A method for the accurate measurement of isotope ratios of chenodeoxycholic and cholic acids in serum

A method for the extraction of bile acids from serum is described that enables the stable isotopic content of chenodeoxycholic acid and cholic acid to be determined accurately to levels as low as the natural 13C abundance. The method uses Sep-Pak C18 reverse phase cartridges both for extraction and purification procedures. Free bile acids, bile acid conjugates, and 3-monosulfated bile acid conjugates are recovered in high yield from the Sep-Pak in methanol-water 75:25 after first removing impurities with hexane and methanol-water 40:60 washes. Other important features of the method include the use of enzymatic rather than alkaline hydrolysis of bile acid conjugates, the use of ammonia as the reagent gas for chemical ionization mass spectrometric measurement of isotopic ratios, and the exclusion of all extraneous components in the final sample from the ion source. This method should be applicable to kinetic studies of bile acids using bile acids labeled with stable isotopes and serum measurements, and provides an alternative sampling point in the enterohepatic circulation to conventional duodenal bile samples requiring intubation.