Rapid, Robust and Reliable Mycotoxins Analysis

Introduction

Fungal toxins known as aflatoxins pose a substantial risk to human health, particularly in some developing nations where there is a high prevalence of aflatoxin-related health consequences and significant food contamination. There are numerous methods available for testing of food mycotoxins. Although expensive, challenging to operate, and sensitive, modern chromatographic techniques enable for quantitative measurement with great accuracy and sensitivity. Rapid tests offer a more affordable option than screening several samples at once, but they still require validation on all food matrices that are evaluated. Making sure there are appropriate detection and quantification technologies that are quick, sensitive, accurate, reliable, and affordable for food surveillance in resource-constrained contexts is crucial to combating aflatoxin contamination and exposure. According to recent estimates, 60-80% of crops contain measurable levels of mycotoxins. Co-contamination with more than one toxin occurs frequently, and this varies geographically depending on climate and farming practices.

Techniques for general mycotoxin analysis

The analysis of mycotoxins is extremely difficult. They are heterogeneously distributed at various quantities in a variety of agricultural commodities, foods, feeds, and biological samples, as well as including a variety of chemical components, necessitating extraction, cleanup, separation, and detection techniques. As a result of plant metabolism, some mycotoxins, particularly deoxynivalenol and zearalenone, are conjugated, and these "masked" mycotoxins may contribute 20% of the total amount of the parent mycotoxin but are undetectable by standard examination [1]

Quantification of mycotoxins necessitates the use of costly laboratory equipment that must be operated by highly trained individuals, as well as a sequence of stages and procedures that can be difficult and time-consuming. The need for high sensitivity tests to detect the lowest possible levels of mycotoxin for regulatory purposes, combined with rapidity, high accuracy, simplicity, robustness, and selectivity, has been the primary driving force behind the development and improvement of new mycotoxin analytical protocols. Mycotoxin analysis is required to quantify the toxin for risk assessment, diagnosis, and mitigation techniques.

Aflatoxin testing in food items is particularly difficult due to uneven toxin distribution and the low levels at which mycotoxins exist [1]. As a result, some national and international food safety authorities and organisations have prescribed sampling methods for a variety of food commodities to obtain representative samples that can be used to determine concentrations of various mycotoxins in foodstuffs for official control purposes; sampling is potentially the most significant source of error in mycotoxin testing [2]. Many commodities have thorough sampling plans in place. To produce a representative sample from a grain storage facility, for example, incremental samples must be collected from various locations across the facility [3], with the entire primary sample pulverised, blended, and subsampled to assure consistency.

Mycotoxins are extracted from the matrix using a suitable solvent, cleaned of co-extracted matrix components, and identified/quantified using appropriate analytical facilities. Some unique approaches, such as infrared spectroscopy, may detect mycotoxin contamination directly in ground samples without prior solvent extraction or clean up, but are limited to screening applications due to significant matrix interference and a lack of acceptable calibration materials. Although additional purification is required for chromatographic determination, the diluted extracts can be employed directly with immunoanalytical procedures.

Determination of toxins

Conventional analytical methods for mycotoxin analysis in food typically involve chromatographic separation techniques such as liquid chromatography (LC), thin layer chromatography (TLC), and gas chromatography (GC). High-performance liquid chromatography (HPLC) is commonly used, often combined with immunoaffinity cleanup, to quantitatively determine regulated mycotoxins. Detection systems such as fluorescence detection (FLD) or mass spectrometry (MS) are frequently employed for enhanced sensitivity and selectivity. These methods help ensure the safety and quality of food by identifying and measuring mycotoxins present in the samples.

1. Thin layer chromatography

Earlier, thin layer chromatography (TLC) was commonly used as a mycotoxin screening technique due to its affordability and ability to process many samples quickly. However, TLC has limitations in terms of separating power, which makes it difficult to distinguish between the mycotoxin of interest and other interfering substances present in the sample. To address this issue, modern cleanup techniques have been developed that effectively remove impurities, thereby enhancing the reliability and accuracy of TLC analysis. These advancements have helped overcome the limitations of TLC and improved its usefulness in mycotoxin analysis.

2. High performance liquid chromatography

Currently, the most used method for mycotoxin determination is high-performance liquid chromatography (HPLC) due to its advantages in sensitivity, precision, and automation. After extracting and cleaning up the samples, they are injected into the HPLC column. In this technique, individual mycotoxin compounds are separated based on their interaction with the column matrix and the solvent used in the mobile phase [4].

For better quantification of mycotoxins using the HPLC technique coupled with fluorescence detection (HPLC-FLD), derivatization is important. Derivatization can enhance the fluorescence signal, making it easier to quantify mycotoxins accurately. Different methods of derivatization can be employed, such as pre-column derivatization with trifluoroacetic acid (TFA) or post-column derivatization with bromine or iodine, which can be used to identify aflatoxins [5]. There are also alternative approaches like photochemical post-column derivatization or the incorporation of specific cyclodextrins in the mobile phase to enhance fluorescence without the use of chemical derivatization [6].

While HPLC-FLD offers good sensitivity and specificity in mycotoxin analysis, it does have limitations. It requires expensive equipment and skilled operators to perform the analysis. Additionally, the sample preparation procedures can be time-consuming and laborious.

3. Liquid chromatography /Mass spectrometry

Liquid chromatography with mass spectrometry (LC/MS) is a technique that allows for more sensitive and selective determination of multiple mycotoxins in complex matrices, with improved limits of detection and quantification. Modern LC/MS instruments use interfaces like atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI), and electrospray ionization (ESI) due to their robustness, ease of handling, high sensitivity, accuracy, and ability to work with a wide range of compound polarities.

4. Gas Chromatography

Gas chromatography (GC) is a method used to determine mycotoxins that can easily turn into gas inside the chromatography column. For example, GC combined with electron capture detection (ECD), flame ionization detection (FID), or mass spectrometric detection (MS) can be used to identify mycotoxins like trichothecenes or patulin. However, GC requires the samples to be cleaned up before analysis and treated with chemicals to make them more volatile and sensitive. GC has some disadvantages too. The sample needs to be in a gas form or converted into a gas, there can be losses due to heat, and the equipment for GC is expensive.

5. Rapid screening methods

Quick screening methods, such as immunochemical techniques, offer rapid detection of mycotoxins. These methods range from simple tests like lateral flow assays and enzyme-linked immunosorbent assays (ELISA) to advanced immunosensors. They work by binding a specific antibody to the target mycotoxin, without the need for extensive sample cleanup or enrichment steps.

• ELISA techniques

The enzyme-linked immunosorbent assay (ELISA) technique uses specific antibodies to detect and bind the target molecule. In ELISA, the target molecule can directly bind to the antibody or be linked to an enzyme, which then reacts with a coloured substance to produce a visible result.

However, mycotoxins have a small size and are not easily detected by antibodies alone. To make them detectable in ELISA, they are attached to a carrier molecule to make them more noticeable. ELISA is known for being highly sensitive, accurate, portable, quick, and easy to use, making it suitable for testing many samples efficiently.

Despite its advantages, ELISA also has some limitations. It often requires single-use kits, which can be expensive for large-scale testing. The results of ELISA can be influenced by the composition of the sample being tested, and there can be issues with false positive reactions with other substances. Moreover, ELISA has a limited range of detection due to the specific nature of the antibodies used in the test.

5.2 Lateral Flow devices

Lateral flow strips and dipstick devices, which are also known as immunochromatographic test devices, are simple and disposable tools used to detect mycotoxins. These devices have a toxin or antibody attached to them, which can be labelled with enzymes, liposomes, or colloidal gold. Colloidal gold is commonly used in mycotoxin test strips because it is easily available, simple to produce, and can be easily combined with antibodies.

In these devices, the mycotoxin in the sample interacts with the attached antibodies, which are labelled with colloidal gold, at the base of the strip. The antibodies, whether bound to the mycotoxin or not, move along the strip membrane. As they move, they pass a test line that contains immobilized mycotoxin. If there are any free antibodies, they will bind to the mycotoxin on the test line, forming a visible line that indicates the presence of aflatoxin below a certain limit. The device also has a control line further along the strip, which consists of anti-antibodies. This control line ensures that the sample has moved completely along the strip.

There is another type of device called a membrane-based flow-through device or enzyme-linked immunofiltration assay (ELIFA). In this device, the liquid flows through the membrane in a perpendicular direction and is collected on an absorbent pad on the other side of the membrane. It uses an enzyme label that requires a step of incubating the sample with a substrate. The test and control lines are generated by a colour reaction between the enzyme and the substrate.

Due to their simplicity and ease of use, the development of dipstick and lateral flow assays for mycotoxins is likely to continue. Researchers are exploring the use of stable, non-enzymatic labels in these assays, and there are already several commercially available devices. Additionally, innovative labels based on nanoparticles, such as quantum dots, gold nanoparticles, magnetic nanoparticles, carbon nanoparticles, and time-resolved fluorescent microspheres, have been developed to improve the detection capabilities of lateral flow devices. The use of fluorescence quenching principles in lateral flow immunoassays has also increased the sensitivity of these assays.

• Mycomaster -Trouw Nutrition

Trouw Nutrition has developed a special program that helps feed producers manage the risk of mycotoxin contamination. This program uses a 3-step approach: identifying the risk, ensuring quality control, and applying effective solutions.

• First, the program helps identify the risk of mycotoxin contamination by regularly testing and analysing the feed ingredients. This helps farmers and feed producers understand the level of risk and take necessary measures.
• Next the program focuses on quality control. It ensures that the raw materials used for making feed and the final feed product meet high-quality standards. This involves thorough testing and analysis to detect any mycotoxin presence and taking steps to prevent further contamination.
• Trouw Nutrition provides solutions to mitigate the risk of mycotoxin contamination. These solutions may include special additives or treatments that can neutralize or bind mycotoxins, making them less harmful.

Feed producers can reduce the risk of mycotoxin contamination, maintain the quality of their feed, and protect the health and performance of animals. It is an important step towards ensuring safe and high-quality animal feed production.

Trouw Nutrition has developed a rapid analysis tool called Mycomaster. This device helps farmers to find out if their animal feed contains harmful substances called mycotoxins. Mycomaster is a smart device that uses a simple method called lateral-flow technology to measure the levels of mycotoxin contamination in the feed. Farmers can check for six different types of mycotoxins with Mycomaster: Zearalenone, Deoxynivalenol, Aflatoxins, Fumonisins, Ochratoxin, and T2-HT2. The device gives results in just 15-30 minutes, so farmers can quickly know if their feed is contaminated.

Mycomaster can also connect to Trouw Nutrition's global data system. This means that farmers can see information from around the world about mycotoxin contamination. It helps them understand the situation fast, better and make necessary mitigation strategy. 

Conclusion

It is important to ensure that our food is safe from contamination by aflatoxins and other mycotoxins. Analysing and quantifying these toxins is crucial for feed-to-food safety. There are various methods available for detecting and measuring aflatoxins, each with its own advantages and disadvantages.

Analytical techniques like HPLC coupled with mass spectrometry or fluorescent detectors are accurate but expensive and require trained personnel. TLC is a simpler and more affordable option, especially in developing countries. Screening methods, such as ELISAs and dipstick tests, are rapid and easy to use, making them suitable for low-income countries. However, it is important to ensure that the chosen method is appropriate for the specific food item being tested.

Efforts are being made to develop multi-toxin screening assays, as aflatoxins are often found together with other mycotoxins. This helps us understand the extent of contamination and take necessary measures to protect public health. It is also important to ensure that the results obtained from rapid screening tests align with quantitative analysis conducted in regulatory laboratories.

By employing effective analytical methods and screening techniques, we can identify and address mycotoxin contamination in our food supply chain, ensuring the safety of our food and protecting the health of consumers. 

References

1) Bryden WL. Mycotoxin contamination of the feed supply chain: Implications for animal productivity and feed security. Animal Feed Science and Technology. 2012;173:134-158

2) Whitaker TB. Sampling for mycotoxins. In: Magan N, Olsen M, editors. Mycotoxins in Food. UK: Woodhead Publishing; 2004. pp. 69-87

3) Whitaker TB. Sampling foods for mycotoxins. Food Additives and Contaminants. 2006;23:50-61. DOI: 10.1080/02652030500241587

4) Yao H, Hruska Z, Mavungu DJ. Developments in detection and determination of aflatoxins. World Mycotoxin Journal. 2015;8(2):181-191

5) Pascale M, Visconti A. Overview of detection methods for mycotoxins. In: Leslie JF, Bandyopadhyay R, Visconti A, editors. Mycotoxins Detection Methods, Management, Public Health and Agricultural Trade. Cambridge (MA): CABI International; 2008. pp. 171-183

6) Shephard GS. Aflatoxin analysis at the beginning of the twenty-first century: A review. Analytical Bioanalytical Chemistry. 2009;395(5):1215-1224

7) Turner NW, Subrahmanyam S, Piletsky SA. Analytical methods for determination of mycotoxins: A review. Analytica Chimica Acta. 2009;632(2):168-180

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