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.
College of Engineering, Nihon University, Fukushima, Japan.
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Momose
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Mure
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Iida
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Goto
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Nambara
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