Dionex ultimate 3000 pump

All samples were processed and analyzed as previously described (Lyte et al., 2022 (link)). Briefly, thawed samples were homogenized in a BeadRuptor and then centrifuged at 3,000 × g and 4°C for 15 min. Yolk samples required heating to 37°C and diluting 1:10 with mobile phase in order to pass through the spin filters. Sample supernatant was passed through 2–3 kDa spin filters, and flow-through was stored at −80°C until ultra-high performance liquid chromatography with electrochemical detection (UHPLC-ECD). The UHPLC-ECD consisted of a Dionex Ultimate 3,000 autosampler, a Dionex Ultimate 3,000 pump, and Dionex Ultimate 3,000 RS electrochemical detector (ThermoFisher-Scientific, Sunnyvale, CA). Mobile phase was buffered 10% acetonitrile (Catalog #: NC9777698, ThermoFisher-Scientific) and the flow rate was 0.6 mL/min on a 150 mm (length) 3 mm (internal diameter) 3 µm (particle size) Hypersil BDS C18 column (Catalog #: 28,103-153030, ThermoFisher-Scientific). A 6041RS glassy carbon electrode set at 400 mV was used for electrochemical detection. Data were analyzed using the Chromeleon software package (version 7.2, ThermoFisher-Scientific), and neurochemical identification was confirmed using the retention time of the corresponding analytical standard (Millipore-Sigma, St. Louis, MO) (for norepinephrine, Catalog #: 636-88-4; for L-dopa, Catalog #: 59-92-7).

Ten mg of sample was weighed into a capped tube and then added with 250 µL of anhydrous pyridine. The mixture was cooled to 0 °C before adding 500 µL acetic anhydride, then stirred overnight on an orbital stirrer at room temperature. The mixture was then cooled to 0 °C before adding 1 mL of methanol. Excess reagent and solvents were removed by successive addition of toluene and methanol under a vacuum. The resulting samples were dissolved in 5 mL of tetrahydrofuran (THF) and filtered prior to analysis by HPSEC (0.45 µm GHP Acrodisc microfilters, Merck, Darmstadt, DE, USA). SEC analysis of acetylated samples was performed using THF stabilized with butylated hydroxytoluene (BHT) as eluent at 1 mL min⁻¹ (Dionex Ultimate 3000 Pump, Thermo Fisher Scientific, Waltham, MA, USA). Then, 10 µL was injected (Dionex Ultimate 3000 Autosampler, Thermo Fisher Scientific, Waltham, MA, USA) on a Mixed C column (5 µm, 7.5 × 600 mm; Polymer Laboratories, Long Beach, CA, USA) and the signal was read at 280 nm (Dionex Ultimate 3000 UV/vis detector, Thermo Fisher Scientific, Waltham, MA, USA). Molar mass distributions were determined by a calibration curve based on polystyrene standards (Polymer Laboratories, Church Stretton, UK).

CCM samples were measured on a QSTAR Elite (AB Sciex, Darmstadt, Germany) coupled to a Dionex Ultimate 3000 pump (Thermo Fisher Scientific) as described previously [31 (link)]. TIF samples were analyzed on a Q Exactive Plus (Thermo Fisher Scientific) coupled to an EASY-nLC 1000 liquid chromatograph (Thermo Fisher Scientific) as described before [32 (link)]. Mass spectrometers were operated in data-dependent mode for MS and MS/MS.

DESI-MSI was performed on an orbitrap mass spectrometer (Q Exactive, Thermo Fisher Scientific) equipped with an automated Prosolia 2D DESI source (Prosolia OmniSpray 2D). The DESI source was modified with the following parameters. The spray tip was positioned at 1.5 mm above the sample surface and at an angle of 75°. The distance between the sprayer to mass spectrometer inlet was 7 mm with a collection angle of 10°. The spray solvent was methanol/water (95:5, v/v), delivered at 1.5 μl/min using a Dionex Ultimate 3000 pump (Thermo Fisher Scientific) at a spray voltage of 4.5 kV. Nitrogen was used as the nebulization gas at a pressure of 7 bars. The Q Exactive mass spectrometer was operated using an S-Lens setting of 50 V and using m/z range of 65 to 400 in positive ionization mode, with a spatial resolution of 100 μm for tissue sections and 150 μm for the blood spot. For acquisition of MS/MS spectra, an injection time of 300 ms, automatic gain control target of 5,000,000, and mass resolution of 70,000 were used. MS/MS analysis was performed using various high collision dissociation settings (shown in Results) and a mass isolation window of ±0.3 Da. Data were converted into imzML format using imzML converter version 1.1.4.5, and data were visualized using MSiReader version 0.09 (40 (link)).

The different extracts of R. roseus were analyzed by analytical HPLC-DAD. This analysis was performed with the Dionex Ultimate 3000 pump and Thermos Separation Waters 996 product detector (Thermo Fisher Scientific, USA) using a reverse-phase C18 column (25 cm × 4.6 mm × 5 μm) as described by Yahyaoui et al. [16 (link)], with some modifications.
The mobile phase used for elution is 1.2 mL/min with a limit of detection (LOD) defined between 0.01 and 0.1 mg/L. The mobile phase includes acidified water (pH = 2.65) (solvent A) and acidified water/ACN (20 : 80 v/v) (solvent B). The sample was eluted by the following linear gradient: from 0.1% B to 30% B for 35 minutes, from 30% B to 50% B for 5 minutes, from 50% B to 99.9% B for 5 minutes, and finally from 99.9% B to 0.1% B 15 minutes. Using the acidified water/ACN mixture (80 : 20 v/v), the different extracts were prepared at the same concentration (20 mg/mL) and then filtered through a Millex-HA 0.45 μm syringe filter (Sigma-Aldrich). After injection of twenty microliters (20 μL) of each sample and detection at 280 nm, phenolic compounds were identified by comparison with the retention time of some standards with known retention time.