DETERMINATION OF MONOAMINE-OXIDASE-B ACTIVITY BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH ELECTROCHEMICAL DETECTION

A sensitive high-performance liquid chromatography method with electro chemical detection for measuring monoamine oxidase B activity in blood platelets is described. Dopamine is used as substrate and is incubate d with isolated platelets and aldehyde dehydrogenase to convert dihydr oxyphenylacetaldehyde to dihydroxyphenylacetic acid (DOPAC). The acid and the added internal standard hydrocaffeic acid are separated from d opamine and the incubation mixture by extraction with 5 mi of ethyl ac etate-toluene (5:1, v/v). The organic phase is evaporated under nitrog en stream and the residue dissolved in 0.1 M citric acid. Dihydroxyphe nylacetic acid and the internal standard dihydrocaffeic acid are then separated on a Eurosphere 100-C-18 5 mu m column. The mobile phase use d was a mixture of sodium acetate, citric acid, and acetonitrile at pH 2.5. The standard curve was linear from 125 pg to 10 ng. Absolute rec overy of DOPAC was 85 +/- 3.8% and of hydrocaffeic acid 87 +/- 4.1%. T he method presented is sensitive (detection limit 8.0 pg of DOPAC inje cted) and reproducible (coefficient of variation 0.4-1%) with good acc uracy (94-98%).