Determination of urinary free cortisol by high performance liquid chromatography with sulphuric acid-ethanol derivatization and column switching.
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.
Nozaki
O
O
Department of Clinical Pathology, Kinki University School of Medicine, Osaka, Japan.
Ohata
T
T
Ohba
Y
Y
Moriyama
H
H
Kato
Y
Y
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
D013464:Sulfuric Acids; D000431:Ethanol; D006854:Hydrocortisone