LC-MS analysis of free and esterified fatty acids.

Fatty Acid Extraction

Internal standard mixture consisted of 12.5 µg/mL of (1, 2, 3, 4, 5, 6-¹³C₆) docosanoic acid and 50 µg/mL ¹³C-labeled myristic, palmitoleic, palmitic, margaric, linoleic, oleic, elaidic, and stearic acid in ethanol (Cambridge Isotope Laboratories, Tewksbury, MA, USA). To sample containing tubes, pre-washed with methanol, 32 µL of internal standard mixture, 1 µL of 10 mM   butylated hydroxytoluene in methanol and 1.5 mL of ice-cold methanol was added. Samples were homogenized using a liquid nitrogen cooled Precellys Evolution bead mill homogenizer, vortexed 10 min at room temperature, allowed to sit on ice 10 min then centrifuged at 17,000 (x g), at 4 °C, for 10 min. The supernatants were then transferred to 2 mL vials with Teflon caps and dried using nitrogen gas.

Fatty Acid Picolylamide Derivatization

Extracted free fatty acid acids were converted to acyl chloride intermediates by treatment with 300 µL of 2 M oxalyl chloride in dichloromethane, at 65 °C, for 5 min. The solutions were then dried using nitrogen gas. Dried samples were further derivatized by adding 225 µL of 1% (v/v) 3-picolylamine in acetonitrile. The reaction was incubated at room temperature for 5 min. Finally, the solutions were dried using nitrogen gas. 

Reverse Phase Liquid Chromatography 

Dried derivatization products were reconstituted in 100 µL ethanol, transferred to glass auto-sampler vial inserts and centrifuged at 4,000 g, at 4 °C, for 3 min. Supernatants were next transferred to fresh vial inserts for analysis. The injection volume was 10 µL. Mobile phase A (MPA) was water containing 0.1 % formic acid, and mobile phase B (MPB) was acetonitrile containing 0.1 % formic acid. The chromatographic method included a Thermo Fisher Scientific Accucore C30 column (2.6 µm, 150 x 2.1 mm) maintained at 15 °C, autosampler tray chilling at 8 °C, a mobile phase flowrate of 0.500 mL/min, and a gradient elution program as follows: 0-5 min, 5% MPB; 5-40 min, 5-95% MPB; 40-85 min, 95% MPB; 85-85.1, 95-5% MPB; 85.1-90 min, 5% MPB. 

Tandem Mass Spectrometry

A Thermo Fisher Scientific Orbitrap Fusion Tribrid mass spectrometer with heated electrospray ionization source was operated in data dependent acquisition mode with a scan range of 150 - 550 m/z. Orbitrap resolutions of 120,000 (FWHM) and 30,000 for MS1 and MS2 were used, respectively. The instrument was operated in positive ionization mode with a spray voltage of 3,600 V, and vaporizer and capillary temperatures set at 350 and 325 °C, respectively. The sheath, auxiliary and sweep gas pressures were 50, 10, and 1 (arbitrary units), respectively. Ions were fragmented using assisted HCD with stepped collision energies of 25, 30, and 35%.

Fatty Acid Extraction

For free fatty acid detection, samples were homogenized using a liquid nitrogen cooled Precellys Evolution bead mill homogenizer and mixed with internal standard mixture in ice-cold methanol. Extracted free fatty acid acids were converted to acyl chloride intermediates and further derivatized. Dried derivatization products were reconstituted in 100 µL ethanol, transferred to glass auto-sampler vial inserts and supernatants were next transferred to fresh vial inserts for analysis. Mobile phase A (MPA) was water containing 0.1 % formic acid, and mobile phase B (MPB) was acetonitrile containing 0.1 % formic acid. The chromatographic method included a Thermo Fisher Scientific Accucore C30 column (2.6 µm, 150 x 2.1 mm) maintained at 15 °C, autosampler tray chilling at 8 °C, a mobile phase flowrate of 0.500 mL/min, and a gradient elution program as follows: 0-5 min, 5% MPB; 5-40 min, 5-95% MPB; 40-85 min, 95% MPB; 85-85.1, 95-5% MPB; 85.1-90 min, 5% MPB. A Thermo Fisher Scientific Orbitrap Fusion Tribrid mass spectrometer with heated electrospray ionization source was operated in data dependent acquisition mode with a scan range of 150 - 550 m/z. Orbitrap resolutions of 120,000 (FWHM) and 30,000 for MS1 and MS2 were used, respectively. The instrument was operated in positive ionization mode with a spray voltage of 3,600 V, and vaporizer and capillary temperatures set at 350 and 325 °C, respectively. The sheath, auxiliary and sweep gas pressures were 50, 10, and 1 (arbitrary units), respectively. Ions were fragmented using assisted HCD with stepped collision energies of 25, 30, and 35%.