Synthesis of 8α-hydroxyriboflavin was carried out by the method of McCormick (21). Briefly, riboflavin was added to a solution of acetic acid: acetic anhydride (1:1) and the yellow solution was stirred at room temperature for 24 hours. Tetra-acetylriboflavin (TAR) was extracted from the aqueous reaction mixture with CHCl3, followed by extraction with water and evaporation to give a yellow residue of essentially pure TAR. Dibenzoyl peroxide and dioxane dibromide in dioxane were added to a solution of TAR in dioxane, and the solution was refluxed. The crude bromo-TAR was separated from the reaction mixture on a C-18 Semi-Prep HPLC column (Beckman). The bromo-TAR was hydrolyzed to yield 8α-hydroxyriboflavin, which was separated by HPLC (System Gold, Beckman) through a linear gradient from 0.5% trifluoroacetic acid (TFA) in water to 0.5% TFA in acetonitrile for 50 minutes at a flow rate of 4 ml/minute.
