Separation of double conjugates of bile acids by two-dimensional high-performance thin-layer chromatography with tetra-n-butylammonium phosphate and methyl β-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 glycine or taurine and sulfonated or glucosylated at hydroxyl groups in the 5β-steroid nucleus. The method involves two-dimensional (2D) reversed-phase (RP) HPTLC with the combined use of tetra-n-butylammonium phosphate (TBAP) and methyl β-cyclodextrin (Me-β-CD) as mobile phase additives. Complete separation of the hydrophilic bile acid conjugates, particularly of the recalcitrant pairs of chenodeoxycholic acid and deoxycholic acid conjugates in each group, was achieved by 2D inclusion RPHPTLC by developing with methanol-water-0.5 mol L−1 TBAP, 90:10:5–75:25:5 (ν/ν) in the first dimension and the same mobile phase containing 5mm Me-β-CD in the second dimension. The method could be usefully applied to biosynthetic and metabolic studies of bile acids in biological materials.