In this study, two microwave protocols have been developed and here described as METHOD A and B. Both solutions have as the ultimate target of FAME analysis enabling, during the preparation process, a sequential determination of total fat (Method A) and free fat (Method B). - Milestone’s ETHOS X
- SR-15 with easyTEMP extraction rotor
- RAR-15 evaporation rotor
- Aluminum caps
- SFS-24 filtration system
- Vacuum system with condensation module
- Analytical balance with direct interface to ETHOS X terminal
Depending on reporting needs, laboratories can choose the right microwave protocol that can be applied for the FAME analysis. These approaches aim to provide a faster and simpler sample preparation process delivering to food processors and contract labs a reliable and consistent technique to enhance throughput and to reduce labor costs.
SOLVENTS AND REAGENTS
Solvents and reagents were purchased by Sigma Aldrich. 25% solution of sulphuric acid and ACS reagent grade cyclohexane, pentane, hexane, petroleum ether, methanol, sodium sulphate anhydrous and sodium chloride were used.
For this study, certified reference materials (FAPAS and IFIP) and labeled foodstuff (purchased at the grocery store) were used. Regarding commercial foodstuff, the samples should be homogenized before the weighing step in order to get a representative aliquot of sample. The sample has been used as it is, avoiding any drying step.
Two methods were tested in this study, here described as METHOD A, developed for total fat and FAME determinations, and METHOD B, developed for free fat and FAME determinations. Both methods were compared to certified reference material or to samples analyzed with Reg UE 1833/2015 12/10/2015 GU UE L266 13/10/2015.
METHOD A (Total fat determination)
Approximately from 1 to 3 g of sample was directly weighed into the SR-15 eT extraction vessels; 10 mL of sulphuric acid (25%) and 25 mL of cyclohexane were subsequently added, recording its final mass.
At the end of the program, vessels were opened, and the aliquots of the organic phase were transferred into aluminum caps and then weighed. After a fast solvent evaporation, using the RAR-15 evaporation rotor, the aluminum caps were newly weighed for total fat determination.
METHOD B (Free fat determination)
Approximately from 1 to 3 g of sample was directly weight into the SR-15 eT extraction vessels then; 30 mL of pentane was subsequently added. Magnetic stirring bars were added to each vessel. The SR-15 eT was properly sealed.
At the end of the program, vessels were opened and a suitable amount of sodium sulphate anhydrous was added, while a small amount of the mixture was kept. The solution was then filtered into the Milestone’s SFS-24 on 0.45 µm PTFE filters.
After a solvent volume reduction into the SFS-24, the filtered solutions were transferred into the RAR-15 for a fast evaporation. The final residue inside the aluminum caps was weighed for the determination of free fat.
MICROWAVE ASSISTED FAME PREPARATION METHOD
This procedure was suitable to prepare the methyl esters from the extract residues both obtained with METHOD A and B.
Approximately from 0.1 to 0.2 g of dry fat aliquot was directly weighed into the SR-15 eT vessel; 10 mL of acidic methanolic solution and 25 mL of cyclohexane were subsequently added. Magnetic stirring bars were added to each vessel. The SR-15 eT was properly sealed.
At the end of the program, vessels were opened and the entire mixture was transferred into a centrifuge vial type; 2.5 g of NaCl were subsequently added and the vial was shacked. When the two phases were separated, an aliquot of the upper layer (organic phase) was collected, treated with sodium sulphate anhydrous and filtered (0.45 µm PTFE filter) into a GC vial. The mixture is now ready for the GC analysis.