Simultaneous determination of spironolactone and its metabolites in human plasma

This study describes a specific, precise, sensitive and accurate method for determination of unchanged spironolactone and its major active metabolites in human plasma. After one-step liquid-liquid extraction, analysis of the parent drug and its metabolites was performed in one chromatographic run, using a high performance liquid chromatography (HPLC) method with a programmed switchover of the UV wavelength. Spironolactone and 7 alpha-thiomethyl-spironolactone were detected at 245 nm, while canrenone and internal standard were detected at 280 nm. The column used was an S5 ODS2 (500 mm x 4.6 mm i.d.). The mobile phase was a mixture of acetonitrile-aqueous orthophosphoric acid (pH 3.4). Chromatographic separations were performed at 5 degrees C. The standard curves were linear over the range 10-400 ng ml-1 for spironolactone and 10-600 ng ml-1 for 7 alpha-thiomethyl-spironolactone and canrenone. The precision and accuracy of the method were confirmed by relative standard deviations below 10% for different concentrations, except for the concentration equal to the quantitation limit, where these parameters ranged from 12-15%. The recovery was above 80% for all investigated compounds and for the internal standard. The assay proved to be suitable for pharmacokinetic studies of spironolactone.