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.     

Identification of metabolites of isopropyl 3‐(3,4‐dihydroxyphenyl)‐2‐hydroxypropanoate in rats by high‐performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

Isopropyl 3‐(3,4‐dihydroxyphenyl)‐2‐hydroxypropanoate (IDHP) is an investigational new drug having the capacity for treating ailments in the cardiovascular and cerebrovascular system. In this work, a rapid and sensitive method using high‐performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (HPLC‐ESI‐Q‐TOF‐MS) was developed to reveal the metabolic profile of IDHP in rats after oral administration. The method involved pretreatment of the samples by formic acid–methanol solution (v/v, 5:95), chromatographic separation by an Agilent Eclipse XDB‐C₁₈ column (150 × 4.6 mm i.dx., 5 μm) and online identification of the metabolites by Q‐TOF‐MS equipped with electrospray ionizer. A total of 16 metabolites from IDHP, including four phase I metabolites and 12 phase II metabolites, were detected and tentatively identified from rat plasma, urine and feces. Among these metabolites, Danshensu (DSS), a hydrolysis product of IDHP, could be further transformed to 11 metabolites. These results indicated that DSS was the main metabolite of IDHP in rats and the major metabolic pathways of IDHP in vivo were hydrolysis, O‐methylation, sulfation, glucuronidation and reduction. The results also demonstrated that renal route was the main pathway of IDHP clearance in rat. The present study provided valuable information for better understanding the efficacy and safety of IDHP. Copyright © 2015 John Wiley & Sons, Ltd.     

Chemical UPLC‐ESI‐MS/MS profiling of aconitum alkaloids and their metabolites in rat plasma and urine after oral administration of Aconitum carmichaelii Debx. Root extract

In this paper, an ultra high performance liquid chromatography tandem mass spectrometric (UPLC‐ESI‐MS/MS) method in positive ion mode was established to systematically identify and to compare the major aconitum alkaloids and their metabolites in rat plasma and urine after oral administration of Fuzi extract. A total twenty‐nine components including twenty‐five C19‐diterpenoid alkaloids and four C20‐diterpenoid alkaloids were identified in Fuzi extract. Thirteen of the parent components and five metabolites were detected in rat plasma and sixteen parent compounds and six metabolites in urine. These parent components found in rat plasma and urine were mainly C19‐diterpenoid alkaloids. All of the metabolites in vivo were demethylated metabolites (phase I metabolites), which suggested that demethylation was the major metabolic pathway of aconitum alkaloids in vivo. A comparison of the parent components in rat plasma and urine revealed that 3‐deoxyacontine was found in plasma but not in urine, while kalacolidine, senbusine and 16‐β‐hydroxycardiopetaline existed in urine but not in plasma, which indicated that most alkaloids components were disposed and excreted in prototype form. This research provides some important information for further metabolic investigations of Fuzi in vivo.     

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.     

Use of liquid chromatography hybrid triple‐quadrupole mass spectrometry for the detection of emodin metabolites in rat bile and urine

Emodin is the representative form of rhubarb, which is widely used in traditional Chinese medicine for the treatment of purgative, anti‐inflammatory, antioxidative and antiviral, etc. Previous reports demonstrated that emodin glucuronide was the major metabolite in plasma. Owing to the extensive conjugation reactions of polyphenols, the aim of this study was to identify the metabolites of emodin in rat bile and urine. Neutral loss and precursor ion scan methods of triple‐quadrupole mass spectrometer revealed 13 conjugated metabolites in rat bile and 22 metabolites in rat urine, which included four phase I and 18 phase II metabolites. The major metabolites in rat biosamples were emodin glucuronoconjugates. Moreover, rhein monoglucuronide, chrysophanol monoglucuronide and rhein sulfate were proposed for the first time after oral administration of emodin. Overall, liquid chromatography hybrid triple‐quadrupole mass spectrometry analysis leads to the discovery of several novel emodin metabolites in rat bile and urine and underscores that conjugated with glucuronic acid is the main metabolic pathway.     

Studies on excretion kinetics of ten constituents in rat urine after oral administration of Shensong Yangxin Capsule by UPLC‐MS/MS

A rapid and sensitive ultra‐high performance liquid chromatography–mass spectrometry (UPLC‐MS/MS) method was developed and validated for the quantification of 10 major active constituents in rat urine after oral administration of Shensong Yangxin Capsule (SSYX) using diazepam as an internal standard (IS). The urine samples were pretreated and extracted by solid‐phase extraction prior to UPLC. Chromatographic separation was achieved on a Waters C₁₈ (2.1 × 50 mm, 1.7 µm) column using a gradient elution program with 0.1% formic acid aqueous solution and acetonitrile at a flow rate of 0.4 mL/min. Detection and quantitation were accomplished by a hybrid quadrupole mass spectrometer using electrospray ionization source and multiple reaction monitoring in the positive ionization mode. The mass transition ion‐pairs (m/z) for quantitation were all optimized and the total run time was 4.50 min. The specificity, linearity, accuracy, precision, recovery, matrix effect and stabilities were all validated for the analytes in urine samples. The validation results indicated that this method was simple, rapid, specific and reliable. The proposed method was successfully applied to investigate the urinary excretion kinetics of 10 compounds in rat after oral administration of SSYX. Copyright © 2013 John Wiley & Sons, Ltd.     

Qualitative screening of absorbed indoloquinazoline alkaloids and their metabolites in rat plasma after the oral administration of Wu‐Zhu‐Yu decoction by high‐resolution mass spectrometry with multiple data mining algorithms

A rapid and sensitive liquid chromatography with high‐resolution mass spectrometry method with multiple data processing algorithms was developed and applied for the metabolite profiling of evodiamine and its analogous alkaloids in rat plasma after the administration of Wu‐Zhu‐Yu decoction. All samples were purified using hydrophilic‐lipophilic balanced solid‐phase extraction cartridges and analyzed by a Sciex TripleTOF 5600⁺ mass spectrometer with a 35 min liquid chromatography gradient elution. High‐resolution full‐scan mass spectrometry and information‐dependent acquisition tandem mass spectrometry data were analyzed using multiple data processing approaches. The results indicated that the detected eight prototype alkaloids could be metabolized to 58 metabolites through both phase I and phase II reactions. Oxidation was demonstrated to be the principle metabolic pathway of the parent compounds. The study contributes to the understanding of the absorption and metabolism of the alkaloids in Wu‐Zhu‐Yu decoction and provides a detailed analysis of scientific data.     

Pharmacokinetics of 2,3,5,4′‐tetrahydroxystilbene‐2‐O‐β‐D‐glucoside in rat using ultra‐performance LC‐quadrupole TOF‐MS

2,3,5,4′‐Tetrahydroxystilbene‐2‐O‐β‐D‐glucoside (THSG) from Polygoni multiflori has been demonstrated to possess a variety of pharmacological activities, including antioxidant, anti‐inflammatory and hepatoprotective activities. Ultra‐performance LC‐quadrupole TOF‐MS with MS Elevated Energy data collection technique and rapid resolution LC with diode array detection and ESI multistage MSⁿ methods were developed for the pharmacokinetics, tissue distribution, metabolism, and excretion studies of THSG in rats following a single intravenous or oral dose. The three metabolites were identified by rapid resolution LC‐MSⁿ. The concentrations of the THSG in rat plasma, bile, urine, feces, or tissue samples were determined by ultra‐performance LC‐MS. The results showed that THSG was rapidly distributed and eliminated from rat plasma. After the intravenous administration, THSG was mainly distributing in the liver, heart, and lung. For the rat, the major distribution tissues after oral administration were heart, kidney, liver, and lung. There was no long‐term storage of THSG in rat tissues. Total recoveries of THSG within 24 h were low (0.1% in bile, 0.007% in urine, and 0.063% in feces) and THSG was excreted mainly in the forms of metabolites, which may resulted from biotransformation in the liver.     

Characterization of metabolites of sweroside in rat urine using ultra‐high‐performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry and NMR spectroscopy

Sweroside, a major active iridoid in Swertia pseudochinensis Hara, is recognized as an effective agent in the treatment of liver injury. Based on previous reports, the relatively short half‐life (64 min) and poor bioavailability (approximately 0.31%) in rats suggested that not only sweroside itself but also its metabolites could be responsible for the observed hepato‐protective effect. However, few studies have been carried out on the metabolism of sweroside. Therefore, the present study aimed at identifying the metabolites of sweroside in rat urine after a single oral dose (100 mg/kg). With ultra‐high‐performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (UHPLC/Q‐TOF‐MS), the metabolic profile revealed 11 metabolites in rat urine, including phase I, phase II and aglycone‐related products. The chemical structures of metabolites were proposed based on accurate mass measurements of protonated or deprotonated molecules and their fragmentation patterns. Our findings showed that the aglycone of sweroside (M05) and its glucuronide conjugate (M06) were principal circulating metabolites in rats. While several other metabolic transformations, occurring via reduction, N‐heterocyclization and N‐acetylation after deglycosylation, were also observed. Two metabolites (M05 and M06) were isolated from the rat urine for structural elucidation and identifcation of reaction sites. Both M05 and M06 were characterized by ¹H, ¹³C and two‐dimensional nuclear magnetic resonance (NMR) spectroscopy. UHPLC/Q‐TOF‐MS analysis has provided an important analytical platform to gather metabolic profile of sweroside. Copyright © 2014 John Wiley & Sons, Ltd.     

Study on the pharmacokinetics and metabolism of costunolide and dehydrocostus lactone in rats by HPLC‐UV and UPLC‐Q‐TOF/MS

A method based on high‐performance liquid chromatography coupled with ultraviolet detection was developed for studying the pharmacokinetics of costunolide (Cos) and dehydrocostus lactone (Dehy) in rats after intravenous (i.v.) administration. Following i.v. administration, the maximum plasma concentrations of Cos and Dehy were observed to be 12.29 ± 1.47 and 5.79 ± 0.13 µg/mL, respectively. The bioavailability of Cos was larger than that of Dehy; however, the clearance and the volume of distribution of Dehy were much larger than those of Cos. An ultraperformance liquid chromatography/quadrupole time‐of‐flight mass spectrometry system with automated MS^E (E represents collision energy) data analysis software (MetaboLynx^TM) was used to analyze and identify the metabolites of Cos and Dehy in vivo. Four metabolites of Cos and six metabolites of Dehy were discovered from the plasma, urine and feces of rats. The main metabolic pathway of Cos was phase II biotransformation, but the main metabolic pathways of Dehy was phase ? biotransformation. Two sequential desaturations and N‐acetylcysteine conjugation were the common metabolic pathways of Cos and Dehy in rats. This information may be useful for the further development of the two drug candidates. Copyright © 2014 John Wiley & Sons, Ltd.     

Identification of metabolites in human and rat urine after oral administration of Xiao‐Qing‐Long‐Tang granule using ultra high performance liquid chromatography combined with quadrupole time‐of‐flight mass spectrometry

Xiao‐Qing‐Long‐Tang is a traditional Chinese formula used for the treatment of cold syndrome, bronchitis, and nasal allergies for thousands of years. However, the in vivo integrated metabolism of its multiple components and the active chemical constituents of Xiao‐Qing‐Long‐Tang remain unknown. In this study, a method using ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry was established for the detection and identification of the metabolites in human and rat urine after oral administration of Xiao‐Qing‐Long‐Tang. A total of 19 compounds were detected or tentatively identified in human urine samples, including eight prototypes and 11 metabolites. Also, a total of 50 compounds were detected or tentatively identified in rat urine samples, including 15 prototypes and 35 metabolites detected with either a highly sensitive extracted ion chromatogram method or the MS^E determination using Mass Fragment software. Our results indicated that phase Ⅱ reactions (e.g. glucuronidation and sulfation) were the main metabolic pathways of flavones, while phase I reactions (e.g. demethylation and hydroxylation) were the major metabolic reaction for alkaloids, lignans, and ginger essential oil. This investigation provided important structural information on the metabolism of Xiao‐Qing‐Long‐Tang and provided evidence to obtain a more comprehensive metabolic profile.     

Screening and identification of multiple constituents and their metabolites of Zhi‐zi‐chi decoction in rat urine and bile by ultra‐high‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry

Zhi‐zi‐chi decoction (ZZCD) is a classical formula widely used in Chinese clinical application. In the present study, a novel and efficient strategy has been developed for screening and identification of multiple constituents and their metabolites of ZZCD using ultra‐high‐performance liquid chromatography combined with triple time‐of‐flight mass spectrometry. The novel approach of an online data acquisition method dependent on multiple mass defect filter and dynamic background subtraction is combined with multiple data processing techniques. First, a total of 109 potential bioactive compounds were detected in ZZCD. Based on the same instrumental conditions, 100 compounds were found in rat biofluids after oral administration of ZZCD, including 61 original compounds of ZZCD as well as 39 metabolites. Conjugations with sulfate, glucuronate and amino acids were found as the predominant metabolic reaction of ZZCD. As more xenobiotics were detected in urine than those in bile were, it demonstrated that multiple components of ZZCD have undergone comprehensive renal excretion. This study reported the urinary and biliary excretion in rats after oral administration of ZZCD for the first time. The present study expands our knowledge about the constituents and metabolism of ZZCD, which could be very useful for further pharmacological and clinical studies of ZZCD.     

Metabolic study of paeoniflorin and total paeony glucosides from Paeoniae Radix Rubra in rats by high‐performance liquid chromatography coupled with sequential mass spectrometry

A clear understanding of the metabolism of Traditional Chinese Medicines is extremely important in their rational clinical application and effective material foundation research. A novel and reliable strategy was performed to find more metabolites of paeoniflorin, determine the metabolites of total paeony glucosides (TPG) by means of determining those metabolites of paeoniflorin, and compare the metabolism differences between paeoniflorin and TPG by intragastric administration. This strategy was characterized as follows. Firstly, the rats were divided into two groups (the paeoniflorin group and the TPG group) to find differences in metabolism mechanisms between paeoniflorin and TPG. Secondly, UPLC‐FT‐ICR MS and UPLC‐Q‐TOF MS² were applied to obtain accurate molecular weight and structural information, respectively. Thirdly, the metabolites were tentatively identified by a combination of data‐processing methods including mass defect screening, characteristic neutral loss screening and product ion screening. Finally, a comparative study was employed in the metabolism of paeoniflorin and TPG. Based on the strategy, 18 metabolites of paeoniflorin (including four new compounds) and 11 metabolites of TPG (including two new compounds) were identified. In all of the identified metabolites of paeoniflorin, two metabolites in rat plasma, four metabolites in rat urine and six metabolites in rat feces were found for the first time after paeoniflorin administration. The results indicate that hydrolyzation of the ester bond and glucosidic band and conjugation with glucuronide were the major metabolic pathways of paeoniflorin. The metabolites of paeoniflorin and TPG in rat plasma, urine and feces have been detected for the first time after intragastric administration. The results may contribute to a better understanding of the metabolism mechanism and provide a scientific rationale for researching the material basis of paeoniflorin and TPG in vivo.     

Identification of the absorbed components and their metabolites of Tianma‐Gouteng granule in rat plasma and bile using ultra‐high‐performance liquid chromatography combined with quadrupole time‐of‐flight mass spectrometry

Tianma‐Gouteng granule (TGG), a Chinese herbal formula preparation, is clinically used for the treatment of cardio‐cerebrovascular diseases such as hypertension, cerebral ischaemia, acute ischaemic stroke and Parkinson's disease. Although few reports have been published concerning the absorbed prototype components of TGG, the possible metabolic pathways of TGG in vivo remain largely unclear. In this study, a method using UPLC–Q/TOF MS was established for the detection and identification of the absorbed prototype components and related metabolites in rat plasma and bile after oral administration of TGG at high and normal clinical dosages. A total of 68 components were identified or tentatively identified in plasma and bile samples, including absorbed prototypes and their metabolites. The major absorbed components were gastrodin, isorhynchophylline, rhynchophylline, isocorynoxeine, corynoxeine, geissoschizine methyl ether baicalin, baicalein, wogonoside, wogonin, geniposidic acid, leonurine, 2,3,5,4′‐tetrahydroxystilbene‐2‐O‐β‐d ‐glucoside and emodin. The main metabolic pathways of these components involved phase I (isomerization, hydrolysis and reduction) and phase II (glucuronidation and sulfation) reaction, and the phase II biotransformation pathway was predominant. The present study provides rich information on the in vivo absorption and metabolism of TGG, and the results will be helpful for further studies on the pharmacokinetics and pharmacodynamics of TGG.     

Metabolite profiles of epimedin C in rat plasma and bile by ultra‐performance liquid chromatography coupled with quadrupole‐TOF‐MS

Epimedin C is one of the major bioactive constituents of Herba Epimedii. In this study, the metabolite profiles of epimedin C in rat plasma and bile were qualitatively investigated, and the possible metabolic pathways of epimedin C were subsequently proposed. After oral administration of epimedin C at a single dose of 80 mg/kg, rat biological samples were collected and pretreated by protein precipitation. Then these pretreated samples were injected into an Acquity UPLC BEH C₁₈ column and detected by ultra‐performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry. In all, 12 metabolites were identified in the biosamples. Of these, eight, two from plasma and six from bile, are, to our knowledge, reported here for the first time. The results indicated that epimedin C was metabolized via desugarization, dehydrogenation, hydrogenation, dehydroxylation, hydroxylation, demethylation and glucuronidation pathways in vivo. Thus, this study revealed the possible metabolite profiles of epimedin C in rat plasma and bile. Copyright © 2014 John Wiley & Sons, Ltd.     

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.     

Metabolic profiling of quercetin in rats using ultra‐performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry

Quercetin, a kind of major flavonoid found in many traditional chinese medicines, is an effective substance for treatments such as lowering blood lipids. However, the studies on quercetin have been mainly focused on its pharmacological effect; the treatment of diseases on a material basis, particularly the metabolites derived from quercetin in vivo , has not been evaluated. In this study, we determined the levels, distributions and types of quercetin's metabolites in plasma, urine, feces and bile of rats after a single oral administration of quercetin at a dose of 80 mg/kg, using ultra‐performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry (UPLC‐Q‐TOF/MS). A total of 36 metabolites of quercetin were identified, including 11 metabolites in plasma, 34 metabolites in urine, 12 metabolites in feces and 21 metabolites in bile. The results showed that phase I metabolites were reduction metabolites and phase II metabolites mainly included glucuronidation, sulfation and methylation metabolites. These results provide important information on the metabolism of quercetin, which will be helpful for its further development and utilization.     

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.     

Metabolism studies of casticin in rats using HPLC‐ESI‐MSn

Casticin (3′,5‐dihydroxy‐3, 4′,6,7‐tetramethoxyflavone) has been revealed to possess various kinds of pharmacological activities, including immunomodulatory, anti‐hyperprolactinemia, anti‐tumor and neuroprotetective activities. In order to gain an understanding of the biotransformation of casticin in vivo, a systematic method based on liquid chromatography–electrospray ionization tandem mass spectrometry (LC‐ESI‐MSⁿ) was developed to identify the metabolites of casticin in rats after oral administration of single dose of casticin at 200 mg/kg. By comparing their changes in molecular masses (ΔM), retention times and spectral patterns with those of the parent drug, the parent compound and 25 metabolites were identified in rat plasma, urine and six selected tissues. This is the first systematic metabolism study of casticin in vivo. The results indicated that methylation, demethylation, glucuronidation and sulfation were the main biotransformation pathways of casticin in vivo. Copyright © 2012 John Wiley & Sons, Ltd.