High performance liquid chromatography of folates: identification of poly-gamma-glutamate chain lengths of labeled and unlabeled folates.

A sensitive method is described for the identification of the polyglutamate chain lengths of labeled and unlabeled folates in biological extracts. Folates in bacterial or mammalian tissue extracts were quantitatively cleaved to rho-aminobenzoylpolyglutamates. The primary aromatic amines were converted to azo dyes of naphthylethylene diamine and were purified by chromatography on BioGel P2 polyacrylamide columns. The purified azo dyes were reductively reconverted to rho-aminobenzoylpolyglutamates, which were separated according to glutamate chain length by high performance liquid chromatography on a Partisil 10 SAX anionic exchanger. Unlabeled derivatives derived from tissue folates were detected and quantitated by their absorbance at 280 nm. Lactobacillus casei contained folates of glutamate chain length up to 11 with the octa- and nonaglutamates predominating, while tetraglutamates predominated in Streptococcus faecalis. Vitamin in rat liver was a mixture of pteroylmono- to heptaglutamate with the pentaglutamate predominating.