DETERMINATION OF STEROIDS IN URINE BY MICELLAR HPLC WITH DETECTION BY SENSITIZED TERBIUM FLUORESCENCE

Efficient Forster energy transfer from steroid donors to a terbium ion acceptor occurs in aqueous solution when the two species are sequestered in sodium dodecyl sulfate micelles. The microorganized medium provides both proximity between donor and acceptor and protection from quenching of terbium fluorescence by water. Detection limits down to 100 pg have been achieved for steroids with an alpha,beta-unsaturated carbonyl group in the A-ring. This detection strategy has been used in HPLC separations, using either normal- or reversed-phase chromatography with postcolumn addition of detergent and/or terbium, or micellar chromatography with the detergent solution as the mobile phase. This last method proved to be superior, requiring no sample preparation and allowing direct injection of urine onto the HPLC column.