Amperometric detection of aldosterone by high-performance liquid chromatography with copper(II) bis-phenanthroline.

Abstract

A sensitive method for the determination of aldosterone in human urine has been developed. The method uses high performance liquid chromatography (HPLC) with amperometric detection for the estimation of urinary aldosterone. The method utilizing electrochemical detection has been developed to overcome problems of low sensitivity and low selectivity that are often encountered in HPLC, particularly in the analysis of complex matrices such as biological fluids. Essential steps to the method include acid hydrolysis of the aldosterone-18 conjugate in order to release the aldosterone in a 24 hr. urine sample, followed by solid-phase extraction with cyclohexyl and silica cartridges. This was followed by high performance liquid chromatography using a C18 silica based reversed phase column with detection of current in an electrochemical cell by the oxidation of the synthesized copper (II) bis-phenanthroline reagent. There was a linear correlation between the amount of aldosterone and the area under the aldosterone representative peak on the chromatogram. The method was able to detect aldosterone concentrations as low as 20 ng. This technique provided an accurate means of assaying urinary aldosterone.