Nuts About Christmas … And Mycotoxin Detection

By December 21, 2020


A bowl of nuts, with polished or carved nutcrackers, is part of many traditional winter holiday feasts. Growing up in England in the 1970s, I enjoyed hazelnuts, almonds, walnuts and Brazil nuts as special treats for the Christmas holiday. Once I’d wrestled with a nutcracker, seemingly designed to send nuts and shards of shell across the room, I recall noticing that some of those nuts just didn’t seem “right,” covered in a powdery coating and with a distinctively sharp mold smell. Today, I recognize those signs as indication of fungal infection and I am thankful for the advanced liquid chromatography and mass spectrometry technologies available for the detection of mycotoxin contamination that enable future generations, grappling with the dreaded nutcracker, to remain healthy and enjoy the holiday festivities.

Nuts and food safety: The not-so-sweet smell of spoilage and mycotoxins

Tree nuts are rich in proteins and unsaturated fat, and contain low amounts of water, which restrict spoilage by microorganisms, but can support mold growth, some of which can produce mycotoxins. Mycotoxins are secondary metabolites of filamentous fungi that can occur in food and agricultural products via many contamination pathways, including production, processing, transport and storage.

Fungal growth and mycotoxin production depend on biological (susceptible crop) and environmental factors, with the emphasis on regional climatic conditions during plant development and crop harvest. Consumption of nut fruits is one of the most important contributors to human exposure to mycotoxins. Mycotoxins are responsible for significant losses in revenue and the potential erosion of brand and reputation.

The most common mycotoxins are regulated in many countries of the world after thorough risk assessment, considering toxicity, occurrence, and consumption data as well as economic and political considerations. Regulations typically include values that are specified in various food and feed commodities, which are either maximum allowed levels or guidance values. The applied regulatory levels and standards of mycotoxins vary in the different regions of the world.1 The maximum levels for mycotoxins in food are very low due to their severe toxicity.

Increased food safety through the prevention and monitoring of mycotoxin occurrence

Food safety is addressed through the analysis, control, and monitoring of physical, chemical, and biological hazards, from raw material manufacturing, supply, and handling, to production, distribution, and consumption of the finished product. Implementation of a Hazard Analysis and Critical Control Point (HACCP) food management system by food business operators, including analytical testing, is an effective strategy for prevention, control, and periodic monitoring of mycotoxin in all stages from field to the consumer.

The mycotoxins most frequently found in nuts are aflatoxins, which are a group of related bisfuranocoumarin compounds produced by the Aspergillus fungi. A variety of testing solutions exists for determination of these mycotoxins, ranging from easy to use, rapid tests, which can be used at the point of production, to lab-based reference methods that are more time-consuming, but can be used to provide a more comprehensive view of the level of contamination.

Nut mycotoxins analysis is a challenging task, due to the

  • Complexity of the matrix (high lipid content),
  • Low concentrations in which these compounds are usually present, and
  • Strict regulatory limits.

VICAM’s portfolio of quantitative strip tests provide validated monitoring solutions for companies seeking to minimize the costly consequences of mycotoxin contamination in a wide range of agricultural products and feedstuffs.

Lateral flow devices are the most economical choice for situations where immediate, onsite decision support is critical, including determining the acceptability of shipments at buying and distribution points and routine quality control checks at points during production of dried fruits.  These tests have limited analytical scope, as they are designed to detect and sometime quantify a specific key mycotoxin of interest and they provide an ideal option for focused analytical testing of tree nuts.

The Afla-V strip tests utilize the proven sensitivity and selectivity of VICAM’s monoclonal antibodies to accurately detect and measure total aflatoxins B1, B2, G1, and G2 at levels as low as 2 ppb and as high as 100 ppb. The single dilution sample preparation procedure saves time and materials, and the test takes 5 minutes to develop. Digital readings are clearly displayed on the screen of the Vertu Lateral Flow Reader, eliminating any guesswork about the results.

Laboratory-based methods for the quantitative determination of mycotoxins are typically based on high-performance liquid chromatography (HPLC) with different detection systems.

The stringent regulatory limits for aflatoxins make it difficult to determine them at the required limits with a UV detector so fluorescence detection (FLD) is preferred. The combination of clean-up using immunoaffinity columns (IAC), based on antibodies, and analysis by HPLC-FLD has been a pre-requisite step to achieve the desired sensitivity and selectivity to be successfully used to check compliance with the strict regulatory maximum limits for aflatoxins and ochratoxins.

Due to their high specificity, immunoaffinity columns produce cleaner extracts when compared to less selective SPE sorbent materials and so are less susceptible to interference from co-extractives. The AflaTest series of IAC columns produce accurate numerical results for the aflatoxins B1, B2, G1, G2, M1, and M2 in a variety of commodities. The procedure can be performed in less than 15 minutes (excluding sample preparations and extraction) and requires only basic HPLC skills. The use of a large volume flow cell within the Waters ACQUITY Fluorescence Detector has nullified the need for any post-column derivatization and provides the necessary low limits of quantification for aflatoxins.  The improvements in chromatographic efficiency offered by using columns with sub 2 µm porous particles (e.g. Waters ACQUITY UPLC columns) or equivalent fused core/ superficially porous columns (e.g. Waters CORTECS 2.7 µm columns), significantly improves sensitivity, resolution and speed; efficient separation for aflatoxins has been achieved in 5 minutes.

Over the last two decades, liquid chromatography with (tandem) mass spectrometry (LC-MS(/MS) has become the most frequently used laboratory-based technology for the determination of mycotoxins, often within a multi-analyte method.

For specific, single analyte analysis, such as aflatoxins, immunoaffinity clean-up using AflaTest columns are attractive options, especially in complex commodities such as tree nuts, even when using mass spectrometry. However, when such toxins are included in a multi-analyte method, specific IAC options are no longer viable. The combination the multifunctional Myco6in1+ column with LC-MS/MS, offers an attractive, cost-effective option for the simultaneous determination of different groups of mycotoxins, including aflatoxins.

Although the additional selectivity from MS/MS has proven a more popular platform than MS only, this kind of multifunctional IAC clean-up remains useful, especially for legacy LC-MS/MS instruments and for more complex samples or when samples are from a wide range of different commodities. Approaches for the determination of multiple mycotoxins in a single method require generic extraction conditions to ensure recovery of the different types of mycotoxin. Due to the sensitivity and selectivity of LC-MS/MS, simple dilution of the extract, typically without clean-up, is often preferred.  Matrix-matched calibration standards and stable isotope analogues are recommended for accurate quantitation. Many laboratories follow or modify the procedure developed and validated by Kryska’s laboratory2 using extraction solvent with a low water content and low pH, or a modified version of QuEChERS3, coupled with LC-MS/MS using ACQUITY UPLC systems and Xevo series mass spectrometers.

Learn more about the determination of mycotoxins:

 

References:

  1. Agriopoulou S et al. (2020). Advances in Occurrence, Importance, and Mycotoxin Control Strategies: Prevention and Detoxification in Foods. Foods 9(2):137. https://doi.org/10.3390/foods9020137
  2. Sulyok et al. (2006). Development and validation of a liquid chromatography/tandem mass spectrometric method for the determination of 39 mycotoxins in wheat and maize. Rapid Commun. Mass Spectrom. 20(18):2649-2659. https://doi.org/10.1002/rcm.2640
  3. Kafouris, D et al. (2017) A validated UPLC-MS/MS multi-mycotoxin method for nuts and cereals: results of the official control in Cyprus within the EU requirements, Food Agr. Immunol. 28(1): 90-108. https://doi.org/10.1080/09540105.2016.1228834