Metabolite extraction, derivatisation, and mass spectrometry analysis were conducted in a randomized order. Serum samples were thawed on ice at 4 °C, and 50 µL was mixed with 750 µL of extraction solvent (methanol:isopropanol:water, 3:3:2, v/v/v)33 (link). Following 5 minutes of sonication and centrifugation, 700 µL of each supernatant was aliquoted into a new 1.5 mL tube. The aliquots were concentrated to complete dryness using a speed vacuum concentrator (SCANVAC, Korea). The dried extracts were then stored at −80 °C until derivatization.
The dried extracts were derivatized with 5 µL of 40 mg/mL methoxyamine hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) in pyridine (Thermo, USA) and with 45 µL of N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA + 1% TMCS; Thermo, USA)34 (link). A mixture of 13 fatty acid methyl esters was added as an internal retention index marker (C8, C9, C10, C12, C14, C16, C18, C20, C22, C24, C26, C28, and C30)35 (link). Chromatographic separation was performed using Agilent 7890B gas chromatography (Agilent Technologies) equipped with an RTX-5Sil MS column (Restek, Gellefonte, PA, USA). Mass spectrometry analysis was performed on a LECO Pegasus HT time-of-flight mass spectrometer controlled by Chroma TOF software 4.50 version (LECO, St. Joseph, MI, USA)36 (link).
The dried extracts were derivatized with 5 µL of 40 mg/mL methoxyamine hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) in pyridine (Thermo, USA) and with 45 µL of N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA + 1% TMCS; Thermo, USA)34 (link). A mixture of 13 fatty acid methyl esters was added as an internal retention index marker (C8, C9, C10, C12, C14, C16, C18, C20, C22, C24, C26, C28, and C30)35 (link). Chromatographic separation was performed using Agilent 7890B gas chromatography (Agilent Technologies) equipped with an RTX-5Sil MS column (Restek, Gellefonte, PA, USA). Mass spectrometry analysis was performed on a LECO Pegasus HT time-of-flight mass spectrometer controlled by Chroma TOF software 4.50 version (LECO, St. Joseph, MI, USA)36 (link).