FIG. 2.

Representative HPLC chromatograms and standard curves of folates. (A) Folate monoglutamates of known concentrations in a standard mixture were analyzed on a C₁₈-reversed phase column on an Agellient HPLC equipped with both absorbance and fluorescence detectors. Peak 1, THF; 2, 5,10-CH₂-THF/THF (This peak was obtained when 5,10-CH₂-THF was dissolved in buffer and injected into HPLC column for analysis); 3, 5-CH₃-THF, 4, 5,10-CH⁺-THF; 5, 10-CHO-THF; 6, 5-CHO-THF; 7, DHF; 8, folic acid. The folate concentration represented by each peak is shown in Table 2. (B) The standard curves show the linearity between folate concentrations and corresponding peak areas. The regression line for 10-CHO-THF represents the correlation between the change of the THF peak area and the 10-CHO-THF concentration in samples, where the THF concentration is maintained at 120 nM. This standard curve has been corrected for the decrease in the THF area as a result of the addition of FDH-N as described in Materials and Methods. DHF, dihydrofolate; 5-CH₃-THF, 5-methyl-tetrahydrofolate; 5,10-CH₂-THF, 5,10-methylene-tetrahydrofolate; 10-CHO-THF, 10-formyl-tetrahydrofolate.