Determination of folates by HPLC‐chemiluminescence using a ruthenium(II)–cerium(IV) system,and its application to pharmaceutical preparations and supplements

A chemiluminescence (CL) reaction of folic acid (FA) with ruthenium (II) and cerium (IV) was applied to quantify FA‐related compounds such as FA, dihydrofolic acid, tetrahydrofolic acid, 5‐methyltetrahydrofolic acid, 5‐formyltetrahydrofolic acid and methotrexate (MTX). Among the FAs, 5‐methyltetrahydrofolic acid provided the highest CL intensity. HPLC‐CL detection of FA was applied to quantify FA in pharmaceutical preparations and supplements. Analytical samples were separated on a semi‐micro ODS column with a mixture of 20 mM phosphate buffer (pH 5.7) and acetonitrile (94 : 6, v/v %). The separated samples were mixed with a post‐column CL reagent consisting of 1.5 mM Ru(bipy)₃²⁺ and 1.0 mM Ce(SO₄)₂, then the generated CL was monitored. The calibration range for FA was 10–100 μM and the limit of detection was 1.34 μM (signal‐to‐noise ratio of 3). Repeatabilities were 4.2, 4.6 and 5.0 RSD% (10, 25, 50 μM), and the recoveries for FA supplement, vitamin B complex supplement and FA‐containing medication (tablet) were 102.4 ± 10.5, 103.3 ± 13.3 and 100.3 ± 8.5%, respectively. The described method is robust against changes in the chromatographic parameters of ± 3.3 or ± 1.5%. The measured FA content corresponded well to the labeled content of FA‐containing products (100.6–104.9%), demonstrating the precision and accuracy of this method for the evaluation of FA pharmaceutical preparations. Copyright © 2014 John Wiley & Sons, Ltd.