DIRECT-INJECTION ANALYSIS OF 6-BETA-HYDROXYCORTISOL AND CORTISOL IN URINE BY HPLC-UV WITH ONLINE ISRP PRECOLUMN

The simultaneous measurement of 6 beta-hydroxycortisol (6 beta-OHF) an d cortisol (FF) is interesting for the evaluation of enzyme induction in man. An on-line HPLC-UV analysis of urinary steroids is described. In a first step, the biological sample was injected onto an ISRP (Inte rnal Surface Reversed Phase) precolumn with water for the elimination of proteins and indesirable products and for the concentration of hydr ophobic molecules. In a second step, a simple gradient of acetonitrile (ACN) in water, by a backflush procedure, eluted the retained analyte s which are analysed by conventional RP-HPLC coupled with UV detection .