Determination of free and amidated bile acids by high-performance liquid chromatography with evaporative...

A simple reverse phase high-performance liquid chromatographic method for a simultaneous analysis of free, glycine- and taurine-amidated bile acids is described. The resolution of ursodeoxycholic, cholic, chenodeocycholic, deoxycholic, and lithocholic acids, either free or amidated with glycine and taurine, is achieved using a C-18 octadecylsilane column (30 cm length, 4 micron particle size) with a gradient elution of aqueous methanol (65----75%) containing 15 mM ammonium acetate, pH 5.40, at 37 degrees C. The separated bile acids are detected with a new evaporative light-scattering mass detector and by absorbance at 200 nm. A complete resolution of the 16 bile acids, including the internal standard nor-deoxycholic acid, is obtained within 55 min. Using the light-scattering mass detector, amidated bile acids and, for the first time, free bile acids can be detected with similar detection limits in the order of 2-7 nmol. The new detector improves the baseline and the signal-to-noise ratio over the UV detection as it is not affected by impurities present in the samples with higher molar absorptivity than bile acids or by the change in the mobile phase composition during the gradient. The method fulfills all the standard requirements of precision and accuracy and the linearity of the mass detector is over 5 decade the detection limit. The new method has been used for the direct analysis of bile acid in stools and bile with only a preliminary clean-up procedure using a C-18 reverse phase extraction.