C30 guard column

Carotenoid extracts were filtered with a PVDF filter (0.22 μm) and injected (50 μL) in the same UHPLC system used for the analysis of phenolic compounds. Carotenoids were separated using a YMC carotenoid C30 analytical column (150 × 4.6 mm i.d., 3 μm) protected with a YMC C30 guard column (10 × 4.0 mm, 3 μm) (YMC CO., LTD, Kyoto, Japan). The chromatographic process was carried out at a flow rate of 1.7 mL/min, sample temperature of 10°C, column temperature of 30°C, and eluates were monitored at 450 nm. The reverse phase elution was performed using a ternary gradient elution with methanol (A), dichloromethane (B), and acetonitrile (C). The initial conditions were A/B/C (76/5/19) from 0 to 3.2 min, then solvent B was increased to 34% at 23.3 min (A/B/C: 52.8/34/13.2), and changed to initial conditions until the 25.1 min. The column was equilibrated for 2.9 more min (total run time of 28 min). The carotenoid peak identification was conducted by comparison of their retention times and spectral characteristics with those of external standards. Lutein and zeaxanthin were quantified using calibration curves (r² ≥ 0.9900) built with pure carotenoid standards. Lutein isomers were quantified as lutein. Results were presented as μg per g sample DW.

The analysis of carotenoid compounds was performed with a YMC carotenoid C30 reverse-phase analytical column (150 × 4.6 mm i.d., 3 μm) coupled to a YMC C30 guard column (10 × 4.0 mm, 3 μm) (YMC CO., LTD, Japan) using the same UHPLC system as previously described for the phenolic compound analyses. Filtered carotenoid extracts (0.22 μl, PVDF filter) were injected at 1.7 ml/min flow rate and monitored at 450 nm. A ternary gradient elution was used (methanol, dichloromethane, acetonitrile) and same reverse-phase chromatographic conditions as those reported by Fuentes-Cardenas et al. (19 (link)) were applied. The retention time and UV–VIS spectra characteristics of external carotenoid standards and the library data were used for the identification of carotenoid compounds in evaluated samples. In addition, the information of carotenoid analyses in other maize samples from reported literature was also useful for the identification of carotenoid isomers. Calibration curves made with external standards were used for the quantification of carotenoids (r² ≥ 0.9900) and results were presented as μg per g sample DW. Lutein and zeaxanthin compounds and their isomers were quantified as lutein and zeaxanthin, respectively. Unidentified carotenoid compounds, neoxanthin, and violaxanthin isomers were expressed as lutein. β-cryptoxanthin isomers were expressed as β-cryptoxanthin.