High Performance Liquid Chromatographic Separation of Cortisol, Cortisone, and Their 20–Reduced Metabolites in Perfusion Media

Abstract

A reversed-phase liquid chromatographic assay to quantitate cortisol, cortisone and their respective 20α- and 20β-dihydro reduced metabolites in tissue culture media from in vitro perfusions of the human placental lobule is described. The internal standard used in this assay was 6α-methyl-prednisolone. Steroids were extracted from the perfusion medium using Sep-Pak reversed-phase cartridges with the average recoveries of each steroid at 150 and 600 nmol/L ranging from 84.4 to 99.1% and 85.6 to 93.5% respectively. The separation was achieved by using two C₁₈ columns linked in series at 40°C with a mobile phase of methanol/water (53/47 v/v) and a flow rate of 1.1 mL/min. The eluant was monitored by UV absorption at 242 nm. The assay was linear for each steroid to a concentration of 750 nmol/L with a lower detectable limit of 5 nmol/L. Intra-assay coefficients of variation were measured at 150 and 750 nmol/L with ranges of 4.0% (cortisone) to 5.5% (cortisol) and 2.8% (cortisol) to 4.0% (cortisone and 20α-dihydrocortisone) respectively. Inter-assay coefficients of variation were 6.0 (20α-dihydrocortisone) to 9.6% (cortisone) and 5.8 (20α-dihydrocortisol and cortisone) to 6.9% (20α-dihydrocortisone) at these concentrations respectively. With this method prednisolone coelutes with cortisol however no other interferences, from endogenous steroids or drugs which may be used in pregnancy, were found.