Estimation of tetrahydro, dihydro and fully oxidised pterins by high-performance liquid chromatography using sequential electrochemical and fluorometric detection

A method is described for the separation and detection of tetrahydro, dihydro and fully oxidised pterins in a single chromatographic run using ion-pair reversed-phase high-performance liquid chromatography. Tetrahydropterins are detected by electrochemical oxidation, dihydropterins by fluorescence following post-column electrochemical oxidation and the fully oxidised pterins by their natural fluorescence. The post-column electrochemical conversion of the non-fluorescent dihydropterins to fluorescent compounds is proportional to the amount injected over three orders of magnitude. Because of the relative selectivity of the fluorescence detection and the low potential required to oxidise the tetrahydropterins, all the oxidation species of the pterins may be measured in biological samples with minimal sample clean-up.