title: A Simple Method for Measuring Carbon-13 Fatty Acid Enrichment in the Major Lipid Classes of Microalgae Using GC-MS.
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 of13C 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, includingcis/transisomers, of the PFB-derivatised fatty acids. The combination of solid-phase extraction and GC-MS enables the accurate determination of13C 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.
authors.affiliation: Centre for Chemistry and Biotechnology, School of Life and Environmental Sciences, Burwood Campus, Deakin University, Melbourne 3125, Australia. nelaheed@deakin.edu.au.
authors.forename: Sheik Nadeem
authors.initials: SN
authors.lastname: Elahee Doomun
authors.affiliation: Centre for Cellular and Molecular Biology, School of Life and Environmental Sciences, Burwood Campus, Deakin University, Melbourne 3125, Australia. stella.loke@deakin.edu.au.
authors.forename: Stella
authors.initials: S
authors.lastname: Loke
authors.affiliation: Bio21 Molecular Sciences and Biotechnology Institute, The University of Melbourne, Parkville 3052, Australia. spoc@unimelb.edu.au.
authors.forename: Sean
authors.initials: S
authors.lastname: O'Callaghan
authors.affiliation: Centre for Chemistry and Biotechnology, School of Life and Environmental Sciences, Burwood Campus, Deakin University, Melbourne 3125, Australia. damien.callahan@deakin.edu.au.
authors.forename: Damien L
authors.initials: DL
authors.lastname: Callahan
keywords: 13C stable isotope measurement; algae; chemical ionisation; gas chromatography; lipids; mass spectrometry; solid-phase extractions