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Journal of Lipid Research, Vol 38, 1685-1701, Copyright © 1997 by Lipid Research, Inc.

ARTICLES

An updated look at the analysis of unsaturated C27 sterols by gas chromatography and mass spectrometry

N Gerst, B Ruan, J Pang, WK Wilson and GJ Schroepfer Jr
Department of Biochemistry, Rice University, Houston, TX 77005-1892, USA.

Gas chromatography-mass spectrometry (GC-MS) and GC are commonly used methods for the identification and quantitation of sterols from samples of biological origin. To investigate the utility and limitations of these methods, we have determined gas chromatographic mobilities and mass spectral properties of 5alpha-cholestan-3beta-ol and 26 unsaturated C27 sterols as their acetate and trimethylsilyl (TMS) ether derivatives by GC and GC-MS. The GC retention data showed that numerous sterols were essentially coeluted on capillary GC columns coated with either 5% phenyl-95% methyl polysiloxane or polyethylene glycol, although the peaks were more widely dispersed on the latter column. Mass spectra of many groups of sterol isomers were also quite similar. Sterol mixtures of any complexity are likely to contain coeluting components, and attempts to establish structures based on mass spectra that may represent a mixture of sterol isomers could easily lead to errors. Our results demonstrate that GC and GC-MS alone cannot generally be used for rigorous structure determinations of individual components in mixtures of unsaturated sterols. However, all but a few of the 26 sterols could be distinguished by their combined chromatographic mobilities on the two GC columns coupled with critical examination of their mass spectra. GC-MS analysis of appropriate sterol subclasses or preferably individual sterol components obtained by prior purification by other methods may provide valuable supporting evidence for the identification of sterol structures. Reliability of identification is dependent upon careful attention to GC and MS conditions, calibration of GC and MS data with authentic sterol standards, and consideration of possible decomposition under GC conditions and of the effect of overloading on GC retention times.
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