Structural Elucidation of in vivo and in vitro Metabolites of Epiberberine in Rat and Its Liver and Intestines by Liquid Chromatography-Tandem Mass Spectrometry

The in vivo and in vitro metabolism of epiberberine was investigated using a highly specific and sensitive liquid chromatography–mass spectrometry (LC–MS/MS) method. In vivo samples including rat urine, feces, and plasma samples were collected individually after ingestion of 35 mg/kg epiberberine to healthy rats. In vitro samples were prepared by incubating epiberberine with homogenized liver and intestinal flora of rats, respectively. As a result, at least 17, 3 and 5 metabolites were found in rat urine, feces, and plasma, respectively. Additionally, 1 and 3 metabolites were found in the rat intestinal flora and homogenized liver incubation mixtures, respectively.     

LC/MS/MS for identification of in vivo and in vitro metabolites of jatrorrhizine

The in vivo and in vitro metabolism of jatrorrhizine has been investigated using a specific and sensitive LC/MS/MS method. In vivo samples including rat feces, urine and plasma collected separately after dosing healthy rats with jatrorrhizine (34 mg/kg) orally, along with in vitro samples prepared by incubating jatrorrhizine with rat intestinal flora and liver microsome, respectively, were purified using a C(18) solid-phase extraction cartridge. The purified samples were then separated with a reversed-phase C(18) column with methanol-formic acid aqueous solution (70:30, v/v, pH3.5) as mobile phase and detected by on-line MS/MS. The structural elucidation of the metabolites was performed by comparing their molecular weights and product ions with those of the parent drug. As a result, seven new metabolites were found in rat urine, 13 metabolites were detected in rat feces, 11 metabolites were detected in rat plasma, 17 metabolites were identified in intestinal flora incubation solution and nine metabolites were detected in liver microsome incubation solution. The main biotransformation reactions of jatrorrhizine were the hydroxylation reaction, the methylation reaction, the demethylation reaction and the dehydrogenation reaction of parent drug and its relative metabolites. All the results were reported for the first time, except for some of the metabolites in rat urine.     

Comparative Metabolism of Mequindox in Liver Microsomes,Hepatocytes, and Intestinal Microflora of Chicken

Drug metabolism studies in vitro were carried out inexpensively and readily to serve as an adequate mechanism to characterize drug metabolites, elucidate their pathways, and make suggestions for further testing in vivo. In this work, the comparative metabolism of mequindox (MEQ) was investigated in vitro by incubation with chicken liver microsomes, hepatocytes, and intestinal microflora, followed by analysis using ultra-performance liquid chromatography coupled with electrospray ionization hybrid quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) for structure identification. There were 12 metabolites detected when MEQ was incubated with liver microsomes, 6 metabolites with the hepatocytes and 4 metabolites with intestinal microflora, respectively. The major metabolites in liver microsomes were bideoxymequindox and 2-isoethanol-N1-deoxymequindox, and that in hepatocytes were 2-isoethanol mequindox and 2-isoethanol-N1-deoxymequindox, but in intestinal incubations, N1-deoxymequindox and bideoxymequindox were the major metabolites. The results indicated that the metabolism of MEQ was active in vitro; meanwhile, revealed the main metabolic pathways of MEQ were N→O group reduction, carbonyl reduction and hydroxylation reaction. The information regarding in vitro metabolism of MEQ provided a better understanding of the role of the liver and intestinal tract in the disposition of MEQ.     

Metabolism of a sulfur-containing heteroarotionoid antitumor agent, SHetA2, using liquid chromatography/tandem mass spectrometry

SHetA2 {[(4-nitrophenyl)amino][2,2,4,4-tetramethylthiochroman-6-yl)amino]methanethione], NSC 726189}, a sulfur-containing heteroarotinoid, selectively inhibits cancer cell growth and induces apoptosis without activation of nuclear retinoic acid receptors (RARs). The objective of this study was to investigate its in vitro metabolism in rat and human liver microsomes and in vivo metabolism in the mouse and rat using liquid chromatography-ultraviolet/multi-stage mass spectrometry (LC-UV/MS(n)) on an ion-trap mass spectrometer coupled with a photo-diode array (PDA) detector. In vitro, in the absence of glutathione (GSH), oxidation of the four aliphatic methyl groups of SHetA2 yielded one mono-, two di-, and one tri-hydroxylated SHetA2 metabolites, which were identified based on their UV and multi-stage mass spectra. In the presence of GSH, in addition to these primary oxidative metabolites, four GSH adducts of SHetA2 and a novel rare form thioether GSH adduct was detected and characterized. In vivo, the monohydroxylated SHetA2 metabolites were also detected in mouse and rat plasma and two GSH adducts were detected in rat liver following intravenous (i.v.) bolus administration of SHetA2 at 40 mg/kg.     

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.     

Determination of piceatannol in rat serum and liver microsomes: pharmacokinetics and phase I and II biotransformation

A method of analysis of piceatannol in biological fluids is necessary to study the kinetics of in vitro and in vivo metabolism and determine its concentration in foodstuffs. A novel and simple high-performance liquid chromatographic method was developed for simultaneous determination of piceatannol and products of its metabolism in rat serum and liver microsomes. Serum, or microsomes (0.1 mL), were precipitated with acetonitrile after addition of the internal standard, 4-methylumbelliferone. Separation was achieved on a phenomenex C(18) column (250 x 4.6 mm i.d., 5 microm) equipped with a phenomenex C(18) (4 x 3.0 mm i.d., 5 microm) guardcolumn with fluorescence excitation at 320 nm and emission at 420 nm. Separation was also possible with UV detection at 310 nm. The fluorescent calibration curves were linear ranging from 0.05 to 100 microg/mL. The mean extraction efficiency was >95%. Precision of the assay was <10% (coefficient of variation), and was within 10% at the limit of quantitation (0.05 ng/mL). Bias of the assay was lower than 7%. The limit of detection was 50 ng/mL for a 0.1 mL sample. The assay was applied successfully to the in vitro kinetic study of metabolism of piceatannol in rat liver microsomes and pharmacokinetics in rats. Three metabolites of piceatannol have been identified. .     

In vitro characterization of borneol metabolites by GC-MS upon incubation with rat liver microsomes

The metabolism of borneol is studied by the analysis of incubations of in vitro-prepared rat liver microsomes. A sensitive gas chromatography (GC)-mass spectrometry (MS) method is developed for the identification of borneol and its metabolites. Four novel metabolites, which have not previously been reported, are isolated and confirmed by comparison of the GC-MS method. The biotransformation pathway of borneol in rat liver microsomes is proposed based on the in vitro results.     

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.     

Characterization of metabolites of meisoindigo in male and female rat kidney microsomes by high-performance liquid chromatography coupled with positive electrospray ionization tandem mass spectrometry

Meisoindigo has been effectively applied for the treatment of chronic myelogenous leukemia (CML). Although the metabolic profile of meisoindigo has been studied in liver, information relevant to extrahepatic metabolism of meisoindigo is absent in kidney so far. In this study, the metabolism of meisoindigo in rat kidney microsomes was qualitatively and quantitatively investigated by liquid chromatography/tandem mass spectrometry (LC/MS/MS), in terms of metabolite identification, metabolic stability, metabolite formation and gender effect. The metabolic profiling was accomplished by integration of multiple reaction monitoring (MRM) with conventional full MS scan followed by MS/MS methodology. The major in vitro metabolites of meisoindigo in rat kidney microsomes were identified as stereoselective 3,3' double-bond reduced meisoindigo, whereas the minor metabolites were regioselective phenyl monohydroxylmeisoindigo. An LC/MS/MS method for quantification of meisoindigo in rat kidney microsomes was also developed and validated. The calculated in vitro half-life (t(1/2)) values of meisoindigo in male and female rat kidney microsomes were 107.8 +/- 17.0 min and 130.0 +/- 12.9 min, respectively. There were no statistically significant differences between different genders in the metabolic stability profiles of meisoindigo. The reductive metabolite-formation profiles of meisoindigo in male and female rat kidney microsomes were plotted semi-quantitatively as well. The information regarding in vitro renal metabolism of meisoindigo provided a better understanding of the role of the kidney in the disposition of meisoindigo.     

Metabolism studies of the anti-tumor agent maytansine and its analog ansamitocin P-3 using liquid chromatography/tandem mass spectrometry

Maytansine, a potent clinically evaluated plant-derived anti-tumor drug, and its microbial counterpart, ansamitocin P-3, showed a substantially higher cytoxicity than many other anti-tumor drugs. Owing to a shortage of material and lack of sufficiently sensitive analytical methods at the time, no metabolism studies were apparently carried out in conjunction with the initial preclinical and clinical studies on maytansine, but some products of decomposition during the period of storage of the formulated drug were reported. In the current study, the in vitro metabolism of maytansine and ansamitocin P-3 was studied after incubation with rat and human liver microsomes in the presence of NADPH, and with rat and human plasma and whole blood, using liquid chromatography/multi-stage mass spectrometry. Unchanged ansamitocin P-3 and 11 metabolites and unchanged maytansine and seven metabolites were profiled and the structures of some metabolites were tentatively assigned based on their multi-stage electrospray ion-trap mass fragmentation data and in some cases accurate mass measurement. The major pathway of ansamitocin P-3 metabolism in human liver microsomes appears to be demethylation at C-10. Oxidation and sequential oxidation/demethylation also occurred, although to a lesser extent. However, the major pathway of maytansine metabolism in human liver microsomes is N-demethylation of the methylamide of the ester moiety. Several minor pathways including O/N-demethylation, oxidation and hydrolysis of the ester bond were also observed. There were no differences in maytansine metabolism between rat and human liver microsomes; however, the rate of metabolism of ansamitocin P-3 was different in rat and human liver microsomes. About 20% of ansamitocin P-3 was converted to its metabolites in rat liver microsomes and about 70% in human liver microsomes under the same conditions. Additionally, 10-O-demethylated ansamitocin P-3 was also detected in the urine after i.v. bolus administration of ansamitocin P-3 to Sprague-Dawley male rats. No metabolites were detected following incubation of maytansine and ansamitocin P-3 with human and rat whole blood and plasma.     

Liquid chromatography-electrospray ionization ion trap mass spectrometry for analysis of in vivo and in vitro metabolites of scopolamine in rats

In vivo and in vitro metabolism of scopolamine is investigated using a highly specific and sensitive liquid chromatography-mass spectrometry (LC-MSn) method. Feces, urine, and plasma samples are collected individually after ingestion of 55 mg/kg scopolamine by healthy rats. Rat feces and urine samples are cleaned up by a liquid-liquid extraction and a solid-phase extraction procedure (C18 cartridges), respectively. Methanol is added to rat plasma samples to precipitate plasma proteins. Scopolamine is incubated with homogenized liver and intestinal flora of rats in vitro, respectively. The metabolites in the incubating solution are extracted with ethyl acetate. Then these pretreated samples are injected into a reversed-phase C18 column with mobile phase of methanol-ammonium acetate (2 mM, adjusted to pH 3.5 with formic acid) (70:30, v/v) and detected by an on-line MSn system. Identification and structural elucidation of the metabolites are performed by comparing their changes in molecular masses (DeltaM), retention-times and full scan MSn spectra with those of the parent drug. The results reveal that at least 8 metabolites (norscopine, scopine, tropic acid, aponorscopolamine, aposcopolamine, norscopolamine, hydroxyscopolamine, and hydroxyscopolamine N-oxide) and the parent drug exist in feces after administering 55 mg/kg scopolamine to healthy rats. Three new metabolites (tetrahydroxyscopolamine, trihydroxy-methoxyscopolamine, and dihydroxy-dimethoxyscopolamine) are identified in rat urine. Seven metabolites (norscopine, scopine, tropic acid, aponorscopolamine, aposcopolamine, norscopolamine, and hydroxyscopolamine) and the parent drug are detected in rat plasma. Only 1 hydrolyzed metabolite (scopine) is found in the rat intestinal flora incubation mixture, and 2 metabolites (aposcopolamine and norscopolamine) are identified in the homogenized liver incubation mixture.     

The Tannins from Sanguisorba officinalis L. (Rosaceae): A Systematic Study on the Metabolites of Rats Based on HPLC–LTQ–Orbitrap MS2 Analysis

Sanguisorba tannins are the major active ingredients in Sanguisorba ofJicinalis L. (Rosaceae), one of the most popular herbal medicines in China, is widely prescribed for hemostasis. In this study, three kinds of tannins extract from Sanguisorba officinalis L. (Rosaceae), and the metabolites in vivo and in vitro were detected and identified by high-pressure liquid chromatography, coupled with linear ion trap orbitrap tandem mass spectrometry (HPLC–LTQ–Orbitrap). For in vivo assessment, the rats were administered at a single dose of 150 mg/kg, after which 12 metabolites were found in urine, 6 metabolites were found in feces, and 8 metabolites were found in bile, while metabolites were barely found in plasma and tissues. For in vitro assessment, 100 μM Sanguisorba tannins were incubated with rat liver microsomes, liver cytosol, and feces, after which nine metabolites were found in intestinal microbiota and five metabolites were found in liver microsomes and liver cytosol. Moreover, the metabolic pathways of Sanguisorba tannins were proposed, which shed light on their mechanism.     

Biotransformation of Timosaponin BII into Seven Characteristic Metabolites by the Gut Microbiota

Timosaponin BII is one of the most abundant Anemarrhena saponins and is in a phase II clinical trial for the treatment of dementia. However, the pharmacological activity of timosaponin BII does not match its low bioavailability. In this study, we aimed to determine the effects of gut microbiota on timosaponin BII metabolism. We found that intestinal flora had a strong metabolic effect on timosaponin BII by HPLC-MS/MS. At the same time, seven potential metabolites (M1–M7) produced by rat intestinal flora were identified using HPLC/MS-Q-TOF. Among them, three structures identified are reported in gut microbiota for the first time. A comparison of rat liver homogenate and a rat liver microsome incubation system revealed that the metabolic behavior of timosaponin BII was unique to the gut microbiota system. Finally, a quantitative method for the three representative metabolites was established by HPLC-MS/MS, and the temporal relationship among the metabolites was initially clarified. In summary, it is suggested that the metabolic characteristics of gut microbiota may be an important indicator of the pharmacological activity of timosaponin BII, which can be applied to guide its application and clinical use in the future.     

Metabolites study on 5‐O‐methylvisammioside in vivo and in vitro by ultra high performance liquid chromatography coupled to quadrupole time‐of‐flight mass spectrometry

5‐O‐Methylvisammioside is one of major chromones of Radix Saposhnikoviae possessing definite pharmacological activities, but there are few reports with respect to the metabolism of 5‐O‐methylvisammioside. In this work, metabolites in vivo were explored in male Sprague‐Dawley rats and in vitro investigated on rat intestinal bacteria incubation model and were identified by using ultra high performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry. An online data acquisition method based on a multiple mass defect filter and dynamic background subtraction was developed to trace all probable metabolites. As a result, 26 metabolites in vivo (including 18, 15, 10, and 10 in rat urine, faece, bile, and blood) and 7 metabolites in vitro were characterized, respectively. Additionally, the main metabolic pathways in vivo and in vitro, including deglycosylation, deglycosylation + demethylation, deglycosylation + oxidation, N‐acetylation, and sulfate conjugation, were summarized by calculating the relative content of each metabolite. The obtained results significantly enriched our knowledge about 5‐O‐methylvisammioside metabolism and will lead to a better understanding of its safety and efficacy.     

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.     

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.     

Role of Human Liver Microsomes in In Vitro Metabolism of Drugs—A Review

Drug metabolism studies are essential and necessary during the evaluation of drugs. This review discusses the in vitro human liver models to estimate the drug metabolic fates in vivo. Different approaches are provided and emphasis is placed on the potential of human liver microsomes for drug metabolism and inhibition studies. The methodology for these studies using human liver microsomes, applications of human liver microsomes, and the drugs studied by human liver microsomes are listed. Human liver microsomes represent a critical experimental model for the evaluation of drug metabolites with a high probability of clinical success.     

Metabolism and excretion of 5-ethyl-2-{5-[4-(2-hydroxyethyl)piperazine-1-sulfonyl]-2-propoxyphenyl}-7-propyl-3,5-dihydropyrrolo[3,2-d]-pyrimidin-4-one (SK3530) in rats

The in vitro and in vivo metabolism of a novel PDE 5 inhibitor, SK3530, was investigated in rats. Bile, plasma, feces, urine and liver samples were collected and analyzed using a high-performance liquid chromatography (HPLC) system equipped with ultraviolet (UV), mass spectrometric and radioactivity detectors. After a single oral administration, the mean radiocarbon recovery was 92.32+/-6.26%, with 91.25+/-6.25 and 1.07+/-0.21% in the feces and urine, respectively. The biliary excretion of radioactivity for the first 24 h period was approximately 38.82%, suggesting that SK3530 is cleared by hepatobiliary excretion. In vitro incubation of SK3530 with rat and human liver microsomes resulted in the formation of twelve and ten metabolites, respectively. SK3530 was extensively metabolized to twenty different metabolites, including three glucuronide and three sulfate conjugates in rats. The structures of these metabolites were elucidated based on MSn spectral analyses. Six major metabolic pathways were identified in the rat: N-dealkylation and oxidation of the hydroxyethyl moiety; N,N-deethylation and hydroxylation of the piperazine ring; hydroxylation of the propyl group and sulfate conjugation. An additional metabolite due to aromatic hydroxylation was also identified in hepatic microsomes.     

Preparative Chromatographic Isolation of Caulophine from Radix caulophylli and its Metabolism In Vivo and In Vitro

Caulophine, as a novel alkaloid, was found in Radix caulophylli and has anti-myocardial ischemia activities. To conduct product development research on Radix caulophylli and caulophine, a preparative high-performance liquid chromatography method for the preparation of caulophine was investigated. Preparative HPLC was optimized to allow fraction I to be separated first and the caulophine was isolated following the second round of preparative HPLC. The purity of caulophine was >98%, which was assessed using analytical HPLC. Then, a highly sensitive and specific liquid chromatography-mass spectrometric method for the excretion and metabolism of caulophine in vivo was investigated. The metabolism to caulophine glucuronide conjugate was studied in rat liver microsomes or dog liver microsomes in vitro systems. Biosamples were pretreated by solid phase extraction (SPE) and analyzed by LC-MS with electrospray ionization (ESI) interface. Method validation results showed within-day and between-day precision was 1.14–6.21% and 5.45–9.78%, respectively, and average recoveries for all matrices were greater than 80%. The limits of detection for this method were determined to be up to 1 ng · mL^?1 of caulophine. Excretion of caulophine in rat results indicated that the total cumulative excretion of caulophine was high, with greater than 50% of the treatment dose being excreted. Two metabolites including glucuronide conjugate and N-oxide of caulophine were found in rat urine and feces by LC-MS. Moreover, the same caulophine glucuronide conjugate was observed in rat liver microsomes system.     

Identification of carbadox metabolites formed by liver microsomes from rats, pigs and chickens using high-performance liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry

Carbadox (methyl-3-(2-quinoxalinylmethylene)-carbazate-N(1),N(4)-dioxide) is a chemotherapeutic growth promoter added to feed for starter pigs. In this work, the metabolism of carbadox in rat, pig and chicken liver microsomes has been studied firstly. The incubation mixtures were then processed and analyzed for metabolites with a sensitive and reliable method based on high-performance liquid chromatography combined with hybrid ion trap/time-of-flight mass spectrometry (LC/MS-IT-TOF). With the help of chromatographic behavior and accurate mass measurements, it is possible to rapidly and reliably characterize the metabolites of carbadox. The structural elucidations of these metabolites were performed by comparing the changes in the accurate molecular masses and fragment ions generated from precursor ions with those of parent drug. The present results showed that the metabolism of carbadox in liver microsomes had qualitative species-difference. A total of seven metabolites were identified in rat liver microsomes. Five metabolites (Cb1-Cb3, Cb5, Cb7) were observed in pig and chicken liver microsomes. In addition, metabolite Cb6 was also detected in chicken liver microsomes. The peak areas of the metabolites in the three species are different. For the formations of Cb1, Cb2, Cb5 and Cb6, the rank order was rat>chicken>pig; Cb3; pig~chicken>rat. Cb1, Cb2 and Cb3 have been previously reported, whereas the other four metabolites were novel. The N→O group reduction and hydroxylation followed by N→O group reduction were the main metabolic pathways for carbadox in the three species.