Development and Validation of RP-HPLC method for Estimation of Olopatadine HCl in Rabbit Plasma: Application to Pharmacokinetic Studies

Abstract

Anew, simple, sensitive, accurate, precise, and stable reverse phase liquid chromatographic method was developed for the estimation of olopatadine HCl in rabbit plasma, using a hypersil C18 (5 µm, 2504.6 mm) column and a mobile phase comprising of methanol and water mixture in the ratio of 75:25 (%v/v). This mixture was adjusted to pH 3.0 with trifluor acetic acid. The detection wavelength and retention time for olopatadine HCl obtained were at 254 nm and 5.6 minutes respectively with the optimized chromatographic conditions and internal standard was not used as resolution is good enough for the study. Calibration curve was established for olopatadine HCl in the range of 1-30µg/mL. A good linear relationship was observed as indicated by correlation coefficient (r=0.9999) and the limit of detection was found to be 0.5 µg/mL. The percentage recovery of the suggested method for olopatadine HCl was found to be in the range of 100–101% with low %CV values (<2%). The precision of the method showed low %CV values (< 2%)at intra-day and inter-day estimation of olopatadine HCl. Solution stability studies indicated that solution was stable for 2 days at room temperature(~26ºC).The method developed for estimation of drug in rabbit plasma was found to be reproducible, accurate and precise with good solution stability. Hence, this method can be successfully used for estimation/quantification of olopatadine HCl in rabbit plasma, for pharmacokinetic studies in animal models.