Dertermination Of Obeticholic Acid Active Metabolites By LC-MS/MS In Human Plasma And Bioequivalence Study

Objective:1.An LC-MS/MS method was established and verified,which can simultaneously determine the concentration of glycine obeticholic acid and taurine obeticholic acid in human plasma.2.Design a reasonable clinical trial program to evaluate the bioequivalence of obeticholic acid tablets（10 mg/tablet）developed by Intercept Pharmaceuticals in the United States and obeticholic acid tablets（10 mg/tablet）developed by a domestic company.Method:1.The LC-MS/MS method for the determination of glycine obeticholic acid and taurine obeticholic acid in human plasmaThe plasma samples should be precipitated with acetonitrile protein,then the supernatant was taken and dried with nitrogen,and finally the dried supernatant was reconstituted with 25%methanol in water,using glimepiride as the internal standard.Chromatographic separation was achieved on a Poroshell 120 EC-C18 colunmn of Agilent at 40°C with the mobile phase of acetonitrile-water containing 0.05%formic acid under gradient elution,and the flow rate is 0.4 m L?min^-1with 20μL of injection volume.The negative ion mode under electrospray ionization source coupled with multiple reaction monitoring was adopted to monitor the transitions of the precursors tothe product ions:m/z 476.3→71.4 for glycine obeticholic acid,m/z 526.3→123.9 for taurine obeticholic acid and m/z 489.3→224.8 for the IS.2.Study on bioequivalence of obeticholic acid tablets in human plasmaThis test adopted an unicentric,random,open,two-agent,three-cycle,three-sequence,and partially repeated designsn to evaluate the bioequivalence after a meal or on an empty stomach.The washout period among different administration periods was21 days,48 healthy volunteers were selected to participate in the test,randomly divided into three groups.Each group received a single oral dose of 10 mg obeticholic acid tablets（test products and reference products）random after a meal or on an empty stomach.The pharmacokinetic parameters and the bioequivalence of two preparations were calculate and evaluate with DAS 3.2.5 software.Results:1.The evaluation results of LC-MS/MS determination methods for glycine obeticholic acid and taurine obeticholic acid in human plasmaThis test established the LC-MS/MS determination method of glycine obeticholic acid and taurine obeticholic acid in human plasma,which has a good selectivity and endogenous substances in plasma did not interfere with the determination of samples.The calibration curves were linear among the range 1.00～120 ng?m L^-1 for glycine obeticholic acid,and the linear relationship is good,and the regression equation is y=0.022240 x+0.003357（correlation coefficient r²=0.9960）.The calibration curves were linear among the range 0.150～18.0 ng?m L^-1 for taurine obeticholic acid,and the linear relationship is good,and the regression equation is y=0.09701 x+0.000923（correlation coefficient r²=0.9955）.The intra-batch and inter-batch precisions of the three concentration levels of high,medium are all below 8.6%,and the intra-batch and inter-batch precision of the lower limit of quantification are less than 11.8%.The intra-batch and inter-batch deviations of the four concentration levels are between-6.7%and 7.2%.At the three concentration levels,the total precision of matrix factor normalized by internal standard of glycine and taurine was less than 8.1%.At both concentration levels,the hemolytic matrix had a precision of less than 3.0%and the high-fat matrix had a precision of less than 3.2%.The stock solutions of glycine obeticholic acid and taurine obeticholic acid were kept at room temperature for 17.2 hours,and stored at-20°C for 45 days with good stability.The plasma samples of glycine obeticholic acid were placed at room temperature for 16.4 hours,-70°C under 5 freeze-thaw cycles,4 freeze-thaw cycles at-20°C,long-term storage at-70°C for 142 days,long-term storage at-20°C for 51 days,which all have good stability.The plasma samples of taurine obeticholic acid were placed at room for 16.4 hours,5 freeze-thaw cycles at-70°C,4 freeze-thaw cycles at-20°C,and long-term storage stability at-70°C and-20°C for 142 days,which all have good stability.The supernatant of plasma sample after treatment was placed in an autosampler at 8°C for 5 days and 23 hours with good stability.This method meets the analytical requirements of biological sample.2.The bioequivalence research results of obercholic acid tabletThe blood concentration of 48 healthy subjects who have reference preparations or test preparations was tested with a validated analytical method,and the pharmacokinetic parameters was calculated with Phoenix Win Nonlin 6.4 software.Because obeticholic acid has significant hepatointestinal circulation,it is impossible to evaluate AUC_0-∞.So,the main evaluation indicators are C_max and AUC_0-t.Under fasting conditions,the S_WR of C_max and AUC_0-t of obeticholic acid are not less than 0.294,evaluated by RSABE method,and the cutoff values were-0.110 and-0.078,which all are less than 0 and the GMR point estimates are 1.009 and 1.015 between 0.80 and 1.25.The S_WR of C_max and AUC_0-t of glycine obeticholic acid are both less than 0.294,evaluated by ABE method and the 90%confidence intervals are 94.89%～110.66%and 94.66%～104.76%within 80.00%～125%.The S_WR of C_max of taurine obeticholic acid is greater than 0.294,evaluated by RSABE method.The point estimate is 1.016 between 0.80 and 1.25 and the cutoff value is-0.083,which is less than 0.Therefore,The S_WR of AUC_0-t of taurine obeticholic acid is less than0.294,evaluated by ABE method,and the 90%confidence interval is 91.48%～107.87%in the range of 80.00%～125%.Under postprandial conditions,the S_WR of C_max of obeticholic acid is greater than 0.294,evaluated by RSABE method.The point estimate is 1.009 between 0.80 and 1.25 and the cut-off value is-0.110,which less than 0.The S_WRof AUC_0-tof obeticholic acid is less than 0.294,evaluated by ABE method,and the 90%confidence interval is 94.85%～105.13%within the range of 80.00%～125%.The S_WR of C_max and AUC_0-t of glycine obeticholic acid are both less than 0.294,which are evaluated by ABE method,and the 90%confidence intervals are 96.23%～108.52%and97.98%～107.86%within the range of 80.00%～125%.The S_WR of C_max of taurine obeticholic acid is greater than 0.294,which is evaluated by RSABE method.The point estimate is 1.150,between 0.80 and 1.25,and the limit value is-0.003,which is less than0.The S_WR of AUC_0-t is less than 0.294,which is evaluated by ABE method.The 90%confidence interval is 101.97%～117.74%in the range of 80.00%～125%.According to the above data,the test and reference preparations are bioequivalent under fasting and postprandial conditions.Conclusion:This study is the first report of the LC-MS/MS method for the simultaneous determination of glycine obeticholic acid and taurine obeticholic acid in human plasma.The method is simple,sensitive and specific,which meets the requirements of"Guidelines for the Validation of Quantitative Analysis Methods of Biological Samples"and can be used for the analysis of plasma samples in clinical trials.The test and reference preparations of obeticholic acid tablets are bioequivalent under fasting and postprandial conditions.