Abstract
We describe an improved determination of conjugated bile acids hydroxylated at 1β-, 2β-, 4β- or 6α-position in urine sample of newborn by high-performance liquid chromatography employing labeling with 1-anthroyl cyanide and fluorescence detection. After extraction of bile acids from urine with a Bond Elut C18 cartridge, conjugated bile acids were separated into the three types of conjugates by ion-exchange chromatography on a lipophilic gel, piperidinohydroxypropyl Sephadex LH-20. The sulfated bile acids were solvolyzed by treatment with trifluoroethanol in acetone containing hydrochloric acid, followed by fractionation into the modes of conjugation with amino acids. The individual conjugated bile acids were labeled with 1-anthroyl cyanide in acetonitrile and triethylenediamine, and then separated on a C18 reversed phase column of high-performance liquid chromatography. The developed method showed the lowest limit of detection at 0.1pmol for the individual bile acids. It was available for the simultaneous analysis of double conjugated bile acids in urine of newborns.
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