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Compound-specific stable isotope analysis of nitrogen-containing intact polar lipids
Svensson, E.; Schouten, S.; Stam, A.; Middelburg, J.J.; Sinninghe Damsté, J.S. (2015). Compound-specific stable isotope analysis of nitrogen-containing intact polar lipids. Rapid Comm. Mass Spectrom. 29(23): 2263–2271.
In: Rapid Communications in Mass Spectrometry. Wiley: London. ISSN 0951-4198, more
Peer reviewed article  

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  • Middelburg, J.J., more
  • Sinninghe Damsté, J.S., more

    RATIONALE: Compound-specific isotope analysis (CSIA) of nitrogen in amino acids has proven a valuable tool in manyfields (e.g. ecology). Several intact polar lipids (IPLs) also contain nitrogen, and their nitrogen isotope ratios have thepotential to elucidate food-web interactions or metabolic pathways. Here we have developed novel methodology forthe determination of d15N values of nitrogen-containing headgroups of IPLs using gas chromatography coupled withisotope-ratio mass spectrometry. METHODS: Intact polar lipids with nitrogen-containing headgroups were hydrolyzed and the resulting compoundswere derivatized by (1) acetylation with pivaloyl chloride for compounds with amine and hydroxyl groups or(2) esterification using acidified 2-propanol followed by acetylation with pivaloyl chloride for compounds withboth carboxyl and amine groups. The d15N values of the derivatives were subsequently determined using gaschromatography/combustion/isotope-ratio mass spectrometry. RESULTS: Intact polar lipids with ethanolamine and amino acid headgroups, such as phosphatidylethanolamine andphosphatidylserine, were successfully released from the IPLs and derivatized. Using commercially available purecompounds it was established that d15N values of ethanolamine and glycine were not statistically different from theoffline-determined values. Application of the technique to microbial cultures and a microbial mat showed that themethod works well for the release and derivatization of the headgroup of phosphatidylethanolamine, a common IPLin bacteria. CONCLUSIONS: A method to enable CSIA of nitrogen of selected IPLs has been developed. The method is suitable formeasuring natural stable nitrogen isotope ratios in microbial lipids, in particular phosphatidylethanolamine, and will beespecially useful for tracing the fate of nitrogen in deliberate tracer experiments. Copyright © 2015 JohnWiley & Sons, Ltd.

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