Hss c18 sb column

¹H-, ¹³C-, and ³¹P-NMR spectra were recorded on Bruker DPX or Bruker AV NMR spectrometers operating at 400, 101, and 162 MHz, respectively. Small molecules were analyzed using a Waters TQD mass spectrometer equipped with a triple quadrupole analyzer. Samples were introduced to the mass spectrometer via an Acquity UltraPerformance Convergence Chromatography (UPC²) system, including a UPC² Waters HSS C18 SB column (100 mm × 3.0 mm × 1.8 μm) gradient 90% CO₂:10% methanol modifier (25 mM ammonium acetate) to 60% CO₂:40% methanol modifier (25 mM ammonium acetate) in 3 min at a flow rate of 1.5 mL/min. The makeup flow (methanol/1% formic acid) was pumped at a flow rate of 0.45 mL/min into the mass spectrometer. Mass spectra were recorded using positive ion electrospray ionization.

The protocol was based on the work of Bijttebier et al. (2013) [13 (link)]. One µL of extract was injected on a 2.1 mm × 100 mm, 1.8 μm HSS C₁₈ SB column (Waters, Milford, MA, USA) at 35 °C in a Waters Acquity UPLC^®. Mobile phase A consisted of water +5 mM ammonium acetate:methanol:acetonitrile:ethylacetate (50/22.5/22.5/5 v/v/v/v) and mobile phase B consisted of acetonitrile:ethylacetate (50/50 v/v) with a flow rate of 500 μL·min⁻¹. The following gradient (%/min B) was applied: 10/0; 10/0.1; 30/0.8; 91/20.0; 100/20.1; 100/20.4; 10/20.5; 10/23. For detection, an orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) operating with an APCI was used. A full scan of the ions was acquired at m/z range of 200 to 1400 at a resolution of 70,000 full width at half-maximum.
As the internal standard, 36.25 ng·mL⁻¹ of trans-β-Apo-8′-carotenal was used. The concentration of the standards (α-carotene, antheraxanthin, β-cryptoxanthin, β-carotene, fucoxanthin, trans-lutein, trans-violaxanthin, zeaxanthin) ranged between 0.034 and 26.55 µg·mL⁻¹ (average). In addition, 3 quality controls (corresponding to the standard 5) were placed in between the injections of the samples. The lowest calibration point was set as the limit of quantification. The relative standard deviation of these controls was on average 6.35 ± 4.21%.

Carotenoid extracts^{80 (link)} from approximately 200 mg frozen pericarp were dried at reduced pressure and reconstituted in ethylacetate containing 50 μg/ml of diindolylmethane (internal standard). Carotenoids were separated and quantified using an ultra performance convergence chromatography (UPC²) system and a 2.1 × 150 mm HSS C18 SB column packed with 1.8 µm diameter particles (Waters, Milford, MA). The column effluent was monitored between 250 and 500 nM. See Supplementary Table 4 for details. β-carotene was used to construct a 5-point calibration curve (10–140 ng/μl) and the relative concentrations of each carotenoid are reported in β-carotene equivalents.
Ethylene production by the fruit was measured with seven biological replications using a gas chromatograph (Hewlett-Packard 5890 series II) with standard curve method and normalized by fruit mass^{14 (link)}.