Dionex ultimate 3000 autosampler

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).