3.4. Quantification of Carotenoids

The ethanolic ultrasonic extracts were evaporated, resuspended in chromatographic methanol, and filtered (Ø = 0.22 µm filter) together with the conventional extracts (in methanol). Thereafter, they were transferred into a chromatography vial and immediately used to quantify carotenoids via liquid chromatography. HPLC was performed on the Beckman System Gold binary delivery system equipped with a UV–vis photodiode array detector (Beckman Instruments, Fullerton, CA, USA) using a C18 column (150 mm × 4.6 mm i.d., 5 µm, SunFire ^TM column; Waters, Milford, MA, USA). The flow rate was maintained at 1 mL/min and injection volume was 40 µL of the algal extracts. Ethyl acetate was used as mobile phase A and acetonitrile/water (9:1 v/v) was used as mobile phase B. The mobile phase gradient was as follows: 0–16 min, 0–60% solvent A; 16–30 min, 60% A; and 30–35 min, 100% A. The selected carotenoids were detected at a wavelength of 450 nm and by comparing the peak areas obtained from the methanolic C. onubensis extracts with those obtained from the injected standards (Sigma-Aldrich, 0–50 ppm, ~R² = 0.998). Lutein concentration was referred to as dry biomass or weight of the extract, whereas lutein recovery was calculated using Equation (9):

where Wc is the mass of lutein (mg) extracted under the ultrasonic conditions described in this study and Wt is the mass of lutein conventionally extracted (using solvent extraction with an average lutein concentration of 3.24 ± 0.11 mg/g, expressed as mg of lutein/g of the dry weight of C. onubensis, benchmark extraction).