Separation of pteridines from blood cells and plasma by reverse-phase high-performance liquid chromatography

In order to determine free and conjugated pteridines, the pteridine-releasing procedure for biological materials (e.g., plasma, blood cells, bone marrow) was optimized and the parameters which influence pteridine releasing are discussed. Using reverse-phase high-performance liquid chromatography (rp-HPLC) on 5-micron ODS (C18; mobile phase, K2HPO4 buffer, pH 7.0-7.8), up to 10 pteridines (pterin-6-carboxylic acid, xanthopterin, neopterin, monapterin, isoxanthopterin, lumazine, biopterin, 6-hydroxymethylpterin, pterin) could be separated in the extracts of blood samples and determined fluorometrically (femtomolar range). In addition, three sepiapterins (sepiapterin, deoxysepiapterin, 3'-hydroxysepiapterin) could be separated with aqueous methanol as eluent (uv and fluorometric detection, respectively). The methods described are suited to compile pteridine patterns for plasma and individual cell fractions (erythrocytes, lymphocytes, buffy coat) from blood. Moreover, it was shown that the pteridine concentrations in dog blood (beagles) were in some cases substantially higher than in human blood, and that neopterin is lacking in the blood cell fractions of beagle dogs or occurs only in very low concentrations.