Determination of pilocarpine in aqueous humour by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.

A new method has been developed for rapid analysis and determination of pilocarpine in aqueous humour using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry. The chromatography was carried out on a reversed-phase phenyl column with 0.1% acetic acid-acetonitrile (95:5, v/v). Pilocarpine and its analogues, isopilocarpine, 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 application of 2% pilocarpine (w/v) eye-drops. The main metabolite, pilocarpic acid, was easily identified. The protonated molecular ion of pilocarpine was used for the determination. The calibration curve had a good linearity within the concentration range investigated (2 ng to 10 micrograms/ml). The limit of determination was estimated to be an aqueous humour concentration of ca. 2 ng/ml. The method was applied to the determination of unchanged pilocarpine after the topical application of 2% pilocarpine (w/v) eye-drops.