Simplex optimization of the blue tetrazolium assay procedure for alpha-ketol steroids

Two independent, simplex procedures were employed to optimize the official blue tetrazolium reaction. The net absorbance for a fixed amount of hydrocortisone was maximized by the simultaneous variation of five factors of the pharmacopeial method, i.e.: concentrations of tetramethylammonium hydroxide, blue tetrazolium, and water, as well as temperature and time for color development. An initial simplex was used to determine an optimum response, while a second was performed to minimize the blank absorbance obtained at the optimum. A series of pharmaceutically important steroids: prednisone, prednisolone, hydrocortisone acetate, and cortisone acetate were also analyzed by the optimized method. The results indicated improvements in sensitivity ranging from 9.27 to 22.95% for the corticosteroids studied. The optimized method also decreased the assay time in relation to the official procedure by one-sixth.