Edited by: Javier Carballo, University of Vigo, Spain
Reviewed by: Daniela Jakšić, University of Zagreb, Croatia; Masoomeh Shams-Ghahfarokhi, Tarbiat Modares University, Iran
This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Aflatoxins, produced mainly by filamentous fungi
Mycotoxins are secondary metabolites of filamentous fungi and their presence indicates biological contamination. These compounds may enter the human and animal bodies directly by the consumption of contaminated agricultural products or ready-to-eat products or indirectly through the consumption of animal products (mainly milk, eggs, and offal), deriving from animals that consumed contaminated feed (
Aflatoxins are the first known mycotoxin group, described as a result of turkey “X” disease in the 1960s (
More than ten types of aflatoxins exist naturally, of which AFB1 is the most toxic. AFB1 and AFB2, AFG1, and AFG2 occur in the contaminated feed. AFM1 and AFM2 are present in ruminant milk after the digestion of feed contaminated by AFB1 and AFB2. In order to analyze aflatoxins, various analytical methods are required. Transformation of aflatoxins can be seen in
Transformation of aflatoxins.
There is a wealth of scientific information with respect to aflatoxins and their acute and chronic effects and numerous research groups have worked on this topic recently. According to Web of Science, there are nearly 16,000 publications since 1975 to this day in connection with aflatoxins, of which over 7,000 have been published in the last decade. These numbers and legal restrictions across the world regarding the highly carcinogenic aflatoxins indicate the importance of the topic.
This publication gives a complex and transparent summary of the regulatory environment and the diverse measurement techniques of aflatoxins from rapid methods through seemingly simple separation techniques to complex hyphenated techniques. Sample preparation methods associated with the different measurement techniques are also covered.
Free trade of food and feed is getting more and more common around the world. In order to keep the product flow under control, there is a need for harmonized regulation and control systems both in exporting and importing countries. Because of this, many countries have already established common regulations and maximum levels for different contaminants, including aflatoxins. Nonetheless, some non-community countries (
Worldwide aflatoxin regulations, allowed maximum levels.
Communities | Countries | Organization | Reference of regulation | Aflatoxin B1 (μg/kg) (food) | Total Aflatoxin (μg/kg) (food) | AflatoxinM1 (μg/kg) | Aflatoxin B1 (μg/kg) (feed) | Total Aflatoxin (μg/kg) (feed) |
African Union (AU) | South Africa | South Africa Department of Health | 5 | 10 | x | x | x | |
ASEAN (Association of Southeast Asian Nations) | Brunei | Department of Health Services, Ministry of Health | 0 | 0 | 0 | x | x | |
Cambodia | x | x | x | x | x | |||
Democratic Republic of Laos, Myanmar | x | x | x | x | x | |||
Indonesia | National Agency of Drug and Food Control (NADFC) | 15 | 0.5–5 | 0.5–5 | x | 20–50 | ||
Malaysia | Food Safety and Quality Division, Ministry of Health Malaysia | 0.1 | 5–35 | 0.025–0.5 | x | x | ||
Philippines | Department of Agriculture | 10 | 10–50 | 0.5 | 20 | x | ||
Singapore | Food Regulations | 0.1–5 | 5 | 0.025–0.5 | x | x | ||
Thailand | Bureau of Quality and Safety of Food (BQSF) | x | 15–50 | x | x | x | ||
Vietnam | National Institute for Food Control | 0.1–12 | 4–15 | 0.025–0.5 | x | x | ||
CODEX | x | 15 | x | x | x | |||
Codex GCC (Gulf Cooperation Council) | Bahrain, Yemen, Kuwait, Oman, Qatar, Saudi Arabia, United Arab Emirates | 5–12 | 0.05–20 | 0.05 | x | x | ||
EU (European Union) | European Food Safety Authority | Food: Commission Regulation |
0.1–12 | 4–15 | 0.025–0.050 | 5–20 | x | |
MERCOSUR (Mercado Común del Sur) (Southern Common Market) | Argentina, Paraguay, Uruguay, Venezuela (suspended since 2016) | x | 20 | x | x | x | ||
Brazil | 0.5–2.5 | 1–20 | 0.5–2.5 | x | x | |||
USA (United States of America) | Food and Drug Administration | x | 20 | 0.5 | x | x | ||
Algeria | 8 | x | x | x | ||||
Australia, New-Zealand | Australian New Zealand Food Standards Code (ANZFA) | x | 15 | 0.02 | x | x | ||
Bosnia and Herzegovina | 8–12 | 10–15 | x | x | x | |||
Canada | Canadian Food Inspection Agency | x | 15 | x | x | x | ||
China | 0.5–20 | only for: Chinese medicine: Chen pi, suan zao ren, jiang can, pang da hai, tao ren: 10 | 0.5–20 | ≤10–≤50 | 0.5 | |||
Egypt | 0.1–12 | 4–15 | x | x | x | |||
India | APEDA (Agricultural and Processed Food Products Export Development Authority) | x | 10–15 | x | x | x | ||
Japan | Food Safety Commission; Feed: MAFF (Ministry of Agriculture, Forestry and Fisheries) | 10 | 10 | 0.5 | 10–20 | 10–1000 | ||
Korea | 0.1–10 | x | 0.1–10 | 15 | 0.5 | |||
Mexico | x | 20 | x | x | x | |||
Nigeria | National Agency For Food And Drug Administration And Control (NAFDAC) | 20 | x | x | x | x | ||
Peru | Codex | x | 15 | x | x | x | ||
Russia | 5 | x | x | x | x | |||
Turkey | 8–12 | 12–15 | x | x | x | |||
Ukraine | 8–12 | 10–15 | x | x | x |
As
In
Mycotoxins are toxic chemical compounds with low molecular weight (MW < 1000), and due to their diverse chemical structure, there exists no single standard technique for their analysis and/or detection (
Most of the methods used are based on appropriate extraction and clean-up. Sample preparation is one of the most important steps in the determination of mycotoxins. It may add up to two-thirds of the time of the full analysis and could significantly affect the accuracy and precision of the results. The most commonly used clean-up methods applied in aflatoxin analysis are liquid–liquid extraction (LLE), solid-phase extraction (SPE) and QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) methods. In addition, there are a number of other extraction methods in the literature that are less widely used in routine analysis at present.
This is a simple and cheap method for the extraction of aflatoxins. It is based on the solubility properties of the toxin in the aqueous or organic phase or in their mixture. The disadvantage of this method is that it does not provide sufficiently clean analyte in all cases. Researchers have tested AFB1, AFB2, AFG1, AFG2, and AFM1 in breast milk with LLE, then high-pressure liquid chromatography (HPLC) with photochemical derivatization (PHRED) and fluorescence detection (FLD). The limits of the quantification (LOQ) were between 0.005 and 0.03 μg/kg (
Liquid–solid extraction is a simple method for the extraction of aflatoxins from solid matrices of different consistency. The extraction steps include the weighing of homogenized sample of the appropriate particle size, adding the suitable extraction agent and then disintegrating the mixture applying, e.g., shaker, ultra-turrax, blender, vortex, or other methods to extract the components of interest. The extract, before analysis, is filtered and cleaned if necessary. An important step in the process is to select the most effective extraction solvent. The most commonly used extraction agents are mixtures of acetonitrile/water or methanol/water in different ratios (
The use of ultrasound can substantially increase the efficiency of LSE. Ultrasound extraction is most often implemented by immersing the vessel (e.g., Erlemeyer flask, centrifuge tube or vial) containing the sample to be extracted and the extraction solvent into an ultrasonic bath that contains water. During a few-minute treatment, the acoustic cavitation induced by the ultrasound significantly increases the transfer of the analytes and matrix components from the sample to the extraction solvent, thereby increasing the efficiency of extraction (
The PLE procedure, also known as accelerated solvent extraction (ASE), is actually the same as LSE performed under increased pressure and temperature in a suitable pressure-resistant vessel. By selecting a vessel of appropriate size, samples of 1 to 100 g can be extracted. Naturally, in the case of test portions of a few grams, it is important to investigate the magnitude of the random and systematic errors resulting from the reduction of sample size, in order to avoid subsequent inadequate results. The advantages of the procedure are that the extraction process can be automated, and higher extraction efficiency can be achieved in shorter time and with lower amount of extraction solvent (
Supercritical fluid extraction uses a supercritical CO2 fluid for the extraction of the required compound from the matrix. The SFE procedure is mainly used efficiently for the extraction of apolar organic molecules (
Solid phase extraction is a popular clean-up method before qualitative and quantitative measurements of the components that have already been dissolved. Two types of SPE are used. In the case of the multi-step process (conditioning, sample application, washing, elution), either the measurand or the matrix component(s) is bound or removed from the sample (
Special types of SPE procedures are
Compared to other extraction techniques, SPME has a number of benefits. Among others, it requires only sorption and desorption steps, it is a method easy to be automated, compatible with chromatographic systems, allows to achieve high enrichment, appropriate specificity can be assured, and it has very small sample requirements. The SPME method has been tested on the extraction of the aflatoxin content of nuts, spices, cereals and dried fruits. The result of the 8-min LC-MS measurement after clean-up with SPME method showed a sensitivity of 2.1–2.8 pg/ml for aflatoxins, which is more than 23 times greater than that achieved by the direct injection method (10 μl injection volume) (
A specific application of SPE is the so-called immuno-affinity clean-up columns (IAC). They are applicable for the selective binding of mycotoxins as well. These columns contain selective antibodies produced against the mycotoxin to be analyzed and placed in the gel in the column.
Others combined different IAC columns with hyphenated methods for selective clean-up of rye flour, maize and morning cereal samples (
Matrix solid phase dispersion clean-up was used for aflatoxin analysis in olive oil samples with liquid chromatography electrospray ionization tandem mass spectrometric (LC/ESI-MS/MS) detection giving LOQ values between 0.04 and 0.12 μg/kg (
The
TurboFlowTM technology is an automatic online sample preparation method for mass spectrometric analysis of complex matrices (
Magnetic nanoparticles based solid phase extraction based on the use of magnetic or magnetizable adsorbents can be used for the preconcentration of target analytes from large sample volumes (
At present, TLC is the best-known separation technique, but it may not be the most widely used anymore. Its popularity can be associated with its simplicity and low price, since its instrumental requirements at basic level are small. In preparative chemical laboratories TLC can be used to monitor the progress of reactions, determine the purity of a substance or identify compounds present in a given mixture.
In planar chromatography techniques, the stationary phase is an adsorbent material with different thicknesses through which the liquid mobile phase migrates via capillary forces. The most commonly used porous layers are silica gel, chemically modified silica gel, aluminum oxide (alumina), cellulose, chemically modified cellulose, polymer or ion-exchange resin. According to the phases we can differentiate between normal-, reversed- or mixed-phase plates.
HPTLC allows more selective and accurate quantitative measurements. The main differences between the techniques (TLC and HPTLC) can be derived from the differences in the particle size of the stationary phases, their sensitivity and data processing methods (
Over-pressured layer chromatography was developed by Hungarian scientists in the mid-70s (
Over-pressured layer chromatography is carried out on a TLC or HPTLC plate, applying forced flow in a pressurized ultramicro (UM) chamber, based on the principle of liquid chromatography (
Over-pressured layer chromatography integrates the advantages of classical TLC and HPLC, namely the possibility of parallel analysis in thin layer chromatography and the application of forced flow used in HPLC (
The applicability of OPLC for aflatoxins was proven in a validation procedure carried out by the scientists who developed the technology. As a result, the following LODs were defined for aflatoxins: 0.018, 0.100, 0.15, and 0.14 μg/kg for AFG2, AFG1, AFB2 and AFB1, respectively (
The reference methods for the detection of aflatoxins are based on chromatography, more precisely on HPLC/UPLC. During the determination of aflatoxins HPLC-fluorescent detection (FLD) and HPLC-MS/MS systems can be used in most cases. If the separated components are detected with fluorescent detector, there is a need for post-column derivatization (PCD) in order to increase the natural fluorescence properties of AFB1 and AFG1. This derivatization can be based on electrochemical or photochemical principles. For electrochemical derivatization trifluoroacetic acid (TFA), potassium bromide (KBr) or iodine can be used as reagent.
After MultiSep # 228 column clean-up
Post-column derivatization (PCD) including electrochemical bromination is considered as a widely used method for the analysis of aflatoxins. PCD can be achieved with either pyridinyl hydrobromide perbromide (PBPB) or with an electrochemical cell (KobraCell) where KBr is added to the mobile phase. Both derivatization techniques were used in several laboratories to analyze baby foods. When evaluating the results, no significant differences were found between the two PCD techniques. The recoveries ranged from 92 to 101%. During the laboratory analyses the technique resulted in an LOD of 0.02 μg/kg, LOQ of 0.1 μg/kg for AFB1 in baby food (infant formula) samples (
For enhancing the fluorescence properties/response of aflatoxins, PCD using iodine can also be considered as a method for aflatoxin detection. A great disadvantage of PCD using iodine is that the derivatization capability of iodine constantly reduces over time and, consequently, there is a parallel decrease in the sensitivity of the technique. The method yielded reproducible results at 1 μg/kg LOD for peanut butter samples.
Aggressive chemicals (e.g., KBr), however, which shorten the lifespan of instruments and capillaries, can be replaced by PHRED. Significant features of detection of aflatoxins with PHRED and FLD are 0.004 μg/kg (LOD) and 0.015 μg/kg (LOQ) (
There are further possibilities for the fluorescence-based detection of aflatoxins, e.g., HPLC-LIF. Laser-induced fluorescence (LIF) is based on the analysis of fluorescent light emitted during laser irradiation. Sensitivity of the method is 0.1 μg/kg for AFB1 and AFG1, and 1.2 μg/kg for AFB2 and AFG2 (
Derivatization is not needed for the analysis of AFM1 occurring in milk and dairy products, as this component can be analyzed with HPLC-FLD with sufficient sensitivity. AFM1 determination was performed in milk and milk powder samples by using OASISTM Hydrophilic-Lipophilic Balance (HLB) SPE clean-up column, C-18 reversed-phase HPLC column and FLD detection, which is a simple and not the most expensive method. The detection limit/quantification limit of this method was 0.006/0,026 μg/kg for milk and 0.026/0.087 μg/kg for milk powder (
Capillary electrophoresis (CE) is in fact a range of separation techniques based on different separation principles: capillary zone electrophoresis – CZE (based on differences between electrophoretic mobilities of analyses), micellar electro-kinetic capillary chromatography – MEKC (partition of neutral compounds with surface active micelles), capillary gel electrophoresis – CGE (filtration of analytes through a gel network), capillary isoelectric focusing – CIEF (separation of zwitterionic analytes with pH gradient), capillary electrochromatography – CEC (separation of compounds on a column packed with silica gel particles using electric field) (
The classic CZE method, which is based on the differences between the electrophoretic mobilities of the analytes, is unfit for the separation of neutral compounds, which migrate with the same rate as the electro-osmotic flow (EOF) (
Based on a hybrid method combining chromatographic and electrophoretic separation principles, micellar electro-kinetic capillary chromatography (MEKC) extends the applicability of capillary electrophoretic methods to neutral analytes. In the case of MEKC, surface-active compounds are added to the buffer solution in a concentration exceeding their critical micellar concentration. Consequently, they form micelles, which affect the electrophoretic migration, like any other charged particle. The separation is based on the differential distribution of the analyte between the two phases of the system: the mobile liquid phase and the micellar pseudostatic phase (
Hyphenated techniques usually mean separation procedures connected to a mass spectrometer. Of these, LC/UPLC-MS, SFC-MS, CE-MS and Chip-MS techniques have been used to determine aflatoxins. These procedures are presented below.
Until the early 1990s, thermospray, particle beam and fast atom bombardment interfaces were used for the LC/MS measurement of mycotoxins (
It needs to be mentioned, however, that the wider proliferation of these methods is hindered by their high price and the costs of training personnel for their professional operation and method development.
LC-MS analysis of aflatoxins is possible with the application of all three commonly used atmospheric pressure ion sources. Review publications reveal that the atmospheric pressure electrospray (ESI) source is used predominantly for the LC-MS determination of aflatoxins (
If only aflatoxins need to be determined in samples to be tested, APPI can be considered to be the best choice among atmospheric pressure ion sources, as it has considerably lower background noise and ion suppression compared to ESI and APCI. The reason is that in the case of direct photoionization (direct APPI), only components with ionization potential (IP) value below the energy of photons emitted by the vacuum UV lamp of the ion source (10 eV) are ionized in the ion source. In other words, significant portion of matrix components and potential contaminants in the mobile phase will not give noise during photoionization (signal enhancement/ion suppression). It was found that a mass spectrometer will be 2–3 times more sensitive during aflatoxin measurement, if equipped with APPI instead of ESI ion source (
Leaving the atmospheric pressure ion source, the ionized molecules enter the vacuum chamber of the mass spectrometer, and they reach the actual mass filter/mass analyzer through an iontransporting and focusing region. The mass analyzer can be single-stage or multi-stage (MS/MS) (
Simple schematic of LC-MS system.
Due to the lack of collision-induced dissociation (CID), the fragmentation of molecular ions is not possible in mass spectrometers equipped with single-stage mass analyzers (e.g., single quadrupole) (with the exception of in-source CID), which would be prerequisite to the MS/MS spectrum based identification and exact determination of components eluting from the LC/UPLC column. Single-stage type mass analyzers are not compliant with EU requirements of residue analysis, requiring a precursor ion, two product ions and their ratio for the MS identification of a component (
The most widespread and one of the best solutions for the quantitative determination of organic compounds with hyphenated techniques (e.g., LC/UPLC-MS/MS) is certainly the application of mass spectrometers equipped with triple quadrupole (QqQ) mass analyzer. LC/UPLC-QqQ-MS procedures are the most widespread among multitoxin methods (including aflatoxins, too) (
If necessary, this information can be acquired by the application of a hybrid mass spectrometer such as a quadrupole-linear ion trap (QTRAP®) equipment, which enables both quantitative determination and confirmation based on the mass spectrum (
LC-MS/MS having QTRAP® mass analyzer has been applied for multi-toxin measurement of aflatoxins in baby food. LOD and LOQ values ranged between 0.05–0.4 and 0.1–1 μg/l for the four aflatoxins (AFB1, AFB2, AFG1, and AFG2); the recovery was 78% (
For aflatoxin analysis, LC-MS instruments including the so-called 3D iontrap (IT) mass analyzer have already been used.
The ion suppression/enhancement caused by the matrix effect can rarely be avoided even by these sophisticated multi-stage mass analyzers, particularly, when the raw sample extract is analyzed by LC/UPLC-MS/MS without clean-up (“extract and shoot” method). To avoid such problems and reduce the LOD/LOQ values, the sample clean-up procedures discussed above are extensively used before the LC/UPLC-MS/MS measurement of mycotoxins, including aflatoxins. Prominent procedures of these are the IAC clean-up (
The SFC technique combines the numerous advantages of liquid and gas chromatography. Its application is beneficial for non-volatile, heat sensitive, reactive and multicomponent samples. SFC provides results faster than HPLC, because diffusion of the substance is 10 times faster in the supercritical solvent (CO2) than in liquid phase. The analysis is usually performed in environmentally benign manner without the use of organic solvents; however, MeOH or a 1:2 MeOH:ACN mixture is added to CO2 as a polar modifier if necessary (
The SFC procedure combined with a tandem mass spectrometer containing ESI ion source (SFC-MS/MS) has been used for the simple, fast and sensitive determination of aflatoxins in edible oil (
In the first chip-MS-based system for AFB1 determination, a plastic microfluidic chip was used for the automatic affinity dialysis, concentration and subsequent ESI-MS determination of reaction mixtures containing AFB1 antibodies and aflatoxins (
For the determination of aflatoxins in peanut products, a procedure was also developed, where a nano LC pump was coupled to a QqQ-MS through a chip-ESI-MS ion source (chip-nano LC) (
Beside the sensitivity of determination and the low amounts of sample needed, the significance of the chip-MS procedure is its environmentally benign manner resulting from low solvent consumption. Due to decreasing prices of the chips and instruments, the spreading of these methods is to be expected.
Rapid tests developed for the analysis of aflatoxins are built upon several different technologies. The most common ones are the enzyme-linked immunosorbent assay (ELISA), lateral flow devices (LFD) and chemical methods. Rapid tests are indispensable to provide analytical results within a short time. These procedures enable the analysis to be easily performed with lower prices, even at the location of sampling.
The vast majority of the rapid methods used for aflatoxin measurement are immunoassays based on the reaction of a special antibody and the antigen of the analyte, which can be detected by various markers.
Many markers have been developed over the years, including enzymes, radioisotopes, fluorophores, gold nanoparticles and other sensitive optical and electrochemical components (
The aim of the ELISA technique is the qualitative or quantitative determination of mycotoxins found in the analytical sample, based on the application of antibodies, which are specific to compounds to be analyzed. The method is based on an enzyme-linked color reaction. For the detection of mycotoxins, competitive-type ELISA tests are typically used. Consequently, the measured color intensity is inversely proportional to the concentration of the measured compound (
Structure of a competitive ELISA.
These ELISA analytical systems are excellent screening devices, provide quantitative results in a short time period, and as previously mentioned, they can often be used at the location of sampling, too. However, cross-reactions with molecules very similar to the analyzed substance and matrix effects found during the analysis of different products may influence the results. Naturally, quantitative determination of AFB1, AFT and AFM1 can also be performed with the ELISA technique (
The sensitivity of the ELISA kit depends on the manufacturer. For instance Romer Labs Inc. United States reported an LOD of 0.018 μg/kg and LOQ of 0.025 μg/kg with recoveries ranging between 80 and 120% for the determination of AFM1 in milk.
An improved version of ELISA is
Radioimmunoassay applies radioactively labeled molecules during the stepwise formation of immunocomplexes. RIA is a highly specific and very sensitive method. In the case of agricultural samples (maize, soybean, wheat and rice), the LOD/LOQ of the method was 0.2/0.5 μg/kg for AFB1. The recovery was between 92 and 107% (
RIA requires the application of an expensive, special equipment to minimize the adverse effects caused by gamma rays (
For this reason, in order to avoid health risks, other types of marker compounds might be more beneficial for the analysis of aflatoxins (
Immuno reagents with probes based on fluorescent labeling are already used widely. By combining the highly sensitive fluorescence method with the sensitivity of the measuring instrument, a simple and rapid analytical procedure can be achieved, where the concentration of the analyte can be directly measured in the reaction mixture. The problem with FIA methods was the low sensitivity caused largely by the high background noise of the fluorometric measurement (
Chemiluminescence immunoassay is an immunoanalytical technique, where the marker is a luminescent molecule. Luminescence is usually the emission of visible or near visible (λ = 300–800 nm) radiation. The advantage of luminescence in spectrophotometry over absorption is that its signal is absolute, while the latter one is relative. Chemiluminescence methods can be direct, by using luminophores as markers or indirect, by using enzyme markers. Each of them can be competitive or non-competitive.
In some areas of analytics, color label markers (e.g., gold nanoparticles, colored latex) are the most widely used for rapid and qualitative determination. In addition to the above mentioned markers, aflatoxins can also be made fluorescent by irradiation with UV or laser light. However, they may also be derivatized with various chemical agents (e.g., iodine, bromine, etc.) (
The most widely used immunological devices are microplate-based immunoassays, lateral flow immunoassays (LFIAs) and different biosensors (immunosensors) (
When analyzing aflatoxins, microtiter plate and reader-based immunoassays allow simultaneous analysis of many samples, since the plates used have multiple wells. Most widely used microplate-based immunoassays are ELISA, fluorescence and chemiluminescence based analyses (
Immunochromatographic dipsticks are another appropriate technology on the market of rapid mycotoxin tests. The basis of the method is the detection of the analyzed component by linking to a specific antibody in the test zone, which is placed on a membrane fixed on the dipstick. In addition to the test zone there is the control zone on the membrane verifying the correct functioning of the test. When the sample extract flows on the membrane, it passes the test and control zones and, depending on the concentration of the toxin, both (test and control) lines or only the control line will become visible. The dipstick can be evaluated visually by the naked eye or with the help of a reading device. When quantitative results are needed, the evaluation is performed by an instrument (reflectance photometer), which measures the intensity of the test and control lines and evaluates the results on the basis of data determined. The immunochromatographic dipstick is a rapid, easy-to-perform technique, which is ideal and cost-effective even for the analysis of a single sample. Similar to the ELISA technique, cross-reactions and matrix effects occurring during the analysis of certain products limit the applicability of the dipstick. For the determination of aflatoxins, qualitative, and quantitative immunochromatographic dipsticks are available (
However, results are available from the analysis of certain more complex matrices as well. The visual detection limit for AFB1 in this case was 5 μg/kg (
Structure of lateral flow immunoassay.
Practical application of the lateral flow immunoassay.
The detection options of LFIA depends on the type of the marker. In case of color label markers (e.g., gold nanoparticles, colored latex), besides instrumental reader, there is a possibility of visual evaluation, while in case of fluorescence (e.g., quantum dots, ruthenium complexes) or other markers (e.g., enzyme labels or paramagnetic labels), only readers or expensive detectors can be used for quantification (
A portable immunosensor based on chromatographic time-resolved fluoroimmunoassay has been developed for fast on-site sensitive determination of AFB1 in food and feed samples. CTRFIA provides an increased positive signal and low signal-to-noise ratio in time-resolved mode.
LFIA is considered as a fast and sufficiently sensitive screening method. The need for the development of multi mycotoxin analysis has arisen in this research area as well, as this method was previously only applicable for one mycotoxin analysis at a time. The publication of
Chemical sensors are small-size devices, which convert the chemical information characterizing the composition of the compound into electronic or optical signal by continuous tracking, in real time. Such sensors represent modern analytical devices of our days. They take over the role of traditional analytical methods in several areas, since they can be well miniaturized due to their robust structure, can be integrated in automatic systems, and can be applied in
Biosensors, a sub-group of chemical sensors, are special selective analytical devices, which are closely linked to or integrated into a physico-chemical transducer (e.g., electrochemical, optical, piezoelectric, etc.) and contain a substance of biological origin (e.g., enzyme, tissue, microorganism, antibody, etc.) or an imitating substance (e.g., molecularly imprinted polymers, MIP) (
Detection is based on the linking of the analyte to its specific complementary biological element (bioreceptor), which is fixed on a suitable portable surface (
Techniques based on labeling molecules are increasingly lagging behind in the area of measurement of interactions between different molecules in biological and biochemical systems. Surface plasmon resonance (SPR) is a distinguished method among label-free analytical methods, which can analyze the interactions near surfaces, based on the SPR phenomenon. It can indicate not only the endpoint, but the whole process can be monitored.
Mass-change-based sensors most often use mechano-acoustic sensors based on the change of resonance frequency, with label-free techniques of quartz crystal microbalance (QCM) and optical waveguide light-mode spectroscopy (OWLS). Similar to other label-free detection methods, OWLS enables the real-time inspection of molecular-level processes at the interface. This can be achieved by the application of the two-part integrated optical waveguide sensor (chip), which is the basis of the technique. A sensitive method could be developed for mycotoxins including aflatoxins from pepper, applying gold nanoparticles of different sizes and origin (
Lab-on-a-chip is a device, which integrates one or more laboratory functions into one chip, having a size of only a few square centimeters. LOCs are able to manage extraordinarily small amounts of liquid below pico-liter quantities (
Biosensors enable real-time detection of AFB1 in foods with a fast, sensitive, completely automated and miniaturized system (
Flow injection immunoassays is an automatic method for chemical analyses, where the sample is injected into a flowing carrier solution, which is mixed with the reagents before reaching the detector. The automated system can be combined with several different detectors, e.g., biosensor, spectrophotometer, or even with mass spectrometer. For the determination of AFM1 in milk, a FI-IA method was developed with amperometric detection (
Currently, several other analytical procedures are under development, which can be grouped in several ways. Some procedures are exceptions regarding the groupings as they may be allocated into more than one group such as direct analysis in real-time-mass spectrometry (DART-MS), near infrared spectroscopy (NIRS), Luminex xMAP® technology and Biochip Array Technology (BAT) as a new technological direction.
Since there is no chromatographic or eletrophoretic separation in MALDI-TOF-MS, it is not in the group of hyphenated techniques.
The DART-MS procedure includes no
Near-infrared spectroscopy is an innovative technology used in the food-, chemical-, pharmaceutical- and petrochemical industries. Coupled with the development of chemometric techniques, this technology is an efficient, fast, reliable and non-destructive analytical method to measure the qualitative and quantitative characteristics of organic substances. Results of earlier studies showed that the application of the NIRS technique was successful in the detection and to some extent the determination of chemical contaminants, for example mycotoxins (
The xMAP technology enables the multiplexing of biological tests, and the reduction of time, human resources and costs spent, compared to traditional methods such as ELISA, Western blot or PCR techniques (Luminex, Austin, TX, United States). Microbeads are labeled with a special mixture of dyes, resulting in color-coded microbeads. The different microbead clusters can be mixed. As each microbead carries an individual recognition signal, the xMAP system can detect which microbead belongs to which cluster. With the aid of several lasers or LEDs, a high-speed digital signal processing system reads the processes taking place on the surface of each color-marked microbead. Red laser excites both the red and infrared dyes found in the microbeads, enabling the grouping of the microbead into one of the potential 100 clusters. Green laser induces fluorophore linked to the surface of the microbeads, enabling the determination of the substance contained in the sample. Theoretically, 100 different measurements can be performed in one sample at the same time.
Biochip Array technology is an immunoassay based technology enabling the simultaneous semi-quantitative detection of various mycotoxins from various cereals and cereal based products. The immunoassays define discrete test regions on the biochip surface on which the immunoreactions take place. Applying specific Myco 7 kit, the screening decision levels were for aflatoxin B1 and ochratoxin A (0.25 μg/kg); aflatoxin G1, deoxynivalenol, zearalenone, T2-toxin, fumonisin B1 0.5, 100, 2.5, 5, and 10 μg/kg, respectively. The within laboratory reproducibility was 11.6% and the overall average recovery was 104%. With multiplex Myco arrays, results can be obtained within 3 h, which is comparable to that required when using a single ELISA kit. The chemiluminescence reactions can be monitored with digital picture imaging technology. such as Evidence Investigator. The flexibility of the technology allows extension of analytical profile and implementation of new assays. It should be noted that the cost of the instrument is in the range of HPLC systems, though its operation cost is lower (
As aflatoxins pose danger to both humans and animals, researchers are continuously searching for analytical methods most suitable for specific tasks. Due to the development of analytical and IT techniques, increasingly faster and more sensitive have come into focus in the last decades, but only a few of them have gained applicability in routine analysis.
Immunoanalytical methods (e.g., LFIA, ELISA) proved to be promising to detect the aflatoxin present in low levels in feed and food. Immunoanalyses with portable devices are simple, fast, sensitive, and cost-effective. Occasionally they are even capable of quantification with the aid of a reader. However, application of these methods provides only informative data on the given analyzed product. Their disadvantage is that despite their general suitability for the analysis of raw materials, interferences may occur at the measurement of more complex matrices. Therefore, the areas of future research are primarily including the removal or compensation of matrix components or compensating their adverse effects, application of nanoparticle technology, specific antibody production, automation and the miniaturization of instruments.
Several immunological methods including ELISA and other fast antibody-based tests can be used for screening purposes. However, confirmatory analyses with more robust methods are needed in these cases as well.
Analytical methods for the accurate quantitative determination of aflatoxins are under constant development.
Future developments will be directed to lab-on-a-chip miniaturized technologies, chip-based biosensors and multitoxin detection by immuno-based techniques, where some analytical steps will be partly or fully replaced by micro/nanotechnology. An important goal for the research of chip-based technology is to achieve simple, fast and cost-effective methods, which can be combined with other devices and methods (e.g., immunochemical analyses) in a flexible way. It can be expected that methods and technologies, recently or further developed, will be more user-friendly and will provide better results.
Nowadays, ELISA is the most commonly used fast method in the laboratories. Using test strips for solid matrices in the fields is a technology which needs to be developed before practical application. There are many publications regarding this topic. Sample homogenization and extraction needs more development. Under industrial laboratory circumstances, methods based on test strips are mainly used as they provide faster results than ELISA.
For the confirmation of screening methods and the exact quantitative determination of aflatoxins, HPLC-FLD, combined with pre- or post-column derivatization is still the most commonly used procedure.
The best method for the exact, reproducible, qualitative and quantitative determination of aflatoxins today is HPLC-MS-MS technique using triple quadrupole mass analyzer.
However, in industrial and smaller laboratory circumstances, regarding screening tests the future is pointing toward fast and micro methods with low solvent-need, such as immuno flow cytometry.
This publication summarizes the analytical techniques that were or can be used for aflatoxin measurement or detection. The major deficiency of the majority of published methods is that they do not include the processes applied for reduction of large laboratory samples to the few grams of test portions to be extracted. Moreover, the evaluation of repeatability or reproducibility of the results, if reported, was based on a few spiked samples. Materials contaminated naturally have rarely been used to evaluate the performance of the developed methods. Much more attention is needed in the future to characterize the contribution of sample size reduction and test portion size to the overall uncertainty of the results, which are required for the correct interpretation of the measured concentration in relation to the legal limits and estimating the exposure of consumers.
In the future, when methods are evaluated from technical point of view, sources of errors must be indicated, and potential limitations of the performance parameters must be pointed out. The spike levels and the number of replicates applied must be indicated together with the reported repeatability and if possible reproducibility data. Finally, it is a must to indicate, whether repeatability and or reproducibility of mycotoxin concentration was investigated in naturally contaminated samples or not.
GM and TB contributed to create the conception and design of the review. CA, AN, and VK organized the database. GM and TB wrote the first draft of the manuscript. ÁA reviewed the first and revised drafts of the manuscript. ZF, AZ, KK, and ÁJ have done the language verification. GM, TB, and ÁA finalized the manuscript and prepared for publication. All authors contributed to manuscript revision, read, and approved the version to be submitted.
CA and TB were employed by Fumizol Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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