Spe columns

Antibiotic standards, Disodium EDTA (OmniPur®), Oxalic acid (ReagentPlus®), Sodium sulphate (≥99%), n-hexane (Emsure®), Dichloromethane (LiChrosolv®), Methanol (LiChrosolv®) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile (≥99.9%) was sourced from Fisher Scientific, Loughborough, Leicestershire, while the SPE columns (200 mg x8 mL tubes) were purchased from Supelco™, Bellefonte, USA. The 0.45 μm GHP ACRODISC 13 mm disposable syringe filter unit was purchased from Agilent, whereas the deionised water (Ultrapure water) used in this study was produced using an Integral 10 Elix Milli-Q system with an LC (Biopak) polisher (Massachusetts, USA). Other apparatuses used include the Waring laboratory blender – Z272205 (Sigma-Aldrich, St. Louis, MO, USA), and the Vortex mixer VM18 (Schiltern Scientific, Beds, UK).

Plasma samples (200 µL) were thawed slowly in cold water, vortexed, and mixed with 50 µL of internal standard (fatty acid 17:0, 1 mg/mL). Fatty acids were extracted by adding 4 mL of chloroform:methanol 1:1 (v/v) and 2 mL of 0.5% NaCl. After vortexing and centrifugation, 1 mL of the chloroform phase was collected and evaporated at 40 °C under nitrogen gas. The remaining precipitate was dissolved in 200 μL chloroform and run through solid-phase extraction (SPE) columns (Supelco, Bellefonte, PA, USA, NH₂, 500 mg/3 mL), preconditioned with 2 × 2 mL hexane. The columns were rinsed with 2 × 2 mL chloroform:isopropanol 2:1 (v/v) and 2 × 2 mL of 2% acetic acid in diethyl ether. Thereafter, the adsorbed phospholipids were eluted with 2 × 2 mL of methanol. Methylation of fatty acids was carried out as follows: the methanol was evaporated and the precipitate was dissolved in 2 mL of toluene and 2 mL of 10% acetyl chloride in methanol and vortexed for 1 min. The mixture was incubated at 70 °C for 2 h with vortexing every 30 min. Thereafter, 1 mL of Milli-Q water and 2 mL of petroleum ether were added to the samples. After vortexing and centrifugation, 3 mL of the organic phase was collected and evaporated at 40 °C under nitrogen gas.