Bioeauivalence Study Of Paracetamol In Healthy Human Subjects Using HPLC-MS/MS

Objective:To develop a sensitive and selective high performance liquid chromatography-tandem mass spectrometric(HPLC-MS/MS)assay for the quantitative determination of paracetamol in human plasma,and apply it to a pharmacokinetic and bioequivalence study of two paracetamol formulations in human,for bioequivalence evaluation of the two formulations and ensuring safe and effective use of them in clinicMethods:Paracetamol and tinidazole(used as internal standard,IS)were liquid-liquid extracted from 200 ?L human plasma with ethyl acetate.Chromatographic separation was achieved on an Waters XBridge? C18 column under isocratic elution condition.Then with the mass spectrometer operated in positive electrospray ionization mode,both paracetamol and IS were quantitatived by detecting MS/MS ion transition at m/z 152?110 and m/z 248?121 by multiple reaction monitoring,respectively.The validated method was employed in the pharmacokinetic and bioequivalence study of two paracetamol formulations,which conducted in 19 healthy,adult human subjects following single dose oral administrations of 500 mg paracetamol under fasting condition.The main pharmacokinetic parameters such as the maximum concentration(Cmax)and area under the curve(AUC0-t,AUC0-?)were calculated for both formulations using Phoenix WinNonlin software for the bioequivalence evaluation.Results:The method was validated to be highly selective and the impurity such as plasma matrix didn't interfere with the detection of paracetamol and IS.A good linearity was showed over the concentration range of 0.1-8.0 ?g/mL(r2>0.99)for paracetamol in human plasma.The lower limit of quantitation achieved was 0.1 ?g/mL and the extraction recovery was in the range of 91.0%-98.7%.Intra-day accuracy ranged from 98.8%to 111.3%(precision:CV ?9.03%)and inter-day accuracy ranged from 94.9%to 102.6%(precision:CV ?10.68%)respectively,which demonstrated that the method was accurate and precise.Additionally,in the bioequivalence study,about the main pharmacokinetic parameters of the test and reference formulations,the median Tmax was 0.99 h and 0.89 h;the mean Cmax was 7.53±1.91 ?g/mL and 8.09±2.00 ?g/mL;the mean AUC0-t was 28.97±7.35 ?g·h/m and 29.46±8.24 ?g·h/mL;the AUC0-? was 30.41±7.59 ?g·h/mL and 31.29±9.10 ?g·h/mL,respectively.The calculated 90%confidential intervals of geometric mean(test/reference)ratios for Cmax,AUC0-t and AUC0-? of the two formulations were 83.50%-105.79%,94.25%-101.54%and 93.24%-101.02%,respectively,which all fell within the bioequivalence acceptance range of 80.00-125.00%.Conclusion:The developed sensitive and selective method coupled with a high recovery rate of the extraction procedure and a low matrix effect was fully validated and successfully applied to the pharmacokinetic and bioequivalent study of paracetamol in healthy human subjects.The results showed that the test formulation was similar to the reference formulations in terms of absorption rate and extent,and the two studied formulations were bioequivalent.