Selecting an HPLC method for chemotaxonomic analysis of phytoplankton community in Mediterranean coastal lagoons

1 - Phytoplankton observations are commonly used to contribute to the assessment of aquatic ecosystem health and their trophic status. Compared to other methods, chemotaxonomic analysis based on High Pressure Liquid Chromatography (HPLC) presents many advantages (e.g., rapidity, reproducibility, and capacity to include pigments from all cell sizes), but its use in coastal lagoons studies is still not very common. The method of Wright et al., (1991) recommended by the UNESCO (Jeffrey et al., 1997) and most frequently used for phytoplankton analysis in coastal lagoons, so far, was selected and compared to the more novel method of Hagerthey et al. (2006). 2 - The two methods that differed slightly with respect to their solvent gradients during chromatography (mobile phase) and column (stationary phase), were tested using a pigment mix from DHI Water and Environment comprising 30 different pigments. Extraction methods were tested using replicates of 1l of sub-surface water from the Thau lagoon (South of France), sampled in June 2013. Optimization of the extraction was performed by testing different volumes of solvent (2 to 5 ml), different solvents based on a mix of methanol, acetone, dimethylformamide, water, compared to acetone 90% and pure methanol, as well as different extraction times (10 min to 2 h), and the addition of the ion-pairing agent tetrabutyl ammonium acetate hydroxide (TBAA). 3 - The second method of analysis allowed better separation and resolution of most of the pigments, especially of lutein and zeaxanthin. The early-eluting most polar pigments and the more hydrophobic pigments eluting in the end of the chromatogram (chlorophylls and carotenoids) showed also better separation and peak shapes. 5 mL of the mix of acetone/ methanol/ water (45:45:10) allowed the best extraction of the pigments. The use of TBAA showed negative effects. 4 - For pigment analysis in coastal lagoon, our final protocol used 1 h extraction with 5 mL of acetone/ methanol/ water, and analysis with the gradient from Hagerthey et al. (2006). On our analytical equipment it needed some adjustments. It uses a longer chromatography run and quantified the phytoplankton pigment markers better than the method of Wright et al. (1991)