Determination of aldosterone secretion rate utilizing mixed mode and high performance liquid chromatography

A simpler method for determining aldosterone secretion rate (ASR) has several applications. High performance liquid chromatography (HPLC) has several advantages over traditional chromatographic methods for purification to constant specific activity of aldosterone liberated from its 18-glucuronide by acid hydrolysis. We found it necessary to introduce several modifications to remove urochromes before HPLC. Two methods for determining ASR were developed. With Method A a more traditional initial procedure was followed, and Sephadex LH-20 chromatography allowed removal of considerable urochromes before HPLC. However, aldosterone recovery was improved with Method B, which employed several bonded phase silica derivatives (Sepralytes) and a PBE 94 column to remove urochromes before HPLC. With this procedure the Sephadex LH-20 chromatography was not required. Aldosterone purification to constant specific activity was achieved by HPLC on a diol column with a normal phase system, and quantification was performed by RIA. ASR determinations were equivalent with both methods. This methodology should be applicable to other steroid secretory rate determinations and to applications involving purification of steroid conjugates.