不同产地桔梗HPLC指纹图谱及化学模式识别研究

为建立桔梗HPLC指纹图谱并对其质量控制方法进行探讨。研究采用YMC Hydrosphere C_(18)分析色谱柱(250×4.6 mm,5μm),以水和乙腈为流动相进行梯度洗脱,检测波长210 nm。建立不同产地15批桔梗药材HPLC指纹图谱。采用相似度评价、聚类分析、主成分分析和正交偏最小二乘判别分析等化学模式识别方法对不同产地桔梗质量及其控制方法进行分析和评价。建立的桔梗HPLC指纹图谱共标定21个共有峰,并通过对照品指认其中7个成分;不同产地桔梗相似度在0.927～0.991之间;聚类分析和主成分分析结果都将15批桔梗分为3类,利用正交偏最小二乘判别分析筛选出造成桔梗差异的13个色谱峰。本研究建立的HPLC指纹图谱结合化学模式识别的方法简便准确,能够为桔梗的质量控制和品质评价提供依据。     

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。     

UPLC指纹图谱结合化学模式识别评价白芷药材质量

为建立白芷药材UPLC指纹图谱测定方法,并用于白芷药材的质量评价。研究采用Waters UPLC超高效液相色谱仪,ACQUITY UPLC BEH C_(18)(50 mm×2.1 mm,1.7μm)色谱柱,以乙腈-水为流动相进行梯度洗脱,检测波长254 nm,建立10批白芷药材的UPLC指纹图谱。采用对照品化学指认,并结合相似度评价、聚类分析(CA)、主成分分析(PCA)和正交偏最小二乘法-判别分析(OPLS-DA)等模式识别技术,对不同产地和处理方式的白芷药材的总体质量进行分析评价。建立的指纹图谱方法符合方法学要求,指纹图谱共标定22个共有峰,经对照品进行化学指认共鉴定了其中10个色谱峰;通过相似度评价、CA、PCA结果可知不同产地白芷药材质量有差异,且硫磺熏蒸对白芷药材品质有较大影响;最后采用OPLS-DA和组间差异性分析,筛选出了导致白芷药材质量差异的主要标志性成分。本研究建立的指纹图谱结合化学模式识别方法可用于白芷药材的质量控制和品质评价。     

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。     

广西产拳卷地钱HPLC指纹图谱研究

为了对广西产拳卷地钱药材进行质量控制,采用HPLC指纹图谱法对8批药材进行了研究,色谱条件为安捷伦ZORBAX SB-C18色谱柱(4.6 mm×250 mm,5μm),流动相为乙腈(B)-0.1%磷酸水溶液(D),梯度洗脱,检测波长为230 nm,柱温为25℃。结果表明:建立了参考指纹图谱,共有峰6个,8批广西产拳卷地钱药材HLPC指纹图谱相似度均大于0.75,其精密度、重复性、稳定性实验均符合指纹图谱技术要求。HPLC指纹图谱分析,数据稳定可靠,方法简便高效,可为广西产拳卷地钱质量评价提供参考。     

基于HPLC指纹图谱结合化学模式识别以及多指标成分定量评价前胡药材质量

基于HPLC指纹图谱和多指标成分含量测定,并结合化学模式识别,评价不同产地前胡药材质量。采用Agilent SB-C₁₈(4.6 mm×250 mm, 5μm)色谱柱,以甲醇-0.5%甲酸水为流动相进行梯度洗脱,流速为0.5 mL/min,检测波长321 nm,建立指纹图谱并对4个香豆素类成分进行定量,采用聚类分析(hierarchical clustering analysis, HCA)、主成分分析(principal component analysis, PCA)、偏最小二乘法-判别分析(partial least squares discriminant analysis, PLS-DA)对不同产地前胡药材进行质量评价。结果显示16批前胡药材HPLC指纹图谱相似度为0.966～0.999,确定了21个共有峰,共指认了9个成分。通过化学模式识别分析,将16批样品聚类为三类：以安徽宁国、亳州等为主的道地产区前胡,以湖南、贵州、重庆等为主的前胡,以及高海拔云南产区的前胡,并确立8个差异性质量标志物。多指标含量测定不同产地前胡的佛手柑内酯、白花前胡甲素、白花前胡...     

不同产地野生与栽培伊贝母药材水溶性成分指纹图谱研究

目的:建立伊贝母药材水溶性成分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:采用HPLC-DAD方法,以Kromasil C18色谱柱(4.6 mm×150 mm,5μm),流动相为甲醇和水,梯度洗脱,流速0.5 mL·min-1,柱温25℃,检测波长:260 nm。采用药典委员会颁布的中药色谱指纹图谱相似度评价系统2004 A版软件,对29批野生与栽培伊贝母药材进行指纹图谱分析。结果:29批伊贝母药材中有14个共有特征峰,建立了HPLC指纹图谱共有模式。各批次伊贝母药材相似度都在0.714以上,29批野生与栽培伊贝母药材可通过系统聚类分成5类,不同产地野生与栽培药材组成质量相似性较好,并定量测定了样品中的β-胸苷和腺苷。结论:所建立的HPLC指纹图谱及定量分析均具有良好的精密度、重复性、稳定性,可用于伊贝母药材的质量综合评价。     

基于HPLC指纹图谱和多成分定量评价不同品种百合鲜品质量

目的：建立3种不同品种百合鲜品HPLC指纹图谱，并同时测定其中5种成分。利用聚类（CLA）、主成分分析（PCA）等方法考察不同来源百合质量，为其质量控制评价提供参考。方法：HPLC梯度洗脱法建立了20批百合鲜品的指纹图谱，通过中药指纹图谱相似度评价软件进行相似度评价，采用聚类、主成分分析等进行分析。并同时测定5种成分的含量。结果：标定15个色谱共有峰，20批药材共有峰相似度为0.930～0.997；通过聚类分析分为2类；PCA筛出累积方差贡献率达86.70%的2个主成分；偏最小二乘法-判别分析（OPLS-DA）结果表明不同来源百合药材存在明显差异，对样品有显著影响的色谱峰分别为3号、6号（王百合苷C）和10号。三种分析方法所得结果基本一致。结论：所建立的HPLC指纹图谱和多指标成分定量方法可行，可为系统评价不同品种百合提供一些参考。     

钩藤UHPLC指纹图谱及化学模式识别研究

利用UHPLC指纹图谱结合化学模式识别技术对钩藤药材进行质量评价。采用ACQUITY UPLC HSS T_3色谱柱(2.1 mm×100 mm, 1.8μm),以乙腈-0.1%甲酸水溶液为流动相梯度洗脱,检测波长254 nm,流速0.3 mL/min,进样量2μL,柱温30℃,采集并记录不同来源的钩藤UHPLC指纹图谱,然后利用相似度评价、聚类分析、主成分分析和正交偏最小二乘判别分析等化学模式识别方法,对不同来源的钩藤药材质量进行分析和评价。所建立的钩藤UHPLC指纹图谱共识别、标定了15个共有峰,经鉴定其中的7个共有峰分别为绿原酸、异帽柱木菲碱、异钩藤碱、钩藤碱、缝籽嗪甲醚、去氢毛钩藤碱、毛钩藤碱;30批不同市售来源钩藤的相似度在0.850～0.995之间。经正交偏最小二乘判别分析筛选出了导致不同批次药材质量差异的5个共有峰。本研究建立的UHPLC指纹图谱结合化学模式识别的方法简单、可靠,可为钩藤的质量评价提供参考。     

吉林12种蒲公英HPLC指纹图谱研究

通过比较吉林产不同种蒲公英HPLC指纹图谱,探讨吉林产蒲公英HPLC指纹图谱的特点,为蒲公英药材的质量控制提供理论参考。采用HPLC方法,以Agilent Extend-C18(250 mm×4.6 mm,5μm)为色谱柱,以甲醇-0.5%冰醋酸水溶液梯度洗脱;检测波长为323 nm,流速为1.000 mL·min-1,柱温35℃,进样量为20μL,检测了12种吉林产不同种类蒲公英药材,确立了9个共有峰,建立了蒲公英对照指纹图谱,计算各被测样品的HPLC指纹图谱的整体相似度,并指认了绿原酸、咖啡酸、总黄酮3个特征峰,比较了上述成分在不同药材中的含量,定量结果表明,3种成分平均含量分别为0.027%、0.026%、0.128%。所建立的指纹图谱具有良好的精密度、重现性和稳定性,可作为吉林产蒲公英药材的质量控制标准。     

金线莲HPLC指纹图谱研究

以14批不同来源金线莲药材为原料,采用高效液相色谱法(HPLC),乙腈-甲酸水溶液为流动相,梯度洗脱,建立栽培及野生金线莲(Anoectochilus roxburgii)的指纹图谱,结合相似度分析,探讨金线莲与银线莲(Goodyera hachijoensis)的差异。结果表明,金线莲HPLC指纹图谱标定了24个共有峰,并指认了金线莲苷和葫芦巴碱、鼠李素-3-O-芸香糖苷、槲皮素、山柰酚、异鼠李素、Batatasin-Ⅲ等6个共有峰。除3批药材外,其余11批相似度均高于0.910,相似度总体较好。银线莲图谱相似度仅达0.529、0.721、0.698,均低于14批金线莲样品间的相似度。根据相似度评价,认为金线莲与银线莲存在差异,该结果可为分析评价金线莲与银线莲差异提供参考。     

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.     

广西铁皮石斛HPLC指纹图谱研究

采用HPLC法建立广西铁皮石斛提取液的指纹图谱,运用相似度评价药材质量。结果表明,在13个共有峰构成的铁皮石斛指纹图谱中,有11批药材相似度均在0.8以上,2批的相似度较差,未达到0.8,表明相似度大小与药材的产地有关。通过对栽培铁皮石斛不同部位药材的指纹图谱峰面积的比较,不提倡对药材提早采收。3批组织培养铁皮石斛的图谱相似度较低,说明培养到不同阶段的铁皮石斛材料组织培养物,其化学成分相差较大。     

酢浆草的UPLC指纹图谱研究

酢浆草(Oxalis corniculata)为酢浆草科植物,资源分布广泛,全草均可入药。该研究为建立酢浆草超高效液相色谱指纹图谱,采用乙腈-0.05%醋酸水溶液梯度洗脱,以Agilent ZORBAX RRHD Eclipse Plus C18(2.1 mm×100 mm,1.8μm)色谱柱,流速0.3 mL·min~(-1),进样量为10μL,检测波长280 nm,柱温25℃等色谱条件,研究了15个不同产地30批酢浆草药材的指纹图谱。结果表明:确立了24个共有峰,相似度评价软件计算30批样品的相似度为0.839~0.987;应用聚类分析对各样品指纹图谱的共有峰峰面积统计分析得出,除贵州剑河、贵州印江及贵州天柱8月3批样品外,其余27批样品共有化学成分的含量较为接近。该研究结果表明建立的酢浆草UPLC指纹图谱方法简便、迅速、可靠,较其HPLC指纹图谱有更高的灵敏度和分辨率,极大地缩短了检测时间,可用于酢浆草质量评价的快速分析。     

太子参商品药材及其四倍体植株块根的高效液相指纹图谱

对不同产地太子参〔Pseudostellaria heterophylla(Miq.)Pax〕商品药材及其四倍体植株的块根进行了高效液相指纹图谱分析。结果表明,10个批次的不同产地太子参商品药材与经选育获得的6个株系太子参同源四倍体植株块根的HPLC-UV指纹图谱相似度较高,均在0.9以上。选取15个特征峰并大致判断其峰位和比例关系,构成太子参特有的HPLC色谱指纹图谱,为太子参药材鉴别、品质评价及优良品种的选育提供可靠依据。     

Fingerprint analysis of Psoralea corylifolia L. by HPLC and LC-MS

High-performance liquid chromatography (HPLC) was developed for fingerprint analysis of Psoralea corylifolia. Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MSn) technique was first employed to identify the components of the fingerprint. The samples were separated with an Alltima C18 column (250 mm x 4.6 mm, 5 microm) by linear gradient elution using water-acetic acid (A; 100:0.1, v/v) and acetonitrile (B; 0 min, 40%; 15 min, 50%; 35 min, 60%; 45 min, 70%; 55 min, 80%; and maintained for 5 min) as mobile phase at a flow rate of 1.0 ml/min and detector wavelength at 245 nm. A standard procedure was developed for HPLC fingerprint analysis. Average chromatogram of 10 batches of P. corylifolia L. from Sichuan and Henan Provinces, PR China, which has been considered as the original and genuine herbal medicine for a long time, was first established as the characteristic fingerprint. There are 12 common peaks in this fingerprint. Ten of these common peaks were identified by MS data. This profile was then used to identify and assess the differences among the herb grown in various areas of China. The HPLC fingerprint analysis is specific and may serve for quality identification and comprehensive evaluation of P. corylifolia.     

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.     

A multi-strategy platform for quality control and Q-markers screen of Chaiqin chengqi decoction

BackgroundAcute pancreatitis (AP) is an inflammatory disorder of the pancreas that is associated with substantial morbidity and mortality. Chaiqin chengqi decoction (CQCQD) has been proven clinically to be an effective treatment for AP for decades in West China Hospital. Quality control for CQCQD containing many hundreds of characteristic phytochemicals poses a challenge for developing robust quality assessment metrics.PurposeTo evaluate quality consistency of CQCQD with a multi-strategy based analytical method, identify potential quality-markers (Q-markers) based on drug properties and effect characteristics, and endeavor to establish CQCQD as a globally-accepted medicine.MethodsA typical analysis of constitutive medicinal plant materials was performed following the Chinese Pharmacopoeia. The extraction process was optimized through an orthogonal array (L₉(3⁴)) to evaluate three levels of liquid to solid ratio, soaking time, duration of extraction, and the number of extractions. An ultra-high-performance liquid chromatography (UHPLC) fingerprinting combined with absolute quantitation of multi chemical marker compounds, coupled with similarity, hierarchical clustering analysis (HCA), and principal component analyses (PCA) were performed to evaluate 10 batches of CQCQD. On the basis of systematic analysis of fundamental features of CQCQD in treating AP, the potential Q-marker screen was proposed through detection of quality transfer and efficacy for chemical markers. UHPLC coupled with quadrupole orbitrap mass spectrometry were used to determine compounds in medicinal materials, decoctions and plasma. Network pharmacology and taurolithocholic acid 3-sulfate induced pancreatic acinar cell death were used to evaluate the correlation between chemical markers and anti-pancreatitis activity. A cerulein induced AP murine model was used to validate quality assessed CQCQD batches at clinically-equivalent dose. The effective content of chemical markers was predicted using linear regression analysis on quantitative information between validated batches and the other batches.ResultsThe chemical markers and other physical and chemical indices in the original materials met Chinese Pharmacopoeia standards. A total of 22 co-existing fingerprint peaks were selected and the similarity varied between 0.946 and 0.990. Batch D10 possessed the highest similarity index. HCA classified the 10 batches into 2 main groups: 7 batches represented by D10 and 3 batches represented by D1. During the initial Q-marker screen stage, 22 compounds were detected in both plant materials and decoctions, while 13 compounds were identified in plasma. Network pharmacology predicted the potential targets and pathway of AP related to the 22 compounds. All 10 batches showed reduced necrosis below 60% with the best effect achieved by D10 (~40%). The spectrum-efficacy relationship analyzed by Pearson correlation analysis indicated that emodin, rhein, aloe emodin, geniposide, hesperridin, chrysin, syringin, synephrine, geniposidic acid, magnolol, physcion, sinensetin, and baicalein showed positive correlation with pancreatic acinar cell death protection. Similar to the in vitro evaluation, batch D10 significantly reduced total histopathological scores and biochemical severity indices at a clinically-equivalent dose but batch D1 did not. The content of naringin, narirutin and baicalin in batches D1, D5 and D9 consistently exceeds the upper limit of the predicted value. Eight markers whose lower limit is predicted to be close to 0 contributed less to the material basis for AP protection.ConclusionDespite qualified materials used for CQCQD preparation, the clinical effect depends on appropriate content range of Q-markers. Emodin, rhein, aloe emodin, magnolol, hesperidin, synephrine, baicalein, and geniposide are considered as vital Q-markers in the primary screen. This study proposed a feasible platform for producing highly consistent batches of CQCQD in future study.