Gc 2030af

Total lipids in Caco-2 cells, AC and BLC were extracted, and relative fatty acids methylated as previously described [18 (link)]. Before methylation, pentadecanoic acid was added as internal standard. The qualitative and quantitative content of fatty acid methyl esters (FAMEs) was determined by fast-GC (GC-2030AF; Shimadzu, Kyoto, Japan) using a capillary column (30 mt, 0.2 μm film thickness) with a programed temperature gradient (50–250 °C, 10 °C/min). The gas chromatographic peaks were identified based on their retention time ratios relative to methyl stearate and predetermined by use of authentic samples [19 (link)]. Gas chromatographic traces and quantitative evaluations were obtained using a Lab Solution software (Shimadzu, Kyoto, Japan).

The fatty acid content of the microalgae was estimated as the amount of fatty acid methyl esters (FAMEs), which was obtained using the direct esterification method. The obtained crude lipid was saponified with KOH-CH₃OH and then FAMEs were prepared by methylene esterification with boron trifluoride in methanol and extracted with n-hexane [31 (link)].
The qualitative and quantitative analyses of the lipid sample were carried out using a GC-2030AF gas chromatograph (Shimadzu, Tokyo, Japan) equipped with a DB-Wax capillary column (30 cm, 0.32 mm ID, 0.5 μm film thickness) and a flame ionization detector (FID). The temperature program was as follows: initially kept 120 °C for 3 min; then increased to 190 °C at 10 °C/min, where the temperature was maintained for 1 min; then increased to 230 °C at 2 °C/min and held there for 15 min with a split ratio of 10:1. Nitrogen was used as a carrier gas at a flow rate of 3 mL/min. Fatty acids were identified by comparing the peak and retention time of the reference standard 21-component fatty acid methyl ester mix (NU-CHEK-PREP, INC). The relative and absolute contents of fatty acids were quantified by the area normalization method using methyl heptadecanoate purchased from Sigma-Aldrich (Merck & Co., Kenilworth, NJ, USA) as an internal standard.

Total lipids were extracted according to Folch et al. [55 (link)]. After methyl-esterification [56 (link)], the quantitative and qualitative content of fatty acid methyl esters (FAMEs) was determined by fast-GC (GC-2030AF; Shimadzu, Kyoto, Japan) using a capillary column (30 mt, 0.2 μm film thickness) with a programmed temperature gradient (50–250 °C, 10 °C/min). The gas chromatographic peaks were identified using authentic samples based on their retention time. FAMEs from chemicals added during in vitro digestion were subtracted and quantitative evaluations were normalized for the dilution factor due to the addition of digestive fluids. Bioaccessibility was assessed as FAME content in digested sample/FAME content in the corresponding sample before digestion × 100 [57 (link)].