β carotene and lutein standards

Acetonitrile HPLC grade (assay 99.9%) was purchased from Panreac Química S.A. (Barcelona, Spain); trifluoroacetic acid for HPLC (assay 99%) and formic acid for HPLC (assay 98%) were purchased from Sigma–Aldrich (Madrid, Spain). Folin–Ciocalteu reagent was purchased from Merck (Darmstadt, Germany). Water was purified by a Milli-Q water purification system from Millipore (Bedford, MA, USA).
The β-carotene and lutein standards used in this study was purchased from the Sigma Chemical Company (St. Louis, MO, USA). Other reagents including methanol, chloroform, n-hexane, petroleum ether (PE), diethyl ether (DE), acetone, ethanol, sodium chloride, potassium hydroxide, tri-ethyl amine and butylated hydroxyltoluene (BHT) were of analytical or HPLC grade.

The extraction and analysis of carotenoids were performed in triplicate, according to Martínez-Zamora et al. [35 (link)], based on the method developed by Gupta et al. [36 (link)]. Carotenoids were quantified with an Ultra High Performance Liquid Chromatography (UHPLC) instrument (Shimadzu, Kyoto, Japan) equipped with a C32 column, DGU-20A degasser, LC-30CE quaternary pump, SIL-30AC autosampler, CTO-10AS column heater, and SPDM-20A photodiode array detector. The carotenoid content (9-cis-β-carotene, 13-cis-β-carotene, all-trans-β-carotene, and lutein) was expressed as mg per L of sample. For that, β-carotene and lutein standards were used (Sigma-Aldrich, St. Louis, MO, USA).