Analysis of Aldosterone in Urine by Double Isotope Dilution and Gas-Liquid Chromatography

Abstract

The instability of corticosteroids, including aldosterone, during GLC was recognized by VandenHeuvel and Horning (1). Protection of aldosterone was provided by prior derivative formation as the gamma lactone (2, 3) and as the 18,21-diacetate (3, 4) Elimination of the 18 acetate group was found by Kliman and Foster (3) using 2%, 6 ft SE-30 columns, but not by Wotiz, Naukarinen and Carr who used 3%, 2-4 ft SE-30 columns (4). The application of GLC to the assay of biological samples containing aldosterone was reported by Kittinger (5), using gamma lactone formation after extraction of incubation fluid from rat adrenal glands. Because of prolonged retention time and low sensitivity of detection, 0.05-0.10 µg of aldosterone derivatives were required for analysis. The low concentration of aldosterone in biological fluids further limited the application of this new technique. The purpose of this investigation was to develop preparative and analytical methods allowing analysis of submicrogram amounts of aldosterone in human urine. Results using GLC were compared to the standard, sensitive, double isotope derivative method of Kliman and Peterson (6). The sensitivity of detection and identification of fractions eluting from the chromatograph were enhanced by simultaneous mass detection and collection of fractions for analysis of carbon-14 and tritium, by a modification of the method of Karmen et al. (7). The accuracy, precision, sensitivity, and utility of several methods of analysis were compared. The method of choice allows analysis in the millimicrogram range when radioactivity measurement is substituted as a detection system for GLC of aldosterone diacetate.

Supported by U.S.P.H.S. Grant AM-06731 from the National Institute of Arthritis and Metabolic Diseases, Bethesda, Md.