Simultaneous determination of cefatrizine and clavulanic acid in dog plasma by HPLC

A rapid and specific high-performance liquid chromatographic method was developed and validated for the simultaneous determination of cefatrizine and clavulanic acid in the plasma of beagle dog. The sample pretreatment procedure involved reaction of clavulanic acid with 1,2,4-triazole, which readily produced a derivative with its maximum UV absorption at 314nm. This derivative was separated in a reverse-phase C-18 column without being interfered by other components present in plasma. Cefatrizine, however, was not derivatized and, therefore determined directly at 269nm. Sulfanilamide was used as an internal standard. The retention times of sulfanilamide, the derivative, and cefatrizine were, 3.5, 4.9, and 6.0min, respectively. The assay showed linearity from 2 to 100μg/ml for cefatrizine and from 1 to 50μg/ml for clavulanic acid. Precision expressed as R.S.D. ranged from 4.2 to 18.2% for cefatrizine and 5.5 to 15.8% for clavulanic acid. Accuracy ranged from 97.9 to 120% (lower limit of quantitation) for cefatrizine and from 97.7 to 119.2% for clavulanic acid. Extraction efficiencies for cefatrizine, clavulanic acid, and internal standard from dog plasma averaged 79.8±5.8%, 84.8±6.2%, and 89.0±3.8%, respectively. This method was employed successfully to follow the time course of the concentration of cefatrizine and clavulanic acid in beagle dogs following oral administration of cefatrizine and clavulanic acid.