QUALITY-CONTROL IN THE DETERMINATION OF CORTISOL IN PLASMA SERUM BY USING, ON EVERY SAMPLE, 2 DIFFERENT 3-STEP SEPARATION METHODS INCLUDINGULTRAFILTRATION, RESTRICTED-ACCESS HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY AND REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY, AND CONTRASTING RESULTS TO IMMUNOASSAYS/

Tests of HPLC columns with restricted access, polymer covered alumina, polymer, and different ODS phases showed that base-acid compatible OD S columns gave the best peak shapes of cortisol, internal standard, as well as of plasma/serum (P/S) matrix components. Further trials with cortisol in P/S showed that three separation steps were essential in o rder to obtain chromatographic data which were superior to immunoassay data. Also, sufficient confidence in results required determination o f each sample with two newly developed separation methods: (a) pre-sep aration with a restricted access column, concentration of the desired cut with a 20 mm base-acid compatible ODS column, and analysis with a 250 mm column filled with the same ODS; (b) pre-separation with an ult rafilter followed by the last two steps in (a), For detection UV was p referred over fluorescence. This twin multistep chromatography showed that immunoassays were very treacherous in that they produced a spectr um of results, ranging from good to untenable without any warning what ever about functionality. The measurement of official controls, with r eference values derived via gas chromatography-isotope dilution mass s pectrometry, also demonstrated the superiority of the double HPLC meth od.