A Simplified and Rapid High-Performance Liquid Chromatographic Assay for Pilocarpine Hydrochloride

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Abstract
A high-performance liquid chromatographic (HPLC) procedure for determination of pilocarpine hydrochloride in aqueous-based pharmaceutical formulations has been developed. Previously reported HPLC assays for pilocarpine hydrochloride involve the use of very low pH (pH 2.5) and a high buffer concentration (5% w/v), both of which can shorten HPLC column life. The present assay for pilocarpine hydrochloride utilizes a reversed-phase ODS column and ultraviolet detection at 220 nm. Clonidine hydrochloride is used as an internal standard in the procedure. The mobile phase consists of pH 3.5, 0.5% w/v phosphate buffer—methanol (70:30) used at a flow rate of 1.25 mL/min. The calibration curves are linear (r ≥ 0.995) over the 4 to 100 μg/mL range. The retention times for pilocarpine hydrochloride and the internal standard are 2.51 and 4.25 min, respectively. The run time per sample is 6 min. The minimum detectable concentration of pilocarpine hydrochloride is 100 ng/mL. The assay is simple, rapid, and reproducible. Details of the specificity, accuracy, reproducibility, and linearity of the HPLC assay are described.

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