Rapid and sensitive method for high-performance liquid chromatographic analysis of pterins in biological fluids

A rapid and sensitive high-performance liquid chromatographic (HPLC) method for the analysis of the most important urinary pterins is described. The method involves a preliminary sample oxidation to stabilize and convert pterins into their fluorescent forms and a purification by anion-exchange chromatography, followed by a short reversed-phase HPLC separation with fluorometric detection and quantitation of the different pterins. A complete HPLC analysis is accomplished in as little as 15 min. The sensitivity of the method allows the detection of as little as 20 pg of each pterin with a mean recovery greater than 99% for all pterins analysed. Reference values were obtained from 50 normal babies aged between 1 and 120 days. A significant correlation was found between urinary biopterin levels and the age of the babies (r = 0.445), while neopterin did not show any significant correlation with age. The "biopterin neopterin creatinine ratio" (BNCR index) was also significantly correlated with the age of the babies (r = 0.428). This rapid and sensitive method for pterin determination in biological fluids may be useful in the differential diagnosis of the various hyperphenylalaninemic conditions identified by neonatal mass screening programmes.