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.     

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.     

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.     

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.     

Simultaneous determination of niacin and pyridoxine at trace levels by using diode array high‐performance liquid chromatography and liquid chromatography with quadrupole time‐of‐flight tandem mass spectrometry

A highly sensitive and simple diode‐array high‐performance liquid chromatography and liquid chromatography with quadrupole time‐of‐flight tandem mass spectrometry method was developed for the simultaneous determination of niacin and pyridoxine in pharmaceutical drugs, tap water, and wastewater samples. To determine the in vivo behavior of niacin and pyridoxine, analytes were subjected to simulated gastric conditions. The calibration plots of the diode‐array high‐performance liquid chromatography and liquid chromatography with quadrupole time‐of‐flight tandem mass spectrometry method showed good linearity over a wide concentration range with close to 1.0 correlation coefficients for both analytes. The limit of detection/limit of quantitation values for liquid chromatography quadrupole time‐of‐flight tandem mass spectrometry analysis were 1.98/6.59 and 1.3/4.4 μg/L for niacin and pyridoxine, respectively, while limit of detection/limit of quantitation values for niacin and pyridoxine in high‐performance liquid chromatography analysis were 3.7/12.3 and 5.7/18.9 μg/L, respectively. Recovery studies were also performed to show the applicability of the developed methods, and percentage recovery values were found to be 90–105% in tap water and 94–97% in wastewater for both analytes. The method was also successfully applied for the qualitative and quantitative determination of niacin and pyridoxine in drug samples.     

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.     

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.     

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.     

Multi‐constituent determination and fingerprint analysis of Scutellaria indica L. using ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry

An ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry method integrating multi‐constituent determination and fingerprint analysis has been established for quality assessment and control of Scutellaria indica L. The optimized method possesses the advantages of speediness, efficiency, and allows multi‐constituents determination and fingerprint analysis in one chromatographic run within 11 min. 36 compounds were detected, and 23 of them were unequivocally identified or tentatively assigned. The established fingerprint method was applied to the analysis of ten S. indica samples from different geographic locations. The quality assessment was achieved by using principal component analysis. The proposed method is useful and reliable for the characterization of multi‐constituents in a complex chemical system and the overall quality assessment of S. indica.     

Urinary metabolomic study of non‐small cell lung carcinoma based on ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry

Metabolic profiles from human urine reveal the significant difference of carnitine and acylcarnitines levels between non‐small cell lung carcinoma patients and healthy controls. Urine samples from cancer patients and healthy individuals were assayed in this metabolomic study using ultra high performance liquid chromatography coupled to quadrupole time‐of‐flight mass spectrometry. The data were normalized by the sum of all intensities and creatinine calibration, respectively, before orthogonal partial least squares discriminant analysis. Twenty differential metabolites were identified based on standard compounds or tandem mass spectrometry fragments. Among them, some medium‐/long‐chain acylcarnitines, for example, cis‐3,4‐methylene heptanoylcarnitine, were found to be downregulated while carnitine was upregulated in urine samples from the cancer group compared to the control group. Receiver operating characteristic analysis of the two groups showed that the area under curve for the combination of carnitine and 11 selected acylcarnitines was 0.958. This study suggests that the developed carnitine and acylcarnitines profiling method has the potential to be used for screening non‐small cell lung carcinoma.     

Comparative analysis of nucleosides,nucleobases, and amino acids in different parts of Angelicae Sinensis Radix by ultra high performance liquid chromatography coupled to triple quadrupole tandem mass spectrometry

A rapid and sensitive ultra high performance liquid chromatography coupled to triple quadrupole tandem mass spectrometry method was established and employed to determine 21 nucleosides, nucleobases, and amino acids in 60 samples from different parts of Angelicae Sinensis Radix. The established methods were validated by good linearity (r² > 0.9937), limits of detection (0.12–77.75 ng/mL), limits of quantitation (0.31–272.13 ng/mL), intra‐ and interday precisions (RSD ≤ 4.84%, RSD ≤ 6.26%), stability (RSD ≤ 5.92%), repeatability (RSD ≤ 7.14%), recovery (91.4–103.4%), and matrix effects (0.92–1.03). Chemical comparative analysis revealed that the content of total analytes in four parts of Angelicae Sinensis Radix were different, and exhibited the order: Head (14.89 mg/g) > Body (10.15 mg/g) > All (8.22 mg/g) > Tail (6.23 mg/g). Principal component analysis showed that the samples could be classified into four groups in accord with four different parts of Angelicae Sinensis Radix. The results could provide a scientific basis and reference for the quality control of Angelicae Sinensis Radix, and may be conducive to further research on the pharmacological activities of Angelicae Sinensis Radix.     

Structural characterization and identification of flavonoid aglycones in three Glycyrrhiza species by liquid chromatography with photodiode array detection and quadrupole time‐of‐flight mass spectrometry

Flavonoids, including flavones, isoflavones, flavanones, chalcones, and isoflavans, have long been recognized as the main active ingredients in licorice. A method combining liquid chromatography with photodiode array detection and quadrupole time‐of‐flight mass spectrometry was developed to characterize components in three Glycyrrhiza species, and to expound the characteristic fragmentation behaviors in the positive ion mode. Based on the fragmentation patterns of reference compounds, a total of 39 compounds, including 37 flavonoid aglycones and two coumestans, were identified or tentatively identified. Besides, some common features, such as H₂O, CO, and CH₂O₂ losses, together with retro‐Diels–Alder fragmentation, were observed in these compounds. Furthermore, diagnostic fragmentations of C‐ring cleavages and UV absorption on the skeleton groups were observed to structurally characterize flavonoid aglycones. In addition, typical losses of different substituent groups were detected: Neutral losses of 56 (C₄H₈) and 68 Da (C₅H₈) were yielded from a prenyl chain; neutral losses of 42 (C₃H₆), 54 (C₄H₆), and 70 Da (C₄H₆O) were generated by a pyran ring. Particularly, neutral losses of 18 (H₂O), 16 (CH₄), 112 (C₈H₁₆), and 98 Da (C₇H₁₄) predicted a hydroxyl, a methoxyl, double prenyl chains, and a prenyl chain with a pyran ring, respectively.     

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 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.     

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.     

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.     

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.