Characterization of protostane triterpenoids in Alisma orientalis by ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry

A reliable and sensitive ultra‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry (UPLC/Q‐TOF‐MS) method has been optimized and established for analysis of protostane triterpenoids in a commonly used traditional Chinese herbal medicine Alisma orientalis (Sam.) Juzep. The separation of crude extract of A. orientalis was achieved on a Waters ACQUITY HSS T3 column (100 mm × 2.1 mm, 1.8 µm) eluting with 0.1% (v/v) formic acid/acetonitrile. A total of 20 protostane triterpenoids including 19 known compounds and a new one were well separated within 7 min. The collision‐induced dissociation (CID) tandem mass spectrometric (MS/MS) fragmentation patterns of protostane triterpenoids was firstly reported in this study. The hydrogen rearrangement at the C‐23‐OH leads to dissociation of the bond between C‐23 and C‐24 in the protostane triterpenoid skeleton during the CID process. This dissociation was the characteristic CID fragmentation pathway of this class of triterpenoids, and was useful for further differentiation of some positional isomers which contain an acetyl unit on the C‐23 or C‐24 position. The identities of isolated compounds were identified by comparing their retention times and CID fragmentation behaviors with those of reference standards or tentatively assigned by matching the empirical molecular formulae with those reported in the literature. It is concluded that this newly established UPLC/Q‐TOF‐MS method is a powerful approach for structural elucidation of protostane triterpenoids isolated from A. orientalis. Copyright © 2010 John Wiley & Sons, Ltd.     

Metabolic profiles of dioscin in rats revealed by ultra‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry

Dioscin (DIS), one of the most abundant bioactive steroidal saponins in Dioscorea sp., is used as a complementary medicine to treat coronary disease and angina pectoris in China. Although the pharmacological activities and pharmacokinetics of DIS have been well demonstrated, information regarding the final metabolic fates is very limited. This study investigated the in vivo metabolic profiles of DIS after oral administration by ultra‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry method. The structures of the metabolites were identified and tentatively characterized by means of comparing the molecular mass, retention time and fragmentation pattern of the analytes with those of the parent compound. A total of eight metabolites, including seven phase I and one phase II metabolites, were detected and tentatively identified for the first time. Oxidation, deglycosylation and glucuronidation were found to be the major metabolic processes of the compound in rats. In addition, a possible metabolic pathway on the biotransformation of DIS in vivo was proposed. This study provides valuable and new information on the metabolism of DIS, which will be helpful for further understanding its mechanism of action. Copyright © 2015 John Wiley & Sons, Ltd.     

Rapid screening and identification of lycodine‐type alkaloids in Lycopodiaceae and Huperziaceae plants by ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry

Lycodine‐type alkaloids have gained significant interest owing to their unique skeletal characteristics and acetylcholinesterase activity. This study established a rapid and reliable method using ultra‐performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (UPLC‐ESI‐Q/TOF‐MS/MS) for comprehensive characterization of lycodine‐type alkaloids for the first time. The lycodine‐type alkaloids were detected successfully from Lycopodiastrum casuarinoides, Huperzia serrata and Phlegmarirus carinatus in seven plants of the Lycopodiaceae and Huperziaceae families, based on the established characteristic MS fragmentation of five known alkaloids. Furthermore, a total of 13 lycodine‐type alkaloids were identified, of which three pairs of isomers were structurally characterized and differentiated. This study further improves mass analysis of lycodine‐type alkaloids and demonstrates the superiority of UPLC with a high‐resolution mass spectrometer for the rapid and sensitive structural elucidation of other trace active compounds. Copyright © 2016 John Wiley & Sons, Ltd.     

Serum metabolomics of laryngeal cancer based on liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry

The discovery of new laryngeal cancer‐related metabolite biomarkers could help to facilitate early diagnosis. A serum metabolomics study from laryngeal cancer patients and healthy individuals was conducted using liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry. Univariate and multivariate statistics were used to discriminate laryngeal cancer patients and healthy individuals. 1‐Palmitoyl‐sn‐glycero‐3‐phosphocholine (LysoPC 16:0), 1‐o‐hexadecyl‐2‐acetyl‐sn‐glycero‐3‐phosphocholine (PAF) and 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine were found to be significantly different between the laryngeal cancer group and the healthy group. They are mainly involved in phospholipids catabolism, linoleic acid metabolism, α‐linoleic acid metabolism and arachidonic acid metabolism. The area under the curve of the biomarker combined by two metabolites (LysoPC 16:0 and PAF) was 0.935, the sensitivity was 0.962 and the specificity was 0.825. LysoPC 16:0 and PAF may show diagnostic potential for laryngeal carcinoma.     

Identification of four cornua by ultra‐performance liquid chromatography with time‐of‐flight mass spectrometry coupled with principal component analysis

An ultra‐high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry method coupled with principal component analysis was developed and applied to the identification of Cornu Antelopis, Cornu Bubali, Cornu Naemorhedi, and Cornu Bovis. The data obtained from the trypsin‐digested samples were subjected to principal component analysis to classify these four cornua. Additionally, marker peptides of the cornua were determined by orthogonal partial least‐squares discriminant analysis, and fragmentation tandem mass spectra of these marker peptides were evaluated. The results from this study indicate that the proposed method is reliable, and it has been successfully applied to the identification of variants of cornua commonly used in traditional Chinese medicine.     

Tissue distribution and excretion study of neopanaxadiol in rats by ultra‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry

Neopanaxadiol (NPD), a major ginsenoside in Panax ginseng C. A. Meyer (Araliaceae), was reported to have neuroprotective effect. In this study, a method of ultra‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry (UPLC/QTOF‐MS) was developed and validated for quantitative analysis of NPD in tissues, urine and feces, using liquid–liquid extraction (LLE) to isolate NPD from different biological samples, and chromatographic separation was performed on an Agilent Zorbax Stable Bond C₁₈ (2.1 × 50 mm, 1.8 µm) column with 0.1% formic acid in water and acetonitrile. All standard calibration curves were linear (all r² > 0.995) within the test range. After oral administration, NPD was extensively distributed to most of the tissues without long‐term accumulation. The higher levels were observed in stomach and intestine, followed by kidney and liver. Approximately 64.56 ± 20.32% of administered dose in feces and 0.0233 ± 0.0356% in urine were found within 96 h, which indicated that the major elimination route was fecal excretion. This analytical method was applied to the study of NPD distribution and excretion in rats after oral intake for the first time. The results we found here are helpful for us to understand the pharmacological effects of NPD, as well as its toxicity. Copyright © 2014 John Wiley & Sons, Ltd.     

Pharmaceutical metabolite profiling using quadrupole/ion mobility spectrometry/time‐of‐flight mass spectrometry

The use of hybrid quadrupole ion mobility spectrometry time‐of‐flight mass spectrometry (Q/IMS/TOFMS) in the metabolite profiling of leflunomide (LEF) and acetaminophen (APAP) is presented. The IMS drift times (T_d) of the drugs and their metabolites were determined in the IMS/TOFMS experiments and correlated with their exact monoisotopic masses and other in silico generated structural properties, such as connolly molecular area (CMA), connolly solvent‐excluded volume (CSEV), principal moments of inertia along the X, Y and Z Cartesian coordinates (MI‐X, MI‐Y and MI‐Z), inverse mobility and collision cross‐section (CCS). The correlation of T_d with these parameters is presented and discussed. IMS/TOF tandem mass spectrometry experiments (MS² and MS³) were successfully performed on the N‐acetyl‐p‐benzoquinoneimine glutathione (NAPQI‐GSH) adduct derived from the in vitro microsomal metabolism of APAP. As comparison, similar experiments were also performed using hybrid triple quadrupole linear ion trap mass spectrometry (QTRAPMS) and quadrupole time‐of‐flight mass spectrometry (QTOFMS). The abilities to resolve the product ions of the metabolite within the drift tube and fragment the ion mobility resolved product ions in the transfer travelling wave‐enabled stacked ring ion guide (TWIG) demonstrated the potential applicability of the Q/IMS/TOFMS technique in pharmaceutical metabolite profiling. Copyright © 2009 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.     

A strategy for screening and identifying mycotoxins in herbal medicine using ultra‐performance liquid chromatography with tandem quadrupole time‐of‐flight mass spectrometry

The objective of this study was to develop an effective strategy for screening and identifying mycotoxins in herbal medicine (HM). Here, Imperatae Rhizoma, a commonly used Chinese herb, was selected as a model HM. A crude drug contaminated with fungi was analyzed by comparing with uncontaminated ones. Ultra‐performance LC coupled to tandem quadrupole TOF‐MS (UPLC–Q‐TOF‐MS) with collision energy function was applied to analyze different samples from Imperatae Rhizoma. Then, MarkerLynx^TM software was employed to screen the excess components in analytes, compared with control samples, and those selected markers were likely to be the metabolites of fungi. Furthermore, each of the accurate masses of the markers obtained from MarkerLynx^TM was then searched in a mycotoxins/fungal metabolites database established in advance. The molecular formulas with relative mass error between the measured and theoretical mass within 5 ppm were chosen and then applied to MassFragment^TM analysis for further confirmation of their structures. With the use of this approach, five mycotoxins that have never been reported in HM were identified in contaminated Imperatae Rhizoma. The results demonstrate the potential of UPLC–Q‐TOF‐MS coupled with the MarkerLynx^TM software and MassFragment^TM tool as an efficient and convenient method to screen and identify mycotoxins in herbal materials and aid in the quality control of HM.     

Identification and metabolite profiling of alkaloids in aerial parts of Papaver rhoeas by liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry

Papaver plants can produce diverse bioactive alkaloids. Papaver rhoeas Linnaeus (common poppy or corn poppy) is an annual flowering medicinal plant used for treating cough, sleep disorder, and as a sedative, pain reliever, and food. It contains various powerful alkaloids like rhoeadine, benzylisoquinoline, and proaporphine. To investigate and identify alkaloids in the aerial parts of P. rhoeas, samples were collected at different growth stages and analyzed using liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry. A liquid chromatography with mass spectrometry method was developed for the identification and metabolite profiling of alkaloids for P. rhoeas by comparing with Papaver somniferum. Eighteen alkaloids involved in benzylisoquinoline alkaloid biosynthesis were used to optimize the liquid chromatography gradient and mass spectrometry conditions. Fifty‐five alkaloids, including protoberberine, benzylisoquinoline, aporphine, benzophenanthridine, and rhoeadine‐type alkaloids, were identified authentically or tentatively by liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry in samples taken during various growth stages. Rhoeadine alkaloids were observed only in P. rhoeas samples, and codeine and morphine were tentatively identified in P. somniferum. The liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry method can be a powerful tool for the identification of diverse metabolites in the genus Papaver. These results may help understand the biosynthesis of alkaloids in P. rhoeas and evaluate the quality of this plant for possible medicinal applications.     

Characterization of tropane and cinnamamide alkaloids from Scopolia tangutica by high‐performance liquid chromatography with quadrupole time‐of‐flight tandem mass spectrometry

Scopolia tangutica is a traditional Chinese medicine used for antispasmodic, anesthesia, analgesia, and sedation. Its medicinal activity is associated to alkaloid constituents, including tropane and cinnamamide types. Low content of alkaloids in plant makes them difficult to be isolated and identified. The present work developed an effective method to quickly characterize alkaloids from Scopolia tangutica by high‐performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry. Thirteen reference compounds were studied for their fragmentation pathways, including five tropane alkaloids and eight cinnamamide ones. Alkaloid constituent was analyzed by an optimized high‐performance liquid chromatography method and mass spectrometry analysis to achieve systematic characterization of alkaloids from Scopolia tangutica. As a result, 53 compounds were identified, including 21 tropane alkaloids (eight new ones), 18 caffeoyl ones (ten new ones) and 14 dicaffeoyl ones (seven new ones). It was important to provide rich information in phytochemical study and structure‐guided isolation of important compounds from this plant.     

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.     

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.     

Metabolic profiling comparison of isovitexin in normal and kidney stone model rats by ultra‐high‐performance liquid chromatography coupled to quadrupole time‐of‐flight mass spectrometry

Isovitexin, a bioactive flavonoid constituent isolated from Desmodii Styracifolii, is considered an adjuvant for antiurolithiasis diseases. In this study, an ultra‐high‐performance liquid chromatography coupled with hybrid triple quadruple time‐of‐flight mass spectrometry method was developed to characterize and compare the metabolic profiling of isovitexin experimented on normal and kidney stone model rats. The comparative research indicated that 28 metabolites (18 phase I and 10 phase II) in normal rats and 33 metabolites (20 phase I and 13 phase II) in kidney stone model rats were initially identified. The results of relative quantitative determination reflected that the contents of metabolites produced by deglycosylation, reduction, and isomerization in kidney stone model rats were greater than those in healthy rats. Instead, the levels of oxidative and dehydrogenated metabolites in normal groups were higher than those in kidney stone model groups. The results of this study are valuable and important for understanding the metabolic process of isovitexin in clinical application, and especially the metabolism study in kidney stone model rats could provide a beneficial reference for the further search of effective substances associated with the treatment of kidney stones.     

Identification of metabolites of Helicid in vivo using ultra‐high performance liquid chromatography‐quadrupole time‐of‐flight mass spectrometry

Helicid is an active natural aromatic phenolic glycoside ingredient originating from a well‐known traditional Chinese herbal medicine and has the significant effects of sedative hypnosis, anti‐inflammatory analgesia and antidepressant. In this study, we analyzed the potential metabolites of Helicid in rats by multiple mass defect filter and dynamic background subtraction in ultra‐high‐performance liquid chromatography–quadrupole time‐of‐flight mass spectrometry (UHPLC‐Q‐TOF‐MS). Moreover, we used a novel data processing method, ‘key product ions’, to rapidly detect and identify metabolites as an assistant tool. MetabolitePilot™ 2.0 software and PeakView™ 2.2 software were used for analyzing metabolites. Twenty metabolites of Helicid (including 15 phase I metabolites and five phase II metabolites) were detected by comparison with the blank samples. The biotransformation route of Helicid was identified as demethylation, oxidation, dehydroxylation, hydrogenation, decarbonylation, glucuronide conjugation and methylation. This is the first study simultaneously detecting and identifying Helicid metabolism in rats employing UHPLC‐Q‐TOF‐MS technology. This experiment not only proposed a method for rapidly detecting and identifying metabolites, but also provided useful information for further study of the pharmacology and mechanism of Helicid in vivo. Furthermore, it provided an effective method for the analysis of other aromatic phenolic glycosides metabolic components in vivo.     

Evaluation and validation of an ion mobility quadrupole time‐of‐flight mass spectrometry pesticide screening approach

An ion mobility quadrupole time‐of‐flight mass spectrometry‐based pesticide suspect screening methodology was developed and validated covering 20 plant‐derived food matrices deriving from six commodity groups of different complexity according to the actual European Commission document SANTE/11813/2017 applying a QuEChERS sample preparation protocol. The method combines ultra‐performance liquid chromatography, traveling wave ion mobility, and quadrupole time‐of‐flight mass spectrometry. Besides the determination of the physicochemical property collision cross‐section and the establishment of a corresponding scientific suspect screening database comprising 280 pesticides for several pesticides, different protomers, sodium adducts, as well as dimers were identified in ion mobility spectrometry traces. Additionally, collision cross‐section values were included in the validation requirements regarding chromatography and mass spectrometry for the detection of pesticides. A collision cross‐section value window was analyzed within a tolerable error of ±2%. For this cross‐matrix validation, screening detection limits were determined at concentration levels of 0.100 mg/kg (84% of the original pesticide scope), 0.010 mg/kg (56%), and 0.001 mg/kg (21%). By application of ion mobility spectrometry, the compound identification was improved due to independence of commodity of concern and concentration levels of analyte molecules, as false assignments are reduced by application of a collision cross‐section range.