Fatty acids analysis of HSO and nanoemulsions

The fatty acid profile of samples was determined according to procedures reported earlier¹⁴. Fatty acid methyl esters (FAMES) were analyzed by GC/MS using a PerkinElmer model operated with Clarus 680-GC-SQ8T Mass Spectrometer (MA. USA). Helium (99.99%) was used as carrier gas with a flow rate of 1 mL/min. A volume of 1 μL prepared sample was injected at 250 °C in a split mode (50:1). The temperature was programmed according to: 150 °C initial temperature held for 2 min; increased to 185 °C at a rate of 1.5 °C/min; reached a final temperature of 220 °C at a rate of 5.0 °C/min. MS conditions were 70 eV ionization energy with 250 °C ion source temperature. The mass to charge (m/z) range was set as 20–450 atomic mass units. TurboMass Ver. 2.3 (NIST 2011, Waltham, MA, USA) software was used to determine the fatty acids profile of HSO and nanoemulsions.

We used NMR analysis to confirm the polyunsaturated and saturated fatty acid content (PUFA and SFA) of HSO. Samples for analysis were prepared as follows: HSO (200 μL) was dissolved in 800 μL of CDCl₃. ¹H NMR spectra were recorded on a Bruker Avance Cryoprobe 600 MHz spectrometer (MA, USA). Chemical shifts are reported in parts per million downfield from TMS. Spectra were processed using MestReNova Ver. 12.0.4 (Mestrelab Research, Santiago de Compostela, Spain).