Clinical Chemistry, Vol 17, 161-165, Copyright © 1971 by the American Association for Clinical Chemistry

A Simple Method for Separate Determination of Delta⁴- and Delta^1,4-Corticosteroids in Urine

¹ Kitano Hospital, Tazuke Kofukai Medical Research Institute, Kita-ku, Osaka City, Japan.

Ethanolic solutions of ⁴- and ^1,4-3-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 ⁴-3-ketosteroids show an absorption peak at about 290 nm, giving an isosbestic point in the region of 275 nm. However, ^1,4-3-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 ⁴- and ^1,4-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 ^1,4-corticosteroids such as prednisolone or prednisone; at 250, 290, and 330 nm for ⁴-corticosteroids. The readings are corrected by Allen’s method [J. Clin. Endocrinol. Metab. 10, 71 (1950)] and the amount of steroid is estimated.

Submitted on June 1, 1970
Accepted on December 29, 1970