Separation of C-25 Epimers of 5β-Cholestanoic Acids by High Performance Liquid Chromatography with Precolumn Fluorescence Labeling

Abstract

A method for the separation of C-25 epimers of 3α,7α-dihydroxy- and 3α,7α, 12α-trihydroxy-5β-cholestanoic acids by reversed-phase high performance liquid chromatography (HPLC) is described. The 5β-cholestanoic acids and their glycine and taurine conjugates were derivatized quantitatively into fluorescent compounds through the 3a-hydroxyl group by treatment with 1-anthroyl nitrile in the presence of quinuclidine in acetonitrile. Subsequent resolution into each epimer was attained by HPLC on a Cosmosil 5C18 column using a 0.3% potassium phosphate buffer (pH 7.0)-methanol (1:4 or 1:5) as a mobile phase. The 3-( 1-anthroyl) derivatives of C-25 epimeric 5β-cholestanoic acids were monitored by fluorescence detection (excitation wavelength 370 nm; emission wavelength 470 nm), the limit of detection being 40 fmol. The effect of pH of the mobile phase on chromatographic behaviors of 5β-cholestanoic acids is also discussed.