HPLC fingerprinting and LC-TOF-MS analysis of the extract of Pseudostellaria heterophylla (Miq.) Pax root

High-performance liquid chromatographic (HPLC) was developed for fingerprint analysis of Pseudostellaria heterophylla (Miq.) Pax. Liquid chromatography-electrospray ionization-time-of-flight mass spectrometry (LC-TOF-MS) technique was first employed to identify the components of the fingerprint. Twelve major peaks in chromatographic fingerprint were analyzed by on-line LC-TOF-MS analysis; one cyclic peptide was unequivocally identified and five cyclic peptides were tentatively assigned based on their MS data. These cyclic peptides served as the marker peaks in the HPLC fingerprints. The chromatographic fingerprints have been analyzed by similarity index calculations and hierarchical clustering analysis (HCA). The result showed that the HPLC fingerprints could be used to determine the optimal harvest time for P. heterophylla (Miq.) Pax and to authenticate the species of the herb.     

桂南地区苦玄参药材RP-HPLC指纹图谱研究

采用反相HPLC法测定了广西梧州、龙州、苍梧、越南等多产地12批苦玄参药材指纹图谱,并对不同产地的苦玄参指纹图谱进行比较。色谱条件为:LunaC18(4.6×250mm,5μm)色谱柱,乙腈-水梯度洗脱,流速1.0mL/min,检测波长254nm,柱温25℃。结果12批苦玄参样品指纹图谱共标定了16个分离度良好的共有峰,方法的精密度、稳定性、重复性均符合国家相关规定,可作为控制苦玄参药材质量的定性标准。     

化学模式识别结合HPLC指纹图谱的僵蚕质量评价研究

建立HPLC指纹图谱结合化学模式识别技术对僵蚕药材进行质量评价。采用Insertsil ODS-3(4.6 mm×250 mm,5μm)色谱柱,以乙腈-0.1%磷酸水溶液为流动相梯度洗脱,检测波长365 nm,流速0.8 mL/min,进样量10μL,柱温30℃。采用相似度评价、聚类分析、主成分分析等化学模式识别方法,对不同来源的20批僵蚕药材进行质量分析和评价。建立的指纹图谱标定了9个共有峰,指认了5个峰,1号峰为芦丁,2号峰为金丝桃苷,4号峰为紫云英苷,6号峰为槲皮素,7号峰为山奈酚,并将9个共有峰峰面积与其性状、总灰分、酸不溶性灰分、浸出物及白僵菌素含量进行相关性分析。结果表明本研究建立的僵蚕HPLC指纹图谱结合化学模式识别的方法可靠、易行,方法重复性好、专属性强,与药材性状、总灰分、浸出物及白僵菌素含量等质控指标均有一定的相关性,为后续僵蚕药材的质量控制提供依据和参考。     

Development of the fingerprints for the quality of the roots of Salvia miltiorrhiza and its related preparations by HPLC-DAD and LC-MS(n)

High-performance liquid chromatographic (HPLC) fingerprints were developed for identification of both lipophilic and hydrophilic components of the roots of Salvia miltiorrhiza and four related preparations. These samples were separated with an Agilent Zorbax Extend C(18) reserved-phase column (5 microm, 250 mm x 4.6 mm) by linear gradient elution using water-phosphoric acid (100:0.026, v/v) and acetonitrile as mobile phase. The flow rate was 0.8 ml/min and the detector wavelength was set at 280 nm. Mean chromatograms and correlation coefficients of samples were calculated by the software "Similarity Evaluation System for Chromatographic Fingerprint of TCM". The correlation coefficients of Danshen and Fufang Danshen tablets (FDT) samples were in the range of 0.352-0.993 and 0.768-0.987, respectively. The correlation coefficients of Compound Danshen dripping pills (CDDP), Danshen injection (DSI) and Xiangdan injection (XDI) samples were higher than 0.928, 0.850 and 0.960, respectively. It was the first time to identify 34 peaks by comparing with standard compounds and using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS(n)) technique. All results indicated that the developed fingerprint assay could be readily utilized as a quality control method for S. miltiorrhiza and its related preparations.     

芦荟的HPLC指纹图谱建立、化学模式识别分析及其含量测定

采用Phenomenex C18色谱柱(250 mm×4.6 mm,5 μm),以甲醇-乙腈-0.3％磷酸水为流动相进行梯度洗脱,建立芦荟的指纹图谱.运用化学模式识别方法对不同产地芦荟药材质量控制方法进行评价.结果表明:12批芦荟HPLC指纹图谱共标定23个共有峰,并通过对照品指认其中6个成分;除了广西的3批药材之外,...     

Development of capillary electrophoresis fingerprint for quality control of Rhizoma Smilacis Glabrae

Introduction – Rhizoma Smilacis Glabrae (RSG) is a Chinese herbal medicine used for detoxication and as a diuretic. However, in some regions of China, RSG is used confusedly with some other herbs. Objective – To develop a capillary electrophoresis (CE)‐DAD fingerprint method for quality evaluation, species differentiation and product identification of RSG. Methodology – The CE separation conditions and extraction procedure were optimised. Eighteen batches of RSG samples were analysed and the standard fingerprint used for authentication was simulated by the average of all tested samples. Results – The optimal CE separation conditions were developed with running buffer of 20 mm borax containing 3 mm β‐cyclodextrin at pH 9.4, voltage of 25 kV and temperature of 25°C. The separation could be completed within 8 min. Nine peaks were found in the electropherogram of RSG and five peaks were identified as astilbin, taxifolin, 5‐O‐caffeoylshikimic acid, shikimic acid and trans‐resveratrol, respectively. Methanol and sonication were recommended for the sample preparation. All RSG samples showed similar chromatographic profile and six ‘held in common’ peaks were found. By the standard fingerprint, RSG could be well distinguished from its two confusable species, Rhizoma Smilacis Chinae and Rhizoma Heterosmilacis. Conclusion – A CE‐DAD fingerprint analysis method was developed for the quality control of RSG. The standard fingerprint could represent the chemical profile of RSG and be used for its authentication. Copyright © 2010 John Wiley & Sons, Ltd.     

紫花地丁HPLC指纹图谱研究

目的:建立紫花地丁药材HPLC指纹图谱,提供药材质量控制的可靠方法。方法:采用HPLC方法,以Agi-lent C18(4.6 mm×250 mm,5μm)为色谱柱,甲醇-0.5%醋酸水溶液进行梯度洗脱;检测波长353 nm,流速1.0mL/min。结果:检测了12批不同来源的紫花地丁药材,确立了18个共有峰,建立了紫花地丁对照指纹图谱,计算各被测样品的HPLC指纹图谱的整体相似度,并指认了菊苣苷、七叶内酯、东莨菪素、早开堇菜苷4个特征峰,比较了上述成分在不同药材中的含量。结论:所建立的指纹图谱具有良好的精密度、重现性和稳定性,可作为紫花地丁药材质量控制标准。     

陕西秦艽药源植物及秦艽药材HPLC的比较研究

目的:系统比较研究秦艽药材与其药原植物间主要化学成分的差异。方法:高效液相色谱法,色谱柱:Diamonsil C18(200 mm×4.6 mm,5um);洗脱条件:乙腈-水溶液梯度洗脱;检测波长:240 nm;柱温:30℃;流速:0.5 mL.min-1。结果:陇县、太白秦艽中龙胆苦苷含量明显高于市售秦艽药材;通过确定7个共有峰建立陕西秦艽HPLC指纹图谱,所测定的29批秦艽药材具有很高的相似度,而秦艽伪品相似度较低。采用HPLC各峰面积及其相对之比进行聚类分析结果显示,采自太白的10个样本和陇县的8个样本分别聚为两支,表明秦艽化学成分含量与其地理分布存在密切关系。结论:本实验建立的指纹图谱可用于秦艽及其药材鉴别和质量控制;聚类分析可以鉴别秦艽产地。     

毛脉酸模不同生长发育期HPLC色谱指纹图谱研究

采用HPLC-DAD方法,梯度洗脱,对10批不同生长发育期毛脉酸模根样品进行主成分分析。色谱条件为:Planetsil C18分析柱(5μm, 200 mm×4.6 mm),预柱Phenomenex ODS-C18(4×3.0 mm ID),柱温40℃,流动相A为甲醇;流动相B为水(磷酸调pH值为2.0),流动相A从30%甲醇到100%甲醇,时间为0~50 min,检测波长254 nm。10批毛脉酸模样品得到的色谱指纹图谱有27个共有峰,其特征峰指纹图谱可分为Ⅰ, Ⅱ, Ⅲ三组:第Ⅰ组包括0~17 min(1~14号峰),第Ⅱ组包括17~35 min(15~24号峰),第Ⅲ组包括35~50 min(25~27号峰)。HPLC-DAD方法分析毛脉酸模主成分,方法准确可靠,其本身具有多成分同时定性的优势;27个共有峰的出峰先后顺序及相对含量极具特征性、专属性,重复性好,形成了毛脉酸模特有的HPLC色谱指纹图谱,可作为毛脉酸模内在质量评价、鉴定及其最佳采收期确定提供科学依据。     

高良姜等五味山姜属中药乙酸乙酯部位的HPLC指纹图谱

以95%乙醇作为提取的溶媒,将加热回流提取得到的提取液水浴挥干得到浸膏,并用甲醇配制成1 mg·m L~(-1)供试液。采用phenomenex-C18(250 mm×4.6 mm,5μm)色谱柱,流动相为乙腈-0.1%磷酸水溶液,梯度洗脱,流速为1.0 m L·min~(-1),柱温为30℃,检测波长为260 nm HPLC色谱法测定,采用国家药典委员会相似度评价软件进行分析。结果表明:建立了精密度、稳定性和重现性均较好的5味山姜属中药乙酸乙酯部位HPLC指纹图谱,确定了9个共有峰,6个强峰;其中高良姜、大高良姜含有5个共有峰,高良姜和草豆蔻含有3个共有峰,大高良姜和红豆蔻含有2个共有峰,高良姜和益智含有3个共有峰。对5味山姜属中药乙酸乙酯部位化学成分进行相似度分析,得出其相似度分别为0.955、0.805、0.371、0.794、0.345。所建立的5味山姜属中药乙酸乙酯部位的指纹图谱方法稳定、简便、可靠,其特征峰所代表的化学成分的相似性不尽相同,高良姜、大高良姜、草豆蔻的相似性较大,红豆蔻、益智的相似性较小,说明同基原中药之间的化学成分有一定的相关性。该研究结果为探讨山姜属中药亲缘关系与化学成分相关性提供了思路与基础。     

罗汉果祛痰作用谱效关系研究

为了研究罗汉果祛痰作用与活性成分的相关性,该研究首先测定了19批罗汉果提取物高效液相(HPLC)指纹图谱和小鼠气管酚红排泌量,找出了指纹图谱共有峰,采用灰色关联度法确定各共有峰对祛痰效果贡献度,利用偏最小二乘法(PLS)得出了各共有峰的正负相关及贡献度。结果表明:共有峰为14个,其中关联度在0.8以上的有12个峰(正相关峰为3、11、12、13号峰,负相关峰为1、2、4-10、14号峰);11、12号峰分别为氧化罗汉果苷Ⅴ和罗汉果苷Ⅴ。因此,罗汉果祛痰作用不是单一成分起作用,而是多种成分综合作用的结果。其中,氧化罗汉果苷Ⅴ和罗汉果苷Ⅴ有祛痰作用,贡献率较大。     

High-performance liquid chromatographic determination of lamivudine in human serum using liquid-liquid extraction; application to pharmacokinetic studies

A simple, fast, and sensitive high performance liquid chromatographic (HPLC) assay was developed for quantitation of lamivudine in human serum. Lamivudine is polar compound and its extraction from the human serum in previously published HPLC methods involved either protein precipitation or solid phase extraction techniques. However, existence of endogenous peaks which interfere with the drug or appeared as late eluting peaks and lead to long run time of analysis has been reported. Application of either an ion pairing agent in the mobile phase or time consuming column purge has been used in the published methods. Present paper describes liquid - liquid extraction of lamivudine and internal standard (famotidine) using dichloromethane-isopropyl alcohol (1:1, v/v) as an extracting solvent and salting out approach. The mobile phase was a mixture of phosphate buffer (0.05 M) containing triethylamine (1 mL/L, v/v; pH 3.5) and methanol (91:9, v/v) at a flow rate of 2.2 mL/min. The analysis was performed on a column (150 mm x 6 mm i.d.) which was packed with 5 microm particles of ODS packing material. Under these conditions no interference in the assay from any endogenous substance was observed. The limit of quantification was evaluated to be 5 ng/mL. Accuracy and precision of the method were also studied and the technique was shown to be selective and linear into the concentration range of 5-2500 ng/mL. This method has been used in two randomized crossover bioequivalence studies of 100 and 150 mg lamivudine preparations in 12 and 24 healthy volunteers, respectively.     

木瓜药材HPLC指纹图谱研究

以熊果酸为参照物,利用高效液相色谱（HPLC）梯度洗脱,测定了19批木瓜（Chaenomeles speciosa（Sweet） Nakai）样品。建立了药用木瓜的高效液相指纹图谱,为评价控制药用木瓜的质量提供了依据。色谱柱为YPW—Kromasil TM—C18柱（250mm×4．6mm,5μm）（美国迪马公司）;流动相：甲醇（A）一1％冰醋酸（B）,流动相A为甲醇,流动相B为1％冰醋酸水溶液。检测波长290nm,柱温30℃,流速10mL／mjn,进样量20μL。通过分析19批木瓜样品得到的高效液相指纹图谱有11个共有峰,多数峰都可以达到较好分离且19批次相似度符合。因此药用木瓜的指纹图谱特征性及专属性强,可用于药用木瓜的质量控制。     

猫须草HPLC指纹图谱研究

为建立猫须草药材HPLC指纹图谱分析方法,采用高效液相色谱法,以Phenomenex Synergi 4u hydro-RP 250×4.60 mm为色谱柱,以甲醇-0.1%甲酸溶液为流动相梯度洗脱,检测波长254 nm,流速1.0 mL·min^-1,柱温40 ℃。结果表明,建立的猫须草药材HPLC指纹图谱,确定了15个共有峰,各猫须草样品指纹图谱与对照指纹图谱的相似度均在0.9以上。该方法简单、准确、重复性好,为更好地控制猫须草药材质量提供有效可靠的方法。     

萆薢类药材的HPLC指纹图谱研究

应用HPLC方法测定了薯蓣属根状茎组10种1亚种1变种植物23个样本,建立了萆薢类药材总皂苷元粗提物的HPLC指纹图谱.色谱柱为Zorbax Eclipse XDB-C_(18)柱(4.6×250 mm,5 μm),流动相为乙腈-水,柱温30℃,检测波长为203 nm.结果表明,用上述条件所建立的指纹图谱共标示出7个共有峰,且可较全面地反映萆薢类药材的皂苷元类成分,为萆薢类药材薯蓣属根状茎组植物鉴别及质量控制提供一种方法.     

不同品种玫瑰花药材的HPLC指纹图谱研究

目的:建立中药玫瑰花的高效液相色谱指纹图谱,为科学评价和有效控制其质量提供可靠的方法。方法:用HPLC-UV方法测定了11份玫瑰花样品,采用中国药典委员会颁布的"中药色谱指纹图谱相似度评价"2004A系统建立了玫瑰花药材指纹图谱,并进行相似度评价和聚类分析。结果:11份玫瑰花样品的HPLC指纹图谱有18个共有峰,有10份样品相似度都在0.8以上,聚类分析时归为一类。结论:建立了玫瑰花不同品种的HPLC指纹图谱,并比较了其之间的差异,该方法稳定、可靠、精密度高、重复性好,可为中药玫瑰花的质量评价体系的建立提供科学依据。     

朝鲜淫羊藿药材HPLC指纹图谱研究

目的:建立朝鲜淫羊藿药材HPLC指纹图谱分析方法。方法:采用Agilent C18(4.6 mm×250 mm,5μm)色谱柱,柱温:25℃,流速:1 mL/min,以乙腈、水为流动相进行梯度洗脱,检测波长为270 nm,以淫羊藿苷、朝藿定A、朝藿定B、朝藿定C为对照品。结果:对同一批次朝鲜淫羊藿药材样品进行测定,标定了4个特征峰,通过"中药色谱指纹图谱相似度评价系统2004A版软件"计算其相似度。结论:该方法建立的朝鲜淫羊藿药材指纹图谱方法精密度高、重现性好,可为更好的控制朝鲜淫羊藿药材的内在质量提供科学依据。     

柘荣太子参HPLC指纹图谱的研究

建立太子参的 HPLC指纹图谱分析条件,为太子参药材内在质量评价积累数据.方法：应用RP-HPLC法;Cosmosil C18分析柱;乙腈-水二元梯度洗脱;流速为1.0 mL/in;检测波长203 nm;分析时间60 min.结果：建立太子参药材指纹图谱,特征共有峰有15个.结论：该方法准确可靠,重复性好,可用于太子参的HPLC的指纹图谱分析.     

Analysis of pharmaceutical samples of Resina Draconis by HPLC-PAD

Introduction: The quality evaluation of traditional Chinese medicine (TCM) represents a particular challenge owing to the complexity of the matrix, which renders separation and identification of the individual components extremely difficult. In recent years, fingerprinting of TCMs has played a dominant role in quality control. Resina Draconis was authorised as a new TCM in 1991, but a satisfactory HPLC fingerprint method for this preparation has not yet been published. Objective: To develop a simple and reliable protocol for the quality control of Resina Draconis using an HPLC‐PAD method. Methodology: The TCM was extracted with methanol at room temperature. Chromatography was carried out using a Lichrospher C₁₈ column eluted with a linear gradient of acetonitrile (A) and water containing 0.1% phosphoric acid (B), initially at 30:70 (A:B) and changing to 60:40 in 90 min. UV (PAD) spectra were acquired in the range 210–400 nm. Results: Four chromatograms of samples of Resina Draconis obtained from different pharmaceutical factories showed 20 peaks in common. The average chromatogram was taken as a template from which the correlation coefficients and cosine ratios of the samples were determined. Whereas the contents of individual components in each sample were different, overall the samples were extremely similar one to another, and the products from different pharmaceutical factories were consistent. Conclusion: A reliable and validated HPLC method has been developed for the fingerprint analysis of Resina Draconis that can be applied for the quality control of this TCM. Copyright © 2008 John Wiley & Sons, Ltd.