DETECTION AND QUANTIFICATION OF CIMICOXIB, A NOVEL COX-2 INHIBITOR, IN CANINE PLASMA BY HPLC WITH SPECTROFLUORIMETRIC DETECTION: DEVELOPMENT AND VALIDATION OF A NEW METHODOLOGY

Cimicoxib (CX) is a selective COX-2 inhibitor recently launched on the veterinary market. No analytical method to detect CX in biological samples has been published to date. The chromatographic separation was performed with a Kinetex C18 analytical column (100 x 4.6 mm, 2.6 μm particle size) at 25° C. The mobile phase consisted of acetonitrile:buffer (10 mM AcONH4, pH 4.5) (35:65 v/v) at a flow rate of 1 mL/min. Excitation and emission wavelengths were 268 and 430 nm, respectively. The extraction used 500 μL of plasma added to 100 μL of IS (5 μg/mL) and 100 μL of 10% CF3COOH, extracted with 600 μL of C2H2:Et2O (3:7, v/v). The organic phase was evaporated and reconstituted with 200 μL of mobile phase. The CX recovery ranged from 74.5% to 82.6%. The limit of quantification was 25 ng mL−1. The chromatographic runs were specific with no interfering peaks at the retention times of the analytes, as confirmed by HPLC–mass spectrometry experiments. The other validation parameters were in agreement with the international guidelines. The method was successfully tested on two dogs treated at two dose rates. It facilitated tracking of the plasma concentration for 24 h and calculation of the main pharmacokinetic parameters. In conclusion, this method (extraction, separation and applied techniques) is simple, effective and specific. This is the first time that a method for the quantification of CX in plasma has been reported. This technique may have applications for further pharmacokinetic studies