A specific, accurate, precise and reproducible HPLC method has been developed and subsequently validated for the simultaneous determination of timolol maleate and pilocarpine nitrate in ophthalmic formulation. The proposed HPLC method utilizes BDS hypersil (thermo scientific) C18 column (250 mm × 4.6 mm id, 5 μm particle size) and mobile phase consisting of methanol: 0.02 M phosphate buffer [60: 40, v/v] and pH adjusted to 5.0 with NaOH at a flow rate of 1.0 ml/min. Quantitation was achieved with UV detection at 230 nm based on peak area with linear calibration curves at concentration ranges 5-15 μg/ml for timolol maleate and 20-60 μg/ml for pilocarpine nitrate. The retention time of timolol maleate and pilocarpine nitrate were found to be 3.467 min and 6.08 min respectively. The method was validated in terms of accuracy, precision, linearity, limits of detection, limits of quantitation and robustness. This method has been successively applied to ophthalmic formulation and no interference from the formulation excipients was found.
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