Sirhan, Ala’ y. A. (2012) Development and validation of chromatographic methods for the determination of mycotoxins in food / Ala’ Y.A. Sirhan. PhD thesis, University of Malaya.
Abstract
The development and validation of analytical techniques to be applied for the determination of naturally occurring mycotoxins has been the focus of this study. The structure of this work is divided into four main parts, from which independent conclusions are drawn. The first part is in the development of a rapid, reliable and confirmatory method to determine the levels of aflatoxins B1, B2, G1, and G2 in barley, wheat, soybeans and corn. This method is based on a single extraction step followed by liquid chromatography coupled with electrospray ionization quadrupole time of flight mass spectrometry (LC-ESI-QTOF-MS/MS). The sensitivity of the ESI interface was significantly enhanced by optimizing the chromatographic conditions and the fragmentor voltage in the interface. By using the mycotoxin database table, the mycotoxins were confirmed by their retention times, the accurate mass measurements of the TOF analyzer and the products ions, thus avoiding false-positive results. The quantification of the analytes was carried out by performing low-energy collision induced tandem mass spectrometry (CID-MS/MS) using the multiple reaction monitoring (MRM) mode. Secondly, the implementation and validation of the optimized LC-ESI-QTOF-MS/MS method and the development of a new method based on Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) technique for the determination of eight(8) type-A and type-B trichothecenes in cereal samples are carried out. The recovery results showed that the developed QuEChERS method was effective in removing unwanted interfering components without any further clean-up procedure. Therefore, the use of dispersive solid-phase extraction (dSPE) cleanup step was excluded to reduce the time and cost of analysis. The third part is focused on optimizing the sample pretreatment conditions of the developed method and the optimization of the chromatographic conditions of the HPLC-FLD method with postcolumn photochemical derivatization for the quantification of four(4) aflatoxins B1, B2, G1 and G2 in food. The extraction solvent was found to be the most important factor as it significantly affects the extraction efficiency. On the other hand, it was found that the wavelengths setting at 365nm excitation and 440nm emission could be used as the optimum wavelengths for all aflatoxins. The developed QuEChERSHPLC method was then validated and compared with the standard fluorometric determination method. It was found that the fluorometric determination method showed a poorer precision and a positive bias when compared to the QuEChERS-HPLC method. The QuEChERS-HPLC method was then used for the analysis of the selected aflatoxins in a total of 669 domestic and imported food samples in Jordan. Peanut and peanut butter samples showed the highest incidence of contamination (10 contaminations) followed by pistachio nut samples (6 contaminations) and sesame seed samples (2 contaminations). The final component of this study is the implementation and validation of the optimized QuEChERS-HPLC method for the determination of ochratoxin A in cereal samples. Excellent linearity, high recoveries and acceptable precision with the LOQ values, which are lower than the stipulated Maximum Residue Level (MRL), were achieved indicating the suitability of the proposed methods for the determination of mycotoxins in foods could be implemented for routine analysis.
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