【作者】 孙丽；

【导师】 邓远辉；

【作者基本信息】 广州中医药大学 ， 中药学， 2008， 硕士

【摘要】 目的:建立液相色谱-质谱（LC-MS/MS）联用法测定人尿液中人参皂苷A浓度,以尿排泄数据法研究人参皂苷A注射液在健康受试者体内的药代动力学规律。并进一步研究其在健康人体内的药物代谢规律。方法:1、招募12名健康受试者,采用开放、随机、交叉的试验方法,进行三个周期、三个剂量（10、40、75mg）的单次给药试验。2、建立LC-MS/MS法检测尿样品中人参皂苷A的浓度。色谱条件为:色谱柱为CAPCELL PAK C₈（5μm,2.0×150mm）;流动相:甲醇-10mmol·L_-1乙酸铵（加1%乙酸）-乙腈（40:20:40 v/v）;流速250μL·min^-1;柱温:室温:进样体积为5μL。质谱条件为:电喷雾ESI源;喷雾电压IS为5000V;雾化温度300℃;雾化气NEB（GAS1）为10:加热辅助气AUX（GAS2）为7 L·min^-1;帘气CUR为6;碰撞气CAD为2;检测方式为正离子多离子反应监测（MRM）,以龙胆苦苷为内标,用于定量分析的离子为m/z 964.7/767.6（人参皂苷A）、m/z374.1/195.1（内标龙胆苦苷）,采用稀释法处理尿样品。3、应用所建立的方法检测各剂量组（10mg、40mg、75mg）受试者尿样品中人参皂苷A的浓度。根据不同时间段的尿样品的体积及药物浓度计算出各时间段的药物排泄量,采用孙瑞元教授编制的DAS药代动力学软件处理数据,并以SPSS13.0软件进行进一步的统计分析。4、人参皂苷A在健康人体内的药物代谢研究:分段收集给药前和给药后的尿样品,以固相萃取法及直接稀释法处理尿样品,采用LC-TOF/MS及Agi lent’s ChipLC/IonTrap MS系统进行检测分析,根据所检测到的人参皂苷A的代谢产物推测其在健康人体内可能发生的代谢转化规律。结果:1、所建立的LC-MS/MS法,通过了方法学确证实验。人参皂苷A和龙胆苦苷保留时间分别为2.10min和1.49min,内源性物质不干扰样品的测定。人参皂苷A的线性范围为30.3-10100ng·mL^-1;定量下限为30.3±2.16 ng·mL^-1;批内、批间变异系数均＜15%。2、所得各剂量组的药代动力学参数如下:半衰期依次是21.07±7.99、18.52±2.81、23.23±8.61h,清除速率常数依次是0.038±0.014,0.038±0.005,0.033±O.01lh^-1,累积排泄率（%）依次是7.64±2.53、7.38±2.83、6.37±1.78。以SPSS13.0统计软件对所得参数进行统计分析,结果这三个药代动力学参数在三个剂量组间的差别均无统计学意义。3、人参皂苷A在健康人体内的药物代谢研究:采用LC-TOF/MS技术检测不同时间段的尿样品,结果只能检测到人参皂苷A,并未检测出其代谢产物。采用Agilenz’sChipLC/Ion Trap MS系统进行分析,结果发现4种代谢产物,其准分子离子[M-H]^-分别是m/z1005、m/z931、m/z915、m/z899。结论:1、本实验首次以尿排泄数据法进行人参皂苷A注射液在健康人体内的药代动力学规律研究。所建立的方法灵敏、准确、简便,特异性强,适用于大量生物样品中人参皂苷A浓度的测定。2、通过本实验的研究主要得出了三个药代动力学参数:半衰期、清除速率常数、累积排泄率。结果表明,人参皂苷A注射液在健康人体内的消除是较慢的,且只有较少部分药物以原形的形式排出体外。对所得参数进一步统计分析发现在静脉给药10、40、75mg后,药物在健康人体内的消除符合线性药代动力学特征。3、人参皂苷A注射液在健康人体内的药物代谢研究:本实验首次采用LC-TOF/MS及Agilent’s ChipLC/Ion Trap MS技术对该药物在健康人体内的药物代谢情况进行研究。在尿样品中共发现4种代谢产物,并进一步推测出人参皂苷A在健康人体内可能发生的代谢反应是氧化、还原、结合反应。更多还原

【Abstract】 ObJective:Establish the method of LC/MS/MS for the determination of Ginsenoside-A in human urin for pharmacokinetics studies.And furtherly study the metabolism of Ginsenoside-A in health human.Mathods:1.Ten healthy volunteers were involved to participate single administration trials in this study.They were injected for three dosages （lOmg,40mg,75mg）based on opened,randomized,crossover trial method in three periods.2.Establish the method of LC/MS/MS for the determination of ginsenoside -A in human urine.The chromatographic condition is:the separations were carried out using a CAPCELL PAK TYPEUG 120 column（5μm,2.0×150mm）;the mobile phase is methanol-ammonium acetate-（1%of ammonium acetate v/v）-acetonitrile （40:20:40 v/v）;Chromatography was performed at a flow rate of 0.25 ml/min;the column temperature is room temperature and the injection volume was 5μL.The mass spectrogram condition is:Electrospmy ionization source was applied;the ionspray voltage were set 500oV;Probe temperature was set at 300℃with ultrahigh-purity nitrogen as curtain gas（CUR,6 psi）,nebulizer gas（NEB, GAS1,10）and Auxiliary gas（nitrogen,AUX,GAS2,about 7 L/min）;the fragmentation transitions for the multiple reaction monitoring（MRM）were m/z 964.6-767.5 for Ginsenoside-A and m/z 374.1-195.1 for the IS:use the gentiopicroside as internal standard;adopt the dilution method to prepare urine samples.3.The LC-MS/MS method was applied for the quantification of ginsenoside-A in human urine,then calculate the accumulated excretion of different period. Adopt DAS and spss13.0 software to processe data.4.The metabolism study of ginsenoside-A:Urine samples were collected before and after intravenous administration of 40 mg and 75mg of ginsenoside-A.Samples were preparated with solid-phase extraction and dilution。Then,adopt LC-TOF/MS and Agilent’s ChipLC/Ion Trap MS to analyze.Rusults:1.A HPLC-MS/MS method for the quantification of ginsenoside-A in human urine was developed and validated.The retention time of ginsenoside-A and internal standard is 2.10min and 1.49min respectively,endogenous substances didn’t affect to detect the object under test.By this method the linearity limit of ginsenoside-A is 30.3-10100 ng·ml^-1;the lower quantitative limit is 30.3±2.16ng·ml^-1;The inter-and intra-day precision（RSD）was less than 15%.2.The half life of the three dose group is 21.07±7.99、18.52±2.81、23.23±8.61;the elimination rate constant is 7.64±2.53、7.38±2.83、6.37±1.78;the accumulation excretion rate（%）is 7.64±2.53、7.38±2.83、6.37±1.78 respectively.The difference of the parameters among groups was not statistically significant.3.The metabolism study of ginsenoside-A:adopt HPLC-TOF/MS to analysis" ginsenoside-A can be detected but its metabolites;Adopt ChipLC/Ion Trap MS to analysis:quasi-molecular ion of four metabolites detected are m/z1005、m/z931、m/z915、m/z899 respectively.Conclusions:1.The pharmacokinetics of the ginsenoside-A injection in healthy human body was studied for the first time by the method of rate of urine drug in this experiment.The LC-MS/MS method for the quantification of ginsenoside -A in healthy human urine is an accurate、sensitiveand、convenient method, Which can be applicated.2.Three parameters were gained from this study:half life;elimination rate constant and ccumulative excretion rate.Statistics indicate that the ginsenoside-A may be metabolized slowly after intravenous administration, and only a few of the medicines were evacuated by origin.This drug showed linear kinetics over the dosage of 10mg,40mg,75mg.2.The metabolism study of ginsenoside-A:the metabolism of ginsenoside-A was studied by LC-TOF/MS and Agilent’ s ChipLC/Ion Trap MS for the first time. Four metabolites were identified in healthy human urin.Its main metabolic pathwayrs are elucidated:Oxidation,combination and reduction reaction were found to be the major metabolic pathway of ginsenoside-A in healthy human.更多还原