**6. Toxin determination**

## **6.1 Conventional analytical techniques**

Conventional analytical methods employ chromatographic separation, particularly, liquid chromatography (LC), thin layer chromatography (TLC) and gas chromatography (GC) coupled to a detection system, with high-performance liquid chromatography (HPLC) after immunoaffinity cleanup combined with fluorescence detection (FLD) or mass spectrometry (MS) frequently employed for the quantitative determination of regulated mycotoxins in food [11, 18].

### *6.1.1 Thin layer chromatography*

Thin layer chromatography (TLC) was traditionally the most widely used mycotoxin screening technique that did not require expensive equipment and enabled high sample throughput [23]. The TLC techniques lack separating power that limits discrimination of co-extracted interference from the analyte of interest. This may, however, be overcome through improved modern cleanup techniques that remove impurities [12].

### *6.1.2 High-performance liquid chromatography*

High-performance liquid chromatography (HPLC) is now most commonly used for mycotoxin determination offering good sensitivity and precision, coupled with ease of automation [12, 19]. After extraction and cleanup, samples are injected into the HPLC column, individual compounds are separated based on their affinity for the column matrix and the mobile phase solvent [27]. To enhance fluorescence for better mycotoxin quantification using the HPLC-FLD technique, derivatization is important [12]. Pre-column derivatization with trifluoroacetic acid (TFA), or post-column derivatization, with bromine or iodine, can be used to identify aflatoxins [17]. Photochemical post-column derivatization may provide a cheaper alternative, whereas specific cyclodextrins may be incorporated in the mobile phase for non-chemical enhancement of fluorescence [28]. Despite offering good sensitivity and specificity, HPLC-FLD techniques are limited by expensive equipment requiring operation by experienced staff and may require laborious sample preparation procedures [12, 19].

#### *Aflatoxin and Mycotoxin Analysis: An Overview Including Options for Resource-limited… DOI: http://dx.doi.org/10.5772/intechopen.106834*

Recent utilization of ultra-HPLC (UHPLC) methods that are based upon increasing the mobile phase pumping pressure up to 1000 bar and above and reduction of particle size from 5 μm (HPLC) to 1–2 μm (UHPLC) improves resolution, sensitivity, and achieves rapid chromatographic separation as a result of increased speed and resolution between analytes [39, 40]. However, to avoid the high cost of the UHPLC system, columns packed with materials having solid core particles, coated with an outer layer of porous material can achieve more efficient separations at a much faster rate than with standard columns eliminating the need for expensive high-pressure facility because they are able to work at standard pressures (up to 600 bar) and can be used on all HPLC systems [41].

#### *6.1.3 Liquid chromatography/mass spectrometry*

Liquid chromatography with mass spectrometry (LC/MS) is a technique that allows more sensitive and selective determination of multiple mycotoxins in complex matrices with improved limits of detection and quantification [27]. Atmospheric pressure chemical ionization (APCI), atmospheric pressure photo ionization (APPI), and electrospray ionization (ESI) interfaces are currently employed in modern LC/MS instrumentation owing to their robustness, easy handling, high sensitivity, accuracy, and analyte selectivity and compatibility to a wide range of compound polarities [22, 42].

Liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) equipment has been developed that can significantly increase the sensitivity and specificity of multi-mycotoxin assays [37].

Liquid chromatography with tandem mass spectrometry (LC-MS/MS) has been increasingly used to provide a simultaneous determination of different classes of mycotoxins, including the regulated mycotoxins, thus affording to increase sample throughput and decreasing the cost per analysis [11]. However, given the complexity of matrixes to be analyzed and the wide-ranging physical and chemical mycotoxin properties, such methods require great skill to develop [11, 22, 42].

Mass spectrometry detectors linked to HPLC, UHPLC, and GC systems can increase their separation and identification power. However, the MS facilities are expensive with complex laboratory requirements, require skilled operators, and may suffer solvent limitations [23].

#### *6.1.4 Gas chromatography*

Gas chromatography (GC) may be used to determine mycotoxins that are volatile within the column [19]. For example, GC coupled with electron capture detection (ECD), flame ionization detection (FID) or mass spectrometric detection (MS) may be used for trichothecene or patulin determination. However, when compared to alternative methods, GC requires prior cleanup of extracts and pre-column derivatization to increase the volatility and sensitivity of the toxins [11, 17]. Although having successful applications, GC has several disadvantages that include the analyzed sample to be volatile or converted into a volatile sample, problems with the thermal stability of the sample leading to losses, and the high cost of the equipment [23].

#### **6.2 Rapid screening methods**

Rapid screening methods, that include immunochemical techniques, varying from simple lateral flow and enzyme-linked immunosorbent assays (ELISA) to highly

sophisticated immunosensors, are based upon binding of an antigen, for example a mycotoxin to a specific antibody, and often do not require any cleanup or analyte enrichment steps [11, 18, 43].

#### *6.2.1 ELISA techniques*

The enzyme-linked immunosorbent assay (ELISA) technique utilizes a specific antibody to bind the target molecule directly or conjugated with the enzyme and interaction with a chromogenic substrate to give a measurable result [18, 23]. However, due to the low molecular weight of mycotoxins, they are not immunogenic and must be conjugated to a carrier molecule to achieve immunogenicity. The ELISA technique can be highly sensitive and specific, portable, rapid, and simple to use with high sample turnover. However, ELISA has a number of disadvantages that include single-use kits that can increase the cost of bulk screening, high matrix dependence, cross-reactivity, and limited detection range due to the narrow sensitivity of the antibodies [13, 23].

#### *6.2.2 Immunosensor/biosensor techniques*

Biosensors are based upon the interaction of a mycotoxin with a recognition system fabricated as a layer onto the surface of a matrix substance that induces a change that is converted into a measurable electronic signal by a transducer. This provides great sensitivity and selectivity, easy application, low cost, and portability [27, 44]. Biosensors are often classified by the type of toxin-binding element (e.g., antibody, aptamer, imprinted polymers, etc.) as well as by the technology used for signal transduction and detection (e.g., optical, electrochemical, piezoelectric, etc.) [33].

A number of biosensor/immunosensor assays and techniques have been developed for mycotoxin determination, including fiber optic devices, surface plasmon resonance (SPR), dip-stick and lateral flow devices, fluorescence polarization, timeresolved fluorescence, microbead, capillary electrophoresis (CE), and electrochemical and piezoelectric immunoassays [45, 46]. These techniques are outlined in the following subsections.

#### *6.2.2.1 Optical biosensors and fiber optic devices*

Optical sensors, based on a variation of optical signals generated by a transducer from molecular recognition events on a sensing element are divided into many subclasses depending on the type of signal generated, including calorimetric, fluorescent, chemiluminescent, and surface plasmon resonance [35]. Photoelectrochemical optical biosensors use light as an excitation source and photocurrent as the recognition signal, whereas another subset of optical biosensors uses total internal reflection ellipsometry with localized surface plasmon resonance for detection with an optical planar waveguide polarization interferometer [33]. For example, fluorescent-based fiber optic devices can capture fluorescence emission from the fluorescently labeled mycotoxin or the naturally fluorescent mycotoxin, for example, aflatoxin when they bind to the fiber optic surface and transmit it to a sensitive detector [45]. A commercial device "Octet" based on biolayer interferometry to detect changes in the interference pattern of light reflected from the surface of optical fiber when materials bind to the tip of the fiber has been developed and available from ForteBio (Menlo Park, Calif., USA) [47, 48].

*Aflatoxin and Mycotoxin Analysis: An Overview Including Options for Resource-limited… DOI: http://dx.doi.org/10.5772/intechopen.106834*

#### *6.2.2.2 Surface plasmon resonance*

The SPR technique is based upon the property that the binding of materials to a surface, for example, the binding of antibodies to the mycotoxin, can alter the refractive index near that surface. The SPR device measures the small changes in the angle, or intensity, of internally reflected light that results from the binding event, and the magnitude of the response is influenced by the amount of material adhering to the surface. Alternatively, surface plasmons may be used to excite fluorophores captured on a surface, a technique is known as surface plasmon-enhanced fluorescence spectroscopy (SPFS). With this technique, light is used to excite plasmons (electron charge density waves) in a thin film of gold foil attached to the surface of a glass prism, the resonance of which enhances the fluorescence of the captured fluorophores, for example, the labeled antibody [45, 46, 49]. Using imaging, SPR (iSPR) allows multiple binding events on different regions of the sensor surface to be monitored simultaneously (multiplexing), hence capable of measuring multiple antigen-antibody interactions simultaneously in a single injection [49, 50].

The advantages of SPR include rapid and simple cleanup procedures, short analysis times, reusable sensor chips, and not necessarily requiring competition or labeled reagents for detection. It has great potential for multiplexing, with a wide variety of commercially available devices [46, 49]. However, like most immunoassays, SPR can be influenced by matrix effects that can be dealt with by increasing the dilution of the sample extract or by cleanup of the extract before the detection step [46].

#### *6.2.2.3 Lateral flow devices*

Lateral flow strip and dipstick devices (immunochromatographic test devices) use rapid disposable devices that may be attached with the toxin or the antibody that can bear enzymatic, liposome associated, or colloidal gold labels to detect the presence of mycotoxins [45]. Colloidal gold is frequently used as a label in test strips developed for mycotoxins due to availability, ease of production, and ease of conjugate formation with antibodies [51]. "Mycotoxin in the sample extract interacts with colloidal gold conjugated anti-mycotoxin antibodies at the base of the stick, with both bound and unbound antibodies moving along the stick membrane, passing a test line composed of immobilized mycotoxin, which will bind free antibody to form a visible line indicating a level of aflatoxin contamination below the test cut-off value. The control line further along the stick is composed of anti-antibodies to ensure complete extract migration along the strip" [28].

The related, membrane-based flow-through device, also known as enzyme-linked immunofiltration assay (ELIFA) differs from lateral flow devices, in that the applied liquid flows perpendicularly through the membrane rather than laterally, where it is collected on an absorbent pad on the opposite side of the membrane. It uses an enzymatic label that requires a substrate-incubation step, with the test and control lines being generated by an enzyme-substrate color reaction [28, 45].

Because of their easy application, efforts to develop dipstick and lateral flow assays for mycotoxins are likely to continue, particularly using stable, nonenzymatic labels [45], with a number of devices already being commercially available [17]. Also, innovative labels based on nanoparticle applications, such as quantum dots (QDs), gold nanoparticles (AuNPs), magnetic nanoparticles (Fe3O4), carbon nanoparticles (CNPs), time-resolved fluorescent microspheres (TRFM), have been developed for signal amplification in LFD, which can improve detection. Moreover, the advent of a

fluorescence quenching principle in lateral flow immunoassays (LFIA) in contrast to traditional competitive LFIA increases the sensitivity of the LFIA [35, 52].

#### *6.2.2.4 Fluorescence polarization and time-resolved fluorescence*

Fluorescence polarization (FP) immunoassays are solution-phase assays that rely on the measurement of change in the rate of rotation of a fluorescent-labeled mycotoxin (tracer) when it forms an immune complex with the added antitoxin antibody after competing with unlabeled mycotoxin in the sample extract [28, 45, 46]. FP can be used to measure the rate of association of the toxin with the antibody (kinetic assays) or the equilibrium point in a competition reaction (equilibrium assays). Critically, FP relies on the proper selection of antibody and tracer pairs [45, 46].

Unlike FP immunoassays, time-resolved fluorescent immunoassays (TR-FIA) use the property of fluorescence lifetime to measure the rate of decay of a fluorophore that is associated with a mycotoxin [45]. The newer fluorescent materials known as lanthanides, such as Eu (III) and Tb (III), have much longer fluorescence lifetimes that can eliminate the background fluorescence interference from the matrix, thus improving the sensitivity of methods based on TR-FIA [35].

The fact that FP is a homogeneous assay that does not require the separation of the free and bound tracer, may eliminate additional steps, such as washing, in competitive ELISA, thus increasing method rapidity [53]. However, like most immunoassays, it can be affected by the presence of a matrix, which can be controlled through dilution, cleanup, matrix-matched calibration curves, or data normalization [46, 53]. Although the available FP immunoassay readers are not capable of multi-mycotoxin detection, the potential speed of FP assays combined with the portability of the devices, suggests this technology has a promising future [46].

#### *6.2.2.5 Microbead assays*

Microbead assays use antibodies or antigens attached to the microbeads in miniaturized IAC assays, often with the cleanup and detection steps performed on a single instrument. It can be affected by poor re-usability of the columns due to fouling and reduced functional capacity of antibodies [45].

#### *6.2.2.6 Capillary electrophoretic immunoassays*

Capillary electrophoresis (CE) employs capillaries that are injected with the cleaned sample extracts in aqueous buffer solutions where they are separated in an electrical field before detection, typically using fluorescence or UV absorbance [23, 45]. The CE methods have comparable sensitivity, precision, and accuracy to HPLC methods, use less expensive capillaries, eliminate the use of organic solvents and take shorter analysis times, thus making them viable alternatives to HPLC [17].

#### *6.2.2.7 Electrochemical immunosensor assays*

Electrochemical immunosensors for mycotoxin determination are based on the high-affinity interaction between antigen and specific antibodies that can be transformed into a measured electrochemical signal based on a variety of electrochemical techniques [54]. They can be categorized into amperometric,

#### *Aflatoxin and Mycotoxin Analysis: An Overview Including Options for Resource-limited… DOI: http://dx.doi.org/10.5772/intechopen.106834*

potentiometric, conductometric, impedimetric, and voltammetric sensors according to the types of detectable electrical signals [35]. In their simplest format, the immobilized antibody is bound to the surface of a screen-printed electrode, and the final enzymatic stage develops a reaction product that can be measured by its electrical properties [28].

These electrochemical assays can be affected by factors that influence the interface between antigen and antibody, including solvent-matrix interactions and the reduction/oxidation potential of the diluent. The extent of testing using this technology, the accessibility of components, and the capacity for miniaturization, suggest future utility of these devices in the detection of aflatoxins [46].

#### *6.2.2.8 Piezoelectric sensors*

Piezoelectric sensors often called quartz crystal microbalance (QCM) are based upon piezoelectric quartz crystals and they work through the application of an alternating current to a quartz crystal, which induces oscillations of the crystal, the frequency of which depends in part on the thickness of the crystal, for example, after mycotoxin binding on immobilized antibodies [46]. Mass change on the sensory layer of the surface of the gold-plated crystal quartz transducer causes specific measurable vibrations of the crystal in response to an electrical signal [20]. The advantage of QCM is that they do not require the use of labeled reagents [46].

In general terms, immunochemical techniques are affected by high matrix dependence, cross-reactivity, and loss of antibody stability under the extreme environment, such as pH, organic solvents, and high temperature. Moreover, the cost of their development may be high and requires a stable source of antibodies to ensure continuity of analytical performance and stability. Therefore, the development of synthetic receptors can solve some of these challenges, particularly, problems associated with antibody stability in an extreme environment [18, 44, 54]. As an example, [55] developed an aptamer-based assay for the detection of AFB1 in corn samples that exhibited a wide dynamic range from 0.1 to 10 ng/mL, limit of detection of 0.11 ng/mL, and recovery values between 60.4 and 105.5% that were described as promising results.

It is worthy to note that, chemical and biochemical sensor devices are increasingly developed based on advanced microchip technology, including microfluidic chips and microarrays for portability, easy on-site field application, robustness, reliability, reduced cost, rapidity, high throughput, and increased sensitivity. Also, the advent of innovative labels based on nanoparticle application has led to a significant improvement in their detection capability. Examples of these include the microfluidic devices based on flow-through (capillary electromigration) and lateral flow formats and the emerging microchip-based sensing methods, such as surface plasmon resonance (SPR) and magnet nonotag-based detection [35, 56, 57].

#### **6.3 Noninvasive techniques**

Noninvasive techniques, such as spectroscopic and imaging techniques [27], DNA microarrays, electronic chemical sensors (electronic nose and tongue), and polymerase chain reaction-based methods [27, 44] provide a potential approach for rapid nondestructive detection of fungal infection and mycotoxin contamination on grains. However, many of these techniques may either be expensive and/or may need further validation studies.
