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.