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.