黄芩素和七种代谢物液—质联用分析方法的建立及应用

发布时间：2018-06-10 02:11

本文选题：黄芩素 + 代谢物　；参考：《吉林大学》2017年硕士论文

【摘要】：黄芩素是由中药黄芩提取出的有效成分之一,为黄酮类化合物,具有广泛的药理作用,具有抗炎,抗氧化,神经保护,抗过敏细菌,抗病毒感染,抗心血管疾病,抗癌等多种药理作用。黄芩素在体内发生广泛的代谢作用,产生多种代谢产物,目前对黄芩素与一种代谢物黄芩苷的测定及药代动力学研究比较多,对其他代谢物鉴定、定量分析研究比较少。本文主要目的是对黄芩素在人体内代谢产物进行鉴定,建立同时测定黄芩素与七种代谢物的LC-MS/MS分析方法,并将分析方法进行实际应用研究。研究方案:使用高分辨质谱仪,分别对服用黄芩素片前和服用后的血浆样品进行测定,对比分析两者色谱图,结合代谢分析软件对代谢产物初步筛选,再根据黄芩素结构,裂解规律,二级碎片等信息对代谢产物进行推测鉴定,最后合成可能代谢产物标准品,并对代谢产物进行色谱分离,根据保留时间区分鉴定同分异构体。采用两套独立液相系统,黄芩素采用液液萃取方法提取,使用乙腈-0.5%甲酸溶液作为流动相,SUPELCO Ascentis-C18(50 mm×4.6 mm I.D.,5μm粒径)色谱柱对其进行分离。七种代谢物采用沉淀蛋白方法提取,使用乙腈-10 mM醋酸铵0.1%甲酸溶液作为流动相,Agilent HC-C18(150 mm×4.6 mm I.D.,5μm粒径)对代谢物进行分离。流速为1.2 mL/min,梯度洗脱进行分析。使用电喷雾电离源,正离子扫描模式,多反应监控模式进行检测。黄芩素的定量分析离子反应为m/z 271.0→m/z 122.8,内标柯因为m/z 255.0→m/z 153.0,代谢物6BG、7BG为m/z 447.1→m/z 271.1,6BS、7BS为m/z 351.0→m/z 271.0,BGG为m/z 623.2→m/z 271.0,6-MeBG为m/z 461.1→m/z 284.9,BGGlu为m/z 609.1→m/z 271.1,内标木犀草素为m/z 287.0→m/z 135.0。黄芩素的线性范围为1-500 ng/m L,定量下限为1 ng/m L。七种代谢物:6BG选定1-300 ng/m L为其定量的线性范围,选定1 ng/m L为其定量下限;7BG选定1-500 ng/m L为其定量的线性范围,选定1ng/m L为其定量下限;6BS选定10-500 ng/mL为其定量的线性范围,10 ng/m L为其定量下限;7BS选定10-3000 ng/mL为其定量的线性范围,选定10 ng/m L为其定量下限;BGG选定50-5000 ng/m L为其线性范围,选定50 ng/m L为其定量下限;6-MeBG的线性范围为1-200 ng/m L,定量下限为1 ng/mL;BGGlu的线性范围为1-300 ng/mL,定量下限为1 ng/m L。对方法的专属性,线性范围,灵敏度,准确度和精密度,基质效应,提取回收率,样品在多种条件下的稳定性进行了验证,以检验分析方法的可靠性。应用建立的分析方法,对口服200mg黄芩素片的志愿者血浆中的黄芩素及七种代谢物进行了分析测定,并对相关药代参数进行了计算与分析。研究结果:对黄芩素血浆中的代谢物进行了查找鉴定,鉴定出血浆中的M1为黄芩素6,7-二-O-葡萄糖醛酸苷(BGG),M2为6-O-葡萄糖-7-O-葡萄糖醛酸苷(BGGlu),M3为黄芩素7-O-葡萄糖醛酸苷(7BG),M4为黄芩素6-O-葡萄糖醛酸苷(6BG),M5为黄芩素7-O-硫酸酯苷(7BS),M6为黄芩素6-O-硫酸酯苷(6BS),M7为黄芩素6-OCH3-7-O葡萄糖醛酸(6-MEBG),M8为黄芩素7-OCH3-6-O葡萄糖醛酸(7-MEBG)八种代谢物。建立了采用两套独立液相系统同时分析黄芩素及七种代谢物的液相色谱-质谱联用方法。该方法可将同分异构体代谢物完全分离,方法可适用于分析实际生物样品高通量要求,分析方法灵敏度高,专属性强,基质效应小,提取回收率平行一致,精密度和准确度高,分析可用于药代动力学研究。血浆样品分析测定结果表明黄芩素发生广泛且快速的代谢,黄芩素代谢产物与黄芩素呈现多相消除,血浆浓度-时间曲线有多峰现象,经历肝肠循环。多种黄芩素代谢产物体内水平较高,黄芩素及代谢物药代参数如消除半衰期存在个别受试者与其他受试者有较大差异现象。
[Abstract]:Baicalein, one of the effective ingredients extracted from Scutellaria baicalensis, is a kind of flavonoids, and has extensive pharmacological effects. It has many pharmacological effects, such as anti-inflammatory, antioxidation, neuroprotection, antiallergic bacteria, antiviral infection, anti cardiovascular disease, anticancer and so on. Baicalein has extensive metabolic effects in the body and produces a variety of metabolites. There are many studies on the determination and pharmacokinetics of baicalein and a metabolite baicalin, and there are few quantitative studies on the identification of other metabolites. The main purpose of this paper is to identify the metabolites of baicalein in human body, to establish a LC-MS/MS method for the simultaneous determination of baicalein and seven metabolites, and to analyze the methods in the analysis. Practical application research. Research scheme: using high resolution mass spectrometer, the plasma samples before and after taking baicalein were measured, compared and analyzed the two chromatograms, combined with metabolic analysis software to screen the metabolites, and then according to the structure of baicalein, the law of fragmentation, two grade fragments and other information to speculate on the metabolites. Finally, the standard product of the possible metabolites was synthesized, and the metabolites were separated by chromatographic separation, and the isomers were identified according to the retention time. Using two sets of independent liquid phase systems, baicalein was extracted by liquid liquid extraction and acetonitrile -0.5% formic acid solution was used as a mobile phase, SUPELCO Ascentis-C18 (50 mm * 4.6 mm I.D., 5 m particle size) color. The seven metabolites were separated by the precipitation protein method. The metabolites were separated by using acetonitrile -10 mM ammonium acetate 0.1% formic acid solution as the mobile phase, Agilent HC-C18 (150 mm * 4.6 mm I.D., 5 micron m particle size). The flow rate was 1.2 mL/min, gradient elution was used to analyze. The electrospray ionization source, the positive ion scanning mode, and the multiple reaction were used. The quantitative analysis of baicalein is m/z 271 to m/z 122.8, the internal standard is m/z 255 to m/z 153, the metabolite 6BG, 7BG is m/z 447.1 to m/z 271.1,6BS, 7BS is m/z 351, m/z 271, 461.1, 284.9, 284.9, 609.1, 271.1, internal standard wood The linear range of m/z 287 to m/z 135.0. baicalein is 1-500 ng/m L, and the quantitative lower limit is 1 ng/m L. seven metabolites. The selected 1-300 ng/m L is the quantitative linear range of the selected 1-300 ng/m L, and the selected 1 ng/m L is the quantitative lower limit. For its quantitative linear range, 10 ng/m L is its quantitative lower limit; 7BS selected 10-3000 ng/mL as its quantitative linear range, and selected 10 ng/m L as its quantitative lower limit; BGG selected 50-5000 ng/m L is its linear range, and the selected 50 ng/m L is its quantitative lower limit; the linear range of 6-MeBG is 1 and the quantitative lower limit is 1. 1-300 ng/mL, a quantitative lower limit of 1 ng/m L. for the specificity of the method, linear range, sensitivity, accuracy and precision, matrix effect, extraction recovery, and the stability of the sample under a variety of conditions to verify the reliability of the analytical method. The application established analysis method for the volunteers in the volunteers' plasma of the oral 200mg baicalein tablets The analysis and analysis of scutellarin and seven metabolites were carried out, and the related drug generation parameters were calculated and analyzed. The results: the metabolites in the baicalein plasma were found and identified. The M1 in the bleeding pulp was baicalein 6,7- two -O- glucuronide (BGG), M2 6-O- glucose -7-O- glucuronide (BGGlu) and M3 7-O- of baicalein 7-O-. Glucuronide (7BG), M4 is baicalein 6-O- glucuronide (6BG), M5 is baicalein 7-O- sulphate glycoside (7BS), M6 is baicalein 6-O- sulfate glycoside (6BS), M7 is baicalein 6-OCH3-7-O glucuronic acid, and it is the eight metabolites of baicalein glucuronic acid. The simultaneous separation of two sets of liquid phase systems is established. The method for the analysis of baicalein and seven metabolites by liquid chromatography mass spectrometry. This method can completely separate the isomer metabolites. The method can be used to analyze the high throughput requirements of the actual biological samples. The analytical method has high sensitivity, strong specificity, small matrix effect, parallel and high recovery rate, high precision and accuracy, and can be used for analysis. Pharmacokinetic study. The results of plasma sample analysis show that baicalein has extensive and rapid metabolism. The metabolites of baicalein and baicalein have multiphasic elimination. The plasma concentration time curve has multiple peaks, experienced the hepatic intestinal circulation. The levels of various baicalein metabolizing objects are higher, and the parameters of baicalein and metabolites are eliminated. The half-life is different between individual subjects and other subjects.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285

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