Novel strategy for the determination of illegal adulterants in health foods and herbal medicines using high‐performance liquid chromatography with high‐resolution mass spectrometry

The detection, confirmation, and quantification of multiple illegal adulterants in health foods and herbal medicines by using a single analytical method are a challenge. This paper reports on a new strategy to meet this challenge by employing high‐performance liquid chromatography coupled with high‐resolution mass spectrometry and a mass spectral tree similarity filter technique. This analytical method can rapidly collect high‐resolution, high‐accuracy, optionally multistage mass data for compounds in samples. After a preliminary screening by retention time and high‐resolution mass spectral data, known illegal adulterants can be detected. The mass spectral tree similarity filter technique has been applied to rapidly confirm these adulterants and simultaneously discover unknown ones. By using full‐scan mass spectra as stem and data‐dependent subsequent stage mass spectra to form branches, mass spectrometry data from detected compounds are converted into mass spectral trees. The known or unknown illegal adulterants in the samples are confirmed or discovered based on the similarity between their mass spectral trees and those of the references in a library, and they are finally quantified against standard curves. This new strategy has been tested by using 50 samples, and the illegal adulterants were rapidly and effectively detected, confirmed and quantified.     

Similarity of High-Resolution Tandem Mass Spectrometry Spectra of Structurally Related Micropollutants and Transformation Products

High-resolution tandem mass spectrometry (HRMS2) with electrospray ionization is frequently applied to study polar organic molecules such as micropollutants. Fragmentation provides structural information to confirm structures of known compounds or propose structures of unknown compounds. Similarity of HRMS2 spectra between structurally related compounds has been suggested to facilitate identification of unknown compounds. To test this hypothesis, the similarity of reference standard HRMS2 spectra was calculated for 243 pairs of micropollutants and their structurally related transformation products (TPs); for comparison, spectral similarity was also calculated for 219 pairs of unrelated compounds. Spectra were measured on Orbitrap and QTOF mass spectrometers and similarity was calculated with the dot product. The influence of different factors on spectral similarity [e.g., normalized collision energy (NCE), merging fragments from all NCEs, and shifting fragments by the mass difference of the pair] was considered. Spectral similarity increased at higher NCEs and highest similarity scores for related pairs were obtained with merged spectra including measured fragments and shifted fragments. Removal of the monoisotopic peak was critical to reduce false positives. Using a spectral similarity score threshold of 0.52, 40% of related pairs and 0% of unrelated pairs were above this value. Structural similarity was estimated with the Tanimoto coefficient and pairs with higher structural similarity generally had higher spectral similarity. Pairs where one or both compounds contained heteroatoms such as sulfur often resulted in dissimilar spectra. This work demonstrates that HRMS2 spectral similarity may indicate structural similarity and that spectral similarity can be used in the future to screen complex samples for related compounds such as micropollutants and TPs, assisting in the prioritization of non-target compounds.

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Unraveling the metabolic transformation of tetrazepam to diazepam with mass spectrometric methods

The metabolic transformation pathways of the 1,4-benzodiazepine tetrazepam (C(16)H(17)ClN(2)O, average mass: 288.772) were studied with capillary LC-QqTOF-MS and -MS/MS by analyzing human plasma and urine samples collected from healthy volunteers. Each volunteer took 50 mg of tetrazepam, given in the form of one tablet of Myolastan (Sanofi-Synthelabo, Vienna, Austria). Accurate molecular mass measurements in full-scan mode (scan range: 50-700) were used to survey the collected samples for putative metabolic transformation products. Full-scan fragment ion mass spectra were collected in subsequent LC/MS/MS experiments. Each spectrum was matched to a spectral library containing 3759 MS/MS-spectra of 402 compounds, including eighteen different benzodiazepines, to prove the structural relatedness of a tentative metabolite to tetrazepam. This "similarity search" approach provided a rapid and powerful tool to exclude non-drug-related species, even without any knowledge of the fragmentation chemistry. Interpretation of tandem mass spectrometric data was only required in order to elucidate the site of transformation. Using this strategy, 11 major classes of tetrazepam metabolites were identified. Possible metabolic routes from tetrazepam to diazepam (C(16)H(13)ClN(2)O, average mass: 284.740) via repeated hydroxylation and dehydration of the cylohexenyl moiety were discovered. No evidence for extensive hydroxylation of tetrazepam at position 3 of the diazepine ring was found. In contrast to what is commonly believed, this distinct transformation reaction may be of only minor importance. Furthermore, the occurrence of demethylation, hydration, and glucuronidation reactions was proven.     

Tentative identification of new metabolites of epimedin C by liquid chromatography-mass spectrometry

Epimedin C is one of the major bioactive constituents of Herba Epimedii. The aim of this study is to characterize and elucidate the structure of metabolites in the rat after administration of epimedin C. Metabolite identification was performed using a predictive multiple reaction monitoring-information dependent acquisition-enhanced product ion (pMRM-IDA-EPI) scan in positive ion mode on a hybrid triple quadrupole-linear ion trap mass spectrometer. A total of 18 metabolites were characterized by the changes in their protonated molecular masses, their MS/MS spectrum and their retention times compared with those of the parent drug. The results reveal possible metabolite profiles of epimedin C in rats; the metabolic pathways including hydrolysis, hydroxylation, dehydrogenation, demethylation and conjugation with glucuronic acid and different sugars were observed. This study provides a practical approach for rapidly identifying complicated metabolites, a methodology that could be widely applied for the structural characterization of metabolites of other compounds.     

Rapid characterization of chlorogenic acids in Duhaldea nervosa based on ultra‐high‐performance liquid chromatography–linear trap quadropole‐Orbitrap‐mass spectrometry and mass spectral trees similarity filter technique

Duhaldea nervosa (Wallich ex Candolle) A. Anderberg has been traditionally used as a food spice and also in folk medicine for treating traumatic injury and relieving rheumatism, especially accelerating the healing of a fracture. However, so far as we are aware, the chemical constituents have not been fully investigated. In this study, a practical method of mass spectral trees similarity filter, a data‐mining technique, was developed and evaluated for the rapid detection and identification complicated constituents based on ultra‐high‐performance liquid chromatography–linear trap quadropole‐Orbitrap‐mass spectrometry. Finally, a total of 47 chlorogenic acids, including 19 monoacyl‐quinic acids, 22 diacyl‐quinic acids, and six triacyl‐quinic acids, were unambiguously or tentatively identified based on their accurate mass measurement, chromatographic retention, MSⁿ spectra, and bibliography data. To our best knowledge, it is the first time to report the chlorogenic acids of D. nervosa, which would be beneficial for the further material basis and quality research. Meanwhile, this mass spectral trees similarity filter method could be envisioned to exhibit a wide application for the identification of complicated components from botanical extracts.     

Rapid detection and structural characterization of verapamil metabolites in rats by UPLC–MSE and UNIFI platform

High-resolution mass spectrometry (HRMS) is an important technology for studying biotransformations of drugs in biological systems. In order to process complex HRMS data, bioinformatics, including data-mining techniques for identifying drug metabolites from liquid chromatography/high-resolution mass spectrometry (LC/HRMS) or multistage mass spectrometry (MSⁿ) datasets as well as elucidating the detected metabolites’ structure by spectral interpretation software, are important tools. Data-mining technologies have widely been used in drug metabolite identification, including mass defect filters, product ion filters, neutral-loss filters, control sample comparisons and extracted ion chromatographic analysis. However, the metabolites identified by current different technologies are not the same, indicating the importance of technique integration for efficient and complete identification of metabolic products. In this study, a universal, high-throughput workflow for identifying and verifying metabolites by applying the drug metabolite identification software UNIFI is reported, to study the biotransformation of verapamil in rats. A total of 71 verapamil metabolites were found in rat plasma, urine and faeces, including two metabolites that have not been reported in the literature. Phase I metabolites of verapamil were identified as N-demethylation, O-demethylation, N-dealkylation and oxidation and dehydrogenation metabolites; phase II metabolites were mainly glucuronidation and sulfate conjugates, indicating that UNIFI software could be effective and valuable in identifying drug 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.     

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 urinary metabolites of imperatorin with a single run on an LC/Triple TOF system based on multiple mass defect filter data acquisition and multiple data mining techniques

The detection of drug metabolites, especially for minor metabolites, continues to be a challenge because of the complexity of biological samples. Imperatorin (IMP) is an active natural furocoumarin component originating from many traditional Chinese herbal medicines and is expected to be pursued as a new vasorelaxant agent. In the present study, a generic and efficient approach was developed for the in vivo screening and identification of IMP metabolites using liquid chromatography-Triple TOF mass spectrometry. In this approach, a novel on-line data acquisition method mutiple mass defect filter (MMDF) combined with dynamic background subtraction was developed to trace all probable urinary metabolites of IMP. Comparing with the traditionally intensity-dependent data acquisition method, MMDF method could give the information of low-level metabolites masked by background noise and endogenous components. Thus, the minor metabolites in complex biological matrices could be detected. Then, the sensitive and specific multiple data-mining techniques extracted ion chromatography, mass defect filter, product ion filter, and neutral loss filter were used for the discovery of IMP metabolites. Based on the proposed strategy, 44 phase I and 7 phase II metabolites were identified in rat urine after oral administration of IMP. The results indicated that oxidization was the main metabolic pathway and that different oxidized substituent positions had a significant influence on the fragmentation of the metabolites. Two types of characteristic ions at m/z 203 and 219 can be observed in the MS/MS spectra. This is the first study of IMP metabolism in vivo. The interpretation of the MS/MS spectra of these metabolites and the proposed metabolite pathway provide essential data for further pharmacological studies of other linear-type furocoumarins.     

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.     

Application of stable isotope labeled glutathione and rapid scanning mass spectrometers in detecting and characterizing reactive metabolites

The formation of reactive metabolites from a number of compounds was studied in vitro using a mixture of non-labeled and stable isotope labeled glutathione (GSH) as a trapping agent. GSH was labeled by incorporating [1,2-(13)C(2),(15)N]glycine into the tripeptide to give an overall increase of 3 Da over the naturally occurring substance. Detection and characterization of reactive metabolites was greatly facilitated by using the data-dependent scanning features of the linear ion trap mass spectrometers to give complimentary and confirmatory data in a single analytical run. A comparison was made by analyzing the samples simultaneously on a triple-stage quadrupole mass spectrometer operated in the constant neutral loss mode. The compounds studied included 2-acetamidophenol, 3-acetamidophenol, 4-acetamidophenol (acetaminophen), and flufenamic acid. GSH adducts for each of these compounds produced a characteristic pattern of 'twin ions' separated by 3 Da in the mass spectral data. This greatly facilitated the detection and characterization of any GSH-related adducts present in the microsomal extracts. Furthermore, characterization of these adducts was greatly facilitated by the rapid scanning capability of linear ion trap instruments that provided full-scan, MS/MS and MS(3) data in one single analysis. This method of detecting and characterizing reactive metabolites generated in vitro was found to be far superior to any of the existing methods previously employed in this laboratory. The combination of two techniques, stable isotope labeled glutathione and linear ion traps, provided a very sensitive and specific method of identifying compounds capable of producing reactive metabolites in a discovery setting. The complimentary set of mass spectral data (including full-scan, MS/MS and MS(3) mass spectra), obtained rapidly in a single analysis with the linear ion trap instruments, greatly accelerated identification of metabolically bioactivated soft spots on the molecules. This in turn enabled chemists to rapidly design out the potential metabolic liability from the back-up compounds by making appropriate structural modifications.     

Strategy of using microsome-based metabolite production to facilitate the identification of endogenous metabolites by liquid chromatography mass spectrometry

One of the challenges in metabolomic profiling of complex biological samples is to identify new and unknown compounds. Typically, standards are used to help identify metabolites, yet standards cannot be purchased or readily synthesized for many unknowns. In this work we present a strategy of using human liver microsomes (HLM) to metabolize known endogenous human metabolites (substrates), producing potentially new metabolites that have yet to be documented. The metabolites produced by HLM can be tentatively identified based on the associated substrate structure, known metabolic processes, tandem mass spectrometry (MS/MS) fragmentation patterns and, if necessary, accurate mass measurements. Once identified, these metabolites can be used as references for identification of the same compounds in complex biological samples. As a proof of principle, a total of 9 metabolites have been identified from individual HLM incubations using 5 different substrates. Each metabolite was used as a standard. In the analysis of human urine sample by liquid chromatography MS/MS, four spectral matches were found from the 9 microsome-produced metabolite standards. Two of them have previously been documented as endogenous human metabolites, the third is an isomer of a microsome-metabolite and the fourth compound has not been previously reported and is also an isomer of a microsome-metabolite. This work illustrates the feasibility of using microsome-based metabolism to produce metabolites of endogenous human metabolites that can be used to facilitate the identification of unknowns in biological samples. Future work on improving the performance of this strategy is also discussed.     

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.     

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

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

Differentiation of sulfate and phosphate by H/D exchange mass spectrometry: application to isoflavone

Often phosphorylation or sulfation is an important step which occurs in the signal transduction and cascade of metabolic pathways. Some natural products and metabolites contain one or more sulfate or phosphate groups. Isoflavone sulfate has been identified from high-resolution mass spectrometry (HRMS) and enzymatic digestion by sulfatase. We previously reported the new water-soluble isoflavone analogs, daidzein 7-O-phosphate and genistein 7-O-phosphate, which were surprisingly hydrolyzed by sulfatase. In this previous study, we could not determine the phosphate from the results of HRMS and enzymatic digestion, that is, HRMS and enzymatic digestion did not provide clear evidence. In this case, we drew conclusions from NMR analysis. HRMS has been ineffective with a regular fast atom bombardment (FAB) mass spectrometer to distinguish between phosphate and sulfate since the mass difference is only 0.009 mass units. There was, however, no conventional method of microanalysis to distinguish phosphate from sulfate owing to the same nominal mass. It is still very difficult to determine by negative FABMS [--O--P(==O)(OH)(2)] = 80 and [--O--S(==O)(2)OH] = 80. In this paper, we report a method to distinguish between these groups by using a popular low-resolution mass instrument; thus, phosphate and sulfate were measured by H/D exchange mass spectrometry at the picomole level to differentiate [--O--P(==O)(OD)(2)] = 82 and [--O--S(==O)(2)OD] = 81, respectively. This method is applicable not only to the isoflavone, but also to other phospho and sulfo compounds.     

The application of high-resolution mass spectrometry-based data-mining tools in tandem to metabolite profiling of a triple drug combination in humans

Patients are usually exposed to multiple drugs, and metabolite profiling of each drug in complex biological matrices is a big challenge. This study presented a new application of an improved high resolution mass spectrometry (HRMS)-based data-mining tools in tandem to fast and comprehensive metabolite identification of combination drugs in human. The model drug combination was metronidazole-pantoprazole-clarithromycin (MET-PAN-CLAR), which is widely used in clinic to treat ulcers caused by Helicobacter pylori. First, mass defect filter (MDF), as a targeted data processing tool, was able to recover all relevant metabolites of MET-PAN-CLAR in human plasma and urine from the full-scan MS dataset when appropriate MDF templates for each drug were defined. Second, the accurate mass-based background subtraction (BS), as an untargeted data-mining tool, worked effectively except for several trace metabolites, which were buried in the remaining background signals. Third, an integrated strategy, i.e., untargeted BS followed by improved MDF, was effective for metabolite identification of MET-PAN-CLAR. Most metabolites except for trace ones were found in the first step of BS-processed datasets, and the results led to the setup of appropriate metabolite MDF template for the subsequent MDF data processing. Trace metabolites were further recovered by MDF, which used both common MDF templates and the novel metabolite-based MDF templates. As a result, a total of 44 metabolites or related components were found for MET-PAN-CLAR in human plasma and urine using the integrated strategy. New metabolic pathways such as N-glucuronidation of PAN and dehydrogenation of CLAR were found. This study demonstrated that the combination of accurate mass-based multiple data-mining techniques in tandem, i.e., untargeted background subtraction followed by targeted mass defect filtering, can be a valuable tool for rapid metabolite profiling of combination drugs in vivo.     

Identification of absorbed constituents and metabolites in rat plasma after oral administration of Shen‐Song‐Yang‐Xin using ultra‐performance liquid chromatography combined with quadrupole time‐of‐flight mass spectrometry

In this study, a rapid and sensitive method by ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry, and Metabolynx^TM software with mass defect filter technique was developed for screening and identification of the metabolites in rat plasma after oral administration of Shen‐Song‐Yang‐Xin capsule (SSYX). A total of 92 SSYX‐related xenobiotics were identified or characterized, including 45 prototypes and 47 metabolites. The results indicated that the absorbed constituents and metabolites mainly came from benzocyclooctadiene lignans, tanshinones, isoquinoline alkaloids and triterpenic acids, while phase I reactions (e.g. hydrogenation, hydroxylation, demethylation) and phase II reaction (glucuronidation) were the main metabolic pathways of these ingredients in SSYX. This is the first study on metabolic profiling of SSYX in rat plasma after oral administration. Furthermore, these findings provide useful information on the potential bioactive compounds, and enhance our understanding of the action mechanism of SSYX. Copyright © 2015 John Wiley & Sons, Ltd.     

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.     

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.