Studies on steroids. CCXXXXV. Determination of 5 beta-cholestanoic acids in human urine by gas chromatography-mass spectrometry with negative ion chemical ionization detection.

A method for the determination of 3 alpha,7 alpha-dihydroxy-5 beta-cholestanoic acid (DHCA) and 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) in human urine by gas chromatography (GC) in combination with negative ion chemical ionization (NICI) mass spectrometry is described. Unconjugated, glycine- and taurine-conjugated DHCA and THCA labelled with 18O and 2H were used as internal standards. 5 beta-Cholestanoic acids in urine were extracted with a Sep-Pak C18 cartridge, separated into the unconjugated, glycine- and taurine-conjugated fractions by ion-exchange chromatography on piperidinohydroxypropyl Sephadex LH-20 and, following alkaline hydrolysis of conjugated forms, derivatization into the pentafluorobenzyl ester-dimethylethylsilyl ethers. Subsequent resolution of each fraction into DHCA and THCA was attained by GC on a cross-linked 5% phenylmethylsilicone fused-silica capillary column where 5 beta-cholestanoic acids were monitored with a characteristic carboxylate anion [M-181]- in the NICI mode using isobutane as a reagent gas. The method was applied to separation and determination of 5 beta-cholestanoic acids in urine from a patient with Zellweger syndrome and from healthy volunteers.