Method for the separation of the unconjugates and conjugates of chenodeoxycholic acid and deoxycholic acid by two-dimensional reversed-phase thin layer chromatography with methyl beta-cyclodextrin.
A simple and efficient method for the separation of individual unconjugated bile acids and their glycine- and taurine-amidated, 3-sulfated, 3-glucosylated and 3-glucuronidated conjugates is described. The method involves the use of a two-dimensional (2D) reversed-phase (RP) high-performance thin-layer chromatographic (HPTLC) technique with methyl beta-cyclodextrin (Me-beta-CD). Five major unconjugated bile acids, chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), ursodeoxycholic acid and lithocholic acid, and their conjugates were examined as the solutes. A high degree of separation of individual bile acids in each homologous series was achieved on a RP-HPTLC plate by developing with aqueous methanol in the first dimension and the same solvent system containing Me-beta-CD in the second dimension. In particular, all of the six 'difficult-to-separate' pairs, unconjugated CDCA and DCA and their conjugated forms with glycine, taurine, sulfuric acid, D-glucose and D-glucuronic acid, were effectively resolved by adding Me-beta-CD in the aqueous mobile phases with the formers having larger mobilities than the latter. The application of this 2D inclusion RP-HPLC method to the separation of glycine-conjugated bile acids in human bile is also described. The present method would be useful for separating and characterizing these bile acids present in biological materials.
Momose
T
T
College of Engineering, Nihon University, Fukushima, Japan.
Mure
M
M
Iida
T
T
Goto
J
J
Nambara
T
T
D001646:Bile / Q000737:chemistry; D002635:Chenodeoxycholic Acid / Q000737:chemistry / Q000302:isolation & purification*; D002855:Chromatography, Thin Layer; D003505:Cyclodextrins; D003840:Deoxycholic Acid / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D007202:Indicators and Reagents; D012997:Solvents; D047392:beta-Cyclodextrins
D003505:Cyclodextrins; D007202:Indicators and Reagents; D012997:Solvents; D047392:beta-Cyclodextrins; D003840:Deoxycholic Acid; D002635:Chenodeoxycholic Acid; C031215:betadex