Determination of mycotoxins

As a result of the global marketplace, the safety of food and feed has become of increasing concern for consumers, governments and producers. Several highly publicised worldwide incidents related to chemical contaminants such as mycotoxins in food and feed have also attracted much media attention¹. Mycotoxins are toxic natural secondary metabolites produced by fungi on agricultural commodities in the field and during storage under a wide range of climatic conditions. Mycotoxins are potent toxins and have a wide range of actions on animals and humans, e.g. cyto-, nephro- and neurotoxic, carcinogenic, mutagenic, immunosuppressive and estrogenic effects². Most of the occurrence studies have emphasised ‘traditional’ mycotoxins, such as aflatoxins, ochratoxin A, zearalenone, fumonisins and trichothecenes³.

However, in addition to conjugated (masked) mycotoxins, which can occur after metabolisation by living plants, fungi or mammals and after food processing³⁷, concerns have also been raised recently about so-called emerging mycotoxins. These are usually referred to as (unusual) toxins especially produced by Fusarium spp. which have become quantifiable due to the availability of high performance mass spectrometric tools, such as LC-MS/MS⁴. These include e.g. enniatins, beauvericin, fusaproliferin, moniliformin and even ergot alkaoids have been referred to as this ‘new’ group of toxins.

To provide information about the levels of mycotoxins in foods and feeds and to support food safety standard setting activities it is essential to analyse food and feed samples using sensitive, fast and accurate analytical methods. The chemical diversity of the mycotoxins and the wide range of agricultural commodities and foods however pose a challenge in method development. Concentration levels in food and mixed feed may also vary considerably. These requirements, in conjunction with the increasing number of sample matrices and analytes of interest, have led to the development of both rapid screening methods for various analytes, mostly based on immunochemical techniques, and of highly sophisticated multi-analyte methods based on liquid chromatography coupled with multiple-stage mass spectrometry (LC-MSⁿ) to allow identification and simultaneous determination of a wide range of secondary fungal contaminants^5,6.

This paper is based on a recent book edited by the authors⁷ and is dedicated to the determination of mycotoxins including proper extraction and clean-up procedures, separation and detection techniques with a special emphasis on rapid strip tests and LC-MS/MS multi-toxin methods.

Extraction and clean-up

Various combinations of solvents, sometimes with the addition of modifiers (e.g. acids, bases, etc.), are used for extraction, depending on the physicochemical properties of the mycotoxins, the sample matrix and the type of clean-up used afterwards^7,27. Following extraction, the resulting raw extract is usually further processed to remove unwanted substances such as lipids, carbohydrates and peptides and often concentrated to make determination of toxins at low concentrations possible.

In the last 20 years, tedious clean-up procedures such as liquid/liquid separation have been replaced by less labour intensive techniques such as Solid Phase Extraction (SPE), the use  of immunoaffinity columns and the QuEChERS approach (Quick, Easy, Cheap, Effective, Rugged and Safe approach). Besides the use of C₈ and C₁₈ bonded silica columns, Mycosep™ multifunctional clean-up columns⁸ have been employed as SPE technique which consist of adsorbents, which are packed in a plastic tube. On top of the plastic tube, the purified extract appears within seconds. Columns are usually suitable for one analyte only and are available for a range of mycotoxins  such as aflatoxins, deoxynivalenol (DON) and patulin⁹. Mycosep™ multifunctional have also been successfully employed in a multi-analyte-LC-MS/MS method for the simultaneous determination of nine Fusarium mycotoxins.

Immunoaffinity columns (IACs) for clean-up purposes have become increasingly popular in recent years because they offer high selectivity¹⁰. They are easy to use for purification of samples that are contaminated with different mycotoxins. The mycotoxin is bound selectively to the antibodies on the column after a preconditioning step. The successful use of IACs in combination with subsequent LC-MS determination has recently been demonstrated within an interlaboratory comparison study for the determination of fumonisins B1 and B2 in corn¹¹. Immunoaffinity columns have also become available for the simultaneous determination of aflatoxins, ochratoxin A, fumonisins, deoxynivalenol, zearalenone, T-2 and HT-2 toxins¹².

Another option, only recently introduced in multi-mycotoxin analysis, is the use of the various modifications of the QuEChERS approach, currently widely used in multi-pesticide analysis, for very fast extraction and purification. The key principle is partitioning of an acetonitrile-water mixture induced by addition of inorganic salts¹³. There is an increase in the number of publications on mycotoxin determination using the QuEChERS method¹⁴.

Rapid strip tests

In the last decade, rapid immuno-assay based tests have increasingly been used in the food and feed sector¹⁵. Tests for mycotoxins which allow for the screening of agricultural commodities with results within less than 20 minutes are gaining acceptance and are increasingly being integrated into routine quality monitoring procedures¹⁶. Enzyme linked immunosorbent assays (ELISAs) have become a standard tool for rapid monitoring of mycotoxins¹⁷. Microtitre plate ELISAs offer the advantage of speed, ease of operation, sensitivity and high sample throughput. Rapid disposable assay tests have been developed in multiple formats such as flow through tests¹⁸, dip sticks¹⁹ and strip tests²⁰ also called lateral flow devices (LFDs).

Driven through a strong demand from industry, a trend can be seen from qualitative towards (semi-) quantitative strip tests²⁰ as well as to multi-mycotoxin approaches such as a LFDs for the rapid simultaneous detection several mycotoxins. Strip test based test kits are commercially available for several mycotoxins including aflatoxins (qualitative and quantitative), deoxynivalenol (semi-quantitative and quantitative), fumonisins (qualitative and quantitative), ochratoxin A (quantitative), and zearalenone (quantitative). Very recently a multiplex dipstick approach based on an indirect competitive immunoassay for the simultaneous semi-quantitative determination of major Fusarium toxins, namely zearalenone, T-2 and HT-2 toxins, deoxynivalenol and fumonisins in wheat, oats and maize has been developed²¹. Also very recently, a membrane and a gel based flow-through enzyme immunoassay for the detection of four mycotoxins (ochratoxin A, fumonisin B1, deoxynivalenol and zearalenone) in peanut cake, maize and cassava flour samples have been developed and their performance compared²². Both assays provided a yes/no response indicating whether the toxins were present or not above the maximum permitted levels of these analytes.

Problems with reproducibility and sensitivity especially with complex food and feed matrices, however, often limit the application of strip tests¹⁵. Moreover, differences observed between spiked samples and naturally contaminated samples contribute to calibration and validation problems with shifts of the cut-off level in the strip test or shifts of relative reflectance readings of the test line in a quantitative strip test.

LC-MS(/MS) based methods

Until the mid 1990s, most instrumental analytical methods for the determination of mycotoxins used HPLC in combination with FLD²³ or UV detection and GC-ECD (e.g. trichothecenes)²⁴ or GC-FID. During the last 10 years, online coupling of HPLC to tandem mass spectrometry (LC-MS/MS) has developed to become one of the most powerful techniques for the determination of mycotoxins^25,26.

Until recently, most of the available analytical methods only covered single mycotoxin classes (e.g. aflatoxins, type-B trichothecenes or fumonisins)^27,28. An LC-MS/MS method for the quantification of several type-A and type-B trichothecenes and ZEA in wheat and maize was developed in 2005^14,29. In addition to conventional SPE, multi-immunoaffinity columns were used in combination with LC-MS/MS for the simultaneous determination of all regulated mycotoxins plus T-2 and HT-2 toxin in maize¹².

In contrast to single class methods which apply some kind of clean-up³⁰, in a comprehensive approach for the quantification of 87 mycotoxins, as developed by Sulyok et al²⁶, a clean-up is not possible as a consequence of the wide range of toxin properties. This ‘dilute-and-shoot’ method included the most relevant members of the following toxin classes: type-B- and type-A trichothecenes, zearalenone (ZEA) and derivatives, fumonisins, beauvericin, enniatins, moniliformin, ochratoxins, aflatoxins, ergot alkaloids and patulin. Additionally, further bioactive fungal metabolites produced by Penicillium, Claviceps and Alternaria spp. are covered by this method. The conditions for extraction, HPLC separation and detection in such an approach cannot be optimal for each target analyte. For some of the acidic toxins, e.g. moniliformin and citrinin extraction efficiencies were only 55 per cent and 30 per cent respectively²⁸. The method detection limits of the procedure described by Sulyok et al²⁶ ranged from below 1 µg/kg (e.g. for the enniatins and ergot alkaloids) to about 200 µg/kg for neosolaniol. In most cases, the limits of detection were below the regulated values of mycotoxins in food and the apparent recoveries complied with official guidelines with few exceptions²⁶. This method has recently been extended to the determination of 187 fungal and bacterial metabolites³¹. A typical LC-MS/MS chromatogram obtained from such a multi-mycotoxin method is displayed in Figure 1.

Figure 1: LC-MS/MS chromatogram obtained from a multi-analyte method covering 223 mycotoxins

One of the major problems in the quantification by LC-MS and LC-MS/MS is that compound and matrix-dependent signal response suppression or enhancement may occur³². Ion suppression and its negative effects on method accuracy and precision can be reduced or eliminated by improvement of sample clean-up and/or the change of HPLC conditions³² . However, the ‘dilute and shoot approach’ without any clean-up  used for multi-toxin-determination often requires matrix calibration for accurate quantification. The need for accurate methods in mycotoxin analysis has also led to the introduction of stable isotope dilution assays (SIDA) in which the use of internal standards (ISTD) compensates these matrix effects and other random variations and systematic errors.  In a SIDA, isotopologues of the same mycotoxins are mixed and the quantification is based on the evaluation of the signal ratio of the isotopologues. The use of SIDAs in mycotoxin analysis has been reviewed by Rychlik et al³³. Most recently, a  SIDA for the accurate determination of 11 mycotoxins currently regulated in maize and other cereal-based food products in Europe by UHPLC-MS/MS was developed and validated by Varga et al³⁴.

Validated multianalyte LC-MS/MS methods for the quantification of numerous mycotoxins have in the meantime been successfully applied to a variety of different food and feed matrices³⁵ and revealed a great variety of mycotoxins in theses commodities. Also recently, novel approaches in the analysis of mycotoxins in cereals employing ultra performance liquid chromatography coupled with high resolution mass spectrometry have been introduced³⁶. In this context, an LC-high-resolution FT-Orbitrap mass spectrometric technique was evaluated for the quantification of selected mycotoxins and the simultaneous screening of fungal metabolites in food³⁷.

Outlook

It can be expected that further improvements of LC-MS/MS instrumentation and its availability at lower price will further contribute to LC-MS/MS becoming the major tool for the analysis of multi-contaminants, such as mycotoxins in grains, food and biological samples. LC-MS-based screening has also been playing a vital role in the discovery of novel mycotoxin conjugates so-called masked-forms of mycotoxins in the past and it is believed that this will also continue in the future³⁷. Employing LC-MS/MS for the determination of mycotoxin biomarkers can lead to improved exposure assessment of humans and animals to mycotoxins.

The established state-of-the-art chromatography-based methods for the determination of mycotoxins are increasingly being complemented by an growing number of methods for fast and cost efficient analysis, including rapid strip tests. The optimisation and validation of rapid test systems including strip tests will contribute to meeting contract or legislative specifications for maximum acceptable levels of mycotoxins in foods and feeds through effective screening.

References

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Biographies

Associate Professor Rainer Schuhmacher is head of Working Group for Metabolomics and Bioactive Substances at the Department IFA‑Tulln of the University of Natural Resources and Life Sciences, Vienna (BOKU). He has received two scientific awards and is author of 95 SCI publications.

Professor Rudolf Krska is head of the Department IFA‑Tulln at the University of Natural Resources and Life Sciences, Vienna (BOKU). From 2009 to 2010, he worked for a year as A/Chief of Health Canada´s Food Research Division in Ottawa. He has received six scientific awards and is one of the most cited authors in the area of mycotoxins worldwide.

[email protected]