We have developed a new method for the fluorometric determhmtion of A'-fketosteroids.
In a methanolic solution of aluminum salt, A'-3-ketosteroids reacted with isonicotinylhydraxine (INH), and the resulting hydrazone fluoresced through complex formation with the aluminum ion in the solution. As little as 0.25 nmol of cortisol can be determined by this method.
In order to determine A4-3-ketosteroids Umberger (1) introduced isonicotinylhydrazine as a calorimetric reagent which reacted to form yellow-colored hydrazones in weakly acidified alcoholic solution. Later Weichselbaum and Margraf (2) observed that a greenish-yellow fluorescence appeared under ultraviolet illumination when the above reaction was carried out on filter paper. This was applied by Smith and Foell(3) with some modifications to a differential fluorescence detection of A4-3ketosteroids and A1,4-3-ketosteroids on paper chromatograms. But this fluorogenic reaction was only observed on the chromatograms, and no attempt was made to describe the feasibility of using the reaction for quantitative analysis.
In this paper we describe the fluorogenic reaction between the isonicotinylhydrazones of A4-3-ketosteroids and aluminum salts in methanol and its application to the determination of A4-3-ketosteroids. EXPERIMENTAL Materials.
All chemicals used were reagent grade obtained from commercial sources. Metal salts were hydrated except antimony, zinc, and mercuric chloride. Isonicotinylhydrazone of testosterone (INH-T) was prepared following the procedure of Umberger (1) and aluminum perchlorate hexahydrate by the procedure of Weinland and Ensgraber (4). INH was recrystallized from hot ethanol.
Apparatus. Fluorescence was measured with a Hitachi spectrofluorometer Model MPF-2A, and absorption spectra were measured with a Shimadzu uv-spectromonitor Model UV-202.