Analysis of some folate monoglutamates by high-performance liquid chromatography-mass spectrometry. I

The use of high-performance liquid chromatography for the determination of folates is well documented. The methods used are based on reversed-phase chromatography with UV and/or fluorescence detection. In many instances it is difficult to reach the required detection limits and many of the methods lack specificity. High-performance liquid chromatography-mass spectrometry (LC-MS) has become a well established analytical tool in: modern laboratories. It can offer superior specificity and often lower detection limits than conventional LC detection methods and thus is ideally suited to the analysis of folates. A system capable of separating the four main folates [folic acid (pteroylglutamic acid, PGA)I, 5-methyltetrahydrofolic acid, terahydrofolic acid and 5- and/or 10-formyltetrahydrofolic acid [folinic acid (CHOTHF)] using LC-MS with negative ion electrospray has been developed. After optimisation, a system using a 12.5 cmx3 mm, 3 mu m Hypersil ODS column and a mobile phase of 2.5 mM acetic acid-acetonitrile (88:12, v/v) was developed which was capable of separating the four folates in under 10 min without the use of a gradient system. Extraction of the folates is by heat treatment and sample clean-up is by solid-phase extraction (C-18). On column limits of confirmation are 0.6 ng for PGA and CHOTHF. (C) 1999 Elsevier Science B.V. All rights reserved.