Identification of chemical constituents and rat metabolites of Kangxianling granule by HPLC‐Q‐TOF‐MS/MS

A high‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass tandem mass spectrometry method was established to characterize the chemical constituents of Kangxianling granule (KXL), a traditional Chinese medicine formula, and the metabolic profile in rat urine and plasma after oral administration of KXL. A total of 27 compounds in KXL extract and 13 prototype compounds with 12 metabolites in rat urine and plasma were identified. Among the 27 detected compounds, 15 were identified by comparing the retention time and MS data with that of reference compounds and the other 12 compounds were tentatively assigned based on the MS data and reference literature. The main prototype components absorbed in rat were amygdalin, salvianolic acid B, tanshinones and anthraquinones. Hydroxylation, glucuronidation and sulfation were the principal metabolic pathways in rat. The results revealed that the 25 compounds identified in rat urine and plasma were the potential active ingredients of KXL, which provides helpful chemical information for further study of the pharmacology mechanism of KXL. Copyright © 2015 John Wiley & Sons, Ltd.     

Characterization of chemical constituents and rats metabolites of Shuanghua Baihe tablets by HPLC‐Q‐TOF‐MS/MS

A high‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry method was established to detect as many constituents in rat biological fluids as possible after oral administration of Shuanghua Baihe tablets (SBT). An Agilent Poroshell 120 EC‐C₁₈ column was adopted to separate the samples, and mass spectra were acquired in positive and negative modes. First, the fingerprints of SBT were established, resulting in 32 components being detected within 40 min. Among these compounds, 12 were tentatively identified by comparing the retention times and mass spectral data with those of reference standards and the reference literature; the other 20 components were tentatively assigned solely based on the MS data. Furthermore, metabolites in rat plasma and urine after oral administration of SBT were also analyzed. A total of 19 compounds were identified, including 13 prototypes and six metabolites through metabolic pathways of demethylation and glucuronide conjugation. Glucuronidated alkaloids were the main constituents in the plasma, and were then excreted from urine. This is the first systematic study on the metabolic profiling of SBT. Copyright © 2014 John Wiley & Sons, Ltd.     

Investigation of the in vivo metabolism of harpagoside and distribution of its metabolites in rats by HPLC‐IT‐TOF‐MSn

Harpagoside, an iridoid glycoside, is the major bioactive constituent of the traditional Chinese medicine Scrophulariae Radix. High‐performance liquid chromatography with a diode array detector combined with electrospray ionization ion trap time‐of‐flight multistage mass spectrometry (HPLC‐ESI‐IT‐TOF‐MSⁿ) was used to profile and identify the metabolites of harpagoside in rats in vivo and to study the distribution of these metabolites in rats for the first time. A total of 45 metabolites were identified, 37 of which were postulated to be new compounds. The number of detected metabolites in the heart, liver, spleen, lung, kidney, stomach and small intestine was 2, 9, 6, 16, 4, 16 and 6, respectively, which indicated that the target organs of harpagoside should be spleen, lung and stomach. The main types of metabolic reactions of harpagoside in rats are hydrolysis, reduction, sulfuric acid addition, hydroxylation, methoxylation, sulfate substitution, methylation, glucose conjugation and amino acid conjugation. Furthermore, 23 metabolites were determined to have bioactivities based on the literature and ‘PharmMapper’ analysis. These findings are useful for better comprehension of the effective forms, target organs and pharmacological effects of harpagoside. Moreover, these findings provide a reference for studying the metabolism and distribution of iridoid compounds.     

HPLC‐Q‐TOF‐MS/MS for analysis of major chemical constituents of Yinchen–Zhizi herb pair extract

The Yinchen–Zhizi herb pair (YZHP) consists of Herba Artemisiae Scopariae (Yinchen in Chinese) and Fructus Gardeniae (Zhizi in Chinese), and is mainly used to treat icteric hepatitis, itching skin and eczema. However, the bioactive constituents responsible for the pharmacological effects of YZHP are still unclear to date. In this work, a rapid and sensitive method was established to comprehensively study the constituents in YZHP extract by HPLC‐Q‐TOF MS/MS. The analysis was performed on an HPLC system equipped with an Agilent poroshell 120 EC‐C₁₈ column (100 × 2.1 mm, 2.7 mm) working in a gradient elution program coupled to quadrupole‐time‐of‐flight mass spectrometry operating in the negative ion mode. As a result, a total of 46 compounds including 17 from Herba Artemisiae Scopariae and 36 from Fructus Gardeniae were detected and tentatively identified in YZHP extract by comparing the retention time and mass spectrometry and retrieving the reference literature. More importantly, a series of constituents, such as many iridoid glycosides, were reported for the first time in this formula. The HPLC‐Q‐TOF MS/MS method was developed and utilized successfully to identify the major constituents in YZHP extract and would be helpful for further metabolism and pharmacology research on YZHP. Copyright © 2013 John Wiley & Sons, Ltd.     

Chemical identification and quality evaluation of Lycopus lucidus Turcz by UHPLC‐Q‐TOF‐MS and HPLC‐MS/MS and hierarchical clustering analysis

Lycopus lucidus Turcz has been used as a traditional phytomedicine for menstrual disorder, amenorrhea, menstrual cramps, inflammation and cardiovascular diseases. However, there is not enough information about identification and quantification for the chemical constituents of L. lucidus Turcz. In this work, a simple, rapid and sensitive UHPLC‐Q‐TOF‐MS method was developed for characterization and identification of the phytochemical compositions in L. lucidus Turcz in negative ion mode. A total of 37 compounds, including 15 phenolic acids, 12 flavonoids, three triterpenoids and seven organic acids were tentatively characterized and identified by means of the retention time, accurate mass and characteristic fragment ions. Thirteen compounds were reported for the first time in L. lucidus Turcz. Among of them, 11 compounds were further quantified by multiple reactions monitoring. The results showed good performance with respect to linearity (r > 0.9959), repeatability (RSD < 2.6%), intra‐ and inter‐day precision (RSD < 3.2%), recovery (93.1–104.9%), and lower limit of quantification (5–50 ng/mL). Subsequently, the results were analyzed and classified by hierarchical cluster analysis. The research could be applied for identification and quality evaluation for L. lucidus Turcz.     

Metabolic profiling study on potential toxicity and immunotoxicity‐biomarker discovery in rats treated with cyclophosphamide using HPLC‐ESI‐IT‐TOF‐MS

Despite the recent advances in understanding toxicity mechanism of cyclophosphamide (CTX), the development of biomarkers is still essential. CTX‐induced immunotoxicity in rats by a metabonomics approach was investigated using high‐performance liquid chromatography coupled with ion trap time‐of‐flight mass spectrometry (HPLC‐ESI‐IT‐TOF‐MS). The rats were orally administered CTX (30 mg/kg/day) for five consecutive days, and on the fifth day samples of urine, thymus and spleen were collected and analyzed. A significant difference in metabolic profiling was observed between the CTX‐treated group and the control group by partial least squares‐discriminant analysis (PLS‐DA), which indicated that metabolic disturbances of immunotoxicity in CTX‐treated rats had occurred. One potential biomarker in spleen, three in urine and three in thymus were identified. It is suggested that the CTX‐toxicity mechanism may involve the modulation of tryptophan metabolism, phospholipid metabolism and energy metabolism. This research can help to elucidate the CTX‐influenced pathways at a low dose and can further help to indicate the patients' pathological status at earlier stages of toxicological progression after drug administration. Copyright © 2014 John Wiley & Sons, Ltd.     

Fragmentation patterns study of iridoid glycosides in Fructus Gardeniae by HPLC‐Q/TOF‐MS/MS

Iridoid glycosides (IGs), the major constituents in Fructus Gardeniae, have demonstrated various pharmacological activities, but there is no systematic chemical profile of IGs in Fructus Gardeniae in the published literature until now. Therefore, it is imperative that a rapid and sensitive high‐performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry (HPLC‐Q/TOF‐MS/MS) method is established for comprehensive characterization of IGs in Fructus Gardeniae. Firstly, the fragmentation patterns of six known IGs were investigated and proposed and further concluded the diagnostic fragment ions and characteristic fragmentation pathways. Then, based on the summarized fragmentation patterns and the known compounds in the literatures, the other IGs in Fructus Gardeniae were identified successively. As a result, a total of 20 IGs were identified, of which three pairs of epimers were structurally characterized and differentiated. More importantly, one compound, the isoshanzhiside methyl ester, was tentatively identified as a new compound. The results of this study demonstrate the superiority of HPLC‐MS with a high‐resolution mass spectrometer for the rapid and sensitive structural elucidation of the multiple groups of constituents in Fructus Gardeniae. Copyright © 2014 John Wiley & Sons, Ltd.     

Metabolic profile of phillyrin in rats obtained by UPLC‐Q‐TOF‐MS

Forsythia suspensa Vahl (Oleaceae) is an important original plant in traditional Chinese medicine. The air‐dried fruits of Forsythia suspensa have long been used to relieve respiratory symptoms. Phillyrin is one of the main chemical constituent of Forsythia suspensa. A clear understanding of the metabolism of phillyrin is very important in rational clinical use and pharmacological research. In this study, the metabolism of phillyrin in rat was investigated for the first time using an ultra‐high‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry (UPLC‐Q‐TOF‐MS) method. Bile, urine and feces were collected from rats after single‐dose (10 mg/kg) orally administered phillyrin. Liquid–liquid extraction and ultrasonic extraction were used to prepare samples. UPLC‐Q‐TOF‐MS analysis of the phillyrin samples showed that phillyrin was converted to a major metabolite, M26, which underwent deglucosidation, further dehydration and desaturation. A total of 34 metabolites were detected including 30 phase I and four phase II metabolites. The conjugation types and structure skeletons of the metabolites were preliminarily determined. Moreover, 28 new metabolites were reported for the first time. The main biotransformation route of phillyrin was identified as hydrolysis, oxidation and sulfation. These findings enhance our understanding of the metabolism and the real active structures of phillyrin. Copyright © 2015 John Wiley & Sons, Ltd.     

Analysis of the chemical components of Qixianqingming granules and their metabolites in rats by UPLC‐ESI‐Q‐TOF‐MS

Qixianqingming granules (QXQM) comprise a traditional Chinese medicine (TCM) formula that was developed based on the combination of TCM theory and clinical practice. This formula has been proven to effectively treat asthma. In this study, an analytical procedure using ultraperformance liquid chromatography, coupled with electrospray ionization quadrupole time‐of‐flight mass spectrometry, was established for the rapid separation and sensitive identification of the chemical components in QXQM and its metabolites in serum of rats. Seventy‐two compounds were systematically identified in QXQM, including flavonoids, terpenoids, anthraquinones, phenylethanoid glycosides, stilbenes, alkaloids, and organic acids. Thirteen prototype compounds and 29 metabolites were detected in the serum of rats. The results provided fundamental information for further studying the mechanisms and clinical application of QXQM.     

Screening free radical scavengers in Xiexin Tang by HPLC‐ABTS‐DAD‐Q‐TOF/MS

Xiexin Tang (XXT) is a traditional Chinese medicine (TCM) that has been used in herbal clinics for more than 1800 years. Many studies have shown that XXT has therapeutic effects on patients with arteriosclerosis owing to its antioxidant activity. However, there is little information about the relationship between the chemical composition of XXT and its antioxidant activity. In this study, the HPLC‐ABTS‐DAD‐Q‐TOF/MS method, which can simultaneously identify individual components and rapidly screen for antioxidant compounds, was used to screen and identify antioxidant components in XXT. The 15 compounds identified were gluco‐syringic acid, adenine, gallic acid, biflorin, cularine, 6‐C ‐arabinose‐8‐C ‐glucose‐chrysin, 6‐C ‐glucose‐8‐C ‐arabinose–chrysin, baicalin, rhein‐8‐O‐β ‐d ‐glucopyranoside, coptisine, epiberberine, jatrorrhizine, norwogonin, 5,7,2′‐trihydroxy‐6‐ methoxyflavone and baicalein. In addition, the data showed that the antioxidant activity of peaks 4, 6, and 11 was lower in XXT than in its constituent herbs, while the activity of peaks 1, 2, 3, 5, 7, 8, 10, 12, 13, 14 and 15 was higher in XXT. Compound 5 had the strongest antioxidant activity in XXT, while compound 1 showed the strongest antioxidant activity among its constituent herb. The differences between antioxidant activities of major components of XXT and those of its constituent herbs might be due to the interaction of crude drugs that changes the solubility of active components during the decoction process. The results show that the HPLC‐ABTS‐DAD‐Q‐TOF/MS method can successfully combine on‐line mass spectrometry with activity detection system. It is a useful tool for the rapid detection and identification of antioxidants, and for quantitative analysis of individual antioxidants in complex mixtures such as plant extracts. Furthermore, this method does not require extensive extract purification and fraction collection.     

Identification of the constituents and metabolites in rats after oral administration of Zi Shen Formula by UPLC‐Q‐TOF/MS combined pattern recognition analysis

An ultra‐high‐performance liquid chromatography mass spectrometry method was established to detect and identify the chemical constituents of Zi Shen Formula (ZSF) and its metabolites in serum, urine and feces, after oral administration to rats. A total of 68 compounds were characterized in ZSF extracts. In vivo, 38 prototype components and 32 metabolites of ZSF were tentatively identified in rat serum, urine and feces. Seven metabolic pathways including demethylation, hydroxylation, oxidation, sulfation, glucuronidation, methylation and de‐caffeoyl were proposed to be involved in the generation of these metabolites. It was found that glucuronidation, methylation and demethylation were the major metabolic processes of alkaloids, while demethylation, methylation, sulfation and de‐caffeoyl were the major metabolic pathways of phenylethanoid glycosides. The main metabolic pathways of steroidal saponins were oxidation and isotype reactions. These findings are significant for our understanding of the metabolism of ZSF. The proposed metabolic pathways of bioactive components might be crucial for further studies of the mechanisms of action and pharmacokinetic evaluations of ZSF.     

Studies on metabolism of total glucosides of paeony from Paeoniae Radix Alba in rats by UPLC‐Q‐TOF‐MS/MS

Total glucosides of paeony are the active constituents of Paeoniae Radix Alba. In this study, a novel strategy was proposed to find more metabolites and the differences between paeoniflorin, albiflorin and total glucosides of paeony (TGP). This strategy was characterized as follows: firstly, the animals were divided into three groups (paeoniflorin, albiflorin and TGP) to identify the source of TGP metabolites from paeoniflorin or albiflorin; secondly, a generic information‐dependent acquisition scan for the low‐level metabolites was triggered by the multiple mass defect filter and dynamic background subtraction; thirdly, the metabolites were identified with a combination of data‐processing methods including mass defect filtering, neutral loss filtering and product ion filtering; finally, a comparative study was used in the metabolism of paeoniflorin, albiflorin and TGP. Based on the strategy, 18 metabolites of TGP, 10 metabolites of paeoniflorin and 13 metabolites of albiflorin were identified respectively. The results indicated that the hydrolysis, conjugation reaction and oxidization were the major metabolic pathways, and the metabolic sites were the glycosidic linkage, the ester bond and the benzene ring. This study is first to explore the metabolism of TGP, and these findings enhance our understanding of the metabolism and the interactions of paeoniflrin and albiflorin in TGP. Copyright © 2015 John Wiley & Sons, Ltd.     

Method for rapidly discovering active components in Yupingfeng granules by UPLC‐ESI‐Q‐TOF‐MS

Yupingfeng granules (YPFG) were isolated from a traditional Chinese medicine (TCM) formulation composed of three herbs (Astragali Radix, Atractylodis Macrocephalae Rhizoma, and Saposhnikoviae Radix). This formulation is used in TCM to tonify qi, and it can help strengthen exterior and reduce sweating. Nevertheless, the active components of YPFG remain unclear. In this study, the chemical constituents of YPFG were systematically characterized by ultra‐performance liquid chromatography coupled with electrospray ionization/ quadrupole time‐of‐flight mass spectrometry (UPLC‐ESI‐Q‐TOF‐MS). Fifty‐eight compounds, namely, 20 flavonoids, 19 saponins, nine organic acids, four volatile coumarins, three lactones, one alkaloid, and two other components, were identified. In addition, the constituents of YPFG with the potential for in vivo bioactivities following oral administration were investigated in Sprague–Dawley rats. Thirteen compounds, namely, 11 flavonoid‐related and 2 saponin‐related components, were detected in rat plasma. After enriching flavonoids and saponins in YPFG by extraction, the extracts and YPFG were administrated to immunosuppressed rats, respectively. Plasma samples were analyzed by UPLC‐ESI‐Q‐TOF‐MS, and principal component analysis (PCA) confirmed that the extracts had similar effects to YPFG. This method could discover active ingredients in YPFG quickly and provide a scientific basis for quality control and mechanism research.     

Fragmentation pathways of deprotonated amide‐sulfonamide CXCR4 inhibitors investigated by ESI‐IT‐MSn,ESI‐Q‐TOF‐MS/MS and DFT calculations

Amide‐sulfonamides provide a potent anti‐inflammatory scaffold targeting the CXCR4 receptor. A series of novel amide‐sulfonamide derivatives were investigated for their gas‐phase fragmentation behaviors using electrospray ionization ion trap mass spectrometry and quadrupole time‐of‐flight mass spectrometry in negative ion mode. Upon collision‐induced dissociation (CID), deprotonated amide‐sulfonamides mainly underwent either an elimination of the amine to form the sulfonyl anion and amide anion or a benzoylamide derivative to provide sulfonamide anion bearing respective substituent groups. Based on the characteristic fragment ions and the deuterium–hydrogen exchange experiments, three possible fragmentation mechanisms corresponding to ion‐neutral complexes including [sulfonyl anion/amine] complex ( INC‐1 ), [sulfonamide anion/benzoylamide derivative] complex ( INC‐2 ) and [amide anion/sulfonamide] complex ( INC‐3 ), respectively, were proposed. These three ion‐neutral complexes might be produced by the cleavages of S–N and C–N bond from the amide‐sulfonamides, which generated the sulfonyl anion (Route 1), sulfonamide anion (Route 2) and the amide anion (Route 3). DFT calculations suggested that Route 1, which generated the sulfonyl anion (ion c ) is more favorable. In addition, the elimination of SO₂ through a three‐membered‐ring transition state followed by the formation of C–N was observed for all the amide‐sulfonamides.     

Qualitative and quantitative analysis of major constituents from Dazhu Hongjingtian capsule by UPLC/Q‐TOF‐MS/MS combined with UPLC/QQQ‐MS/MS

In this work, a sensitive and efficient method was established and validated for qualitative and quantitative analysis of major bioactive constituents in Dazhu Hongjingtian capsule by liquid chromatography tandem mass spectrometry. A total of 32 compounds were tentatively identified using ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry. Furthermore, 12 constituents, namely gallic acid, 3,4‐dihydroxybenzoic acid, salidroside, p‐ coumaric acid‐4‐O ‐β ‐d ‐glucopyranoside, bergeninum, 4‐hydroxybenzoic acid, 4‐hydroxyphenylacetic acid, syringate, 6′′‐O ‐galloylsalidroside, rhodiosin, rhodionin and kaempferol‐7‐O ‐α ‐l ‐rhamnoside, were simultaneously quantified by the developed ultra‐performance liquid chromatography coupled with a triple quadrupole mass spectrometry method in 9 min. All of them were analyzed on an Agilent ZorBax SB‐C₁₈ column (3.0 × 100 mm, 1.8 μm) with linear gradient elution of methanol–0.1% formic acid water. The proposed method was applied to analyze three batches of samples with acceptable linearity (R , 0.9979–0.9997), precision (RSD, 1.3–4.7%), repeatability (RSD, 1.7–4.9%), stability (RSD, 2.2–4.9%) and recovery (RSD, 0.6–4.4%) of the 12 compounds. As a result, the analytical method possessing high throughput and sensitivity is suitable for the quality control of Dazhu Hongjingtian capsule.     

Identification of the absorbed components and metabolites of modified Huo Luo Xiao Ling Dan in rat plasma by UHPLC‐Q‐TOF/MS/MS

To reveal the material basis of Huo Luo Xiao Ling Dan (HLXLD), a sensitive and selective ultra‐high performance liquid chromatography coupled with quadrupole‐time‐of‐flight mass spectrometry (UHPLC‐Q‐TOF/MS) method was developed to identify the absorbed components and metabolites in rat plasma after oral administration of HLXLD. The plasma samples were pretreated by liquid–liquid extraction and separated on a Shim‐pack XR‐ODS C₁₈ column (75 × 3.0 mm, 2.2 μm) using a gradient elution program. With the optimized conditions and single sample injection of each positive or negative ion mode, a total of 109 compounds, including 78 prototype compounds and 31 metabolites, were identified or tentatively characterized. The fragmentation patterns of representative compounds were illustrated as well. The results indicated that aromatization and hydration were the main metabolic pathways of lactones and tanshinone‐related metabolites; demethylation and oxidation were the major metabolic pathways of alkaloid‐related compounds; methylation and sulfation were the main metabolic pathways of phenolic acid‐related metabolites. It is concluded the developed UHPLC‐Q‐TOF/MS method with high sensitivity and resolution is suitable for identifying and characterizing the absorbed components and metabolites of HLXLD, and the results will provide essential data for further studying the relationship between the chemical components and pharmacological activity of HLXLD.     

Identification of astilbin metabolites produced by human intestinal bacteria using UPLC‐Q‐TOF/MS

Astilbin, mainly isolated from a commonly used herbal medicine, Smilax glabra Roxb (SGR), exhibits a variety of pharmacological activities and biological effects. It is metabolized by intestinal bacteria after oral administration which leads to the variation of ethnopharmacological profile of this traditional medicine. However, little is known on the interactions of this active compound with intestinal bacteria, which would be very helpful in unravelling how SGR works. In this study, different pure bacteria from human feces were isolated and were used to investigate their conversion capability of astilbin. Ultra‐performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry (UPLC‐Q‐TOF/MS) technique combined with Metabolynx^TM software was used to analyze astilbin and its metabolites. The parent compound and two metabolites (quercetin and eriodictyol) were detected in the isolated bacterial samples compared with blank samples. Quercetin was present in Enterococcus sp. 8B, 8–2 and 9–2 samples. Eriodictyol was only identified in Enterococcus sp. 8B sample. The metabolic routes and metabolites of astilbin produced by the different intestinal bacteria are reported for the first time. This will be useful for the investigation of the pharmacokinetic study of astilbin in vivo and the role of different intestinal bacteria in the metabolism of natural compounds. Copyright © 2014 John Wiley & Sons, Ltd.     

Identification of isoquercitrin metabolites produced by human intestinal bacteria using UPLC‐Q‐TOF/MS

In this paper, ultraperformance liquid chromatography/quadrupole time‐of‐flight mass spectrometry (UPLC‐Q‐TOF/MS) and the MetaboLynx? software combined with mass defect filtering were applied to identity the metabolites of isoquercitrin using an intestinal mixture of bacteria and 96 isolated strains from human feces. The human incubated samples collected for 72 h in the anaerobic incubator and extracted with ethyl acetate were analyzed by UPLC‐Q‐TOF/MS within 10 min. The parent compound and five metabolites were identified by eight isolated strains, including Bacillus sp. 17, Veillonella sp. 23 and 32 and Bacteroides sp. 40, 41, 56, 75 and 88 in vitro. The results indicate that quercetin, acetylated isoquercitrin, dehydroxylated isoquercitrin, hydroxylated quercetin and hydroxymethylated quercetin are the major metabolites of isoquercitrin. Furthermore, a possible metabolic pathway for the biotransformation of isoquercitrin was established in intestinal flora. This study will be helpful for understanding the metabolic route of isoquercitrin and the role of different intestinal bacteria in the metabolism of natural compounds. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification of ilaprazole metabolites in human urine by HPLC‐ESI‐MS/MS and HPLC‐NMR experiments

Ilaprazole is a new proton pump inhibitor designed for the treatment of gastric ulcers, and limited data is available on the metabolism of the drug. In this article, the structural elucidation of urinary metabolites of ilaprazole in human was described by HPLC‐ESI‐MS/MS and stopped‐flow HPLC‐NMR experiments. Urinary samples were precipitated by sodium carbonate solution, and then extracted by liquid–liquid extraction after adding ammonium acetate buffer solution. The enriched sample was separated using a C₁₈ reversed‐phase column with the mobile phase composed of acetonitrile and 0.05 mol/L ammonium acetate buffer solution in a gradient solution, and then directly coupled to ESI‐MS/MS detection in an on‐line mode or ¹H‐NMR (500 MHz) spectroscopic detection in a stopped‐flow mode. As a result, four sulfide metabolites, ilaprazole sulfide (M1), 12‐hydroxy‐ilaprazole sulfide (M2), 11,12‐dihydroxy‐ilaprazole sulfide (M3) and ilaprazole sulfide A (M4), were identified by comparing their MS/MS and NMR data with those of the parent drug and available standard compounds. The main biotransformation reactions of ilaprazole were reduction and the aromatic hydroxylation of the parent drug and its relative metabolites. The result testified that HPLC‐ESI‐MS/MS and HPLC‐NMR could be widely applied in detection and identification of novel metabolites. Copyright © 2010 John Wiley & Sons, Ltd.