High performance liquid chromatographic determination of 3α, 5β‐tetrahydroaldosterone and cortisol in human urine with fluorescence detection

A simple and sensitive method for the simultaneous determination of 3 alpha, 5 beta-tetrahydroaldosterone (THALD) and cortisol in human urine is described. The method uses high performance liquid chromatography with fluorescence detection. THALD and cortisol, released by enzyme hydrolysis, and fludrocortisone (internal standard) are isolated by a Sephadex G-25M column and a Bond-Elut C18 cartridge, and then oxidized by cupric acetate to form the corresponding glyoxal derivatives. The glyoxal derivatives are converted into the fluorescent quinoxalines by reaction with 1,2-diamino-4,5-methylenedioxybenzene. The quinoxalines are successfully separated on a reversed phase column (L-column ODS) with isocratic elution and monitored fluorimetrically. The detection limits for THALD and cortisol are 0.45 and 1.18 ng/mL urine (0.65 and 2.65 pmol/100 microL injection volume), respectively, at a signal-to-noise ratio of 3 in a 100 microL injection volume. This method permits the precise and sensitive determination of THALD and cortisol in human urine (2 mL).