Micromethod for the analysis of evans blue in plasma using ion-pair high-performance liquid chromatography.

A micromethod for the determination of Evans Blue in plasma has been developed. Effective release of the dye from the dye-albumin complex was accomplished using a solution of urea and 2-mercaptoethanol. Ion-pairing techniques were used to extract the dye as well as the internal standard (Naphthol Blue Black B) from plasma. Separation involved ion-pair high-performance liquid chromatography. Eluent was monitored at 605 nm. The technique results in a linear response over the concentration range of approximately 1.0-30.0 micrograms/ml. Assay performance was verified by estimating plasma volume in the rat.