Simultaneous determination of ZLR-8 and its active metabolite diclofenac in dog plasma by high-performance liquid chromatography.

ZLR-8 is a nitric oxide releasing derivative of diclofenac for the treatment of inflammation. In this paper, a sensitive and reliable high-performance liquid chromatography method for simultaneous determination of ZLR-8 and its active metabolite diclofenac in the plasma of beagle dogs has been developed and validated. After the addition of ketoprofen as the internal standard (IS), plasma samples were extracted with n-hexane-isopropanol (95:5, v/v) mixture solution and separated by HPLC on a reversed-phase C(18) column with a mobile phase of gradient procedure. Analytes were determined by the UV detector which was set at 280 nm. The method was proved to be sensitive and specific by testing six different plasma batches. Calibration curves of ZLR-8 and diclofenac were linear over the range 0.05-4.0 microg/mL. The within- and between-batch precisions (RSD%) were lower than 10% and accuracy ranged from 85 to 115%. The lower limit of quantification was identifiable and reproducible at 0.05 microg/mL. The proposed method has been readily implemented in preclinical pharmacokinetics studies of ZLR-8 and its active metabolite diclofeance. Representative plasma concentration vs time profiles resulting from administration of ZLR-8 to beagle dogs are presented in this communication.