*2.2. Reference Analysis*

GC-FID was employed as the reference method to analyze the fat profile of the fish oils. To extract the FAs from the oils and transform them into fatty acid methyl esters (FAMEs), the methylation process described in Commission Regulation (EC) No. 796/2002 (2002), method B, was used with some modifications [27]. In this procedure, 80 mg of sample were transferred to a flat-bottom flask, where 8 mL of sodium methylate in methanol (0.6 mol/L) and some pumice stones were added. The mixture was boiled with a reflux condenser for 10 min. Once the mixture was chilled, two drops of phenolphthalein were incorporated, and a solution of hydrochloric acid in methanol (3.5%) was added until the solution became colorless, a sign of complete acidification. The sample mixture was boiled again under the same conditions, and when cooled, 8 mL of n-hexane was added with 5 mL of a concentrate solution of sodium chloride, shaking the mixture vigorously for 1 min. Finally, the same concentrate solution of sodium chloride was added to elevate the organic phase, which contained the FAMEs, and it was transferred to a gas chromatograph vial before injection.

The solution with the FAMEs was analyzed in a gas chromatograph (Agilent 5890 from Agilent Technologies Inc., Santa Clara, CA, USA) with a DB 23 column (60 m × 0.25 mm id × 0.25 μm from J&W scientific, Santa Clara, CA, USA), a flame ionization detector (FID) and helium as carrier gas (at 30 psi and a flow rate of 1.2 mL/min). To conduct the chromatographic analysis, 2 μL of sample was injected in split mode (split of 80 mL/min) at 220 ◦C. The initial temperature of the chromatograph oven was 40 ◦C, which was maintained for 3 min. The temperature was increased at a rate of 25 ◦C per minute, up to 125 ◦C, where it was maintained for 2 min. Next, the temperature was increased again, this time at a rate of 4 ◦C per minute, and maintained at 180 ◦C for 1 min. The last temperature increase was at a rate of 1 ◦C per minute, up to 215 ◦C, where it was maintained for 10 min. Finally, the temperature of the detector was increased to 250 ◦C. Each GC-FID analysis was conducted 3 times.

Data from the chromatogram were collected with ChemStation Software (version A.10.02) from Agilent, (Santa Clara, CA, USA). The FAMEs of the oils, which were equal to their respective FAs, were identified with FAMEs chromatographic external standards from Sigma-Aldrich (PUFA No. 3 From Menhaden Oil and Supelco 37 Component FAME Mix). Then, the area under each FA peak was integrated in the chromatogram, and the percentage of the total oil represented by each area was calculated. Afterwards, the percentage of FAs that belonged to the same group was summed to obtain the final percentage of all the categories (SFAs, MUFAs, PUFAs, ω-3 and ω-6) for each oil. The FAs of each group are presented in Table 2.

**Table 2.** Identified FAs in each category.


SFAs: saturated fatty acids, MUFAs: monounsaturated fatty acids, PUFAs: polyunsaturated fatty acids, ω-3: omega-3 fatty acids, ω-6: omega-6 fatty acids.

Due to the complexity of the chromatographic method (time-consuming) and the need to analyze many samples to create a robust chemometric model, only the initial pure oils were analyzed (from A to I), and the composition of each oil mixture was calculated afterwards. To ensure that the composition of the mixtures was correct, a few were chosen randomly and analyzed.
