生物样品中新疆紫草脂溶性成分的HPLC分析

发布时间：2018-05-13 01:39

本文选题：新疆紫草 + 大鼠　；参考：《新疆医科大学》2016年硕士论文

【摘要】：目的:建立检测新疆紫草脂溶性成分的HPLC法,并应用于体内外药物浓度的测定,考察新疆紫草脂溶性成分在体内的处置情况。方法:(1)通过比较不同流动相的分离效果及影响因素,建立了用于测定新疆紫草脂溶性成分体内外浓度的HPLC法。(2)考察了方法学指标;(3)比较分析了不同有机溶剂萃取、酸化和碱化处理血液等方法对血中药物萃取效果的影响。(4)考察了血浆与血清样品的适用性和影响。(5)考察新疆紫草脂溶性成分在不同酸碱环境及模拟胃液、模拟肠液中的稳定性试验。(6)给于大鼠灌胃给药,分别于给药前及给药后不同时间采血,分离血清和血浆,收集给药前及给药后的48小时内的尿液和粪便,进行HPLC分析,测定这些样品中左旋紫草素、乙酰紫草素、去氧紫草素、β-β二甲基丙烯酰紫草素等单一成分的浓度。结果:(1)色谱条件为:色谱柱COSMOIL C18-AR-Ⅱ谱柱(250mm×4.6mm,5um);流速:1.0m L/min;柱温:(25)℃;检测波长:275nm,流动相A液-乙腈,B液-甲酸:水(0.5:300)(v/v)溶液,进样量20μl,梯度洗脱,0~10min(60-40)%;15~30min(70-30)%;30~35min(60-40)%;检测时间35min。在该色谱条件下,新疆紫草脂溶性提取物中左旋紫草素、乙酰紫草素、β-β二甲基丙烯酰紫草素得到较好的分离。(2)方法学指标符合规定:日间精密度试验中各目标峰保留时间RSD1%,样品12h内稳定相对峰面积RSD3%,重复性试验相对峰面积RSD3%;(3)血浆和血清样品中药物提取分离条件为,样品先用等体积的稀盐酸酸化,用乙酸乙酯萃取3次,乙腈沉淀蛋白。血浆样品在5μg/ml~500μg/m浓度范围内与峰面积呈良好线性关系,回归方程为左旋紫草素:y=20.613x+979.88;乙酰紫草素:y=24.254x-57.209;β-β二甲基丙烯酰紫草素:y=48.406x+3180.5;相关系数分别为0.997、0.9957、0.9935。(4)样品的适用性方面,血浆样品较适合于疆紫草脂溶性提取物成分的测定,在血清样品中添加适量的肝素后,峰形和峰面积得到了改善。(5)新疆紫草脂溶性成分(左旋紫草素、乙酰紫草素、β-β二甲基丙烯酰紫草素)在pH2.5磷酸盐缓冲液及人工胃液中相对稳定,但随着时间的延长,含量呈缓慢下降趋势;在pH5.8磷酸盐缓冲液呈明显的下降趋势;随着pH值的上升,含量下降迅速。(6)经大鼠灌胃给药后0~48小时内血样、尿液中均未检测到新疆紫草脂溶性提取物所含有原型物质特有峰。而在粪便中发现有左旋紫草素、乙酰紫草素、β-β二甲基丙烯酰紫草素等成分的峰。结论:大鼠灌胃后的血液样品及尿液中未检测到新疆紫草脂溶性提取物,而在粪便发现有该提取物的相关成分,说明新疆紫草脂溶性提取物在胃肠道几乎不吸收或吸收很少,其吸收、分布、代谢、排泄过程有待于进一步深入研究。
[Abstract]:Objective: to establish a HPLC method for the determination of liposoluble components in Xinjiang, and to determine the treatment of liposoluble components in Xinjiang in vivo. Methods: (1) a HPLC method was established to determine the concentration of liposoluble constituents in Xinjiang by comparing the separation effect and influencing factors of different mobile phases. (2) the methodological indexes were investigated, and (3) the effects of different organic solvent extraction, acidification and alkaline treatment of blood on the effect of drug extraction in blood were compared and analyzed. (4) the applicability and influence of plasma and serum samples were investigated. (5) the stability of lipid soluble fractions in different acid base environment and simulated gastric juice in Xinjiang and the stability of simulated intestinal fluid were investigated. (6) (6) the rats were given the medicine by gavage, the blood was collected before and after the administration, the serum and plasma were separated, the urine and feces were collected for 48 hours before and after the administration, and the HPLC analysis was carried out to determine the single formation of levoshikonin, acetoxin, deoxyshioxin, beta - beta two methacyleylephrin, etc. Results: (1) the chromatographic conditions are: chromatography column COSMOIL C18-AR- II column (250mm x 4.6mm, 5um); flow rate: 1.0m L/min; column temperature: (25) C; detection wavelength: 275nm, liquid phase A liquid acetonitrile, B liquid formic acid: water (0.5:300) (v/v) solution, the sample volume is 20 mu, gradient elution, 60-40%; 70-30%; 60-40%; detection time Under this chromatographic condition, a better separation was obtained from levopurple, acetyl, beta two methacrolein in the liposoluble extracts of Xinjiang purple grass. (2) the methodological indexes were in accordance with the regulations: the retention time of each target peak in the day precision test was RSD1%, the stable relative peak area within the sample 12h was RSD3%, and the relative peak area of the repeatability test was RSD3%; 3) the extraction and separation condition of plasma and serum samples is that the sample is acidified by dilute hydrochloric acid with equal volume first, extract 3 times with ethyl acetate and precipitate acetonitrile protein. The plasma sample has a good linear relationship with the peak area in the range of 5 mu g/ml~500 g/m, and the regression equation is levoviolet herb: y=20.613x+979.88; acetyl purplicin: y=24.254x-57.209 The beta beta two methacryl purple herb: y=48.406x+3180.5; the correlation coefficient is 0.997,0.9957,0.9935. (4), respectively. The plasma sample is suitable for the determination of the composition of the liposoluble extract of the purple herb. After adding a proper amount of heparin in the serum samples, the peak shape and peak area have been improved. (5) the liposoluble composition of Xinjiang purple grass (left) It was relatively stable in pH2.5 phosphate buffer solution and artificial gastric juice, but the content decreased slowly in pH2.5 phosphate buffer solution and artificial gastric juice, while pH5.8 phosphate buffer solution decreased obviously with the rise of pH value. (6) 0~48 was small after gavage of rats. In the urine and urine, there were no specific peaks in the lipid soluble extracts of Xinjiang herb, and the peaks were found in the feces, such as levopurple, acetylenetin, beta - beta two methacryrodacysin. Conclusion: the blood samples and urine of rats after the gavage of the stomach were not detected in the liposoluble extract of Xinjiang. The related components of the extract were found in the feces, indicating that the liposoluble extract of Xinjiang herb was hardly absorbed or absorbed in the gastrointestinal tract, and its absorption, distribution, metabolism, and excretion process need to be further studied.

【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R29;O657.72

【参考文献】