UPLC-MS/MS法测定犬血浆中阿莫西林和克拉维酸片剂的生物等效性研究.

Abstract：[Objectives]The paper aimed to establish a UPLC-MS/MS method for the determination of amoxicillin and clavulanic acid in dog plasma, and to evaluate the bioequivalence of domestic amoxicillin-clavulanate potassium tablets and reference preparations orally in dogs. [Methods]Multi reaction monitoring (MRM) of acetonitrile and aqueous formic acid were used as mobile phases and gradient elution was performed on a HSS T3 column. Amoxicillin and clavulanic acid were monitored by electrospray in positive and negative ion modes, respectively. Plasma samples were precipitated with acetonitrile and extracted with dichloromethane. For the plasma sample pretreatment method of clavulanic acid, the step of n-hexane degreasing was added. After methodological validation, the concentrations of amoxicillin and clavulanic acid in plasma samples were detected respectively. The bioequivalence study adopted a single-dose, two-treatment, and two-cycle randomized crossover trial design. Twenty-six healthy adult beagle dogs were selected. The reference preparation or the test preparation 250 mg were orally adminstrated on an empty stomach in order, respectively. The concentrations of amoxicillin and clavulanic acid in plasma were detected by the established UPLC-MS/MS method. Pharmacokinetic parameters of the drug were calculated using the non-compartmental model of the Phoenix Winnonlin 8.1 pharmacokinetic software. The bioequivalence of Cmax,AUC0-t and AUC0-∞ was evaluated after logarithmic conversion. [Results]The specificity of the method was suitable for detecting amoxicillin and clavulanic acid. The signal response values of amoxicillin in the concentration range of 20-10 000 ng·mL-1 and clavulanic acid in the concentration range of 50-5 000 ng·mL-1 had a good linear relationship with concentration (R~2＞0.99) . Intra-batch and inter-batch accuracy deviations and precision were both within 15%. The matrix effect met the requirements. Amoxicillin and clavulanic acid were stable under the test conditions. In the reference preparation and test preparation, the Cmax of amoxicillin was (10 934.87±3 118.79) ng·mL-1 and (11 101.66±3 185.09) ng·mL-1,AUC0-t was (30 983.24±9 395.40) ng·h·mL-1 and (31 646.91±9 319.93) ng·h·mL-1,AUC0-∞ was (31 093.42±9 425.85) ng·h·mL-1 and (31 843.77±9 290.25) ng·h·mL-1,respectively; the Cmax of clavulanic acid was (3 494.90±1 574.11) ng·mL-1 and (3 694.26±1 701.65) ng·mL-1,AUC0-twas (5 652.61±2 409.17) ng·mL-1 and (6 041.81±2 458.42) ng·h·mL-1,AUC0-∞ was (5 730.52±2 392.78) ng·h·mL-1 and (6 122.80±2 469.20) ng·h·mL-1,respectively. The 90% confidence intervals of geometric mean ratio of Cmax,AUC0-t and AUC0-∞ after log conversion all fell within 80.00%-125.00%. There was no significant difference in Tmax by nonparametric test. [Conclusions]The established UPLC-MS/MS method met the requirements of biological sample analysis, and amoxicillin-clavulanate potassium tablet and reference preparation were bioequivalent in dogs.MoreReset [ABSTRACT FROM AUTHOR]