Determination of pterins in urine by HPLC with UV and fluorescent detection using different types of chromatographic stationary phases (HILIC, RP C8, RP C18)

Pterins are a class of potential cancer biomarkers. New methods involving hydrophilic interaction liquid chromatography (HILIC) and reversed phase (RP) high-performance liquid chromatography have been developed for analysis of eight pterin compounds: 6,7-dimethylpterin, pterin, 6-OH-methylpterin, biopterin, isoxanthopterin, neopterin, xanthopterin, and pterin-6-carboxylic acid. The effect of mobile phase composition, buffer type, pH and concentration on retention using HILIC, C-8 and C-18 RP stationary phases were examined. Separation of pterins on RP and HILIC stationary phase was performed and optimized. Eight pterins were successfully separated on HILIC Luna diol-bonded phases, Aquasil C-18 RP column and LiChrospher C-8 RP column. Determination and separation of the pterins from urine samples were performed on HILIC Luna and LiChrospher C-8 RP columns which were chosen as the most appropriate ones. Finally, LiChrospher C-8 RP column with fluorescence detection was selected for further validation of the method. The optimum chromatographic condition was mobile phase methanol (A)/phosphoric buffer pH 7, 10 mM (B), isocratic elution 0-15 min 5% A flow = 0.5 ral/min 15-17 min. 5% A, flow = 0.5-1 ml/min the linearity (R-2 >0.997) and retention time repeatability (RSD% <1) were at satisfactory level. The precision of peak areas expressed as RSD in % was between 0.55 and 14. Pterins detection limits varied from 0.041 ng/ml to 2.9 ng/ml. Finally, HPLC method was used for the analysis of pterins in urine samples with two different oxidation procedures. Concentration levels of pterin compounds in bladder cancer patients and healthy subjects were compared. (C) 2013 Elsevier B.V. All rights reserved.