The fatty acid composition of TG in canola oil was analyzed by GC. Fresh canola oil (5 mg) was mixed with an internal standard (50 µg of heptadecanoic acid) in a screw cap test tube. Then, 2 mL of methanolic hydrogen chloride (5%, v/v) with 0.2 mL of benzene and 2 µL of BHT methanolic solution (0.01%, wt%) were added. The sample was methyl esterified at 100 °C under a nitrogen atmosphere. After 1 h, 5 mL of aqueous potassium carbonate (6%, wt%) and 1 mL of hexane were added. The mixture was partitioned by centrifugation (1000 × g for 5 min at 4 °C) into two layers. The upper hexane layer was collected, and remaining aqueous layer was re-extracted with 1 mL of hexane. The combined hexane layer was evaporated under a nitrogen gas stream, and the residue was dissolved in 1 mL of hexane. A 2 µL aliquot was analyzed with GC-4000 (GL Sciences Inc., Tokyo, Japan) equipped with a DB-225 column (length, 30 m; internal diameter, 0.32 mm; film thickness, 0.25 µm; Agilent Technologies, Santa Clara, CA, USA). Helium gas was used as the mobile phase. The injector and detector temperatures were set at 220 and 250 °C respectively. The gradient profile was as follows: 140–180 °C (8 °C/min linear), 180–220 °C (3 °C/min linear), and 220 °C (for 25 min).
TG molecular species were analyzed by Q1 mass- and product ion-scan mode using LC–MS/MS. A Prominence liquid chromatography system (Shimadzu, Kyoto, Japan) was equipped with a 4000 QTRAP mass spectrometer (SCIEX, Tokyo, Japan). Fresh canola oil was 10,000-fold diluted with methanol/2-propanol (100:1, v/v) and analyzed using an ODS column (5C18-MS-II, 5 µm, 4.6 × 250 mm, nacalai tesque, Kyoto, Japan) with a binary gradient consisting of solvent A (methanol containing 0.1 mM sodium acetate) and solvent B (2-propanol containing 0.1 mM sodium acetate). The gradient profile was as follows: 0–20 min, 50–70% B linear. The flow rate was 1.0 mL/min, and the column temperature was 40 °C. Elution was split at the post-column and one of the split eluents was sent to the MS/MS system at 0.2 mL/min. The MS parameters are shown in SI5-Method 1 and 2 . A 10 µL sample was injected into the LC–MS/MS system.
TG molecular species were analyzed by Q1 mass- and product ion-scan mode using LC–MS/MS. A Prominence liquid chromatography system (Shimadzu, Kyoto, Japan) was equipped with a 4000 QTRAP mass spectrometer (SCIEX, Tokyo, Japan). Fresh canola oil was 10,000-fold diluted with methanol/2-propanol (100:1, v/v) and analyzed using an ODS column (5C18-MS-II, 5 µm, 4.6 × 250 mm, nacalai tesque, Kyoto, Japan) with a binary gradient consisting of solvent A (methanol containing 0.1 mM sodium acetate) and solvent B (2-propanol containing 0.1 mM sodium acetate). The gradient profile was as follows: 0–20 min, 50–70% B linear. The flow rate was 1.0 mL/min, and the column temperature was 40 °C. Elution was split at the post-column and one of the split eluents was sent to the MS/MS system at 0.2 mL/min. The MS parameters are shown in SI
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