Simplified HPLC methodology for quantifying biological pterins by selective oxidation.

Tetrahydrobiopterin (BH) has become a potential therapeutic tool to treat cardiovascular diseases, since it is an essential cofactor of nitric oxide synthase. In order to quantify the amount of BH and its related biopterins, a procedure that involves differential oxidation is currently used, which measures biopterin (the product of the oxidation of BH and BH) at two different pH conditions to calculate the quantity of BH and BH, using high performance liquid chromatography (HPLC). In this work, a method was established in order to quantify BH and BH by adapting previously described procedures. Several chromatographic conditions were evaluated to define the most convenient methodology. Four types of mobile phases and two different analytical columns were used for HPLC. Additionally, calibration curves were made in acid and basic pH compatible with the differential oxidation method. Each method was suitable for quantification purposes, but the choice was based on an economic factor. The selected condition was a mobile phase of 95% water/5% methanol using a C18 column at 35°C at a flow rate of 0.9mL/min. Then, it was calculated the recovery rate, which was about 80% using the chosen method. The aim of this work was to establish a simplified method of differential oxidation, compatible with matrixes such as cardiac tissue in order to facilitate the assessment of the BH/BH ratio in biological samples.