DETERMINATION OF PILOCARPINE IN AQUEOUS-HUMOR BY LIQUID-CHROMATOGRAPHY ATMOSPHERIC-PRESSURE CHEMICAL-IONIZATION MASS-SPECTROMETRY

A new method has been developed for rapid analysis and determination o f pilocarpine in aqueous humour using liquid chromatography-atmospheri c pressure chemical ionization mass spectrometry. The chromatography w as carried out on a reversed-phase phenyl column with 0.1% acetic acid -acetonitrile (95.5, v/v). Pilocarpine and its analogues, isopilocarpi ne, pilocarpic acid and isopilocarpic acid, were separated. An aqueous humour sample was deproteinized with methanol. After evaporation, the residue was dissolved in the mobile phase. The method was applied to the analysis of the metabolite in aqueous humour after the topical app lication of 2% pilocarpine (w/v) eye-drops. The main metabolite, piloc arpic acid, was easily identified. The protonated molecular ion of pil ocarpine was used for the determination. The calibration curve had a g ood linearity within the concentration range investigated (2 ng to 10 mu g/ml). The limit of determination was estimated to be an aqueous hu mour concentration of ca. 2 ng/ml. The method was applied to the deter mination of unchanged pilocarpine after the topical application of 2% pilocarpine (w/v) eye-drops.