Abstract
Retention characteristics of ultra‐HPLC (UHPLC) hybrid stationary phases (bridged ethyl hybrid (BEH) and BEH Amide) were studied in hydrophilic interaction chromatography system in the group of polar basic pteridines (neopterin, biopterin, dihydroneopterin and dihydrobiopterin). The effect of mobile phase composition, buffer type, pH and concentration on retention of pteridines were examined in detail under UHPLC‐fluorescence detection and UHPLC‐MS conditions. The selectivity, retention properties and column performance were examined. BEH HILIC did not provide sufficient retention and selectivity for the separation of four pteridines under any tested conditions. BEH Amide provided strong retention for all pteridines especially at high pH values such as 9.8. However, at pH 9.8 the selectivity of separation for the pairs neopterin‐dihydroneopterin and biopterin‐dihydrobiopterin substantially decreased and resulted in very long analysis time. The best separation of four pteridine derivatives was obtained in the pH range 4.8–7.8 within a reasonable analysis time up to 8 min for UHPLC‐fluorescence detection using higher concentrations of ammonium acetate buffer and up to 4 min for UHPLC‐MS using lower concentrations of ammonium acetate.