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    A Simple Method for Measuring Carbon-13 Fatty Acid Enrichment in the Major Lipid Classes of Microalgae Using GC-MS

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    Author
    Doomun, SNE; Loke, S; O'Callaghan, S; Callahan, DL
    Date
    2016-12-01
    Source Title
    Metabolites
    Publisher
    MDPI
    University of Melbourne Author/s
    O'Callaghan, Sean; CALLAHAN, DAMIEN
    Affiliation
    School of BioSciences
    Bio21
    Metadata
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    Document Type
    Journal Article
    Citations
    Doomun, S. N. E., Loke, S., O'Callaghan, S. & Callahan, D. L. (2016). A Simple Method for Measuring Carbon-13 Fatty Acid Enrichment in the Major Lipid Classes of Microalgae Using GC-MS. METABOLITES, 6 (4), https://doi.org/10.3390/metabo6040042.
    Access Status
    Open Access
    URI
    http://hdl.handle.net/11343/257167
    DOI
    10.3390/metabo6040042
    Open Access at PMC
    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5192448
    Abstract
    A simple method for tracing carbon fixation and lipid synthesis in microalgae was developed using a combination of solid-phase extraction (SPE) and negative ion chemical ionisation gas chromatography mass spectrometry (NCI-GC-MS). NCI-GC-MS is an extremely sensitive technique that can produce an unfragmented molecular ion making this technique particularly useful for stable isotope enrichment studies. Derivatisation of fatty acids using pentafluorobenzyl bromide (PFBBr) allows the coupling of the high separation efficiency of GC and the measurement of unfragmented molecular ions for each of the fatty acids by single quadrupole MS. The key is that isotope spectra can be measured without interference from co-eluting fatty acids or other molecules. Pre-fractionation of lipid extracts by SPE allows the measurement of 13C isotope incorporation into the three main lipid classes (phospholipids, glycolipids, neutral lipids) in microalgae thus allowing the study of complex lipid biochemistry using relatively straightforward analytical technology. The high selectivity of GC is necessary as it allows the collection of mass spectra for individual fatty acids, including cis/trans isomers, of the PFB-derivatised fatty acids. The combination of solid-phase extraction and GC-MS enables the accurate determination of 13C incorporation into each lipid pool. Three solvent extraction protocols that are commonly used in lipidomics were also evaluated and are described here with regard to extraction efficiencies for lipid analysis in microalgae.

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