SIMULTANEOUS DETERMINATION OF SPIRONOLACTONE AND ITS METABOLITES IN HUMAN PLASMA

This study describes a specific, precise, sensitive and accurate metho d for determination of unchanged spironolactone and its major active m etabolites in human plasma. After one-step liquid-liquid extraction, a nalysis of the parent drug and its metabolites was performed in one ch romatographic run, using a high performance liquid chromatography (HPL C) method with a programmed switchover of the UV wavelength. Spironola ctone and 7 alpha-thiomethylspironolaclone were detected at 245 nm, wh ile canrenone and internal standard were detected al 280 nm. The colum n used was an S5 ODS2 (500 mm x 4.6 mm i.d.). The mobile phase was a m ixture of acetonitrile-aqueous orthophosphoric acid (pH 3.4). Chromato graphic 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. Th e precision and accuracy of the method were confirmed by relative stan dard deviations below 10%, for different concentrations, except for th e concentration equal to the quantitation limit, where these parameter s ranged from 12-15%, The recovery was above 80% for all investigated compounds and for the internal standard. The assay proved to be suitab le for pharmacokinetic studies of spironolactone.