Analysis of pteridines and creatinine in urine by HPLC with serial fluorimetric and photometric detectors

Although pteridines are excreted in the urine of normal healthy persons, levels of some of these compounds seem to be modified by several diseases. Creatinine is considered to be the best reference for study of the excretion of these markers in urine. A fast, very sensitive, and precise HPLC method with ultraviolet and fluorimetric detection has been established for the simultaneous determination of pterin-6-carboxylic (CAR), neopterin (NEO), xanthopterin (XAN), isoxanthopterin (ISO), biopterin (BIO), and creatinine (CREA) in urine. The influence of conditions such as mobile-phase composition and flow rate, have been studied. 0.015m Tris-HCl-10−3m NaCl buffer solution, pH 6.8, was selected as mobile phase. These five pteridine derivatives are well resolved in less than six minutes. CREA is determined by photometric detection at 230 nm and pteridines by fluorimetric detection at 444 nm, with excitation at 280 nm. The detection limits are below 25 pg for all pteridines and 0.30 ng for creatinine. The method has been applied to the determination of these compounds in urine. A non-clean-up concentration step is necessary before the separation. The urine sample is oxidized before HPLC injection. Values for the pteridine-to-creatinine ratios found for two groups of healthy persons (younger and older than 30 years) are reported.