Separation of Dietary Folates by Gradient Reversed-Phase HPLC: Comparison of Alternative and Conventional Silica-Based Stationary Phases

A study of ten silica-based stationary phases and gradient elution conditions to separate dietary folates by reversed-phase HPLC was performed. Alkyl-bonded stationary phases (both conventional and alternative) were found to be the most promising for the separation of different folate monoglutamates in terms of selectivity and peak shape. These phases were better than phenyl-bonded phases which lacked selectivity when separating 10-formyl-folic acid and 5-formyl-tetrahydrofolate. Polar-bonded (cyano) stationary phase showed similar retention characteristics as the conventional alkyl-bonded phases, but ranked below those in terms of peak shape. Overall, alternative stationary phases exhibited slightly higher retention of late-eluted folates and greater retention variability for early-eluting tetrahydrofolate and 5-methyl-tetrahydrofolate. Best selectivity was achieved on alternative polar endcapped Aquasil C₁₈ followed by conventional Synergy MAX C₁₂ and Genesis C₁₈ stationary phases.