Simultaneous chromatographic analysis of eight fat-soluble vitamins in plasma

A high-performance liquid chromatographic (HPLC) method for the simultaneous analysis of eight fat-soluble vitamins including A acid, retinol and retinal, vitamins D2 and D3 together with menadione (vitamin K3) is described. The eight vitamins extracted from plasma were analyzed by reversed-phase HPLC with UV detection at 245 nm. The mobile phase was composed of methanol and ethanol (85:15, v/v) with 0.1% triethylamine. The extraction efficiency of different solvents, namely, dichloromethane, benzene, methanol, ethanol, chloroform and hexane, with or without the addition of detergents was studied. There is no single solvent system that could extract all eight vitamins in one step. A recovery study of the vitamins was performed using rabbit plasma. An average recovery for individual vitamins was about 40% or more with the described extraction methods. The absorbance response was linear at the nanogram level. The present method may be broadly applied with alternative extraction methods for the eight vitamins from different sources.