Rapid identification of efavirenz metabolites in rats and humans by ultra high performance liquid chromatography combined with quadrupole time‐of‐flight tandem mass spectrometry

An in vivo study of efavirenz metabolites in rats and human patients with ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry combined with MetabolitePilot^MT software is reported for the first time. Considering the polarity differences between the metabolites, solid‐phase extraction and protein precipitation were both applied as a part of the sample preparation method. The structures of the metabolites and their fragment ions were identified or tentatively characterized based on the accurate mass and MS² data. As a result, a total of 15 metabolites, including 11 from rat samples and 13 from human samples, were identified or tentatively characterized. Two metabolites and several new metabolism pathways are reported for the first time. This study provides a practical approach for identifying complicated metabolites through the rapid and reliable ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry technique, which could be widely used for the investigation of drug metabolites.     

Characterization of the multiple components of Acanthopanax Senticosus stem by ultra high performance liquid chromatography with quadrupole time‐of‐flight tandem mass spectrometry

Acanthopanax Senticosus Harms. has been used widely in traditional Chinese medicine for the treatment of chronic bronchitis, neurasthenia, hypertension and ischemic heart disease. However, the in vivo constituents of the stem of Acanthopanax Senticosus remain unknown. In this paper, ultra high performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight mass spectrometry and the MarkerLynx^TM software combined with multiple data processing approach were used to study the constituents in vitro and in vivo. The aqueous extract from the Acanthopanax Senticosus stem and the compositions in rat serum after intragastric administration were completely analyzed. Consequently, 115 compounds in the aqueous extract from Acanthopanax Senticosus stem and 41 compounds absorbed into blood were characterized. Of the 115 compounds in vitro, 54 were reported for first time, including sinapyl alcohol, sinapyl alcohol diglucoside, and 1‐O‐sinapoyl‐β‐d ‐glucose. In the 41 compounds in vivo, 7 were prototype components and 34 were metabolites which were from 21 components of aqueous extract from Acanthopanax Senticosus stem, and the metabolic pathways of the metabolites were elucidated for first time. The results narrowed the range of screening the active components and provided a basis for the study of action mechanism and pharmacology.     

Metabolite identification of tanshinol borneol ester in rats by high‐performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight mass spectrometry

Tanshinol borneol ester (DBZ) is a potential drug candidate composed of danshensu and borneol. It shows anti‐ischemic and anti‐atherosclerosis activity. However, little is known about its metabolism in vivo. This research aimed to elucidate the metabolic profile of DBZ through analyzing its metabolites using high‐performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight mass spectrometry. Chromatographic separation was performed on an Agilent TC‐C₁₈ column (150 × 4.6 mm, 5.0 μm) with gradient elution using methanol and water containing 0.2% (v/v) formic acid as the mobile phase. Metabolite identification involved analyzing the retention behaviors, changes in molecular weights and MS/MS fragment patterns of DBZ and its metabolites. As a result, 20 potential metabolites were detected and tentatively identified in rat plasma, urine and feces after administration of DBZ. DBZ could be metabolized to O‐methylated DBZ, DBZ‐O‐glucuronide, O‐methylated DBZ‐O‐glucuronide, hydroxylated DBZ and danshensu. Danshensu, a hydrolysis product of DBZ, could further be transformed into 12 metabolites. The proposed method was confirmed to be a reliable and sensitive alternative for characterizing metabolic pathways of DBZ and providing valuable information on its druggability.     

Comprehensive metabolite profiling of Plantaginis Semen using ultra high performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry coupled with elevated energy technique

Plantaginis Semen is commonly used in traditional medicine to treat edema, hypertension, and diabetes. The commercially available Plantaginis Semen in China mainly comes from three species. To clarify the chemical composition and distinct different species of Plantaginis Semen, we established a metabolite profiling method based on ultra high performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry coupled with elevated energy technique. A total of 108 compounds, including phenylethanoid glycosides, flavonoids, guanidine derivatives, terpenoids, organic acids, and fatty acids, were identified from Plantago asiatica L., P. depressa Willd., and P. major L. Results showed significant differences in chemical components among the three species, particularly flavonoids. This study is the first to provide a comprehensive chemical profile of Plantaginis Semen, which could be involved into the quality control, medication guide, and developing new drug of Plantago seeds.     

Rapid characterization of the chemical constituents and rat metabolites of the Wen‐Jing decoction by ultra high performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

The Wen‐Jing decoction, a traditional Chinese medicine formula, has been used as a blood‐activating and stasis‐eliminating drug to treat gynaecological syndromes, such as dysmenorrhea, amenorrhea, and menstrual disorders. However, its pharmacodynamic material basis and mechanism of action have not been thoroughly elucidated to date. The goal of this study was to characterize and identify multiple constituents and metabolites in Wen‐Jing decoction. An ultra high performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry method was established and validated in the present study for the first time. A total of 101 compounds, including 11 monoterpene glycosides, 19 flavonoids, 49 triterpene saponins, 5 phthalides, 3 phytoecdysones, and 14 others, were unambiguously or tentatively characterized by comparing their retention times and MS data with reference standards or with data reported in the literature. After oral administration of Wen‐Jing decoction, 27 compounds, including nine prototype compounds and 18 metabolites were detected in rat plasma. Thus, the ultra high performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry method was found to be efficient for in‐depth structural elucidation of chemical compounds in complex matrices of herbal medicines, which will provide useful chemical information for quality control and mechanism‐of‐action research.     

Characterization of chemical constituents in Zhi–Zi–Da–Huang decoction by ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry

A sensitive and reliable ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry method was established to separate and identify the chemical constituents of Zhi–Zi–Da–Huang decoction, a classic traditional Chinese medicine formula. The chromatographic separation was achieved on a Shim‐pack XR‐ODS C₁₈ column (75  × 3.0 mm, 2.2 μm) using a gradient elution program. The detection was performed on a Waters Xevo G2 Q‐TOF mass spectrometer equipped with electrospray ionization source in both positive and negative modes. With the optimized conditions, a total of 82 compounds were identified or tentatively characterized. Of the 82 compounds, 21 compounds were identified by comparing the retention time and MS data with reference standards, the rest were characterized by analyzing MS data and retrieving the reference literature. In addition, 31 compounds were identified from Gardenia jasminoides Ellis, ten compounds were identified from Rheum palmatum L., 33 compounds were identified from Citrus aurantium L., and eight compounds were identified from Sojae Semen Praeparatum. Results indicated that iridoids, anthraquinones, flavonoids, isoflavonoids, coumarins, glycosides of crocetin, monoterpenoids, and organic acids were major constituents in Zhi–Zi–Da–Huang decoction. It is concluded that the developed ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry method with high sensitivity and resolution is suitable for identifying and characterizing the chemical constituents of Zhi–Zi–Da–Huang decoction, and the analysis provides a helpful chemical basis for further research on Zhi–Zi–Da–Huang decoction.     

Characterization and identification of saponins in Achyranthes bidentata by rapid‐resolution liquid chromatography with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

A rapid‐resolution liquid chromatography (RRLC) method coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (Q‐TOF MS/MS) has been developed for analysis of oleanane‐type triterpenoid saponins in Achyranthes bidentata. Collision‐induced dissociation techniques were used to fragment the precursor molecular ions and the resulting product ions. A retro‐Diels‐Alder rearrangement from the oleanane aglycone skeleton in the MS/MS process yielded characteristic fragment ions in positive ion mode. These characteristic ions were helpful in predicting the aglycone structure. Losses of monosaccharide sequences, presence of sugar‐chain fragment ions, and cleavage of CO₂ were observed for important information on sugar types and attachment sequences. Fragmentation rules of three major groups of saponins from A. bidentata were summarized, and the possible fragmentation pathways were proposed. A total of 22 compounds including both the target and unknown oleanane‐type triterpenoid saponins were rapidly screened and predicted in the herbal extract by the developed method. The RRLC‐Q‐TOF MS/MS method has provided a powerful approach for rapid separation, target screening and structural elucidation of oleanane‐type saponins, and also opened perspectives for similar studies on other herbal medicines. Copyright © 2010 John Wiley & Sons, Ltd.     

Chemical fingerprint of Ganmaoling granule by double‐wavelength ultra high performance liquid chromatography and ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry

A method incorporating double‐wavelength ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry was developed for the investigation of the chemical fingerprint of Ganmaoling granule. The chromatographic separations were performed on an ACQUITY UPLC HSS C₁₈ column (2.1 × 50 mm, 1.8 μm) at 30°C using gradient elution with water/formic acid (1%) and acetonitrile at a flow rate of 0.4 mL/min. A total of 11 chemical constituents of Ganmaoling granule were identified from their molecular weight, UV spectra, tandem mass spectrometry data, and retention behavior by comparing the results with those of the reference standards or literature. And 25 peaks were selected as the common peaks for fingerprint analysis to evaluate the similarities among 25 batches of Ganmaoling granule. The results of principal component analysis and orthogonal projection to latent structures discriminant analysis showed that the important chemical markers that could distinguish the different batches were revealed as 4,5‐di‐O‐caffeoylquinic acid, 3,5‐di‐O‐caffeoylquinic acid, and 4‐O‐caffeoylquinic acid. This is the first report of the ultra high performance liquid chromatography chemical fingerprint and component identification of Ganmaoling granule, which could lay a foundation for further studies of Ganmaoling granule.     

Characterisation of oxazepam degradation products by high‐performance liquid chromatography/electrospray ionisation mass spectrometry and electrospray ionisation quadrupole time‐of‐flight tandem mass spectrometry

Oxazepam has been subjected to controlled degradation at 100°C for 3 h in 0.5 M HCl and 0.5 M NaOH. Following neutralisation of the degradation mixture and removal of salts by solid‐phase extraction (SPE), isocratic high‐performance liquid chromatography/mass spectrometry (HPLC/MS) using water/methanol (25:75 v/v) as the mobile phase was carried out using a flow diverter to collect fractions prior to their characterisation by electrospray ionisation multi‐stage mass spectrometry (ESI‐MSⁿ) and proposal of the corresponding fragmentation patterns. The elemental compositions of the degradation products and their MS fragments were evaluated using electrospray ionisation quadrupole time‐of‐flight tandem mass spectrometry (ESI‐QTOF‐MS/MS) which was then used to support the proposed fragmentation patterns. Copyright © 2010 John Wiley & Sons, Ltd.     

Rapid separation and identification of major constituents in Pseudolarix kaempferi by ultra‐performance liquid chromatography coupled with electrospray and quadrupole time‐of‐flight tandem mass spectrometry

A rapid and reliable method based on ultra‐performance liquid chromatography (UPLC) coupled with photodiode‐array detection (PDA) and electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (ESI‐Q‐TOF‐MS/MS) has been developed for separation and identification of major constituents in extracts of root bark of Pseudolarix kaempferi Gordon (PKG). Identification of the constituents was carried out by interpretation of their retention times, UV absorption spectra, MS and MS/MS spectra, as well as the data provided by authentic standards and literatures. A total of 20 components were separated in only 8.0 min on a small particle size C₁₈ column (1.7 µm). These components included nine diterpene acids, seven glycosides and four triterpenoids, among which pseudolaric acid C‐O‐β‐D‐glucopyranoside and pseudolaric acid C₂‐O‐β‐D‐glucopyranoside were separated and identified for the first time in this study. Furthermore, the fragmentation patterns of the three types of compounds were elucidated for the first time. This established UPLC‐PDA/Q‐TOF‐MS/MS method is reliable and effective for the separation and identification of the 20 compounds and will be useful for quality control of the crude materials of Pseudolarix kaempferi Gordon and their related preparations. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification of metabolites of rupestonic acid in rat urine by liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

Rupestonic acid, a potential anti‐influenza agent, is an important and characteristic compound in Artemisia rupestris L., a well‐known traditional Uighur medicine for the treatment of colds. In the present study, high‐performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry was used to detect and identify the metabolites in rat urine after oral administration of rupestonic acid. A total of 10 metabolites were identified or partially characterized. The structure elucidations of the metabolites were performed by comparing the changes in accurate molecular masses and fragment ions with those of the parent compound. The results showed that the main metabolites of rupestonic acid in rat urine were formed by oxidation, hydrogenation and glucuronidation. A metabolism pathway was proposed for the first time based on the characterized structures. This metabolism study can provide essential information for drug discovery, design and clinical application of rupestonic acid. Copyright © 2014 John Wiley & Sons, Ltd.     

Identification of the metabolites of gigantol in rat urine by ultra‐performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

Gigantol is a typical bibenzyl compound isolated from Dendrobii Caulis that has been widely used as a medicinal herb in China for the treatment of diabetic cataract, cancer and arteriosclerosis obliterans and as a tonic for stomach nourishment, saliva secretion promotion and fever reduction. However, few studies have been carried out on its in vivo metabolism. In the present study, a rapid and sensitive method based on ultra‐performance liquid chromatography/electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (UPLC‐Q/TOF‐MS) in positive ion mode was developed and applied to identify the metabolites of gigantol in rat urine after a single oral dose (100 mg/kg). Chromatographic separation was performed on an Acquity UPLC HSS T3 column (100 × 2.1 mm i. d., 1.8 µm) using acetonitrile and 0.1% aqueous formic acid as mobile phases. A total of 11 metabolites were detected and identified as all phase II metabolites. The structures of the metabolites were identified based on the characteristics of their MS, MS² data and chromatographic retention times. The results showed that glucuronidation is the principal metabolic pathway of gigantol in rats. The newly identified metabolites are useful to understand the mechanism of elimination of gigantol and, in turn, its effectiveness and toxicity. As far as we know, this is the first attempt to investigate the metabolic fate of gigantol in vivo. Copyright © 2014 John Wiley & Sons, Ltd.     

Separation and identification of phenolic compounds in Bidens pilosa L. by ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry

A validated method based on ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry was established to separate and identify phenolic compounds in Bidens pilosa L. Mass spectrometry experiments were performed both in positive and negative ion modes. A total of 35 compounds were detected, and 26 phenolic compounds were unequivocally identified or tentatively assigned based on retention time, maximum UV absorption, molecular formula, and fragments. The ultra high performance liquid chromatography method was validated and showed good linearity (R² ≧ 0.9996) over the test range. The limits of detection and quantification were above 0.072 and 0.162 μg/mL, respectively. The relative standard deviations of intraday and interday precision were below 0.3 and 1.6%, respectively.     

Analysis of ascorbic acid based black powder substitutes by high‐performance liquid chromatography/electrospray ionization quadrupole time‐of‐flight mass spectrometry

Black powder substitutes are an important sub‐group of explosive propellants in the United States because they are readily accessible, and can be used as fillers for improvised explosive devices. Many brands of black powder substitutes incorporate an ascorbic acid fuel source with potassium nitrate (KNO₃) and/or potassium perchlorate (KClO₄) oxidizer(s). A gradient high‐performance liquid chromatography/electrospray ionization quadrupole time‐of‐flight mass spectrometry (HPLC/ESI‐QToFMS) method has been developed for the analysis of both the organic and the inorganic constituents. The HPLC/ESI‐QToFMS method was utilized to examine aqueous extracts of intact samples and post‐burn residues from six different brands of ascorbic acid based powders. Aqueous extracts of the post‐blast residues from two brands of ascorbic acid based propellant were also analyzed. The results showed that both the ascorbic acid fuel and the inorganic oxidizer(s) KClO₄ and/or KNO₃ were successfully detected via the [M–H]^? ion of ascorbic acid and the anions (ClO and NO) of the oxidizers. This method was proven to be a rapid and efficient procedure for the analysis of this class of explosives. The high mass resolution provided by the QToFMS instrument fulfills the degree of certainty required in a court of law. Published in 2010 by John Wiley & Sons, Ltd.     

Characterization of ginsenoside compound K metabolites in rat urine and feces by ultra‐performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

Ginsenoside compound K (CK) is an active metabolite of ginsenoside and has been shown to have ameliorative property in various diseases. However, the detailed in vivo metabolism of this compound has rarely been reported. In the present study, a method using liquid chromatography quadrupole time‐of‐flight tandem mass spectrometry together with multiple data processing techniques, including extracted ion chromatogram, multiple mass defect filter and MS/MS scanning, was developed to detect and characterize the metabolites of CK in rat urine and feces. After oral administration of CK at a dose of 50 mg/kg, urine and feces were collected for a period of time and subjected to a series of pretreatment. A total of 12 metabolites were tentatively or conclusively identified, comprising 11 phase I metabolites and a phase II metabolite. Metabolic pathways of CK has been proposed, including oxidation, deglycosylation, deglycosylation with sequential oxidation and dehydrogenation and deglycosylation with sequential glucuronidation. Relative quantitative analyses suggested that deglycosylation was the main metabolic pathway. The result could offer insights for better understanding of the mechanism of its pharmacological activities.     

Global chemical profiling based quality evaluation approach of rhubarb using ultra performance liquid chromatography with tandem quadrupole time‐of‐flight mass spectrometry

A global chemical profiling based quality evaluation approach using ultra performance liquid chromatography with tandem quadrupole time‐of‐flight mass spectrometry was developed for the quality evaluation of three rhubarb species, including Rheum palmatum L., Rheum tanguticum Maxim. ex Balf., and Rheum officinale Baill. Considering that comprehensive detection of chemical components is crucial for the global profile, a systemic column performance evaluation method was developed. Based on this, a Cortecs column was used to acquire the chemical profile, and Chempattern software was employed to conduct similarity evaluation and hierarchical cluster analysis. The results showed R. tanguticum could be differentiated from R. palmatum and R. officinale at the similarity value 0.65, but R. palmatum and R. officinale could not be distinguished effectively. Therefore, a common pattern based on three rhubarb species was developed to conduct the quality evaluation, and the similarity value 0.50 was set as an appropriate threshold to control the quality of rhubarb. A total of 88 common peaks were identified by their accurate mass and fragmentation, and partially verified by reference standards. Through the verification, the newly developed method could be successfully used for evaluating the holistic quality of rhubarb. It would provide a reference for the quality control of other herbal medicines.