1-Cyanoisoindole derivative as a highly sensitive fluorescence labelling reagent for steroidal primary alcohols in liquid chromatography

A sensitive and reactive labelling reagent for steroidal primary alcohols, m-(1-cyano-2-isoindole)benzoyl azide, was prepared from the corresponding carboxylic acid, which was synthesized from o-phthalaldehyde and m-aminobenzoic acid in the presence of potassium cyanide in one step. A hydroxysteroid, such as cortisol, can react with the reagent, when kept in benzene at 80 °C for 40 min, resulting in the formation of a urethane derivative which shows a single peak on the chromatogram and provides high sensitivity. The detection limit of the cortisol in reversed-phase chromatography is 80 fmol.