• Glycerides (glycerolipids, such as triglycerides, diglycerides, monoglycerides and lecithin)

* Cannot be used with free fatty acids, sterol esters or sphingolipids.
This kit is suitable for measuring the fatty acid composition of glycerides. For other fatty acids (except sphingolipids), please use the regular Fatty Acid Methylation Kit, which can also methylate free fatty acids and sterol esters.

Methylation is done with a transesterification (methanolysis) reaction between the glyceride and methanol.

Reaction was confirmed by methylating 10 of a triglyceride (triolein) using the Fatty Acid Methylation Kit for Glycerides and performing TLC using a silica gel plate.

• Developing solution: Hexane / t-butyl methyl ether / acetic acid = 92 / 8 / 0.4
• Coloring: Sprayed with 50% sulfuric acid, then heated at 137°C for 15 minutes

(Results)
The triglyceride was successfully methylated.

Fatty acid analysis of oil extracted from sardines

(Sample preparation)

1. Using a mortar and pestle, about 10 g of a small sardine was extracted with 30 ml of acetone, then 20 ml of hexane. After extraction, the mixture was filtered.
2. 30 ml of water was added to the filtrate and the solution was mixed.
3. The hexane layer was collected and evaporated in a rotary evaporator.
4. From the resulting oil, about 20 mg was collected and methylated using the Fatty Acid Methylation Kit for Glycerides.

(Results)
In addition to saturated fatty acids, the sample contained large amounts of unsaturated fatty acids, such as DHA and EPA.

Analysis of fatty acids in milk

(Sample preparation)

1. 0.5 ml of milk was combined with 2 ml of reagent A from the Fatty Acid Methylation Kit for Glycerides.
2. 1 ml of the organic layer was methylated with the Fatty Acid Methylation Kit for Glycerides.

(Results)
Methylation using the Fatty Acid Methylation Kit for Glycerides can be done quickly at room temperature, so volatile short-chain fatty acids, such as methyl acetate (4:0) and methyl hexanoate, can be analyzed.

Fatty acid analysis of rice (Nipponbare) for producing sake (effect of different degrees of polishing)

(Sample preparation)

1. The samples (100 mg each of brown rice, 10% polished rice and 30% polished rice) were crushed.
2. 1 ml of acetone was added to the crushed samples and stirred with a vortex mixer. The acetone solution was collected.
3. 1 ml of hexane was added to the residues, stirred with a vortex mixer, collected and mixed with the acetone solutions.
4. 1 ml of ultra-pure water was added to the mixtures obtained in (3), and the mixtures were stirred lightly.
5. The mixtures obtained in (4) were centrifuged at 2000 rpm for 3 minutes, and the upper hexane layers were collected.
6. The hexane solutions were evaporated, leaving an oil sample.
7. Half of the oil (equivalent to 50 mg of rice) was methylated using the Fatty Acid Methylation Kit for Glycerides.

Fatty acid analysis of refined sake yeasts

(Sample)

Yeast - parent cell line 1103
Yeast - mutant cell line 0101 (cerulenin-resistant)

(Sample preparation)

1. Yeast incubation (YPD, 30°C, approx. 24 hours)
2. After harvesting, the cultures were washed twice with distilled water.
3. The bacteria were lyophilized overnight.
4. Lipids were extracted with acetone and hexane.
5. The sample was methylated using the Fatty Acid Methylation Kit for Glycerides.

Data courtesy of Gakkeikan Sake Company, Ltd.

Soybean oil

Extracted oil from tuna

Wild boar fat

Potato chips

Extracted lipid from green leaf of tabacco

Main components: Galactolipids (glycolipids), phospholipids

Extracted lipid from yolk of chicken

Main components: Phosphatidylcholines, phosphatidylethanolamines

1. Place your glyceride sample (50 mg or less) in a suitable container.
2. Add 1.0 ml of reagent A (solvent), and dissolve the sample.
3. Add 0.1 ml of reagent B (reaction solution), and stir with a vortex mixer for 3 seconds.
4. Let the mixture stand for about 10 seconds, then add 1.0 ml of reagent C (stop solution). Stir with a vortex mixer for 5-10 seconds.
5. Let stand until two layers form. Move the upper layer to a different container, such as an autosampler vial.
6. Analyze with GC.
   * This kit does not use the Fatty Acid Methyl Ester Purification Kit.