CONVENIENT CHROMATOGRAPHIC PREPURIFICATION STEP BEFORE MEASUREMENT OFURINARY CORTISOL BY RADIOIMMUNOASSAY

We applied various prepurification protocols (extraction with differen t solvents, liquid/solid separation on bonded silica media, Celite, an d Sephadex LH20 chromatography) with a range of commercially available RIA kits to measure cortisol in urine samples. We then compared the r esults with the concentrations measured by a HPLC method validated wit h reference to isotope dilution gas chromatography-mass spectrometry. We conclude that chromatography on a commercial, prepacked diol minico lumn (Waters(TM) Sep-Pak Vac RC) in combination with dichloromethane e xtraction is a convenient and very effective purification step before RIA of urinary cortisol in patients not receiving corticoid medication . We tested numerous steroids for interference and found that free pol ar cortisol derivatives (hydroxylated or hydrogenated) could only part ially account for the overestimations routinely encountered when free urinary cortisol concentrations are measured by direct RIA.