A comparative assessment of cell disruption and extraction solvent composition for the analysis of pigments and lipids in phytoplankton using liquid chromatography mass spectrometry

Efficient and reproducible extraction of pigments and lipids from microalgae remains a challenge for comprehensive LC–MS–based profiling. This study systematically compared two cell lysis methods and five commonly used solvents in algal extractions with different polarities (100% methanol; acetone:water (9:1 v /v); ethanol/hexane (2:1 v/v); chloroform/ methanol/water (8:4:3 v/v/v), ethanol/hexane (1:1)) and compared the pigment and lipid class profiles from three microalgal species ( Haematococcus pluvialis, Desmodesmus subspicatus, and Chlorella variabilis). The analytes were measured using a fast, sensitive LC-MS method with a C18 column and a gradient of water (A) and water:acetonitrile:isopropanol (5:20:75 v /v/v). These conditions separated 16 pigments, phosphatidylcholines (PCs), and triacylglycerols (TAGs) in 25 min. Mechanical disruption by bead beating was found to be essential for achieving higher extraction yields compared with sonication alone; accordingly, bead beating was subsequently applied across the five extraction solvent systems. It was found that the 9:1 v /v acetone:water mixture provided the broadest pigment and lipid coverage, while ethanol/hexane yielded the highest efficiency for chlorophyll a nd non-polar TAGs. These results highlight the significant influence of solvent polarity as no single solvent mixture can be considered optimal for all compound classes. In summary, among the five solvent systems evaluated, 9:1 v /v acetone:water combined with bead beating represented the least toxic extraction strategy and enabled broad recovery of pigments and polar lipids across the three microalgal species examined; however, it was less effective for highly non-polar triacylglycerols.