Aflatoxin and Mycotoxin Analysis: An Overview Including Options for Resource-limited Settings

*Godfrey Z. Magoke, Robyn G. Alders, Mark Krockenberger and Wayne L. Bryden*

#### **Abstract**

Aflatoxins are fungal toxins of serious human health concern, more so in some developing countries where significant contamination of staple foods occurs and the prevalence of aflatoxin-related health effects is high. A plethora of techniques for food mycotoxin testing has been developed. Modern chromatographic techniques allow quantitative determination with high accuracy and sensitivity, but are expensive and difficult to operate and maintain. Rapid tests provide a cheaper alternative for screening large numbers of samples, although they need validation on all food matrices that are tested. One important aspect of tackling aflatoxin contamination and exposure is to ensure the availability of suitable methods for detection and quantification that are rapid, sensitive, accurate, robust, and cost-effective for food surveillance in resource-limited settings.

**Keywords:** mycotoxins, analysis, food safety, rapid tests

#### **1. Introduction**

Food contamination with mycotoxins is a serious human health concern worldwide and of greatest significance in developing countries [1, 2]. Of all the mycotoxins, aflatoxins are more toxic, widespread in nature, and have been associated with significant health effects in humans and reduced productivity in farmed animals [3–5]. Recent estimates suggest that 60–80% of crops contain detectable concentrations of mycotoxins. In many instances, there is co-contamination with more than one toxin and this is geographically dependent on climate and farming practices [6, 7].

Aflatoxins affect approximately 4.5 billion people in developing countries, causing acute fatal hepatitis in individuals exposed to highly contaminated grains. Low level, chronic exposure to aflatoxins is associated with the development of liver cancer in adults, reduced immunity, and lowered growth and stunting in infants and children [8–10]. Monitoring food for contamination with aflatoxins is essential, although a number of challenges must be faced, including low concentrations and variable distribution of the toxin in contaminated grains within storage facilities. These factors

will contribute to variable test results as well as issues related to test sensitivity and specificity in varied food matrices [5, 11].

This chapter provides an overview of sample extraction and cleanup procedures, together with analytical techniques developed for mycotoxins, including aflatoxins. The advantages and disadvantages of the different approaches affecting suitability for use in aflatoxin food surveillance and quantitative confirmation are outlined. In developing countries, rapid tests make a significant contribution to aflatoxin control and a perspective on their application in resource-limited settings is given.

#### **2. General mycotoxin analytical techniques**

Mycotoxins present a great analytical challenge. Not only do they include a diversity of chemical compounds, but are heterogeneously distributed at varying concentrations in a wide range of agricultural commodities, foods, feeds, and biological samples that require specific extraction, cleanup, separation, and detection methods [11]. Some mycotoxins, especially deoxynivalenol and zearalenone, are conjugated as a result of plant metabolism, and these "masked" mycotoxins may contribute 20% of total of the parent mycotoxin but are not detected during conventional analysis [5, 11].

Quantification of mycotoxins requires expensive laboratory equipment that needs well-trained personnel to operate [12], as well as involving a series of steps and procedures that may be laborious and time-consuming [11]. The need for high sensitivity tests to detect the minimum levels of the mycotoxin possible for regulatory purposes, coupled with rapidity, high accuracy, simplicity, robustness, and selectivity have been the main driving forces behind the improvement and development of new mycotoxin analytical protocols [11, 13]. Mycotoxin analysis is essential to quantify the toxin for risk evaluation, diagnosis, and monitoring mitigation strategies [5].

#### **3. Sampling**

Sampling for aflatoxin determination in food commodities poses a particular challenge given uneven toxin distribution and the low levels at which mycotoxins occur [5]. As a result, some national and international food safety authorities and organizations have prescribed sampling methods for various food commodities for the purpose of achieving representative samples that may be used to determine concentrations of various mycotoxins in foodstuffs for official control purposes; sampling is potentially the biggest source of error in mycotoxin testing [14]. For many commodities, detailed sampling plans have been devised [15]. To obtain a representative sample from a grain storage facility, for example, incremental samples have to be taken from different places of the facility [11] with the entire primary sample ground, mixed, and subsampled to ensure that the analyzed portion has a similar toxin concentration as the original sample [11, 16].

#### **4. Analytical procedures**

Analytical procedures for mycotoxins entail extraction from the matrix with a suitable solvent, cleanup of co-extracted matrix components, and identification/

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

quantification of the toxin using suitable analytical facilities [12, 17]. Some exceptional techniques, such as infra-red spectroscopy, may detect mycotoxin contamination directly in ground samples without prior solvent extraction or cleanup but are limited to screening purposes because of high matrix interference and lack of suitable calibration materials [11]. Although additional cleanup is essential for chromatographic determination, the diluted extracts may be directly used with immunoanalytical methods [13].

#### **5. Sample extraction**

Extraction liberates the mycotoxin from the sample matrix with subsequent extract cleanup to reduce matrix interference, hence improving the sensitivity and robustness of the technique [11, 18]. Depending on the physicochemical properties of the mycotoxins and sample matrix, various combinations of extraction solvents may be used [11]. Relatively polar solvents, such as methanol, acetone, acetonitrile, ethyl acetate, diethyl ether, 1-octanol, toluene, dichloromethane, chloroform, or a mixture of them, may provide efficient extraction of mycotoxins, with minimal addition of water and acid solution helping to enhance extraction efficiency [19, 20]. A suitable extraction solvent should only remove the mycotoxins from the sample with high efficiency as well as being cheap, safe to use, and reduce matrix interference. For this purpose, mixtures of methanol-water and acetonitrile-water at appropriate ratios are the most frequently used extraction solvents for mycotoxin analysis [20].

Other parameters, such as sample/extractive solvent ratio, temperature, and time of extraction, may affect the extraction process; therefore, need to be carefully controlled to achieve accurate quantification [16]. High temperature and pressure instruments, such as accelerated solvent extraction/pressurized liquid extraction and microwave-assisted extraction methods hasten the process by speeding up and automating the extraction, use less solvent and provide better extraction efficiencies (in terms of extraction yield and/or recovery) compared to classical solvent extraction techniques. However, they are limited by the high cost of the equipment and may not be suitable for thermally unstable analytes [11, 16]. Non-polar solvents, such as hexane and cyclohexane, may be used before or following the extraction procedure to remove lipids in certain sample types, for example, groundnuts and maize [16, 19]. The presence of pigments, essential oils, and fatty acids in some samples may make extraction difficult and necessitate the use of different extraction solvents, such as a mixture of ethyl acetate-formic acid [20]. Chlorinated solvents are considered to be toxic and ecologically harmful, hence should be avoided, where possible in the extraction process [12]. Deep eutectic solvent has been recently reported as an environmentally safe extraction solvent limiting the use of traditional solvents and derivatization reagents [20].

Extraction is usually enabled by the high-speed blending of ground sampleextraction solvent mixture or employing a mechanical shaker followed by filtration before subsequent purification step, where applicable [19]. Evaluation of extraction procedures based on methanol-water and acetone-water in maize found [21] that the acetone-water mixture (6 + 4 v/v) showed the best extraction efficiency for all aflatoxins (B1, B2, G1, and G2) compared to the commonly used mixtures of methanolwater (8 + 2 v/v) or acetone-water (85 + 15 v/v).

Purification of sample extracts is required to reduce matrix-induced signal suppression or enhancement in mycotoxin detection [11, 19, 22]. Immunoaffinity

columns (IAC), solid-phase extraction (SPE), column chromatography, multifunctional columns, and liquid-liquid partitioning (LLE) may be used for purification purposes with the purified sample reconstituted in a suitable solvent before chromatographic analysis [13].

#### **5.1 Solvent extraction methods**

#### *5.1.1 Liquid extraction/partitioning*

Liquid extraction or partitioning is a common and arguably the simplest method of sample purification relying on the solubility of the target compounds in a particular solvent, and the insolubility of competing or interfering compounds in the same solvent [18].

#### *5.1.2 Liquid-liquid partitioning/extraction*

Liquid-liquid extraction (LLE) is used repetitively to extract analytes quantitatively by concentrating those analytes that migrate between two partitioned immiscible solvents [19]. In LLE, traditional solvents with a low dielectric constant (those that tend to be immiscible with water) are poor at extracting polar compounds, including most mycotoxins. Suitable solvents, such as methanol or acetonitrile, should be mixed with water in the presence of salts to reduce the mutual miscibility, allowing the polar analytes to move selectively into the polar organic phase from the aqueous phase [18]. Solvents, such as hexane and cyclohexane, for example, may be used to remove non-polar contaminants, for example, lipids and cholesterol through liquid-liquid extraction [23]. However, the method is used infrequently because it is labor intensive, uses vast amounts of solvent, leads to losses, and is time-consuming [13, 19].

#### *5.1.3 Dispersive liquid-liquid microextraction*

Dispersive liquid-liquid microextraction (DLLME) is a recently introduced miniaturized extraction procedure. The technique is based on the formation of a cloudy solution consisting of fine droplets of the extractant solvent dispersed entirely in the aqueous (continuous) phase. This occurs following the rapid addition of a mixture of a water-immiscible extractant solvent, and a water-miscible dispersive solvent into an aqueous solution containing the analytes. As a result of a very large surface area formed by the dispersed extractant micro-droplets, the analytes are rapidly and efficiently enriched in the extractive solvent and, after centrifugation, can be separated in the sediment phase [16, 24].

This technique is cheap, environmentally safe, simple, fast, and efficient [16]. However, it is difficult to automate and necessitates using a third component (disperser solvent), which commonly decreases the partition coefficient of analytes into the extractant solvent [24].

#### *5.1.4 Vortex-assisted liquid-liquid microextraction*

Vortex-assisted liquid-liquid microextraction is a new equilibrium-based solvent microextraction technique. It is based on the dispersion of micro-droplets of the extraction solvent into the aqueous sample and is achieved by vortex agitation,

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

forming a mild emulsification process [24, 25]. Separation of the two phases occurs upon centrifugation, with the floating extractant phase restoring its original single micro drop shape; it is easily collected with the help of a microsyringe and used for HPLC analysis [24, 26].

Several experimental parameters, namely, organic solvent, agitation time, rotational speed of the vortex agitator, acceptor phase volume, aqueous sample volume, pH, and salt addition may affect the extraction process, and these need to be controlled and optimized for optimum performance of the procedure [24]. Surfactants, such as Triton X-114, Tween-20, Triton X-100, and cetyltrimethylammonium bromide (CTAB), may be used to enhance extraction efficiency [25].

This technique is rapid, as the fine droplets formed, extract target analytes toward equilibrium faster because of the shorter diffusion distance and larger specific surface area compared to the DLLME where the need for a disperser solvent is mandatory [24, 26].

#### *5.1.5 Dilute and shoot method*

The dilute-and-shoot (DaS) method utilizes the improved sensitivity and robustness of modern equipment. It is based on dilution followed by direct injection of samples that are presumed to be inherently clean enough to not require full preparation, thus reducing cost. It has the benefits of rapidity, can work with multiple analytes, and limits the potential loss of analyte due to pretreatment, although it still has a risk of matrix interference that can overwhelm instrument sensitivity [18].

#### **5.2 Solid-phase extraction methods**

#### *5.2.1 Solid-phase extraction*

The solid-phase extraction (SPE) technique utilizes small disposable cartridges packed with silica gel or bonded phases that are in the stationary phase to bind impurities or target analytes. The impurities can be washed off, and the analyte recovered using a suitable rinse solution [19, 23, 27].

In SPE, the aqueous sample extract is applied to the conditioned column followed by rinsing to remove matrix compounds, with the analyte eluted from the column using an organic solvent. Evaporation of excessive solvent can be employed for further concentration [13, 19].

Compared to LLE, SPE has the advantage of rapidity, efficiency, reproducibility, uses considerably less solvent, and offers a wide range of selectivity, however, it is limited by the fact that there is no single fit-for-all cartridge [19, 23].

#### *5.2.2 Ion-exchange columns*

Ion-exchange columns use ionic materials, such as SAX (strong anion exchange) in SPE to extract mycotoxins that present as ions, such as moniliformin, in aqueous solutions. The target molecule is bound to charged groups on the silica material and removed by the addition of a strong ionic solution because of its higher affinity to the sorbent or by the altered pH [19, 23].

#### *5.2.3 Matrix solid-phase dispersion*

Matrix solid-phase dispersion (MSPD) utilizes some SPE sorbent materials (usually octadecyl silica, silica gel, or alumina) that is ground typically with 1 g of a homogenized sample using a pestle and mortar. The solid mixture is then transferred to a glass column or cartridge containing a lower layer of co-sorbent material, such as carbon black, with the adsorbed residues selectively eluted with an appropriate solvent [16, 28, 29]. This technique has the advantages of flexibility and versatility and can be used in a single step with small amounts of sorbent and solvent, thus reducing the cost and time of analysis. However, it is not easily automated, often requiring an additional cleanup step that could be time-consuming for a large number of samples [16, 29].

#### *5.2.4 Solid-phase microextraction*

Solid-phase microextraction (SPME) combines extraction and concentration of analytes in a single step and is based on the extraction of analytes by adsorption to a thin fiber coated with different stationary phases. This is followed by thermal desorption into a heated injector for gas chromatography or with a solvent when liquid chromatography is used [16]. It is simple, safe, and has a wide application on polar and non-polar compounds [30]. However, it has the disadvantage of high cost, fiber fragility, and is susceptible to experimental conditions that can affect reproducibility and sensitivity [16, 31].

#### *5.2.5 Micro-solid phase extraction*

The recently introduced micro-solid phase extraction (μ-SPE) uses a sorbent material trapped in a porous membrane sheet to extract the analyte diffusing through it, the μ-SPE device tumbling to stir the process facilitating the mass transfer. Following extraction, desorption is carried out by ultrasonification with the extraction device immersed in a suitable organic solvent. The technique is simple as extraction and cleanup steps are carried out simultaneously and it uses less solvent and sorbent materials [30].

#### *5.2.6 Magnetic solid-phase extraction*

Magnetic solid-phase extraction (MSPE) is a new SPE technique that is based on the use of magnetic nanoparticles that are dispersed into the sample solution with separation effected by applying an external magnetic field outside the sample solution [31]. The technique avoids time-consuming column or filtration operations encountered in SPE with the large contact area between the adsorbent and the analyte ensuring a fast mass transfer, which guarantees high extraction efficiency compared to the SPME technique [31, 32].

#### *5.2.7 Immunoaffinity columns*

Immunoaffinity columns (IACs) are increasingly used for the cleanup and enrichment of sample extracts [11]. The column containing mycotoxin-specific antibodies bound to solid phase support within the cartridge selectively binds the mycotoxin in the extract. Mycotoxin desorption is achieved using a miscible solvent or by antibody denaturation [16, 19].

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

Compared to traditional solid-phase cleanup techniques, IAC is more sensitive, specific, easy to use, rapid, safe (minimizes use of hazardous solvents), and robust in terms of applicability to different matrices. However, columns are single-use, more expensive, suffer from storage limitations and stability problems regarding organic solvents, and the possibility of nonspecific interactions due to cross-reactivity with other mycotoxins [11, 12, 16, 19]. However, there is now a commercially available immunoaffinity column ('Myco 6in1'; Vicam, Milford, MA, USA) that may be used in a cleanup procedure for simultaneous determination of multiple mycotoxins [33] that helps mitigate the single use of these IACs.

#### *5.2.8 MycoSep®/Multisep® columns*

Mycosep® /Multisep® columns contain selected adsorbents packed in a plastic tube to recover individual mycotoxins from a sample extract [23]. Despite the practicability of the method, the columns are designed per analyte, hence not suitable for multi-toxin determination and may not provide effective purification for some matrixes [16, 23].

#### *5.2.9 Molecular imprinted polymers and aptamers*

Synthetic systems, such as molecular imprinted polymers (MIPs), aptamers, and peptides, have been developed to counter shortcomings related to the use of antibodies in IACs [20].

The molecular Imprinted Polymer (MIP) is a synthetic material providing an artificially generated three-dimensional network that is able to specifically rebind a target molecule. It is a cheaper alternative for mycotoxin cleanup and preconcentration as well as affording chemical and thermal stability and solvent compatibility, which is contrary to immunoaffinity columns [11, 34].

During molecular imprinting, cross-linked polymers are formed by free-radical copolymerization of functional monomers. The cross-linking occurs in the presence of an analyte serving as a template followed by template removal by liquid extraction (washing). This leaves highly selective three-dimensional binding pockets complementary in size, shape, and functionality to the imprinted molecule remaining in the polymer matrix [13]. Despite offering promise for future application, MIP may still be affected by the low specificity and robustness of the technique in terms of kinetics, reuse, ability to withstand unfavorable solvents, and potential sample contamination by template bleeding [23].

On the other hand, aptamers are small fragments of oligonucleotide sequences (single-stranded DNA or RNA), usually containing 10 to 100 bases that bind to their targets by folding into specific three-dimensional structures [35]. Compared to antibodies, they are cheap, stable, reversible, not limited by immunogenicity of targets, and do not require immunization of animals during production [35]. Although difficult to develop, they provide an important avenue for exploitation in mycotoxin cleanup procedures and in sensing instruments [20, 35].

#### *5.2.10 QuEChERS extraction/cleanup*

QuEChERS (quick, easy, cheap, effective, rugged, and safe) as a sample pretreatment technique entails solvent extraction, partitioning with magnesium sulfate and other salts, such as NaCl, and cleanup using a dispersive solid-phase extraction

(d-SPE) sorbent, especially the primary secondary amine (PSA) and extract centrifugation before analysis [36]. Magnesium sulfate along with NaCl is used to reduce water in the sample during extraction, while PSA retains co-extracted compounds during cleaning [16]. This procedure is simple, rapid, cost-effective, and enables multi-residue determination [16, 36]. The use of QuEChERS is becoming a popular alternative to the dilute-and-shoot approach for multi-mycotoxin determination using LC/MS-based techniques to reduce matrix interference [37]. However, it should be noted that the several QuEChERS commercial kits or QuEChERS-like protocols differ in extraction, partitioning, or dispersive solid-phase extraction (dSPE) steps. They, therefore, may show different cleanup efficiencies, and for optimization an additional cleanup step may be needed to improve the performance of QuEChERS protocols [38].
