Simplified HPLC methodology for quantifying biological pterins by selective oxidation

- The methodology developed is based on a principle of differential oxidation, which presents the best performance with mobile phase water 95%/methanol 5% and C18 column.
- It is emphasized the use of calibration curves to pH 3 and 9, and 2 calibration curves to these pH because calibration curves vary according to pH and therefore also the limits of detection/quantification.
- The performance of the methodology developed, according to “spiked” method, scored an average of 80%.
- The proposed methodology for quantification of BH4, can be used to assess levels of tetrahydrobiopterin in pathologies where any alteration of this cofactor exists.

Tetrahydrobiopterin (BH4) 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 BH4 and its related biopterins, a procedure that involves differential oxidation is currently used, which measures biopterin (the product of the oxidation of BH4 and BH2) at two different pH conditions to calculate the quantity of BH2 and BH4, using high performance liquid chromatography (HPLC).In this work, a method was established in order to quantify BH4 and BH2 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 0.9 mL/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 BH4/BH2 ratio in biological samples.