A Simple Method for Separate Determination of Delta4- and Delta1,4-Corticosteroids in Urine

Abstract

Abstract Ethanolic solutions of Δ4- and Δ1,4-3-ketosteroids absorb ultraviolet light most strongly in the region of 240 nm, but, as sulfuric acid is added in increasing concentrations, absorption decreases. With sulfuric acid concentrations greater than 40 ml/100 ml of ethanol, the Δ4-3-ketosteroids show an absorption peak at about 290 nm, giving an isosbestic point in the region of 275 nm. However, Δ1,4-3-ketosteroids develop the absorption maximum at around 260 nm with the isosbestic point in the region of 250 nm. This bathochromic shift in ultraviolet absorption was used to separately determine Δ4- and Δ1,4-corticosteroids in urine. Urine is extracted with chloroform or methylene chloride. The extract is purified by chromatography and dissolved in H2SO4:ethanol(1:1, by volume). Ultraviolet absorption is measured at 220, 260, and 300 nm for Δ1,4-corticosteroids such as prednisolone or prednisone; at 250, 290, and 330 nm for Δ4-corticosteroids. The readings are corrected by Allen’s method [J. Clin. Endocrinol. Metab. 10, 71 (1950)] and the amount of steroid is estimated.