Simultaneous determination of twelve polar pteridines including dihydro- and tetrahydropteridine in human urine by hydrophilic interaction liquid chromatography with tandem mass spectrometry.

Pteridines and their derivatives are important cofactors in the process of cell metabolism, and the level of urinary excretion of these compounds is considered as an important clinical criterion. In this work, a new separation method involving hydrophilic interaction chromatography (HILIC) with tandem mass spectrometric detection has been developed for the simultaneous analysis of 12 pteridines including oxidized, di- and tetrahydroforms, namely neopterin, 7,8-dihydroneopterin, biopterin, 7,8-dihydrobiopterin, 5,6,7,8-tetrahydrobiopterin, dimethylpterin, dimethyltetrahydropterin, pterin, isoxanthopterin, xanthopterin, sepiapterin and pterin-6-carboxylic acid, in human urine without oxidative pretreatments. The stabilizing agent (dithiothreitol) at various concentrations and the stability of oxidized, di- and tetrahydroforms during the sample's short-term storage and processing and of the extracts were tested. In the developed method, 12 pteridines were chromatographically separated on an ZIC-HILIC column by gradient elution, and the run time was 20 min. Matrix effect was evaluated and several dilutions of urine were tested in order to study the evolution of signal suppression. Spiked recovery studies demonstrated that the technique was both accurate (83.1-116.7%) and precise (RSD 1.4-15.6%). Finally, several clinical urine specimens without oxidative pretreatments were examined with the new technique and compared with previous reports.