Identification and evaluation of the chemical similarity of Yindan xinnaotong samples by ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry fingerprinting

Yindan xinnaotong, a compound preparation used in traditional Chinese medicine, is composed of eight herbs: Ginkgo biloba leaf (yinxingye), Salvia miltiorrhizae (danshen), Herba gynostemmatis (jiaogulan), Erigerontis herba (dengzhanxixin), Allii sativi bulbus (dasuan), Notoginseng radixe rhizoma (sanqi), Crataegi fructus (shanzha), and Borneolum (tianranbingpian). Yindan xinnaotong is primarily used to treat cardiovascular and cerebrovascular diseases. However, to date, no scientific methods have been established to assess the quality of Yindan xinnaotong. Therefore, a combinatorial method was developed based on chemical constituent identification and fingerprint analysis to assess the consistency of Yindan xinnaotong quality. In this study, ultra high performance liquid chromatography coupled with time‐of‐flight mass spectrometry was used to identify the chemical components of Yindan xinnaotong soft capsules. Approximately 74 components were detected, of which 70, including flavonoids, ginkgolide, phenolic acid, diterpenoid tanshinones, and ginsenoside, were tentatively identified. A fingerprint analysis was also conducted to evaluate the uniformity of the quality of Yindan xinnaotong soft capsules. Ten batches of Yindan xinnaotong soft capsules were analyzed. All of the resulting chromatograms were imported into the “Similarity Evaluation System for Chromatographic Fingerprints of TCM” (Chinese Pharmacopoeia Commission, version 2004A). The similarity scores of common peaks from these samples ranged from 0.903–1.000, indicating that samples from different batches were highly correlated.     

Quality evaluation of Salvia miltiorrhiza Bge. by ultra high performance liquid chromatography with photodiode array detection and chemical fingerprinting coupled with chemometric analysis

An ultra high performance liquid chromatography with photodiode array detection method is developed for the simultaneous quantitative determination of five water‐soluble compounds including danshensu, protocatechualdehyde, rosmarinic acid, salvianolic acid B, and salvianolic acid A in Salvia miltiorrhiza Bge. samples. Through method optimization, the five compounds all expressed good linearity (R² > 0.9990) in a wide concentration range together with satisfactory accuracy, precision, and stability. Moreover, through qualitative analysis of the chemical fingerprint combined with similarity analysis, hierarchical cluster analysis, principle component analysis, and partial least‐squares discriminate analysis, we determined that the 13 batches of Salvia miltiorrhiza Bge. were similar in internal quality and the differences resulted from various cultivation environments, recovery elements, and others. Seen from the results of hierarchical cluster analysis and principle component analysis, the classification of 13 batches was in accordance, and partial least‐squares discriminate analysis technique was more suitable than the principle component analysis model to provide a distinct classification of test samples on the basis of their different components. Moreover, a permutation test verified the rationality of partial least‐squares discriminate analysis and variable importance plot showed that peaks 37 and 38 were the most significant variables in distinguishing the Salvia miltiorrhiza Bge. samples. The idea of the quantitative and qualitative analysis of Salvia miltiorrhiza Bge. was convenient, sensitive, and comprehensive, which could be applied to evaluate the quality of more traditional Chinese medicines.     

Quantification and semiquantification of multiple representative components for the holistic quality control of Allii Macrostemonis Bulbus by ultra high performance liquid chromatography with quadrupole time‐of‐flight tandem mass spectrometry

Allii Macrostemonis Bulbus (named Xiebai in China) is a folk medicine with medicinal values for the treatment of thoracic obstruction and cardialgia, and a food additive as well. However, there is even no quantitative standard for Allii Macrostemonis Bulbus recorded in the current edition of the Chinese Pharmacopeia. Hence, simultaneous assay of multiple components is urgent. In this study, chemometric methods were firstly applied to discover the components with significant fluctuation among multiple Allii Macrostemonis Bulbus samples based on optimized fingerprints. Meanwhile, the major components and main absorbed components in rats were all selected as its representative components. Subsequently, a sensitive method was established for the simultaneous determination of 54 components (15 components for quantification and 39 components for semiquantification) by ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry. Moreover, the validated method was successfully applied to evaluate the quality of multiple samples on the market. It became known that multiple Allii Macrostemonis Bulbus samples varied significantly and showed poor consistency. This work illustrated that the proposed approach could improve the quality of Allii Macrostemonis Bulbus, and it also provided a feasible method for quality evaluation of other traditional Chinese medicines.     

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.     

Simultaneous detection of eight active components in Radix Tinosporae by ultra high performance liquid chromatography coupled with electrospray tandem mass spectrometry

A rapid and sensitive ultra high performance liquid chromatography with electrospray ionization tandem mass spectrometry method was developed and validated for the simultaneous determination of eight major active components (magnoflorine, menisperine, 20‐hydroxyecdysone, cepharanthine, columbamine, jatrorrhizine, columbin, and palmatine) in Radix Tinosporae. The separation was performed on an InterSustainSwift C₁₈ column (1.9 μm, 2.1 id × 100 mm) at 40 °C with a gradient elution. A mixture of acetonitrile and methanol (v/v = 1:1) and ammonium acetate buffer (25 mmol/L ammonium acetate with 0.2% formic acid) were used as mobile phases, and the flow rate was set at 0.4 mL/min. The recovery was tested in real samples and calculated to be 86.97–111.28%, and all the compounds showed good linearity (r > 0.998) in relatively wide concentration ranges. The developed method was applied to the determination of eight active compounds in real herb samples, which were collected from four different places. It has been demonstrated that the proposed method has great potential for the quality control of the traditional Chinese medicine Radix Tinosporae.     

Ultra high performance liquid chromatography with quadrupole time-of-flight tandem mass spectrometry and liquid chromatography with tandem mass spectrometry combined with chemometric analysis as an approach for the quality evaluation of Mume Fructus

Mume Fructus is an important traditional Chinese medicine that has been widely used in the treatment of intestinal diseases and asthma for thousands of years. In order to evaluate the quality of Mume Fructus in different processing methods, the main chemical components in Mume Fructus were investigated and a method was established for simultaneous quantification of organic acids of Mume Fructus. First, an optimized ultra-performance liquid chromatography-quadrupole-time of flight tandem-mass spectrometry method was used to identify the structures of main components in Mume Fructus. A total of 41 chemical compounds were identified, including 11 organic acids, 13 flavonoids, and three fatty acids. The contents of 11 organic acids in 18 batches of Mume Fructus from different processing methods were simultaneously determined by a liquid chromatography with tandem mass spectrometry method. The results of quantitative and hierarchical cluster analysis indicated that Mume Fructus under different processing methods were rich in the above 11 organic acids and the contents were obviously different. Taken together, the proposed quality evaluation method was fast and comprehensively reflects the content of the main chemical components in Mume Fructus under different processing methods, and provides a useful reference for the quality control and evaluation of Mume Fructus.     

Determination and Fingerprint Analysis of Steroidal Saponins in roots of Liriope muscari (Decne.) L. H. Bailey by ultra high performance liquid chromatography coupled with ion trap time‐of‐flight mass spectrometry

Liriope muscari (Decne.) L. H. Bailey is a well‐known traditional Chinese medicine used for treating cough and insomnia. There are few reports on the quality evaluation of this herb partly because the major steroid saponins are not readily identified by UV detectors and are not easily isolated due to the existence of many similar isomers. In this study, a qualitative and quantitative method was developed to analyze the major components in L. muscari (Decne.) L. H. Bailey roots. Sixteen components were deduced and identified primarily by the information obtained from ultra high performance liquid chromatography with ion‐trap time‐of‐flight mass spectrometry. The method demonstrated the desired specificity, linearity, stability, precision, and accuracy for simultaneous determination of 15 constituents (13 steroidal glycosides, 25(R)‐ruscogenin, and pentylbenzoate) in 26 samples from different origins. The fingerprint was established, and the evaluation was achieved using similarity analysis and principal component analysis of 15 fingerprint peaks from 26 samples by ultra high performance liquid chromatography. The results from similarity analysis were consistent with those of principal component analysis. All results suggest that the established method could be applied effectively to the determination of multi‐ingredients and fingerprint analysis of steroid saponins for quality assessment and control of L. muscari (Decne.) L. H. Bailey.     

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.     

Rapid characterization of the constituents in Jigucao capsule using ultra high performance liquid chromatography with quadrupole time-of-flight mass spectrometry

Jigucao capsule is a well-known Chinese patent medicine for the treatment of acute and chronic hepatitis and cholecystitis. The chemical components of Jigucao capsule were not clear resulting from the paucity of relevant studies, which hindered the research of the pharmacological mechanism, the comprehensive development, and utilization of Jigucao capsule in clinical studies. By establishing a high-throughput ultra-performance liquid chromatography quadrupole time of flight mass spectrometry in combination with intelligent UNIFI software data processing platform to automatically characterize and identify the chemical profile of Jigucao capsule, 144 compounds were determined rapidly, including 34 terpenoids, 25 flavonoids, 22 steroids, 21 phenylpropanoids, 10 glycosides, six alkaloids, 13 organic acids, and other 13 components. These compounds may be the active components of Jigucao capsule. In this study, a rapid and robust method for comprehensively analyzing the chemical composition of Jigucao capsule was described and established for the first time. The results will provide a reference for the quality control of Jigucao capsule and the establishment of a higher quality standard, as well as for the pharmacodynamic material basis research.     

Multiple analytical methods for identification and quality evaluation of Fritillariae Thunbergii Bulbus based on biological single molecules by high‐performance liquid chromatography

Quantitative analysis for biological single molecules in Fritillariae Thunbergii Bulbus and identification method by chemometrics was established by using high‐performance liquid chromatography. Amino acids, nucleosides, and nucleobases were quantitatively analyzed, and 11 peaks were selected for species identification. A Common pattern was established for chemometrics, and then similarity analysis, principal component analysis, and hierarchical cluster analysis heatmap were applied, and the results indicated that species were ideally identified from the adulterants as Fritillariae Cirrhosae Bulbus. This evaluation method was valuable for further quality control to select the characteristic components.     

Influence of different processing times on the quality of Polygoni Multiflora Radix by metabolomics based on ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry

A metabolomics method based on ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry was developed to evaluate the influence of processing times on the quality of raw and processed Polygoni Multiflora Radix . Principal component analysis and partial least‐squares discriminant analysis was used to screen the potential maker metabolites that were contributed to the quality changes. Then these marker metabolites were selected as variables in Fisher's discriminant analysis to establish the models that were used to distinguish the raw and processed Polygoni Multiflora Radix in the markets. Additionally, 36 compounds were identified. Twelve raw Polygoni Multiflora Radix samples and 23 processed Polygoni Multiflora Radix samples were distinguished. The results showed that the 12 raw Polygoni Multiflora Radix samples belonged to the group of processing time of 0 h, and two processed Polygoni Multiflora Radix samples were part of the group of processing times of 4 h, 12 samples belonged to group of processing times of 8 to 16 h, and nine samples were the group of processing times of 24 to 48 h. The results demonstrated that the method could provide scientific support for the processing standardization of Polygoni Multiflora Radix .     

Chemical fingerprint and quantitative analysis for the quality evaluation of Vitex negundo seeds by reversed‐phase high‐performance liquid chromatography coupled with hierarchical clustering analysis

A simple and efficient method was developed for the chemical fingerprint analysis and simultaneous determination of four phenylnaphthalene‐type lignans in Vitex negundo seeds using high‐performance liquid chromatography with diode array detection. For fingerprint analysis, 13 V. negundo seed samples were collected from different regions in China, and the fingerprint chromatograms were matched by the computer‐aided Similarity Evaluation System for Chromatographic Fingerprint of TCM (Version 2004A). A total of 21 common peaks found in all the chromatograms were used for evaluating the similarity between these samples. Additionally, simultaneous quantification of four major bioactive ingredients was conducted to assess the quality of V. negundo seeds. Our results indicated that the contents of four lignans in V. negundo seeds varied remarkably in herbal samples collected from different regions. Moreover, the hierarchical clustering analysis grouped these 13 samples into three categories, which was consistent with the chemotypes of those chromatograms. The method developed in this study provides a substantial foundation for the establishment of reasonable quality control standards for V. negundo seeds.     

Qualitative and quantitative determination of YiXinShu Tablet using ultra high performance liquid chromatography with Q Exactive hybrid quadrupole orbitrap high‐resolution accurate mass spectrometry

To clarify and quantify the chemical profile of YiXinShu Tablet rapidly, a feasible and accurate strategy was developed by applying ultra high performance liquid chromatography with Q Exactive hybrid quadrupole orbitrap high‐resolution accurate mass spectrometry. A total of 105 components were identified, including 25 phenanthraquinones, 11 lactones, 19 lignans, 24 acids, and 26 other compounds. Among them, 26 major compounds were unambiguously detected by comparing with reference standards. And 19 of these compounds in three batches of YiXinShu Tablet were selected for quantitative determination. (Z )‐Ligustilide, salvianic acid A, salvianolic acid A, salvianolic acid B, and rosmarinic acid were abundant in these three batches with contents over 1 mg/g. The established analysis methods were examined to be accurate and feasible. The results show that the ultra high performance liquid chromatography with Q Exactive hybrid quadrupole orbitrap high‐resolution accurate mass spectrometry method has a powerful qualitative ability and promising quantitative application.     

Fingerprint analysis and quality consistency evaluation of flavonoid compounds for fermented Guava leaf by combining high‐performance liquid chromatography time‐of‐flight electrospray ionization mass spectrometry and chemometric methods

Guava leaves are used in traditional herbal teas as antidiabetic therapies. Flavonoids are the main active of Guava leaves and have many physiological functions. However, the flavonoid compositions and activities of Guava leaves could change due to microbial fermentation. A high‐performance liquid chromatography time‐of‐flight electrospray ionization mass spectrometry method was applied to identify the varieties of the flavonoids in Guava leaves before and after fermentation. High‐performance liquid chromatography, hierarchical cluster analysis and principal component analysis were used to quantitatively determine the changes in flavonoid compositions and evaluate the consistency and quality of Guava leaves. Monascus anka Saccharomyces cerevisiae fermented Guava leaves contained 2.32‐ and 4.06‐fold more total flavonoids and quercetin, respectively, than natural Guava leaves. The flavonoid compounds of the natural Guava leaves had similarities ranging from 0.837 to 0.927. The flavonoid compounds from the Monascus anka S. cerevisiae fermented Guava leaves had similarities higher than 0.993. This indicated that the quality consistency of the fermented Guava leaves was better than that of the natural Guava leaves. High‐performance liquid chromatography fingerprinting and chemometric analysis are promising methods for evaluating the degree of fermentation of Guava leaves based on quality consistency, which could be used in assessing flavonoid compounds for the production of fermented Guava leaves.     

Simultaneous quantitation of 14 active components in Yinchenhao decoction by using ultra high performance liquid chromatography with diode array detection: Method development and ingredient analysis of different commonly prepared samples

We developed a novel quantitative analysis method based on ultra high performance liquid chromatography coupled with diode array detection for the simultaneous determination of the 14 main active components in Yinchenhao decoction. All components were separated on an Agilent SB‐C18 column by using a gradient solvent system of acetonitrile/0.1% phosphoric acid solution at a flow rate of 0.4 mL/min for 35 min. Subsequently, linearity, precision, repeatability, and accuracy tests were implemented to validate the method. Furthermore, the method has been applied for compositional difference analysis of 14 components in eight normal‐extraction Yinchenhao decoction samples, accompanied by hierarchical clustering analysis and similarity analysis. The result that all samples were divided into three groups based on different contents of components demonstrated that extraction methods of decocting, refluxing, ultrasonication and extraction solvents of water or ethanol affected component differentiation, and should be related to its clinical applications. The results also indicated that the sample prepared by patients in the family by using water extraction employing a casserole was almost same to that prepared using a stainless‐steel kettle, which is mostly used in pharmaceutical factories. This research would help patients to select the best and most convenient method for preparing Yinchenhao decoction.     

Multi‐component determination and chemometric analysis of Paris polyphylla by ultra high performance liquid chromatography with photodiode array detection

Multi‐source analysis of traditional Chinese medicine is key to ensuring its safety and efficacy. Compared with traditional experimental differentiation, chemometric analysis is a simpler strategy to identify traditional Chinese medicines. Multi‐component analysis plays an increasingly vital role in the quality control of traditional Chinese medicines. A novel strategy, based on chemometric analysis and quantitative analysis of multiple components, was proposed to easily and effectively control the quality of traditional Chinese medicines such as Chonglou. Ultra high performance liquid chromatography was more convenient and efficient. Five species of Chonglou were distinguished by chemometric analysis and nine saponins, including Chonglou saponins I, II, V, VI, VII, D, and H, as well as dioscin and gracillin, were determined in 18 min. The method is feasible and credible, and enables to improve quality control of traditional Chinese medicines and natural products.     

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.     

Fingerprint analysis and multi‐ingredient quantitative analysis for quality evaluation of Xiaoyanlidan tablets by ultra high performance liquid chromatography with diode array detection

A rapid and sensitive ultra high performance liquid chromatography method with diode array detection was developed for the fingerprint analysis and simultaneous determination of seven active compounds in Xiaoyanlidan (XYLD) tablets. The chromatographic separations were obtained on an Agilent Eclipse plus C₁₈ column (50 × 2.1 mm id, 1.8 μm) using gradient elution with water/formic acid (1%) and acetonitrile at a flow rate of 0.4 mL/min. Within 63 min, 36 peaks could be selected as the common peaks for fingerprint analysis to evaluate the similarities among several samples of XYLD tablets collected from different manufacturers. In quantitative analysis, seven compounds showed good regression (R > 0.9990) within test ranges and the recovery of the method was within the range of 95.9–104.3%. The method was successfully applied to the simultaneous determination of seven compounds in six batches of XYLD tablets. These results demonstrate that the combination of chromatographic fingerprint analysis and simultaneous multi‐ingredient quantification using the ultra high performance liquid chromatography method with diode array detection offers a rapid, efficient, and reliable approach for quality evaluation of XYLD tablets.     

Fingerprint analysis,multi‐component quantitation,and antioxidant activity for the quality evaluation of Salvia miltiorrhiza var. alba by high‐performance liquid chromatography and chemometrics

Salvia miltiorrhiza Bge. var. alba C.Y. Wu and H.W. Li has wide prospects in clinical practice. A useful comprehensive method was developed for the quality evaluation of S. miltiorrhiza var. alba by three quantitative parameters: high‐performance liquid chromatography fingerprint, ten‐component contents, and antioxidant activity. The established method was validated for linearity, precision, repeatability, stability, and recovery. Principal components analysis and hierarchical clustering analysis were both used to evaluate the quality of the samples from different origins. The results showed that there were category discrepancies in quality of S. miltiorrhiza var. alba samples according to the three quantitative parameters. Multivariate linear regression was adopted to explore the relationship between components and antioxidant activity. Three constituents, namely, danshensu, rosmarinic acid, and salvianolic acid B, significantly correlated with antioxidant activity, and were successfully elucidated by the optimized multivariate linear regression model. The combined use of high‐performance liquid chromatography fingerprint analysis, simultaneous multicomponent quantitative analysis, and antioxidant activity for the quality evaluation of S. miltiorrhiza var. alba is a reliable, comprehensive, and promising approach, which might provide a valuable reference for other herbal products in general to improve their quality control.     

Qualitative and quantitative analysis in quality control of traditional Chinese medicines

Separation techniques with high efficiency and sensitive detection have been widely used for quality control of traditional Chinese medicines (TCMs). High-performance liquid chromatography, gas chromatography, and capillary electrophoresis are commonly used to separate various components in TCMs. Ultraviolet detection, fluorescence detection, evaporative light-scattering detection, mass spectrometry and nuclear magnetic resonance can be applied to separation techniques for qualitative and quantitative analysis of TCMs. The development of quality control for TCMs based on quantitative and qualitative analysis from 2000 to 2007 are reviewed; the fingerprint technique is also discussed due to its broad application in the quality control of TCMs. Prospects for further research based on our primary results are also discussed.