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Analysis of corticosteroids in urine by HPLC and thermospray LC/MS.

The method for simultaneous determination and confirmation of nine corticosteroids in urine by high-performance liquid chromatography with a diode array detector and thermospray liquid chromatography-mass spectrometry was studied. HPLC was performed on a C18 column with a gradient mobile phase system of water and acetonitrile. The calibration curve was linear from 20 ng/mL to 1.0 micrograms/mL for each corticosteroid, and the detection limit was 10 ng/mL in 5 mL of urine. The extraction recovery of each corticosteroid from the spiked urine was equal to or greater than 85% by liquid-liquid extraction (LLE) at pH 9 using diethyl ether and approximately 79% or more by solid-phase extraction (SPE) with a Sep-Pak C18 cartridge. The mass spectra obtained with the positive ion mode showed protonated molecular species [M + H]+, ammonium adduct ion [M + NH4]+, [MH-60]+, [MH-30]+, and [MH-18]+. The LC/MS detection limits ranged from 10 to 50 ng in the scan mode and from 1 to 5 ng in the selected ion monitoring (SIM) mode.