Conventional methods for mycotoxin analysis

As the presence of mycotoxins is presently unavoidable, management strategies have been implemented to prevent the contaminated commodities from entering into the food chain. In 2003, the Council for Agricultural Science and Technology stated that the development of new technologies for mycotoxin analysis and improvement of detection (with specificity) of mycotoxins in prepared foods should be one important area of research that needs to be addressed in order to provide a safer food and feed supply in the 21st _century.10 _{Moreover, the} requirement to meet the regulatory limits set by national and international authorities has prompted the development of a vast number of analytical methods for the identification and quantification of mycotoxins.25

Mycotoxins comprise a variety of different structures and occur in varying concentration ranges in a wide range of agricultural commodities, foodstuffs, and biological samples, and therefore, it is rather impossible to develop a single standard protocol for their analysis and detection. Thus, most methods target only individual mycotoxins, or at best a group of closely related mycotoxins. In principle, the detection methods can be divided into chromatographic methods, which are based on separation and detection of the toxins by different detectors, and bioanalytical assays or biosensors which rely on specific biorecognition elements capable of recognizing the target mycotoxin. The method chosen for each particular application should consider the sensitivity required, available instrumentation and expertise of the person performing the analysis, as well as the cost and analysis time.25,67

On the other hand, mycotoxin monitoring includes not only the analysis or quantification step but also the sampling and sample preparation.16 _{Sampling refers to all the operations that} are applied to a lot of an agricultural product and lead to a laboratory sample. This step specifies the size of the sample and how it is selected or taken from the lot, and it is often a major source of variability in mycotoxin analysis. Sample preparation includes grinding, homogenizing and sub-sampling in order to obtain an analytical portion for the actual analysis. This test portion is further solvent-extracted, most commonly with organic solvents, such as methanol, acetonitrile or acetone, depending on the physical properties of the analyte, and finally, the sample is analyzed to determine the mycotoxin concentration.16,25

Thin-layer chromatography (TLC) was the first chromatographic method to be applied to mycotoxin detection, and although nowadays it has been mainly replaced by more advanced techniques, TLC is still routinely used to some extent, for example, in developing countries. Additionally, TLC methods have been approved as AOAC International official methods for several mycotoxins, such as aflatoxins, OTA, DON, ZEA, and patulin. Improvements to the basic TLC include the use of high-performance TLC plates, two-dimensional and bi-directional TLC, quantification by fluorescence densitometry, or combined with surface-enhanced Raman spectroscopy.16,68,69

The predominant chromatographic method for mycotoxin detection is high-performance liquid chromatography (HPLC). The small size and polarity of most mycotoxins make them ideally suited for separation by reverse-phase HPLC using a variety of mobile phase compositions made up of water, methanol and acetonitrile or mixtures of these.16 _{Most commonly HPLC separation} is coupled with UV and fluorescence detectors which use either the natural UV absorption or fluorescence of the analyzed mycotoxins or suitable derivatization methods. For example, fumonisins and type A trichothecenes which naturally lack a UV absorption band require

derivatization before HPLC analysis.16 _{Coupling of HPLC to mass spectrometry (MS) detection} has enabled the development of analytical methods for the simultaneous quantification of mycotoxins, and it is considered as the reference method for unequivocal mycotoxin identification.16,70 _{Recent developments in this field have provided interesting alternatives for} the simultaneous detection of multiple mycotoxins, and moreover, MS-based detection has also contributed to the discovery unknown mycotoxins.16,71,72 _{Also, some methods based on gas} chromatography (GC) have been introduced, mainly for trichothecenes,73,74 _{but the application} of this technique for mycotoxin analysis is limited by the polarity of mycotoxins and often requires derivatization before injection into the GC column.16 _{Chromatographic methods are useful for} the separation and quantification of highly similar compounds, such as different analogs of aflatoxins or fumonisins. However, they usually require labor-intensive sample preparation, for example using immunoaffinity columns (IAC)75,76 _{or solid-phase extraction sorbents (SPE).}77,78 Moreover, despite the high sensitivity and specificity that can be achieved with these methods, they require expensive and bulky instrumentation and skilled personnel to perform the analysis.69

Biosensors and bioanalytical assays are usually more appropriate for applications where fast and simple analysis are preferred, perhaps with the cost of slightly lower sensitivity. Immunochemical methods which rely on the high sensitivity and specificity achieved by mycotoxin-specific antibodies might not reach the accuracy and precision of chromatographic methods but have several advantages, such as simplicity, speed, and applicability to analyses in field conditions.25 _{For example, widely used ELISAs, in contrast to chromatographic methods,} do not usually require profound sample clean-up but can be combined with fast extraction step in aqueous methanol or acetonitrile followed by dilution of the extract to avoid the matrix effect.16 _{Moreover, the speed and low cost usually make ELISA a superior option over HPLC} when a large number of samples are screened for a single mycotoxin. In their simplest format, ELISAs can be based on visual detection or colorimetric readers, but higher sensitivity can be achieved with fluorescent or chemiluminescent detection, and the development of biosensors can also offer real-time readout and even perform the entire process automatically.67,79