SIMULTANEOUS DETERMINATION OF TETRAHYDROFOLATE, 10-FORMYLTETRAHYDROFOLATE AND 5-METHYLTETRAHYDROFOLATE IN RAT BILE BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH ELECTROCHEMICAL DETECTION

This report describes an analytical method for the measurement of tetrahydrofolate (H4PteGlu), 10-formyltetrahydrofolate (10-HCO-H4PteGlu) and 5-methyltetrahydrofolate (5-CH3-H-4-PteGlu) in rat bile by high-performance liquid chromatography with electrochemical detection (HPLC-ECD). After diluting the bile sample with 0.2% sodium ascorbate solution, the sample was analyzed under the following conditions; (a) phenyl bonded phase column as an analytical column; (b) mobile phase consisting of 20 mM acetate buffer (pH 5.0) containing 0.1 mM EDTA; (c) an applied ECD potential of +300 mV; (d) 0.8 ml/min of flow rate. Under the above conditions, peaks of H4PteGlu, 10-HCO-H4PteGlu and 5-CH3-H4PteGlu in rat bile were well separated on the ECD-chromatogram. Detection limits of H4PteGlu, 10-HCO-H4PteGlu and 5-CH3-H4PteGlu were 0.13, 0.11 and 0.10 ng/ml, respectively, at S/N = 3. Bile excretion rates for H4PteGlu, 10-HCO-H4PteGlu and 5-CH3-H4PteGlu, which were analyzed by this method in rats, were 314+/-181, 321+/-179 and 449+/-198 ng/hr, respectively. Bile concentrations of the folates were more than 5,000 times higher than the detection limits for this method. This HPLC-ECD method is, therefore, a useful tool for bile folate analysis.