高效毛细管电泳及液质联用技术在中药质量控制过程中的应用

【作者】 李文龙；

【导师】 王小如；

【作者基本信息】 国家海洋局第一海洋研究所 ， 海洋化学， 2007， 硕士

【摘要】 高效毛细管电泳(HPCE)技术是一种与高效液相色谱(HPLC)分离机理不同的分离技术,该技术具有分离效率高,实验消耗较少,柱系统易清洗等优点,在复杂样品的分离分析上可作为液相色谱的一种有力补充。中药材成分复杂,分离分析难度较高,本文探索性地采用高效毛细管电泳技术,对太子参、金银花、龙胆和莲子心四种药材中的有效成分进行分析,为利用该技术对中药材进行质控的可行性提供了相关理论和应用基础。本文还采用高效液相色谱-电喷雾飞行时间质谱(HPLC-ESI-TOF/MS)联用技术对龙胆和莲子心有效成分进行了分析,鉴定出了其中的多种化合物。现将本文主要研究结果归纳如下:1.建立了测定太子参中环肽Pseudostellarin B含量的HPCE分析方法,并对10种不同产地太子参中环肽Pseudostellarin B的含量进行了测定。在此基础上进行了太子参HPCE指纹图谱研究,采用中药指纹图谱相似度计算软件对不同太子参样品的HPCE指纹图谱进行相似度计算。2.建立了测定金银花和山银花药材中绿原酸、槲皮素和芦丁含量的高效毛细管电泳(HPCE)分析方法,并对其进行指纹图谱研究。采用加速溶剂萃取法(ASE)对药材进行提取,在选定的HPCE工作条件对金银花药材进行了含量测定。并在此基础上进行了金银花HPCE指纹图谱研究,采用中药指纹图谱相似度计算软件对HPCE指纹图谱进行相似度计算,以系统生成的对照指纹图谱为对照模板,同一产地金银花药材指纹图谱相似度均在0.93以上,不同产地金银花药材指纹图谱相似度在0.77-0.98之间不等,且可有效区分金银花和山银花药材。3.建立利用大孔吸附树脂对龙胆药材中龙胆苦苷和马钱子甙酸进行富集和分离纯化的方法,对D301,AB-8,D101,XDA-1四种大孔树脂对龙胆苦苷和马钱子甙酸的吸附性能进行了比较,最终确定采用D301型大孔树脂对二者进行富集、纯化。全程采用HPLC进行龙胆苦苷和马钱子甙酸浓度监测,对该过程中的工艺参数进行优化,龙胆苦苷和马钱子甙酸分别富集在8%和55%的乙醇洗脱液中,洗脱液浓缩后冷冻干燥,可得到纯度分别为74.3%和80.9%的粗产物。4.建立了胶束电动毛细管色谱(MEKC)和微乳液毛细管电动色谱法(MEEKC)分析龙胆药材中龙胆苦苷和马钱子甙酸含量的方法。分别考察了两种分离模式下电泳介质的构成和电泳过程中的各操作参数对样品分离过程的影响,优化了MEKC和MEEKC的分析条件,在各自对应的缓冲液体系和选定的工作条件下,龙胆苦苷和马钱子甙酸与其他组分达到了基线分离,对六处不同产地的龙胆药材进行了分析,并对测定结果进行了t检验,结果表明,两种模式下,测定结果之间不存在显著性差异,而不同产地的龙胆药材的龙胆苦苷和马钱子甙酸含量之间存在较大差异。5.建立了采用MEKC,MEEKC及非水毛细管电泳(NACE)三种模式对莲子心中的的莲心碱,异莲心碱和甲基莲心碱进行定量分析的方法。采用正交实验设计方法优化了分析条件。结果表明,三种模式均可用于莲子心药材中生物碱含量的测定及其质量控制,将其用于实际的提取物分析工作中,方法快捷,结果可靠。6.建立HPLC-ESI-TOF/MS联用技术用于中药材龙胆有效成分含量测定及其指纹图谱研究的方法。龙胆药材ASE粗提物通过氯仿萃取分为水溶相和氯仿相,采用HPLC-DAD对龙胆苦苷和马钱子甙酸进行定量测定,并在此基础上建立龙胆药材的HPLC二元特征指纹图谱,采用HPLC-ESI-TOF/MS对指纹图谱中的特征峰进行定性。结合文献,通过各色谱峰紫外光谱和质谱数据,鉴别出了水溶相中的马钱子甙酸,阿魏酸,和龙胆苦苷三种化合物,初步判断氯仿相中的主要组分为三萜类化合物。采用中药指纹图谱相似度计算软件对不同龙胆样品的指纹图谱进行相似度计算,同批次药材指纹图谱相似度均在0.98以上,表明本文所建立的方法重现性好;6处不同产地药材水相指纹图谱相似度均在0.90以上,而氯仿相指纹图谱相似度在0.62-0.99之间不等,说明不同产地龙胆药材的质量差异主要体现在三萜类化合物上。本方法可用于龙胆药材有效成分的含量测定及质量控制。7.发展了HPLC-DAD-ESI-TOF/MS分析莲子心中生物碱类化合物的方法,在此基础上,进行了莲子心药材的指纹图谱研究。采用加速溶剂萃取法(ASE)对莲子心进行提取,采用HPLC-DAD-ESI-TOF/MS联用技术对其中的化学成分进行分离鉴定,鉴定了其中的六种生物碱分别为:前荷叶碱,莲心季铵碱,莲心碱,异莲心碱,荷叶碱和甲基莲心碱。利用指纹图谱相似度计算软件对所得指纹图谱进行分析,产自安徽的12批莲子心药材指纹图谱相似度均在0.98以上,证明本方法重现性较高;六处不同产地的莲子心药材指纹图谱相似度在0.76-0.99之间不等,表明不同产地莲子心药材之间存在着质量差异。本文所建立的方法重现性好,指纹图谱主要特征峰成分明确,可用于莲子心药材的质量控制。以上分析方法的建立和应用,表明高效毛细管电泳技术可应用于中药材活性成分的分离分析,液质联技术是进行中药材中化合物定性分析的有力工具,可利用二者对中药材进行有效成分的含量测定和指纹图谱研究,从而实现中药材的质量控制。更多还原

【Abstract】 High performance capillary electropheresis (HPCE) is an analytical technique whose separation mechanism is distinctively different from high performance liquid chromatography (HPLC). The characteristics of HPCE include high separate efficiency, low solvent consumption and easily cleanable column system. It can be used as a useful supplement to HPLC. The Traditional Chinese medicines (TCM) has complicated composition, and are therefore difficult to analysis. This research explored the potential application of HPCE in TCM analysis using four important Chinese herbs as the target species. HPCE was used as a main instrument in order to supply related theory and application groundwork for the quality control of TCM. In this text, high performance liquid chromatography-electrospray-time-of-flight mass spectrometry has also been used to analyze active components in Radix Gentianae and Semen Nelumbinis. The results can be summarized as follows.1. A modern HPCE analysis method was developed for the determination of cyclic peptide Pseudostellarin B in Pseudostellaria heterophylla (Miq.) Pax samples. Separations and determinations were carried out by HPCE under the following conditions: bared fused silica capillary (50 cm×75μm i.d.), 20 mmol/L borate (pH=9.3)as buffer, the run voltage is +15 kV, detection length of UV at 203 nm, and column temperature of 20℃. The results indicated that the developed method was simple, accurate and reliable for the determination of Pseudostellarin B with a good linearity(Y=0.6357X+2.546, r=0.9985), and quantitative recovery ranging from 93.8%～105.6%. The HPCE fingerprints of Pseudostellaria heterophylla (Miq.) Pax were established using this method. The chromatographic fingerprints were compared by the software of“similarity evaluation system”. The fingerprint congruence coefficients of 8 electropherograms in 10 samples were above 0.90. The HPCE fingerprinting method is thus reliable and accurate.2. Set up a method for the determination of chlorogenic acid and flavonoids in honeysuckle flower by HPCE, and for the study of their fingerprints. Separation and determination were carried out by HPCE under the following conditions: bared fused silica capillary (50 cm×75μm i.d.),buffer: 30 mmol/L borate (pH=10.2), run voltage: +18 kV, detection length: 238 nm, column temperature: 25℃. Under the optimized conditions, the determination of chlorogenic acid and flavonoids showed good linearity, and the recovery ranged from 95.2～104.4%. Based on this method, the HPCE fingerprints of Honeysuckle Flower were established.The fingerprints were compared by the software of the similarity evaluation system. The fingerprint congruence coefficients of 15 samples from the same group were above 0.93, and the congruence coefficients of samples from 7 different areas varied within 0.77-0.98. Honeysuckle Flower and Lonicera Confusa DC can be distinguished effectively by HPCE fingerprint. This method is simple, accurate, rapid and has a good reproducibility, so, it can be used to control the quality of Honeysuckle Flower.3. A method to enrich, separate and purify gentiopicroside and loganic acid from Radix Gentianae by macro porous absorbing resin was developed. The Radix Gentianae was extracted by ASE technique. The performance of four types of macro porous absorbing resin were compared, and D301 was chosen based on its performance. The operating conditions of HPLC was optimized. The best condition are as following: the sample concentration: 0.2 g/mL, the maximum capacity for medicinal materials was 0.25g medicinal material per 1g resin, the best absorbing time:8 hours, after absorbing ,wash down the gentiopicroside and loganic acid with 8% and 55% ethanol respectively. Condense and freeze-dry the washed solution resulted in the production of coarse product of gentiopicroside and loganic acid The respective purities are 74.3% and 80.9%, and the respective recoveries are 70.11% and 67.82%.4. A reliable and rapid HPCE method in MEKC and MEEKC modes was developed for the determination of gentiopicroside and loganic acid in the extracts of Radix Gentianae. The analyte was extracted from Radix Gentianae samples via accelerated solvent extraction, and the extraction conditions were optimized. Separation and determination were carried out by HPCE in a bared fused silica capillary(50 cm×75μm i.d.), with corresponding buffer . The run voltageof MEKC and MEEKC were 30 kV and 22KV respectively, the detection wavelength of DAD was 238 nm, and the column temperature was 25 oC. The developed HPCE method is simple and reliable for the determination of gentiopicroside and loganic acid in Radix Gentianae samples with a wide linear dynamic range, a recovery range of 96.3%～105.16%, and a detection limit of below 10 mg/L. The contents of gentiopicroside and loganic acid in six samples from different regions were determined by the developed method in two modes. T test result indicated that the measured contents of gentiopicroside and loganic acid by MEKC and MEEKC modes were consistent. The method is simple, accurate, rapid and with good reproducibility. It can be used to determine active components in Radix Gentianae.5. A reliable and rapid method by HPCE in MEKC, MEEKC and NACE modes was developed for the determination of alkaloids in extracts of Semen Nelumbinis. The analyte was extracted from Semen Nelumbinis samples via accelerated solvent extraction, and the extraction conditions were optimized. Separation and determination were carried out by HPCE in a bared fused silica capillary(50 cm×75μm i.d.), with corresponding buffer. The run voltage of MEKC, MEEKC and NACE are 30 kV, 22 kV and 30 kV respectively, the detection wavelength of DAD was at 282 nm and column temperature was 25 oC. The developed HPCE method is simple and reliable for the determination of alkaloids in Semen Nelumbinis samples with a wide linear dynamic range, a recovery range of 83%～104%, and a detection limit of below 10mg/L. The contents of liensinine, isoliensinine and neferine in 8 samples from different regions were determined by the developed method in three modes. The method is simple, accurate, rapid and with good reproducibility. It can be used to determine liensinine, isoliensinine and neferine in Semen Nelumbinis.6. An HPLC-DAD-ESI-TOF/MS analysis method was developed for the determination of gentiopicroside and loganic acid in Radix Gentianae samples. Fingerprinting technique for the herbs was also studied. The extract was separated into a water phase and a chloroform phase. Based on MS analysis of the chromatographic peaks and their comparison with standard references, three compounds were identified: gentiopicroside, asafetida acid and loganic acid. In the chloroform phas, DAD/MS data reveal 4 isomeric compounds. The compounds remain to be determined since no literature information is currently available. Based on this method, the HPLC fingerprints of Radix Gentianae were established. The fingerprints were compared by the software of the similarity evaluation system. The fingerprint congruence coefficients of ten samples from the same group were above 0.98, indicated that the method is reliable and accurate. The water phase fingerprint congruence coefficients of samples from six different areas are above 0.90. However, the chloroform phase fingerprint congruence coefficients varies from 0.62-0.99, indicating that Radix Gentianae from different producing area differ widely, and the diversities are embodied in the content of triterpenoids.7. An HPLC-DAD-ESI-TOF/MS method was developed for the analysis and identification of alkaloids in Semen Nelumbinis. The samples were extracted using ASE for 10 min under 100℃and 1400Psi. The extract was analyzed with HPLC-DAD-ESI-TOF/MS, and six compounds were identified based on MS analysis and reference compound comparison. The six compounds identified are pronucifefine, 1otusine, nuciferine, 1iensinine, isolisensinine, and neferine. Based on this method, the HPLC fingerprints of Semen Nelumbinis were established. The fingerprints were compared by the software of the similarity evaluation system for chromatographic fingerprint, the fingerprint congruence coefficients of samples from eight different areas varied in the range of 0.62-0.99, indicating that Semen Nelumbinis from different producing area differ widely.This method can be used for the determination of Alkaloids component in Semen Nelumbinis and its quality control.The establishment and application of those analytical methods demonstarted that the HPCE technology can be used for the quality control of TCMs through compositional or fingerprinting analysis. HPLC-ESI-TOF/MS is also shown to be a powerful tool for compounds identification in TCM.更多还原