Agilent autosampler

To evaluate fatty acid composition of oils obtained from A. vulgare, C. nucifera, and E. guineensis, alkaline trans-methylation of fatty acids was carried out as described by Raes et al.^{100 (link)}. Analysis of methyl esters was performed using gas chromatography (GC) with the HP 6890 chromatograph (Agilent Technologies, Inc., Santa Clara, CA, USA), equipped with a 60 m DB-23 capillary column (60 m × 0.25 mm × 0.25 μm) and a flame-ionization detector (FID); split injections were performed using an Agilent autosampler. A total of 1 µL of standards in hexane were injected in split mode (1:40 ratio) into the injector, heated to 230 °C. The column temperature was initially set at 120 °C for 6 min then programmed to 170 °C at a rate of 15 °C/min. The temperate gradient was further configured to 210 °C at the rate of 3 °C/min and held for 13.5 min. Finally, the temperature was programmed to 230 °C at the rate of 40 °C/min and held for 7 min. Nitrogen was used as the carrier gas, at a flow rate of 0.8 mL/min. Supelco 37 Component FAME Mix, PUFA 1, PUFA 2, PUFA 3, trans-vaccenic acid, and a mixture of conjugated isomers of linoleic acid (Sigma-Aldrich, Prague, CZ) were used as standards. Fatty acids were identified based on retention times with respect to standards.

The concentration of VRP in the brain tissue was measured by HPLC. The brain tissue samples were homogenized with a fourfold excess volume of distilled water. To 1.5 mL of the homogenate, 1 mL methanol was added. The samples were shaken for 5 min and centrifuged at 8000 rpm for 15 min. The organic layer was separated and filtered using a 0.45 μm membrane filter (Millipore, Ireland) prior to HPLC acquisition.
The HPLC system was consisted of a liquid chromatograph [Agilent™ 1260 Infinity Quaternary HPLC System equipped with Agilent High Performance ALS (G1367E), Agilent Quaternary Pump (G1311B/C), thermostat Agilent autosampler with reliable injections from 0.1 to 100 μL (G1329B), and Agilent fluorescence detector (G1321C) operated at 231 nm (excitation) and 318 nm (emission)]. The column used for HPLC was ZORBAX^® Eclipse XDB C₁₈ column (100 mm × 3 mm with 3.5 μm particle size) from Agilent. The mobile phase was acetonitrile-25 mM phosphate buffer (pH 4.0), methanol and acetonitrile (25:25:50, vol/vol). The flow rate of the mobile phase was 1.0 mL/min. The limit for quantification was 80 ng/mL in brain tissue.

The water: ACN (1:1, v/v) carrier solution was pumped through the system using an Agilent 1100 binary pump (G1312A, Agilent Technologies, Palo Alto, CA, USA) at a flow rate of 0.4 mL min⁻¹. Samples and standards were injected with an Agilent autosampler (G1379A); the injection volume was 20 μL. Analytes were detected by multiple reaction monitoring (MRM) using a hybrid triple quadrupole/linear ion trap mass spectrometer (QTRAP 4000, from Applied Biosystems, AB Sciex, Framingham, MA, USA). Electrospray (ESI) was the ionization source, working in positive mode. Nitrogen was used as a nebulizer and auxiliary gas as well as the collision gas. The source conditions are as follows: Source voltage, 4500 V; source temperature, 500 °C; gas 1 pressure (heating gas at the source), 50 psi; auxiliary gas pressure (drying gas), 50 psi; capillary temperature, 350 °C; curtain gas pressure, 10 psi. Transitions selected and other MS conditions have been detailed in Table S3 (Supplementary Materials). Analyst 6.2 software (AB Sciex) was used to control the instrument and quantify the analytes.
BA standards for calibration were injected at the beginning and end of the FIA-MS sequence. Samples were subsequently injected randomly. Three independent replicates of each sample were analyzed.