Quantification of Neurotransmitters via UHPLC-ECD

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).