Determination of monoamine oxidase B activity by high-performance liquid chromatography with electrochemical...

A sensitive high-performance liquid chromatography method with electrochemical detection for measuring monoamine oxidase B activity in blood platelets is described. Dopamine is used as substrate and is incubated with isolated platelets and aldehyde dehydrogenase to convert dihydroxyphenylacetaldehyde to dihydroxyphenylacetic acid (DOPAC). The acid and the added internal standard hydrocaffeic acid are separated from dopamine and the incubation mixture by extraction with 5 ml of ethyl acetate-toluene (5:1, v/v). The organic phase is evaporated under nitrogen stream and the residue dissolved in 0.1 M citric acid. Dihydroxyphenylacetic acid and the internal standard dihydrocaffeic acid are then separated on a Eurosphere 100-C18 5 microns column. The mobile phase used 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 recovery of DOPAC was 85 +/- 3.8% and of hydrocaffeic acid 87 +/- 4.1%. The method presented is sensitive (detection limit 8.0 pg of DOPAC injected) and reproducible (coefficient of variation 0.4-1%) with good accuracy (94-98%).