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Development of an appropriate method for the determination of multiple mycotoxins in pork processed products
Thesis Abstract:
Mycotoxins, secondary metabolites produced by mycotoxigenic fungi, are common contaminants worldwide and pose substantial health risks to human beings and animals. Due to their stable physical and chemical characteristics, common food processing methods hardly clear these compounds. Additionally, mycotoxins have been found in various processed foods, including pork products, highlighting their widespread presence. Pork products are among the most consumed meat products in Taiwan, as recorded in the database of the Nutrition and Health Survey in Taiwan (NAHSIT). In the Philippines, pork is also one of the primary meats for production and is widely consumed by Filipinos, according to Philippine Statistics. There is no established method for detecting multiple mycotoxins, specifically in processed meat products in these countries. Thus, this study aimed to develop an appropriate method for determining 8 mycotoxins, namely, AFM1, AFG2, AFG1, AFB2, AFB1, CIT, ZEN, and OTA, in processed pork products. The research initially optimized the mobile phases for chromatographic separation of 8 mycotoxins using 50% acetonitrile and 50% ddH2O, adjusting the formic acid concentration and adding 2% formic acid produced clear, distinguishable peaks, particularly for CIT, ZEN, and OTA. The QuEChERS extraction method was then optimized through experiments to test recovery rates to meet regulatory standards. Six solvent and powder combinations were evaluated, with 80% acetonitrile, 19% ddH2O, 1% acetic acid, and 4g MgSO4 achieving the highest recovery rates. The optimal sample mixing time was 5 min, and sonication was 40 min. The time spent on the QuEChERS extraction method was approximately 2-3 h, including air-drying the samples with nitrogen gas. Results also showed that n-hexane washing significantly improved sensitivity, particularly for ZEN (110.10 µg/kg to 29.37 µg/kg) and CIT (31.4 µg/kg to 8.65 µg/kg). The limits of detection for AFMI (1.37 µg/kg to 1.19 µg/kg) and OTA (1.04 µg/kg to 0.34 µg/kg). Recovery rates for the 7 target mycotoxins showed no significant difference, except for AFMl, which dropped from 104.94% to 83.82%. Regardless of washing, recovery rates of above 8 target mycotoxins remained within acceptable limits set by regulatory agencies. The results showed that 50% methanol had the highest recovery rates and was the best for recovering mycotoxins, with no over- or under-recovery issues. The QuEChERS method was validated for linearity, detection quantification limits, and quantification limits were successfully applied to commercial products (pork jerky and meat floss). This study developed a method to detect and quantify 8 mycotoxins (AFM1, AFG2, AFG1, AFB2, AFB1, CIT, ZEN, and OTA) simultaneously in pork products using optimized QuEChERS extraction combined with UHPLC-TCFLD. This approach is more time-efficient and practical compared to immunoaffinity columns. By using our established detection method, 8 target mycotoxins can be quantified simultaneously within approximately 30 min, and the entire process, including washing and extraction, takes only about 2-3 h. Additionally, our method is more cost-effective compared to HPLC-MS/MS. This detection method can also be used for analyzing actual samples instead of the official detection methods currently in use.