Two-cycle liquid-chromatographic quantitation of cortisol in urine.

When the free cortisol in urine was analyzed by liquid chromatography after pretreatment with the usual organic extraction, interfering substances having chromatographic behavior similar to that of cortisol and the internal standard precluded accurate measurement. To remove these materials, we processed the organic extract by liquid chromatography with a normal-phase column for clean-up; the specific cortisol fraction and the internal standard collected in the effluent were then subjected to liquid chromatography with a reversed-phase column. The "two-cycle" chromatograms thus obtained were sufficiently specific for the quantitation of urinary cortisol. The method clearly detects cortisol in urine at a minimum concentration of about 5 micrograms/L when 1 mL of urine is extracted. The mean urinary free cortisol in apparently healthy individuals was 40.9 (SD 17.7) micrograms/day (n = 32) by this method.