Lipidomics pp 413-441 | Cite as

OnLine Ozonolysis Methods for the Determination of Double Bond Position in Unsaturated Lipids

  • Michael C. Thomas
  • Todd W. Mitchell
  • Stephen J. Blanksby
Part of the Methods in Molecular Biology book series (MIMB, volume 579)


Modern lipidomics relies heavily on mass spectrometry for the structural characterization and quantification of lipids of biological origins. Structural information is gained by tandem mass spectrometry (MS/MS) whereby lipid ions are fragmented to elucidate lipid class, fatty acid chain length, and degree of unsaturation. Unfortunately, however, in most cases double bond position cannot be assigned based on MS/MS data alone and thus significant structural diversity is hidden from such analyses. For this reason, we have developed two online methods for determining double bond position within unsaturated lipids; ozone electrospray ionization mass spectrometry (OzESI–MS) and ozone-induced dissociation (OzID). Both techniques utilize ozone to cleave C–C double bonds that result in chemically induced fragment ions that locate the position(s) of unsaturation.

Key words

Ozonolysis Double bond position Structural characterization Unsaturated lipids Mass spectrometry OzESI–MS OzID 



S.J.B. and T.W.M. acknowledge the financial support of the University of Wollongong and the Australian Research Council (Grants DP0452849 and LP0455472). M.C.T. acknowledges the support of an Australian Postgraduate Award. The authors acknowledge Dr. David Harman for providing details of the LTQ modification. The authors also acknowledge MS Jane Deeley for Providing the human lens spectra and Miss Jessica Nealon for the Preparation of the cow kidney sample.


  1. 1.
    Han X, Gross RW. (2005) Shotgun lipidomics: multidimensional MS analysis of cellular lipidomes. Expert Rev Proteomic. 2(2):253–64.CrossRefGoogle Scholar
  2. 2.
    Pulfer M, Murphy RC. (2003) Electrospray mass spectrometry of phospholipids. Mass Spectrom Rev. 22(5):332–64.PubMedCrossRefGoogle Scholar
  3. 3.
    Moe MK, Anderssen T, Strom MB, Jensen E. (2004) Vicinal hydroxylation of unsaturated fatty acids for structural characterization of intact neutral phospholipids by negative electrospray ionization tandem quadrupole mass spectrometry. Rapid Comm Mass Spectrom. 18(18):2121–30.CrossRefGoogle Scholar
  4. 4.
    Cook HW. (1996) Fatty acid desaturation and chain elongation in eukaryotes. In: Vance DE, Vance J, eds. Biochemistry of lipids, lipoproteins and membranes. Amsterdam: Elsevier:129–52.CrossRefGoogle Scholar
  5. 5.
    Fatty Acids and Eicosanoids. (Accessed at .)
  6. 6.
    Pariza MW, Park Y, Cook ME. (2001) The biologically active isomers of conjugated linoleic acid. Prog Lipid Res. 40(4):283–98.PubMedCrossRefGoogle Scholar
  7. 7.
    Ackman RG. (2002) The gas chromatograpy in practical analyses of common and uncommon fatty acids for the 21st century. Anal Chim Acta. 465:175–92.CrossRefGoogle Scholar
  8. 8.
    Bryant DK, Orlando RC, Fenselau C, Sowder RC, Henderson LE. (1991) 4-Sector tandem mass-spectrometric analysis of complex-mixtures of phosphatidylcholines present in a human-Immunodeficiency-virus preparation. Anal Chem. 63(11):1110–4.PubMedCrossRefGoogle Scholar
  9. 9.
    Moe MK, Anderssen T, Strom MB, Jensen E. (2005) Total structure characterization of unsaturated acidic phospholipids provided by vicinal di-hydroxylation of fatty acid double bonds and negative electrospray ionization mass spectrometry. J Am Soc Mass Spectrom. 16(1):46–59.PubMedCrossRefGoogle Scholar
  10. 10.
    Moe MK, Strom MB, Jensen E, Claeys M. (2004) Negative electrospray ionization low-energy tandem mass spectrometry of hydroxylated fatty acids: a mechanistic study. Rapid Commun Mass Spectrom. 18(15):1731–40.PubMedCrossRefGoogle Scholar
  11. 11.
    Harrison KA, Murphy RC. (1996) Direct mass spectrometric analysis of ozonides: Application to unsaturated glycerophosphocholine lipids. Anal Chem. 68(18):3224–30.PubMedCrossRefGoogle Scholar
  12. 12.
    Van Pelt CK, Carpenter BK, Brenna JT. (1999) Studies of structure and mechanism in acetonitrile chemical ionization tandem mass spectrometry of polyunsaturated fatty acid methyl esters. J Am Soc Mass Spectrom. 10(12):1253–62.PubMedCrossRefGoogle Scholar
  13. 13.
    Van Pelt CK, Brenna JT. (1999) Acetonitrile chemical ionization tandem mass spectrometry to locate double bonds in polyunsaturated fatty acid methyl esters. Anal Chem. 71(10):1981–9.PubMedCrossRefGoogle Scholar
  14. 14.
    Xu Y, Brenna JT. (2007) Atmospheric pressure covalent adduct chemical ionization tandem mass spectrometry for double bond localization in monoene- and diene-containing triacylglycerols. Anal Chem. 79(6):2525–36.PubMedCrossRefGoogle Scholar
  15. 15.
    Thomas MC, Mitchell TW, Blanksby SJ. (2006) Ozonolysis of phospholipid double bonds during electrospray ionization: a new tool for structure determination. J Am Chem Soc. 128(1):58–9.PubMedCrossRefGoogle Scholar
  16. 16.
    Thomas MC, Mitchell TW, Harman DG, Deeley JM, Murphy RC, Blanksby SJ. (2007) Elucidation of double bond position in unsaturated lipids by ozone electrospray ionization mass spectrometry. Anal Chem. 79(13):5013–22.PubMedCrossRefGoogle Scholar
  17. 17.
    Grimm RL, Hodyss R, Beauchamp JL. (2006) Probing interfacial chemistry of single droplets with field-induced droplet ionization mass spectrometry: Physical adsorption of polycyclic aromatic hydrocarbons and ozonolysis of oleic acid and related compounds. Anal Chem. 75:3800–6.CrossRefGoogle Scholar
  18. 18.
    Thomas MC, Mitchell TW, Harman DG, Deeley JM, Nealon JR, Blanksby SJ. (2008) Ozone-induced dissociation: elucidation of double bond position within mass-selected lipid ions. Anal Chem. 80(1):303–11.PubMedCrossRefGoogle Scholar
  19. 19.
    Ozone compatible materials – Ozone solutions website (Accessed at
  20. 20.
    Ozone compatible materials – Ozone services website. (Accessed at
  21. 21.
    Harman DG, Blanksby SJ. (2007) Investigation of the gas phase reactivity of the 1-adamantyl radical using a distonic radical anion approach. Org Biomol Chem. 5:3495–503.PubMedCrossRefGoogle Scholar
  22. 22.
    Limits for air contaminants – Occupational Safety and Health Administration. (Accessed at = STANDARDS&p_id = 9992.)
  23. 23.
    IDLH documentation – Centres for Disease Control and Prevention. (Accessed at
  24. 24.
  25. 25.
    Baba S, Satoh S, Yamabe C. (2002) Development of measurement equipment of half life of ozone. Vacuum. 65:489–95.CrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Michael C. Thomas
    • 1
  • Todd W. Mitchell
    • 2
  • Stephen J. Blanksby
    • 3
  1. 1.School of Chemistry and Health SciencesUniversity of WollongongWollongongAustralia
  2. 2.School of Health SciencesUniversity of WollongongWollongongAustralia
  3. 3.School of ChemistryUniversity of WollongongWollongongAustralia

Personalised recommendations