Direct determination of folate monoglutamates in plasma by high-performance liquid chromatography using an automatic...

A simple and rapid technique for the simultaneous isolation and analysis of folate monoglutamates (folic acid, 7,8-dihydrofolic acid, 5,6,7,8-tetrahydrofolic acid and 5-formyl-, 5-methyl- and 10-formyl-5,6,7,8-tetrahydrofolic acids) was developed using reversed-phase high-performance liquid chromatography with an automatic precolumn-switching system. The plasma or the dissolved diet samples were directly injected onto a short precolumn flushed with 50 mM phosphate buffer. The folate vitamers absorbed on the precolumn were backflushed onto the analytical column with a 25 mM phosphate buffer containing 5% methanol and then detected by UV absorption at 280 nm. A linear response was found between the injected sample amounts and the integrated areas for all vitamers analysed. The detection limit was 1-10 pmol and the precision ranged from 1.6 to 10%, depending on the metabolite studied. The recovery rates of folates in plasma were 90-95%. Decomposition of the unstable folates was avoided. Our method was applied to the analysis of mouse plasma and animal diets.