Reaction of vitamin K and dithiothreitol on reversed-phase C18 high-performance liquid chromatographic columns

A non-enzymatically catalyzed side reaction between dithiothreitol and vitamin K occurs on reversed-phase C18 high-performance liquid chromatographic columns. Excess dithiothreitol may be effectively removed from enzymatic reaction mixtures with N-ethylmaleimide. A low polarity gradient elution system is reported which gives quantitative recoveries (greater than 95%) of the vitamins in a concentration range useful for analysis of enzymatically catalyzed reactions.