Comprehensive identification and structural characterization of target components from Gelsemium elegans by high‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry based on accurate mass databases combined with MS/MS spectra

This study reports an applicable analytical strategy of comprehensive identification and structure characterization of target components from Gelsemium elegans by using high‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry (LC‐QqTOF MS) based on the use of accurate mass databases combined with MS/MS spectra. The databases created included accurate masses and elemental compositions of 204 components from Gelsemium and their structural data. The accurate MS and MS/MS spectra were acquired through data‐dependent auto MS/MS mode followed by an extraction of the potential compounds from the LC‐QqTOF MS raw data of the sample. The same was matched using the databases to search for targeted components in the sample. The structures for detected components were tentatively characterized by manually interpreting the accurate MS/MS spectra for the first time. A total of 57 components have been successfully detected and structurally characterized from the crude extracts of G. elegans , but has failed to differentiate some isomers. This analytical strategy is generic and efficient, avoids isolation and purification procedures, enables a comprehensive structure characterization of target components of Gelsemium and would be widely applicable for complicated mixtures that are derived from Gelsemium preparations. Copyright © 2017 John Wiley & Sons, Ltd.     

Metabolic mapping of Schisandra chinensis lignans and their metabolites in rats using a metabolomic approach based on HPLC with quadrupole time‐of‐flight MS/MS spectrometry

Schisandra chinensis lignans are the main active components of the traditional Chinese medicine Schisandra chinensis in East Asia. At present, there are more and more medicines and health foods in which the total S. chinensis lignans extracts are considered as the main active components, but little research has been done on the active components of S. chinensis lignans in the blood and main target organs. In this study, the components of S. chinensis lignans in the blood, liver and brain tissues of rats at different time points after the intragastrical administration of S. chinensis lignans were determined by a metabolomic method based on high‐performance liquid chromatography with quadrupole time‐of‐flight tandem mass spectrometry spectrometry. Twelve Schisandra chinensis lignans and 15 metabolites in the blood, liver, and brain of rats were identified. The results showed that the main metabolic ways of S. chinensis lignans in rats were hydroxylation, demethylation, and demethylation‐hydroxylation, and some of them might undergo demethylation, dehydrogenation, epoxidation, and elimination reaction. The time‐dose characteristics of S. chinensis lignans and their metabolites in the blood and target organs were analyzed, which may be helpful to elucidate the active substances that really exert the pharmacodynamic effects of S. chinensis lignans in organisms.     

Characterization of the chemical constituents in Da‐Huang‐Gan‐Cao‐Tang by liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry and liquid chromatography coupled with ion trap mass spectrometry

In this work, the chemical constituents in Da‐Huang‐Gan‐Cao‐Tang, a traditional Chinese formula, were studied by liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry and liquid chromatography coupled with ion trap mass spectrometry for the first time. Among the 146 compounds detected in Da‐Huang‐Gan‐Cao‐Tang, 104 compounds were identified unambiguously or tentatively based on their accurate molecular weight and multistage MS data, including one potential novel compound and two reported in Glycyrrhiza genus for the first time. The possible fragmentation pathways were proposed and fragmentation rules of the major types of compounds were concluded. This study provided an example to facilitate the tedious identification of chemical composition in traditional Chinese medicine, and maybe a promising reference approach to research the analogous formulae.     

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

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

Qualitative analysis of major constituents from Xue Fu Zhu Yu Decoction using ultra high performance liquid chromatography with hybrid ion trap time‐of‐flight mass spectrometry

Xue Fu Zhu Yu Decoction, a famous formula that has been used for treating many blood stasis‐caused diseases for many centuries, comprises 11 kinds of traditional Chinese medicines. A convenient, efficient, and rapid analytical method was developed to simultaneously determine the major compounds in this decoction. An ultra‐high performance liquid chromatography with hybrid ion trap time‐of‐flight mass spectrometry method was used to rapidly separate and detect the major constituents of the decoction. Using this technique, we identified or tentatively identified 34 compounds, including 21 flavonoids, 5 terpenoids, 3 organic acids, 2 lactones, 1 alkaloid, 1 amino acid, and 1 cyanogenic glycoside. The MS analysis of these constituents was described in detail. Findings may contribute to future metabolic and pharmacokinetic studies of this medicine.     

Solid acids assisted matrix solid‐phase dispersion microextraction of alkaloids by capillary electrophoresis coupled with quadrupole time‐of‐flight mass spectrometry

The quantification of three alkaloids is important because quantitative study is a means of assessing the reliability of the experimental method, and three alkaloids of peimine, peiminine, and peimisine are main active ingredients in Chinese Pharmacopoeia 2015. An effective method based on the matrix solid‐phase dispersion microextraction was developed for the extraction of alkaloid compounds in Fritillariae Thunbergii Bulbus. Target analytes were analyzed by capillary electrophoresis coupled with quadrupole time‐of‐flight mass spectrometry. The optimized experimental condition was that 50 mg Fritillariae Thunbergii Bulbus was blended homogeneously with 10 mg citric acid for 5 min. Two hundred microliters of water acidized by 1 mol/L hydrochloric acid (pH = 4.5) was selected to elute tested alkaloids. The results demonstrated that the investigated method had low limits of detection (1.32–1.59 ng/mL), good recoveries (86.63–98.12%), and reproducibility (relative standard deviations of peak areas < 0.87%). The proposed matrix solid‐phase dispersion microextraction coupled with capillary electrophoresis combined with quadrupole time‐of‐flight mass spectrometry was successfully applied for the extraction of alkaloids in plants.     

Metabolomics approach based on ultra‐high‐performance liquid chromatography coupled with quadrupole‐time‐of‐flight mass spectrometry to identify the chemical constituents of the Traditional Chinese Er‐Zhi‐Pill

Er‐Zhi‐Pill, which consists of Ligustri lucidi fructus and Ecliptae prostratae herba , is a classical traditional Chinese medicinal formulation widely used as a liver‐nourishing and kidney‐enriching tonic. To identify the bioactive ingredients of Er‐Zhi‐Pill and characterize the variation of chemical constituents between co‐decoction and mix of individually decocted L. lucidi fructus and E. prostratae herba , a novel metabolomics approach based on ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry in both positive and negative ion modes, was established to comprehensively analyze chemical constituents and probe distinguishable chemical markers. In total, 68 constituents were unambiguously or tentatively identified through alignment of accurate molecular weights within an error margin of 5 ppm, elemental composition and fragmentation characteristics, including eight constituents, which were confirmed by comparing to reference standards. Furthermore, principal component analysis and partial least squares discriminant analysis using Simca‐p+ 12.0 software were applied to investigate chemical differences between formulations obtained by co‐decoction and a mixture of individual decoctions. Global chemical differences were found in samples of two different decoction methods, and 16 components, including salidroside, specneuzhenide and wedelolactone, contributed most to the observed differences. This study provides a basic chemical profile for the quality control and further mechanism research of Er‐Zhi‐Pill.     

Development of an analytical strategy to identify and classify the global chemical constituents of Ziziphi Spinosae Semen by using UHPLC with quadrupole time‐of‐flight mass spectrometry combined with multiple data‐processing approaches

According to traditional Chinese medical theory, Ziziphi Spinosae Semen needs to be stir‐fried before clinical application for its sedative‐hypnotic effect enhancement. A rapid and comprehensive analysis strategy of ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry and multiple data analysis platforms was developed for the efficient and sensitive identification of components in crude and parched Ziziphi Spinosae Semen to explore the composition changes that happen during the stir‐frying process. Both positive and negative ion modes were applied for mass spectrometry detection, and 40 components were identified from crude and parched Ziziphi Spinosae Semen, respectively. Principal component analysis and t‐test were applied to find differences between crude and parched Ziziphi Spinosae Semen. As a result, Ziziphi Spinosae Semen samples could be clearly divided into two groups according to their processing methods, and 19 key markers that contributed to the classification significantly (P < 0.05) were found. This kind of change in contents of components might be responsible for the recommended clinical application of parched Ziziphi Spinosae Semen.     

Characterization of the multiple chemical components of Glechomae Herba using ultra high performance liquid chromatography coupled to quadrupole‐time‐of‐flight tandem mass spectrometry with diagnostic ion filtering strategy

Glechomae Herba is a traditional Chinese medicine used for the treatment of urolithiasis, cholelithiasis, and urinary tract infections in China. Identification of chemical constituents is helpful to discover the potential active ingredients. However, this significant work is stymied by complex chemical constituents. Therefore, an ultra high performance liquid chromatography coupled to quadrupole‐time‐of‐flight tandem mass spectrometry analysis with diagnostic product ions and neutral loss filtering strategy was established for chemical profiling of Glechomae Herba. The diagnostic product ions and neutral loss filtering strategy simplified spectral elucidation. A total of 120 compounds, including 10 chlorogenic acids, 10 gallic acids, 21 phenylpropionic acids, and 77 flavonoids, were reasonably identified in Glechomae Herba. Sixty‐five constituents were first discovered in Glechomae Herba. Four types of chlorogenic acids (caffeoylquinic acid, feruloylquinic acid, p‐coumaroylquinic acid, and di‐caffeoylquinic acid), three types of galloylglucoses (di‐O‐galloyl‐glucose, tri‐O‐galloyl‐glucose, and tetra‐O‐galloyl‐glucose), three types of phenylpropionic acid skeletons (p‐coumaric acid, caffeic acid, and rosmarinic acid) and five types of flavonoid aglycone skeletons (apigenin, kaempferol, luteolin, quercetin, and chrysin) were identified in Glechomae Herba. The results indicated that the developed strategy was feasible and rational technique for identifying the complex chemical constituents in Glechomae Herba.     

Simultaneous determination of seven compounds in Chinese patent medicines Chenxiangqu by matrix solid‐phase dispersion coupled with ultra high performance liquid chromatography and quadrupole time‐of‐flight mass spectrometry

A simple, efficient, and sensitive strategy by coupling matrix solid‐phase dispersion with ultra high performance liquid chromatography quadrupole time‐of‐flight mass spectrometry was proposed to extract and determine three types of components (including seven analytes) in Chinese patent medicines Chenxiangqu. The highly ordered mesoporous material Fe‐SBA‐15 synthesized under weakly acidic conditions was selected as a dispersant in matrix solid phase dispersion extraction for the first time. Several parameters including the mass ratio of sample to dispersant, the type of dispersant, the grinding time, and the elution condition were investigated in this work. Under the optimized conditions, 20 compounds were identified by quadrupole time‐of‐flight mass spectrometry and seven analytes were quantified. The results demonstrated that the developed method has good linearity (r > 0.9995), and the limits of detection of the analytes were as low as 0.55 ng/mL. The recoveries of all seven analytes ranged from 97.6 to 104.6% (relative standard deviation < 3.4%). Finally, the improved method was successfully applied to determination of five batches of Chenxiangqu samples, which provided a robust method in quality control of Chinese patent medicines Chenxiangqu. The developed strategy also shows its great potential in analysis of complex matrix samples.     

Homoisoflavonoids profiling of Ophiopogon japonicus by off‐line coupling high‐speed countercurrent chromatography with high‐performance liquid chromatography–diode array detector‒quadrupole time‐of‐flight tandem mass spectrometry

Roots of Ophiopogon japonicus have been used as a functional food ingredient and traditional Chinese medicine for a long time in China. Homoisoflavonoids are one of the major kinds of bioactive compounds in O. japonicus; however, literature data about its homoisoflavonoids profile are scarce because of the complex ingredients with low abundance. Here, homoisoflavonoid fraction was prepared by petroleum ether extraction. Then, a high‐speed countercurrent chromatography off‐line coupling with high‐performance liquid chromatography–diode array detector?quadrupole time‐of‐flight tandem mass spectrometry was developed for systematic identification of homoisoflavonoids. After that, 39 homoisoflavonoids, including 29 homoisoflavanone and 10 homoisoflavone, were unambiguously or tentatively identified, while 12 of them were reported in O. japonicus for the first time. Finally, eight available homoisoflavonoids were sensitively, precisely, and accurately determined by standard calibration curves, with limit of detection and limit of quantification in the range of 0.05–0.30 μg/mL and 0.12–0.66 μg/mL, relative standard deviation less than 7.3% for intra‐ and interday variations, and recovery at 94.5–105.2%. Collectively, our developed method is efficient, reliable, and valuable to profile chemical components of complex natural products.     

Study on the metabolites of betulinic acid in vivo and in vitro by ultra high performance liquid chromatography with time‐of‐flight mass spectrometry

Betulinic acid is a triterpenoid organic acid with remarkable antitumor properties and is naturally present in many fruits, condiments and traditional Chinese medicines. Currently, a strategy was developed for the identification of metabolites following the in vivo and in vitro biotransformation of Betulinic acid with rat intestinal bacteria utilizing ultra high performance liquid chromatography with time‐of‐flight mass spectrometry with polymeric solid‐phase extraction. As a result, 46 metabolites were structurally characterized. The results demonstrated that Betulinic acid is universally metabolized in vivo and in vitro, and Betulinic acid could undergo general metabolic reactions, including oxidation, methylation, desaturation, loss of O and loss of CH₂. Additionally, the main metabolic pathways in vivo and in vitro were determined by calculating the relative content of each metabolite. This is the first study of Betulinic acid metabolism in vivo, whose results provide novel and useful data for better understanding of the safety and efficacy of Betulinic acid.     

Analysis of Chuanxiong Rhizoma substrate on production of ligustrazine in endophytic Bacillus subtilis by ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry

Ligustrazine was the active ingredient of the traditional Chinese medicine Chuanxiong Rhizoma. However, the content of ligustrazine is very low. We proposed a hypothesis that ligustrazine was produced by the mutual effects between endophytic Bacillus subtilis and the Ligusticum chuanxiong Hort. This study aimed to explore whether the endophytic B. subtilis LB5 could make use of Chuanxiong Rhizoma fermentation matrix to produce ligustrazine and clarify the mechanisms of action preliminarily. Ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry analysis showed the content of ligustrazine in Chuanxiong Rhizoma was below the detection limit (0.1 ng/mL), while B. subtilis LB5 produced ligustrazine at the yield of 1.0268 mg/mL in the Chuanxiong Rhizoma‐ammonium sulfate fermentation medium. In the fermented matrix, the reducing sugar had a significant reduction from 12.034 to 2.424 mg/mL, and rough protein content increased from 2.239 to 4.361 mg/mL. Acetoin, the biosynthetic precursor of ligustrazine, was generated in the Chuanxiong Rhizoma‐Ammonium sulfate (151.2 mg/mL) fermentation medium. This result showed that the endophytic bacteria B. subtilis LB5 metabolized Chuanxiong Rhizoma via secreted protein to consume the sugar in Chuanxiong Rhizoma to produce a considerable amount of ligustrazine. Collectively, our preliminary research suggested that ligustrazine was the interaction product of endophyte, but not the secondary metabolite of Chuanxiong Rhizoma itself.     

Comparison of the origin and phenolic contents of Lycium ruthenicum Murr. by high‐performance liquid chromatography fingerprinting combined with quadrupole time‐of‐flight mass spectrometry and chemometrics

The fruits of Lycium ruthenicum Murr. have long been used in folk medicine. Nevertheless, detailed information related to its phenolic composition and its quality control remains scarce. In this study, a simple and reproducible method, based on high‐performance liquid chromatography combined with chemometrics, was developed to authenticate 18 samples of L. ruthenicum Murr. collected from different parts of China through fingerprint analysis. The main peaks were identified by quadrupole time‐of‐flight electrospray ionization mass spectrometry. Four phenolics were quantified, and the most abundant phenolic compound in almost all samples was kukoamine A. Hierarchical cluster analysis and principal component analysis were applied to classify these samples. Also, a total of 26 compounds, which were mainly phenolic compounds and anthocyanins, were identified or tentatively identified based on the available literature and standard references. Among these, 16 were reported for the first time in the extract. The results showed that there was no significant difference between L. ruthenicum fruits from different provinces in terms of chemical composition. Also, the fingerprint together with chemometric analyses and quadrupole time‐of‐flight electrospray ionization mass spectrometry are promising methods for evaluating the quality consistency, identification, and comprehensive evaluation of L. ruthenicum .     

Comprehensive characterization of multiple components and metabolites of Xiaojin Capsule based on ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry

Xiaojin Capsule, a classic traditional Chinese medicine formula, has been used to treat mammary cancer, thyroid nodules, and hyperplasia of the mammary glands. However, its systematic chemical information remained unclear, which hindered the interpretation of the pharmacology and the mechanism of action of this drug. In this research, an ultra high performance liquid chromatography coupled with a quadrupole time‐of‐flight mass spectrometry method was developed to identify the complicated components and metabolites of Xiaojin Capsule. Two acquisition modes, including the MS^Energy mode and fast data directed acquisition mode, were utilized for chemical profiling. As a result, 156 compounds were unambiguously or tentatively identified by comparing their retention times and mass spectrometry data with those of reference standards or literature. After the oral administration of Xiaojin Capsule, 53 constituents, including 24 prototype compounds and 29 metabolites, were detected in rat plasma. The obtained results were beneficial for a better understanding of the therapeutic basis of Xiaojin Capsule. A high‐resolution and efficient separation method was firstly established for systematically characterizing the compounds of Xiaojin Capsule and the associated metabolites in vivo, which could be helpful for quality control and pharmacokinetic studies of this medicine.     

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.     

Systematic characterization of flavonoids from Siraitia grosvenorii leaf extract using an integrated strategy of high‐speed counter‐current chromatography combined with ultra high performance liquid chromatography and electrospray ionization quadrupole time‐of‐flight mass spectrometry

The chemical constituents of the Siraitia grosvenorii leaf extract were studied. Firstly, high‐speed counter‐current chromatography was applied to the one‐step separation of four compounds from S. grosvenorii leaf extract with the solvent system composed of 0.01% acetic acid water/n‐butanol/n‐hexane/methanol (5:3:1:1, v/v/v/v). In this work, 270 mg of crude sample yielded four compounds, a new kaempferol O‐glycoside derivative, kaempferol 3‐O‐α‐L‐[4‐O‐(4‐carboxy‐3‐hydroxy‐3‐methylbutanoyl)]‐rhamnopyranoside‐7‐O‐α‐L‐rhamnopyranoside, named kaempferitrin A (2.1 mg, 90%), and three known compounds, grosvenorine (3.4 mg, 93%), kaempferitrin (14.4 mg, 99%) and afzelin (4 mg, 98%), and the structures of these compounds were identified by NMR spectroscopy and mass spectrometry. Then, ultra high performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight mass spectrometry was used to illustrate the dominant flavonoids in S. grosvenorii leaf extract. 34 flavonoids including 19 kaempferol O‐glycosides, 4 quercetin O‐glycosides, 6 flavanone derivatives, and 5 polymethoxyflavones, were accurately or tentatively identified by carefully comparing their retention times, UV data, precise masses, the typical fragments of the standards and literature data. Most of these compounds were reported for the first time. This study establishes a foundation for the further development and utilization of S. grosvenorii leaves in future.