Carotenoid standards

The analysis of carotenoids refers to the methods previously reported in our lab [38 (link)]. Waters high-performance liquid chromatography (HPLC) system (Waters Corporation, Milford, MA, USA), a YMC carotenoid 30 column (5 μm, 4.5 × 250 mm) with 25 °C temperature and a photodiode array detector were applied in the measurements. The mobile phase A was 0.1% (w/v) BHT and 0.05 M ammonium acetate in 97% (w/v) methanol–water, and the mobile phase B was 0.1% (w/v) BHT in methyl tert-butyl ether. The gradient elution sequence was as follows: 0 to 18 min, 0−20% B; from 18 to 20 min, 20−50% B; from 20 to 25 min, 50–90% B; from 29 to 29.5 min, 90−10% B; from 29.5 to 40 min, 10−0% B. The flow rate was one milliliter per minute. The UV absorbance was set at 450 nm. Carotenoid standards were purchased from CaroteNature (CaroteNature, Münsingen, Switzerland). Qualitative and quantitative analysis of carotenoids was carried out by comparing the retention time and peak area with the standards. Data were expressed as micrograms per gram in the dry weight of the sample and showed as µg/g DW (means ± SD).

Ethanol (≥ 99.9%, LiChrosolv^®), tetrahydrofuran (THF, ≥ 99.9%, LiChroSolv^®), n-hexane (SupraSolv^®), dichloromethane (< 99.8%, SupraSolv^®), chlorophyll a and b (analytical standards) and norflurazone (Pestanal^®, analytical standard) were obtained from Merck KGaA (Darmstadt, Germany). Tert-butyl methyl ether (≥ 99.9%, Rotisolv^®), 2-propanol (≥ 99.9%, Rotisolv^®), ammonium acetate (≥ 98%) and acetic acid (100%, Supra Quality) were purchased from Carl Roth GmbH (Karlsruhe, Germany). MEthanol (Chemsolute^®) and acetonitrile (Chemsolute^®) were purchased from Th. Geyer GmbH & Co. KG (Renningen, Germany). Carotenoid standards were obtained from CaroteNature GmbH (Münsingen, Switzerland) and flavonol glycosides from PhytoLab GmbH & Co. KG (Vestenbergsgreuth, Germany). Abscisic acid (ABA) standard was purchased from Sigma Aldrich Chemie GmbH (Taufkirchen, Germany) and (+)-abscisic acid-d6, ≥ 98%) from Toronto Research Chemicals (North York, Canada). All solvents were of LC-MS quality and the water was of ultra-pure quality.

The extraction of pigments was performed according to Frede et al. (2019) (link). In brief, 5 mg of plant material was dissolved in 0.5 ml of methanol/tetrahydrofuran (1:1, v/v) and incubated for 5 min in a shaker (1,400 rpm, 20°C) followed by centrifugation for 5 min (4,500×g, 20°C). The supernatant was collected in a vial, and the extraction was performed five times. The solution was evaporated to dryness under a nitrogen stream, dissolved in dichloromethane/isopropanol (1:5, v/v), sonicated (3 min, 20°C), filtered (PTFE filter tubes), and transferred to an HPLC vial. The analysis was performed using Agilent Technologies 6530 QToF-DAD-UHPLC-MS (Agilent Technologies Sales and Services GmbH & Co. KG, Germany) according to Frede et al. (2017) (link). Identification was achieved using mass spectra and UV/VIS spectra (Figure S1), and quantification was achieved using external calibration with carotenoid standards (CaroteNature GmbH, Switzerland) of all-trans-isomers from β-carotene, lutein, and zeaxanthin and 9-cis-neoxanthin as well as chlorophyll standards (Sigma Aldrich Chemie GmbH, Germany) of chlorophyll a and b at a wavelength of 450 nm. Data analysis was performed using the TOF Quantitative Analysis (Quant-My-Way) 10.2 (MassHunter, USA).

Serum carotenoid levels were assessed using high-performance liquid chromatography (HPLC). Serum carotenoids were extracted using 3 consecutive hexane extraction processes using a previously published protocol [24 (link)]. Briefly, the hexane layers were combined, dried under nitrogen, taken up into 90% MTBE, 8% methanol, and 2% ammonium acetate in water solution (1.5% solution) and then analyzed for carotenoid concentrations using the Alliance HPLC system (e2695 Separation Module) equipped with 2998 photodiode array detector (Waters, Milford, MA, USA) and a reverse-phase C30 column (4.6 × 150 nm, 3 micron, YMC, Wilmington, NC, USA). Serum levels of carotenoids previously found in human neural tissue were assessed including lutein, zeaxanthin, beta-carotene, and cryptoxanthin.
Carotenoid standards were obtained from Carotenature, Ostermundigen, Switzerland. For quantification, standard curves were run for each carotenoid, and serum carotenoids were quantified by use of the following extinction coefficients (all 1% solution): lutein 2550 in ethanol; zeaxanthin 2540 in ethanol; beta carotene 2592 in hexane; cryptoxanthin 2565 in hexane. The Erdman laboratory routinely participates in the National Institutes for Standards and Testing micronutrient proficiency testing program, and our serum carotenoid values for blinded serum samples consistently were within 1–2 SD of the medians.