Quantification of Carotenoids and Fatty Acids

The amount of astaxanthin and lutein in the extracts was measured by u-HPLC (Agilent 1290 Infinity II with Agilent Zorbax Eclipse plus C18 column 1.8 μm) [3 (link)]. The u-HPLC was equipped with a quaternary pump, thermostated oven column, and UV diode array detector (DAD) (measuring absorbance at 444–450–478 nm). A mixture of methanol/water (95:5%) was used as the mobile phase solvent in isocratic flow, while the sample was dissolved in a mixture of methanol/chloroform (90:10 containing 0.1% BHT as antioxidant agent). The flow rate and column temperature were kept constant at 0.4 mL/min and 28 °C, respectively. Gas chromatograph was used for the analysis of FAs, which was equipped with Flame Ionization detector (FID), a column HP-88 100 mt × 0.25 mm × 0.2 μm. This chromatographic column produced by Agilent is composed of a high polarity bis (Cyanopropyl) siloxane stationary phase and was chosen for its high resolution of positional and geometric isomers of fatty acid methyl esters. The column was maintained at 150 °C for 5 min and was followed by temperature ramping at 1.6 °C/min to 180 °C, then at 1.4 °C/min to 190 °C, and finally holding the temperature at 190 °C for 10 min. Nitrogen (purity ≥ 99.9999%) was used as carrier gas with a linear velocity of 30 cm/s and split ratio of 1:100. The injection port and detector were maintained at 250 °C. To quantify the concentration of the astaxanthin, lutein, and FAs compounds, the calibration curves were built by using chromatographic standards bought by Sigma Chemical Co., St Louis, MO, USA.