Application of high-performance liquid chromatography-electrospray ionization time-of-flight mass spectrometry for analysis and quality control of Radix Astragali and its preparations

A high-performance liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry (HPLC-ESI-TOF-MS) method has been developed for qualitative and quantitative analysis of isoflavonoids and saponins, as well as for the quality control of Radix Astragali and its preparations. The selectivity, reproducibility and sensitivity are compared with HPLC with diode array detection (DAD) and evaporative light scattering detection (ELSD). Limits of detection and quantification fell in ranges of 9-40 and 23-103 ng/mL for 13 analytes with a injection of 10 microL samples, and all calibration curves showed good linear regression (r(2)>0.9938) within the test range. The assay was successfully utilized to analyze the 13 marker components in 20 samples of Radix Astragali products. The quantitative results demonstrated that samples from different localities and manufacturers showed different quality. Advantages, in comparison with conventional HPLC-DAD-ELSD, are that reliable identification of target compounds could be achieved by accurate mass measurements (<3 ppm) along with characteristic retention time, extracted ions chromatograms using a narrow mass window for quantification ensure that the chromatographic peaks are free from background or co-elutes interference, and the great enhancement in selectivity and sensitivity allows identification and quantification of low levels of constituents in complex Radix Astragali matrixes.     

黄芪药材的指纹图谱研究方法的建立

用反相高效液相色谱－紫外－质谱联用技术对黄芪药材进行指纹图谱研究，为阐明不同产地药材的异同性，建立黄芪质量的国际统一标准奠定基础，黄芪的总提取物各类成分得到很好分离，紫外和质谱两种检测器可对不同特性化合物的检测进行互补，获得相对充分的指纹图谱信息。     

体外细胞模型和高效液相质谱联用分析预测黄芪中的活性成分

利用体外细胞模型模拟体内细胞对中药有效成分的特异性吸收,结合高效液相色谱/质谱分析筛选中药黄芪中的生物活性成分。将中药黄芪提取液与Caco-2细胞及红细胞分别混合培养,破碎与药材结合后的细胞,使之释放出结合的药材中的成分。运用高效液相色谱/飞行时间质谱(HPLC-ESI/TOFMS)分析中药黄芪提取液与活性细胞有结合的成分,并对其进行结构鉴定。结果显示:黄芪中有10个化合物与Caco-2细胞结合,14个化合物与红细胞结合。本方法可用于预测口服药物在体内的吸收以及与特定靶细胞的结合情况,特异性地筛选中药复杂体系中的药效物质基础。     

Identification and determination of flavonoids in astragali radix by high performance liquid chromatography coupled with DAD and ESI-MS detection

A method for the analysis of flavonoids in Astragali Radix by high-performance liquid chromatography (HPLC) combined with photodiode-array detection (DAD) and an electrospray ionization (ESI)--mass spectrometry (MS) was developed. After the samples were extracted with ethanol, the optimum separation conditions for these analytes were achieved using a gradient elution system and a 2.0 x 150 mm Shimadzu VP-ODS column. Eight flavonoids were identified to exist in Astragali Radix based on their characteristic UV data and mass spectra. The concentrations of three major components in this herb--ononin, calycosin and formononetin--were determined by LC/ESI-MS in positive selective ion monitoring (SIM) mode. The calibration curves were linear in the range of 0.9~180.0 μg·mL?1 for ononin, 1.8~360.0 μg·mL?1 for calycosin and 1.4~280 μg·mL?1 for formononetin, respectively. The limits of quantification (LOQ) and detection (LOD) were 0.9 μg· mL?1 and 0.2 μg mL?1 for ononin, 1.8 μg mL?1 and 0.5 μg·mL-1 for calycosin, 1.4 μg mL?1 and 0.5 μg·mL?1 for formononetin, respectively. The standard recoveries were between 95.4~104.7%. The developed method was proven to be useful for the quantitative and qualitative analysis of flavonoid constituents in various resources of Astragali Radix.     

Simultaneous determination of bioactive constituents in Danggui Buxue Tang for quality control by HPLC coupled with a diode array detector,an evaporative light scattering detector and mass spectrometry

Danggui Buxue Tang (DBT), a classical traditional Chinese formula comprising Radix Angelicae Sinensis (RAS) and Radix Astragali (RA), has been widely used to treat menopausal irregularity in Chinese women for nearly 800 years. In this study, a comprehensive analytical method of simultaneously determining the main types of bioactive constituents, eighteen in all from the formula, involving flavonoids, saponins, organic acid and some volatile compounds, was developed. This method was based on HPLC coupled to a diode array and evaporative light scattering detectors (HPLC-DAD-ELSD) on a common reverse-phase C(18) column. Liquid chromatography coupled with on-line electrospray ionization mass spectrometry (LC-ESI-MS) was also used to further validate and analyze the constituents. It was found that 0.3% aqueous formic acid and acetonitrile was the optimum mobile phase for gradient elution. This method, which showed good precision and accuracy, was successfully used to quantify the bioactive constituents in six products. As a result, the validated HPLC method, together with the LC-ESI-MS analysis, provided a new basis for assessing the quality of traditional Chinese medicinal compound preparations (TCMCPs) consisting of many bioactive components.     

Exploring the In Vivo Existence Forms (23 Original Constituents and 147 Metabolites) of Astragali Radix Total Flavonoids and Their Distributions in Rats Using HPLC-DAD-ESI-IT-TOF-MSn

Astragali Radix total flavonoids (ARTF) is one of the main bioactive components of Astragali Radix (AR), and has many pharmacological effects. However, its metabolism and effective forms remains unclear. The HPLC-DAD-ESI-IT-TOF-MSⁿ technique was used to screen and tentatively identify the in vivo original constituents and metabolites of ARTF and to clarify their distribution in rats after oral administration. In addition, modern chromatographic methods were used to isolate the main metabolites from rat urine and NMR spectroscopy was used to elucidate their structures. As a result, 170 compounds (23 original constituents and 147 metabolites) were tentatively identified as forms existing in vivo, 13 of which have the same pharmacological effect with ARTF. Among 170 compounds, three were newly detected original constituents in vivo and 89 were new metabolites of ARTF, from which 12 metabolites were regarded as new compounds. Nineteen original constituents and 65 metabolites were detected in 10 organs. Four metabolites were isolated and identified from rat urine, including a new compound (calycoisn-3’-O-glucuronide methyl ester), a firstly-isolated metabolite (astraisoflavan-7-O-glucoside-2’-O-glucuronide), and two known metabolites (daidzein-7-O-sulfate and calycosin-3’-O-glucuronide). The original constituents and metabolites existing in vivo may be material basis for ARTF efficacy, and these findings are helpful for further clarifying the effective forms of ARTF.     

Detection and identification of diterpenoid alkaloids, isoflavonoids and saponins in Qifu decoction and rat plasma by liquid chromatography-time-of-flight mass spectrometry

A liquid chromatography-time-of-flight mass spectrometric (LC-TOFMS) method has been developed for analysis of components in Qifu decoction (QFD), a traditional Chinese medical formula consisting of Radix Astragali and Acontium carmichaeli, and in rat plasma after oral administration. Based on accurate mass measurements within 3 ppm error for each molecular ion and subsequent fragment ions of TOFMS, as well as matching of empirical molecular formulae with those of published components in the in-house chemical library, a total of 44 major components including 21 diterpenoid alkaloids, 12 flavonoids and 11 saponins were identified in QFD. After oral administration of QFD, 22 components in rat plasma were detected and identified by comparing and contrasting the constituents measured in QFD with those in the plasma samples. The results provided valuable chemical information for further pharmacology and active mechanism research on QFD. LC-TOFMS was also applied for the comparison of relative peak area of major active components between QFD and the single herb extracts. The concentration ratios of major saponins detected in the crude herb Radix Astragali were found to be different from those in QFD. The experimental data indicated that the decocting process could result in differences in the amounts of active components.     

Comparative chemical analysis of Radix Astragali and Radix Hedysari by HPLC

Radix Astragali and Radix Hedysari, two famous traditional Chinese medicines (TCM), were considered to possess the same efficacy in TCM traditionally. However, modern pharmacological and phytochemical investigations showed that they were different in efficacy and chemical constituents to some degree. In this study, the principal constituents of the two drugs, isoflavonoids and saponins, were comparatively analysed using HPLC-UV and HPLC-ELSD methods. The results showed that there were some common compounds, especially flavonoids, in both drugs, while the differences in the types and contents of their chemical constituents, especially in saponins, were also obvious. The similarities and differences of the chemicals may be responsible, at least partially, for the similarities and differences in their efficacies or bioactivities.     

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.     

Determination and quantification of astragalosides in Radix Astragali and its medicinal products using LC–MS

In the present study, a liquid chromatography–tandem mass spectrometry method was developed for the separation and simultaneous quantification of astragalosides I–IV in samples of Radix Astragali and a medicinal product thereof (Jinqi Jiangtang tablets). Chromatographic separation was achieved on an Agilent Eclipse XDB (ODS)‐C18 column with a mobile phase consisting of acetonitrile and 0.05% formic acid aqueous solution by use of an efficient 17‐min program. A triple quadrupole mass spectrometer was operated in positive ionization mode with multiple reaction monitoring for the detection of four astragalosides. The saponin ginsenoside Rg1 (similar structure to astralagosides) was used as an internal standard. All calibration curves showed excellent linear regressions (r² ? 0.9912) within the range of tested concentrations. The intra‐ and inter‐day variations were below 4.57% in terms of RSD. The recoveries were 94.38–103.53% with RSD of 1.39–3.58% for spiked Radix Astragali samples. The method was successfully used for the analysis of samples of Radix Astragali and Jinqi Jiangtang tablets. In conclusion, we have developed a rapid, efficient, and accurate LC–MS/MS method for the detection of astragalosides, which can be applied for quality control of Radix Astragali and related medicinal products.     

Concordance between antioxidant activities in vitro and chemical components of Radix Astragali (Huangqi)

Five Radix Astragali (RA) extracts were prepared and their antioxidant activities were measured in vitro using ABTS [2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid diammonium salt)], DPPH?·?(1,1-diphenyl-2-picrylhydrazyl radical), reducing power, ?O?? and ?OH assays. Their chemical contents were then determined, including total phenolics, total flavonoids, total saponins and total sugars. The 1/IC?? values of the various antioxidant assays were used to evaluate the level of antioxidant activity of the RA extracts (plot showing 1/IC?? values vs. chemical contents), and the average R values (correlation coefficients) of total phenolics and total flavonoids were 0.762 and 0.638. In contrast, the average R values of the total saponins and total sugars were -0.0386 and -0.132, respectively. This large difference clearly demonstrates that the antioxidant effects of RA in vitro might be generally considered to be a result of the presence of phenolic compounds (including flavonoids) but not astragalosides and polysaccharides.     

联用色谱及多组分光谱相关色谱用于当归补血汤与单味药成分的比较研究

摘要利用HPLC-DAD提供的二维化学数据信息, 通过多组分光谱相关色谱方法及化学计量学分辨技术, 对相同试验条件下所得的当归补血汤及其单味药的色谱组分进行了比较和归属分析. 结果表明， 在当归补血汤色谱响应值较高的21个色谱峰中, 8个色谱峰的组分来自当归, 6个色谱峰的组分来自黄芪, 7个色谱峰的组分同时来源于当归和黄芪. 其中一些色谱峰簇中的其它组分发生了消长变化， 既有新物质的产生, 又有原有化学成分的消失， 说明中药复方配伍的药效物质基础不仅是各单味药化学成分的总和, 还有单味药在复方配伍过程中各种成分的相互作用. 本方法为中药复方与单味药化学组分的比较及归属分析, 尤其对重叠色谱峰簇的组分比较分析提供了方便、 快捷及可行的研究方法.     

Quality analysis combined with mass spectrometry imaging reveal the difference between wild and cultivated Phyllanthus emblica Linn.: From chemical composition to molecular mechanism

In this paper, the quality of Phyllanthus emblica was evaluated by establishing a comprehensive taste index and odor component analysis method, and the formation mechanism of quality difference was revealed by mass spectrometry imaging and metabolomics. Studies have found that the representative substances of bitterness and astringency in cultivated PE, such as amlaic acid, kaempferol, quercetin and their derivatives, have higher content. Compared with wild PE, cultivated PE has a very lower 2-Isobutyl-3-methoxypyrazine content, a kind of odor molecule that destroys the fruits flavor. Biological activity studies showed that the biological activity of wild PE was stronger than cultivated PE. Mass spectrometry imaging revealed significant differences in the distribution of multiple components between the two types of PE. Metabonomic studies show that their quality differences may be related to the difference of flavonoids and flavonol biosynthesis in PE.     

Simultaneous determination of 15 marker constituents in various radix Astragali preparations by solid-phase extraction and high-performance liquid chromatography

An improved quality control method was developed to simultaneously determine 15 major constituents (eight flavonoids and seven saponins) in various radix Astragali preparations, using SPE for pretreatment of samples, HPLC with diode-array and evaporative light scattering detectors (DAD-ELSD) for quantification in one run, and HPLC-ESI-TOF/MS for definite identification of compounds in preparations. Optimum separations were obtained with a ZORBAX C(18) column, using a gradient elution with 0.3% aqueous formic acid and ACN. This established method was fully validated with respect to linearity, precision, repeatability, and accuracy, and was successfully applied to quantify the 15 compounds in 19 commercial samples, including 3 dosage forms, i. e., oral solution, injection, concentrated granule, and its processed products of radix Astragali. The results demonstrated that many factors might result in significant differences in quality of the final preparations, including crude drugs, pretreatment processes, manufacturing procedure, storage conditions, etc. Then the developed method provided a reasonable and powerful manner to ensure the efficacy, safety, and batch-to-batch uniformity of radix Astragali products by standardizing each procedure, and thus should be proposed as quality control for the clinical use and modernization of herbal preparations.     

Characterization of chemical composition variations in raw and processed Codonopsis Radix by integrating metabolomics and glycomics based on multiple chromatography-mass spectrometry technology

Codonopsis Radix, a popular food homology medicine, is widely used in clinical traditional Chinese medicine and food supplement, raw products and three types of processed products are the main forms of decoction pieces in China. However, there is no scientific basis for comprehensive chemical characterization of raw and three types of processed products. Herein, we investigated qualitatively and quantificationally secondary and primary metabolites in raw Codonopsis Radix and three types of processed products by metabolomics and glycomics employing multiple chromatography-mass spectrometry technology combined with chemometric analysis further to look for differential compounds and propose the processing-induced chemical mechanisms. The results indicated that Codonopsis Radix became dark-colored and the smell of burnt incense odor was observed after processing. The principal component analysis demonstrated that secondary metabolome and glycome were significantly altered between raw and processed products, and 36 differential secondary metabolites and 11 differential primary metabolites were finally screened through orthogonal partial least-squares-discriminant analysis. The main types of compounds are alkaloids, terpenoids, glycosides, amino acids, monosaccharides, oligosaccharides, and furfural derivatives. Meanwhile, Chemical mechanisms could be involved, including oxidation, glycosidic hydrolysis, esterification, dehydration, and Maillard reaction. This work supplies a chemical basis for the application of various types of Codonopsis Radix decoction pieces.     

Combining network pharmacology with chromatographic fingerprinting and multicomponent quantitative analysis for the quality evaluation of Astragali Radix

Nowadays, cultivated variants and adulterants of Astragali Radix (AR) have flooded the market, causing the quality assurance of AR to be challenging. To address this issue, we combined network pharmacology with chromatographic fingerprinting and multicomponent quantitative analysis for the quality evaluation of AR. Specifically, through network pharmacology, a complete understanding of the active components and pharmacological activities of AR was established. In addition, establishing fingerprint profiles and multicomponent quantitation by high-performance liquid chromatography (HPLC) is convenient and comprehensive, and can more fully reflect the overall situation of the distribution of various chemical components. To evaluate and differentiate AR from different origins, hierarchical cluster analysis and principal component analysis were performed. The result showed that AR acts synergistically through multiple targets and pathways. The content of chemical components in AR from different origins varied significantly. Combining network pharmacology and multicomponent quantification results, astragaloside II and IV and formononetin can be used as quality markers for the quality control of AR. This study provides a comprehensive and reliable strategy for the quality evaluation of AR and identifies its quality markers to ensure the quality of the herb.     

Chemical profiling and quantification of ShenKang injection,a systematic quality control strategy using ultra high performance liquid chromatography with Q Exactive hybrid quadrupole orbitrap high‐resolution accurate mass spectrometry

ShenKang injection is traditional Chinese medicine used to treat chronic renal failure in China. It is a compound preparation that consists of four herbs: Rhubarb, Salvia miltiorrhiza, Safflower and Radix Astragali . We developed an ultra high pressure liquid chromatography coupled with Q Exactive hybrid quadrupole‐orbitrap high resolution accurate mass spectrometry tandem mass spectrometry method to analyze its chemical compositions, and a total of 90 compounds were identified from ShenKang injection. Among them, 19 major compounds were unambiguously detected by comparing with reference standards. Meanwhile, 13 representative compounds selected as quality control markers were simultaneously quantified in ShenKang injection samples. Chromatographic separation was accomplished on a Waters ACQUITY HPLC® HSS C₁₈ column using gradient elution. The method was validated with respect to linearity, sensitivity, accuracy and precision, reproducibility and stability. And the validated method was successfully applied for simultaneous determination of 13 bioactive compounds in ShenKang injection from ten batches of samples by liquid chromatography with mass spectrometry. The results were analyzed by principal components analysis method, and three compounds had a significant relationship with the quality control of ShenKang injection. This research established a rapid and reliable method for the integrating quality control, including qualitation and quantification of ShenKang injection.     

Quantitative Analysis and Differential Evaluation of Radix Bupleuri Cultivated in Different Regions Based on HPLC-MS and GC-MS Combined with Multivariate Statistical Analysis

The quality of Radix Bupleuri is greatly affected by its growing environment. In this study, Radix Bupleuri samples that were harvested from seven different regions across northwest China were examined by high-performance liquid chromatography (HPLC) and gas chromatography (GC) coupled with mass spectrometry (MS) to reveal significant differences in quality contributed by the cultivation region. An HPLC-MS method was firstly established and used in the multiple reaction monitoring mode for the quantitative analysis of five saikosaponins in Radix Bupleuri so as to evaluate the difference in the absolute content of saikosaponins attributable to the cultivation region. The effect on the components of Radix Bupleuri was further investigated based on the profiles of the representative saponins and volatile compounds, which were extracted from the Radix Bupleuri samples and analyzed by HPLC-MS and GC-MS. Multivariate statistical analysis was employed to differentiate the Radix Bupleuri samples cultivated in different regions and to discover the differential compositions. The developed quantitative method was validated to be accurate, stable, sensitive, and repeatable for the determination of five saikosaponins. Further statistical tests revealed that the collected Radix Bupleuri samples were distinctly different from each other in terms of both saponins and volatile compounds, based on the provinces where they were grown. In addition, twenty-eight saponins and fifty-eight volatile compounds were identified as the differentially accumulated compositions that contributed to the discrimination of the Radix Bupleuri samples. The Radix Bupleuri samples grown in Shouyang county showed the highest content of saikosaponins. All of the results indicated that the cultivation region significantly affected the accumulation and diversity of the main chemical components of Radix Bupleuri. The findings of this research provide insights into the effect of the cultivation region on the quality of Radix Bupleuri and the differentiation of Radix Bupleuri cultivated in different regions based on the use of HPLC-MS and GC-MS combined with multivariate statistical analysis.     

Holistic quality evaluation of Saposhnikoviae Radix (Saposhnikovia divaricata) by reversed-phase ultra-high performance liquid chromatography and hydrophilic interaction chromatography coupled with ion mobility quadrupole time-of-flight mass spectrometry-based untargeted metabolomics

Untargeted metabolomics more suits the quality evaluation of TCM because of its holistic property. To assess the holistic quality difference of Saposhnikoviae Radix (the roots of Saposhnikovia divaricata), we integrate ultra-high-performance liquid chromatography coupled with ion mobility/quadrupole time-of-flight mass spectrometry (UHPLC/IM-QTOF-MS)-based untargeted metabolomics and quantitative assay. A BEH C18 column in the reversed-phase mode and a BEH Amide column in Hydrophilic Interaction Chromatography (HILIC) mode were utilized for metabolites profiling, which enabled high coverage of the non-polar to polar components in Saposhnikoviae Radix. Moreover, the application of major components knockout strategy enlarged the exposure of those minor components. Integrated use of high-definition MS^E (HDMS^E) and data-dependent acquisition (DDA) could enhance the metabolites characterization by providing reliable fragmentation information and collision cross section values. Computational in-house library-driven automated peak annotation of the HDMS^E and DDA data assisted to characterize 104 components from Saposhnikoviae Radix. Chemometric analyses of the commercial Saposhnikoviae Radix samples (64 batches collected from 11 cultivars aging from 1 to 4 years), based on the positive MS^E data, in general could indicate large discrimination between Guan-Fang-Feng (from Heilongjiang) and the others, but negligible difference among Saposhnikoviae Radix from the other ten provinces of China and with different ages. Quantitative assays of prim-O-glucosylcimifugin and 4′-O-β-D-glucosyl-5-O-methylvisamminol, by a rapid and fully validated UHPLC-UV method, could primarily deduce that Guan-Fang-Feng aging 2 and 3 years exhibited better quality. The methods established can holistically assess the quality of TCM with wide spans of plant metabolites covered.