Determination of Fatty Acid Methyl Esters in olive oil using GCFID


The determination of Fatty Acid Methyl Esters (FAME) is a commonly performed analysis, to determine the quality of extra virgin olive oil (EVOO). Natural EVOO is made by pressing or centrifuging olives, without exposing the olives to any chemical processing. A way of producing cheaper olive oil is to mix them with cheaper oils, such as sunflower oil and refined olive oils. This mixing with refined olive oils results in a divergent fatty acid content in the end product.

The determination of fatty acids for quality control in EVOO’s is performed by Gas Chromatography (GC).  Therefore, the fatty acids need to be trans esterified into FAME because of the thermally instable volatile behaviour of the fatty  acids. This results in a more reliable analysis. In the transesterification process free and bonded fatty acids react with an alcohol in the presence of a catalyst, forming a mixture of FAME compounds and an alcohol.1 The composition of FAME compounds in EVOO can say much about the quality and origin of the olive oil, which makes this analysis very valuable for quality control.

This application will study the quantification and qualification of FAME within EVOO’s, by GC with a Flame Ionization Detector.(FID)

This application can be performed on either the SCION Instruments 8300 GC & 8500 GC platform with the SCION 8400PRO Autosampler, shown in figure 1. A SCION-FAME column is used for obtaining the best separation of the cis and trans-isomers of most FAME’s.

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Figure 1 SCION Instruments 8300 & 8500-GC equipped with the 8400PRO Autosampler

Table 1 details the GC parameters used throughout this analysis.


For this application a FAME standard was purchased for the qualification and quantification of unknown samples.

The FAME standard contained 37 FAME compounds with a concentration range from 150 – 400 µg/mL.

The components present in the FAME standard are shown in Table 2, along with their concentrations, and displayed in elution order for this application.

The SCION-FAME column provides a perfect resolution between the cis and trans FAME isomers that are present in the FAME-mix, seen in the chromatogram in figure 2. Therefore all 37 FAME components were successfully identified according to the Retention time match gained from analyzing the FAME standard with known concentrations.

Table 2: Components and concentrations of the FAME-standard

Sample preparation

A calibration set from the FAME standards (diluted with n-heptane) prepared from 0.20 µg/mL up to 50 µg/mL.  Dodecane (50 µg/mL) is used as an internal standard (IS) and added to all standards and samples. An IS working solution 1000 µg/ml is prepared by dissolving 20 µL dodecane into n-heptane, in a 15 mL volumetric flask.

The quality control sample (QC) is prepared2 by dissolving 50 µL extra virgin olive oil in n-Heptane in a 25 mL volumetric flask. 0.1 mL of the diluted olive oil is transferred into a test tube. To this tube 100µL of the  IS work solution is added plus 1900 µL n-heptane, followed by shaking. Then, 200 µL of 2M KOH dissolved in methanol is added to perform the esterification process, by shaking vigorously for 30 seconds. The solution is left to stratify until the upper layer of the solution  becomes clear. The upper layer is transferred into a GC sample vial and is ready for injection.

Blank injections of n-Heptane are performed in between samples to determine RMS Noise, and to ensure that the system is not contaminated after sample injections.

Figure 2 Example chromatogram of the FAME-standard, close up for resolution between isomers Methyl Elaidate(Trans) & Methyl Oleate(Cis)


Selected compounds for showing results are FAME compounds that were found in the EVOO sample: Methyl Palmitate (#12), Methyl Palmitoleate (#13), Methyl Stearate (#16),  Methyl Oleate (#18), Methyl Linoleate (#20), Methyl Arachidate (#21), Methyl cis-11-Eicosenoate (#23) and Methyl Linolenate (#24). If necessary, the results from the other FAME components are accessible by request.

The calibration curves for the FAME standards were prepared from 0.2 µg/mL up to 50 µg/mL.

The precision of the instrument and method was obtained by ten consecutive injections of FAME standard #4 (range 10-25 µg/ml).
The results of the precision of the selected FAME compounds can be found in Table 3, along with the linearity results (R2) obtained by the calibration curves.

Table 3: Summary of Results – Linearity and repeatability

For all FAME components an R2 of 0.99 or higher was achieved, which is an excellent result,  with many regulations requiring an R2 value of only ≥0.98. Repeatability results show that for most FAME components the relative standard deviations (RSD%) are below 1%. For only 2 FAME components RSD ≤1.2%. This is a good precision for the method, since most acceptance criteria for method validation are requiring an RSD ≤2%.

The limit of detection (LOD) and limit of quantitation (LOQ) were calculated according to equations 1 and 2:

Where the RMSNoise (in µV) is generated from a blank injection and calculated by CompassCDS, by root-mean-square (RMS) of the baseline over a selected time window. The concentration of the components is from the lowest calibration standard  (800x dilution of FAME standard, in µg/mL) with corresponding peak heights (in µV).

The calculated LOD’s were found to be ≤0.34 µg/mL, these are shown in Table 4. This table also displays that the LOQ’s are ≤1 µg/mL. If lower LOD’s and LOQ’s are required it is recommended to use this application with the SCION GC-SQMS configuration.

Identified FAME components found in the sample are shown in Table 4. According to regulations3 an EVOO FAME content (summation of total FAMES) should be ≤75 mg/kg. If compensated for the dilution in n-heptane the ƩFAME content this is ≤51.3 µg/mL. In the olive oil sample 8 FAME compounds are found, the concentration of each component is calculated from the calibration curves equations. The total Ʃ FAME-content from all identified components is 34.51 µg/mL, and therefore within the specification mentioned in regulations.

Table 4: Summary of Results – LOD, LOQ and Amount found in extra virgin olive oil sample.

Figure 3 Shows an example chromatogram generated in CompassCDS of the EVOO sample.

The composition range (%) of FAMEs mentioned in regulations4 for EVOO are shown in Table 5.

The composition of identified FAMES from the EVOO sample (% peak Areas Ratio) are  compared with the set up specifications for a genuine extra virgin olive oil, and all components are passed specifications criteria. #23 Methyl cis-11-Eicosenoate wasn’t mentioned in the regulations and is therefore marked as N.D.

Another confirmation that be sure an EVOO is not adulterated is to check the presence of trans-isomers. Trans-isomers (for example #17 Methyl Elaidate) in double bonded fatty acids are in nature not present in EVOO, but can be formed after heating processes. In this case the analyzed EVOO does not contain any trans-FAME compounds which indicates that the sample has not been exposed to any heating processes.

With the total FAME content mentioned in Table 4, the composition of FAME components in Table 5, and no presence of trans-isomers in the sample it can be concluded that the analyzed sample is a genuine EVOO, which has not been exposed to dilution with cheaper oils, or any heating or chemical processes.

Table 5: Results FAME composition in EVOO sample compared to specifications mentioned in regulations4


Figure 3: Example chromatogram and peak results for EVOO sample


The SCION 8500 GC platform equipped with a split/spitless injector, SCION FAME column and Flame Ionization Detector with 8400PRO sampler is a perfect solution for the determination of FAME in olive oil. Good system precision, good linearity results and low LOQ’s and LOD’s are easily achieved for this application.

The SCION-FAME column shows good resolution between cis and trans FAME isomers, which is an easy way to verify if there are no trans-FAME isomers present in an EVOO that could indicate heating in the production process.

The analyzed  EVOO sample  is, according to multiple results obtained by this application, most likely a natural EVOO that has not    been exposed to adultery in the production process.

This method is also applicable on the SCION Instruments 8300 or 8500 GC-platform with 8700 Single Quad Mass Spectrometer (SQ-MS). This technique is achieving lower LOD’s and LOQ’s while measuring in SIM-mode, in comparison to the GC-FID application. The use of the MSWS-software unknown FAME components can be easily identified with our NIST-Libraries. This application note ‘Determination of FAME in olive oil using GC-SQMS’ can be found our Application Library.



2. COMMISSION IMPLEMENTING REGULATION (EU) Characteristics of olive oil and olive-residue oil and on the relevant methods of analysis 2015, (accessed 14-02-2024)

3.  COMMISSION REGULATION (EU) Characteristics of olive oil and olive-residue oil and on the relevant methods of analysis 2011, (accessed 18-02-2024)


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Download The Application Note

Download the application note: AN174v1 – Determination of FAME’s in olive oil using GC-FID.2