HPLC determination of serum pteridine pattern as biomarkers

Abstract

Pteridinic derivatives are important biomolecules considered as biomarkers for several diseases, especially in cancer and infectious pathologies. A new fluorimetric-HPLC method for the analysis of nine pteridines in human serum has been reported. Two analytical columns composed by C18 porous and fused core particles were assayed and the results compared. Fused core particle column allows us adequate separation, in only one run and in 15min. Acid precipitation step of the proteins and clean-up process with an Isolute ENV+ (hydroxylated polystyrene-divinylbenzene copolymer) cartridge of the serum samples have been optimized. Analytes were determined by fluorimetric detection, exciting at 272nm and measuring the fluorescence emission at 410nm for isoxanthopterin, at 465nm for xanthopterin, and at 445nm for the analysis of the other pteridines. Detection limits between 0.07 and 0.61ngmL−1 were calculated according to Clayton criterium. Intraday precision varied from 1.2 to 5.3 and interday precision between 1.2 and 7.4, both expressed as RSD (%). External standard and standard addition calibrations were compared in the analysis of serum samples. The pteridine amounts in serum (expressed as ngmL−1±confidence interval) were 3.69±1.78; 1.35±0.24; 0.46±0.14; 0.54±0.24; 0.84±0.55; 2.10±0.51 and 0.23±0.11 for XAN, NEO, MON, ISO, BIO and 6HMPT, respectively, using the external standard method. Comparable results were obtained by the standard addition method. It is noticeable that 7BIO was not detected in the healthy serum samples analyzed.