2.4.4. Fatty Acid Composition
The fatty acid composition was measured using the fatty acid methyl esters (FAME) method described by Taghvaei et al. (2021). In brief, an aliquot of oleoresin (1 mL) was transferred into glass test tube (15 mL) with boron trifluoride (14%) in methanol (3 mL) and heated at 100oC for 40 minutes. Saturated NaCl solution (15 mL) and hexane (3 mL) was added, and the tube was inverted 3 times. The mixture was allowed to settle for a few minutes for the separation of organic and aqueous phases. The organic top layer was collected using a Pasteur pipette into a clean test tube, where a small amount (1 g) of anhydrous sodium sulfate was added to remove any residual moisture. The methylated organic layer (1 µL) was injected into a GC-MS system (HP 5890–5973 MS, Agilent) equipped with an HP-23 FAME capillary column (30 m length x 0.25 mm i.d. x 0.25 μm film thickness) (Agilent Technologies, Santa Clara, CA, USA). Helium (1 mL/min) was used as the carrier gas. The initial oven temperature was 60 oC ld for 0.5 min), then brought to 150 oC at a rate of 5oC / min, followed by an increase of 15oC / min to a final temperature of 230oC. The program was finalized with a hold at 230oC for 4 min. In addition to the National Institute of Standards and Technology (NIST) 2014 database, a FAME standard (> 95 %, FAME Standard Mixture, St, Louis, MO, USA) over a concentration range of 1.25 -10 mg/mL was used for identification and quantification. The quantification was performed by using the peak areas.