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 a nalysis of bile acids. Good separation of free as well as glycine- and taurine-conjugated bile acids was achieved with a C-18 reversed-phase column (3 mu m particle size, 70 x 4.6 mm I.D.) employing methanol-15 mM ammonium acetate as the mobile phase for both isocratic and gradie nt 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 mu l/min; and (2 ) a complementary evaporative light scattering mass detector. When bil e salts were ionized in the electrospray interface operating in the ne gative-ion mode, only [M - H](-) molecular ions were generated; the de tection limit was 15 pg injected for all bile acids studied. In the se cond system, a semi-micro pre-column splitting apparatus (Acurate, LC Packings) was utilized: with this device the flow-rate from the HPLC p ump was reduced to 1.4 mu l/min and bile acids were separated with a m icro-bore C-18 column (3 mu m particle size, 150 x 0.30 I.D.), using t he 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 t o 5 pg injected. Application of the HPLC-electrospray-mass spectrometr ic method to bile and serum bile acid analysis is described; prelimina ry data on the ability of the first system to determine the C-13/C-12 isotope ratio in C-13-labeled bile acid enriched serum is also critica lly discussed.