Determination of 3-oxo-delta4- and 3-oxo-delta4,6-bile acids and related compounds in biological fluids of infants with cholestasis by gas chromatography-mass spectrometry.

A method has been developed for the determination of 3-oxo-delta4- and 3-oxo-delta4,6-bile acids and related bile acids in biological fluids of infants by gas chromatography-mass spectrometry (GC-MS) of the methyl ester-dimethylethylsilyl ether-methoxime derivatives. The 7alpha-hydroxylated 3-oxo-delta4-bile acids were partially dehydrated to give the 3-oxo-delta4,6-bile acids by trimethylsilyl or dimethylethylsilyl derivatization and other pretreatments under acidic or alkaline conditions for GC-MS analysis. To prevent dehydration, the 3-oxo-delta4-bile acids were derivatized to the oximes by treatment with O-methylhydroxylamine prior to pretreatments such as solid-phase extraction, enzymatic solvolysis and hydrolysis of the conjugates, and silylation with dimethylethylsilylimidazole. Calibration curves for the bile acids were linear over a range of 5-250 ng and the detection limit was 100 pg for each 3-oxo-delta4-bile acid. Recoveries of the bile acids and their glycine and taurine conjugates from bile acid-free urine and serum ranged from 94.2 to 105.9% of their added amounts. The bile acids in urine and serum of four patients with severe cholestatic liver disease were measured by the analytical method, and the 3-oxo-delta4-bile acids were determined to be the major bile acids (59-68%) in the urines associated with 3-oxo-delta4-steroid 5beta-reductase deficiency or dysfunction.