Studies on steroids. CCLII. Separation and characterization of 3-oxobile acids in serum by high-performance liquid chromatography with fluorescence detection.
The separation of unconjugated and glycine- and taurine-conjugated bile acids with a C-3 oxo group has been carried out by high-performance liquid chromatography on a reversed-phase column. The chromatographic behaviour of these 3-oxobile acids was dependent on the number and positions of hydroxyl groups and the structure of the side-chain. The newly developed method has been applied to the characterization of 3-oxobile acids in biological fluids. The bile acid fraction was obtained from a serum specimen by passing it through a Sep-Pak C18 cartridge. 3-Oxobile acids were derivatized quantitatively to fluorescent oximes through the oxo group by treatment with O-(2-anthrylmethyl)hydroxylamine. The derivatives were separated into the unconjugated and glycine- and taurine-conjugated fractions by ion-exchange chromatography on a lipophilic gel, piperidinohydroxypropyl Sephadex LH-20. Subsequent resolution of each fraction into individual 3-oxobile acids was achieved by chromatography on a Nova-Pak Phenyl column using 3% methanol in 0.3% potassium phosphate buffer (pH 7.0)-acetonitrile (8:5, v/v) as the mobile phase. The derivatized 3-oxobile acids were monitored by fluorescence detection (excitation wavelength 260 nm and emission wavelength 405 nm), the limit of detection being 20 fmol. Glycine- and taurine-conjugated 7 alpha,12 alpha-dihydroxy- and 7 alpha-hydroxy-3-oxo-5 beta-cholanoic acids in human serum were unambiguously idenitified on the basis of their chromatographic behaviour using mobile phases of different pH values.
Pharmaceutical Institute, Tohoku University, Sendai, Japan.
J
J
Goto
Y
Y
Saisho
T
T
Nambara
D001647:Bile Acids and Salts / Q000097:blood* / Q000302:isolation & purification; D002021:Buffers; D002851:Chromatography, High Pressure Liquid; D005998:Glycine / Q000097:blood; D006801:Humans; D006863:Hydrogen-Ion Concentration; D013050:Spectrometry, Fluorescence; D013256:Steroids / Q000032:analysis*; D013654:Taurine / Q000097:blood
D001647:Bile Acids and Salts; D002021:Buffers; D013256:Steroids; D013654:Taurine; D005998:Glycine