Determination of unmetabolized folic acid in human plasma using affinity HPLC

Background: Folic acid (FA) fortification of food created the need to determine whether fortification elevated concentrations of unmetabolized FA in plasma and whether this form of the vitamin in blood is associated with adverse health outcomes. Objective: The objective of this research was to devise a simple, rapid method for the measurement of unmetabolized plasma FA in epidemiologic studies. Design: We previously used the affinity/HPLC with electrochemical detection method to measure folate distribution in human plasma and red blood cells (RBCs). We modified this method with the inclusion of synthetic ethyltetrahydrofolate as an internal standard and with the use of 2 affinity columns connected in parallel to the analytic column through a switching valve to allow one column to be loaded while the other column was eluted into the analytic column. Results: We identified FA and 5-methyltetrahydrofolate (5-mTHF) by retention time and characteristic response across the channels of the electrochemical detector. Limits of detection were 0.034 pmol for 5-mTHF and 0.027 pmol for FA per injection, and the recovery was 92.2% (5-mTHF) and 98.9% (FA). CVs for samples were 8.1% for (within day) and 6.8% (between day) for 5-mTHF and 3.2% (within day) and 5.9% (between day) for FA. Total folate with the use of this method correlated highly (r(2) = 0.98, P < 0.001) with values from the microbial assay. The run time for the method was 30 min per sample. Researchers can use this method with longer run times to measure the distribution of folate forms in RBCs. Conclusion: This updated method allows efficient analysis of folate forms in human plasma and tissues without the loss of sensitivity or precision. Am J Clin Nutr doi: 10.3945/ajcn.111.013433.