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.
Goto
J
J
Pharmaceutical Institute, Tohoku University, Sendai, Japan.
Miura
H
H
Nambara
T
T
D001647:Bile Acids and Salts / Q000378:metabolism; D002777:Cholestanols / Q000652:urine*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D006801:Humans; D006868:Hydrolysis; D013256:Steroids / Q000652:urine*
C000986:3,7-dihydroxycholestan-26-oic acid; D001647:Bile Acids and Salts; D002777:Cholestanols; D013256:Steroids; C022069:3,7,12-trihydroxycholestan-26-oic acid