A simple method for the separation of (6R)- and (6S)-5,6,7,8-tetrahydrofolic acid by reversed-phase HPLC with hydroxypropyl-β-cyclodextrin as the mobile phase additive

(6R)- and (6S)-5,6,7,8-tetrahydrofolic acid were resolved by reversed-phase HPLC on a C-18 column with hydroxypropyl-β-cyclodextrin (HP-β-CD) in the mobile phase. As the concentration of HP-β-CD increased from 0 to 45 mM, resolution increased and retention times decreased dramatically. The influence of different mobile phase pH, column temperature, and some other factors were evaluated, and we obtained the optimal HPLC separation conditions.