Advertisement

Identification and quantitation of dietary and fecal neutral sterols by mass spectrometry

      This paper is only available as a PDF. To read, Please Download here.

      Abstract

      A procedure is described for the identification and quantitation of dietary and fecal neutral sterols by the combination of thin-layer chromatography, mass spectrometry and Liebermann-Burchard colorimetry. The neutral sterols in the diet and feces were extracted by the method of Ho and Taylor and Abell el al. respectively. The extracted neutral sterols were then further separated from other compounds by thin-layer chromatography with silica gel H as adsorbent and a solvent system composed of light petroleum:ethyl ether:glacial acetic acid (80:20:1, by volume). The isolated neutral sterols consist of stigmastanol, sitosterol, stigmasterol, campesterol, coprostanol and cholesterol. The relative quantitaty of each sterol in such a mixture can be measured from its mass spectrum. Their relative absorbance of the Liebermann-Burchard reaction can also be determined. Thus, the absolute quantity of each individual neutral sterol in a mixture can be computed from the data obtained from the mass spectrum and colorimetry of the sample. The method is accurate and reproducible and of great value in the study of cholesterol metabolism when the diet and feces contain a variety of plant sterols.

      Keywords

      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'

      Subscribe:

      Subscribe to Atherosclerosis
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect

      References

        • Copius-Peereboom J.W.
        • Beekes H.W.
        The analysis of mixtures of animal and vegetable fats, Part 5 (Separation of sterol acetates by thin-layer chromatography in reversed-phase systems and on silica gel G-silver nitrate layers).
        J. Chromatog. 1965; 17: 99-113
        • Ikan R.
        • Harel S.
        • Kashman J.
        • Bergmann E.D.
        The separation of sterols and corresponding stanols by thin-layer chromatography.
        J. Chromatog. 1964; 14: 504-506
        • Werbin H.
        • Chaikoff I.L.
        • Imada M.R.
        5α-Cholestan-3β-ol: Its' distribution in tissues and its synthesis from cholesterol in the guinea pig.
        J. Biol. Chem. 1962; 237: 2072-2077
        • Copius-Peereboom J.W.
        • Beekes H.W.
        The analysis of mixtures of animal and vegetable fats, Part 3 (Separation of some sterols and sterol acetates by thin-layer chromatography).
        J. Chromatog. 1962; 9: 316-320
        • Ikan R.
        • Cudzinovski M.
        Separation of sterols and corresponding stanols on thin layers of silica impregnated with silver nitrate.
        J. Chromatog. 1965; 18: 422-423
        • Desouza N.J.
        • Nes W.R.
        Improved separation of sterols by reversed-phase thin-layer chromatography.
        J. Lipid Res. 1969; 10: 240-243
        • Ditullio N.W.
        • Jacobs C.S.
        • Holmes W.L.
        Thin-layer chromatography and identification of free sterols.
        J. Chromatog. 1965; 20: 354-357
        • Beerthuis R.K.
        • Recourt J.H.
        Sterol analysis by gas chromatography.
        Nature (Lond.). 1960; 186: 372
        • Lipsky S.R.
        • Landowne R.A.
        Effects of varying the chemical composition of the stationary phase on the separation of certain C19, C21 and C27 steroids by gas chromatography.
        Anal. Chem. 1961; 33: 818
        • Luukkainen T.
        • Van Den Heuvel W.J.A.
        • Haahti E.O.A.
        • Horning E.C.
        Gas chromatographic behavior of trimethylsilylesters of steroids.
        Biochim. Biophys. Acta. 1961; 52: 599
        • Wells W.W.
        • Makita M.
        The quantitative analysis of fecal neutral sterols by gas-liquid chromatography.
        Anal. Chem. 1962; 4: 204
        • Miettinen T.A.
        • Ahrens Jr., E.H.
        • Grundy S.M.
        Quantitative isolation and gasliquid chromatographic analysis of total dietary and fecal neutral steroids.
        J. Lipid Res. 1965; 6: 411
        • Bloomfield D.K.
        The relationship between solid support, column efficiency and sterol quantitation by gas chromatography.
        J. Chromatog. 1962; 9: 411
        • Eneroth P.
        • Hellstrom K.
        • Ryhage R.
        Identification and quantitation of neutral fecal steroids by gas-liquid chromatography and mass spectrometry: studies of human excretion during two dietary regimens.
        J. Lipid Res. 1964; 5: 245
        • Rosenfeld R.S.
        • Lebeau M.C.
        • Shulman S.
        • Seltzer J.
        Analysis of fecal sterols by gas chromatography.
        J. Chromatog. 1962; 7: 293
        • Abell L.L.
        • Liese L.
        • Mosbach E.H.
        • Kendall F.E.
        Cholesterol metabolism in the dog.
        J. Biol. Chem. 1956; 220: 527
        • Ho K.J.
        • Taylor C.B.
        Comparative studies on tissue cholesterol.
        Arch. Pathol. 1968; 8: 86
        • Liebermann C.
        Über das Oxychinoterpen.
        Ber. 1885; 18: 1803
        • Burchard H.
        Beiträge zur Kenntniss der Cholesterine.
        Thesis. 1889; (Rostock): 26
        • Martin R.P.
        Reversed phase paper chromatography and detection of steroids of the cholesterol class.
        Biochim. Biophys. Acta. 1957; 25: 408
        • Cooeman D.L.
        • Wells W.W.
        • Baumann C.A.
        Intestinal sterols, Part 2 (Determination of coprostanol and certain related sterols).
        Arch. Biochem. Biophys. 1956; 60: 412
        • Cook R.P.
        • Rattray B.M.
        Methods of isolation and estimation of sterols.
        in: Cook R.P. Cholesterol, Chemistry, Biochemistry and Pathology. Academic Press, New York1958: 117-143
        • Grundy S.M.
        • Ahrens Jr., E.H.
        • Miettinen T.A.
        Quantitative isolation and gasliquid chromatographic analysis of total fecal bile acids.
        J. Lipid Res. 1965; 6: 397
        • Forman D.T.
        • Phillips C.
        • Eiseman W.
        • Taylor c.B.
        Fluorometric measurement of fecal bile acids.
        Clin. Chem. 1968; 14: 348
        • Talalay P.
        Enzymic analysis of steroid hormones.
        Meth. Biochem. Anal. 1960; 8: 119
        • Gould R.G.
        • Jones R.J.
        • Leroy C.G.
        • Wissler R.W.
        • Taylor C.B.
        Absorbability of β-sitosterol in humans.
        Metabolism. 1969; 18: 652
        • Grundy S.M.
        • Ahrens Jr., E.H.
        • Salen G.
        Dietary β-sitosterol as an internal standard to correct for cholesterol losses in sterol balance studies.
        J. Lipid Res. 1968; 9: 374
        • Grundy S.M.
        • Ahrens Jr., E.H.
        Measurements of cholesterol turnover, synthesis, and absorption in man, carried out by isotope kinetic and sterol balance methods.
        J. Lipid Res. 1969; 10: 91