| 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. |
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