LC determination of biopterin reduced forms by UV-photogeneration of biopterin and fluorimetric detection

An off-line photoirradiation LC fluorimetric method to determine tetrahydrobiopterin (BH4), by photogeneration of biopterin (BIO), is described, as an alternative way to the chemical oxidation procedure. To minimize the uncontrolled BH4 oxidation, due to environmental oxygen, an antioxidant, dithiothreitol (DTT), was used. The acidity of the medium, as well as the presence of hydrogen peroxide, affects the rate of the photoreaction and the nature of the obtained fluorescent photoproducts. The best conditions were achieved by irradiation in hydrochloric acid (0.2 M) medium, in presence of 100 mM hydrogen peroxide, and using an irradiation time of 20 min. The method was tested in the analysis of serum samples containing BH4, and recoveries between 89 and 105% were found. Also, the proposed method allows the resolution of BH4 and BIO, in the same sample, by injection of non-irradiated and irradiated sample aliquots in the chromatographic system.