ANALYSIS OF BILE-ACIDS AND BILE ALCOHOLS IN URINE BY CAPILLARY COLUMNLIQUID-CHROMATOGRAPHY MASS-SPECTROMETRY USING FAST-ATOM-BOMBARDMENT OR ELECTROSPRAY-IONIZATION AND COLLISION-INDUCED DISSOCIATION

Solid-phase extraction and group separation by anion exchange chromato graphy were combined with capillary column liquid chromatography-mass spectrometry (LC/MS) to permit a thorough characterization of bile aci ds and intact conjugates of bile alcohols in human urine, Groups of co mpounds were separated according to acid strength and mere analysed on a capillary column, 0.25 x 500 mm, packed with 5 mu m particles of Ch romasil C-18, and connected via a fused silica capillary to the contin uous-flow fast atom bombardment (CF-FAB) or electrospray (ES) sources of an AutoSpec-TOFFPD hybrid mass spectrometer. Acetonitrileiwater mix tures containing 30 mM ammonium acetate pH 7.2 were used as mobile pha ses, with 5% glycerol added for FAB ionisation. Bile acids were analys ed directly or after derivatization of carboxyl groups with 4-aminoben zenesulphonic add, Negative-ion spectra (m/z 1000 or 800 to 300 or 100 ) were recorded using the point detector or,in the case of ES ionizati on, the focal plane array detector (FPD). Deprotonated molecules of bi le acids containing a sulphonic acid group were detected with a spectr al signal to noise ratio of 5:1 when about 90 fmol were injected onto the column of the LC/CF-FAB system. The corresponding peak in the reco nstructed ion chromatogram gave a signal-to-noise ratio of about 25:1. The sensitivity could be increased 20-50 times by using ES ionization and the FPD. Bile acids without a sulphonic acid group gave about 70% of the signal of sulphonic acids using ES ionization. The capillary c olumn LC/MS systems mere evaluated by analyses of urine from an infant with cholestatic liver disease, More than 150 different bile acids an d bile alcohol conjugates were detected, some of which mere partially characterized using collision induced dissociation (CID) of the deprot onated molecules and B/E linked scans. A number of compounds were dete cted for the first time, e.g. di, tri-, and tetra-hydroxycholestanoic acids conjugated with N-acetylhexosamine and cholestenediol, cholesten etriol and cholestanetriol doubly conjugated with sulphuric acid and g lucuronic acid. The relative merits of ES and FAB ionization are discu ssed. (C) 1997 by John Wiley & Sons, Ltd.