9
as Triglycerides and the saponification of free fatty acids. The following acidic derivatisation finally transfers the remaining saponified fatty acids in FAMEs.
The reaction equations are displayed in Figures 1 and 2.
Microwave procedure:
The new microwave procedure for determination of fat has been developed for the microwave system Discover SP- X® (CEM, Kamp Lintfort). It consists of a microwave extraction method (MEM) and a microwave derivatisation method (MDM).
The MEM has been developed based on the Weibull-Stoldt method and extracts the total fat in a food sample in a closed system under defined conditions using microwave radiation simultaneously.
Figure 3: Chromatogram of the 37 Standard-Mix identified FAMEs.
In a closed system (including MDM), derivatisation of the extracted fatty acids is performed under microwave radiation. Transformation of fatty acids in fatty acids methyl esters (FAMEs) also requires a sequence of alkaline and acidic derivatisation in water-free methanolic environment for subsequent GC analysis.
GC analysis:
Analysis of FAMEs of the food samples has been done using a gas chromatograph GC - 2010 Plus AF with FID detector (Shimadzu Europa GmbH, Duisburg, Germany).
Figure 4: Chromatogram of fatty acids in a sausage sample, derivatisation according to ISO procedure and identified FAMEs.
correlates with the extraction method of Weibull-Stoldt for the determination of total fat content in food samples.
The ISO extraction method according to Weibull-Stoldt is based on a saponified digestion using hydrochloric acid and water, which releases fat bound to proteins in food samples. In the following hot filtration, the fat released remains in the filter. The filter including the fat is washed to neutral and then dried. Finally, the fat is extracted with a proper solvent in a Soxhlet extractor, taking several hours. As soon as the extraction is
finished, the solvent is removed using a rotation evaporator and the remainder is dried. Determination of total fat is performed by weighing the dried fat.
Based on the ISO procedure on sample preparation for GC analysis, the transformation of fatty acids in fatty acids methyl esters (FAMEs) requires a sequence of alkaline and acidic derivatisation in water-free methanolic environment.
The alkaline derivatisation generates the FAMEs out of the fatty acids bound
Influence of the method of sample preparation and derivatisation on the content of unsaturated fatty acids in the total fat content of food samples needs to be investigated. In order to compare the contents of unsaturated and saturated fatty acids in both methods, the total amounts of all fatty acid peak areas are calculated (= 100%). Peak areas of unsaturated and saturated fatty acids are then summarised, and the ratio of unsaturated vs. saturated fatty acids is calculated. This procedure has been applied for both the ISO procedure and the microwave procedure.
Experimental Part ISO procedure: Extraction method:
The food sample is weighed in a 600 mL glass beaker, in the expectation that a total fat weight of 2 to 3 g will be present. The sample is treated with 100 mL water and 150
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