Effect of tetrahydrofuran on reversed-phase liquid chromatographic separation of dansylhydrazones of oxo-steroids

The oxo-steroids corticosterone, testosterone and progresterone were converted with 5-N,N-dimethylaminonaphthalene- 1 -sulphonohydrazide (DNS-hydrazine) into DNS-hydrazones, which were separated by reversed-phase liquid chromatography and detected fluorimetrically. The anti and syn conformers of the hydrazones were recognized on a C₁₈ column with eluents consisting of imidazole buffer (nitrate, pH 6.0) containing acetonitrile, methanol, acetone or dioxane, and thus two peaks were observed for each oxo-steroid derivative. On the other hand, a single peak was obtained when an eluent containing tetrahydrofuran was used, affording highly sensitive detection of oxo-steroids. A further improvement in the detection of oxo-steroids down to subpicomole level was achieved by adding a gel permeation chromatographic clean-up procedure to remove the excess reagent before the analysis by reversed-phase liquid chromatography.