A review of the methods available for the separation of bile acids is presented, highlighting the most recent developments. The major chromatographic techniques (TLC, GC, HPLC) and combined detection systems for the determination of bile acids are critically evaluated and their advantages and disadvantages discussed. Moreover, future directions in which progress might occur are also indicated. Capillary GC-MS is the more established method since it provides higher efficiency combined with greater sensitivity and specificity and has proven crucial in identifying unusual bile acids. However, it requires deconjugation and derivatization and hence the conjugated species must be inferred from the initial isolation procedure. HPLC is directly amenable to the different forms of bile acids, but it suffers from insufficient resolving power which can be enhanced by exploiting the mobile-phase selectivity. The development of HPLC detection systems with higher sensitivity and specificity than conventional HPLC-UV is reported. In particular, methods for the direct coupling of HPLC to MS are examined with special emphasis on soft ionization processes (thermospray, fast atom bombardment, ion spray). Finally, the analytical potential for bile acid assays of more recent techniques including supercritical fluid chromatography and capillary electrophoresis is evaluated.
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