Fatty acid composition

Principle: Total lipids are extracted from wet tissue by homogenizing with a chloroform–methanol (2:1) mixture. This solvent system initially forms a single-phase extract, effectively solubilizing all lipid classes. Upon phase separation, lipids preferentially partition into the chloroform layer. The extracted lipids can be dried and weighed for gravimetric fat estimation, or further derivatized to Fatty Acid Methyl Esters (FAMEs) for compositional analysis using Gas Chromatography–Mass Spectrometry (GC-MS).

Lipid Extraction Procedure
  1. Homogenization:
    • Mince fish muscle and homogenize 30 g of tissue using a motor and pestle with chloroform–methanol (2:1) in a 20:1 solvent-to-tissue ratio.
  2. Filtration:
    • Filter the homogenate using gentle vacuum.
    • Repeat the extraction three times using fresh solvent each time.
  3. Solvent Pooling & Evaporation:
    • Pool the chloroform extracts and evaporate to dryness in a rotary evaporator.
    • Weigh the dried total lipids.
    • Dissolve the lipids in 10 mL chloroform and store in amber glass bottles.
  4. Fat Estimation (Gravimetric):
    • Take 1 mL aliquot in a pre-weighed petri dish.
    • Dry, cool in desiccator, and weigh.
Fat Content Calculation

Fat content (g/100g meat) = W2×V1×100
                                                        V2×W1

Where:

  • W₂ = Weight of dried lipid
  • V₁ = Total volume of lipid extract
  • V₂ = Volume of extract taken for drying
  • W₁ = Weight of sample used for extraction
Preparation of Fatty Acid Methyl Esters (FAMEs)
  1. Weigh approximately 150 mg of extracted fish oil.
  2. Add 4 mL methanolic NaOH and reflux for 5 minutes (to saponify fats).
  3. Add 5 mL BF₃-methanol solution and reflux for another 5 minutes (to methylate fatty acids).
  4. Add 16 mL saturated NaCl solution to separate methyl esters.
  5. Transfer to a separating funnel, add 20 mL petroleum ether, and shake vigorously.
  6. Collect the upper petroleum ether layer.
  7. Wash with 20 mL petroleum ether twice more.
  8. Combine ether fractions, dry over anhydrous sodium sulfate, evaporate to dryness, and redissolve in 5 mL hexane for GC-MS analysis.
GC-MS Analysis of FAMEs
  • Instrument: GC (Trace GC Ultra) with MS detector (ITQ 900), Thermo Scientific
  • Column: TR-FAME capillary column (30 m × 0.25 mm i.d., 0.25 µm film)
  • Injection volume: 1 µL (30:1 split ratio)
  • Carrier gas: Helium, 1.0 mL/min flow rate
Temperature Program
  • Initial hold: 50 °C for 1 min
  • Ramp to 150 °C at 20 °C/min, hold for 15 min
  • Ramp from 150 to 240 °C at 20 °C/min, final hold for 2 min at 240°C
MS Conditions
  • Ionization: Electron impact (EI), 70 eV
  • Mass range: 40–500 m/z
  • Scan time: Equal to GC run time
Identification and Quantification
  • Fatty acids were identified by comparing retention times and mass spectra to certified FAME standards (ME-14-KT and ME-19-KT, SUPELCO).
  • Quantification was based on peak area integration.

References

  1. Folch, J. Lees, M., SloaneStanley, G.H (1957). A simple method for the isolation and purification of total lipids from animal tissues. The Journal of Biological Chemistry, 226, 1, 497-509.
  2. Metcalfe, L.D., Schmitz A. A., Pelka, J.R. (1966) Rapid preparation of fatty acid esters from lipids for gas chromatographic analysis. AnalyticalChemistry, 38,3, 514-515.
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