title: Determination of urinary free cortisol by high performance liquid chromatography with sulphuric acid-ethanol derivatization and column switching.
abstract: A 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 microgram/dL (signal-to-noise ratio = 3). The automated HPLC operation resulted in good reproducibility and recovery of the stable cortisol derivative at 5 degrees C.
authors.affiliation: Department of Clinical Pathology, Kinki University School of Medicine, Osaka, Japan.
authors.forename: O
authors.initials: O
authors.lastname: Nozaki
authors.forename: T
authors.initials: T
authors.lastname: Ohata
authors.forename: Y
authors.initials: Y
authors.lastname: Ohba
authors.forename: H
authors.initials: H
authors.lastname: Moriyama
authors.forename: Y
authors.initials: Y
authors.lastname: Kato
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D000431:Ethanol; D016231:Fluorescence Polarization Immunoassay; D006801:Humans; D006854:Hydrocortisone / Q000652:urine*; D013050:Spectrometry, Fluorescence; D013464:Sulfuric Acids
chemical_list: D013464:Sulfuric Acids; D000431:Ethanol; D006854:Hydrocortisone