Comparison of different solvent extraction compositions for the analysis of phytoplankton pigments and lipids using a rapid and sensitive LC-MS method

Background: Carotenoids and chlorophylls are important pigments as they have antioxidant properties and some such as lutein, astaxanthin and β-carotene are used as nutraceuticals. Pigment profiles are also used as taxonomic markers for phytoplankton. There are many solvent compositions published for the extraction of phytoplankton pigments. Here we report the testing of five different commonly used solvents and compare the overall pigment and lipid profiles and abundances using liquid chromatography-mass spectrometry (LC-MS) for three microalgal species with different pigment profiles. Results: : A fast and sensitive LC-MS method was developed using a C18 column with a gradient consisting of isopropanol, acetonitrile and water. A total of 18 pigments along with phosphatidylcholines (PCs), triacylglycerols (TAGs) were separated in 25 mins. An extraction solvent composition of 90% acetone provided the broadest-coverage of pigments and lipids, while ethanol/hexane resulted in the highest extraction efficiency for chlorophyll and the non-polar triacylglycerol lipids. Overall, the result for the different mixtures shows the significant impact that solvent polarity has on the overall pigment and lipid profile and that no single mixture was optimal for all targets. Complete cell lysis using mechanical disruption such as bead beating is also an important step for quantitative extraction as chemical extraction on its own does not provide reproducible results. Conclusion: A 90% acetone extraction solvent mixture provides the broadest extraction of pigments from the species Haematococcus pluvialis, Desmodesmus subspicatus and Chlorella variabilis when coupled with bead beating. This extraction solvent is comparatively safe, polar and less toxic than other solvents. It was also found that none of the common solvent mixtures tested here provided the highest extraction efficiencies across all compound groups