Characterization of the multiple chemical components of Glechomae Herba using ultra high performance liquid chromatography coupled to quadrupole‐time‐of‐flight tandem mass spectrometry with diagnostic ion filtering strategy

Glechomae Herba is a traditional Chinese medicine used for the treatment of urolithiasis, cholelithiasis, and urinary tract infections in China. Identification of chemical constituents is helpful to discover the potential active ingredients. However, this significant work is stymied by complex chemical constituents. Therefore, an ultra high performance liquid chromatography coupled to quadrupole‐time‐of‐flight tandem mass spectrometry analysis with diagnostic product ions and neutral loss filtering strategy was established for chemical profiling of Glechomae Herba. The diagnostic product ions and neutral loss filtering strategy simplified spectral elucidation. A total of 120 compounds, including 10 chlorogenic acids, 10 gallic acids, 21 phenylpropionic acids, and 77 flavonoids, were reasonably identified in Glechomae Herba. Sixty‐five constituents were first discovered in Glechomae Herba. Four types of chlorogenic acids (caffeoylquinic acid, feruloylquinic acid, p‐coumaroylquinic acid, and di‐caffeoylquinic acid), three types of galloylglucoses (di‐O‐galloyl‐glucose, tri‐O‐galloyl‐glucose, and tetra‐O‐galloyl‐glucose), three types of phenylpropionic acid skeletons (p‐coumaric acid, caffeic acid, and rosmarinic acid) and five types of flavonoid aglycone skeletons (apigenin, kaempferol, luteolin, quercetin, and chrysin) were identified in Glechomae Herba. The results indicated that the developed strategy was feasible and rational technique for identifying the complex chemical constituents in Glechomae Herba.     

Chemical Fingerprint Analysis for Quality Control of Herba Ephedrae Based on HPLC-DAD Combined with Chemometrics Methods

A method based on high performance liquid chromatography with photodiode array detector (HPLC-DAD) was developed for chemical fingerprinting analysis of Herba Ephedrae. The index of chromatographic fingerprint's information content was utilized to optimize the fingerprint detection conditions, which reduced the time of analysis and increased the veracity of analysis greatly. Then, the similarity analysis of fingerprints was used in quality consistency evaluation of Herba Ephedrae samples. Moreover, hierarchical clustering analysis (HCA) was applied to classify the samples according to their sources and varieties. In addition, the overlapped chromatographic peaks were resolved with the help of heuristic evolving latent projection (HELP) method in order to gain the better quantitative evaluation. The results indicated that the samples could be successfully grouped in accordance with their varieties and sources. Furthermore, five marker constituents were firstly screened out to be the main chemical markers, which importantly contribute to the classification of Herba Ephedrae samples. This investigation shows that the developed methodology can be generalized to the research of quality control of herbal medicines.     

Qualitative Analysis and Componential Differences of Chemical Constituents in Taxilli Herba from Different Hosts by UFLC-Triple TOF-MS/MS

Taxilli Herba (TH) is a well-known traditional Chinese medicine (TCM) with a wide range of clinical application. However, there is a lack of comprehensive research on its chemical composition in recent years. At the same time, Taxillus chinensis (DC) Danser is a semi parasitic plant with abundant hosts, and its chemical constituents varies due to hosts. In this study, the characterization of chemical constituents in TH was analyzed by ultra-fast liquid chromatography coupled with triple quadrupole-time of flight tandem mass spectrometry (UFLC-Triple TOF-MS/MS). Moreover, partial least squares discriminant analysis (PLS-DA) was applied to reveal the differential constituents in TH from different hosts based on the qualitative information of the chemical constituents. Results showed that 73 constituents in TH were identified or tentatively presumed, including flavonoids, phenolic acids and glycosides, and others; meanwhile, the fragmentation pathways of different types of compounds were preliminarily deduced by the fragmentation behavior of the major constituents. In addition, 23 differential characteristic constituents were screened based on variable importance in projection (VIP) and p-value. Among them, quercetin 3-O-β-D-glucuronide, quercitrin and hyperoside were common differential constituents. Our research will contribute to comprehensive evaluation and intrinsic quality control of TH, and provide a scientific basis for the variety identification of medicinal materials from different hosts.     

A study for quality evaluation of Taxilli Herba from different hosts based on fingerprint-activity relationship modeling and multivariate statistical analysis

In this study, a fingerprint-activity relationship modeling between chemical fingerprints and antirheumatic activity was established, and multivariate statistical analysis was used to evaluate the quality of Taxilli Herba (TH) from different hosts. Characteristic fingerprints of 20 batches of TH samples were generated by high-performance liquid chromatography coupled with triple quadrupole-time of flight tandem mass spectrometry (HPLC-Triple TOF-MS/MS), and the similarity analysis was calculated based on thirteen common characteristic peaks by hierarchical clustering analysis (HCA). Subsequently, nine efficacy markers were discovered by combining fingerprints and antirheumatic activity through grey correlation analysis (GCA) and bivariate correlation analysis (BCA). Meanwhile, the content of 5 constituents in 9 markers was determined by high-performance liquid chromatography coupled with triple quadrupole-linear ion trap tandem mass spectrometry (HPLC-QTRAP-MS/MS). The comprehensive quality of TH was assessed using multivariate statistical analysis, including principal components analysis (PCA) and technique for order preference by similarity to ideal solution (TOPSIS). The results showed that a high dose of TH extract could markedly ameliorate arthritis damage compared to other doses, with flavonoids playing an important role in the antirheumatic activity. The comprehensive quality of samples from Morus alba L. (SS) was superior to those from Liquidambar formosana Hance (FXS). The present study will demonstrate the markers associated with efficacy, and provide an applicable strategy for more comprehensive quality control and evaluation of TH.     

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.     

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.     

Analysis of volatile compounds in Herba Asari by single-drop micro-extraction gas chromatography mass spectrometry

A rapid headspace single-drop micro-extraction(mix) gas chromatography mass spectrometry(SDMEGC -MS) for the analysis of the volatile compounds in Herba Asari was developed in this study.A mixed solvent of n-tridecane and butyl acetate(1:1) was finally used for the extraction at 70 C for 15 min with sample amount of 0.750 g and 100 mesh particle size.Under the determined conditions,the pound samples of Herba Asari were directly applied for the analysis.SDME-GC-MS,SPME-GC-MS and SD-GCMS methods were compared and the results showed that SDME-GC-MS method was a simple, inexpensive and effective way to measure the volatile compounds in Herba Asari and could be used for the analysis of volatile compounds in complex samples.     

Simultaneous quantification of 25 active constituents in the total flavonoids extract from Herba Desmodii Styracifolii by high‐performance liquid chromatography with electrospray ionization tandem mass spectrometry

A sensitive and selective high‐performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry method has been developed and validated for the simultaneous determination of 25 active constituents, including 21 flavonoids and four phenolic acids in the total flavonoids extract from Herba Desmodii Styracifolii for the first time. Among the 25 compounds, seven compounds including caffeic acid, acacetin, genistein, genistin, diosmetin, diosmin and hesperidin were identified and quantified for the first time in Herba Desmodii Styracifolii. Chromatographic separation was accomplished on a ZORBAX SB‐C₁₈ (250 mm×4.6 mm, 5.0 μm) column using gradient elution of methanol and 0.1‰ acetic acid v/v at a flow rate of 1.0 mL/min. The identification and quantification of the analytes were achieved using negative electrospray ionization mass spectrometry in multiple‐reaction monitoring mode. The method was fully validated in terms of limits of detection and quantification, linearity, precision and accuracy. The results indicated that the developed method is simple, rapid, specific and reliable. Furthermore, the developed method was successfully applied to quantify the 25 active components in six batches of total flavonoids extract from Herba Desmodii Styracifolii.     

Quality assessment of Herba Leonuri based on the analysis of multiple components using normal‐ and reversed‐phase chromatographic methods

A novel, improved, and comprehensive method for quality evaluation and discrimination of Herba Leonuri has been developed and validated based on normal‐ and reversed‐phase chromatographic methods. To identify Herba Leonuri , normal‐ and reversed‐phase high‐performance thin‐layer chromatography fingerprints were obtained by comparing the colors and R _f values of the bands, and reversed‐phase high‐performance liquid chromatography fingerprints were obtained by using an Agilent Poroshell 120 SB‐C18 within 28 min. By similarity analysis and hierarchical clustering analysis, we show that there are similar chromatographic patterns in Herba Leonuri samples, but significant differences in counterfeits and variants. To quantify the bio‐active components of Herba Leonuri , reversed‐phase high‐performance liquid chromatography was performed to analyze syringate, leonurine, quercetin‐3‐O‐robiniaglycoside, hyperoside, rutin, isoquercitrin, wogonin, and genkwanin simultaneously by single standard to determine multi‐components method with rutin as internal standard. Meanwhile, normal‐phase high‐performance liquid chromatography was performed by using an Agilent ZORBAX HILIC Plus within 6 min to determine trigonelline and stachydrine using trigonelline as internal standard. Innovatively, among these compounds, bio‐active components of quercetin‐3‐O‐robiniaglycoside and trigonelline were first determined in Herba Leonuri . In general, the method integrating multi‐chromatographic analyses offered an efficient way for the standardization and identification of Herba Leonuri .     

Simultaneous determination of 20 bioactive components in Chuanxiong Rhizoma from different production origins in Sichuan province by ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry combined with multivariate statistical analysis

Chuanxiong Rhizoma is a commonly used in traditional Chinese medicine. Chuanxiong Rhizoma is widely distributed in Sichuan province, China, including the cities of Dujiangyan, Pengzhou, Meishan, Qionglai, and Shifang. However, reports on the comparisons of quality of Chuanxiong Rhizoma of different production origins are limited. Therefore, an ultra-HPLC with triple quadrupole MS method was developed for the determination of 20 bioactive components (12 aromatic acids and eight phthalides) in 36 samples from different production origins and further assessed its quality. The contents of these 20 constituents of samples were analyzed by hierarchical cluster analysis and orthogonal partial least squares discrimination analysis; the result indicated that Chuanxiong Rhizoma of different production origins had some differences. Thirteen constituents of quality difference markers were acquired by variable importance for the project. Furthermore, the sum of the contents of these quality difference markers was different from various production origins of Chuanxiong Rhizoma. Meanwhile, Z-ligustilide and senkyunolide A as main constituents of quality difference markers, the rate of various production origins of Chuanxiong Rhizoma was different. This study provides a foundation for the quality assessment of Chuanxiong Rhizoma.     

Investigating sub-2 μm particle stationary phase supercritical fluid chromatography coupled to mass spectrometry for chemical profiling of chamomile extracts

Roman and German chamomile are widely used throughout the world. Chamomiles contain a wide variety of active constituents including sesquiterpene lactones. Various extraction techniques were performed on these two types of chamomile. A packed-column supercritical fluid chromatography–mass spectrometry method was designed for the identification of sesquiterpenes and other constituents from chamomile extracts with no derivatization step prior to analysis. Mass spectrometry detection was achieved by using electrospray ionization. All of the compounds of interest were separated within 15 min. The chamomile extracts were analyzed and compared for similarities and distinct differences. Multivariate statistical analysis including principal component analysis and orthogonal partial least squares-discriminant analysis (OPLS-DA) were used to differentiate between the chamomile samples. German chamomile samples confirmed the presence of cis- and trans-tonghaosu, chrysosplenols, apigenin diglucoside whereas Roman chamomile samples confirmed the presence of apigenin, nobilin, 1,10-epioxynobilin, and hydroxyisonobilin.     

Comparison of Multiple Bioactive Constituents in the Corolla and Other Parts of Abelmoschus manihot

Abelmoschus manihot (L.) Medic (AM), called Huangshukui in Chinese, is a widely used medicinal plant. Each part of AM has medicinal value, including Abelmoschi Radix (AR), Abelmoschi Herba (AH), Abelmoschi Folium (AF), Abelmoschi Corolla (AC), and Abelmoschi Semen (AS). However, only AC is documented in the Chinese Pharmacopoeia. In order to investigate whether there is any difference between AC and the other parts of AM, an analytical method based on ultra-fast performance liquid chromatography coupled with triple quadrupole-linear ion trap mass spectrometry (UFLC-QTRAP-MS/MS) was established for the simultaneous determination of 35 constituents in different parts of AM. Moreover, principal components analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were applied to classify and evaluate the different parts of AM based on the content of the 35 constituents. The total contents of the 35 constituents in AC were significantly higher than in the other parts of AM and the results revealed significant differences between AC and the other parts of AM. Eight constituents were remarkably related to the sample classifications. This research does not just provide the basic information for revealing the distribution patterns in different parts of AM from the same origin, but also complements some of the scientific data for the comprehensive quality evaluation of AC.     

An MC3T3-E1 Cell Line Biomembrane Extraction and HPLC–ESI-MS n Method for Simultaneous Analysis of Potential Anti-Osteoporosis Components of Epimedium koreanum

Aerial parts of Epimedium koreanum Nakai have been used as Herba Epimedii in China. Its anti-osteoporosis effect has attracted much attention in recent years. In this study, a method involving osteoblastic cell (MC3T3-E1 cell line) extraction and high-performance liquid chromatography–electrospray ionisation–mass spectrometry (HPLC–ESI-MS ⁿ ) was developed for screening of potential anti-osteoporosis agents in E.?koreanum. Four compounds identified as epimedin?A, epimedin?B, epimedin?C and icariin were found to interact with MC3T3-E1 cells and possessed potent osteoblast-stimulating activity as evaluated by cell proliferation [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay] and differentiation [alkaline phosphatase (ALP) activity and Ca content] in?vitro. The results suggest that these four flavonoids are the anti-osteoporosis constituents of Herba Epimedii and that the method combining MC3T3-E1 cell extraction with HPLC–ESI-MS ⁿ is rapid and effective in screening anti-osteoporosis agents from traditional Chinese medicines.     

Fatty Acid Analysis,Chemical Constituents,Biological Activity and Pesticide Residues Screening in Jordanian Propolis

Propolis is a resinous natural product collected by honeybees (Apis mellifera and others) from tree exudates that has been widely used in folk medicine. The present study was carried out to investigate the fatty acid composition, chemical constituents, antioxidant, and xanthine oxidase (XO) inhibitory activity of Jordanian propolis, collected from Al-Ghour, Jordan. The hexane extract of Jordanian propolis contained different fatty acids, which are reported for the first time by using GC-FID. The HPLC was carried out to identify important chemical constituents such as fatty acids, polyphenols and α-tocopherol. The antioxidant and xanthine oxidase inhibitory activities were also monitored. The major fatty acid identified were palmitic acid (44.6%), oleic acid (18:1∆⁹cis, 24.6%), arachidic acid (7.4%), stearic acid (5.4%), linoleic acid (18:2∆^9–12cis, 3.1%), caprylic acid (2.9%), lignoceric acid (2.6%), cis-11,14-eicosaldienoic acid (20:2∆^11–14cis, 2.4%), palmitoleic acid (1.5%), cis-11-eicosenoic acid (1.2%), α–linolenic acid (18:3∆^9–12–15cis, 1.1%), cis-13,16-docosadienoic acid (22:2∆^13–16cis, 1.0%), along with other fatty acids. The major chemical constituents identified using gradient HPLC-PDA analysis were pinocembrin (2.82%), chrysin (1.83%), luteolin-7-O-glucoside (1.23%), caffeic acid (1.12%), caffeic acid phenethyl ester (CAPE, 0.79%), apigenin (0.54%), galangin (0.46%), and luteolin (0.30%); while the minor constituents were hesperidin, quercetin, rutin, and vanillic acid. The percentage of α-tocopherol was 2.01 µg/g of the lipid fraction of propolis. Antioxidant properties of the extracts were determined via DPPH radical scavenging. The DPPH radical scavenging activities (IC₅₀) of different extracts ranged from 6.13 to 60.5 µg/mL compared to ascorbic acid (1.21 µg/mL). The xanthine oxidase inhibition (IC₅₀) ranged from 75.11 to 250.74 µg/mL compared to allopurinol (0.38 µg/mL). The results indicate that the various flavonoids, phenolic compounds, α-tocopherol, and other constituents which are present in propolis are responsible for the antioxidant and xanthine oxidation inhibition activity. To evaluate the safety studies of propolis, the pesticide residues were also monitored by LC-MS-MS 4500 Q-Trap. Trace amounts of pesticide residue (ng/mL) were detected in the samples, which are far below the permissible limit as per international guidelines.     

A Validated LC Method for Simultaneous Determination of Three Major Components in Entada phaseoloides

A high performance liquid chromatography method was developed for the simultaneous determination of three major active constituents in Entada phaseoloides, namely phaseoloidin (1), entadamide A (2), entadamide A-β-d-glucopyranoside (3), respectively. The samples were separated on an Aglient Eclipse XDB-C₁₈ column with gradient elution of acetonitrile and 0.3% phosphoric acid (v/v) at a flow rate of 1.0 mL min^?1 and detected at 280 nm. The three target compounds were completely separated within 10 min. All calibration curves showed good linearity (r > 0.9999) within test ranges. The reproducibility was evaluated by intra- and inter-day assays and RSD values were less than 1.04%. The recoveries were between 97.15 and 101.95%. The method was successfully applied to the analysis of three compounds in 22 commercial samples of E. phaseoloides. The results indicated that the developed LC assay was readily utilized as a quality control method for E. phaseoloides.     

Comparative Analysis of the Major Constituents in the Traditional Tibetan Medicinal Plants Saussurea laniceps and S. medusa by LC–DAD–MS

Liquid chromatography coupled with diode array detector and electrospray ionization mass spectrometry was developed for the qualitative and quantitative comparison of the main constituents in Saussurea laniceps (SL) and S. medusa (SM), two species of plants used under the name “Xuelianhua” in traditional Tibetan medicine. A method validation including linearity, limit of detection, precision and recovery was performed. The results showed that a good linearity with R ² > 0.99 was achieved, and the limit of detection of the quantified constituents was reported to be between 0.8 and 3.3 ng. The relative standard deviation value was below 3.82% for repeatability, and recovery studies for the quantified compounds were found to be within the range 90.92–103.12%. The unique properties of the present method were evaluated by analyzing twelve related herbal samples including five S. laniceps samples and seven S. medusa samples. Twenty-two compounds including phenolic acids, cumarins, lignanoids and flavonoids were identified by online ESI–MS and by comparison with literature data and standard compounds, and seven of them were quantified by LC–DAD simultaneously. The results demonstrated that the common constituents in the two herbs were protocatechuic acid, syringoside, chlorogenic acid, isoquercitroside, 1,5-dicaffeoylquinic acid, apigenin 7-O-β-d-glucoside, chrysoeriol 7-O-β-d-glucoside, acacetin 7-O-β-d-glucoside, apigenin and chrysoeriol. In the present study, it was found that the characteristic constituents were umbelliferone, scopoletin and their glucosides in S. laniceps, as well as arctiin and arctigenin in S. medusa. It was feasible to choose these characteristic compounds for the quality evaluation as well as chemical authentication of the two related herbs. The results also support discrimination between the two species when using them in folk medicine.     

Systematically Exploring the Chemical Ingredients and Absorbed Constituents of Polygonum capitatum in Hyperuricemia Rat Plasma Using UHPLC-Q-Orbitrap HRMS

Polygonum capitatum as an ethnic medicine has been used to treat urinary tract infections, pyelonephritis and urinary calculi. In our previous study, P. capitatum was found to have anti-hyperuricemia effects. Nevertheless, the active constituents of P. capitatum for treating hyperuricemia were still unclear. In this study, an ultra-high-performance liquid chromatography coupled to quadrupole/orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) was used to comprehensively detect the chemical ingredients of P. capitatum and its absorbed constituents in the plasma of hyperuricemia rats for the first time. Xcalibur 3.0 and Compound Discoverer 2.0 software coupled to mzCloud and ChemSpider databases were utilized for qualitative analysis. A total of 114 chemical components including phenolics, flavonoids, tannins, phenylpropanoids, amino acids, amides and others were identified or tentatively characterized based on the exact mass, retention time and structural information. Compared to the previous P. capitatum study, an additional 66 different components were detected. Moreover, 68 related xenobiotics including 16 prototype components and 52 metabolites were found in the plasma of hyperuricemia rats. The metabolic pathways included ring fission, hydrolysis, decarboxylation, dehydroxylation, methylation, glucuronidation and sulfation. This work may provide important information for further investigation on the active constituents of P. capitatum and their action mechanisms for anti-hyperuricemia effects.     

HPLC-DAD and TLC-Densitometry for quantification of hypericin inHypericum perforatum L. Extracts

Summary Hypericum perforatum L. is a spontaneous perennial herbaceous plant, widely distributed in Europe, Asia, Northern Africa, and North America. The dried flowers or dried aerial parts are used to prepare the drug Hyperici Herba or St. John's Wort. Nowadays this drug is largely used as a natural antidepressant; hypericin and hypericin-like substances are considered the main active ingredients. In this work the hypericin and pseudohypericin content of hydroalcoholic extracts both of Hyperici Herba and ofHypericum perforatum L. dried flowers were measured by two techniques, TLC-densitometry with fluorescence detection and reversed-phase HPLC-DAD (diode-array detection). The quantitative data obtained by applying these techniques were compared and the identification of the main flavonoid constituents was performed by HPLC-DAD for characterization of the extracts. The quantitative data obtained by use of the two techniques were in good agreement and statistical analysis of the findings was indicative of the equivalence of the techniques.     

Analysis of Biologically Active Constituents in Centella asiatica by Microwave-Assisted Extraction Combined with LC–MS

Centella asiatica (L.) Urban is a traditional herbal medicine used in Asiatic countries, and is commonly used to treat various wounds, leprosy, tuberculosis and lupus diseases. In this work, a new method based on microwave assisted extraction followed by liquid chromatography–diode array detection–electrospray ionization multistage tandem mass spectrometry analysis has been developed for the identification and quantification of biologically active constituents in C. asiatica, including asiatic acid, asiaticoside and madecassoside. The separation was performed on an Agilent Eclipse C₁₈ column (150 mm × 4.6 mm i.d., 5 μm) with gradient elution of acetonitrile and 0.1% aqueous acetic acid within 50 min. Detection was performed at 205 nm. The calibration curves showed good linearity (r ² > 0.9992). The limits of detection ranged from 1.2 to 1.6 μg mL^?1. The intra- and inter-day precision was less than 3% and the recovery of the assay was in the range of 95.4–106.8%. The method was successfully applied to the quantification of the three constituents in different samples of C. asiatica. The results indicated that the developed method could be considered to be a simple, rapid and reliable method for the quality evaluation of C. asiatica. The samples were also analyzed on a liquid chromatography–electrospray ionization–time-of-flight mass spectrometry system to confirm the identification results.