Quality evaluation of Radix Astragali through a simultaneous determination of six major active isoflavonoids and four main saponins by high-performance liquid chromatography coupled with diode array and evaporative light scattering detectors

A method, high-performance liquid chromatography coupled with diode array and evaporative light scattering detectors (HPLC-DAD-ELSD), was developed to evaluate the quality of Radix Astragali through a simultaneous determination of six major active isoflavonoids and four main saponins. The wavelength at 280 nm was chosen to determine six isoflavonoids: calycosin-7-O-beta-D-glucoside (1), ononin (2), (6alphaR, 11alphaR)-9,10-dimethoxypterocarpan-3-O-beta-D-glucoside (3), (3R)-2'-hydroxy-3',4'-dimethoxyisoflavan-7-O-beta-D-glucoside (4), calycosin (5), and formononetin (6); and ELSD connected after DAD was employed to determine four saponins: astragaloside IV (7), astragaloside II (8), astragaloside I (9), and acetylastragaloside I (10). This assay was fully validated with respect to precision, repeatability and accuracy. The proposed method was successfully applied to quantify the ten components in eleven samples from different localities in China; significant variations were demonstrated in the content of these compounds in the samples from different areas. This simple, rapid, low-cost and reliable HPLC-DAD-ELSD method is suitable for routine quantitative analysis and quality control of traditional Chinese medicines (TCMs) consisting of bioactive multi-components with different structures such as Radix Astragali.     

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

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

Determination of fifteen bioactive components in Radix et Rhizoma Salviae Miltiorrhizae by high-performance liquid chromatography with ultraviolet and mass spectrometric detection

A high-performance liquid chromatographic (HPLC) method coupled with ultraviolet (UV) and electrospray ionization time-of-flight mass spectrometry (ESI-TOF/MS) was established for simultaneous qualitative and quantitative determination of nine phenolic acids and six diterpenoids in Radix et Rhizoma Salviae Miltiorrhizae (RRSM). The optimal chromatographic conditions were achieved on a Zorbax C(18) column by gradient elution with 0.1% (v/v) aqueous formic acid and acetonitrile as mobile phase at the flow rate of 1.0 mL/min. The detection wavelength at 281 nm was chosen to determine the 15 bioactive components, namely danshensu (1), protocatechuic acid (2), protocatechuic aldehyde (3), caffeic acid (4), rosmarinic acid (5), lithospermic acid (6), salvianolic acid B (7), salvianolic acid A (8), salvianolic acid C (9); dihydrotanshinone I (10), cryptotanshinone (11), tanshinone I (12), methylene tanshiqunone (13), tanshinone IIA (14) and miltirone (15). Additionally, LC-ESI-TOF/MS was used to make definite identification of the constituents in samples in comparison with those reference compounds. The validation of the method included tests of linearity, sensitivity, repeatability, stability and recovery. The proposed method was successfully applied to quantify the 15 components in 21 samples; significant variations were demonstrated in the contents of the samples from diverse species and origins. The developed method could be used to effectively and comprehensively evaluate the quality of RRSM for its clinical safety and efficacy.     

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.     

Simultaneous chemical fingerprinting and quantitative analysis of crude and processed Radix Scrophulariae from different locations in China by HPLC

A validated liquid chromatography method was first developed to evaluate the quality of crude and processed Radix Scrophulariae extracts through establishing chromatographic fingerprint and simultaneous determination of five bioactive compounds, namely 5-hydroxymethylfurfural (5-HMF), acteoside, angroside C, harpagoside and cinnamic acid. The chromatographic were separated on an Agilent Zorbax Extend C(18) column (250 mm × 4.6 mm, 5 μm) and detected by diode array detector (DAD). Mobile phase was composed of (A) aqueous phosphoric acid (0.03%, v/v) and (B) acetonitrile using a gradient elution. Analytes were performed at 30 °C with a flow rate of 1.0 mL/min and UV detection at 280 nm. All calibration curves showed good linear regression (r(2) ≥0.9996) within the tested ranges, and the recovery of the method was in the range of 98.12-103.38%, with RSD values ranging from 0.6 to 2.8%. In addition, the contents of those five bioactive compounds in crude and processed Radix Scrophulariae prepared by different locations of China were determined to establish the effectiveness of the method. The results demonstrate that the developed method is accurate and reproducible and could be readily utilized as a suitable quality control method for the quantification of Radix Scrophulariae.     

Simultaneous qualitation and quantification of four phytoecdysones in Radix Achyranthis Bidentatae by high-performance liquid chromatography with diode array detection

A high-performance liquid chromatographic method with diode array detection was developed and validated to simultaneously identify and quantify four phytoecdysones,i.e., polypodine B (1), ecdysterone (2), 25-R inokosterone (3) and 25-S inokosterone (4), in Radix Achyranthis Bidentatae. The analysis was performed using a C(18) column with 6% tetrahydrofuran aqueous and acetonitrile isocratic elution, and the detection was carried out by DAD at 242 nm. The identities of the analytes were determined by comparing the retention time and UV spectrum with those of reference compounds. The validation of the method included linearity, sensitivity, recovery and stability. All calibration curves of the four phytoecdysones showed good linear regression (r(2) >or= 0.9993). The limit of detection (S/N = 3) and limit of quantification (S/N = 10) were less than 7.5 and 12.3 ng, respectively. The method provided desirable repeatability with overall intra- and inter-day variations of less than 4.67%. The obtained recoveries varied between 95.1 and 104.4% while the relative standard deviations were below 4.85% (n = 3). The analysis results indicated that the contents of the investigated phytoecdysones in Radix Achyranthis Bidentatae from different locations were highly variant, and the genuine crude drug indigenous to Henan province did not possess the highest concentration of phytoecdysones.     

纹党参与白条党参红外光谱的SIMCA聚类鉴别方法研究

以纹党参和白条党参的红外光谱为聚类分析的对象,研究了红外光谱结合SIMCA聚类分析法对纹党参和白条党参进行识别与分类的可行性.选取400 ～2 000 cm~(-1)范围内的光谱,通过基线补偿(Offset)和散射校正(MSC)等预处理后,采用SIMCA聚类分析法建立识别模型.结果表明,所建模型对纹党参和白条党参的识别率分别达92%和96%,拒绝率均为100%.用盲样对所建模型进行了测试,测试结果全部正确.该法可实现对纹党参和白条党参的快速鉴别.     

Rapid simultaneous determination of isoflavones in Radix puerariae using high-performance liquid chromatography-triple quadrupole mass spectrometry with novel shell-type column

A high-performance liquid chromatography (HPLC) coupled with triple quadrupole mass spectrometry (MS/MS) method was developed for rapid determination of 13 isoflavones in Radix puerariae. A novel shell-type column, namely Kinetex core-shell C(18) column (50 mm×2.1 mm id, 2.6 μm), and gradient elution were used during the analysis. The chromatographic peaks of 13 investigated compounds were identified by comparing their retention time and MS data with the related reference compounds. Multiple-reaction monitoring (MRM) was employed for the quantitative analysis with negative ionization mode. All calibration curves showed good linearity (r(2)>0.9990) within test ranges. The LOD and LOQ were lower than 0.017 and 0.873 μg/mL on column, respectively. The intra- and inter-day precisions for 13 analytes were <1.17 and 2.17%, respectively, and the recoveries were 93.1-104.4%. The validated method was applied for quantitative analysis of 13 isoflavones in 7 species of Radix puerariae. The result demonstrated that HPLC-MS/MS system with Kinetex column could be a promising analytical tool for the determination of isoflavones in traditional Chinese medicines, which is helpful for comprehensive evaluation of quality of R. puerariae.     

Purification of polar compounds from Radix isatidis using conventional C18 column coupled with polar-copolymerized C18 column

Regarding hydrophilic interaction chromatography and normal phase liquid chromatography, RPLC is another choice used to separate polar compounds with the improvement of polar-modified C18 stationary phase. In this study, a method using conventional C18 column coupled with polar-copolymerized C18 column was successfully developed for the separation and purification of polar compounds from Radix isatidis, which is one of the most commonly used traditional Chinese medicines (TCMs). An XTerra MS C18 column was used to fractionate the extract of R. isatidis and a homemade polar-copolymerized C18 column was utilized for the final purification due to its good separation selectivity and high resolution for polar compounds. The established purification system demonstrated good orthogonality for the polar compounds. As a result, ten compounds were purified and three of them were identified as 3-methyl-5-vinyloxazolidin-2-one (compound A), 5-hydroxymethyl-2-furaldehyde (compound B) and 3-methylfuran-2-carboxylic acid (compound G) based on the MS, IR and extensive NMR data, respectively. It was demonstrated to be a feasible and powerful technique for the purification of polar compounds under RPLC mode and more chemical information of TCMs will be obtained to interpret the efficiency of TCMs.     

高效液相色谱法同时测定黄芪中的五种异黄酮类成分

对蒙古黄芪中5种异黄酮类成分的含量进行了反相高效液相色谱法测定。色谱柱为Diamonsil C18柱,流动相为乙腈-水系统,梯度洗脱,检测波长230 nm,柱温35 ℃。毛蕊异黄酮-7-O-β-D-葡萄糖苷在20.12～201.2 mg/L、芒丙花素-7-O-β-D-葡萄糖苷在4.62～46.2 mg/L、9,10-二甲氧基紫檀烷-3-O-β-D-葡萄糖苷在4.86～48.6 mg/L、毛蕊异黄酮在9.24～92.4 mg/L、芒丙花素在6.92～69.2 mg/L时峰面积与浓度呈良好的线性关系,相关系数分别为0.9992,0.9997,0.9997,0.9995和0.9995。5种成分的加样回收率均高于94%,相对标准偏差（RSD）小于3.2%(n=9)。该法简便快速,重复性良好,结果准确可靠,可用于黄芪药材中5种主要异黄酮类成分的含量测定。     

Rapid method for simultaneous determination of flavonoid,saponins and polyacetylenes in Folium Ginseng and Radix Ginseng by pressurized liquid extraction and high-performance liquid chromatography coupled with diode array detection and mass spectrometry

A rapid pressurized liquid extraction (PLE) and high-performance liquid chromatography coupled with diode array detection and mass spectrometry (HPLC-DAD–MS) method for the simultaneous determination of one flavonoid (panasenoside), nine saponins (ginsenoside Rg1, Re, Rf, Rg2, Rb1, Rc, Rb2, Rb3 and Rd) and two polyacetylenes (panaxydol and panaxynol) in Folium Ginseng and Radix Ginseng was developed. A Prevail C₁₈ rocket column (33 mm × 7 mm, 3.0 μm) and gradient elution were used during the analysis. Flavonoid was quantified at 355 nm, and saponins and polyacetylenes were determined at 203 nm. The chromatographic peaks of 12 investigated compounds in samples were unambiguously identified by compared their UV spectra and/or MS data with the related reference compounds. All calibration curves showed good linearity (r > 0.999) within the test ranges. The intra- and inter-day variations for 12 analytes were less than 1.17% and 2.17%, respectively. The developed method was successfully applied to determine the investigated compounds in 10 samples of Radix Ginseng and Folium Ginseng, respectively. The result showed that PLE combined with rocket column HPLC analysis could provide a rapid method for analysis of compounds in traditional Chinese medicines (TCMs), which is helpful to comprehensive evaluation of quality of Radix Ginseng and Folium Ginseng.     

One single LC-MS/MS analysis for both phenolic components and tanshinones in Radix Salviae Miltiorrihizae and its medicinal products

A LC-MS/MS method was developed for the separation and simultaneous determination of phenolic components including danshensu, protocatechuic acid, protocatechuic aldehyde and caffeic acid as well as tanshinones including cryptotanshinone, tanshinone I and tanshinone IIA in samples of Radix Salviae Miltiorrhizae and Salviae Miltiorrhizae tablet. Triple quadrupole mass spectrometry was optimized in both positive and negative ion multiple reaction monitoring modes for the simultaneous quantitative analysis of the two different types of active components by using a time-segment program. The method gave recoveries of 85.4-106.4% with relative standard deviations of 2.4-8.0% for the spiked herb samples. The limits of detection were 0.30-0.83 μg/g for the analysis of 1.0 g Radix Salviae Miltiorrhizae or tablet samples.     

高效液相色谱-二极管阵列检测法测定白首乌中的4种苯乙酮类成分

建立了白首乌中4种苯乙酮类化合物(Ⅰ:4-羟基苯乙酮,Ⅱ:2,5-二羟基苯乙酮,Ⅲ:白首乌二苯酮,Ⅳ:2,4-二羟基苯乙酮)的高效液相色谱检测方法。采用Symmetry-C18色谱柱(4.6 mm×250 mm,5 μm)分离,流动相为甲醇-水(体积比为26∶74),流速1.0 mL/min;采用二极管阵列检测器检测,化合物Ⅰ、Ⅲ、Ⅳ的检测波长为280 nm,化合物Ⅱ的检测波长为224 nm;测定温度为30 ℃。4种苯乙酮类化合物Ⅰ～Ⅳ的线性范围分别为0.080～0.560 μg,0.080～0.560 μg,0.100～0.700 μg和0.092～0.644 μg (r=0.9996～0.9999);平均加样回收率(n=3)为98.0%～104.0%,相对标准偏差为0.8%～2.6%。该方法简便快速、结果准确、重现性好,可作为白首乌药材质量控制的一个有效方法。     

A rapid method for simultaneous determination of 14 phenolic compounds in Radix Puerariae using microwave-assisted extraction and ultra high performance liquid chromatography coupled with diode array detection and time-of-flight mass spectrometry

A microwave-assisted extraction (MAE) and ultra high performance liquid chromatography coupled with diode array detection and time-of-flight mass spectrometry (UHPLC-DAD-TOF-MS) method was developed for simultaneous determination of 14 phenolic compounds in the root of Pueraria lobata (Wild.) Ohwi and Pueraria thomsonii Benth. Operational conditions of MAE were optimized by central composite design (CCD). The optimized result was 65% ethanol as extraction solvent, 17 mL of extraction volume, 100 °C of extraction temperature and 2 min of hold time. A Zorbax SB C₁₈ (50 mm × 4.6 mm I.D., 1.8 μm) and gradient elution were used during the analysis. The chromatographic peaks of 14 investigated compounds in samples were successfully identified by comparing their retention time, UV spectra and TOF mass data with the reference substances. All calibration curves showed good linearity (r > 0.9997) within the test ranges. The intra-day and inter-day variations were less than 1.77% and 2.88%, respectively. The developed method was successfully applied to determine the investigated compounds in 10 samples of Radix Puerariae Lobatae and Radix Puerariae Thomsonii, respectively. The result indicated that MAE and UHPLC-DAD-TOF-MS system might provide a rapid method for the quality control of Radix Puerariae.     

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。     

Liquid chromatography-electrospray ionization mass spectrometry study of the flavonoids of the roots of Astragalus mongholicus and A. membranaceus

High-performance liquid chromatography-electrospray ionization mass spectrometry has been applied to analyze the flavonoids of Huangqi, the roots of Astragalus mongholicus and A. membranaceus. Eight flavonoids were identified as calycosin-7-O-beta-D-glucoside, calycosin-7-O-beta-D-glucoside-6"-O-malonate (2), ononin, (6aR,11aR)-3-hydroxy-9,10-dimethoxypterocarpan-3-O-bet a-D-glucoside, calycosin, (3R)-7,2'-dihydroxy-3',4'-dimethoxyisoflavan-7-O-beta-D-glucoside, formononetin-7-O-beta-D-glucoside-6"-O-malonate and formononetin by direct comparison with the isolated standards from Huangqi. The existence of (6aR,11aR)-3-hydroxy-9,10-dimethoxypterocarpan, (3R)-7,2'-dihydroxy-3',4'-dimethoxyisoflavan, astrapterocarpanglucoside-6'-O-malonate and astraisoflavanglucoside-6'-O-malonate was detected. This is the first report of flavonoid glycoside malonates in these two Astragalus species, and malonate 2 is a structurally completely identified new compound.     

Quantitative analysis of nine coumarins in rat urine and bile after oral administration of Radix Glehniae extract by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry

Coumarins are the primary bioactive ingredients in Radix Glehniae, named Beishashen in China, which possesses many pharmacological activities, including anticancer, anti‐inflammation and antivirus activities. In the present study, we employed a sensitive and selective high‐performance liquid chromatography–electrospray ionization–tandem mass spectrometry (HPLC‐ESI‐MS/MS) method for the quantification of nine coumarins in rat urine and bile: scopoletin ( 1 ), xanthotoxol ( 2 ), xanthotoxin ( 3 ), psoralen ( 4 ), isoimpinellin ( 5 ), bergapten ( 6 ), oxypeucedanin ( 7 ), imperatorin ( 8 ) and isoimperatorin ( 9 ). Pimpinellin ( 10 ) was used as the internal standard (IS). The urine and bile samples were pretreated by liquid–liquid extraction with ethyl acetate (EtOAc). The chromatographic separation was carried out on a C₁₈ column with gradient elution. The detection of analytes was performed on a tandem mass system equipped with a turbo ion spray interface in positive mode using multiple‐reaction monitoring (MRM). The specificity, linearity, accuracy, precision, recovery, matrix effect and several stabilities were validated for coumarins in rat urine and bile samples. The results showed that this method is robust, specific and sensitive and it can successfully fulfill the requirements of the excretion study of the nine coumarins in Radix Glehniae. Copyright © 2010 John Wiley & Sons, Ltd.     

Authentication of Radix Aucklandiae and its substitutes by GC-MS and hierarchical clustering analysis

Radix Aucklandiae (Muxiang in Chinese), the dried root of Aucklandia lappa, is used as a medicinal material for digestive system disorders in traditional Chinese medicine for centuries. Owing to the similarity of morphologies and trade names, Radix Vladimiriae (Chuan-Muxiang), the roots of Vladimiria souliei and V. souliei var. cinerea, and Radix Inulae (Tu-Muxiang), the roots of Inula helenium and Inula racemosa, as well as the renal toxic aristolochic acid containing Radix Aristolochiae (Qing-Muxiang), the roots of Aristolochia debilis and Aristolochia contorta, are often used confusedly as the substitutes of Radix Aucklandiae. In order to ensure the effective and safe utility of Radix Aucklandiae, a GC-MS method was developed to generate the chemical profiles of essential oils of Radix Aucklandiae and its substitutes. In addition, hierarchical clustering analysis was used to compare the similarities of these chemical profiles. It was found that all the samples of A. lappa have similar chemical profiles and were clustered into one group, while the samples of Radix Vladimiriae, Radix Inulae, and Radix Aristolochiae were clustered into their own independent groups, respectively, suggesting that together with hierarchical clustering analysis, chemical profiles of essential oils generated by GC-MS could objectively discriminate Radix Aucklandiae from its common substitutes.