Determination of 21-hydroxycorticosteroids in human urine by high-performance liquid chromatography with fluorescence detection.

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Abstract
A simple and sensitive high-performance liquid chromatographic method with fluorescence detection for the determination of nineteen 21-hydroxycorticosteroids is described. The corticosteroids are oxidized by cupric acetate to form the corresponding glyoxal derivatives. The derivatives are converted into fluorescent quinoxalines by reaction with 1,2-diamino-4,5-methylenedioxybenzene, a fluorogenic reagent for .alpha.-dicarbonyl compounds. The quinoxalines are separated within 70 min on a reversed-phase column (TSK gel ODS-120T) by stepwise elution with mixtures of methanol, acetonitrile, and 1.0 M ammonium acetate. The detection limits are 0.14-29.4 pmol at a signal-to-noise ratio of 3 in a 50-.mu.l injection volume. This sensitivity permits precise determination of hydrocortisone, cortisone, corticosterone, and their tetrahydro derivatives in 500 .mu.l of normal human urine.