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.     

Qualitative and Quantitative Analysis of 17 Different Types of Tetra- and Pentacyclic Triterpenic Acids in Boswellia papyrifera by a Semi-Automatic Homomodal 2D HPLC method

A semi-automatic two dimensional high-performance liquid chromatography (HPLC) gradient method with photo diode array detection was developed, capable of separating and quantifying up to 17 different triterpenic acids in the gum resin of the frankincense species Boswellia papyrifera. The here reported quantitation of 14 of the possible 17 compounds contains boswellic, tirucallic and lupeolic acids. All compounds were isolated from B. papyrifera and used as external standards. Quantitation of these compounds was performed after minimizing the matrix by liquid?Cliquid separation, using alkaline, acidic and organic media to separate the acids from interfering matrix compounds. Therefore, two different extraction procedures were tested, giving two different extraction profiles. Within the first run (1st dimension) 13 compounds could be quantified. Quantitation of ??-boswellic acid, which was proved to elute as inhomogeneous peak, was achieved by introduction of a second dimension, leading to a fully validated semi-automatic homomodal 2D chromatography. The method is applicable for determination of compounds occurring in different types of frankincense and their pharmaceutical products. It also can be applied to distinguish between different kinds of frankincense. Moreover, it is the first published method feasible of separating and quantifying five different types of tirucallic acids.     

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.     

Analysis of frankincense from various Boswellia species with inhibitory activity on human drug metabolising cytochrome P450 enzymes using liquid chromatography mass spectrometry after automated on-line extraction

In our search for herbal remedies with inhibitory activity on cytochrome P450 (CYP) enzymes, we identified extracts of the gum-resin of Boswellia carteri, Boswellia frereana, Boswellia sacra and Boswellia serrata as equally potent, non-selective inhibitors of the major drug metabolising CYP enzymes 1A2/2C8/2C9/2C19/2D6 and 3A4. LC/LC/ESI-MS fingerprint analyses of the boswellic acids 11-keto-beta-boswellic acid, alpha-boswellic acid, beta-boswellic acid and their 3-O-acylated derivatives were used for the authentication of the commercially obtained frankincense samples. Although the boswellic acids could be identified as moderate to potent inhibitors of the applied CYP enzymes, they are not the major CYP inhibitory principle of frankincense.     

Comparative pharmacokinetic study of two boswellic acids in normal and arthritic rat plasma after oral administration of Boswellia serrata extract or Huo Luo Xiao Ling Dan by LC‐MS

Huo Luo Xiao Ling Dan (HLXLD), a Chinese herbal formula composed of 11 different herbs, has been used traditionally for the treatment of arthritis and other chronic inflammatory diseases. However, the pharmacokinetic profile of its anti‐inflammatory bioactive compounds has not been elucidated. Boswellic acids are the bioactive compounds with potent anti‐inflammatory activity isolated from Boswellia serrate which is one of the 11 herbs of HLXLD. The objective of the study was to compare the pharmacokinetics of the two bioactive bowsellic acids: 11‐keto‐β‐boswellic acid and 3‐O‐acetyl‐11‐keto‐β‐boswellic following oral administration of HLXLD or Boswellia serrata extract alone in normal and arthritic rats. An LC‐MS method was developed and validated for the determination of 11‐keto‐β‐boswellic acid and 3‐O‐acetyl‐11‐keto‐β‐boswellic in the comparative pharmacokinetic study. The results showed that there were significant differences in pharmacokinetic parameters between normal and arthritic groups. Interestingly, the absorptions of two boswellic acids were significantly higher in HLXLD than Boswellia serrata extract alone, indicating the synergistic effect of other herbal ingredients in HLXLD. This comparative pharmacokinetic study provided direct evidence supporting the notion that the efficacy of a complex mixture such as HLXLD is better than that of single components in treating human diseases. Copyright © 2014 John Wiley & Sons, Ltd.     

Quantitative analysis of multicomponents by a single marker and quality evaluation of Venenum Bufonis from different geographical origins

Bufadienolides are the main bioactive components of Venenum Bufonis (VB) and have been widely used to treat different types of human cancers for decades. The bufadienolide content in VB varies significantly in materials from different geographical origins. In this work, a new strategy for the quality assessment of VB was developed through quantitative analysis of multi‐components by single marker (QAMS). Cinobufagin was selected as the internal reference substance; seven bufadienolides were separated and simultaneously determined based on relative correction factors. The correlation coefficient value (r ≥ 0.9936) between QAMS and the normal external standard method proved the consistency of the two methods. According to the outcomes of 30 batches of VB samples, the contents of the seven bufadienolides were used for further chemometric analysis. All of the samples of VB from various geographical origins were divided into three categories based on hierarchical cluster analysis and radar plot, which indicated the crucial influence of geographical origins on VB. This study showed that QAMS combined with chemometristry could be used to comprehensively evaluate and effectively control the quality of VB from different geographical origins.     

Qualitative identification and quantitative comparison of Physochlainae Radix from different regions based on chemometric methods

Physochlainae Radix (PR) is an essential herbal medicine that has been generally applied for treating cough and asthma. In this study, a comprehensive strategy for quality evaluation of PR from different origins was established by integrating qualitative identification, quantitative analysis, and chemometric methods. A total of 58 chemical components were identified by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF-MS/MS), and a sensitive and rapid UHPLC-QqQ-MS/MS method was established for the simultaneous determination of 12 compounds. In addition, multivariate statistical analysis was applied for discriminant analysis to compare the differences among 30 batches of PR samples. The results showed that the 30 batches of PR collected from four provinces could be clustered into three categories, in which scoparone, protocatechuic acid, tropic acid, and scopolin were important components to distinguish the primary and non-primary producing areas, as well as superior and inferior products of PR. Chemometric results were consistent and validated each other, and systematically explained the intrinsic quality characteristics of PR. This study first demonstrated that LC-MS combined with multivariate statistical analysis, provided a comprehensive and effective means for quality evaluation of PR.     

Qualitative and quantitative analysis of multiple components for quality control of Deng‐Zhan‐Sheng‐Mai capsules by ultra high performance liquid chromatography tandem mass spectrometry method coupled with chemometrics

Deng‐Zhan‐Sheng‐Mai capsules are a well‐known traditional Chinese patent medicine that was developed in China for the treatment of ischemic stroke. Its quality control focuses on Erigerontis Herba but ignores the contributions of Ginseng Radix et Rhizoma, Schisandrae Chinensis Fructus, and Ophiopogonis Radix. To improve the quality standards for this medicine, this work reports the application of a systematic ultra high performance liquid chromatography tandem mass spectrometric method coupled with chemometrics. Three qualitative and quantitative parameters are established for the evaluation of quality: chemical profiling, the relationship between the contents of 18 compounds and the antioxidant activity, and chemometric analysis. A total of 55 compounds, including 20 phenolic acids, 10 flavonoids, 15 saponins, and 10 lignans, were identified. The method for the quantitative determination of the aforementioned 18 compounds was validated. The limit of quantification ranged from 0.13 to 9.60 ng/mL. The overall recoveries ranged from 95.31 to 103.54%. Hierarchical cluster analysis and principal component analysis were applied to the data of 18 components in ten batches of samples. Nine compounds, including scutellarin, 3,5‐O‐dicaffeoylquinic acid, 4,5‐O‐dicaffeoylquinic acid, ginsenoside Rb1, ginsenoside Re, ginsenoside Rg1, ophiopogonin D, schisandrin, and schisandrol B, are suggested as chemical markers for evaluating the quality.     

Fast determination of ginsenosides in ginseng by high‐performance liquid chromatography with chemometric resolution

Ginseng is a well‐known traditional Chinese medicinal herb, and ginsenosides are its major active components. A method for the fast determination of ginsenosides in ginseng samples by high‐performance liquid chromatography was developed and used for the quantitative analysis of four ginsenosides in three different ginseng samples. In this method, instead of time‐consuming gradient elution, isocratic elution was used to speed up the analysis. Under strong isocratic elution, all the ginsenosides are eluted in 2.3 min. Although the measured signal is composed of overlapped peaks with the interferences and background, the signal of ginsenosides can be extracted by chemometric resolution. A non‐negative immune algorithm was employed to obtain the chromatographic information of the target components from the data. Compared with conventional chemometric approaches, the method can perform the extraction for one‐dimensional overlapping signals. The method was validated by the determination of four ginsenosides in three different ginseng samples. The recoveries of the spiked samples were in the range of 94.08–107.3%.     

Quality assessment of cortex cinnamomi by HPLC chemical fingerprint, principle component analysis and cluster analysis

HPLC fingerprint analysis, principle component analysis (PCA), and cluster analysis were introduced for quality assessment of Cortex cinnamomi (CC). The fingerprint of CC was developed and validated by analyzing 30 samples of CC from different species and geographic locations. Seventeen chromatographic peaks were selected as characteristic peaks and their relative peak areas (RPA) were calculated for quantitative expression of the HPLC fingerprints. The correlation coefficients of similarity in chromatograms were higher than 0.95 for the same species while much lower than 0.6 for different species. Besides, two principal components (PCs) have been extracted by PCA. PC1 separated Cinnamomum cassia from other species, capturing 56.75% of variance while PC2 contributed for their further separation, capturing 19.08% variance. The scores of the samples showed that the samples could be clustered reasonably into different groups corresponding to different species and different regions. The scores and loading plots together revealed different chemical properties of each group clearly. The cluster analysis confirmed the results of PCA analysis. Therefore, HPLC fingerprint in combination with chemometric techniques provide a very flexible and reliable method for quality assessment of traditional Chinese medicines.     

Comparative analysis of volatile components from Clematis species growing in China

The volatile components between stems and roots and also among five Clematis species from China were studied and analyzed by gas chromatography-mass spectrometry (GC-MS) combined with alternative moving window factor analysis (AMWFA), a new chemometric resolution method. Identification of the compounds was also assisted by comparison of temperature-programmed retention indices (PTRIs) on HP-5MS with authentic samples included in our own laboratory database under construction. A total of 153 different compounds accounting for 86.6-96.5% were identified and significant qualitative and quantitative differences were observed among the samples. The major volatile components in different essential oils from Clematis species were n-hexadecanoic acid and (Z,Z)-9,12-octadecadienoic acid. Our work further demonstrated chemometric resolution techniques upon the two-dimensional data and PTRIs can provide a complementary and convenient method for fast and accurate analysis of complex essential oils.     

Structural analysis of pentacyclic triterpenes from the gum resin of Boswellia serrata by NMR spectroscopy

3α‐Acetyl‐β‐boswellic acid ( 1 ), 3α‐acetyl‐α‐boswellic acid ( 2 ), 3α‐acetyl‐9,11‐dehydro‐β‐boswellic acid ( 3 ), 3α‐acetyl‐9,11‐dehydro‐α‐boswellic acid ( 4 ) and 3α‐acetyl‐11‐keto‐β‐boswellic acid ( 5 ) were isolated from the gum resin of Boswellia serrata. 1D and 2D NMR (COSY45, HMQC, HMBC, ROESY) spectra at 500 MHz were used for shift assignments and structure verification. All boswellic acids investigated share the cis conformation at ring D/E and the 3α orientation of the acetyl ester group. Owing to high‐order spectra, NMR could not determine the exact conformation of H‐20/H‐30 of the β‐boswellic acids. 3α‐Acetyl‐β‐boswellic acid methyl ester ( 1 ^′) was synthesized for experiments with a shift reagent, Eu(fod)₃, that enhanced the resolution considerably. The oxygen atoms of the 3α‐acetyl group form the apparent complex binding site for the shift reagent. Copyright © 2003 John Wiley & Sons, Ltd.     

Exploiting the versatility of vacuum‐assisted headspace solid‐phase microextraction in combination with the selectivity of ionic liquid‐based GC stationary phases to discriminate Boswellia spp. resins through their volatile and semivolatile fractions

The frankincense resins, secreted from Boswellia species, are an uncommon example of a natural raw material where every class of terpenoids is present in similar proportions. Diterpenoids (serratol, incensole, and incensole acetate) are used to discriminate samples from different species and origins. Headspace solid‐phase microextraction has been used for frankincense analysis, although it requires long sampling time for medium‐ to low‐volatility markers; headspace solid‐phase microextraction under vacuum can overcome this limit. Gas chromatography is used for analysis but the separation of incensole and serratol needs polar stationary phases. In this study, we develop a method to discriminate frankincenses based on vacuum‐assisted headspace solid‐phase microextraction combined with fast gas chromatography‐mass spectrometry with ionic liquid–based stationary phases. The optimized conditions for solid samples were: air evacuation below 0°C, 15 min of incubation time, and 15 min of extraction time. Losses of volatiles due to vial air‐evacuation in the presence of the sample were minimized by sample amount above 100 mg and low sample temperature. Fast gas chromatography provides the baseline separation of all markers in 20 min. By applying vacuum sampling and fast gas chromatography, the total analysis was reduced to 50 min compared to 120 min (60 min sampling plus 60 min analysis) as previously reported. The method was successfully applied to commercial frankincense samples.     

Total detection of Tianma Toutong tablets for quality consistency by a five‐wavelength fusion fingerprint and chemometrics

A fingerprint method was developed and combined with chemometrics for quality evaluation of Tianma Toutong tablets, which are herbal medicine tablets used to treat migraine. Samples were analyzed by high‐performance liquid chromatography, where five single‐wavelength profiles (203, 232, 254, 280 and 310 nm) were fused to generate a five‐wavelength fusion fingerprint and were also used for the quantitative analysis of seven chemical markers (gastrodin, caffeic acid, hesperidin, isoimperatorin, chlorogenic acid, ferulic acid and imperatorin). A systematic quantitative fingerprint method and principal component analysis were used to analyze the generated data. Samples could be well distinguished from different manufacturers by analyzing the chromatographic data sets. In addition, the partial least squares model can serve as an antioxidant activity evaluation of Tianma Toutong tablets, as well as a reference for the selection of active constituents to analyze the spectrum–activity relationship. In summary, the integrated use of the fingerprint and chemometric analysis provides a reliable method for the identification of markers and the quality control of Tianma Toutong tablets.     

Authentication and quantitative analysis on the chemical profile of Xanthium fruit (Cang-Er-Zi) by high-performance liquid chromatography-diode-array detection tandem mass spectrometry method

A high-performance liquid chromatographic method using diode-array detection and electrospray ionization tandem mass spectrometry (HPLC-DAD-ESI-MS/MS) was developed for the qualitative and quantitative analysis on Xanthium fruit, a commonly used traditional Chinese medicine. In this study, 7 characteristic components, 1-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, chlorogenic acid, 1,5-O-di-caffeoylquinic acid, 1,3-O-di-caffeoylquinic acid, 4,5-O-di-caffeoylquinic acid and 1,3,5-O-tri-caffeoylquinic acid were identified and quantified by a validated HPLC-DAD method, and a fingerprint comprised of 12 markers was established under the same operating conditions. Furthermore, HPLC-ESI-MS/MS method was successfully used to deduce the structure of three main constituents. On the basis of the established chromatographic profiles, 30 populations of cocklebur samples including 3 related species and 1 unknown species were divided into 3 chemotypes, indicated that place of origin significantly influences the kinds and content of components in cocklebur, and hence affects their quality. The simultaneous determination of 7 caffeoylquinic acids in the 30 samples showed a great variety in the amounts of caffeoylquinic acids present. The study indicated that some species such as Xanthium mongolicum of the genus Xanthium might be suitable for development as new alternative sources of caffeoylquinic acids to supplement the officially listed Xanthium species, and the abundant constituents such as chlorogenic acid perhaps should be recorded in some authorized publications and applied to the quality control or quality evaluation for Xanthium in China. The entire analytical procedure is reproducible and suitable for the authentication and quantification of Xanthium fruits.     

Chemometrics in near-infrared spectroscopy

Spectroscopy methods of chemical analysis are excellent for the application of chemometric methods, because the measurements at many different wavelengths provide inherently multivariate data. The chemist generally requires three categories of information from specimens under investigation: quantitative data, qualitative data, and fundamental information on the properties of the material. Spectroscopy has long been used for all three purposes; the recent application of chemometric algorithms has assisted greatly in these endeavors. Although there is some overlap, three chemometric methods correspond to the three types of information: multiple regression, discriminant analysis, and principal components analysis. The basis of these chemometric methods and some of their strengths and limitations in application to near-infrared spectroscopy are discussed.     

Beneficial Effect of Methanolic Extract of Frankincense (Boswellia Sacra) on Testis Mediated through Suppression of Oxidative Stress and Apoptosis

Boswellia sacra oleo gum resin (Burseraceae) commonly known as frankincense is traditionally used in many countries for its beneficial effect on male fertility. This study explores its effect on the male reproductive system after a 60-day repeated administration at two different doses to rats (in vivo) and on human Leydig cells (in vitro). The methanolic extract of B. sacra was analyzed for the presence of various constituents by preliminary phytochemical analysis and gas chromatography-mass spectrometry (GC-MS) while quantitative analysis of boswellic acids was done by high-performance liquid chromatography (HPLC). Administration of B. sacra extract to rats elevated the serum testosterone levels with an associated reduction in serum levels of FSH and LH. An increase in the activity of antioxidant enzymes, superoxide dismutase and catalase, was seen. A dose-dependent increase in the sperm count and sperm motility was also observed. The in vivo results were supported by changes in the expression of the Bcl-2 gene and caspase-3 gene in human Leydig cells in vitro. The results of this study support the traditional use of B. sacra to increase male fertility.     

High‐performance liquid chromatography based chemical fingerprint analysis and chemometric approaches for the identification and distinction of three endangered Panax plants in Southeast Asia

Among Panax genus, only three endangered species Panax notoginseng, P. vietnamensis, and P. stipuleanatus that have a similar morphology are mainly distributed in Southeast Asia. These three plants are usually misidentified or adulterated. To identify them well, their chemical chromatographic fingerprints were established by an effective high‐performance liquid chromatography method. By comparing the chromatograms, the three Panax species could be distinguished easily using the 22 characteristic peaks. Besides, the data of the chromatographic fingerprints aided by chemometric approaches were applied for the identification and investigation the relationship of different samples and species. Using similarity analysis, the chemical components revealed higher similarity between P. vietnamensis and P. stipuleanatus. The results of hierarchical clustering analysis indicated that samples belonging to the same species could be clustered together. The result of principal component analysis was similar with hierarchical clustering analysis and the three principal components accounted for >80.5% of total variability.     

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

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

Quantitative analysis of Cistanches Herba using high‐performance liquid chromatography coupled with diode array detection and high‐resolution mass spectrometry combined with chemometric methods

We aim to determine the chemical constituents of three species of Cistanches Herba using HPLC coupled with diode array detection and high‐resolution MS. Ten phenylethanoid glycosides were identified and further quantified as marker substances by HPLC coupled with diode array detection method. The separation was conducted using an Agilent TC‐C18 column with 0.1% formic acid and methanol as the mobile phases under gradient elution. The analytical method was fully validated in terms of linearity, sensitivity, precision, repeatability as well as recovery, and subsequently applied to evaluate the quality of 36 batches of Cistanche plants. The chemometric procedures (i.e., hierarchical clustering analysis and principal component analysis) were used to compare different species of Cistanches Herba, leading to successful classification of the Cistanche samples in accordance with their origins. In conclusion, this study provides a chemical basis for quality control of Cistanches Herba.