Abstract
A method for the separation and characterization of bile acid 7- and 12-sulfates without prior deconjugation by means of high performance liquid chromatography (HPLC) is described. Bile acid 7- and 12-sulfates were derivatized quantitatively into the fluorescent compounds through the hydroxyl group at C-3 by treatment with 1-anthroyl nitrile in the presence of quinuclidine in acetonitrile. Subsequent resolution into each individual sulfate was attaind by HPLC on a Cosmosil 5C18 column using 0.3% potassium phosphate buffer (pH 4.0)-methanol (1:3) as a mobile phase. The 3-(1-anthroyl) derivatives of 7- and 12-sulfates were monitored by fluorescence detection (excitation wavelength 370nm; emission wavelength 470nm), the limit of detection being 30 fmol.
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