Simultaneous determination of tetrahydrofolate and N5-methyltetrahydrofolate in pig plasma by high-performance liquid chromatography with electrochemical detection.

An analytical method for measurement of tetrahydrofolate (THF) and N5-methyltetrahydrofolate (5MF) in pig plasma by the high performance liquid chromatography and electrochemical detection is described. The plasma sample was deproteinized by perchloric acid and the supernatant was analyzed using the following conditions; (a) phenyl bonded phase column as an analytical column; (b) mobile phase consisting of 20 mM acetate buffer (pH 3.6) containing 0.1 mM EDTA and acetonitrile (96.5:3.5, v/v); (c) an applied potential of +300 mV. Under the above condition, both peaks of THF and 5MF in the plasma were well separated. The detection limits of THF and 5MF were 0.15 and 0.13 ng/ml, respectively, at S/N = 3. The recoveries of THF and 5MF from the plasma spiked with standard THF and 5MF were 77.6 +/- 2.1% and 83.0 +/- 1.7% (mean +/- S.D.), respectively.