For HPLC analysis, the procedure was as described by Kildegaard et al. (2017 (link)). A total of 100 μL of ethyl acetate extract was evaporated on SpeedVac, and the dry extracts were redissolved in 1 mL 99% ethanol + 0.01% BHT. Then, the extracts were analyzed by HPLC (Thermo Fisher Scientific) equipped with a Discovery HS F5 150 mm x 2.1 mm column (particle size 3 mm). The column oven temperature was set to 30°C. All organic solvents used were HPLC grade (Sigma Aldrich, St. Louis, MO). 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. β-carotene was detected at a retention time of 7.6 min by measuring absorbance at 450 nm. The β-carotene standard (C4582–5 mg) was purchased from Sigma-Aldrich.
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