Spotlight Food & Beverage Analysis


Mycotoxins are a group of toxic metabolites produced naturally by certain species of fungi. They contaminate food and feed products and pose a potential threat to human and animal health through ingestion. Most Asian and Australasian countries are located in the tropics and subtropics, where there is increased growth of fungi due to the high temperatures and humidity. As a result, these areas face serious mycotoxin contamination issues. In response, regulatory bodies in the region have introduced strict legislation to set mycotoxin limits for foods and feed. However, most mycotoxins are toxic in very low concentrations, requiring sensitive and reliable methods for their detection in order to ensure regulatory compliance.


This article provides a brief overview of current legislation enforced in Asia and Australasia and presents benchtop high- resolution mass spectrometry (MS) as the simplest and most powerful available method for monitoring the presence of mycotoxins in food and feed. Recently collected data demonstrates the benefits of the technology for reliable mycotoxin screening analyses.


Examining the Benefits of High Resolution Mass Spectrometry for High Throughput Screening of Priority Mycotoxins in Food Samples


INTRODUCTION


Mycotoxin-producing organisms can infect and colonise various agricultural crops in the field and during storage. Environmental factors such as temperature and humidity influence the occurrence of these toxins on grains, nuts and other commodities susceptible to mould infestation. In addition, any crop that is stored for more than a few days is a target for mould growth and therefore mycotoxin formation. Mycotoxins present a health risk because most mycotoxins are relatively stable compounds that are not destroyed by food processing such as cooking or freezing. Although the generating organisms might not survive processing, the toxin can still be present.


Recently, mycotoxins have been identified by the World Health Organisation (WHO) as significant sources of food- borne illnesses. They can cause acute health conditions, such as immediate toxic response, immunosupression, necrosis of liver cells, sickness, vomiting and abdominal pain, as well as having chronic teratogenic, estrogenic, hepatotoxic, nephrotoxic and carcinogenic effects. Some of the most dangerous mycotoxin groups are aflatoxins found in cereals and nuts, ochratoxins found in cereals, patulin found in fruits and vegetable and fusarium toxins found in corn and wheat.


“Positive and negative


acquisition can be performed in a single run while also allowing for post acquisition data mining.”


A recently published survey [1] about the occurrence of mycotoxins in Asia, conducted by Biomin GmbH together with Romer laboratories in Singapore, reported that 58% of 960 raw feed samples were contaminated with a type of mycotoxins known as fumonisins, produced by several species of fusarium moulds. Reports such as this highlight the need to safeguard human and animal health from mycotoxin contamination. In order to ensure that mycotoxins do not contaminate the food supply, legislation has been introduced across a number of countries worldwide, including in Asia and Australasia.


LEGISLATIVE FRAMEWORK


An international inquiry [2] on mycotoxins, initiated by the Dutch National Institute for Public Health and the Environment in 2002, revealed that a total of 26 countries in Asia and Australasia enforce specific mycotoxin regulations.


Legislation for total aflatoxin compounds is more prevalent for food, whereas regulations for the specific aflatoxin B1 dominate for feed analysis. According to the findings of the inquiry, Australia and New Zealand have harmonised mycotoxin regulations with common limits being applied for total aflatoxins in foods such as peanuts and tree nuts. In addition, the harmonised regulations include limits for another type of mycotoxin, phomopsin, in products derived from lupin seed products as well as for food products containing agaric acid such as those derived from mushrooms and alcoholic beverages.


Author Details:


Michal Godula, Food Safety Group, Thermo Fisher Scientific, Czech Republic


Milena Zachariášová and Jana Hajšlová, Institute of Chemical Technology Prague, Department of Food Chemistry and Analysis, Czech Republic


This inquiry further revealed that China and the Islamic Republic of Iran have the most extensive and detailed regulations for acceptable mycotoxin levels. In addition, most of the member countries of the Association of Southeast Asian Nations (ASEAN) have now set specific regulations for mycotoxins. While harmonised regulations are not yet established by ASEAN, an ASEAN Task Force on Codex Alimentarius has taken a common position to support a unified level of 0.5mg/kg for aflatoxin M1 in milk.


In order to comply with the different regulations for acceptable levels of various types of mycotoxins, it is


essential that the food and feed safety industries in Asia and Australasia have access to an analytical method that can efficiently screen and accurately reveal adulteration of products. The chosen analytical technique will play a key role in the typical mycotoxin method workflow.


TYPICAL MYCOTOXIN METHOD WORKFLOW


The typical method that most laboratories use to perform mycotoxin screening analyses consists of four main steps. The first step is the extraction of mycotoxins from a food sample using different approaches. The most common approach involves using a mixture of acetonitrile and water, with a ratio of 80-20%. Following extraction and prior to liquid chromatography/mass spectrometry (LC/MS) analysis, it is typically necessary to carry out a clean up of the sample, which involves the separation of mycotoxins from the matrix that could later interfere with the final determination. This is usually done using immunoaffinity clean-up columns. These columns specifically attract mycotoxins while all the other compounds are flushed away. Mycotoxins that are retained in the columns are eluted after the matrix has been removed.


For HPLC or UHPLC analysis of mycotoxins with conventional detectors like UV or FL typically a derivatisation step has to be performed. This is of particular importance for some mycotoxins, such as fumonisins, trichothecenes and aflatoxins B1 and G1. The final step is detection. This can be done using a variety of methods, however since most mycotoxins are toxic in very low concentrations, a sensitive and reliable detection method is required.


TRADITIONAL ANALYTICAL APPROACHES


A wide range of analytical tools exist for the food and feed safety industry, but the real challenge is finding a technique that can efficiently screen and accurately reveal adulteration. Liquid chromatography (LC) with fluorescent detection (FLD) has been efficiently used for more than two decades, and in the last five years LC has been increasingly coupled with MS/MS. Quick screening methods based on immunoassay analysis, such as ELISA, are also very popular. Immunoassays are simple and rapid, but they lack sensitivity compared to chromatographic methods and may lead to false negatives. In addition, cross-reactivity of compounds can result in false positives.


The last few years have seen a growing trend towards full scan MS experiments in screening analysis compared to the target LC/MS/MS analysis. Such approaches are performed using high performance time-of-flight (TOF) instruments, with typical mass accuracies of 5 ppm and resolving power of a maximum of 15,000 FWHM, coupled to U-HPLC. However, in complex matrices such as food and feed, this rather limited mass resolving power leads to the risk of inaccurate mass measurements caused by unresolved background matrix interferences [1, 2].


In response, latest technological advancements have focused on the development of an innovative high resolution MS technique using a benchtop LC/MS system capable of achieving routine ultra-high mass accuracy below 2 ppm and mass resolving power of up to 100,000 FWHM.


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