Multistep mass spectrometry methodology for direct characterization of polar lipids in green microalgae using paper spray ionization.

Paper spray ionization, an ambient ionization method, has been applied for the identification of polar lipids in green microalgae with no sample preparation. A multistep experimental protocol was employed to characterize the lipid species of two microalgae strains, Kyo-Chlorella in tablet form and Nannochloropsis in paste form by mass spectrometry (MS). Tandem mass spectrometry (MS/MS) experiments using collision induced dissociation (CID) were employed for initial characterization of the detected lipid species, which were dominated by polar glycolipids and phospholipids. Product ion scan experiments were performed to determine the lipid head groups and fatty acid composition. Precursor ion scan experiments using fragment ions such as m/z 184, which is characteristic of the phosphocholine headgroup, were then used to confirm the lipid classification. Lipid elemental compositions were determined by exact mass measurements using high resolution mass spectrometry. Finally, the position of unsaturation was determined using reactive paper spray ionization experiments with ozone used as a reagent to cleave double bonds. Ozone was produced in situ using dielectric barrier discharge from a low temperature plasma, and it reacted in ambient air with the spray of ions produced by paper spray ionization. Using the precursor ion scan experiment, the resulting ozone cleavage product ions were used to determine the position of unsaturation for some of these species. By applying this experimental protocol, the molecular formulas and key aspects of the structures of glycerophosphocholines (PCs) such as 9Z-16:1/9Z,12Z-16:2 PC and 6Z,9Z-18:2/6Z,9Z,12Z-18:3PC and monogalactosyldiacylglycerols (MGDGs) such as 18:3/16:3MGDG were identified in the positive ion mode, while glycerophosphoglycerols (PGs) such as 18:3/16:0 PG and sulfoquinovosyldiacylglycerols (SQDGs) such as 18:3/16:0 SQDG were identified in the negative ion mode.