The right mycotoxin testing method for you

A variety of testing solutions exists for mycotoxin analysis, ranging from rapid tests that are easy to conduct, to reference methods that are more time-consuming but yield more detailed results. In today’s technologically fast-moving world, ELISA, Lateral Flow Test, HPLC and LC/MS-MS are the most common testing methods.

When to choose what

In general, testing of raw feed ingredients for a few of the main mycotoxins can be done with on-site rapid tests. For compound feed, a broader range of analytes or fulfilling legal requirements, then an analytical service tends to be the best fit. However, the distinction is not binary. In some cases, rapid tests may deliver inconclusive results though symptoms of mycotoxin ingestion in animals can still be observed. If so, it is recommended to conduct further testing using state-of-the-art LC-MS/MS to get a clearer picture of a broader range of potential culprits e.g. masked mycotoxins not detectable by conventional methods or emerging mycotoxins that are less well understood and not yet regulated.

Lateral Flow Test

Lateral flow devices (LFD) allow for a rapid on-site analysis of a wide range of feed ingredient samples and deliver results in under ten minutes. Speed here is the clear advantage, though the scope of LFD is restricted to testing raw materials for aflatoxins (Afla), deoxynivalenol (DON), zearalenone (ZEN) and fumonisins (FUM). While conventional LFD kits use an organic solvent, more environmentally-friendly kits that use water instead offer you a way to avoid the expense and hassle of managing chemical inventory and disposal. The main advantages of lateral flow devices are that they are very fast and inexpensive, they don’t need special equipment and on-field testing is possible. Some disadvantages include that only raw materials can be tested, only some of the main mycotoxins can be detected (Afla, DON, Zen and FUM). Another disadvantage is that other substances are present in the solution that can alter results (matrix interferences).

ELISA

ELISA (enzyme-linked immunosorbent assays) test kits are accurate and reliable. Up to 6 mycotoxins can be analysed from 1 extraction. ELISA test kits are the ideal solution for a parallel measurement of multiple samples with incubation times of as low as 15 minutes for up to 42 samples. The main advantages of ELISA are that they are fast, inexpensive and good to get a first estimation. Some drawbacks include that only raw materials and the main mycotoxins can be detected. Another disadvantage is maintaining the cost effectiveness as it requires a minimum of 30 samples tested per ELISA set.

HPLC

The method relies on pumps that circulate a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material. Different components in the sample, e.g. mycotoxins, interact with the adsorbent material in different ways, therefore crossing the column at different rates and allowing a separation as they flow out of the column. Afterwards, a detector quantifies the mycotoxins by comparing their signal to the signal of selected standards. A variety of detectors are available such as spectrophotometric detectors (UV-VIS, diode array), refractometers (RI), fluorescence detectors (FLD), and mass spectrometers (MS). The main advantages of High Performance Liquid Chromatography (HPLC) are the high sensitivity, only small amounts of samples are need, it is applicable to complex matrixes and it is reliable and highly accurate. The drawbacks include – these tests are time consuming, expensive, compounds must have UV absorption or fluorescence properties and highly skilled technicians are needed to carry out the analysis.

LC-MS

This technique combines the physical separation proprieties of the HPLC with the mass analysis capabilities of the mass spectrometer (MS). The two analytical methods work synergistically. Chromatography separates mixtures with multiple components (e.g. mycotoxins), before the mass spectrometer then provides the structural features of individual components with high sensitivity and specificity. The most common variations of the method are either liquid chromatography coupled to mass spectrometry (LC-MS) or tandem mass spectrometry (LC-MS/MS). Due to the extreme sensitivity, the latter method is the reference method of choice in many laboratories and it currently represents state-of-the-art of analytical chemistry. The technology lends itself to a variety of applications based on the number of target analytes, including: an overview of all regulated metabolites in fulfilment of legal requirements, detection of most common masked and emerging mycotoxins and identification of the full toxic load: mycotoxins, phytoestrogens, pesticides and veterinary drugs. The advantages of LC-MS are the low detection limits, it generates structural information, minimal sample treatment is required, it can cover a wide range of analyses and it is applicable to complex matrices. However, these tests are time consuming, expensive and highly skilled technicians are needed to carry out the analysis.

NIR

Near Infrared Spectrometry (NIR) measures the interaction between infrared electromagnetic radiation and chemical bonds. By analysing the radiation reflected or transmitted by the sample, one can determine the energy of the molecular overtones and the vibrations of the chemical bonds present. From this, the nature of the chemical bonds and, by extension, the functional groups of the molecules themselves can be derived. NIR uses the near-infrared region of light (800-1200 nm). The interpretation of near-infrared spectra requires complex correlation curve to provide reliable data. For the use of analysing samples for mycotoxins, the calibration samples for any NIRS method must first be obtained via high-performance liquid chromatography (HPLC) or mass spectroscopy. After obtaining infrared spectra, mathematical pre-processing is applied to eliminate noise and drifts. A model correlating spectral values and reference values is proposed and then validated. NIRS maybe best thought of as a sorting method: a coarse screening method to classify samples as either extremely high in concentration or not extremely high in concentration. NIR can only measure changes in the matrix under analysis and cannot measure the mycotoxins themselves due to their low molecular mass. Rather, it measures the change in the matrix in question, e.g. protein structure, changes in carbohydrates. Recent research has highlighted the limited sensitivity, the lack of regulatory precision and the need to tailor to specific crop varieties and regions inherent in an indirect measurement system. Given the fact that many fungal metabolites are extremely toxic at very low concentrations, NIRS is currently not very effective for measuring mycotoxin concentrations.

Conclusion

Regular mycotoxin testing of raw commodities is the first and arguably most important step in an effective mycotoxin risk management program. Each analytical method offers its own set of advantages and drawbacks. In practice, it may be useful to use a variety of methods for different purposes in order to strike the right balance between the needed level of visibility and cost.

Author: Renata Olejniczak, Product Manager At Biomin