Determination of urinary free cortisol by high performance liquid chromatography with sulphuric acid–ethanol derivatization and column switching

摘要

AbstractA rapid and highly sensitive determination method for urinary free cortisol has been developed using reversed phase high performance liquid chromatography (HPLC) with a precolumn for sulphuric acid‐ethanol fluorescence derivatization and column switching. Urinary cortisol, eluted from the octadecylsilane‐bonded silica (ODS) minicolumn with 90 aqueous ethanol, was derivatized with the addition of sulphuric acid only at ambient temperature. Cortisol derivatives injected directly onto the ODS precolumn were purified on‐line. After switching the columns, the cortisol derivative was separated on an ODS analytical column with a retention time of 15.3 min and monitored at an emission wavelength of 520 nm (exitation wavelength of 365 nm) to decrease the detection limit to 0.26 μg/dL (signal‐to‐noise ratio = 3). The automated HPLC operation resulted in good reproducibility and recovery of the stable cortisol derivat