基于液质联用技术的清开灵注射液中栀子和金银花色素成分的研究

【作者】 高凤阳；

【导师】 卢建秋；

【作者基本信息】 北京中医药大学 ， 中药学， 2012， 硕士

【摘要】 本实验采用UV-Vis.HPLC-MS/MS相结合的技术对栀子、金银花中的植物色素成分进行研究,并对清开灵注射液的色素成分进行相关分析。通过对注射液组成成分进行筛选,认为应该对可能导致注射液有色的成分一栀子、金银花、黄芩苷和板蓝根进行分析,本实验选择对其中两种药材一栀子和金银花进行研究。实验最终对药材中的两大色素类化合物一藏红花素类成分和类黄酮成分进行快速分析鉴定,同时对多种成分进行定量分析,为清开灵注射液不良反应及注射液的澄明度的改善提供基础数据。本论文研究的具体内容包括以下三个部分：(?)栀子中色素成分的分析鉴定藏红花素类成分的研究采用高效液相色谱-二极管阵列检测器-质谱联用技术,建立栀子中藏红花素类成分1的指纹图谱。实验在正负离子模式下,对藏红花素类色素成分进行较为细致的研究。掌屋该类化合物的结构特征与质谱裂解行为的相关性,重点归纳及总结了这一类化合物在电喷雾电离负离子模式条件下的质谱裂解特征,并根据其紫外光谱特征对质谱检测结果进行验证。色谱条件为：Dikma Diamonsil C18色谱柱(4.61mm×250mm,5μm)；流动旧为0.3%乙酸-乙腈,梯度洗脱；流速I mL·min-1;柱温30℃；二极管阵列检测器记录90～700nm紫外-可见光谱,检测波长设定为440nm；质谱条件为：ESI离子源负离子模式；干燥气流速11L·min-1;雾化室压力50psi；干燥温度350℃；毛细管电压3500eV；扫描范围m/z100-1300；流动相采用检测器后分流进入检测器为0.25mL-min-1。最终获得了34个藏红花素类化合物的紫外光谱数据,精确分子离子峰和多级质谱数据分析,基于对标准品的多级质谱裂解规律的推测,以及文献数据对照,鉴定了26个藏红花素类化合物的结构；藏红花素类成分的含量测定利用高效液相色谱建立栀子中藏红花素类成分的指纹图谱,并对栀子中的藏红花素类色素成分进行了快速鉴定,对其中主要的两种藏红花素类成分—rocin Ⅰ和crocin Ⅱ进行了含量测定；类黄酮成分的研究通过对现有的类黄酮标准品进行HPLC-DAD-MS/MS分析,根据这些标准品的质谱碎片结合文献资料,归纳总结这类化合物的质谱裂解特征。在正负离子检测模式下对栀子中的类黄酮色素成分进行详细的分析研究,栀子获得了9个类黄酮化合物的数据,最终确定了其中6个化合物的结构。2.金银花类黄酮色素成分的分析鉴定通过对现有的类黄酮标准品进行HPLC-DAD-MS/MS分析,根据这些标准品的质谱碎片结合文献资料,归纳总结这类化合物的质谱裂解特征。这类化合物结构分析的重点主要为黄酮苷元和糖基的种类以及糖基与苷元连接位置的确认。黄酮苷中的大多数糖为已知,且糖的种类不多,糖链部分的结构测定通常是对已知糖基进行鉴定,所以鉴定重点是对苷化位置和苷的构型进行确认。根据紫外光谱图也可以有效分析此类化合物的结构类型。本研究根据MS数据结合UV谱以及保留时间等信息,通过与标准品分析数据对比及文献提供数据,可以快速有效的鉴定化合物的化学成分结构,此方法有时还可发现新化合物。在正负离子检测模式下分别对金银花中的类黄酮色素成分进行详细的分析研究,色谱条件为：Dikma Diamonsil C18色谱柱(4.6mm×250mm,5μm)；流动相为0.3%乙酸溶液-乙腈梯度洗脱；流速1mL·min-1;柱温30℃；二极管阵列检测器记录190～400n/n紫外光谱,检测波长设定为355nm；质谱条件为：ESI离子源负离子模式；干燥气流速11L·min-1;雾化室压力50psi；干燥温度350℃；毛细管电压3500eV；扫描范围m/z100～1400；流动相采用检测器后分流进入检测器为0.25mL·min-1。金银花获得18个类黄酮化合物的数据,最终确定了4个化合物的准确结构,推断了金银花13个化合物的基本结构。3.清开灵注射液中色素类成分的分析按照已建立的藏红花素类化合物和黄酮类化合物的检测方法,对清开灵注射液中的这两类色素成分进行综合分析,共推断出3个藏红花素类色素成分和3个类黄酮色素成分的可能结构,同时与对照品进行比较,确认了其中2个藏红花素类色素成分和2个类黄酮类色素成分的结构,为进一步研究清开灵注射液中的色素类物质提供了综合信息。通过本项研究建立了简便、快速分析与鉴定色素类成分的质谱分析方法,并以此作为分析注射液中相关色素成分的主要依据。此种研究方法既可以对原药材进行常规的色素类指纹图谱分析,也可以对以中药材为原料药的注射剂进行色素类成分的定量分析,该方法为中药类注射液的质量评价提供了参考信息。更多还原

【Abstract】 This paper focused on the study of phytochrome in Qingkailing injection systemat ically according to using the combination of UV-VIS, HPLC-MS/MS". Through the scr eening of crude drug, Fructus Gardenia, Flos lonicerae and Radix Isatidis could lead to the injection of the colored medicinal-Fructus Gardenia, Flos lonicerae and Radix Isatidis carries on the analysis, Finally this experiment choose Fructus Gardenia and Flos lonicerae for research. Finally the research aimed at two chief chemical compone nts-crocin and flavonoid, and the made quantitative analysis for multiingredient. It co uld play an important part for adverse reaction resulting from Qingkailing injection an d prepare for solving the clear level problem of Chinese Medicine injection.This paper studies the specific content of include the following three parts:1. The identification of the pigment components in Fructus Gardenia The research of crocetin componentsHigh performance liquid chromatography (HPLC)-photodiode array detector (DAD)-mass spectrometry (MS) was used to build the fingerprint of crocin in Fructus Garde nia. In t-he positive and negative mode, the components of crocin were studied in det ail. Grasp-ing the relation of the structure of such compounds and cracking behavior i n mass sp-ectrometry, and summarizing fragmentation pathways about this kind of co mpound in electrospray ionization and negative ion model, we could test and verify it according t-o its ultraviolet spectrum characteristics. The separation was perfoemed on a Dikma Diamonsil C18column (4.6mm x250mm,5μm); Mobile phase for0.3%acetic acid-acetonitrile gradient elution; Flow:1mL·min-1, diversion ratio for4:1; colu mn temperature:30℃; Photodiode array detector:190～700nm uv-vis spectra, detecte d at440n-m; Mass spectrometry conditions:ESI ion source:negative mode; Dry gas11L·min-1;Spray chamber pressure:50psi; Atomization temperature:350℃; Capillary voltage:35-00eV; Scanning range m/z100～1300; Mobile phase after the detector shunt into d-etector for0.25mL·min-1. Finally we could get ultraviolet spectrum data, precise mol-ecular weight and multi-stage mass spectrum data analysis about34kinds of crocins.Based on the inference to the standard of multistage mass spectrometry an alysis of thelaw that cracking, and literature data control, We could identify structure of26kinds of crocetin.The determination of components of crocinThe fingerprint of components of crocin in Fructus Gardenia was established by using hig-h performance liquid chromatography (HPLC) and make fast identification. T he prima-ry components-crocetin I and II crocin were determined.Flavonoid ingredientsAccording to analysis of the existing flavonoids standard product by HPLC-DAD-MS/MS and mass spectrometry of the standard of the product with debris literature m-aterial, we summarized the class of compounds of mass spectrometry cracking charact-eristics. Finally structures of six compounds were identified by a detailed analysis of the research for the Fructus Gardenia flavonoids ingredients pigment in the positive a nd negate-ve mode.2. The identification of the flavonoids components in Flos loniceraeSummarizes the mass spectrometry cracking characteristics of the flavonoids by an-alyzing the fragments of the standards using HPLC-DAD-MS/MS and integrating with references. The focus of the structure analysis of these compounds is the identification of flavone aglycone and glycosyl, including the identification of connection structure o-f glycosyl and aglycone. Most of the sugar in flavonoid glycoside is known and the kinds of sugar are not much. Structure determination of the sugar chains is usually ba-sed on the identification of known saccharide grous. So the key point is to identify t-he position of glycosidation and the configuration of glycoside. It is an effective way to analyze the structure type of these compounds using UV spectra. This study could identify the structure of the compounds quickly and effectively by MS data, UV spec-trum, retention time, and so on. Meanwhile, analytical data of the standards and datas upplied by references are helpful. Sometimes, this method could find new compounds.In this study, we analyze the Flavonoid pigments in Flos lonicerae in detail by p ositive and negative mode. The separation was perfoemed on a Dikma Diamonsil C18column chromatography (4.6mm x250mm,5μm); Mobile phase for0.3%acid solu tion-acetonitrile gradient elution; Velocity1mL·min-1. diversion ratio for4:1; Thecolu mn temperature30℃; Photodiode array detector record190-400nm ultraviolet spe ctrum, detected wavelength set for355nm;Mass spectrometry conditions for:ESI ion source anion mode; Dry gas velocity and L·min-1; Spray chamber pressure50psi; At omization temperature350℃; Capillary voltage3500eV; Scanning range m/z100～1400; Mobile phase after the detector shunt into detector for0.25mL·min-1. Finall-y, we got18flavonoid data and identified4compounds’ structure. Also, we inferred13compounds, basic structure of Flos lonicerae.3. Analysis of the components of pigment in Qingkailing injectionAccording to the established testing method of the detection to the components of crocins and flavonoids and comprehensive analysis of little content of the two components in Qingkailing injection, three kinds of the components of crocin and the structure of two flavonoids were inferred. At the same time two kinds of the components of crocin and the structure of two flavonoids were confirmed. In addition, this could supply a comprehensive analysis for the study of pigments in Qingkailing injection.A simple and fast analysis and identification of the mass spectrometry pigment composition analysis methods were established through the research, and these methods were regarded as the judgement of pigments in Qingkailing injection. This method could not only analyze fingerprint of regular pigment of the original medicinal materials, but also make quantitive analysis to the original medicinal materials of the injections, and provides references to the envaluation of the traditional Chinese medicine injection.更多还原