THE COLORIMETRIC MICRODETERMINATION OF CORTICOIDS BY USE OF 4,7-DIPHENYL-1, lo- PHENANTHROLINE

With the rapid development of information concerning the isolation, identification, and physiological levels of corticosteroids, a relatively simple microchemical met,hod for their quantitative estimation appears ta be desirable. Since the known biologically active adrenal cortical hormones contain a primary cu-ketol function, an cr,@msaturated %-ketone group, or both, several methods based upon the reducing capacity of the cortical hormones have been proposed for their quantitative analysis. Thus, in 1945, Talbot et al. (1) used the reduction of cupric ion, in 1946 Heard and Sobel (2) used the reduction of phosphomolybdic acid, and, in 1953, Chen and collaborators (3) applied the use of tetrazolium salts. In general, in addition to other limitations, the prevailing methods lack sensitivity. In view of the relatively small concentrations of corticosteroids in normal human urine, processing of large amounts of urine has been necessary for reducing methods previously described. Since the possible presence of non-alcoholic reducing lipides has been reported in urine extracts (4), it is felt that a very sensitive method for corticosteroids will minimize the discrepancies reported. Preliminary work in the field of analytical reagents used in oxidationreduction systems indicated that the heterocyclic nitrogen compounds of the phenanthroline type might be more sensitive and useful reagents, and possibly an improvement over the use of existing reagents for estimating cortical steroids. The present investigation concerns the development of a sensitive and simple method based on the reduction of ferric chloride by corticosteroids, and the estimation of the ferrous iron by certain het,erocyclic nitrogen compounds, to yield a stable red color (ferroin reaction). Of the various phenanthroline derivatives that could be used for reduction study, the reagent 4,7-diphenyl-l , lo-phenanthroline prepared by Case (5) w&6 selected as the analytical reagent of choice. This reagent is also known as bathophen-