Vitamin D pp 497-521 | Cite as

Measurement of Vitamin D and its Metabolites by Gas Chromatography-Mass Spectrometry

  • Hugh L. J. Makin
  • David J. H. Trafford


Gas-liquid chromatography (GC) has been very successfully applied to the analysis of a number of different groups of biologically important compounds. In particular, the analysis of the multiplicity of metabolites of secreted steroid hormones found in human urine has been greatly simplified by the introduction of GC (1). Using conventional packed columns (2) and wall-coated capillary columns (3), ‘profiles’ of varying degrees of complexity have been produced. Plasma steroids have also been analysed by GC using electron capture detection (4) and more recently using gas chromatography-mass spectrometry (GC-MS, 5).


Trimethylsilyl Ether Normalise Mass Spectrum Mass Fragmentography Trimethylsilyl Ether Derivative Heptafluorobutyric Anhydride 
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  1. 1.
    Makin HLJ: Methods of steroid analysis. 1. Group estimations and separation techniques. In: Biochemistry of Steroid Hormones, Makin HLJ (ed), Oxford, Blackwell Scientific Publications Ltd. 1975, p 185–210.Google Scholar
  2. 2.
    Trafford DJH, Makin HLJ: Rapid quantitative gas-chromatographic fractionation of the major urinary 17-oxostreoids and 17-oxogenic steroids. Clin. Chim. Acta 40: 421–430, 1972.PubMedCrossRefGoogle Scholar
  3. 3.
    Shackleton CHL, Taylor NF, Honour JW: An atlas of gas chromatographic profiles of neutral urinary steroids in health and disease, Delft, Packard-Becker B.V., 1980.Google Scholar
  4. 4.
    Mason PA, Fraser R: Estimation of aldosterone, 11-deoxycorticosterone, 18-hydroxy-11-deoxy-corticosterone, corticosterone,cortisol and 11-deoxycortisol in human plasma by gas-liquid chromatography with electron capture detection. J. Endocrinol. 64: 277–288, 1975.PubMedCrossRefGoogle Scholar
  5. 5.
    Setchell KDR: Cas chromatography-mass spectrometry. In: Hormones in Blood, Gray CH, James VHT (eds), London, Academic Press, 1979 (3rd ed., vol. 3 ), p 63–127.Google Scholar
  6. 6.
    Pelc B, Marshall DH: Thermal transformation of cholecalciferol between 100-170°C. Steroids 31: 23–29, 1978.PubMedCrossRefGoogle Scholar
  7. 7.
    Nair P, de Leon S: Stereochemical studies on the microquantitative detection of vitamin D by gas-liquid chromatography. Arch. Biochem. Biophys. 128: 663–672, 1968.PubMedCrossRefGoogle Scholar
  8. 8.
    Murray TK, Day KC, Kodicek E: The differentiation and assay of vitamins D2 and D3 by gas-liquid chromatography. Biochem. J. 98: 293–296, 1966.PubMedGoogle Scholar
  9. 9.
    Sklan D, Budowski P, Katz M: Determination of 25-hydroxycholecalciferol by combined thin layer and gas chromatography. Analyt. Biochem. 56: 606–609, 1973.PubMedCrossRefGoogle Scholar
  10. 10.
    Kobayashi T, Adachi A: Studies on the isomerisation of vitamin D and related compounds with acidic reagents. 1. A simple method for the determination of vitamin D2 isomers in mixtures. J. Nutr. Sci. Vitaminol. (Tokyo) 19: 311–319, 1973.CrossRefGoogle Scholar
  11. 11.
    DeLeenheer AP, Cruyl AAM: Mass fragmentography of vitamin D3 (cholecalciferol) in plasma samples. In: Quantitative Mass Spectrometry in Life Sciences, DeLeenheer AP, Roncucci RR (eds), Amsterdam, Elsevier, 1977, p 165–175.Google Scholar
  12. 12.
    DeLeenheer AP, Cruyl AA: Vitamin D3 in plasma:quantitation by mass fragmentography. Analyt. Biochem. 91: 293–303, 1978.CrossRefGoogle Scholar
  13. 13.
    Wilson PW, Lawson DEM, Kodicek E: Gas-liquid chromatography of ergocalciferol and cholecalciferol in nanogram quantities. J. Chromatog. 39: 75–77, 1969.CrossRefGoogle Scholar
  14. 14.
    Seamark DA, Trafford DJH, Makin HLJ: A new procedure for the formation of isotachysterol derivatives of subnanoaole quantities of ergocalciferol, cholecalciferol and its metabolites prior to gas liquid chromatography. J. Steroid Biochem. 13: 1057–1063, 1980.PubMedCrossRefGoogle Scholar
  15. 15.
    Seamark DA, Trafford DJH, Makin HLJ: The estimation of vitamin D and some metabolites in human plasma by mass fragmentography. Clin. Chim. Acta 106: 51–52, 1980.PubMedCrossRefGoogle Scholar
  16. 16.
    Seamark DA, Trafford DJH, Makin HLJ: The use of isotachysterols in improving the specificity and sensitivity of assays of vitamin D and metabolites. In: Vitamin D: Chemical, Biochemical and Clinical Endocrinology of Calcium Metabolism, Norman AW, Schaefer K, Herrath Dv, Grigoleit H-G (eds), Berlin, Walter de Gruyter, 1982, p 785–787.Google Scholar
  17. 17.
    Edlund DO: Gas-liquid chromatographic determination of vitamin D in multiple vitamin tables and their raw materials. In: Methods in Enzymology, McCormick DB, Wright LD (eds), New York, Wright LD (eds), 1980, vol. 67, p 343–347.Google Scholar
  18. 18.
    Kobayashi T: Gas-liquid chromatographic determination of vitamin D. In: Methods in Enzymology, McCormick DB, Wright LD (eds), New York, Wright LD (eds), 1980, Vol. 67, p 347–355.Google Scholar
  19. 19.
    Okano T, Mizuno K, Matsuyama N, Nobuhara N, Kobayashi T: Gas-liquid chromatography/mass spectrometric identification of previtamin D3 and vitamin D3 in the skin of vitamin D-deficient rats irradiated with ultraviolet light. Rce. Trav. Chim. Pays-Bas 98: 2253–2257, 1979.Google Scholar
  20. 20.
    Seamark DA, Trafford DJH, Makin HLJ: The estimation of vitamin D and its metabolites in human plasma. J. Steroid Biochem. 14: 111–123, 1981.PubMedCrossRefGoogle Scholar
  21. 21.
    Horst RL, Littledike ET, Riley JL, Napoli JL: Quantitation of vitamin D and its metabolites and their plasma concentration in five species of animals. Analyt. Biochem. 116: 189–203, 1981.PubMedCrossRefGoogle Scholar
  22. 22.
    Zagalak B, Curtius HC, Foschi R, Wipf G, Redweik U, Zagalak MJ: Mass fragmentographic determination of cholecalciferol and 25-hydroxy-cholecalciferol in human serum. Experientia 34: 1537–1539, 1978.PubMedCrossRefGoogle Scholar
  23. 23.
    Bjorkhem I, Holmberg I: A novel specific assay of 25-hydroxyvitamin D3. Clin. Chim. Acta 68: 215–221, 1976.PubMedCrossRefGoogle Scholar
  24. 24.
    Bjorkhem I, Holmberg I, Kristiansen T, Pedersen JI: Assay of 1,25-dihydroxyvitamin D3 by isotope dilution-mass fragmentography. Clin. Chem. 25: 584–588, 1979.PubMedGoogle Scholar
  25. 25.
    Bjorkhem I, Larsson A: A specific assay of vitamin D3 in human serum. Clin. Chim. Acta 88: 559–567, 1978.PubMedCrossRefGoogle Scholar
  26. 26.
    Bjorkhem I, Holmberg I, Kristiansen T, Larsson A, Pedersen JI: Use of isotope dilution mass fragmentography in vitamin D research. In: Vitamin D, Basic Research and its Clinical Application, Norman AW, et al (eds), Berlin, Walter de Gruyter, 1979, p 183–187.Google Scholar
  27. 27.
    Bjorkhem I, Holmberg I: Mass fragmentographic assay of 25-hydroxy-vitamin D3. In: Methods in Enzymology, McCormick DB, Wright LD (eds), New York, Wright LD (eds), 1980, vol. 67, p 385–393.Google Scholar
  28. 28.
    Bjorkhem I, Holmberg I: Assay and properties of a mitochondrial 25-hydroxylase active on vitamin D3. J. Biol. Chem. 253: 842–849, 1978.PubMedGoogle Scholar
  29. 29.
    Bjorkem I, Holmberg I: On the 25-hydroxylation of vitamin D3 in vitro studied with a mass fragmentographic technique. J. Biol. Chem. 254: 9518–9524, 1979.Google Scholar
  30. 30.
    Bjorkhem I, Hansson R, Holmberg I, Wikvall K: 25-Hydroxylation of vitamin D3 by a reconstituted system from rat liver microsomes. Biochem. Biophys. Res. Comm. 90: 615–622, 1979.PubMedCrossRefGoogle Scholar
  31. 31.
    Pedersen JI, Holmberg I, Bjorkhem I: Reconstitution of vitamin D3 25-hydroxylase activity with a cytochrome P-450 preparation from rat liver mitochondria. FEBS Let. 98: 394–398, 1979.CrossRefGoogle Scholar
  32. 32.
    Shackleton CHL, Roitman E, Whitney J: Urinary metabolites of vitamin D3. J. Steroid Biochem. 12: 521–528, 1980.PubMedCrossRefGoogle Scholar
  33. 33.
    Halket JM, Lisboa BP: An approach to the recognition and quantitation of vitamin D and its metabolites using gas Chromatography-mass spectrometry. Acta Endocrinol. (Kbh) 87: 120–121, 1978.Google Scholar
  34. 34.
    DeLeenheer AP, Cruyl AAM: A method for the gas chromatographic determination of vitamin D and related structures. J. Chromat. Sci. 14: 434–437.Google Scholar
  35. 35.
    Lisboa BP, Halket JM: A study of some derivatives of vitamin D and related compounds by open tubular capillary column gas chromatography-mass spectrometry. In: Recent Developments in Chromatography and Electrophoresis, Frigerio A, Renoz L (eds), Amsterdam, Elsevier, 1979. p 141–162.Google Scholar
  36. 36.
    Halket JM, Ganschow I, Lisboa BP: Cas chromatographic-mass spectrometric properties of boronate esters of 24R, 25-dihydroxycholecalciferol. J. Chromatog. 192: 434–440, 1980.CrossRefGoogle Scholar
  37. 37.
    Whitney JO, Shackleton CHL, Edmonds CG, Burlingame AL, Piel CF: Synthesis of deuterium-labeled vitamin D3 metabolites: purification from human plasma for selected ion monitoring-mass spectrometry. In: Stable Isotopes: Proceedings of the Third International Conference, Klein ER, Klein PD (eds), New York, Academic Press, 1978, p 47–61.Google Scholar
  38. 38.
    Bjorkhem I, Blomstrand R, Lantto O, Svensson L, Ohman G: Towards absolute methods in clinical chemistry: application of mass fragmentography to high-accuracy analyses. Clin. Chem. 22: 1789–1801, 1976.PubMedGoogle Scholar
  39. 39.
    Holmberg I, Larsson A: Seasonal variation of vitamin D3 and 25-hydroxyvitamin D3 in human serum. Clin. Chim. Acta 100:173–174, 1980Google Scholar
  40. 40.
    Turnbull, H., Trafford DJH, Makin HLJ: A rapid and simple method for the measurement of plasma 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 using Sep-Pak C18 cartridges and a single high-performance liquid chromatographic step. Clin. Chim. Acta 120: 65–76, 1982.PubMedCrossRefGoogle Scholar

Copyright information

© Martinus Nijhoff Publishing, Boston/The Hague/Dordrecht/Lancaster 1984

Authors and Affiliations

  • Hugh L. J. Makin
  • David J. H. Trafford

There are no affiliations available

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