Separation of double conjugates of bile acids by two-dimensional high-performance thin-layer chromatography with tetra-n-butylammonium phosphate and methyl beta-cyclodextrin

This paper describes a method for the separation, by high-performance thin- layer chromatography (HPTLC), of a series of polar, ionic, and hydrophilic double conjugates of bile acids amidated at the C-24 carboxyl group with gl ycine or taurine and sulfonated or glucosylated at hydroxyl groups in the 5 beta-steroid nucleus. The method involves two-dimensional (2D) reversed-ph ase (RP) HPTLC with the combined use of tetra-n-butyl-ammonium phosphate (T BAP) and methyl beta-cyclodextrin (Me-beta-CD) as mobile phase additives. C omplete separation of the hydrophilic bile acid conjugates, particularly of the recalcitrant pairs of chenodeoxycholic acid and deoxycholic acid conju gates in each group, was achieved by 2D inclusion RPHPTLC by developing wit h methanol-water-0.5 mol L-1 TBAP, 90:10:5-75:25:5 (upsilon/upsilon) in the first dimension and the same mobile phase containing 5 mM Me-beta-CD in th e second dimension. The method could be usefully applied to biosynthetic an d metabolic studies of bile acids in biological materials.