An enzymatic assay for the MAO-B inhibitor selegiline in plasma

A sensitive fluorimetric assay based on inhibition of rat brain monoamine oxidase-B (MAO-B) in vitro has been described. The procedure measures the inhibition of MAO activity produced by the addition of selegiline extracted from human plasma. This method uses the substrate kynuramine which is converted by MAO to the product 4-hydroxyquinoline which fluoresces in alkaline solution. Human plasma (500 microliters) containing different concentrations of selegiline was deproteinized and extracted with ethyl acetate-butyl chloride. After reconstitution with 200 microliters phosphate buffer, 50 microliters of rat brain homogenate was added to study the MAO-B inhibition. Selegiline metabolites, amphetamine and methamphetamine (50 ng ml-1), and desmethylselegiline (20 ng ml-1), showed no inhibitory effect on MAO-B inhibition. Selegiline concentrations as low as 0.25 ng ml-1 can be detected. The standard curve was linear from 125 pg (0.25 ng ml-1) to 4000 pg (8.0 ng ml-1) in the incubation tube. This method should be helpful to determine pharmacokinetic parameters of selegiline after i.v. or oral dosing.