Direct Injection Analysis of 6β-Hydroxycortisol and Cortisol in Urine by HPLC-UV With On-Line ISRP Precolumn

The simultaneous measurement of 6β-hydroxycortisol (6β-OHF) and 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 (Internal Surface Reversed Phase) precolumn with water for the elimination of proteins and indesirable products and for the concentration of hydrophobic molecules. In a second step, a simple gradient of acetonitrile (ACN) in water, by a backflush procedure, eluted the retained analytes which are analysed by conventional RP-HPLC coupled with UV detection.