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Enhancing the analysis of mycotoxins in food and feed using SPE

16 January 2015

The globalisation of the food chain has meant that the focus on food safety is greater than ever before and as a result, food manufacturers are facing increasingly stringent regulatory requirements. 

One area that is receiving a growing amount of attention is mycotoxin intake from contaminated food and feed which causes mycotoxin intoxication.

Mycotoxins are secondary fungal metabolites that are typically produced by fungi growing on grains and fruit during shipment or storage. Because mycotoxins are small, heat-stable molecules that can permeate the host, cleaning or heating foods may remove the fungi itself but the mycotoxins are left behind. To ensure against contamination of the food chain, fast and cost-effective sample preparation is essential for the analysis of mycotoxins.

DANGERS OF MYCOTOXINS IN THE FOOD CHAIN
It has been estimated that a quarter of the world’s crops are affected by mould or fungal growth, which means that the threat of the presence of mycotoxins in the food chain is a significant one. In addition, modern global food logistics often leads to crops being stored in conditions during transportation that promote the growth of fungi, which can lead to mycotoxin contamination.

The toxic effects of Fusarium toxins on humans and animals are well documented. Some mycotoxins, such as aflatoxins, are known carcinogens; therefore, their presence renders food unfit for human consumption. When ingested, mycotoxins may cause acute or chronic disease and it has been shown that repeated exposure to low level mycotoxin ingestion can affect growth and development and cause immunosuppression.

Since the discovery of aflatoxins in the 1960s, regulations have been established worldwide to protect consumers from the harmful effects of mycotoxins. For example the European Union legislation regarding mycotoxins determination includes specific maximum levels for certain mycotoxins in individual foodstuffs. The mycotoxins for which there are currently specific maximum levels include aflatoxins, ochratoxin A, patulin and the Fusarium toxins including Deoxynivalenol (DON), zearalenone and fumonisins. Both European and US governmental agencies require the results of mycotoxin analysis to be reported to ensure they do not exceed acceptable levels. Analysts therefore need a method for analysis that delivers accurate results and sufficient resolution.

LIMITATIONS OF EXISTING METHODS FOR THE ANALYSIS OF MYCOTOXINS
LC/MS/MS has become the method of choice for detecting and confirming these toxic compounds and efficient sample preparation is essential to the accurate analysis of mycotoxins.

Traditional sample preparation for mycotoxin analysis typically involves extraction with acetonitrile/water and clean-up via charcoal-alumina columns. As the contaminants can differ considerably in polarity and solubility, recoveries of the more polar analytes can be compromised using this approach. Another technique is the use of immunoaffinity columns (IAC). While these columns provide highly selective extractions with high recoveries, separate IAC columns are required for each toxin, which adds additional costs to the analysis.

In addition, the MS methods that used for the analysis of mycotoxins are expensive to set up and so the more cost effective the sample preparation, the better. To overcome the limitations of these existing sample preparation methods, extraction and clean-up methods can now be used for the simultaneous determination of several mycotoxins with high recoveries by minimising the matrix effects.

BENEFITS OF SOLID PHASE EXTRACTION (SPE) FOR THE ANALYSIS OF MYCOTOXINS
The clean-up process of mycotoxins can be optimized using Agilent Bond Elut Mycotoxin, a solid phase extraction (SPE) sorbent which cleans up food extracts for improved trichothecene and zearalenone analysis.  The sorbent is a proprietary silica-based ion exchange material and the Bond Elut Mycotoxin method for extraction and clean up is successful with a variety of food and grain sample types including wheat, corn, durum, oats and bread.  The method is easy to use because the toxin analytes pass through the cartridge while the food matrix components are retained.

SPE is a reliable technique for sample preparation and is best employed when high selectivity and sensitivity are required, such as when working with food matrices. The benefits of employing the Bond Elut solution for the analysis of mycotoxins include:

  • Faster flow rates
  • Excellent cartridge-to-cartridge reproducibility
  • Reduced sample preparation time
  • Less sample loss
  • More reliable data
  • Greater stability
DON is the most commonly detected Fusarium mycotoxin and is produced by Fusarium fungi on corn and wheat prior to harvest as well as during storage. DON has been associated with reduced milk production in dairy cattle and vomiting in swine that consume contaminated feed or their refusal to eat feed containing the toxin. DON intake from animals also poses a risk to human health since animal products consumed by people may contain toxin residues. The EU recommends guidance values for DON between 0.9 and 12 mg/kg depending on the feed and animal. Suitably fast, sensitive and reliable methods for the determination of Fusarium toxins are therefore required.

When using the Agilent Bond Elut Mycotoxin method, the clean up time for DON is almost half compared to IAC clean up, thus increasing sample throughput. The clean extracts minimize the matrix effects and show high recoveries in LC/MS/MS analysis. As the performance of the SPE cartridge is similar to IAC and the columns are less expensive, the new clean up procedure is a good alternative to IAC methods commonly used.

In addition, typical clean-up methods of trichothecenes, important mycotoxins produced primarily by the genus fusarium from cereals and cereal based foods, use commercially available polar clean up columns. Substances that interfere with the detection of the mycotoxins are retained while trichothsecenes are not. However, this purification method gives low recoveries for the polar toxins: NIV, T-2 tetraol and DON. As shown in the application note, Improved Isolation and Analysis of Mycotoxins from Cereals, Beer and Wine the Bond Elut Mycotoxin SPE cartridge provides a reliable and cost-efficient clean up method for the determination by triple quadrupole LC-MS-MS of 12 type A- and B- trichothecenes in cereals and cereal based foods.

Mycotoxins are an increasingly hot topic within the industry and analysts can now benefit from a functionalised sorbet to optimize their analysis saving both valuable time and money. Bond Elut Mycotoxin has been shown to deliver higher sensitivity and clean up than traditional charcoal alumina fractionation and much faster workflow than immunoaffinity columns. Don’t forget to download the Agilent Mycotoxin Applications Guide which provides a wealth of data to reference for mycotoxin analysis.


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