4.5. Carotenoid Quantification by HPLC

For HPLC measurements, 100 µL of ethyl acetate extract was evaporated in a rotatory evaporator, and the dry extracts were redissolved in 1 mL 99% ethanol + 0.01% BHT. Then, the extracts were analyzed by HPLC (Thermo Fisher Scientific, Waltham, MA, USA ) equipped with a Discovery HS F5 150 mm × 2.1 mm column (particle size 3 mm). For this analysis, the column oven temperature was set to 30 °C. All organic solvents used were HPLC grade (Sigma Aldrich, St. Louis, MO, USA). The flow rate was set to 0.7 mL/min with an initial solvent composition of 10 mM ammonium formate (pH = 3, adjusted with formic acid) (solvent A) and acetonitrile (solvent B) (3:1) until minute 2.0. Solvent composition was then changed at minute 4.0 following a linear gradient until % A = 10.0 and % B = 90.0. The solvent composition was kept until 10.5 min when the solvent was returned to initial conditions and the column was re-equilibrated until 13.5 min. The injection volume was 10 µL. The peaks obtained from the sample analysis were identified by comparison to prepared standards and integration of the peak areas was used to quantify carotenoids from obtained standard curves. The β-carotene and echinenone compounds were detected at retention times of 7.6 min and 6.9 min, respectively, by measuring absorbance at 450 nm, while astaxanthin and canthaxanthin were detected by absorbance at 475 nm and retention times of 5.9 min and 6.4 min, respectively. The results were verified by comparing the samples with the standards. Standards were purchased from Sigma-Aldrich: β-carotene (C4582-5 mg), echinenone (73341-1MG), canthaxanthin (11775-1MG), and astaxanthin (SML0982-50MG).