Separation of unconjugated pteridines by high-pressure cation-exchange liquid chromatography.
In the course of determining the levels of unconjugated pteridines occurring in various biological fluids, such as urines, plasma and tissue culture media, a method has been developed for the separation and quantitative determination in the picomole range of ten 2-amino-4-hydroxy substituted pteridines. This method involves separation by high-pressure cation-exchange liquid chromatography and fluorescence detection of the eluted compounds at 450 nm. Optimal separation was obtained by isocratic elution with 3 mM phosphoric acid-7% methanol-1% acetonitrile at a flow-rate of 2 ml/min or with 1 mM ammonium dihydrogenphosphate pH 2.8-7% methanol-5% acetonitrile at a flow-rate of 1.5 ml/min. With either solvent, the order of elution of the compounds is: isoxanthopterin, pterin-6-carboxylic acid, xanthopterin, pterin-6-carboxaldehyde, D-erythro-neopterin, L-threo-neopterin, biopterin, 6-hydroxymethylpterin, pterin, 6-methylpterin. In addition, a systemic investigation of the effects of ammonium ion concentration and pH of the solvent as well as column temperature on the separation of these compounds was also conducted.
D002411:Cation Exchange Resins; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D008722:Methods; D008832:Microchemistry; D011621:Pteridines / Q000302:isolation & purification*
D002411:Cation Exchange Resins; D011621:Pteridines