Rapid analysis of substituted and unsubstituted pteroyl-oligo-y-L-glutamates at the nanomole level is carried out by high performance liquid chromatography. The use of a siliceous microparticulate anion-exchanger column and gradient elution at pH 6.5 with increasing salt concentration facilitates the separation of the species containing up to seven glutamyl residues without decomposition in 30 min. The column effluent is monitored with a uv detector at 254 nm, and the peaks are conveniently identified by their retention.
The folate coenzyme forms present in biological material, usually in submicromolar concentrations, differ in their state of oxidation, substitution with one carbon units at the N5 and Nl" position, and the number of glutamyl residues present (1). For many years the identification of these folate forms in crude extracts was carried out by means of microbiological assays using the folate-requiring bacteria,l. casei, P. Cerevisiue , and S. fuecalis and involving the prior hydrolysis of any oligoglutamate forms with glutamate carboxypeptidase (EC 3.4.12.10) to the mono-or diglutamyl level. The recent chemical synthesis of authentic oxidized pteroyl polyglutamates
(2,3,4) enables their use as markers on column chromatography through Sephadex gel filtration (5), DEAE and TEAE cellulose (6,7,8), and DEAE-Sephadex (9). This communication presents a rapid, sensitive, analytical method using high performance liquid chromatography for the separation of various folate forms, including oligoglutamates of varying chain lengths, at high resolution within 30 min. The reproducibility of the technique allows the identification of the individual components by their retention times.