Determination of urinary free cortisol by high performance liquid chromatography with sulphuric acid-ethanol derivatization and column switching. |
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Authors: | O Nozaki T Ohata Y Ohba H Moriyama Y Kato |
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Affiliation: | Department of Clinical Pathology, Kinki University School of Medicine, Osaka, Japan. |
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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. |
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