Determination of C-21 ketosteroids in serum using trifluoromethanesulfonicacid catalyzed precolumn dansylation and 1,1 '-oxalyldiimidazole postcolumn peroxyoxalate chemiluminescence detection

A new procedure for the quantitation of C-21 ketosteroids using trifluorome thanesulfonic acid-catalyzed precolumn dansylation and coupled column liqui d chromatographic separation, followed by postcolumn 1,1'-oxalyldiimidazole peroxyoxalate chemiluminescence detection is presented. In the simultaneou s optimization of chromatographic resolution and chemiluminescence intensit y, a coupled column chromatographic system and a stopped-now system were us ed. An eluent containing 20 mM phosphate buffer at pH 6.7 accomplished an e fficient separation of 3 alpha-hydroxy-5 beta-pregnan-20-one from a mixture containing 10 C-21 ketosteroids, Phosphate buffer also proved to be the mo st advantageous, among the six buffers tested, for sensitive detection. Exp erimental design and multivariate data analysis were used to characterize a nd optimize the postcolumn reaction chemistry in the chromatographic system . A valid full factorial design with excellent predictability showed that t he flow rates for both 1,1'-oxalyldiimidazole and hydrogen peroxide were th e factors most strongly affecting the sensitivity of the system. The theore tical plate numbers were above 11 000 for all 10 dansylated ketosteroids, T he 3 sigma detection limit estimated from 3 alpha-hydroxy-5 beta-pregnan-20 -one calibration curve data was 1.6 pmol (n = 4, 125 mu L injected) and spi ked serum containing 0-74 pmol of this compound showed overall recoveries o f 73 +/- 9% (n = 12), Quantitation of 3 alpha-hydroxy-5 beta-pregnan-20-one was finally carried out on 45 serum samples and the results compared to th ose from a radioimmunoassay (RIA) method. The data acquired with the proced ure described in this work compare well with the results from RIA, which co nfirms the reliability of the new analytical procedure.