Fatty acid analysis in Gas Chromatography and Mass Spectrometry (GC/MS)

Fat Extraction

Principle: The wet muscle is homogenized with 2:1 mixture of chloroform and methanol. The chloroform-methanol mixture extracts the total lipid from the tissue in to a single phase of solvent. Disturbing the equilibrium between chloroform and methanol separates the chloroform soluble fat.

Procedure: Minced fish samples were homogenized (using a motor pestle) in the organic solvent mixture (chloroform–methanol 2:1), keeping the solvent/tissue ratio 20:1, and filtered applying little vacuum. The extraction and filtration procedure was repeated thrice with fresh solvent mixture. The chloroform fractions, enriched with lipids, were collected, pooled, and dried in a rotary evaporator. The dried lipids were weighed, dissolved in 10 ml chloroform and stored in small amber glass laboratory bottles. One ml of aliquot was taken from it in a pre weighed petri dish and dried. It was then cooled in a desiccator and weighed.

Calculation

Where,
V1: total volume of extract
V2: volume of extract taken for drying
W2: weight of dried lipid
W1: weight of sample for fat extraction

Preparation of fatty acid methyl ester (FAME): : To about 150 mg of fish oil 4 ml methanolic NaOH was added and refluxed for 5 mins until the fat globules goes into the solution. Then 5 ml BF3- CH3OH solution was added and again refluxed for 5min. 16 ml NaCl was then added to float the methyl esters and transferred in to a separating funnel and 20 ml petroleum ether was added to it and shaked vigorously. The lower layer was collected and 20 ml petroleum ether was added to it twice. The petroleum ether fraction was collected, passed through anhydrous sodium sulfate and evaporated and dissolved in 5ml hexane.

GC/MS Analysis

The FAMEs were quantified by injecting 1 µL (30:1 split ratio) into GC-MS. The fatty acids were identified and quantified using a GC (Trace GC Ultra, Thermo Scientific) equipped with a capillary column (TR-FAME, 30m × 0.25mm, 0.25μm film thickness) and an MS (ITQ 900, Thermo Scientific) attached to it. For separation of saturated fatty acids, the oven temperature programme was set as follows; 1 min initial hold at 50º C, temperature raised from 50-220º C at the rate of 20º C min-1 followed by a hold of 5 min at 220º C, temperature raised from 220-250º C at the rate of 10º C min-1 and a final hold of 10 min at 250º C. For separation of unsaturated fatty acids, the oven temperature programme was as follows: 1 min initial hold at 80º C, temperature raised from 80-150º C at the rate of 20º C/min followed by a hold of 15 min at 150º C, 150-240º C at the rate of 10º C min-1 and a final hold of 2 min at 240º C. Helium was used as a carrier gas with column flow rate of 1.0 mL min-1. The MS conditions were as follows; ionization voltage 70 eV, Mass range of 45-600 and the scan time equal to the GC run time. The individual constituents showed by GC were identified and quantified by comparing the retention times and peak areas to those of standards (ME-14-KT and ME-19-KT, SUPELCO Analytical).

Reference

• 1. Folch J, Lees M and Sloane-Stanley GH (1957) A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemist 226:497-509.

• 2. Metcalfe LD, Schmitz AA and Petha JR (1966) Rapid preparation of fatty acid esters from lipids for gas chromatography analysis.Analytical Chemistry 38: 514-517.

• 3. Mohanty BP, Bhattacharjee S, Paria P, Mahanty A,Sharma AP (2013) Lipid biomarkers of lens aging. Applied Biochemistry and Biotechnology 169:192–200.