Determination of quinonoid dihydrobiopterin by high-performance liquid chromatography and electrochemical detection.

Sodium bisulphite is shown to react with quinonoid dihydrobiopterin to form a stable adduct. Sodium bisulphite does not react with tetrahydrobiopterin. Quinonoid dihydrobiopterin reacts with dithioerythritol to form tetrahydrobiopterin, whereas the quinonoid dihydrobiopterin bisulphite adduct does not. Using these properties we have developed an indirect method for the quantitative measurement of quinonoid dihydrobiopterin. The method requires division of a sample into two. Dithioerythritol is added to one half (a). This converts quinonoid dihydrobiopterin to tetrahydrobiopterin and prevents the oxidation of tetrahydrobiopterin. Measurement of the tetrahydrobiopterin content of this sample by electrochemistry following high-performance liquid chromatographic separation (with dithioerythritol present in the mobile phase to prevent autoxidation of the tetrahydrobiopterin on column), therefore provides a total value of the tetrahydrobiopterin plus quinonoid dihydrobiopterin present within the original sample. Sodium bisulphite is added to the other portion of the sample (b), followed immediately by dithioerythritol which prevents autoxidation of the remaining tetrahydrobiopterin. The bisulphite reacts with the quinonoid dihydrobiopterin present and the quinonoid dihydrobiopterin-bisulphite adduct is no longer detected by electrochemistry at the retention time of tetrahydrobiopterin. Using reversed-phase high-performance liquid chromatography and redox electrochemical detection, measurement of tetrahydrobiopterin in the absence (a) and presence (b) of bisulphite enables the concentration of quinonoid dihydrobiopterin to be calculated by subtraction (a - b). This method is shown to be quantitative and preliminary experiments demonstrate that it can be adapted for biological samples.