Determination of water-soluble vitamins in blood and plasma by coupled-column liquid chromatography

A method is described for the simultaneous determination of riboflavin and the coenzymes related to pyridoxine and thiamine. Pyridoxal-5'-phosphate (PLP) bound to proteins as a Schiff base was liberated and stabilized by reaction with semicarbazide. Blood or plasma proteins were precipitated with acid and the supernatant formed was injected into a liquid chromatographic system consisting of one precolumn and two analytical reversed-phase columns. Riboflavin was detected by its native fluorescence with a separate detector while post-column reactions based on the fluorescence of PLP semicarbazone in alkaline solution and the oxidation of thiamine to thiochrome were combined in the same reaction detector. Chromatographic separation was achieved within 7 min and normal endogenous levels were quantified with a precision of 2-5% (relative standard deviation).