Accela 600 hplc

For LC-MS analysis, a 1 mg/mL sample was prepared in a 1:1 MeOH–ACN solvent system. Experiments were carried out using an Exactive mass spectrometer with an electrospray ionization source attached to an Accela 600 HPLC pump with an Accela autosampler and UV/Vis detector (Thermo Scientific, Bremen, Germany). The mass accuracy was set to less than 3.0 ppm. The Orbitrap mass analyzer can limit the mass error to ±3.0 ppm. Mass spectrometry was carried out over a mass range of 100–2000 m/z in positive and negative ionization modes with a spray voltage of 4.5 kV and capillary temperature of 270 °C. About 10 μL was injected from each vial, at a flow rate of 300 μL/min. The column used was an ACE5 C18 column (5 μm × 75 mm × 3 mm) (Hichrom Limited, Reading, UK). A binary gradient method was utilized using the two solvents, A (water and 0.1% formic acid) and B (MeCN and 0.1% formic acid). The gradient was carried out for 45 min and the program followed; at zero minutes A = 90% and B = 10%, at 30 min A = 0% and B = 100%, at 36 min A = 90% and B = 10% until end at 45 min. The UV absorption wavelength was set at 254 nm, the sample tray temperature was maintained at 4 °C, and the column at 20 °C. The samples were run sequentially, with solvent and media blanks analyzed first. LC-MS data were acquired using Xcalibur version 2.2.

Portions of all oil samples (~ 20 mL) were shipped to a commercial, independent, laboratory (Intertek, Germany). These oils were analyzed by HPLC, with method replication carried out every ten samples on a different sequence (these replicates were used to calculate the inter-assay precision, “Procedure for Assessment of the Methods”). Briefly, oil samples (1 g) were filled up to 10 mL with methyl tert-butyl ether in dibuthylhydroxytoluene (1 g/L). After thoroughly shaking the solution, methanol was added (1:1) and injected (20 µL) to the HPLC. Vitamin A was analyzed on a Thermo Scientific Accela 600 HPLC (Waltham, USA), coupled to a fluorescence detector (λ_ex, 325 nm; λ_em, 480 nm, Thermo Scientific Surveyor FL Plus, Serial number 650212). The vitamin A was separated using a Thermo hypersil GOLD C₁₈ (150 × 4.6 mm, 3 µm). The mobile phase included methanol (solvent A) and methyl tert-butyl ether (solvent B), programmed as follows: 0–10.0 min: 0% B, 10–14 min: 100% B, 14–19 min: 0% B, at a flow rate of 1.0 mL/min. Retinyl palmitate in methanol was used as an external calibrator. The limit of quantification was 0.55 mg RE/kg as defined by the commercial independent laboratory.

Liquid chromatography–mass spectrometry (LC-MS) analyses were performed by electrospray ionization (ESI)-MS with an Exactive mass spectrometer (Thermo Scientific) interfaced with an Accela 600 HPLC (Thermo Scientific). A portion of the sample was loaded onto a Hydrosphere C18 column (4.6 mm, 250 mm, 5 µm; YMC, Kyoto, Japan). Separation was achieved by elution with acetonitrile/0.5% acetic acid aqueous solution (1:1, v/v) at a constant temperature of 30 °C and a flow rate of 1.0 mL/min.
Nuclear magnetic resonance (NMR) spectra were recorded on an ANANCE 400 NMR spectrometer (Bruker Biospin, Billerica, MA, USA). Chemical shifts were reported as δ values (ppm) relative to internal tetramethylsilane (TMS) and the coupling constants (J) were given in Hz.