Optimization of the GC method for routine analysis of the fatty

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This project has been funded with support from the European Commission. This publication reflects the views only of the authors,

and the Commission cannot be held responsible for any use which may be made of the information contained therein.

Optimization of the GC method for routine

analysis of the fatty acid profile in

several food samples

Ph.D. Onejda Kycyk

NETCHEM Remote Access Laboratory Guide

Optimization of the GC method for routine analysis of the fatty acid

profile in several food samples

In this exercise, you will: • Perform sapunification of olive oil,

• Analyze samples taken at certain time intervals on the GS-MS,

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Background

The fatty acids (FAs) composition of food is very important because lipids

are one of the three major constituents of food. Their roles in biological

tissues are: (1) source of energy, (2) components of biological

membranes, (3) precursor for many different molecules and (4) transport

vehicle for vitamin A, D, E and K.

The main sources of lipids in our diet are vegetable oils and different

foodstuffs of animal source.

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GC is the most widely used technique for determining the FA profile (AOAC, 2000;

ISO 5508, 1999; Mendez Antolin et al., 2008). The flame ionization detector is

sufficient for a majority of analytical applications of this kind. GC/MS has been used

for the analysis of trace amounts of FAs especially for identifying FAs when no

standards are available. Electron impact ionization may give us useful information on

a number of double bonds in analyzed FAs but for isomer determination various

deconvolution software has been used (Ramadan et al., 2006). GC x GC technique

is the most useful technique for identification of odd carbon number FAs (de Geus et

al., 2001; Mondello et al., 2006).

Background

Material For this lab exercise, you will need the following material:

Reagents

• Isooctae obtained from Merck (Germany),

• Methanol gradient grade from Merck (Germany),

• Petrol ether, p.a.,

• hydrochloric acid p.a.,

• potassium hydroxide, p.a.,

• sodium hydrogen sulfate monohydrate, p.a.,

Devices

• magnetic stirrer;

• analytical balance.

• Backer,

• fluted filter paper, quantitative,

• GC-MS,

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Material

For this lab exercise, you will need the following material:

Laboratory equipment

• glasses (100, 250 cm3);

• volumetric flask 125 cm3;

• pipette 5 and 20 cm3;

• graduated cylinder 100 cm3;

• two funels;

• micropipette 100-1000 ml;

• micropipette 1000-5000 ml;

• filter paper.

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Procedure:

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Extraction of lipids

Lipids were extracted by standard procedure (ISO 1443, 1973). Approximately 5 g

of homogenized sample was weighted into a conical flask and dried for 1 h at

105ºC. The flask was cooled to room temperature, and 50 mL of 4 M hydrochloric

acid was added. The solution was boiled for 1 h. Then 150 mL of water was added,

the solution was filtered through fluted filter paper and washed until neutral reaction

on litmus paper. Filter paper was dried for 1 h at 105 ºC and inserted in extraction

thimble of Soxhlet apparatus. Lipids were extracted with petrol ether into weighted

round bottom flask for 4 h on the sand bath. After extraction, petrol ether was

evaporated, the flask was dried at 105 ºC and weighted.

Procedure: Preparation of fatty acid methyl esters (FAMEs)

Lipids obtained after extraction of vegetable oil and fat samples were converted

to corresponding FAMEs by trans-esterification with potassium hydroxide (ISO

5509, 2000). Approximately 60 mg of sample was dissolved in 4 mL of isooctane

in test tube and 200 lL of methanolic potassium hydroxide solution (2 mol/L) was

added. Solution was shaken vigorously for about 30 s. The solution was

neutralized by addition of 1 g of sodium hydrogen sulfate monohydrate. After the

salt has settled, 1 mL of upper phase was transferred into 2 mL vial and

analyzed.

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Procedure: GC analysis

The fatty acid profiles of vegetable oil samples were determined using GC–MS

(Varian 431-GC and 220-MS ) equipped with a DB-5MS capillary column (30 m ×

0.25 mm × 0.25 μm) and a time-of-flight mass spectrometer. Helium

(99.99%)was used as the carrier gas at a constant flow rate of 1 mL/min. The

oven temperature was programmed from 60 ºC to 215 ºC at 15 ºC/min, to 250

ºC at 10 ºC/min, and to 260 ºC at 2 ºC/min. Then it was finally increased at 280

ºC at 5 ºC/min and held for 2 min. Injections (1 μL each) used a 40:1 with the

injector inlet temperature is 250 ºC parameters.

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Procedure: Time of flight mass spectrometer was operated by electron ionization (EI), transfer line and

EI temperatures, 270 ºC and 250 ºC, respectively. The solvent was delayed for 10 min.

The masses to display were recorded in total ion chromatogram (TIC). The m/z ranged

from 30 to 500 amu, and the acquisition rate was 20 spectra per second. The electron

energy was 70 eV and detector voltage was 1500 V.

In our experiments, no internal standard was used, but all the oil samples were treated and

repeated for three times according to the described sample preparation procedure, and

GC–MS analyses were also performed three times for every prepared sample to record

the final GC–MS data. We found there were no significant changes between the GC–MS

data obtained from a same oil sample, suggesting the saponification and methylation

procedures.

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Author, Editor and Referee References

This remote access laboratory was created thanks to work done primarily at University of Niš.

Contributors to this material were: Onejda Kycyk

Refereeing of this material was done by: _____________________

Editing into NETCHEM Format and onto NETCHEM platform was completed by: ______________

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References and Supplemental Material The NETCHEM platform was established at the University of Nis in 2016-2019 through the Erasmus Programme.

Please contact a NETCHEM representatives at your institution or visit our website for an expanded contact list.

The work included had been led by the NETCHEM staff at your institution.

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Optimization of the GC method for routine analysis of the fatty