Determination of amoxicillin concentration in human plasma by UPLC-MS/MS and its application in bioequivalence study
Objective To establish a UPLC-MS/MS method with less sample and precipitant and easy operation to determine amoxicillin concentration in human plasma,and to use it in the bioequivalence study of amoxicillin capsules and their reference preparations.Methods A Waters Acquity UPLC BEH C18(2.1 mm × 50 mm,1.7 μm)column was used with the mobile phase consisting of 0.05％formic acid water(A)and 75％methanol solution(containing 0.1％formic acid)(B)in gradient elution.The flow rate was 0.35 mL·min-1 with the injection volume of 1 μL and the column temperature was 35 ℃.Amoxicillin(m/z 366.1 → 114.0)was detected by ESI ion source and positive ion MRM scanning mode,with amoxicillin-d4(m/z 370.2 → 114.0)as the internal standard.After adding the internal standard,the plasma protein was precipitated by methanol,the supernatants were diluted and measured.Results The linearity ranged 20～8000 ng·mL-1 and the lower limit of quantification was 20 ng·mL-1.The intra-day and inter-day precision CV of quality-control samples was≤3.9％,and the accuracy ranged-1.9％～3.9％in terms of relative error.The recovery rate,specificity,matrix effect and stability all met the guiding principles for verification of quantitative analysis of biological samples in the General Rules of Chinese Pharmacopoeia(four edition).The method was successfully applied to a bioequivalence study of amoxicillin orally disintegrating capsules containing 250 mg in healthy volunteers.In the fed test,the Cmax of the test or reference formulation of amoxicillin capsules was(4922.16±967.16)and(5172.19±905.07)ng·mL-1;AUC0～12h was(14 165.22±1686.79)and(13 869.53±1939.31)h·ng·mL-1.In the fasting test,the Cmax of the test or reference formulation of amoxicillin capsules was(6087.36±1766.53)and(5697.95±1768.73)ng·mL-1;AUC0～12h was(15 081.27±2342.12)and(14 564.04±2364.83)h·ng·mL-1.The 90％confidence intervals of the geometric mean ratios of Cmax,andAUC0～12h between the test and the reference formulation completely fell at 80.00％～125.00％.Conclusion The method is highly sensitive,simple and rapid,and is suitable for high throughput analysis of amoxicillin in the plasma sample.The test amoxicillin orally disintegrating capsules are bioequivalent to the reference capsules.
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