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Objectives Corni Fructus is one of the most famous traditional Chinese medicines (TCMs) for the treatment of various chronic kidney diseases. Wine-processed Corni Fructus (WCF) is the main processed form of Crude Corni Fructus (CCF). In this study, potential mechanisms of action of CCF and WCF on chronic renal failure (CRF) model were developed to explore wine-processed mechanism of Corni Fructus. Methods An integrated strategy combining metabolomics, network analysis and bioinformatics analysis has been established to investigate the therapeutic mechanisms of WCF and CCF in rats with CRF. Key findings The histopathological results showed that both WCF and CCF improved kidney injury and dysfunction of CRF rats, but WCF was more effective than CCF. Metabolic pathway analysis indicated that 24 metabolites and 5 major disturbed pathways associated with CCF, while WCF regulated 27 metabolites and 2 metabolic pathways. Bioinformatic analysis and network analysis revealed that 8 genes and 7 genes were regulated by CCF and WCF on CRF rats, respectively. The quantitative real-time polymerase chain reaction experiments verified the regulatory ability of CCF and WCF on the expression of 4 genes. Conclusions An integrated strategy combined metabolomics, network analysis and bioinformatics was established to provide valuable holistic insight to explore the processing mechanism of TCMs.

This paper analyzed the differences between the two harvesting periods (Taiju and Duoju) on the ingredients and pharmacological activity in Chrysanthemum morifolium Ramat.

Rationale: Radix scutellaria-Licorice drug pair (RSLDP), a frequently used herbal pair with the effect of clearing heat and detoxifying, is the commonly employed drug pair in TCM prescription for the treatment of COVID-19. Until now, the metabolism feature and anti-COVID-19 mechanism of RSLDP have not been fully elucidated. Methodology: In this study, a sensitive and rapid method was developed for the separation and identification of the absorbed constituents of RSLDP in the rat plasma by UHPLC-QTOF-MS. Besides, we optimized the conventional process ways of network pharmacology and proposed a new concept called target network pharmacology (T-NP). It used the absorbed constituents and the corresponding targets to generate compound-target network, and compare to the conventional network pharmacology, it could reduce the false positive results. Results: A total of 85 absorbed constituents were identified or tentatively characterized in dosed plasma,including 32 components in Radix Scutellaria,27 components in Licorice and 65 components in RSLDP. The results showed that the compatibility of Radix Scutellaria and Licorice increased the quantity of components in vivo. We found that 106 potential targets of 64 active compounds in RSLDP were related to COVID-19.And 12 targets (MAPK3,IL6,JUN,IL2,STAT3, AKT1,TNF,STAT1,EGFR,HSP9AA1,VEGFA,RELA) could be core targets for RSLDP in treating COVID-19. Conclusions: The metabolism feature of RSLDP in vivo was systematically uncovered. The combining use of the T-NP method could discover potential drug targets and disclose the biological processes of RSLDP, which will clarify the potential mechanisms of RSLDP in the treatment of COVID-19.

Fritillaria cirrhosae bulbus (BFC), a typical traditional Chinese medicine with multiple botanical sources, has been used for relieving cough and reducing sputum. Studies have shown that there were obvious differences in the chemical compositions and clinical efficacy of different sources of BFC. How to characterise BFC from botanical sources accurately and quickly is vital for drug quality evaluation and clinical applications.In the present study, an integrated strategy of plant metabolomics combined with the target network pharmacology was developed to characterise BFC. Plant metabolomics analysis was performed to screen out the chemical markers of six species of BFC. Then, target network pharmacology was applied to explore the relationship between chemical markers and related diseases. Finally, potential Q-markers for species characterization were selected by combined analysis of plant metabolomics and the target network pharmacology.A total of 67 Fritillaria alkaloid compounds were identified. Six species showed clear characterization by multivariate statistical analysis, resulting in 12 chemical markers. Meanwhile, a total of nine components related to asthma were screened out based on the target network pharmacology. Taking content difference and pharmacological activity into consideration, nine constituents were selected as potential Q-markers.The method developed provided not only a standard protocol for characterising different species of BFC directly, but also an effective approach for multisource medicines discrimination.

Tyrosinase inhibitors can alleviate the harm to the liver caused by tyrosinase. How to effectively screen out natural tyrosinase inhibitors becomes a focus. In this study, Isodon excisoides was first extracted with the ultrasound optimized by response surface methodology. Then, a method combined ultrafiltration with ultra-liquid chromatography mass spectrometry (UHPLC/MS) was built to screen and identify tyrosinase inhibitors. The binding energies of active ingredients to tyrosinase were calculated by molecular docking. The reliability of the results was validated by the IC

Co-crystallization of active pharmaceutical ingredients (API) with co-formers can induce synergistic effects on cytotoxicity; however, the underlying mechanism is unclear. Here, cell metabolomics was used to gain insight into the mechanisms of synergistic effect from API and co-former in co-crystal. The 5-Fluorouracil-phenylalanine co-crystal system was selected as the model owing to the apparent difference of cytotoxicity occurring between co-crystal and physical mixture of two components (PM). The cytotoxicity of 5-FU, PM and co-crystal on B16 cells were evaluated by MTT assay. Based on the IC

Peimine, one of the major quality markers in Fritillaria Cirrhosae Bulbus, was expected to become a new anti-asthma drug. However, its metabolic profiles and anti-asthma mechanism have not been clarified previously. In this study, a method was developed for the detection of peimine metabolites in vitro by ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry. The potential anti-asthma mechanism was predicted by an integrated analysis of network pharmacology and molecular docking. A total of 19 metabolites were identified with the aid of software and molecular networking. The metabolic profiles of peimine elucidated that the metabolism was a multi-pathway process with characteristics of species difference. The network pharmacology results showed that peimine and its metabolites could regulate multiple asthma-related targets. The above targets were involved in various regulatory pathways linked to asthma. Moreover, the results of molecular docking showed that both peimine and its metabolites had a certain affinity with the β

Introduction Hederacoside C (HDC) is a bioactive natural triterpenoid saponins constituent originating from traditional Chinese medicines, playing an important role in the treatment of acute respiratory infections and chronic inflammatory bronchitis. Meanwhile, it is recognised by Korea as a botanical drug. Objectives In order to develop an integrated template approach to analysing screening and identification of the metabolites of traditional Chinese medicines. This study will provide available information for further pharmaceutical studies of HDC and other triterpene saponins. Methodology An analysis strategy based on ultrahigh-performance liquid chromatography quadrupole Orbitrap mass spectrometry (UHPLC-Q-Orbitrap-MS) technique combined with automatic fragment ion search (FISh) was firstly exploited for the characterisation metabolites of HDC in vivo and in vitro. Accurate full mass scan combined with an on-line FISh annotations approach was developed to rapidly identify all the potential metabolites of HDC. Furthermore, FISh accurately located the structure of the target compound in a large number of mass spectral data. Results A total of 34 metabolites were detected and tentatively identified by analysing comprehensive biological samples. The results clearly demonstrated that HDC underwent general metabolic reactions including dealkylation, reduction, oxidation, desaturation, dehydration, cysteine conjugation, GSH conjugation, taurine conjugation, and glycine conjugation to produce 26 phase I and eight phase II metabolites. Conclusion In the present study, UHPLC-Q-Exactive Orbitrap MS technique combined with FISh provided a rapid and efficient platform to characterise metabolites of HDC in vivo and in vitro. The proposed method could develop an integrated template approach to screen and identify the constituents and metabolites of traditional Chinese medicines.

The diterpenoids are the most important active constituents that contribute to the pharmacological efficacy of Isodon serra (Maxim.) Hara. Clinical studies have revealed that diterpenoids possess multiple features, e.g. antitumour, antitubercular and anti-ischemic activities. Therefore, the identification and detection of diterpenoids may be equally important for understanding the pharmacological basis of diterpenoids and enhancing the product quality control of I. serra.The purpose of this study was to develop a practical analysis approach of rapid characterisation using ultrahigh-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) for the structure characterisation of the ent-kaurane diterpenoids from I. serra.The analytical strategy was as follows: first, ent-kaurane diterpenoids were detected by a novel on-line data acquisition approach, i.e. sequential window acquisition of all theoretical fragment-ion spectra (SWATH). Second, the MS of eight ent-kaurane diterpenoids was explored, and their mass spectrum cleavage pathways were summarised and determined. Finally, the methanol extract of I. serra was studied using SWATH and identified by extracted ion chromatography (XIC).Compared to the traditional information-dependent acquisition (IDA) method, SWATH significantly improved the hit rate of ent-kaurane diterpenoids. With support from UHPLC separation and specific detection by tandem mass spectrometry (MS/MS), 48 ent-kaurane diterpenoids were successfully characterised and classified as ent-kaurane diterpenoids from a complex matrix.These combined qualitative methods were used to provide a potential approach for the characterisation of traditional Chinese medicine (TCM) and its preparations. Meanwhile, the SWATH provided a novel and reliable method for the structural characterisation of ent-kaurane diterpenoids from other complicated TCMs.

Zingiberis Rhizoma (ZR) is a homologous plant with pungent tastes and aromas, which has unique nutritional value and tremendous application potentiality. Zingiberis Rhizoma Praeparatum (ZRP) and Carbonised Ginger (CG) are processed products of ZR through different processing methods, and they are commonly used ingredients in food supplements. This study used ZR, ZRP and CG from different batches to further understand composition differences after processing. Additionally, we performed non-targeted metabolomics-based profiling of gingerols by ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) in combination with multivariate analysis and compounds identification. In which, we developed a comprehensive SWATH-IDA bi-directionally verified (SIBDV) method integrating the advantages of Sequential Windowed Acquisition of all Theoretical fragment ions (SWATH

Poria cocos (Schw.) Wolf (PCW) is a widely used traditional Chinese medicine and dietary supplement. Its four parts including Poriae Cutis (PC), Rubra Poria (RP), White Poria (WP) and Poria cum Radix Pini (PRP) have different pharmacological effects and clinical applications. It is necessary to establish analytical platforms for differentiating the chemical composition of four botanical parts for the rational utilization. We established a simultaneous qualitative and quantitative method based on UHPLC-MS combined metabolomics approach to give an explanation of the distribution of triterpene compounds in four parts. Eight triterpene compounds were determined absolutely and a total of 51 triterpene compounds were tentatively identified in PCW. PC and PRP showed a quite clear discrimination by the principal component analysis (PCA) and orthogonal projections to latent structures-discriminant analysis (OPLS-DA) and twelve differential compounds were found. Four compounds including poricoic acid D, 16α-hydroxydehydrotrametenolic acid, 3-epidehydrotumulosic acid, 25-hydroxypolyporenic acid C were speculated to be related to diuretic effects.

In this paper, an analytical strategy combined data acquisition with a practical mining strategy aimed at rapid characterization and quantitation of ent-kaurane diterpenoids in Isodon japonica using ultra high-performance liquid chromatography-triple time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS). First, an effective self-built filter template based on drug phase I/II metabolic reaction theory and a components library data set were established. Second, the mass spectra of ent-kaurane diterpenoid standards were studied and their mass spectrum cleavage pathways were summarized. Next, the methanol extract of this herb was studied by data-independent acquisition mode (DIA). With the aid of a self-built filter template, the peaks of ent-kaurane diterpenoids were easily picked out and rapidly classified as ent-kaurane diterpenoids from a complex matrix. A total of 24 ent-kaurane diterpenoids were structurally identified. Meanwhile, the self-built filter template provided a convenient and fast method for the structural characterization and Isodon japonica was used to illustrate this approach for the first time. Furthermore, eight major bioactive diterpenoids were simultaneously quantified by a newly developed superimposed multiple product ion (SMPI) with UPLC-Q-TOF-MS/MS method. Principal component analysis (PCA) revealed significant differences in different batches of samples. These combined qualitative and quantitative methods were used to provide a potential approach for the holistic quality evaluation of traditional Chinese medicine (TCM) and its preparations.

Betulin is an active natural pentacyclic triterpene ingredient with valuable anti-cancer and anti-HIV efficacies. In the present study, an efficient approach was developed to screening and identification of metabolites and to assess the metabolic profiles of betulin in vivo using UHPLC-Q-TOF-MS/MS system based on multiple mass defect filter data acquisition (MMDF) and multiple data processing techniques. Based on the proposed method, 56 phase I and 6 phase II metabolites were detected in rats after oral administration of betulin. The main biotransformation routes of betulin were identified as demethylation, dehydroxylation, deoxidization, dehydration. Conjugation with sulfate, taurine, cysteine and N-acetylcysteine groups produced 6 phase II metabolites. This study not only provided useful information for further study of the pharmacology and mechanism of betulin in vivo, but also provided essential data for further pharmaceutical studies of other pentacyclic triterpenes.

The holistic quality evaluation of Traditional Chinese Medicine (TCM) is confronted with significant challenges due to its extreme chemical complexity. In this study, a sensitive strategy based on ultra-high-performance liquid chromatography-triple/time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS) and chemometric analysis was established and validated for the qualitative and semi-quantitative analyses of characteristic components in Usnea. First, three mass spectrometry fragmentation patterns of phenolic acid standards were studied and summarized. Then, an extract of this herb was analyzed by the full-scan MS spectra and identified by extracted ion chromatography (XIC). Based on the abovementioned methods, a total of 38 compounds (8 dibenzofurans, 11 didepsides, 13 depsidones, and 6 mono-substituted phenyl rings) were identified. Subsequently, the qualities of Usnea samples from different regions were evaluated by the semi-quantitative analysis based on their relative peak areas. Furthermore, principal component analysis (PCA) was performed to compare the Usnea herbs and to find possible diagnostic chemical components. This novel and powerful strategy could provide a potential approach for the holistic quality control of TCM.

A tissue-smashing based ultra-rapid extraction coupled with ultra-performance liquid chromatography tandem-mass spectrometry (UPLC-MS/MS) method was developed to determine 10 major triterpenoid saponins from Pulsatilla herbs. Compound 4 was characterized as betulinic acid glycoside 3-O-α-arabinopyranosyl-28-O-β-glucopyranosyl-23-hydroxy with HR-ESI-MS, 1H-NMR and 13C-NMR experiment. The MS spectra result showed that the ionization of compound 4 was more efficient in the positive mode. Meanwhile, the ions at m/z 789.6 and m/z 627.5 were selected as precursor and product ion for the determination, respectively. The chromatographic separation was carried out on a Phenomenex Kinetex C18 column using a gradient mobile phase system composed of 0.1% formic acid both in methanol and water at a flow rate of 0.4 mL/min. The detection was performed by multiple reaction monitoring mode, using electrospray ionization in the positive and negative mode. The total run time was 6 min. The calibration curves possessed good linearity with all coefficients higher than 0.9987. The intra- and interday precisions were no more than 4.9%, and the average recoveries were from 97.6% to 103.4% with RSD

Genuine regional drugs have played a vital role in clinical use for a long time. There are differences in traditional Chinese medicines (TCM) from different regions based on their chemical composition. Differences in chemical composition may lead to deviations in therapeutic effects. To our knowledge, to date, there are no effective methods for distinguishing genuine regional drugs based on the differences in their chemical composition. Therefore, establishing an analytical platform for distinguishing the compounds used in TCM from various geographical locations is essential. In this work, an integrated platform based on UPLC-Q-TOF-MS/MS combined with plant metabolomics approach was established for comprehensively distinguishing genuine regional drugs. Isodon rubescens (Hemsl.) Hara, a widely used herbal medicine of China, was chosen for this study and 24 samples from four geographical locations in China were collected. A total of 60 ent-kaurane diterpenoids were tentatively identified, and then the samples from four geographical origins were distinguished using PCA (principal component analysis) and PLS-DA (partial least squares discrimination analysis). Different compounds were identified among the samples collected from the four geographical locations, and 12 of them were regarded as marker compounds responsible for the distinction. Our study highlights the essence and predictive ability of metabolomics in detecting minute differences in the same varieties of TCM samples based on the levels and compositions of their metabolites. These results showed that the analysis using UHPLC-Q-TOF-MS/MS combined with metabolomics could be applied to distinguish the geographical origins and varieties of TCM.

Ponicidin is an active natural ent-kaurane diterpenoid ingredient originating from many Isondon herbs and is expected to become a new anticancer agent. In this study, a practical strategy was developed for the identification of ponicidin metabolites in vivo and in vitro utilizing ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). The analytical strategy was as follows: potential ponicidin metabolites were detected by a novel on-line data acquisition approach, i.e., sequential window acquisition of all theoretical fragment-ion spectra (SWATH™). Compared to the traditional information-dependent acquisition (IDA) method, SWATH™ significantly improved the hit rate of low-level or trace metabolites because it could obtain all MS/MS spectra. Moreover, many data post-processing methods were used to deduce the metabolites structures. As a result, a total of 20 metabolites were characterized in vivo and in vitro. The results showed that ponicidin could undergo general metabolic reactions, such as oxidation, reduction, hydrolysis, methylation and glucuronidation. Furthermore, there was an obvious difference in the ponicidin metabolites among four species in vitro. This is the first time that the SWATH™ data acquisition mode has been used to characterize ponicidin metabolites in trace amounts or in a biological matrix. These results not only provided a better understanding of the safety and efficacy of ponicidin but also showed a valuable methodology for the identification of other ent-kaurane diterpenoid metabolites.

C-5-substituted and C-8-substituted furanocoumarin isomers, two important kinds of furanocoumarin, are widely documented as the main active constituents in Angelicae dahuricae radix. Due to the similar polarity and mass fragmentation pathways of such isomers, it is difficult to distinguish them using mass spectrometric methods. To address this issue, we developed a strategy employing combined full scan and product ion scan modes on an ultra high performance liquid chromatography–quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF-MS) platform to differentiate four pairs of furanocoumarin isomer, viz. xanthotoxin and bergapten, imperatorin and isoimperatorin, psoralen and isopsoralen, and impinellin and isoimpinellin. A novel method using the ratio of relative abundance (RRA) of characteristic fragment ions was established to distinguish C-5-substituted and C-8-substituted furanocoumarin isomers, using the formula R = M/N, where M and N represent the ratios of relative abundance of characteristic fragment ions of a pair of furanocoumarin isomers. For R value greater than 1, compound M is substituted at C-5, whereas for R value less than 1, compound M is substituted at C-8. This method with good repeatability was applied to identify five pairs of isomeric furanocoumarins in Angelicae dahuricae radix. This is the first method to distinguish C-5-substituted and C-8-substituted furanocoumarin isomers, and can be used in complex matrix.

Ultra high performance liquid chromatography coupled with mass spectrometry and combining a tissue-smashing extraction technique was developed for the simultaneous quantitative analysis of 12 compounds in the roots of Pulsatilla chinensis. Among them, compound 6 was characterized and accurately quantified in this herb for the first time. The parameters of extraction condition were simultaneously optimized with a Box-Behnken design and Derringer's function. The optimized conditions were as follows: sample quantity of 0.5 g, ethanol concentration of 70%, and extraction time of 200 s. Multiple-reaction monitoring scanning was employed for the quantification between positive and negative mode in a single run of 6 min. Full validation of the method was carried out, and the results indicated that the method was rapid, specific, and reliable. The developed method was successfully applied to quantify the 12 compounds in 33 batches of P. chinensis from different provinces. Moreover, the principal component analysis was performed to compare the P. chinensis collected from different provinces of China based on quantitative data and the results indicated that the content of compounds could be used to differentiate the origins of P. chinensis. These results demonstrated that this method is feasible and reliable for the quality control of P. chinensis.

Screening diagnostic biomarkers can be challenging due to the complexity of traditional Chinese medicine (TCM) and ambiguous pharmacological mechanisms. In this study, we reported an integrated strategy for accurately screening diagnostic biomarkers based on metabolomics coupled with network pharmacology. First, a feasible pharmacological model was established through systems pharmacology and based on metabolomics-based techniques to explore diagnostic biomarkers. While the components satisfying the q-value0.05, fold change (FC) ≥ 1.2 or FC ≤ 0.8, coefficient of variance (CV) ≤ 30%(QC) and the variable importance in the project (VIP) value 1 are considered to be diagnostic biomarkers. Second, the ingredients were retained only when oral bioavailability (OB), Caco-2 permeability, drug half-life, TPSA and drug likeness (DL) satisfied the criteria (OB ≥ 40%; Caco-2 ≥ -0.4; HL ≥ 4 h; TPSA˂140; DL ≥ 0.18) suggested by the TCMSP database. Moreover, ingredients that exhibit extensive biological activity in TCM are also retained. Third, the effect targets of TCM were screened using the TCMSP database, Swiss Target Prediction and STICH online software. Disease targets were gathered from the therapeutic target database (TTD), PharmGkb and TCMSP database. Hub genes were screened by potential protein-protein interaction (PPI) network pharmacology analysis. Finally, a metabolic network pathway is established between the diagnostic biomarker and the hub gene. In the network analysis of metabolic pathways, most of the genes involved in this pathway are the second-step-obtained hub genes, which can explain the accuracy of the identified biomarkers. The proposed integrated strategy was successfully applied to explore the mechanism of action of Pulsatilla decoction (PD) in the treatment of acute ulcerative colitis (UC). Based on this integrated strategy, 23 potential biomarkers of acute UC treated with PD were identified. In conclusion, the integrated strategy provides novel insights into network pharmacology and metabolomics as effective tools to illuminate the mechanism of action of TCM.

Glaucocalyxin A (GLA) belongs to the natural ent-kauranoid diterpenoids family with antitumor, antifibrotic, anticoagulative, antioxidant, and anti-AD effects. In this study, ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) system was applied to observe probable metabolites of GLA

Betulin, as a new type of natural food preservative, is widely used in various kinds of meat products. However, its detailed mechanism of action and metabolism have not been clarified. In this study, for further gain insight of the mechanism of betulin as a preservative, an efficient method has been applied for measuring the antioxidant capacity of betulin, based on the absorbance of the DPPH• and ABTS• radical cation. When the concentration of betulin was more than 2.0 mg/mL, the scavenging rate of ABTS and DPPH radical reached over 90%, which was equivalent to the antioxidant capacity of Trolox. It is indicated that betulin has significant DPPH and ABTS free radical scavenging ability. This should be one of the important mechanisms for betulin as a preservative. A sensitive method using UHPLC-Q-TOF-MS/MS was established to determine the metabolite profile in vivo and in vitro of betulin. 32 phase I and 2 phase II metabolites were structurally characterized. This study will provide theoretical support for the safety and effectiveness of betulin in the field of preservatives and provide theoretical basis for the further study of betulin and the other natural preservatives. This research also contributes to the development of the food industry.

Carbon black nanoparticles (CBNPs) are core component of fine particulate matter (PM2.5) in the atmosphere. It was reported that the particle in the atmosphere with smaller size and the larger the specific surface area are easier to reach the deep respiratory tract or even the alveoli through the respiratory barrier and cause lung injury. Therefore, it has been believed that ultrafine or nanometer particles with more toxic than those with larger particle sizes. Moreover, it was confirmed that CBNPs could induce inflammation, oxidative stress and changes in cell signaling and gene expression in mammalian cells and organs. However, the cytotoxicity mechanism of them has been uncertain so far. The aim of the present study was to explore the underlying mechanism of cytotoxicity induced by CBNPs on A549 cells. In the current research, the viabilities of A549 cells were detected by Cell Counting Kit-8 (CCK-8) assay. The further metabolomics studies were conducted to detect the cytotoxic effect of CBNPs on A549 cells with an IC50 value of 70 μg/mL for 48 h. Potential differential compounds were identified and quantified using a novel on-line acquisition method based on ultra-liquid chromatography quadrupole time-of-flight mass spectrometry(UHPLC-Q-TOF/MS). The cytotoxicity mechanism of CBNPs on A549 cells was evaluated by multivariate data analysis and statistics. As a result, a total of 32 differential compounds were identified between CBNPs exposure and control groups. In addition, pathway analysis showed the metabolic changes were involved in the tricarboxylic acid (TCA) cycle, alanine, aspartate and glutamate metabolism, histidine metabolism and so on. It is also suggested that CBNPs may induce cytotoxicity by affecting the normal process of energy metabolism and disturbing several vital signaling pathways and finally induce cell apoptosis.

Carbon black nanoparticles (CBNPs) are one of atmospheric particles components and have been closely related with a series of lung diseases. It can reach the depths of the respiratory tract or even alveolar more easily than those micro-particles. Although some of its toxicities have been confirmed in animals or human bodies, the subchronic toxicity mechanism of CBNPs has been uncertain so far. Therefore, it is very necessary to establish a novel method and clarify the mechanism of subchronic toxicity caused by concentration adjustments of small molecule metabolites in vivo. In animal experiments, CB exposure, recovery and control group were set up. The concentration of CBNPs in chamber was 30.06 ± 4.42 mg/m3. We developed a UHPLC-Q-TOF-MS/MS-based non-targeted metabolomic analysis strategy to analyze serum samples of rats. Then, differential metabolites in serum were found by multivariate data analysis and 39 potential biomarkers were identified. It was showed that main metabolic pathways associated with CBNPs exposure were hormones metabolism, amino acid metabolism, nucleotide metabolism and lipid metabolism. It is worth noting that long-term exposure to CBNPs had the greatest impact on steroid hormones biosynthesis so that the risk of infertility could increase. The results provided a new mechanistic insight into the metabolic alterations owing to CBNPs induced subchronic toxicity.

Pulsatilla Adans (PSA) herbs (Ranunculaceae) have been widely used in traditional medicine in China and other countries. However, the authentication and quality control of PSA herbs have always been a challenging task due to their similar morphological characteristics and the diversity of the multiple components that exist in the complicated matrix. Herein, a novel integrated strategy combining UHPLC/Q-Orbitrap-MS techniques with chemometrics analysis is proposed for the discrimination of PSA materials. We developed a comprehensive method integrating a nontargeted bidirectionally screened (NTBDS) MS data set and a targeted extraction peak area analysis for the characterization of triterpenoid saponins of PSA from different species. After that, partial least-squares discriminant analysis (PLS-DA) was performed on the obtained MS data set and the parameter variable importance for the projection (VIP) value and P value were employed to screen the valuable MS features to discriminate PSA from different species. In addition, the receiver operating characteristic (ROC) curve is used to verify the reliability of MS features. Finally, heatmap visualization was employed to clarify the distribution of the identified triterpenoid saponins, and four medicinal species of PSA were successfully differentiated. Additionally, 34 constituents were reported in PSAs for the first time, 81 triterpenoid saponins were identified as differential components, and 12 chemical ingredients were characterized as potential chemical markers to differentiate the four officinal PSA herbs. This is the first time that the differences in different PSA herbs have been observed systematically at the chemical level. The results suggested that using the identified characteristic components as chemical markers to identify different PSA herbs was effective and viable. This method provides promising perspectives in the analysis and identification of the ingredients of Chinese herbal medicines, and the identification of similar herbs from the same species.

(+)-usnic acid (UA) is an active natural phenolic acid ingredient originating from Chinese traditional Tibetan herb. Usnea acid is expected to become a new agent for anticancer and remarkable antitumor. To reveal its metabolic profile, metabolites identification of UA in vivo was studied using ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS) in this present study. The chromatographic separation was performed on a C18 column with a mobile phase consisted of methanol and water with a flow rate of 0.4 ml/min. The mass spectral analysis conducted in a negative electrospray ionization mode combined with information-dependent acquirement technology (IDA) was used to trace all the potential UA metabolites. Several sensitive and specific multiple data-mining techniques especially key product ions (KPIs) filter were applied to hunt and identify metabolites rapidly. As a result, a total of 36 metabolites were detected after oral administration of UA, including 33, 8 and 16 in rat urine, plasma and bile, respectively. These results showed that the probable metabolite pathways of UA were oxidation, reduction, dihydroxylation, glycine conjugation, glucuronide conjugation, N-acetylcysteine conjugation and methylation. It is the first time to elucidate the profile of UA in vivo. These results not only provided the basis of UA pharmacological properties, but also gave the guidance in clinical medication. Moreover, the analysis strategy and methodology proposed in this paper could be widely used in characterization of other phenolic acids metabolites.

A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for the simultaneous determination of nine coumarins including aesculin, aesculetin, fraxin, fraxetin, scopoletin, isoscopoletin, 6-hydroxy-7,8-dimethoxy coumarin, 8-hydroxy-6,7-dimethoxy coumarin and umbelliferone in rat plasma using nodakenin as the internal standard (IS). The plasma samples were pretreated by a one-step direct protein precipitation with methanol. The chromatographic separation was carried out on a C18 column with a gradient mobile phase consisting of methanol and water (containing 0.05% acetic acid). All analytes and IS were quantitated through electrospray ionization in negative ion multiple reaction monitoring (MRM) mode. This method was fully validated in terms of the sensitivity, specificity, accuracy, precision (intra- and inter-day), matrix effect, recovery as well as the stability of the analyte under various conditions, and the results satisfied the requirements of biological sample measurement. The validated method was successfully applied to pharmacokinetic study of the nine coumarins in rat plasma after oral administration of Fraxini Cortex aqueous extract, among which the pharmacokinetics of four coumarins including fraxetin, isoscopoletin, 6-hydroxy-7,8-dimethoxy coumarin and 8-hydroxy-6,7-dimethoxy coumarin were studied for the first time.

The detection of drug metabolites, particularly for minor metabolites, continues to be a challenge owing to the complexity of biological samples. Imperatorin is an active natural furocoumarin ingredient originating from many traditional Chinese herbal medicines. In the present study, the metabolites of imperatorin after oral administration were qualitatively investigated, and possible metabolic pathways of it were subsequently proposed. Bile samples were collected after oral administration and pretreated by the application of Waters Ostro. The QTOF-MS/MS data was acquired using ultra high performance liquid chromatography coupled to quadrupole time flight spectrometry (UPLC-QTOF-MS). Based on this analytical strategy, 32 metabolites (23 phase I and 9 phase II metabolites) were identified in rat bile. The results demonstrated that C5H8 could be easily eliminated from imperatorin forming the metabolite M1. It also indicated that imperatorin and M1 underwent extensive metabolic reactions including oxidation, hydrolysis, methylation, glucuronide conjugation, C2H5NO2S conjugation and C3H5NO2S conjugation. This is the first study of imperatorin metabolism in bile samples. The proposed metabolic pathways in this research will provide essential data for further pharmaceutical studies of other linear-type furocoumarins.

Oridonin (ORI) is an active natural ent-kaurane diterpenoid ingredient originating from well-known traditional Chinese herb medicine and is expected to be pursued as a new anticancer agent. In the present study, a novel and efficient approach was developed for in vivo screening and identification of ORI metabolites using ultra high performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UPLC-Triple-TOF-MS/MS). This analytical strategy was as follows: an effective on-line data acquisition method multiple mass defect filter (MMDF) combined with dynamic background subtraction (DBS), was developed to trace all of potential metabolites of ORI. The MMDF and DBS method could trigger an information dependent acquisition scan, which could give the information of low-level metabolites masked by background noise and endogenous components in complex matrix. Moreover, the sensitive and specific multiple data-mining techniques including extracted ion chromatography, mass defect filtering, product ion filtering and neutral loss filtering were employed to identify the metabolites of ORI. Then, structures for the metabolites were successfully assigned based on accurate masses, the mass fragmentation of ORI and metabolic knowledge. Finally, an important parameter Clog P was used to estimate the retention time of isomers. Based on the proposed strategy, 16 phase I and 2 phase II metabolites were detected in rats after oral administration of ORI. The main biotransformation route of ORI was identified as reduction, oxidation, dehydroxylation and glucuronic acid conjugation. This is the first study of ORI metabolism in vivo. This study not only proposed a practical strategy for rapidly screening and identifying metabolites, but also provided useful information for further study of the pharmacology and mechanism of ORI in vivo. At the same time this methodology can be widely applied for the structural characterization of the metabolites of other ent-kaurane diterpenoid.

In this paper, an analytical strategy combined data acquisition with a practical mining strategy aimed at rapid characterization and quantitation of

Betulinic acid is a triterpenoid organic acid with remarkable antitumor properties and is naturally present in many fruits, condiments and traditional Chinese medicines. Currently, a strategy was developed for the identification of metabolites following the in vivo and in vitro biotransformation of Betulinic acid with rat intestinal bacteria utilizing ultra high performance liquid chromatography with time-of-flight mass spectrometry with polymeric solid-phase extraction. As a result, 46 metabolites were structurally characterized. The results demonstrated that Betulinic acid is universally metabolized in vivo and in vitro, and Betulinic acid could undergo general metabolic reactions, including oxidation, methylation, desaturation, loss of O and loss of CH2 . Additionally, the main metabolic pathways in vivo and in vitro were determined by calculating the relative content of each metabolite. This is the first study of Betulinic acid metabolism in vivo, whose results provide novel and useful data for better understanding of the safety and efficacy of Betulinic acid.

A sensitive, specific and rapid liquid chromatography-mass spectrometry (LC-MS) method was developed and validated for analysis of ginsenoside Rb1, naringin, ginsenoside Rb2 and oridonin in rat plasma using sulfamethoxazole as an internal standard (IS). Separation was conducted out on an Agilent Eclipse XDB C18 column with liner gradient elution using acetonitrile (A) and 0.1% aqueous acetic acid (B). A tandem mass spectrometric detection was conducted using multiple reaction monitoring (MRM) via an electrospray ionization (ESI) source. A novel multi-determination-periods program was executed to achieve a higher sensitivity by setting three scanning periods. All analytes exhibited good linearity within the concentration range (r>0.9973). The lower limits of quantitation (LLOQ) of ginsenoside Rb1, naringin, ginsenoside Rb2 and oridonin were 2.64, 4.32, 2.32 and 1.56ng/mL, respectively. Intra-day and inter-day precisions of the investigated components exhibited an RSD within 8.3%, and the accuracy (RE) ranged from -8.6% to 6.0% at all quality control levels. The developed method was successfully applied to a pharmacokinetic study of ginsenoside Rb1, naringin, ginsenoside Rb2 and oridonin in rats after oral administration of a Weifuchun tablet.

Introduction Triterpenoid saponins are the major bioactive constituents of Pulsatilla chinensis, playing an important role in various biological activities such as anti-tumour, cognition-enhancing, anti-biosis, anti-inflammatory, hypoglycemic and immunological adjuvant. Objective To establish a systematic strategy based on ultra-high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) for the efficient characterisation and identification of triterpenoid saponins in crude extracts from Pulsatilla chinensis. Methodology In this work, the strategy includes two aspects: (1) positive mode: by target screening, we can deduce the aglycone type and the composition of sugar moiety according to the fragment ions; untargeted screening includes four steps, find unknown, formula finder, ChemSpider search and MS/MS identification; (2) negative mode: according to the MS/MS spectra, the composition of sugar chain bonded to C-28 is inferred reasonably. The extract of Pulsatilla chinensis was separated within 60 min on a C18 column and eluted with methanol and water both containing 0.1% formic acid. Results As a result, a total of 22 triterpenoid saponins (11 pairs of isomers) with four aglycone skeletons were tentatively identified or elucidated in crude extracts from Pulsatilla chinensis based on their retention times, the mass spectrometric fragmentation patterns, and MS and MS/MS data. Conclusion This study provides an efficient analysis strategy to rapidly identify the triterpenoid saponins in Pulsatilla species even in traditional Chinese medicines.

A novel polarity switching high performance liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC–ESI-MS/MS) approach combining optimization of extraction condition by response surface methodology (RSM) was developed for the simultaneous quantitative analysis of 11 compounds in Cortex Fraxini, a commonly used traditional Chinese medicine. The ultrasonic extraction conditions of the 11 analytes including sample quantity, methanol concentration and extraction time were simultaneously optimized with a Box–Behnken design (BBD) and Derringer's desirability function. Multiple-reaction monitoring (MRM) scanning was employed for quantification with switching electrospray ion source polarity between positive and negative modes in a single run of 16 min. Quantitative parameters of the proposed method with respect to limit of detection (LOD), limit of quantification (LOQ), linearity, precision, accuracy and stability were evaluated under optimum conditions, and the results indicated that the method was sensitive, specific and reliable. The developed method was successfully applied to determine the investigated compounds in four species of Cortex Fraxini and three kinds of its confusable species, and significant differences were found between the official and confusable species.

Diterpenoids are the primary bioactive ingredients in Isodon rubescens named Dong Ling Cao in China which possess many pharmacological activities, including anti-tumor, anti-microbial, anti-inflamm...

In the present study, a microwave-assisted extraction (MAE) and high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was developed for the simultaneous determination of seven triterpenoid saponins in Pulsatilla turczaninovii . Operational conditions of MAE were optimized using a central composite design. Multiple-reaction monitoring was employed for quantification while switching the electrospray ion source polarity between positive and negative modes. Full validation of the method was carried out (linearity, precision, accuracy, limit of detection and limit of quantification). The results indicated that the method was rapid, specific and reliable. The developed method was successfully applied to determine the investigated compounds in nine batches of P. turczaninovii . These samples were collected in different seasons from the Liaoning province and varied greatly. The total triterpenoid saponins content was the highest in April, the seedling stage, and decreased from May to August. The contents of total triterpenoid saponins decreased significantly after the blooming period.

Oridonin (ORI) is an active natural ent-kaurene diterpenoid ingredient with notable anti-cancer and anti-inflammation activities. Currently, a strategy was developed to identify metabolites and to assess the metabolic profiles of ORI in vitro using ultra-high-performance liquid chromatography-Triple/time-of-flight mass spectrometry (UPLC-Triple-TOF-MS/MS). Meanwhile, the metabolism differences of ORI in the liver microsomes of four different species were investigated using a principal component analysis (PCA) based on the metabolite absolute peak area values as the variables. Based on the proposed methods, 27 metabolites were structurally characterized. The results indicate that ORI is universally metabolized in vitro, and the metabolic pathway mainly includes dehydration, hydroxylation, di-hydroxylation, hydrogenation, decarboxylation, and ketone formation. Overall, there are obvious inter-species differences in types and amounts of ORI metabolites in the four species. These results will provide basic data for future pharmacological and toxicological studies of ORI and for other ent-kauranes diterpenoids. Meanwhile, studying the ORI metabolic differences helps to select the proper animal model for further pharmacology and toxicological assessment.

A quick HPLC-ESI-MS/MS method was established for simultaneous determination of four major diterpenoids in Rabdosia japonica var.glaucocalyx, including glaucocalyxin A, oridonin, hebeirubesensin and enmenol. Analysis was performed on an Agilent ZORBAX SB-C18(4.6 mm×250 mm, 5 μm ) column eluted in a gradient program with methanol and water. The flow rate was 0.8 mL•min⁻¹. Multiple reaction monitoring (MRM) scanning mode was performed in negative ion switching mode to apply for the quantitative determination. The calibration curves for the above four compounds were linear in corresponding injection amount. The average recoveries of the compounds ranged from 92.40% to 105.9%, with RSDs of 1.7%-6.5%. The method is simple, rapid, accurate with good repeatability, which can provide a reference for overcalling evaluation the quality of R. japonica var.glaucocalyx. The result of cluster analysis- showed that the quality of R. japonica glaucocalyx var. greatly varied between areas and parts.

A novel method based on high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry was developed for simultaneous determination of the 11 major active components including ten flavonoids and one phenolic acid in Cirsium setosum. Separation was performed on a reversed-phase C(18) column with gradient elution of methanol and 0.1‰ acetic acid (v/v). The identification and quantification of the analytes were achieved on a hybrid quadrupole linear ion trap mass spectrometer. Multiple-reaction monitoring scanning was employed for quantification with switching electrospray ion source polarity between positive and negative modes in a single run. Full validation of the assay was carried out including linearity, precision, accuracy, stability, limits of detection and quantification. The results demonstrated that the method developed was reliable, rapid, and specific. The 25 batches of C. setosum samples from different sources were first determined using the developed method and the total contents of 11 analytes ranged from 1717.460 to 23028.258 μg/g. Among them, the content of linarin was highest, and its mean value was 7340.967 μg/g. Principal component analysis and hierarchical clustering analysis were performed to differentiate and classify the samples, which is helpful for comprehensive evaluation of the quality of C. setosum.

A novel quantitative method using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry was developed for simultaneous determination of the important active constituents including four steroidal saponins, two xanthone glycosides, two isoflavonoids, and one anthraquinone in different parts of Anemarrhena asphodeloides from different habitats. Hierarchical clustering analysis and principal components analysis were performed to differentiate and classify the samples. The separation was performed on a C(18) column with acidified aqueous acetonitrile gradients. Quantification of the analytes was achieved by use of a hybrid quadrupole linear ion-trap mass spectrometer. Multiple-reaction monitoring scanning was employed with switching electrospray ion source polarity between positive and negative modes in a single run. The validation results of the method indicated that the method was simple, rapid, specific, and reliable. The results demonstrated that the quantitative difference in content of nine active compounds was useful not only for chemotaxonomy of many samples from different sources but also for the standardization and differentiation of many similar samples. Simultaneous quantification of bioactive components by HPLC-ESI-MS coupled with chemometric techniques would be a well-acceptable strategy to comprehensively control the quality of A. asphodeloides.

Introduction The Kang-nao-shuai (KNS) capsule is a combined herbal prescription used in the treatment of insomnia, amnesia, neurasthenia, age-related dementia and brain injuries. Multiple constituents are considered to be responsible for the therapeutic effects of this herbal prescription. However, the quality control of the multicomponents is limited. Objective To establish a liquid chromatography–electrospray ionisation–mass spectrometry method for the analysis of 40 constituents in KNS capsules. Methodology The optimal chromatographic conditions were achieved on an Agilent C18-column with a gradient elution that consisted of methanol and 0.1% formic acid in water. The precursor and product ions of analytes were monitored on a hybrid quadrupole linear ion trap mass spectrometer in positive and negative mode respectively using multiple-reaction monitoring. Results A total of 40 constituents including organic acid, flavonoid, quinone, terpene, alkaloid and saponin were quantified, most of the 40 components were determined for the first time in the KNS capsule. A quantitative HPLC–ESI–MS/MS method allowing the quantification of 40 marker compounds was optimised and validated for linearity, precision, accuracy, stability, specificity and limits of detection and quantification. The method was successfully applied to analyse 10 batches of KNS capsule. Conclusion The established method is simple and can be used as a tool for quality evaluation and control of this natural product. Copyright © 2011 John Wiley & Sons, Ltd.

Epimedin C is one of the major bioactive constituents of Herba Epimedii. The aim of this study is to characterize and elucidate the structure of metabolites in the rat after administration of epimedin C. Metabolite identification was performed using a predictive multiple reaction monitoring–information dependent acquisition–enhanced product ion (pMRM-IDA-EPI) scan in positive ion mode on a hybrid triple quadrupole-linear ion trap mass spectrometer. A total of 18 metabolites were characterized by the changes in their protonated molecular masses, their MS/MS spectrum and their retention times compared with those of the parent drug. The results reveal possible metabolite profiles of epimedin C in rats; the metabolic pathways including hydrolysis, hydroxylation, dehydrogenation, demethylation and conjugation with glucuronic acid and different sugars were observed. This study provides a practical approach for rapidly identifying complicated metabolites, a methodology that could be widely applied for the structural characterization of metabolites of other compounds.

Qualitative characterization and quantitative analysis of 15 bioactive constituents in Jiweiling freeze-dried powder (JWL) have been achieved by reversed-phase high performance liquid chromatography coupled with tandem mass spectrometry, including five flavonoids and ten ginsenosides. Chromatographic separation was performed on an Agilent ZORBAX Eclipse XDB-C18 HPLC column, with gradient elution of 0.1% formic acid aqueous solution and acetonitrile as mobile phase. The compounds were detected in the negative ion mode with multiple reaction monitoring (MRM) using a triple quadrupole mass spectrometer equipped with an electrospray ionization interface. By using of this method, 15 bioactive components were unequivocally identified on the basis of their characteristic fragmentation patterns. All calibration curves showed good linearity (r2 > 0.998) within the test ranges. The LOD, LOQ, specificity, precision, and accuracy for the method were validated. The proposed method was successfully applied to analyze t...

A sensitive, specific, and rapid liquid chromatography–mass spectrometry (LC–MS) method was developed and validated for analysis of lasiodonin, oridonin, ponicidin, and rabdoternin A in rat plasma using sulfamethoxazole as an internal standard (IS). The plasma samples were pretreated and extracted by liquid–liquid extraction. Chromatographic separation was performed on a C 18 column with linear gradient elution using water and methanol, both of which were acidified with 0.1% aqueous formic acid, at a flow rate of 0.8 ml/min. Detection was accomplished by scanning with multiple reaction monitoring (MRM) via an electrospray ionization (ESI) source. Higher sensitivity was achieved by setting three scanning periods in a novel detection mode. The optimized mass transition ion pairs ( m / z ) for quantitation were 365.3/347.3 for lasiodonin and oridonin, 361.2/343.2 for ponicidin, 363.2/283.1 for rabdoternin A, and 254.1/156.0 for IS. The total run time was 13.50 min between injections. The specificity, linearity, accuracy, precision, recovery, matrix effect, and several stabilities were validated for all analytes in the rat plasma samples. In conclusion, the validation results demonstrate that this method is robust and specific. The proposed method was further applied to investigate the pharmacokinetics of all analytes after a single oral administration of Isodon rubescens extract to rats.

A new method based on high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry has been developed for simultaneous analysis of 27 components (eighteen diterpenoids, six phenolic acids, and three flavonoids) of Isodon serra, a famous traditional Chinese medicine. Separation on a C18 column was achieved by gradient elution with water and methanol both containing 0.1% formic acid. Identification and quantification of the analytes were achieved by use of a hybrid quadrupole linear ion-trap mass spectrometer. Multiple-reaction monitoring (MRM) was used for quantification, with switching of electrospray ion source polarity between positive and negative modes in the same chromatographic run. An information-dependent acquisition (IDA) method was used to trigger product ion scans above the MRM signal threshold so that the 27 compounds could be identified by use of enhanced product-ion scans (EPI). The method was fully validated (for linearity, precision, accuracy, and limits of detection and quantification). The results indicated that this simple method was rapid, specific, and reliable. The method was successfully applied to analysis of 45 batches of Isodon serra samples from different sources, and quantification of the compounds in Isodon serra was achieved.

Introduction – Isodon nervosa is a commonly used traditional Chinese medicine including diterpenoids, phenolic acids, triterpenoids and volatile oil. Qualitative and quantitative analysis of multi-components is important for its quality control. Objective – To establish a liquid chromatography–electrospray ionisation–mass spectrometry method for simultaneous analysis of 20 bioactive constituents of Isodon nervosa in different places of China and different parts of this herb. Methodology – The optimal chromatographic conditions were achieved on a C18 column (250 × 4.6 mm, 5 µm) with with linear gradient elution with 0.1% aqueous formic acid : methanol containing 0.1% formic acid at a flow-rate of 0.7 mL/min in 15 min. The identification and quantification of those analytes were achieved on a hybrid quadrupole linear ion trap mass spectrometer. Multiple-reaction monitoring scanning was employed for quantification with switching electrospray ion source polarity between positive and negative modes in a single run. Full validation of the method was carried out (linearity, precision, accuracy, limit of detection and limit of quantification). Results – The results indicated that the method was simple, rapid, specific and reliable. The proposed method was successfully applied for the qualitative and quantitative analysis of 20 chemical compositions in Isodon nervosa samples. Conclusion – Twenty chemical compositions in 21 batches of wild and cultivated Isodon nervosa samples from different sources had great variation in the contents. Copyright © 2010 John Wiley & Sons, Ltd.

A novel method, HPLC-MS/MS was developed to qualitatively identify and quantitatively determine the 28 components including 19 diterpenoids, 6 phenolic acids and 3 flavonoids in Isodon rubescens, an important traditional Chinese medicine. The separation was performed on a C(18) column with linear gradient elution with 0.1% aqueous formic acid/methanol containing 0.1% formic acid at a flow rate of 0.7 mL/min. The identification and quantification of those analytes were achieved on a hybrid quadrupole linear ion trap mass spectrometer. Multiple-reaction monitoring scanning was employed for quantification with switching electrospray ion source polarity between positive and negative modes in a single run. Full validation of the method was carried out (linearity, precision, accuracy, LOD and LOQ). The results indicated that the method was simple, rapid, specific and reliable. The proposed method was successfully applied for the qualitative and quantitative analysis of 28 chemical compositions in 21 batches of natural and cultured I. rubescens samples from different sources which had great variation on the contents. The results demonstrated that the method was useful for standardization and differentiation of large numbers of similar samples.