High-performance liquid chromatographic-electrospray mass spectrometric analysis of bile acids in biological fluids

The present work describes the development of HPLC-mass spectrometric systems equipped with an electrospray interface for the quantitative analysis of bile acids. Good separation of free as well as glycine- and taurine-conjugated bile acids was achieved with a C18 reversed-phase column (3 microns particle size, 70 x 4.6 mm I.D.) employing methanol-15 mM ammonium acetate as the mobile phase for both isocratic and gradient mode, at a flow-rate of 0.3 ml/min. This system permits post-column splitting of the eluate for analysis by two different detectors: (1) electrospray-mass spectrometer with a flow-rate of 18 microliters/min; and (2) a complementary evaporative light scattering mass detector. When bile salts were ionized in the electrospray interface operating in the negative-ion mode, only [M-H]- molecular ions were generated; the detection limit was 15 pg injected for all bile acids studied. In the second system, a semi-micro pre-column splitting apparatus (Acurate, LC Packings) was utilized: with this device the flow-rate from the HPLC pump was reduced to 1.4 microliters/min and bile acids were separated with a micro-bore C18 column (3 microns particle size, 150 x 0.30 I.D.), using the same mobile phase as above. With this latter system, a head-column enrichment technique can be used: the amount injected can be increased from 60 to 200 nl, permitting an improvement in the detection limit to 5 pg injected. Application of the HPLC-electrospray-mass spectrometric method to bile and serum bile acid analysis is described; preliminary data on the ability of the first system to determine the 13C/12C isotope ratio in 13C-labeled bile acid enriched serum is also critically discussed.