Routine high-performance liquid chromatographic determination of urinary unconjugated cortisol using solid-phase...

An HPLC method with solid-phase extraction for the specific determination of urinary free cortisol is described. A 3-ml urine sample is cleaned up on a disposable cartridge C 18. After successive washing with 0.025 mol/l borate buffer (pH 7.5) and a mixture of acetone and water, retained cortisol and 6 alpha-methylprednisolone (internal standard) are eluted with ethyl acetate. The recovery of cortisol is 92.9 +/- 4.4%. The reversed-phase chromatographic procedure uses an octadecyl-bonded column with a mobile phase composed of methanol, tetrahydrofuran and water with UV detection at 245 nm. The calibration curve is linear up to a cortisol concentration of 356 nmol/l. Coefficients of variation show good reproducibility of the assay. The detection limit is 9 nmol/l (16 pmol injected). The normal range of urinary free cortisol excretion was found to be 17-132 nmol/24 h. This simple and convenient method is suitable for routine use.