AOAC Official Method 2002.05 Determination of Cholecalciferol (Vitamin D3) in Selected Foods Liquid Chromatography First Action 2002

A. Principle

After the addition of an internal standard (vitamin D₂) and basic hydrolysis, vitamin D₃ is extracted with n-heptane. The fraction that contains vitamin D₂/D₃ is separated by preparative normal-phase liquid chromatography (LC). After evaporation and dilution in acetonitrile–methanol, vitamin D₃ is determined by reversed-phase LC with UV detection at 265 nm. A separate test portion is analyzed in parallel to confirm the absence of endogenous vitamin D₂.