Reversed-phase high-performance liquid chromatography of steroid 3-sulfates and the corresponding unconjugated steroids.

Simultaneous separation of steroid 3-sulfates and the corresponding free steroids has been accomplished by reversed-phase high-performance liquid chromatography (RP-HPLC). Addition of ammonium sulfate to the aqueous component of the mobile phase results in retention of the steroid sulfates to octadecyl silica packings by an apparent ion-pairing mechanism without affecting the retention of the free steroids. The retention time of steroid sulfates was dependent on the ammonium sulfate concentration in the mobile phase. Biologically important C19 delta 5, 3beta-hydroxysteroids and corresponding 3-sulfate conjugates or estrogen and estrogen 3-sulfates have been resolved with this RP-HPLC system.