Pigments analysis was performed on fresh 95% aqueous acetone extracts to characterize lipophilic pigment composition and on MeOH/DCM dried extracts to study the relationship between carotenoids content and antioxidant activity.
Just after extraction, fresh acetone extracts were filtered on a 0.2 µM PTFE filter before immediate HPLC analysis. MeOH/DCM dried extracts were solubilized in ethanol at 0.5 mg mL−1 and filtered on a 0.2 µM PTFE filter before HPLC analysis. The samples were analyzed by HPLC-UV-DAD (Agilent Technologies, Santa Clara, CA, United States, series 1200 HPLC-UV-DAD) using an Eclipse XDB-C8 reverse phase column (150 by 4.6 mm, 3.5 µm particle size, Agilent Technologies) following the method by Van Heukelem and Thomas [87 (link)]. HPLC grade MeOH and water were purchased from Merck Chemicals (Darmstadt, Germany) and tetrabutyl ammonium acetate from Sigma-Aldrich (Darmstadt, Germany). Quantification was carried out using external calibration against pigments standard (lutein, neoxanthin, violaxanthin, antheraxanthin, zeaxanthin, β-carotene, diatoxanthin, diadinoxantin, and fucoxanthin provided by DHI, Denmark). Quantification of siphonaxanthin was done according to fucoxanthin standard as recommended by Roy et al. [88 ].
Just after extraction, fresh acetone extracts were filtered on a 0.2 µM PTFE filter before immediate HPLC analysis. MeOH/DCM dried extracts were solubilized in ethanol at 0.5 mg mL−1 and filtered on a 0.2 µM PTFE filter before HPLC analysis. The samples were analyzed by HPLC-UV-DAD (Agilent Technologies, Santa Clara, CA, United States, series 1200 HPLC-UV-DAD) using an Eclipse XDB-C8 reverse phase column (150 by 4.6 mm, 3.5 µm particle size, Agilent Technologies) following the method by Van Heukelem and Thomas [87 (link)]. HPLC grade MeOH and water were purchased from Merck Chemicals (Darmstadt, Germany) and tetrabutyl ammonium acetate from Sigma-Aldrich (Darmstadt, Germany). Quantification was carried out using external calibration against pigments standard (lutein, neoxanthin, violaxanthin, antheraxanthin, zeaxanthin, β-carotene, diatoxanthin, diadinoxantin, and fucoxanthin provided by DHI, Denmark). Quantification of siphonaxanthin was done according to fucoxanthin standard as recommended by Roy et al. [88 ].