Determination of Steroids from Rat Feces by High Performance Liquid Chromatography with Fluorescence Derivatization and Identification by On-line Mass Spectrometry

A simple and sensitive method for the determination of steroid compounds extracted from rat feces using 1,2-benzo-3,4-dihydrocarbazole-9-ethoxycarbonylhydrazine (BCEC) as the pre-column derivatization reagent by high performance liquid chromatography in combination with a gradient elution with fluorescence detection and mass spectrometer identification has been developed. Separation of the derivatized steroids has been optimized on a Hypersil BDS-C18 column with aqueous acetonitrile in conjunction with a binary gradient elution. BCEC can easily and conveniently label steroid metabolites, such as progesterone, testosterone, cortisol, and corticosterone. Studies on the derivatization conditions indicate that steroid compounds react with BCEC at 65°C within a two-hour period in the presence of trichloroacetic acid (TCA) catalyst with acetonitrile as the reaction co-solvent to provide the corresponding sensitive fluorescence derivatives. The maximum fluorescence excitation and emission wavelengths are 333 nm and 390 nm, respectively. The identification of steroid derivatives is performed under atmospheric-pressure chemical ionization (APCI) source in positive-ion mode. The derivatives are sufficiently stable to be efficiently analyzed by a high-performance liquid chromatography/mass spectrometer (HPLC/MS). Excellent linear responses are observed in the concentration range of 0.016 to 16.7 μM with a correlation coefficient > 0.9999. The detection limits for steroid compounds, at a signal-to-noise ratio of 3:1, range from 47 to 71 fmol. The established method is sensitive and can be reproduced for the determination of steroid compounds from real samples with satisfactory results.