C30 carotenoid column

Sample extraction from freeze-dried supernatant and pellet powders was the same as above.
Carotenoids in the extraction were analyzed using HPLC based on Scott’s method [37 (link)]. Briefly, the injected sample volume was 20 µL and a C-30 YMC Carotenoid column (150 × 4.6 mm, 5 µm; YMC Co. Ltd., Komatsu, Japan) protected by a guard column. The mobile phase was provided by a three-solvent gradient program. The initial composition was 4:81:15 (v/v/v) water:methanol:methyl tert-butyl ether and this was altered with linear gradients to 4:6:90 (v/v/v) by 60 min at a flow-rate of 1 mL min⁻¹, at 30 °C. A PDA detector was used and the scanning range was 200–700 nm at 5 nm increments. The quantification of each compound was calculated based on the standard curves obtained using the authentic standard chemicals.

Analysis was performed in an Orbitrap Exploris 120 mass spectrometer (Thermo Fischer Scientific, Bremen, Germany) controlled by Orbitrap Exploris Tune Application 2.0.185.35 and Xcalibur 4.4.16.14 software. Samples (2 µL) were injected onto a reverse-phase C30 YMC carotenoid column (5 μm, 250 × 4.6 mm; YMC, Kyoto, Japan) with a SecurityGuard ULTRA Cartridge of the same material operating at 30 °C and a flow rate of 0.3 mL/min. The mobile phase consisted of two solvents: methanol (A) and tert-butyl methyl ether (B). It started with 100% A and used a gradient to obtain 30% at 90 min, 30% isocratic from 90 to 12min, and 100% A at 135 min. The capillary voltage of the electrospray ionization source (ESI) was set to 3.5 and 2.5 kV for positive and negative modes. The capillary temperature was 350 °C. The sheath, auxiliary and sweep gases flow rates were 50, 10 and 1 (arbitrary units, as provided by the software settings) with a 70% tube lens setting. The resolution of the MS scan was 60,000. Data-dependent MS/MS was performed on HCD using nitrogen as gas with collision energy settings of 35 V at 1500 of resolution. The range was set from 150 to 2000 Da. The MS system was operated in both positive and negative ion modes with the mass range set at m/z 100–1500 in full scan resolution mode.

Carotenoids were extracted from 2 g juice sacs and then measured according to Liu et al. [20 (link)]. Briefly, 2 g juice sacs were grounded into power in liquid nitrogen for the extraction. Carotenoids profiling were conducted by high-performance liquid chromatography (HPLC, Waters 1525 reverse-phase HPLC with a 2996 photodiode array detector) with a C30 carotenoid column (250 mm × 4.6 mm, YMC, Wilmington, NC, USA). The carotenoids were identified basing on the retention times and the absorption spectra with authentic standards according to our previous studies [20 (link)].

Carotenoid extraction and quantification was performed as previously described with modification [30 (link)]. Carotenoids were analyzed by reversed phase HPLC. Chromatography was carried out with a Waters liquid chromatography system equipped with a model 600E solvent delivery system, a model 2996 photodiode array detection (PAD) system, a model 717 plus autosampler, and an empower Chromatography Manager. Carotenoids were eluted with MeOH- Acetonitrile [75:25 v/v, eluent A] and MTBE [eluent B] using a C30 carotenoid column (15 × 4.6 mm; YMC, Japan). Carotenoids were identified by their characteristic absorption spectra, typical retention time, and comparison with authentic standards (Bern, Switzerland).

β‐cryptoxanthin of the serum and heart was performed with HPLC. The HPLC Waters Alliance 2695 (Waters) was used and consisted of pump and chromatography bound to a 2996 programmable photodiode array detector, a C30 carotenoid column (3 mm, 150 × 3 × 4.6 mm; YMC) and Empower 3 chromatography data software. The HPLC system programmable photodiode array detector was set at 450 nm for carotenoids. The analysis of this compound was performed to confirm SM ingestion. The samples were protected from light and stored at −80°C until analysis. Regarding serum dosage, 200 μL serum aliquot was extracted with β‐cryptoxanthin using the method described by Tang et al.²⁹

Plasma samples of 200 μL were mixed with 10 μL 8-apocarotenal in ethanol [0.0001% (w/v), (Sigma-Aldrich, St. Luis, MO, USA)] as internal standard, 1 mL ethanol containing 0.01% (w/v) butylhydroxytoluene, and 4 mL hexane:dichloromethane [4:1 (v/v)]. The extraction process was repeated if the plasma samples contained precipitated material. After stirring and centrifugation, the supernatants rich in carotenoids were collected and kept in liquid nitrogen. Subsequently, the samples were evaporated to dryness and the residues were re-dissolved in 200 μL hexane:acetone:ethanol:toluene [10:7:6:7 (v/v/v/v)] solution and filtered through a 0.22-μm filter for high-performance liquid chromatography (HPLC) analysis. A C30 carotenoid column [S5 μm, 250 × 2.0 mm I.D. (YMC, Wilmington, NC, USA)] was used at 30 °C, and the SPD-M10 vp diode array detector (Shimadzu, Kyoto, Japan) was set at 460 nm. Carotenoids {[lutein, zeaxanthin, β-cryptoxanthin, α-carotene, β-carotene, and lycopene (cis- and trans-isomers)], Extrasynthese, Genay, France} were quantified by determining peak areas in the HPLC chromatograms, calibrated against known standards.

Brain extracts were analyzed using reverse-phase high performance liquid chromatography. Twenty microliters of the brain extract were injected onto a C30 carotenoid column (3 μm, 150 × 4.6 mm, YMC, Wilmington, NC). The method was described by Yeum et al. in detail [17 (link)]. The lower limit of detection is 0.2 pmol for carotenoids.