C30 reversed phase column

Isomers of lycopene, PTF, and PT were analyzed by reversed-phase HPLC equipped with a diode array detector (Alliance 2695; Waters Corp., Milford, MA, USA). The method was developed by Cooperstone et al. [25 (link)]. The carotenoids were separated using a C30 reversed phase column (150 × 4.6 mm, 3 µm; YMC Co, Kyoto, Japan). For the quantification of lycopene, PTF and PT isomers were performed by peak area at 471, 348, and 286 nm, respectively. The identification of lycopene, PTF, and PT isomers was conducted by comparison of their retention times with that of standards and spectroscopic features (Q-ratio for lycopene, %III/II for PTF, and PT [29 (link)]). The quantification of all-E-lycopene, PTF, and PT isomers was determined by their external standard curve, and Z-lycopene was quantified with the same calibration curve of all-E-lycopene standards.

The extracts were dissolved in a known volume of ethyl acetate and filtered (0.20 μm PTFE filter) in amber vials. The HPLC system (Shimadzu Corporation) was equipped with a SPDM20A diode array detector and an YMC C30 reversed phase column (250 × 4.6 mm i.d., 5 µm particle size). Carotenoid separation was performed applying a gradient elution with methanol/tert‐butyl methyl ether/water (83:15:2, v/v/v) (solvent A) and tert‐butyl methyl ether/methanol/water (90:8:2, v/v/v) (solvent B) in the following conditions: 0 min 0% solvent B, 20 min 0% B; 150 min 82% B; 152 min 0% B, where after the column was equilibrated for 10 min. The flow rate was 0.8 ml/min, and the injection volume was 20 μl. Individual carotenoids were identified by comparing their retention time, elution order on C30 column, UV‐Vis spectra (λ_max, spectral fine structure (%III/II) with those of the available standards (β‐carotene, lutein, zeaxanthin and β‐cryptoxanthin)) and with literature data. Quantification of the three major carotenoids was performed using external ten‐point calibration curves constructed in the range 1–100 μg/ml. The correlation coefficients were as follows: R² = .9912 (β‐carotene), R² = .9991 (lutein), and R² = .9996 (zeaxanthin).

HPLC-DAD separation was performed using a Shimadzu LC20 AT HPLC system (Shimadzu Corporation, Kyoto, Japan) with an SPDM20A diode array detector and a YMC C30 reversed-phase column (250 mm length, 4.6 mm inner diameter and 5 μm particle size). The experimental conditions for the separation and identification by HPLC-DAD were the same as described in a previous study [62 (link)]. Quantification of carotenoids was performed using external calibration with standards of β-carotene, lutein, β-cryptoxanthin, and zeaxanthin purchased from Extrasynthese (Lyon, France) in the range of 1–100 μg/mL. The HPLC-DAD analysis was performed three times for the tested sample and data are expressed in mg/100 g F.W (fresh weight) and presented as the mean ± SD of these three measurements.

HPLC analysis was performed by referring to a previously reported method [2 (link)]. The extract was analyzed with a 2695 HPLC system (Waters Corp., Milford, MA, USA) equipped with a C-30 reversed-phase column (250 × 4.6 mm, 5 μm) and a C-30 guard column (20 × 4.0 mm, 5 μm) (YMC, Inc., Wilmington, NC, USA). Gradient elution was carried out by using methanol/MTBE/distilled water (81:15:4, v/v/v) and MTBE/methanol (90:10, v/v) as the mobile phase. The linear gradient program was set as follows: 0 min: 100% A; 25 min: 75% A; 80 min: 15% per type; 82 min: 100% A. The flow rate was set at 1 mL/min, and the UV–VIS spectra were collected at 450 nm. The column temperature was maintained at 25 °C and a 20 μL sample was injected for analysis.