Analysis of Folate Form Distribution by Affinity Followed by Reversed-Phase Chromatography with Electrochemical Detection

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Abstract
Background: Naturally occurring folates exist in multiple forms, differing in pteridine ring structure and number of glutamate residues. The ability to measure these folate coenzymes in tissues and cells gives important information about in vivo folate metabolism. Methods: Folates were heat-extracted from biological samples. A two-column HPLC system with four-channel coulometric electrochemical detection was used for analysis. An affinity column was used first to purify folates from the extract. Purified folates were eluted from the affinity column onto a phenyl analytical column, utilizing a switching valve, and folate forms were separated using an acetonitrile gradient. Results: Folate forms differing in pteridine ring structure and number of glutamate chain residues were identified by retention time and characteristic response across the channels of the detector. Folates were quantified by comparison to an external calibration mixture. Limits of detection for pentaglutamyl folates ranged from 0.21 pmol for tetrahydrofolate to 0.41 pmol for 5-methyltetrahydrofolate. CVs (n = 5) for peaks containing 9–67 pmol of folate were 0.6–6.4% (within day) and 5.2–8.4% (between days). CVs (n = 5) for peaks containing 0.9–3.5 pmol folate were 5.7–16% (within day) and 8.4–13% (between days). Conclusions: This automated HPLC system allows the simultaneous determination of polyglutamyl forms of folates from biological samples, including tetrahydrofolate, 5-methyltetrahydrofolate, formylated folates, and pteroylglutamate. The low detection limits allow analysis of folate form distribution in human samples such as erythrocytes and lymphocytes.