that needs to be achieved in the future in order to protect the consumer from every corner of the world form the harmful effect of these compounds.
This thesis concerns with the development and the validation of analytical methods for the determination of the mycotoxins aflatoxin B1, zearalenone and patulin, which occur frequently in food and feed. The toxic syndromes produced by them when ingested are known as mycotoxicoses. One of the first reports in history of mycotoxicoses is ergotism, caused by the fungus Claviceps purpurea. Nowadays ergotism is of minor importance;
however the problem of mycotoxicoses and long term sub-acute exposure has not faded.
Therefore, regulations have been established in many countries, and reliable testing methodology is needed to implement and enforce the regulatory limits.
So far, several hundred different mycotoxins have been discovered, exhibiting different structural diversity, with various chemical and physicochemical properties, but only a few present significant food safety challenges. Among these are aflatoxins and ochratoxin A (produced by Aspergillus sp.), fumonisins, trichothecenes such as T-2, HT-2 toxins, deoxynivalenol and zearalenone (produced by Fusarium sp.), patulin (produced by Penicillium sp.) and ergot alkaloids (produced by Claviceps sp.) the most frequent occurring mycotoxins with the highest potential to adverse effects in humans and animals.
The work of this thesis can be clustered into three parts as follows:
(I) Method comparison and collaborative trial for the determination of aflatoxin B1in medicinal herbs. This study was initiated upon the request of the European Pharmacopoeia since the regulatory limits for aflatoxin B1 in medicinal herbs were discussed in that moment.
The methodology used has been adopted from existing methods for the determination of aflatoxin B1 in food. The food is extracted with an organic solvent followed by immunoaffinity clean-up and reversed-phase high performance liquid chromatography with fluorescence detection. The aim was to select the most suitable method parameters in order to obtain a method that allows the precise determination of aflatoxin B1 in a variety of medicinal herbs. Therefore acetone-water and methanol-water were tested as extraction solvents. Further, the influence of different post-column derivatisation options with electrochemically generated bromine, photochemical reaction and chemical bromination was compared. In addition, two different calculation modes peak height versus peak area have
been investigated concerning the precision on the evaluation of the rather small peaks that are obtained for aflatoxin B1 at low contamination levels. The different method parameters were applied in the collaborative study to three matrices: senna pods, ginger root and devil’s claw root.
As a result, the method with all tested variations was found to be fit-for-purpose for the determination of aflatoxin B1 in medicinal herbs at levels of 1 µg/kg and above with mean recoveries from 78% - 92% for the extraction with methanol-water and from 98% - 103%
for the extraction with acetone-water. It could be concluded that the tested derivatisation methods had no influence on the analytical result in a range of 1 - 3 µg/kg for aflatoxin B1in medicinal herbs. This is an interesting conclusion as control laboratories often have a preference for one or the other derivatisation method depending on their experience with one or the other system and its availability.
A second method was adopted by single-laboratory validation for the determination of aflatoxin B1 in tiger nuts. The interest on tiger nuts rose on the fact of recent entries in the Rapid Alert System for Food and Feed regarding contamination with aflatoxin B1 in tiger nuts. This system allows the European Commission, EU member states and other associated countries to share information and take immediate action when potentially dangerous food or feed is detected on the market or at the border. Additionally a small survey of aflatoxin B1
content in chufa, which is a tiger nuts based soft drink in Spain, was conducted with the adopted method. The detection limit and the quantification limit were 0.02 µg/kg and 0.06 µg/kg respectively. The mean recovery at a level of 2 µg/kg was 88 % (n = 6) and the coefficient of variation 9 %.
(II) Development and validation of an analytical method for the determination of zearalenone in infant food as well as in animal feed. For the first time the elaboration of a common method for both infant food and animal feed to determine zearalenone was achieved. The main challenge was to allow the determination of zearalenone at rather low concentrations in infant food, while being also able to deal with complex and more challenging matrices such as compound animal feed, due to their abundant interferences compounds. Previous work performed the determination of zearalenone in cereal grains and at higher levels.
The method was validated in an international interlaboratory trial in which laboratories from EU member states, China, Turkey and Uruguay participated.
Method performance parameters for both baby food and animal feed were calculated based on results for spiked samples blind duplicates at two levels and based on results for naturally contaminated samples blind duplicates at three levels.
Test portions of the samples were spiked at levels of 20 µg/kg and 30 µg/kg zearalenone in baby food and at levels of 100 µg/kg and 150 µg/kg zearalenone in animal feed. Mean recoveries from each participant ranged between 91 - 92 % for baby food and between 72 - 75 % for animal feed. The relative standard deviation for repeatability for baby food ranged between 2.8 - 9.0 % and for animal feed between 5.7 - 9.5 %. The relative standard deviation for repeatability for baby food ranged between 8.2 - 13.3 % and for animal feed between 15.5 - 21.4 %. As a result the method showed acceptable within-laboratory and between-laboratory precision for each matrix, as required by European legislation.
Therefore, the newly developed method allows the enforcement of EU legislative limits for zearalenone in foods for infants at 20 µg/kg.
(III) Development and validation of an analytical method for the determination of patulin in juices and purees for infants. The main challenge was to stress the method to determin patulin reliably at levels of 10 µg/kg in products intended for infants and young children. Previously developed methods for patulin were either collaboratively tested at higher levels, indicating that the lower limit for reliable quantification of patulin in such products was around 25 µg/kg or higher, or no validation data except single-laboratory validation were available.
Method development focussed on improved and simplified extraction and clean-up procedures. A single liquid-liquid extraction in the presence of sodium sulfate as water binding agent showed sufficient extraction recovery rates for patulin in combination with a solid-phase extraction method, which trapped interfering substances and allowed the purification of patulin extracts without any pre-conditioning of the SPE cartridge. As a result, purees can be extracted without previous enzymatic treatment, as it is required by other methods that use multiple liquid-liquid extractions. Patulin was well separated from the main interfering compound 5-hydroxymethylfurfural during chromatography when using RP-HPLC columns that allow the separation of rather polar substances with mobile phases of more 99% of water.
Additionally to this method A and due to the large number of laboratories that intended to participate in the validation process, the participants were split into two groups and a second
method B was validated. This method B is a slightly modified version with the same principle as the one previously published by MacDonald et al. in 2000. The main modifications related to the aliquotation. Patulin is extracted three times from the juice or the de-pectinated puree with neat ethyl acetate. The combined ethyl acetate phases were re-extracted with sodium carbonate solution and evaporated. The residue was then re-dissolved in 0.1 % acetic acid solution and separated by HPLC as in method A. Both methods were tested for the determination of patulin in apple juice and fruit puree at the proposed European regulatory limit of 10 µg/kg.
The methods were validated in an international interlaboratory trial in which laboratories from EU Member States, Japan and Brazil participated. Method performance parameters for both apple juice and fruit puree were calculated based on results for spiked samples blind duplicates at two levels and based on results for naturally contaminated samples blind duplicates at three levels for both methods. Test portions of the samples were spiked at levels of 10 µg/kg and 25 µg/kg patulin for both apple juice and fruit puree.
Apple juice mean recoveries from each participant ranged between 37 - 123 % with an average of 73% for method A and between 47 - 124 % with an average of 88 % for method B. Fruit puree recovery values ranged between 60 - 87 % with an average of 70 % for method A and between 37 - 207 % with an average of 110 % for method B. The relative standard deviation for repeatability in apple juice ranged between 8.0 - 14.3 % for method A and between 5.0 - 37.6 % for method B. For fruit puree these values ranged between 3.5 - 7.5 % for method A and between 8.4 - 27.7 % for method B. The relative standard deviation for repeatability in juice ranged between 19.8 - 39.5 % for method A and between 18.2 - 46.9 % for method B. In puree, these values ranged between 12.5 - 27.7 % for method A and between 16.2 - 57.1 % for method B.
In conclusion, the new developed method A showed acceptable within-laboratory and between-laboratory precision for both juice and puree at all levels, while method B only fulfilled partially the performance parameters as required by current EU legislation.
Therefore, the newly developed method allows the enforcement of EU legislative limits for patulin in foods for infants at 10 µg/kg.
Finally the development and in-house validation of a method for determination of patulin using ultra high pressure liquid chromatography in combination with a mass selective detector, resulting in a better chromatographic and analyte selective separation within a shorter time is described. This is of interest especially for projects in which larger amounts of results need to be generated. A survey with more than 200 samples of baby foods, apple purees and tomato purees from the EU food market was performed with this method. It indicated that during the period of 2006 almost all products were free of patulin and all products were compliant with current EU legislation.