) is undertaken. In vitro and in vivo metabolites of fidarestat have been identified and characterized by using LC/ESI/MS/MS and accurate mass measurements. To identify in vivo metabolites, plasma, urine, and feces samples were collected after oral administration of fidarestat to Sprague–Dawley rats, whereas for in vitro metabolites, fidarestat was incubated in human S9 fraction, human liver microsomes, and rat liver microsomes. Furthermore, in silico toxicity and efficacy of the identified metabolites were evaluated. Eighteen metabolites have been identified. The main in vitro phase I metabolites of fidarestat are oxidative deamination, oxidative deamination and hydroxylation, reductive defluroniation, and trihydroxylation. Phase II metabolites are methylation, acetylation, glycosylation, cysteamination, and glucuronidation. Docking studies suggest that oxidative deaminated metabolite has better docking energy and conformation that keeps consensus with fidarestat whereas the rest of the metabolites do not give satisfactory results. Aldose reductase activity has been determined for oxidative deaminated metabolite (F‐1), and it shows an IC50 value of 0.44 μM. The major metabolite, oxidative deaminated, did not show any cytotoxicity in H9C2, HEK, HEPG2, and Panc1 cell lines. However, in silico toxicity, the predication result showed toxicity in skin irritation and ocular irritancy SEV/MOD versus MLD/NON (v5.1) model for fidarestat and its all metabolites. In drug discovery and development research, it is distinctly the case that the potential for pharmacologically active metabolites must be considered. Thus, the active metabolites of fidarestat may have an advantage as drug candidates as many drugs were initially observed as metabolites.