Standard fatty acid methyl esters

The extracted fat content (50 mg) of the sample was saponified (esterified) for 5 min at 95 0 C with 3.4 mL of the 0.5 M KOH in dry methanol. The mixture was neutralized by using 0.7 M HCl. 3 mL of the 14% boron triflouride in methanol was added. The mixture was heated for 5 min at the temperature of 90 0 C to achieve the complete methylation process. The fatty acid methyl esters were extracted from the mixture with redistilled n-hexane in triplicate. The content was concentrated to 1 mL for GC analysis and 1 µL was injected into the injection port of GC. The injection port and the detector were maintained at 310°C and 350°C, respectively while the initial column temperature was 250°C rising at 5°C/min to a final temperature of 310°C. A polar (HP INNO Wax) capillary column (30 m  0.53 mm  0.25 μm) was used to separate the esters. The peaks were identified by comparison with standard fatty acid methyl esters obtained from Sigma Chemical Co. (St. Louis MO, USA). However, the quantitative evaluation was carried out on the base of GC peak areas of the different methyl esters. The heptadecanoic ester was used to calculate the response factor for FAs which was found to be 0.96 [13] .

A small quantity (5 g) of freshly prepared Hawaijar from each tray was oven dried at 70°C until it became moisture free and was extracted using petroleum ether (boiling point 50°C) for obtaining oil. Fatty acid methyl esters of the oil extracted were prepared using 1N sodium methoxide and were resolved in gas chromatograph Shimadzu GC17A instrument (Kyoto, Japan), fitted with a capillary column (SGEBPX70, 30m×0.32mm×0.25μm).
Programming of the oven was as follows: at 140°C for 3.6 min, then increased to 170°C at a rate of 13.5°C min -1 and maintained for 3.8 min, and finally increased to 182°C at a rate of 5°C min -1 . The flame ionization detector (detection limit: 3×10 -12 g/s for diphenyl, dynamic range: 10 7 , operational temperature range: ~ 450°C in 1°C increment) and injector (number of individually controlled zones: 2, temperature range: ~ 450°C, both heating and cooling step program) were maintained at 240°C. Nitrogen was used as the carrier gas. Peaks obtained for fatty acid methyl esters Indonesian Food and Nutrition Progress were identified by comparing the retention times with those of standard fatty acid methyl esters (Sigma-Aldrich, Bangalore, India) (Rani et al., 2019) . The quantification of fatty acid methyl ester was carried out through Class GC 10 software. The data presented in Table 1 are the average content of triplicate determinations.