Determination of serum cortisol by reversed-phase liquid chromatography using precolumn sulphuric acid-ethanol...

An assay method for serum cortisol, using precolumn sulphuric acid-ethanol fluorescence derivatization and reversed-phase liquid chromatography with a column-switching technique, has been developed. The crude precolumn fluorescence cortisol derivative was prepared by the addition of sulphuric acid to serum deproteinized with ethanol, and directly injected onto an octadecylsilane-bonded silica gel (ODS) precolumn for concentration and purification. After switching columns the samples were separated using an ODS analytical column and monitored fluorimetrically. When the pH of the mobile phase in the analytical separator decreased to 1.85, the emission wavelength of the cortisol derivative changed to 520 nm (excitation of 365 nm) and the fluorescence intensity increased. Among the sulphuric acid-ethanol derivatives of various steroids, cortisol, corticosterone and testosterone emitted fluorescence. However, their retention times differed from those of the cortisol derivatives (12.5 min). The detection limit of cortisol was 0.3 micrograms/dl (signal-to-noise ratio of 3). Use of the fully automated column-switching system contributed to good reproducibility and recovery.