The determination of human urinary aldosterone by high-pressure liquid chromatography

Abstract

The clinical significance of the determination of steroid hormones and their metabolites in urine or in plasma is a means of evaluating adreno-cortical functions. The importance of measuring aldosterone in a clinical laboratory lies in aiding the physician in the diagnosis and treatment of patients with excessive secretion of aldosterone. Measurement of aldosterone presents a technical challenge because of the minute quantities of aldosterone normally present in body fluids. The purpose of this investigation was to develop a prototype method for the determination of human urinary aldosterone by high-pressure liquid chromatography (HPLC). The author proceeded to devise an HPLC method for routine clinical evaluation of aldosterone by attempting to eliminate as many of the tedious, manual time- consuming, extraction and separation steps as possible. The preliminary steps of this procedure are modifications of the gas-liquid chromatography methods as described by Zack, et al 33 and Bravo 34. The specimens for the analyses were 24-h urine collections. Extraction, purification and separation, ,and finally quantification are carried out on duplicated 100-mL aliquots of a 24-h urine specimen. By varying the parameters of reverse-phase HPLC, a method for the determination of human urinary aldosterone was developed. The chromatograms recorded, of pure alosterone standard and pure steroid standards which might interfere with aldosterone determination, are evidence that good resolution and symmetrical peaks can be obtained using reverse-phase HPLC. Chromatograms of urine extracts were obtained, also, which showed a peak with an unresolved shoulder, at the same tr as was obtained with pure aldosterone. The loss of aldosterone was determined in triplicate by adding no aldosterone, 5 g, 26.7 g, and 53.4 g of pure aldosterone stock standard respectively, to 100-mL aliquots of 2x's glass-distilled water. Each 100-mL aliquot of water was then treated as a urine specimen, following all the required steps. The percent recovery was 64-71%. Some modification of the procedure developed, plus further experimentation by varying HPLC parameters should produce a simple, rapid determination of urinary aldosterone by HPLC.