GAS-CHROMATOGRAPHIC MASS-SPECTROMETRIC DETERMINATION OF 4-HYDROXYLATED AND 6-HYDROXYLATED BILE-ACIDS IN HUMAN URINE WITH NEGATIVE-ION CHEMICAL IONIZATION DETECTION .254.

A method for the determination of 4- and 6-hydroxylated bile acids with a vicinal glycol moiety in human urine by gas chromatography-mass spectrometry with negative ion chemical ionization detection is described. The 4-beta-hydroxylated bile acids labelled with O-18 and H-2 were prepared as internal standards by the exchange reaction of the carbonyl group on the steroid nucleus with H-2 O-18, followed by metal deuteride reduction. Bile acids in urine were extracted with a Sep-Pak C18 cartridge and, after solvolysis and hydrolysis of the conjugated forms, transformed into pentafluorobenzyl ester diethylhydrogensilyl ether diethylsilylene derivatives. Subsequent resolution into individual 4- and 6-hydroxylated bile acids was carried out on a cross-linked fused-silica capillary column; a characteristic carboxylate anion, [M - PFB]-, was used to monitor each bile acid in the negative ion mode. The newly developed method was applied to the separation and determination of 4- and 6-hydroxylated bile acids in a urine specimen from a newborn infant.