Simultaneous separation of free and conjugated steroids by micellar electrokinetic chromatography and its clinical application.

We studied the effects of acetonitrile and sodium dodecyl sulfate (SDS) in the running buffer of micellar electrokinetic chromatography (MEKC) on the separation of free and conjugated steroids. When the acetonitrile concentration was increased, the migration time of the conjugated steroids decreased. On the other hand, the migration time of free steroids decreased up to 5--15% acetonitrile, then increased. This behavior of the free steroids was due to the changes of the partition between SDS micelles and the aqueous phase in the running buffer. Increasing the concentration of SDS in the running buffer decreased the migration time of nonpolar free steroids whereas that of polar conjugated steroids was not affected. A practical method for the simultaneous separation of free and conjugated steroids by MEKC was established. The application of this procedure to the analysis of steroids in serum from a patient with Cushing's syndrome and a premature infant is also described.