Fatty Acid Methylation Kit and Fatty Acid Methyl Ester Purification Kit

Features

Product Image

For the analysis of volatile free fatty acids, glycerolipids and sterol esters
Enables reaction at 37°C
Conducts methyl esterification safely and easily
Detects not only long-chain, but also short-chain fatty acids

>> For a faster and simpler method, see Fatty Acid Methylation Kit for Glycerides
>> Fatty Acid Applications (PDF 3.5MB)

Product Information

Methyl esterification of fatty acids is commonly done prior to gas chromatography analysis to prevent peak tailing and increase sample volatility. However, the conventional esterification procedure requires specialized equipment and high technical skill.
By using the Fatty Acid Methylation Kit, which employs a new reaction technique, and the Fatty Acid Methyl Ester Purification Kit, the methyl esterification procedure is greatly simplified.

*Patent applicant: Kyoto Prefectural University and GEKKEIKAN

Targeted fatty acids

Free fatty acids
Glycerolipids such as triglycerides, phospholipids and glycolipids
Sterol esters

Please note this method is not suitable for sphingolipids.

Comparison Data

The methyl esterification efficiencies of the Fatty Acid Methylation Kit and the conventional method are about the same, independent of fatty acid side-chains.

Comparison of methylation efficiency

Table

Quantitative Analysis

The Fatty Acid Methylation Kit and Fatty Acid Methyl Ester Purification Kit offer wide dynamic range, as shown by the results from dried yeast.

Quantitative Analysis

Applications

Application1

GC-MS Analytical Condition
System	6890 GC/5973i MSD (Agilent)
Column	DB-WAX(60 m x 0.25 mm, 0.25 µm)
Column Oven	50 °C(2min) to 250°C at 10°C/min, hold 250°C (8min)
Detection	MSD, 230°C
Carrier gas	He,1.4ml/min
Injection	2.0 µl, split-less, 250°C

Fatty acid analysis of different Yeast strains

It is important to measure the exact amount of various fatty acids in different yeast strains. By using these kits, it was determined that the cerulenin-resistant strain contains more capronic acid than the parent strain.

Application2

Data courtesy of GEKKEIKAN

Protocols

Procedure

Easy and safe without using HCl .

Procedure

Conventional Method

Quantitative capability of the conventional method is questionable due to the high heating requirement. The high temperature causes the degradation of unstable fatty acids (polyunsaturated and cyclopropane fatty acids) and the evaporation of short-chain fatty acid akyl esters.

Procedure

Sample Preparation

Staring Material	Procedure
E. coli or Yeast	Centrifuge cell culture medium of E. Coli or Yeast in a centrifuging tube and then freeze-dry about 20 mg of the pellet. An alternate method for the E. coli sample is to dry in a vacuum desiccator for 1-2 hours.
Blood	Apply 0.04 ml of heparin-treated blood to antioxidant agent BHT-treated filter paper and dry it in a vacuum desiccator for 30 min. or let it dry naturally for more than 2 hours. To get complete methylation, spread the heparin-treated blood on filter paper thinly. [How to make antioxidant agent BHT-treated filter paper] Soak filter paper such as Whatman 3M or ADVANTECH No. 2 into Acetone containing 0.05% BHT for several minutes and then repeat this process. (Prepare an alternative new solvent for the second immersing). Let it dry naturally at room temperature, then put it in a vacuum desiccator for 30 min. or in a desiccator overnight. The paper with a square 1.5 cm on a side is suitable for methylation application. Please note that thicker filter papers, e. g., blood collecting filter papers have low methylation efficiency
Rat Liver	Lyophilization of 15 mg of rat liver. Please note that drying rat liver in a vacuum desiccator decreases methylation efficiency.
Edible Oil	Less than 4 mg of edible oil is suitable for the methylation application.
Soybean Flour	Less than 20 mg of soybean flour is suitable for the methylation application.
Fish	Put 200 mg of fish meat, e.g., Japanese horse mackerel, into a test tube, and add 2 ml of Isolation Solution ,then mesh the fish meat with a glass rod. After vortexing, take 0.5 ml of supernatant to a new test tube, and then dry it in a rotary evaporator, vacuum desiccator, or N2 gas.

For procedure of sample preparation of Glycerolipids analysis, please refer to its instruction.

References

Kanai, M., Yamada, T., Hayashi, M. et al. Soybean (Glycine max L.) triacylglycerol lipase GmSDP1 regulates the quality and quantity of seed oil. Scientific Reports 9, 8924 (2019). [DOI]
Kobayashi H. et al. Environmental Optimization Enables Maintenance of Quiescent Hematopoietic Stem Cells Ex Vivo. Cell Reports 28, 145–158, July 2 (2019). [DOI]