Determination Of Abiraterone In Human Plasma By LC-MS/MS And Its Application In Bioequivalence Study

1 Estabilishment and validation of analysis method for abirateroneObjective:To develop a sensitive and reproducible and specific liquid chromatography coupled with tandem mass spectrometry（LC-MS/MS）method to detect concentrations of abiraterone in human plasma.To validate the method entirely.Methods:Analytes and the internal standard（abiraterone-d4）were extracted from human serum using the supported liquid extraction procedure.The final extract was injected on a XBridge column C18（50×4.6mm ID,particle size 3.5μm,Waters）and separated with gradient elution,using mobile phase A（0.1%formic acid in water）and mobile phase B consisting of acetonitrile-methanol（1:1,v/v）.Analytes were detected by liquid chromat-ography-mass spectrometry.Electrospray ionization in positive mode was applied with multiple reaction monitoring.The MS/MS ion transitions monitored were 350.2→156.0for abiraterone and 354.3→160.3 for the internal standard.Method validation included the evaluation of specificity,the linearity range,LLOQ,precision,accuracy,medium effect,percent recovery of extracion and sample stability.Results:The lower limit of quantitation achieved was 0.2 ng/m L and the linearity range extended from 0.2～200 ng/m L.The intra-and inter-batch precisions of the quality control samples with high,middle,low and LLOQ concentrations in human plasma were in the ranges 1.3-8.7%and 6.3-11.9%.The recovery of extraction of analytes and the internal standard are precise and reproduceable,with the relative standard deviation（RSD）less than15%.On ice and under yellow light,abiraterone were stable in plasma for at least 6 hours,in whole blood for 1 hour.Results for medium effect test met the acceptance criteria.Conclusion:The validated bioassay method is sensitivere,producible,specific and suitable for applying to detect concentrations of abiraterone in human plasma.2 Bioequivalence study of abiraterone acetateObjective:The primary objective of this trial is to assess the bioequivalence between the test formulation abiraterone acetate tablet（250 mg）and the reference formulation ZYTIGA^TM（abiraterone acetate tablet,250 mg）in healthy Chinese male volunteers under fasting condition.To provide support for the generic product to obtain marketing authorization in China.Methods:A single center,randomized-sequence,open-label,single-dose,four-period,crossover study was conducted in thirty-two healthy Chinese male volunteers.Eligible subjects were randomized into two treatment sequence groups（TRTR and RTRT）.In each period,each subject received either an oral dose of one tablet of the test product abiraterone acetate tablet（250 mg）or one tablet of the reference product ZYTIGA^TM（250 mg）in the fasted state.Each study period was separated by a 7-day washout period.Blood samples were collected at 0,0.25,0.5,1,1.5,2,3,4,6,8,12,24,48,60 and 72 h after drug administration.The LC-MS/MS method was applied to determination of the concentration of abiraterone in plasma samples and pharmacokinetic parameters were calculated by using noncompartmental analysis methods through Win Nonlin 7.0software.Tatistical analysis was performed using SAS V9.4（SAS Institute,Cary,North Carolina,USA）.The drug concentration-time curve and the average drug concentration-time curve were plotted based on the plasma concentration-time data to evaluate whether the test preparation and the reference preparation were bioequivalent by a Reference-Scaled Averag Bioequivalence Approach.Results:Thirty-two subjects completed all four periods of the study.The bioavailability of abiraterone from the test product versus the reference product by geometric mean ratio was C_max,100.19%（90%confidence interval[CI]87.05-115.32）;AUC_0-t,105.99%（96.34–116.62）and AUC_0-∞,105.86%（96.49–116.13）.The coefficient of variation（CV）was smaller for T versus R(C_max,CV 40.33 vs.46.58%;AUC ₀–_t,24.02 vs.34.16%;AUC_0-∞,22.61 vs.32.27%,respectively).The 95%confidence upper limit bound for（7）Y_T-Y_R（8）²-?s_W² _R was-0.1079 for C _max and-0.0515 for AUC_0-t.The pharmacokinetics parameters(C_max,AUC_0-t and AUC_0-∞)of the test and reference formulations are bioequivalent by reference-scaled procedure.Conclusion:The study demonstrated that the test abiraterone acetate tablet is bioequivalent to the reference product ZYTIGA^TM.