[58] Separation of folic acid derivatives and pterins by high-performance liquid chromatography

This chapter discusses the separation of folic acid derivatives and pterins, by high-performance liquid chromatography. For this study, folic acid, 7,8-dihydrofolic acid, 5,6,7,8-tetrahydrofolic acid, 5-methyltetrahydrofolic acid, and p-aminobenzoylglutamic acid were purchased from Sigma Chemical Co. 5,10-Methenyltetrahydrofolic acid and 10-formyltetrahydrofolic acid were prepared by the method of Rabinowitz. The 5,10-methylenetetrahydrofolic acid was prepared by the method of Huennekens et al. N(10)‑nitrosofolic acid was prepared by the method of Cosulich and Smith. 2‑Hydroxy-N(10)-nitrosofolic acid and 2-hydroxyfolic acid were prepared by the method of Angier et al. Xanthopterin, pterin, and pterin-6-carboxylic acid were purchased from Sigma. Pterin-6-carboxaldehyde was prepared by the method of Waller et al. Because most reduced derivatives of folic acid are extremely labile to oxidation, by air, 2-mercaptoethanol is normally added to the solutions, containing 7,8-dihydro and 5,6,7,8-tetrahydro derivatives. 2-Mercaptoethanol absorbs at 254 nm and interferes with the detection of chromatographic peaks. This chapter discusses the separation of the three oxidation states of folic acid. Separation of N(5)-and N(10)-substituted tetrahydrofolates is described in the chapter. Separation of the products of nitrosation of folic acid is also elaborated in the chapter in detail.