The automated derivatization of fatty acids (FAs) was performed with the Agilent 7696A Sample Prep WorkBench. Since free fatty acids show tailing in gas chromatography, transformation of fatty acids into fatty acid methyl esters (FAMEs) is widely used.
The automated derivatization of fatty acids (FAs) was performed with the Agilent 7696A Sample Prep WorkBench. Since free fatty acids show tailing in gas chromatography, transformation of fatty acids into fatty acid methyl esters (FAMEs) is widely used. Manual sample derivatization is time-consuming and may lead to poor repeatibility. Automated derivatization shows significant enhancement of reproducibility and saves time. Especially for highly unsaturated fatty acids, slight variations in reaction temperature and time can negatively affect repeatability when using manual procedures.
Salmon oil is an excellent source of polyunsaturated omega-3 fatty acids. The two main fatty acids—eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)—have been identified as important health factors and are correlated with a normal function of the heart. The concentration of EPA and DHA is the crucial quality factor for salmon oil capsules. This application note demonstrates the use of the Agilent 7696A Sample Prep WorkBench for derivatization and subsequent determination of both EPA and DHA from salmon oil capsules.
For sample preparation, 10 mg of salmon oil was weighed into a 2-mL autosampler vial. The sample was diluted in 500 µL of tert-butyl methyl ether (TBME), using the liquid-dispensing module of the Agilent 7696A Sample Prep WorkBench and mixed for 90 seconds with the onboard vortex mixer. A 250-µL aliquot of the prepared sample was transferred to an empty vial and 125 µL of a Trimethylsulfoniumhydroxide (TMSH) derivatization solution [MachereyNagel, Düren] was added and the mixture was again mixed using the vortex mixer of the WorkBench. The mixture was heated for 5 minutes at 80 °C in the single vial heater. The flow diagram for the automated procedure on the Agilent 7696A Sample Prep WorkBench is in shown in Figure 1.
The gas chromatographic conditions were chosen as shown in Table 1.
Table 1. GC/FID Conditions
Figure 2 shows the separation of FAMEs from salmon oil on an Agilent 7696A WorkBench. The separation allows the unequivocal identification of all FAMEs. The two compounds of main interest show retention times of 35.07 minutes (EPA) and 40.55 minutes (DHA). Besides EPA (23.7%) and DHA (20.0%), salmon oil further consists of unsaturated fatty acids oleic (12%), linoic (11 %) and palmitoleic (8%) acid. The content of saturated fatty acids, palmitic and stearic acid, is low, 4% and 5%, respectively.
Figure 2. GC/FID chromatogram of a salmon oil sample, prepared using Agilent WorkBench 7696A
For the repeatability test, 10 individual salmon oil samples were derivatized and analyzed to determinate the reproducibility of the automatic sample preparation and chromatography. As shown in Figure 3, excellent repeatability was obtained. The absolute areas of the EPA and DHA signals showed standard deviations of less than 1% (EPA 0.51%, DHA 0.78%). Moreover, variations of the EPA and DHA relative concentrations were stable. Relative standard deviations of 0.85% for EPA and 1.22% for DHA were achieved. No outliers were observed over the 10 samples.
Figure 3. Repeatability data of of automated FAME determination in salmon oil
The total runtime for sample preparation on the Agilent 7696A Sample Prep WorkBench was only 20 minutes per sample, whereas the time for the manual derivatization depends on the skills of the laboratory technician and can take up to 2 hours.
Figure 4. Structure of EPA methyl ester (left) and DHA methyl ester (right)
The automated sample derivatization is easy, fast, and reliable. For samples with high relative concentrations of polyunsaturated fatty acids especially, the automation is significantly more reliable than manual procedures.
Table 2. Percent distribution of fatty acids in salmon oil sample
(1) Animal and vegetable fats and oils - Gas chromatography of fatty acid methyl esters - Part 3: Preparation of methyl esters using trimethylsulfonium hydroxide (TMSH) (ISO 12966-3:2009)
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