Internal Standard Compounds for Quantitative Determination of Bile Acids by Gas Chromatography

Gas chromatography is well recognized as a useful tool with several advantages for the analysis of bile acids as well as various compounds. In gas chromatographic analysis, bile acids in an analytical sample are subjected to a number of complicated procedures involving many steps such as extraction, fractionation, solvolysis, hydrolysis, derivatization and injection to the gas chromatograph. These procedures result in the loss of bile acids in the analytical sample. The addition of suitable internal standard compound(s) into the analytical sample prior to the extraction of bile acids is indispensable for an accurate determination of bile acids. There are two methods for the quantitative determination of bile acids in a biological sample by gas chromatography: one is the determination of total bile acid amounts in the sample. The other is the determination of bile acid amounts in each fraction after group separation of bile acids in the biological sample using an ion exchange gel column. The addition of 7β,12α-dihydroxy-5β-cholanoic acid or 7β,12β-dihydroxy-5β-cholanoic acid as an internal standard compound is useful for the former method. On the other hand, the addition of 7β,12β-dihydroxy-5β-cholanoic acid, glyco-7α,12α-dihydroxy-5β-cholanoic acid, tauro-7α,12β-dihydroxy-5β-cholanoic acid and glyco-7β,12α-dihydroxy-5β-cholanoic acid 7-sulfate is a suitable combination as internal standard compounds for the latter method.