2.5. Mass Spectrometry Analysis of Free Fatty Acids

The thin layer chromatography spots corresponding to the non-esterified free fatty acid fraction were scraped, redissolved in n-hexane, and analyzed separately in an Agilent 1260 Infinity high-performance liquid chromatograph coupled to an API2000 triple quadrupole mass spectrometer (Applied Biosystems, Carlsbad, CA, USA). The column was a Supelcosil LC-8 (150 × 3 mm, 3 μm particle size), protected with a Supelguard LC-8 (20 × 3 mm) guard cartridge (Sigma-Aldrich). The mobile phase was used on a gradient of solvent A (methanol with 0.01% ammonium hydroxide) and solvent B (water with 0.01% ammonium hydroxide). The gradient was started at 60% solvent A and 40% solvent B. The former was linearly increased to 95% at 10 min, and held at 95% solvent A until 18 min. The initial solvent mixture (60%, 40%B) was recovered at 20 min and the column was re-equilibrated for an additional 5 min before the injection of next sample. The flow rate was fixed at 400 μL/min. Non-esterified fatty acid fraction, extracted from silica plates and filtered, was re-dissolved in 100 μL of methanol/water 60:40 v/v and 90 μL were injected into the high-performance liquid chromatograph. The parameters for electrospray ionization source of mass spectrometer were set as follows: Ion spray voltage, −4500 V; CUR, 20 psi; GS1, 40 psi; GS2, 80 psi; TEM, 525 °C. The analyzer mode was set to Q1MS (DP, −70 V; EP, −10 V; FP, −300 V) performing a m/z scan between 100 and 400 with a step size of 0.1 amu. Non-esterified fatty acids were detected as [M − H]⁻ ions using the Analyst 1.5.2 software version (Applied Biosystems, Carlsbad, CA, USA), and chromatographic peaks were quantified by comparison with peaks of authentic analytical standards.