Gradient high-performance liquid chromatographic analysis of enkephalin peptides, their metabolites, and enzyme inhibitors using combined ultraviolet and electrochemical detection II. Application to ocular permeability studies in vitro.

A gradient HPLC method with combined ultraviolet (UV) and electrochemical detection (ED) was used to study the ocular permeability of [D-Ala2]-methionine enkephalinamide (MEA) in vitro. Coulometric ED was selective for MEA and its tyrosine-containing metabolites with quantitation limits between 20 and 60 nM (1.0-3.0 pmol per 50-microl injection), whereas UV detection at 205 nm allowed the determination of several aromatic metabolites and enzyme inhibitors with quantitation limits between 40 and 500 nM (2.0-25.0 pmol). This method was capable of detecting permeability of MEA and metabolite formation in the cornea and conjunctiva in vitro. Furthermore, effects of aminopeptidase inhibitor bestatin and enkephalinase inhibitor SCH 39370 on permeation and metabolism of MEA could be determined.