Identification of Metabolites of Epimedin A in Rats Using UPLC/Q–TOF–MS

Herba Epimedii (Epimedium) is a kind of tonic herb, widely used in China. Epimedin A is a major component of Herba Epimedii with bioactivities. Analysis of the metabolic profile in vivo plays a pivotal role in understanding how traditional Chinese medicine works. And the metabolites of epimedin A might influence the effects of Herba Epimedii. Moreover, the metabolic routes of epimedin A provide an important basis for safety evaluation. Until now, little has been known about the metabolism of epimedin A. The current study was designed to characterize the metabolic pathways of epimedin A in vivo. The metabolites in rat plasma, bile, feces, and urine were identified by UPLC/Q–TOF–MS analysis. A total of 27 metabolites from epimedin A were detected or tentatively identified. The major metabolic processes were hydrolysis, hydrogenation, hydroxylation, dehydrogenation, demethylation, and conjugation with glucuronic acid and different sugars. The present study revealed the metabolic pathways of epimedin A in rat for the first time, and epimedin A could undergo extensive phase I and phase II metabolism in rat. These findings would provide an important basis for the further study and clinical application of epimedin A. In addition, the results of this work have shown the feasibility of the UPLC/Q–TOF–MS approach for rapid and reliable characterization of metabolites.     

Identification of Metabolites of Epimedin A in Rats Using UPLC/Q–TOF–MS

Herba Epimedii (Epimedium) is a kind of tonic herb, widely used in China. Epimedin A is a major component of Herba Epimedii with bioactivities. Analysis of the metabolic profile in vivo plays a pivotal role in understanding how traditional Chinese medicine works. And the metabolites of epimedin A might influence the effects of Herba Epimedii. Moreover, the metabolic routes of epimedin A provide an important basis for safety evaluation. Until now, little has been known about the metabolism of epimedin A. The current study was designed to characterize the metabolic pathways of epimedin A in vivo. The metabolites in rat plasma, bile, feces, and urine were identified by UPLC/Q–TOF–MS analysis. A total of 27 metabolites from epimedin A were detected or tentatively identified. The major metabolic processes were hydrolysis, hydrogenation, hydroxylation, dehydrogenation, demethylation, and conjugation with glucuronic acid and different sugars. The present study revealed the metabolic pathways of epimedin A in rat for the first time, and epimedin A could undergo extensive phase I and phase II metabolism in rat. These findings would provide an important basis for the further study and clinical application of epimedin A. In addition, the results of this work have shown the feasibility of the UPLC/Q–TOF–MS approach for rapid and reliable characterization of metabolites.

     

Pharmacokinetics and oral bioavailability of epimedin C after oral administration of epimedin C and Herba Epimedii extract in rats

Epimedin C, an ingredient of Herba Epimedii, has potential for treatment of cardiovascular disease and bone loss. However, there is still no sensitive analytical method to monitor epimedin C in biological samples. The goal of this study was to develop a sensitive and reliable method based on a LC‐MS/MS for evaluating the pharmacokinetics of epimedin C after administration of Herba Epimedii in rat. Electrospray ionization in positive‐ion mode and multiple reaction monitoring were used to identify and quantitate active components. Analytes were separated by a reverse‐phase C₁₈ column. Liquid–liquid extraction using ethyl acetate, evaporation and reconstitution was used to plasma sample preparation. Mass transition of precursor ion → product ion pairs were monitored at m/z 823.4 → 313.1 for epimedin C and m/z 237.1 → 178.9 for carbamazepine (internal standard). A calibration curve gave good linearity (r > 0.999) over the concentration range 2.5–500 ng/mL. Pharmacokinetic data demonstrated that there was rapid distribution and slow elimination after epimedin C administration (1 mg/kg, i.v.). Oral bioavailabilities of epimedin C in the pure compound and in the Herba Epimedii were around 0.58% and 0.13%, respectively. The result suggests that other herbal ingredients of Herba Epimedii may suppress the oral bioavailability of epimedin C. Copyright © 2013 John Wiley & Sons, Ltd.     

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.     

A new strategy for the discovery of epimedium metabolites using high-performance liquid chromatography with high resolution mass spectrometry

In this paper, a new strategy of drug metabolite discovery and identification was established using high-performance liquid chromatography with high resolution mass spectrometry (HPLC–HRMS) and a mass spectral trees similarity filter (MTSF) technique. The MTSF technique was developed as a means to rapidly discover comprehensive metabolites from multiple active components in a complicated biological matrix. Using full-scan mass spectra as the stem and data-dependent subsequent stage mass spectra to form branches, the HRMS and multiple-stage mass spectrometric data from detected compounds were converted to mass spectral trees data. Potential metabolites were discovered based on the similarity between their mass spectral trees and that known compounds or metabolites in a mass spectra trees library. The threshold value for match similarity scores was set at above 200, allowing approximately 80% of interference to be filtered out. A total of 115 metabolites of five flavonoid monomers (epimedin A, epimedin B, epimedin C, icariin, and baohuoside I) and herbal extract of epimedium were discovered and identified in rats via this new strategy. As a result, a metabolic profile for epimedium was obtained and a metabolic pathway was proposed. In addition, comparing to the widely used neutral loss filter (NLF), product ion filter (PIF), and mass defect filter (MDF) techniques, the MTSF technique was shown superior efficiency and selectivity for discovering and identifying metabolites in traditional Chinese medicine (TCM).     

Metabolic profile of glyburide in human liver microsomes using LC‐DAD‐Q‐TRAP‐MS/MS

The sulfonylurea urea drug glyburide (glibenclamide) is widely used for the treatment of diabetes milletus and gestational diabetes. In previous studies monohydroxylated metabolites were identified and characterized for glyburide in different species, but the metabolite owing to the loss of cyclohexyl ring was identified only in mouse. Glyburide upon incubation with hepatic microsomes resulted in 10 metabolites for human. The current study identifies new metabolites of glyburide along with the hydroxylated metabolites that were reported earlier. The newly identified drug metabolites are dihydroxylated metabolites, a metabolite owing to the loss of cyclohexyl ring and one owing to hydroxylation with dehydrogenation. Among the 10 identified metabolites, there were six monohydroxylated metabolites, one dihydroxylated metabolite, two metabolites owing to hydroxylation and dehydrogenation, and one metabolite owing to the loss of cyclohexyl ring. New metabolites of glyburide were identified and characterized using liquid chromatography–diode array detector–quadruple‐ion trap–mass spectrometry/mass spectrometry (LC‐DAD‐Q‐TRAP‐MS/MS). An enhanced mass scan–enhanced product ion scan with information‐dependent acquisition mode in a Q‐TRAP‐MS/MS system was used to characterize the metabolites. Liquid chromatography with diode array detection was used as a complimentary technique to confirm and identify the metabolites. Metabolites formed in higher amounts were detected in both diode array detection and mass spectrometry detection. Copyright © 2012 John Wiley & Sons, Ltd.     

A rapid and sensitive UPLC-ESI MS method for analysis of isofraxidin, a natural antistress compound, and its metabolites in rat plasma

Isofraxidin is one of the main bioactive constituents in the root of Acanthopanax senticosus, which has antifatigue, antistress, and immuno-accommondating effects. In this study, an ultraperformance LC (UPLC)-ESI MS method was developed for analyzing isofraxidin and its metabolites in rat plasma. The analysis was performed on a UPLC coupled with ESI MS (quadropole MS tandem TOF MS). The lower LOD (LLOD) for isofraxidin was 0.25 ng/mL, the intraday precision was less than 10%, the interday precision was less than 10%, and the extraction recovery was more than 80%. Isofraxidin and two metabolites (M1 and M2) were detected in rat plasma after oral administration of isofraxidin, and the molecular polarities of M1 and M2 were both increased compared to isofraxidin. The metabolites were identified as 5,6-dihydroxyl-7-methoxycoumarin and 5-hydroxyl-6,7-dimethoxycoumarin when subjected to parent ion spectra, product ion spectra, and extract mass and element composition analyses.     

Feasibility of different mass spectrometric techniques and programs for automated metabolite profiling of tramadol in human urine

The purpose of the study was to determine the advantages of different mass spectrometric instruments and commercially available metabolite identification programs for metabolite profiling. Metabolism of tramadol hydrochloride and the excretion of it and its metabolites into human urine were used as a test case because the metabolism of tramadol is extensive and well known. Accurate mass measurements were carried out with a quadrupole time-of-flight mass spectrometer (Q-TOF) equipped with a LockSpray dual-electrospray ionization source. A triple quadrupole mass spectrometer (QqQ) was applied for full scan, product ion scan, precursor ion scan and neutral loss scan measurements and an ion trap instrument for full scan and product ion measurements. The performance of two metabolite identification programs was tested. The results showed that metabolite programs are time-saving tools but not yet capable of fully automated metabolite profiling. Detection of non-expected metabolites, especially at low concentrations in a complex matrix, is still almost impossible. With low-resolution instruments urine samples proved to be challenging even in a search for expected metabolites. Many false-positive hits were obtained with the automated searching and manual evaluation of the resulting data was required. False positives were avoided by using the higher mass accuracy Q-TOF. Automated programs were useful for constructing product ion methods, but the time-consuming interpretation of mass spectra was done manually. High-quality MS/MS spectra acquired on the QqQ instrument were used for confirmation of the tramadol metabolites. Although the ion trap instrument is of undisputable benefit in MS(n), the low mass cutoff of the ion trap made the identification of tramadol metabolites difficult. Some previously unreported metabolites of tramadol were found in the tramadol urine sample, and their identification was based solely on LC/MS and LC/MS/MS measurements.     

Efficient approach for the detection and identification of new androgenic metabolites by applying SRM GC‐CI‐MS/MS: a methandienone case study

Identification of anabolic androgenic steroids (AAS) is a vital issue in doping control and toxicology, and searching for metabolites with longer detection times remains an important task. Recently, a gas chromatography chemical ionization triple quadrupole mass spectrometry (GC‐CI‐MS/MS) method was introduced, and CI, in comparison with electron ionization (EI), proved to be capable of increasing the sensitivity significantly. In addition, correlations between AAS structure and fragmentation behavior could be revealed. This enables the search for previously unknown but expected metabolites by selection of their predicted transitions. The combination of both factors allows the setup of an efficient approach to search for new metabolites. The approach uses selected reaction monitoring which is inherently more sensitive than full scan or precursor ion scan. Additionally, structural information obtained from the structure specific CI fragmentation pattern facilitates metabolite identification. The procedure was demonstrated by a methandienone case study. Its metabolites have been studied extensively in the past, and this allowed an adequate evaluation of the efficiency of the approach. Thirty three metabolites were detected, including all relevant previously discovered metabolites. In our study, the previously reported long‐term metabolite (18‐nor‐17β‐hydroxymethyl,17α‐methyl‐androst‐1,4,13‐trien‐3‐one) could be detected up to 26 days by using GC‐CI‐MS/MS. The study proves the validity of the approach to search for metabolites of new synthetic AAS and new long‐term metabolites of less studied AAS and illustrates the increase in sensitivity by using CI. Copyright © 2016 John Wiley & Sons, Ltd.     

Rapid characterization and determination of multiple components in Bu‐Shen‐Yi‐Qi‐Fang by high‐performance liquid chromatography coupled to electrospray ionization and quadrupole time‐of‐flight mass spectrometry

In this study, a qualitative and quantitative analysis using high‐performance liquid chromatography coupled to electrospray ionization and quadrupole time‐of‐flight mass spectrometry was performed for the quality control of Bu‐Shen‐Yi‐Qi‐Fang, a traditional Chinese formula used for asthma. Thirty‐four compounds, including flavonoids, isoflavonoids, triterpenoid saponins, and iridoid glycosides were identified or tentatively characterized by comparing their retention times and mass spectra with those of authentic standards or literature data. Sixteen components were considered as the main bioactive constituents of Bu‐Shen‐Yi‐Qi‐Fang and they were chosen as the chemical markers in quantitative analysis, including catalpol, leonuride, calycosin‐7‐O‐β‐d ‐glucoside, hyperoside, acteoside, formononetin‐7‐O‐β‐d ‐glucoside, epimedin A, calycosin, icariin, epimedin B, epimedin C, formononetin, astragaloside IV, astragaloside II, baohuoside‐I, and astragaloside I. The total run time was 20 min. It was found that the calibration curves for all analytes showed good linearity (R² > 0.99) within the test ranges. The relative standard deviations for intra‐ and inter‐day precisions were below 3.9 and 11.7%, respectively. The accuracy was evaluated by the recovery test within the range of 89.20–110.71% with the relative standard deviation < 4.8%. The sample was stable for at least 48 h at 4°C. The results showed that the new approach was effective for the quality control of Bu‐Shen‐Yi‐Qi‐Fang.     

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.     

Rapid separation and identification of Strychnos alkaloids metabolites in rats by ultra high performance liquid chromatography with linear ion trap Orbitrap mass spectrometry

An in vivo study of Strychnos alkaloids metabolites in rats by ultra high performance liquid chromatography with linear ion trap Orbitrap MS is reported for the first time. Two major Strychnos alkaloids compounds including strychnine and brucine were investigated. To obtain optimal extraction efficiency, samples were pretreated by using an SPE plate. The structures of metabolites and their fragment ions were characterized based on the accurate mass and MSⁿ data. Forty‐seven metabolites were identified in rat urine, of which 25 were reported for the first time. Four new metabolism pathways were proposed on the basis of the identified metabolites. This study provides a practical approach for rapidly identifying complicated metabolites, a methodology that could be widely applied not only in forensic and clinically toxicological relevant cases, but also for the structural characterization of metabolites of other compounds.     

超高效液相色谱-四极杆-飞行时间质谱检测和鉴定猪尿中氯丙那林的主要代谢产物

采用超高效液相色谱-四极杆-飞行时间质谱(UPLC/Q-TOF MS)检测和鉴定了猪尿中氯丙那林的主要代谢产物,并讨论了氯丙那林在猪体内的主要代谢途径。按10 mg/kg(b. w.)的剂量口服灌食氯丙那林,分别采集给药前及给药后的猪尿液样品。采用UPLC/Q-TOF MS对样品进行分析,并应用质量亏损过滤和离子色谱峰提取等数据处理技术,在给药后24 h内的猪尿中检测和鉴定了9种氯丙那林的代谢产物,其中,Ⅰ相代谢产物2种,Ⅱ相代谢产物7种。然后,根据氯丙那林原形和代谢产物的碎片离子特征,对代谢产物的结构进行鉴定。最后,根据所鉴定的代谢产物,推测氯丙那林在猪体内的代谢途径包括苯环羟基化、β -羟基和仲氨基的葡萄糖醛酸轭合、羟基化后的葡萄糖醛酸和硫酸轭合等。研究结果表明,羟基化氯丙那林及其轭合产物的相对含量大于60%,明显高于氯丙那林原形及其轭合产物,是尿液中的主要代谢产物。本研究将为确定氯丙那林在动物体内的残留标示物及加强对氯丙那林非法使用的监控提供科学依据。     

Chromatographic Tandem Mass Spectrometric Detection of Physostigmine and its Major Metabolites in Rat Urine

Abstract

A rapid, sensitive, and specific liquid chromatographic‐electrospray ionization (ESI) tandem ion trap mass spectrometric method has been developed for identification of physostigmine and its metabolites in rat urine. 300 µg kg^–1 of physostigmine were used as a safe oral gavage dose for studies on its metabolites. 0–24 h urine was purified using a C18 solid‐phase extraction cartridge, and then detected by an on‐line MS detector. Identification and structural elucidation of the metabolites were performed by comparing their MSⁿ spectra with physostigmine. Six metabolites and unchanged physostigmine existed in rat urine. All of the metabolites were reported for the first time.     

Identification of major metabolites in rat urine and plasma of N(6) -(4-hydroxybenzyl) adenine riboside by LC/MS/MS

N(6) -(4-hydroxybenzyl) adenine riboside, a novel neuroprotective compound found in Gastrodia elata at trace level, is regarded as a potential drug for the treatment of neural degenerative disease. To understand the metabolism of this compound, the metabolites in rat urine and plasma of N(6) -(4-hydroxybenzyl) adenine riboside were analyzed by HPLC-ESI-MS/MS after oral administration of this compound. Beside the parent compound, six phase I metabolites and four phase II metabolites in urine were detected by scanning all possible metabolites in extracted ion chromatograms mode. By comparing their product ion spectra and retention times with those of parent compound, these metabolites were identified and proved to be mainly formed via hydrolysis or hydroxylation in phase I, N-sulfation or N-glucuronidation in phase II or their combinations. Similarly, the parent compound, one phase I metabolite and two phase II metabolites were also identified in rat plasma. Therefore, the in vivo metabolic pathways of N(6) -(4-hydroxybenzyl) adenine riboside in rat were proposed.     

Metabolic profile and pharmacokinetics of polyphyllin I,an anticancer candidate,in rats by UPLC‐QTOF‐MS/MS and LC‐TQ‐MS/MS

Polyphyllin I (PPI), a natural steroidal saponin originating from rihzome of Paris polyphylla , is a potential anticancer candidate. Previous pharmacokinetics study showed that the oral bioavailability of PPI was very low, which suggested that certain amount of PPI might be metabolized in vivo . However, to date, information regarding the final metabolic fates of PPI is very limited. In this study, metabolites of PPI and their pharmacokinetics in rats were investigated using UPLC‐QTOF‐MS/MS and LC‐TQ‐MS/MS. A total of seven putative metabolites, including six phase I and one phase II metabolites, were detected and identified with three exact structures by comparison with authentic standards for the first time. Oxidation, deglycosylation and glucuronidation were found to be the major metabolic processes of the compound in rats. The pharmacokinetics of prosapogenin A, trillin and diosgenin, three deglycosylation metabolites of PPI with definite anticancer effects, were further studied, which suggested that the metabolites underwent a prolonged absorption and slower elimination after intragastric administration of PPI at the dose of 500 mg/kg. This study provides valuable and new information on the metabolic fate of PPI, which will be helpful in further understanding its mechanism of action.     

Rapid screening and characterization of drug metabolites using a multiple ion monitoring-dependent MS/MS acquisition method on a hybrid triple quadrupole-linear ion trap mass spectrometer

A novel LC/MS/MS method that uses multiple ion monitoring (MIM) as a survey scan to trigger the acquisition of enhanced product ions (EPI) on a hybrid quadrupole-linear ion trap mass spectrometer (Q TRAP) was developed for drug metabolite identification. In the MIM experiment, multiple predicted metabolite ions were monitored in both Q1 and Q3. The collision energy in Q2 was set to a low value to minimize fragmentation. Results from analyzing ritonavir metabolites in rat hepatocytes demonstrate that MIM-EPI was capable of targeting a larger number of metabolites regardless of their fragmentation and retained sensitivity and duty cycle similar to multiple reaction monitoring (MRM)-EPI. MIM-based scanning methods were shown to be particularly useful in several applications. First, MIM-EPI enabled the sensitive detection and MS/MS acquisition of up to 100 predicted metabolites. Second, MIM-MRM-EPI was better than MRM-EPI in the analysis of metabolites that undergo either predictable or unpredictable fragmentation pathways. Finally, a combination of MIM-EPI and full-scan MS (EMS), as an alternative to EMS-EPI, was well suited for routine in vitro metabolite profiling. Overall, MIM-EPI significantly enhanced the metabolite identification capability of the hybrid triple quadrupole-linear ion trap LC/MS.     

In vivo biotransformation of 17 alpha-methyltestosterone in the horse revisited: identification of 17-hydroxymethyl metabolites in equine urine by capillary gas chromatography/mass spectrometry

The in vivo phase I biotransformation of 17 alpha-methyltestosterone in the horse leads to the formation of a complex mixture of regio- and stereoisomeric C(20)O(2), C(20)O(3) and C(20)O(4) metabolites, excreted in urine as glucuronide and sulphate phase II conjugates. The major pathways of in vivo metabolism are the reduction of the A-ring (di- and tetrahydro), epimerisation at C-17 and oxidations mainly at C-6 and C-16. Some phase I metabolites have been identified previously by positive ion electron ionisation capillary gas chromatography/mass spectrometry (GC/EI + MS) mainly from the characteristic fragmentation patterns of their methyloxime-trimethylsilyl ether (MO-TMS), enol-TMS or TMS ether derivatives. Following oral administration of 17 alpha-methyltestosterone to two castrated thoroughbred male horses, the glucuronic acid conjugates excreted in post-administration urine samples were selectively hydrolysed by E. coli beta-glucuronidase enzymes. Unconjugated metabolites and the steroid aglycones obtained after enzymatic deconjugation were isolated from urine by solid-phase extraction, derivatised as MO-TMS ethers and analysed by GC/EI + MS. In addition to some of the known metabolites previously identified from the characteristic mass spectral fragmentation patterns of 17 alpha-methyl steroids, some isobaric compounds exhibiting a diagnostic loss of 103 mass units from the molecular ions with subsequent losses of trimethylsilanol or methoxy groups and an absence of the classical D-ring fragment ion were detected. From an interpretation of their mass spectra, these compounds were identified as 17-hydroxymethyl metabolites, formed in vivo in the horse by oxidation of the 17-methyl moiety of 17 alpha-methyltestosterone. This study reports on the GC/EI + MS identification of these novel 17-hydroxymethyl C(20)O(3) and C(20)O(4) metabolites of 17 alpha-methyltestosterone excreted in thoroughbred horse urine.     

An integrated method for metabolite detection and identification using a linear ion trap/Orbitrap mass spectrometer and multiple data processing techniques: application to indinavir metabolite detection

A new strategy using a hybrid linear ion trap/Orbitrap mass spectrometer and multiple post-acquisition data mining techniques was evaluated and applied to the detection and characterization of in vitro metabolites of indinavir. Accurate-mass, full-scan MS and MS/MS data sets were acquired with a generic data-dependent method and processed with extracted-ion chromatography (EIC), mass-defect filter (MDF), product-ion filter (PIF), and neutral-loss filter (NLF) techniques. The high-resolution EIC process was shown to be highly effective in the detection of common metabolites with predicted molecular weights. The MDF process, which searched for metabolites based on the similarity of mass defects of metabolites to those of indinavir and its core substructures, was able to find uncommon metabolites not detected by the EIC processing. The high-resolution PIF and NLF processes selectively detected metabolites that underwent fragmentation pathways similar to those of indinavir or its known metabolites. As a result, a total of 15 metabolites including two new indinavir metabolites were detected and characterized in a rat liver S9 incubation sample. Overall, these data mining techniques, which employed distinct metabolite search mechanisms, were complementary and effective in detecting both common and uncommon metabolites. In summary, the results demonstrated that this analytical strategy enables the high-throughput acquisition of accurate-mass LC/MS data sets, comprehensive search of a variety of metabolites through the post-acquisition processes, and effective structural characterization based on elemental compositions of metabolite molecules and their product ions.     

Metabolite profiling of four major flavonoids of Herba Epimedii in zebrafish

The zebrafish model organism was applied first in a metabolic study of icariin, baohuoside I, epimedin A and epimedin C, which are flavonoids in Herba Epimedii. Metabolites of these compounds in zebrafish after exposure for 24 h were identified by HPLC-ESI-MS, whereby the separation was performed with a Zorbax C-18 column using a gradient elution of 0.05% formic acid acetonitrile-0.05% formic acid water. The quasi-molecular ions of compounds were detected in simultaneous negative and positive ionization modes. Metabolic products of icariin and epimedin C via cleavage of glucose residue instead of rhamnose residues were found, which coincided with the results using regular metabolic analysis methods. In addition, the zebrafish model was used to predict the metabolism of the trace component epimedin A, whose metabolic mechanisms haven't been clearly elucidated with the current metabolism model. The metabolic pathway of epimedin A in zebrafish was similar to those of its homologue icariin and epimedin C. Our study demonstrated that the zebrafish model can successfully imitate the current models in elucidating metabolic pathways of model flavonoids, which has advantages of lower cost, far less amount of compound needed, easy set up and high performance. This novel model can also be applied in quickly predicting the metabolism of Chinese herb components, especially trace compounds.