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Developments of methods for the analysis of antioxidants in a range of foods
Project Code: A01020
Leatherhead Food International
Burch, R ; Ing, B;
Institut fur Chemie, TU Berlin
Bruggemann, O; Visnjevski, A
This project aimed to develop a method or methods for the reliable quantitation of a selection of synthetic antioxidants in the foods in which they are permitted. Antioxidants are added to foods in order to prevent deterioration of the foods through atmospheric oxidation. The antioxidants studied in this project were butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate (PG), octyl gallate (OG) and dodecyl gallate (also known as lauryl gallate, LG). Theseantioxidants are permitted in a limited range of foods, and at well-defined levels, shown in Table 1. There is a need for accurate methods for the measurement of these antioxidants in foods both for regulatory enforcement and for monitoring of the intake of additives by the population. Methods developed must be suitable for use in a general routine or commercial laboratory. Therefore methods must be robust, and ideally use non-specialist equipment.
Initial experimental work was carried out on commercially available samples containing antioxidants, as specified on their labels, but where the level was unknown. A range of different extraction procedures was used, and the antioxidants determined by High Performance Liquid Chromatography (HPLC). Levels of antioxidant found using different extraction methods were compared in order to gain an idea of the methods most suitable to carry forward for further development. Two types of extraction procedure were tested: (i) those that directly extract the antioxidant from the food and (ii) those that extract the fat from the food, followed by extraction of the antioxidant from the fat. It was found in general that the fat extraction methods were too harsh, and caused greater losses of antioxidants than the direct extraction methods. Of the direct extraction methods, one method was found to be suitable only for BHT, therefore was not selected for further development. A cold extraction method, using diethyl ether, was selected for further development.
Foods with known amounts of added antioxidant were prepared at Leatherhead Food International (LFI), in order to properly assess the recovery of antioxidants from foods. The cold extraction method was tested and optimised using these foods. Following this optimisation, ring trial samples were prepared and sent to external laboratories, with detailed protocols of the method to be used, in order to test the reproducibility of the method in other laboratories. Results from the laboratories were inconsistent, for a number of reasons, and therefore cannot be used as validation for the method to be put forward as a standard method. One laboratory had not used the method supplied to them, due to the length of time that would have been required, and had instead used an in-house method, therefore their results cannot be used to assess the method developed here. However, this is perhaps an indication that the method developed would not be attractive to a commercial laboratory or suitable for use as a routine method.
In addition to the use of traditional wet chemistry techniques for the extraction of antioxidants from foods, the project also aimed to develop molecular imprinted polymers against some of the antioxidants under consideration. Molecular imprinted polymers (MIPs) are polymers produced around a template molecule (in this case either BHA, BHT or propyl gallate). The template molecule is then removed by washing, leaving imprints in the polymer that will specifically retain an analyte that is structurally the same as the template molecule. Polymers were produced against BHA, BHT and PG. A control polymer was also prepared, where the same ingredients were used as for the imprinted polymers, except that no template molecule was added. The effect of imprinting was assessed by packing each polymer into an HPLC column, and measuring the extent to which the polymer retained the analyte. This was compared to the retention of the same analyte by the control polymer. It was found that each of the MIPs retained BHA, BHT and PG to a greater extent than the control polymer. However, the MIPs did not show selective retention of their own imprint molecule. This demonstrated that, although imprinting had taken place, the polymers were not specific, and so could not be used for selective clean up of their corresponding template analyte.
For the MIP phase of the project, work was carried out in collaboration with Technical University Berlin (TU Berlin) who have significant experience of this technology. Polymers were produced there, and initial evaluations carried out before the technology was transferred to LFI. Work was carried out at both institutions to assess the possibility of using the polymers for solid phase extraction, but the MIPs were not found to be suitable for this use.
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