Analysis of pilocarpine and isopilocarpine in ophthalmic solutions by normal-phase high-performance liquid chromatography.

A normal-phase high-performance liquid chromatographic separation for pilocarpine and isopilocarpine was developed which is suitable for the routine analysis of ophthalmic preparations. The method utilizes a column packed with 5-micron silica with a mobile phase of hexane-2% ammonium hydroxide in 2-propanol (70:30). Peak detection is by UV at 220 nm. It is suggested that a partition separation mechanism is involved rather than adsorption. A separation factor (alpha) of 1.17 was obtained with a relative separation (Rs) of 2.13. Column lifetimes were typically 6-8 months with daily use. Several standard pilocarpine-isopilocarpine mixtures and five commercially available ophthalmic solutions from different manufacturers were analyzed. The method was specific for pilocarpine and isopilocarpine in the presence of each other and pilocarpine acid and is a significant improvement over the nonspecific colorimetric methods of analysis.