Modernization of Chinese herbal compound and the high performance liquid chromatography tandem mass spectrometry (HPLC-MS)

Chinese herbal compound is playing an important role on curing human diseases.And it has been a trend that Chinese herbal compound is being used all over the world in 21 century.However,our Chinese herbal compound is facing serious challenge for the lack of canonical system of quality criterion for Chinese herbal compound so it has been a urgent problem to set up the quality control standards and reveal therapeutic basis of Chinese herbal compound.In order to give full play to the advantages of Chinese herbal compound,modern scientific and technological is used to research of Chinese herbal compound,especially the high performance liquid chromatography tandem mass spectrometry(HPLC-MS),because it is high sensitive,rapid,and obtain more information.It is very necessary that HPLC-MS is uesed to elucidate the effective components of basic substances of Chinese Herbal Compound,and endow traditional Chinese medicine with modern scientific connotation.

HPLC及LC-MS测定榛子中氯吡苯脲和多菌灵残留

本文采用高效液相色谱法(HPLC)和液相色谱-质谱法(LC-MS)对干果榛子中氯吡苯脲和多菌灵残留量进行含量测定.HPLC和LC-MS均使用C18色谱柱,含量测定采取等度洗脱,流动相:甲醇-水(55∶45,体积比);体积流量:1.0 mL·min-1;测定波长:260 nm;柱温:30℃.LC-MS采取电喷雾离子源,梯度洗脱,体积流量:0.6 mL·min-1,柱温:30℃.结果表明,榛子中氯吡苯脲与多菌灵存在残留,氯吡苯脲质量浓度在1.00~10.00μg·mL-1范围内线性关系良好,加样回收率在95.58%~100.58%;多菌灵质量浓度在1.005~15.075μg·mL-1范围内具有良好的线性关系,加样回收率在95.61%~104.39%.实验证明,HPLC与LC-MS相结合的方法具有操作简便、灵敏度高、检出限低等优点,能有效地检测到榛子样品中膨大剂氯吡苯脲及杀菌剂多菌灵的残留,并确定其残留量,线性关系和回收率结果均令人满意.根据被检测的8批样品中氯吡苯脲和多菌灵两项农药残留量推断,作为一般干果食用榛子是安全的.

HPLC法测定十三味逐瘀合剂中游离大黄酚含量

目的建立十三味逐瘀合剂中大黄酚高效液相含量测定的方法。方法采用高效液相色谱法(HPLC法)对十三味逐瘀合剂中游离大黄酚含量进行测定,从提取溶剂、取样量、提取时间等影响因素中选出最优条件,建立完整的含量测定方法。结果用thermos C18色谱柱(250 mm×4.6 mm,5μm);以甲醇-0.1%磷酸(75∶25,V/V)为流动相,采用等度洗脱法,254 nm波长的色谱条件;以甲醇为浸膏提取溶剂,超声30 min的提取方法;大黄酚质量浓度在1.63~16.32μg/mL(R^(2)=0.9995)范围内具有良好的线性关系;平均回收率为104.17%;测得十三味逐瘀合剂中游离大黄酚平均含量为15.32μg/mL。结论本实验所建立的方法操作简单,专属性强,重现性好,可用于十三味逐瘀合剂中游离大黄酚的含量测定。

HPLC-ELSD同时测定腺梗豨莶草中6个二萜成分的含量

为了研究腺梗豨莶草中6个二萜成分(对映海松烯型二萜:奇任醇、Hythiemoside B和豨莶精醇;对映贝壳杉烷型二萜:对映-17,18-二羟基-贝壳杉烷-19-羧酸、对映-16β,17-二羟基-贝壳杉烷-19-羧酸和对映-16α氢-贝壳杉烷-17,19-二羧酸)的含量,本试验建立了高效液相色谱法-蒸发光散射检测器(HPLC-ELSD)同时测定二萜类化合物含量。样品粉碎过筛加甲醇和乙酸乙酯回流提取,蒸发减压除去溶剂,甲醇溶解,0.45μm滤膜滤过,取续滤液进行测定。色谱条件:色谱柱为Waters Symmetry Shield^(TM)RP18柱(250 mm×4.6 mm,5μm),流动相为0.3%甲酸水溶液-乙腈(v/v),梯度洗脱,流速为1.0 mL/min。蒸发光散射检测器漂移管温度为103℃,雾化气流速为3.0 L/min。应用该方法测定腺梗豨莶草样品不同部位中6个二萜类化合物的含量,同时比较叶、枝、茎中对映海松烯型二萜、对映贝壳杉型二萜和总二萜含量的差异。结果显示,6个二萜成分在其线性范围内线性关系良好(r≥0.9992);日内和日间精密度相对标准偏差(RSD)均小于3.5%;回收率介于96.5%~101.5%,RSD均小于2.3%;腺梗豨莶草不同部位(叶、枝、茎)的二萜类化合物含量差异较大。结果表明,本试验所建立的HPLC-ELSD方法简便、准确、重复性好,为腺梗豨莶草药材全面的质量评价和临床应用中最佳药用部位的选择提供了参考。

Determination of Organic Acids in Root Exudates by High Performance Liquid Chromatography:Ⅱ.Influence of Several Testing Conditions

Effects of column temperature and flow rate on separation of organic acids were studied by determining nine low-molecular-weight organic acids on reversed- phase C18 column, using high performance liquid chromatography (HPLC) with a wavelength of UV (ultraviolet) 214 urn and a mobile phase of 18 mmol L-1 KH2PO4 buffer solution (pH 2.1). The thermal stability of organic acids was determined by comparing the recoveries of organic acids in different temperature treatments. The relationships between column temperature, flow rate or solvent pH and retention time were analyzed. At low solvent pH, separation efficiency of organic acids was increased by raising the flow rate of the solvent because of lowering the retention time of organic acids. High column temperature was unfavorable for the separation of organic acids. The separating effect can be enhanced through reducing column temperature in organic acid determination due to increasing retention time. High thermal stability of organic acids with low concentrations was observed at temperature of 40 ℃-45℃. Sensitivity and separation effect of organic acid determination by HPLC were clearly improved by a combination of raising flow rate and lowering column temperature at low solvent pH.

Determination of Trace Amount of Polycyclic Aromatic Hydrocarbons in Urban Sewage by Solid-phase Extraction Coupled with High Performance Liquid Chromatograph

[Method] This study aimed to determine trace amount of polycyclic aromatic hydrocarbons(PAHs) in urban sewage by using solid-phase extraction(SPE) coupled with high performance liquid chromatograph(HPLC).[Method] From the aspects of solid-phase extraction column,elution solvent,elution volume,elution speed and so forth,the test conditions of SPE-HPLC method were optimized,and trace amount of PAHs in urban sewage was determined.[Result] The optimized solid-phase extraction conditions were SUPELCLEAN LC-18 solid-phase extraction column,methylene dichloride as elution solvent,15 ml elution volume,2 ml/min elution speed,5 ml/min loading speed,1 000 ml water with 200 ml methanol loading volume.Under the optimized extraction conditions,the recovery was high,namely 76.3%-105.2%;relative standard deviation was 3.8%-6.0%,showing good precision;detection limit was low,only 0.000 8-0.048 0 μg/L.[Conclusion] This method is user-friendly,with high sensitivity and good precision,and suitable for continuous determination of a large volume of water samples.

On-line enrichment and determination of polycyclic aromatic hydrocarbons in atmospheric particulates using high performance liquid chromatography with fluorescence as detector

Seven polycyclic aromatic hydrocarbons （PAHs） in atmospheric particulates were determinated by high performance liquid chromatography （HPLC） with fluorescence detector using direction injection and an on-line enrichment trap column. The method simplified the sample pretreatment, saved time and increased the efficiency. With the on-line trap column, PAHs were separated availably even underground injecting 1.0 ml sample with relatively high column efficiency. The recoveries of the seven PAHs were from 85% to 120% for spiked atmospheric particulate sample. The limit of detection was 15.3-39.6 ng/L （S/N=3.3）. There were good linear correlations between the peak areas and concentrations of the seven kinds of PAHs in the range of 1-50 ng/ml with the correlation coefficients over 0.9970. Furthermore, it also indicated that the method is available to determine PAHs in atmospheric particulates well.

高效液相-蒸发光散射检测器(HPLC-ELSD)测定红参中的精氨酸单糖苷(AF)及精氨酸双糖苷(AFG)含量

目的建立一种同时测定人参加工品中精氨酸单糖苷(AF)和精氨酸双糖苷(AFG)含量的方法。[方法]用PrevailTMC18column(4.6 mm×250 mm,5μm)色谱柱进行检测。流动相A:色谱乙腈,流动相B:5.0mmol/L七氟丁酸的0.7%三氟醋酸溶液。色谱条件为[0%A(0 min);0%A(5 min);15%A(8 min);35%A(25 min)];流速0.8 mL/min;漂移管温度为115℃;气体流量3.2 L/min。[结果]AF和AFG的检测限分别为0.015和0.010 mg/mL;线性关系相关系数分别为,AF:0.9997,AFG:0.9999,表明线性关系良好。AF和AFG检测的精密度,重复性,稳定性以及回收率的相对标准偏差分别为0.43%&0.37%,0.43%&0.55%,0.43%&0.49%,0.45%&0.15%。AF和AFG检测的回收率分别为99.5%&100%,表明方法稳定。经检测,高丽红参,中国红参和生晒参中AF含量分别为0.74%,0.91%和1.14%,AFG含量分别为6.69%,5.12%和0.85%。[结论]这种方法成功用于高丽红参,中国红参及生晒参中AF及AFG的检测;且红参中AF和AFG含量明显高于生晒参。该方法测定结果可靠,缩短了测定时间,较少杂质干扰。但因为本研究考察氨基酸种类较少,当衍生物种类较多时,仍需进一步考察其分离度。

HPLC法测定五味子颗粒中7种木脂素成分的含量

建立了同时测定五味子颗粒中7种木脂素成分含量的高效液相色谱(HPLC)法。采用Poroshell 120 SB-C18色谱柱(150 mm×4.6 mm,2.7μm)进行分离,四氢呋喃水-乙腈梯度洗脱,流速为0.6 mL/min,柱温为30℃,检测波长为220 nm,进样量为10μL。结果表明:该方法对五味子醇甲、五味子醇乙、五味子酯甲、五味子酯乙、五味子甲素、五味子乙素和五味子丙素的检测下限(LOD)分别为0.017、0.041、0.156、0.165、0.136、0.166和0.113μg/mL,线性关系良好,相关系数(r)均大于0.9999,平均加样回收率为98.1%~100.9%,相对标准偏差(RSD)均小于3.0%。因此,该方法重复性好、灵敏度高,所得含量关系可对五味子颗粒进行质量控制,特别是可为预防南五味子掺伪提供参考,也为其他含五味子制剂的质量标准修订奠定了基础。

HPLC-DAD测定饲用酸化剂苯甲酸中7种邻苯二甲酸类杂质

为了建立高效液相色谱法测定饲用酸化剂苯甲酸中邻苯二甲酸、2-羟基苯甲酸、3-羟基苯甲酸、4-羟基苯甲酸、2-甲基苯甲酸、3-甲基苯甲酸和4-甲基苯甲酸共7种邻苯二甲酸类杂质的方法。本试验将酸化剂样品溶于甲醇和初始流动相的混合溶液中,采用Agilent ZORBAX SB-C18色谱柱(4.6 mm×250 mm,5.0μm)分离7种邻苯二甲酸类杂质,以水-乙腈-甲基磺酸(85-15-0.1)和乙腈为流动相进行梯度洗脱,流速为1.2 mL/min,采用二极管阵列检测器(DAD),采集波长范围为210~400 nm,记录扫描光谱图和235 nm波长处的色谱图。用外标法定量邻苯二甲酸类杂质的含量。结果显示,7种邻苯二甲酸类杂质的浓度在0.10~40.00μg/mL范围内的线性良好,平均加标回收率在98.5%~102.7%范围内,相对标准偏差(RSD,n=5)介于0.3%~1.5%,检测限和定量限均为4 mg/kg。本试验建立的酸化剂苯甲酸中7种邻苯二甲酸类杂质的高效液相色谱-二极管阵列检测器(HPLC-DAD)检测方法快速且准确,可用于苯甲酸中7种邻苯二甲酸类杂质的定性和定量检测。

Quantification of six bioactive compounds in Zhenqi Fuzheng preparation by high-performance liquid chromatography coupled with diode array detector and evaporative light scattering detector

A simple and accurate high-performance liquid chromatography（HPLC）coupled with diode array detector（DAD）and evaporative light scattering detector（ELSD）was established for the determination of six bioactive compounds in Zhenqi Fuzheng preparation（ZFP）.The monitoring wavelengths were 254,275 and 328 nm.Under the optimum conditions,good separation was achieved,and the assay was fully validated in respect of precision,repeatability and accuracy.The proposed method was successfully applied to quantify the six ingredients in 31 batches of ZFP samples and evaluate the variation by hierarchical cluster analysis（HCA）,which demonstrated significant variations on the content of these compounds in the samples from different manufacturers with different preparation procedures.The developed HPLC method can be used as a valid analytical method to evaluate the intrinsic quality of this preparation.

Biological Fingerprinting Analysis of Interaction Between Taxoids in Taxus and Microtubule Protein by Microdialysis Coupled with High-performance Liquid Chromatography/Mass Spectrometry for Screening Antimicrotubule Agents

Some natural products, such as traditional Chinese medicines（TCMs）, contain compounds with anticancer activity and have attracted a great interest in recent years as alternative anticancer therapies. A quick and convenient assay for screening antimicrotubule compounds in which in vitro microdialysis/high-performance liquid chromatography （HPLC） is used to monitor the binding of the compounds extracted from TCM Taxus cuspidata Siebold ＆ Zucc（Taxus） to microtubules is reported. It was observed that the extract of Taxus contains at least five compounds which have affinity interaction with microtubules by biological fingerprinting analysis, and they were identified as the taxoids of taxol, baccatin III, 10-deacetylbaccatin Ⅲ（10-DAB）, cephalomannine and 7-epi-10-deacetyltaxol （7-epi-10-DAT） based on the comparison of their high-performance liquid chromatographic/mass spectrometric and UV spectra with those of the standard samples, both assembly-promoting and disassembly-inhibiting characteristics of those compounds were evaluated. It was observed that baccatin Ⅲ and 10-DAB bound to microtubules and the binding degrees were influenced by GTP. Competitive binding behavior of taxol with other four taxoids to microtubules was also investigated.

A Sensitive Reversed-Phase High-Performance Liquid Chromatography Method for the Quantitative Determination of Milk Xanthine Oxidase Activity

A new reversed-phase high performance liquid chromatography method was developed to quantitate the activity of xanthine oxidase involved in milk fat globule membrane with xanthine as the substrate and the separation of product (uric acid). The increment of uric acid in the reaction system was used to calculate the total activity of XO. The optimized assay conditions, linearity of detection, recovery of uric acid and chromatogram were developed in text, indicating this method is simple, rapid and efficient. It is an alternative potential method for the determination of the activity of XO in milk.

Study on the Flow Injection Micro-Column Pre-Separation System Coupled With High Performance Liquid Chromatography for the Determination of Ecdysterone in Traditional Chinese Medicine

A flow injection (FI) micro-column system coupled with high performance liquid chromatography (HPLC) was proposed for the pre-separation and determination of active organic component (ecdysterone) in traditional Chinese medicine, Loulu. The factors influencing separation performance were investigated and optimized. Under the optimal conditions, the contents of ecdysterone in Loulu were determined by HPLC system using MeOH-H_2O (40: 60,V/V) as the mobile phase at a flow rate of 1. 0 mL/min. The calibration curve was linear in the range of 0. 5~ 100 mg/L of ecdysterone concentrations. The detection limit of the analyte was 0. 11mol/L(3) with a precision of 0. 38% RSD (n=7 f c= 10. 0 mg/L). The average recovery of the method was 98. 7%. The proposed method has been applied to determine ecdysterone in practical samples, and the determined values by both external standard method and standard addition method were in good agreement. Compared to the traditional solid extraction method, the system proposed has the advantages of simple procedure, good reproducibility, minimum volume requirement, reduction of matrix interference and low contamination risk.

HPLC测定化妆品中二氢燕麦生物碱D的含量

采用高效液相色谱法(HPLC)测定化妆品中二氢燕麦生物碱D的含量。色谱柱:Agilent Zorbax SB-Aq(250 mm×4.6 mm×5µm);流动相为V磷酸(体积分数0.05%)∶V乙腈=67∶33;流速:1.0 mL/min;柱温:30℃;检测波长:220 nm。结果表明,二氢燕麦生物碱D在0.0114~0.2850μg与其峰面积呈良好线性关系(R=0.9999);液态类样品平均回收率为99.01%,相对标准偏差(RSD)为1.96%(n=9);膏霜样品的平均回收率为102.68%,RSD为2.46%(n=9);乳液样品的平均回收率为99.96%,RSD为1.4%(n=9)。该法简便、准确可行,可用于化妆品中二氢燕麦生物碱D的含量测定及质量控制。

基于HPLC测定4种藏茵陈原植物中10个有效成分含量

目的:建立高效液相色谱法(HPLC)测定藏茵陈中獐牙菜苦苷、芒果苷、龙胆苦苷、当药苷、异荭草苷、异牡荆苷、当药黄素、当药醇苷、甲基当药宁、齐墩果酸含量的方法。方法:采用Welch Ultimate XB-C18色谱柱,以乙腈-0.1%磷酸水溶液为流动相,梯度洗脱,体积流量为1.0 mL·min–1,獐牙菜苦苷、芒果苷、龙胆苦苷、当药苷测定波长为240 nm,异荭草苷、异牡荆苷、当药黄素测定波长为270 nm,当药醇苷、甲基当药宁测定波长为250 nm,齐墩果酸检测波长为205 nm,进样量为10μL。结果:该条件下指标成分进样质量分别为獐牙菜苦苷126.7~16040.0 ng(r=0.9998)、芒果苷6.3~801.6 ng(r=0.9996)、龙胆苦苷6.4~814.4 ng(r=0.9997)、当药苷15.9~2016.0 ng(r=0.9999)、异荭草苷7.1~900.0 ng(r=0.9995)、异牡荆苷5.5~699.2 ng(r=0.9997)、当药黄素5.4~681.6 ng(r=0.9998)、当药醇苷5.4~685.6 ng(r=0.9996)、甲基当药宁19.3~2438.0 ng(r=0.9997)、齐墩果酸13.6~1716.0 ng(r=0.9998)时与峰面积具有较好的线性关系;方法学考察结果显示精密度(RSD≤0.95%)和重复性(RSD≤1.86%)良好,供试品溶液在室温条件下24 h内稳定,RSD≤1.85%;平均加样回收率和相应的RSD分别为99.56%(0.58%)、100.37%(1.23%)、98.63%(0.59%)、99.11%(1.02%)、98.64%(0.57%)、102.26%(0.88%)、100.37%(0.59%)、97.05%(1.36%)、96.01%(0.45%)、101.55%(0.49%)。18批藏茵陈原植物中獐牙菜苦苷、芒果苷、龙胆苦苷、当药苷、异荭草苷、异牡荆苷、当药黄素、当药醇苷、甲基当药宁、齐墩果酸的质量分数差异较大,分别为0.111%~6.592%、0.195%~1.959%、0.046%~4.139%、0.058%~0.662%、0.090%~1.421%、0.082%~0.283%、0.061%~0.384%、0.022%~1.589%、0.106%~1.858%、0.160%~0.657%。结论:建立的HPLC同时测定藏茵陈中10个有效成分含量,方法简便、快速、准确,可为藏茵陈质量控制及资源有效开发提供参考。

维生素C片含量测定的HPLC法及溶出度方法的研究

目的建立测定维生素C片含量的高效液相色谱(high performance liquid chromatography,HPLC)法,并通过新建的维生素C片溶出度的测定方法提高其质量控制标准。方法HPLC测定法:色谱柱为菲罗门C_(18)柱(250 mm×4.6 mm,5μm),流动相为0.1 mol·L^(-1)磷酸二氢钾溶液(用磷酸调节pH值至2.5)-甲醇(92∶8),流速为0.8 mL·min^(−1),检测波长为243 nm,柱温为30℃,进样量为10μL;溶出度测定法:按照溶出度与释放度测定法(《中华人民共和国药典》2020年版四部附录0931第一法),以0.1 mol·L^(-1)盐酸溶液1000 mL为溶剂,转速为50 r·min^(−1),20 min时取样。结果维生素C在20~140μg·m L^(-1)范围内与其峰面积线性关系良好,y=37487.239x−6860.622(r=0.99996),平均回收率为100.16%,RSD值为0.48%(n=9);其在水、0.1 mol·L^(-1)盐酸溶液、pH 4.0醋酸盐缓冲液、pH 6.8磷酸盐缓冲液各1000 mL的溶出介质中的溶出曲线差异明显,选择0.1 mol·L^(-1)盐酸溶液为溶出介质对5个企业的样品进行测定,均在10 min全部溶出并进入平台。结论所建立的方法使检测的准确性更高、专属性更强、重复性更好,具有可信、精准的优势,能更有效地对维生素C片进行质量控制。

HPLC法测定云芝菌发酵茶中9种活性成分及抗氧化活性研究

目的:建立同时测定云芝菌发酵茶中9种活性成分含量的高效液相色谱法,并对发酵前后茶叶中9种活性成分含量和抗氧化活性的变化进行研究。方法:采用Agilent C_(18)色谱柱(4.6 mm×250 mm,5μm),流动相为0.05%磷酸溶液和乙腈,梯度洗脱,检测波长为280 nm、流速1.0 mL/min、柱温30℃。结果:与未发酵的对照组绿茶相比,云芝菌发酵茶中咖啡碱的含量无明显变化,没食子酸、可可碱和没食子儿茶素没食子酸酯的含量分别增加了0.4613%、0.3118%和0.5770%,没食子儿茶素、茶碱、表没食子儿茶素、表儿茶素和表没食子儿茶素没食子酸酯含量分别减少了0.0156%、0.0178%、1.2938%、1.1062%和4.0241%。体外抗氧化活性试验结果表明,发酵后茶叶对DPPH·和·OH的清除率均有显著性(P<0.05)降低。当浓度为2.0 mg/mL时,云芝菌发酵茶提取液对DPPH·和·OH的清除率分别为7.79%和9.65%。结论:云芝菌的固体发酵可改变茶叶中活性成分的含量和抗氧化活性,研究为云芝菌发酵茶的开发提供了实验依据。

Determination of Naringin Content in Rhizoma Drynariae by High Performance Liquid Chromatography

[Objectives]To accurately determine the naringin content in Rhizoma Drynariae.[Methods]The high performance liquid chromatography(HPLC)method was applied in the determination of the naringin content in Rhizoma Drynariae.The sample was sonicated at room temperature.The mobile phase was 0.1%phosphoric acid-acetonitrile(75∶25),detected by diode array detector at the wavelength of 284 nm,and quantified by external standard method.[Results]The linearity of naringin was good in the concentration range of 5-500μg/mL with a correlation coefficient of 0.9999.[Conclusions]This method has good linearity,easy operation,correctness and reproducibility as required,and is expected to provide a method for the determination of naringin content in Rhizoma Drynariae.

高效液相色谱-质谱法(HPLC-MS)测定化妆品中达克罗宁的含量

建立了一种高效液相色谱-质谱法(HPLC-MS)测定化妆品中达克罗宁含量的方法。样品经乙腈提取15 min后,用体积分数20%乙腈溶液稀释,经Capcell PAK C_(18)色谱柱(100 mm×2.1 mm×2μm)分离后,多反应监测模式进行检测。结果表明:达克罗宁在1.0~25.0 ng/mL线性关系良好,相关系数(r)大于0.995;加标回收率在89.4%~104.5%。该方法高效、准确,可用于化妆品中达克罗宁的定性和定量检测。