Trace enrichment of biological folates on solid-phase adsorption cartridges and analysis by high-pressure liquid chromatography

Abstract

A procedure involving solid-phase adsorption on bonded silica has been developed for trace enrichment and selective recovery of folate monoglutamates from liver tissue. A variety of reverse-phase (ethyl, octyl, octadecyl, phenyl) and anion-exchange (aminopropyl, quarternary amine, primary/secondary amine) cartridges were tested for their potential to adsorb and elute folate monoglutamates from standard solutions (50 nmol each of H 4-pteroylglutamic acid (H 4PteGlu), 5-CHO-H 4PteGlu, 10-CHO-H 4PteGlu, PteGlu, and 5-CH 3-H 4PteGlu). Quantitative recoveries were obtained from aminopropyl (−NH 2) and all reverse-phase cartridges. For the analyses of rat liver folates, 20 ml of clear supernatant obtained from 5 g of tissue was treated with conjugase, which released folate monoglutamates from endogenous stores. Folate monoglutamates were then separated from nonfolate material by selective adsorption and recovery from −NH 2 extraction cartridges. The procedure also provided a 10-fold concentrate, which allowed direct analysis by HPLC, using C-18 reverse-phase ion-pair columns coupled with uv detection (290 nm). Experiments with standard folates ( n = 3) mixed with liver tissue and carried through the extraction, incubation, and trace-enrichment steps showed the following recoveries: 10-CHO-H 4PteGlu, 55 ± 5.0%; H 4PteGlu, 80 ± 5.0%; 5-CHO-H 4PteGlu, 123 ± 12.0%; and 5-CH 3-H 4PteGlu, 89 ± 3.0%. Endogenous compositions of liver folates ( n = 5) were as follows: 10-CHO-H 4PteGlu, 1.03 ± 0.3 nmol/g (6.7%); H 4PteGlu, 5.70 ± 1.0 (36.4%); 5-CHO-H 4Pte Glu, 1.34 ± 0.4 (8.7%); and 5-CH 3-H 4PteGlu, 7.34 ± 1.2 (48.0%). Chromatographic peaks were identified by their retention times and by comparing their spectral profiles (obtained by a diode array detector) with respective pure folates. We found trace enrichment of biological folates on solid-phase extraction cartridges to be rapid and quantitative. The method allowed, for the first time, direct analysis of tissue folates by HPLC uv methods.