Fluorescence derivatisation of urinary corticosteroids for high-performance liquid chromatographic analysis

Corticosteroids containing a C21 primary hydroxyl group were derivatised with 9-anthroyl cyanide. The reagent was prepared as a solution in acetonitrile, containing 0.1% triethylamine, at a concentration of 2 mg/ml. Approximately 1 microg of corticosteroid was reacted with 100 microl of this reagent, at 45 degrees C for 2 h. The fluorescent derivatives were separated by HPLC on a silica column, 250x4.6 mm I.D., by stepwise elution, with a mobile phase of 2-propanol-hexane (2:98) for 20 min, followed by 2-propanol-hexane (7:93) from 20 to 40 min. The fluorescence detector was set to 370-nm excitation and 470-nm emission. The relatively low temperature for derivatisation avoided reaction with secondary hydroxyl groups and also prevented thermal degradation of the corticosteroids.