OBJECTIVETo develop an RP-HPLC method for assay of pilocarpine in rabbit ocular aqueous humor.

METHODSThe RP-HPLC method was performed on a column of ODS-C(18) with the mobile phase consisting of 0.5% of triethylamine (TEA) of phosphate solutions (10 mmol/L, pH 2.5) and acetonitrile (98/2,v/v). The detection wavelength was 215 nm and flow rate was 1.0 ml/min. Ninety albino rabbits were divided into 3 groups (30 in each):group 1 received 50 microl of eye drops containing 1% generic pilocarpine, group 21% mixture pilocarpine solution consisting of aqueous sample and liposome and group 31% liposome pilocarpine, respectively. The aqueous humor was withdrawn at 5, 10, 30, 40, 60, 90, 120, 180, 240 and 360 min. Pilocarpine was extracted from aqueous humor with dichloromethane.

RESULTThe linear calibration curve was obtained in the concentration range of 0.1 - 20 microg/ml. The average recovery was (68.1+/-2.7)% (n=9). Inter-day and intra-day RSD were 4.33% and 2.87%, respectively. In three formations 1% liposome pilocarpine was the best for the areas under curve and measurable amounts.

CONCLUSIONThe RP-HPLC method is simple and reliable for pilocarpine measurement in ocular aqueous. Liposome formulation can significantly increase the bioavailability of pilocarpine in ocular aqueous.