Hplc Determination Of Active Metabolite Of Aniracetam Aba In The Human Plasma And Bioequivalence Study In Healthy Volunteers

OBJECTIVE:To establish a method to determine active metabolite of Aniracetam ABA concentration in the plasma by HPLC. The method will be used to investigate the bioequivalence of Aniracetam after single oral administration 200 mg test and reference tablets in Chinese healthy male volunteers.METHODS:Section one. Using Hydrochlorothiazide as the internal standard, ABA in the plasma sample was determined by HPLC with liquid-liquid extraction, and achieved by the column of SymmetryShieldTM RP18 C18 (3.9mm×150 mm,5μm) at the temperature of 30℃. The mobile phase consisted of a mixture 20 mM ammonium formate:methanol at the ratio of 83:17 (V/V), pumped at a flow rate of 1.0 mL·min-1. The injection volume was 20μL. The UV detection wavelength was set at 250 nm.In the section two, the main pharmacokinetic parameters of test and reference tablets from twenty Chinese healthy volunteers after single oral administration 200mg test and reference tablets were investigated, and the bioequivalence of two kinds of Aniracetam tablets was evaluated. Twenty Chinese healthy male volunteers were randomly divided into two groups, in cross-over and two period design test. The concentrations of ABA in human plasma of the volunteers after single oral administration 200 mg test and reference tablets were determined by the HPLC method established in the section one. The main pharmacokinetic parameters of two kinds of Aniracetam tablets in test were calculated by DAS, and the bioequivalence were estimated by 90% confidence interval (90% CI).RESULTS:The method was linear in the concentration range of 53.3～13640μg·L-1 for ABA, and the lowest detectable concentratio was 53.3μg·L-1. The intra-day average precision and inter-day average precision was no more than 10.8%. The recovery was 96.9%～103.3%.The main pharmacokinetic parameters of ABA of reference and test tablets were shown as following:Cmax were 6948.8±3016.8 and 7049.3±2586.3 ng·mL-1; Tmax were 0.57±0.34 and 0.48±0.19 h; AUC0â†'4 were 5835.9±1496.9 and 5770.9±1105.3 ng·h·mL-1; AUC0â†'∞were 6008.9±1504.7 and 5770.9±1114.4 ng·h·mL-1. Calculated with AUC0â†'4, the relative bioavailability of the test tablets and the reference tablets was 102.8%±24.9%.Nonparametric tests showed that no significant difference existed in the Tmax between test and reference tablets. ANOVA analysis showed that no significant difference existed in the pharmacokinetics parameters AUC0â†'4,AUC0â†'∞and Cmax between the different tablets and the different periods. Two one-sided t-test showed that both of thigh and tlow are more than one side t-test.90% confidence interval for test formulation/ reference formulation ratios of AUC0â†'4 and AUC0â†'∞were found within the bioequivalence acceptance range of 80%～125%.90% confidence intervals for test formulation/reference formulation ratios of Cmax were found within the bioequivalence acceptance range of 70%～143%.CONCLUSION:The method is accurate and sensitive with no interferences, and is suitable for the determination of active metabolite of Aniracetam ABA in the human plasma. Aniracetam test formulation and reference formulation were bioequivalent in healthy human.