**2.1. Gas Chromatography (GC)**

for instance, European Commission Regulation 2010/165/EC established limits of 8 and 15 µg/kg for AFB1 and total AFs respectively. Several methods have been developed to deter‐ mine AFs in foods, for instance: immunoassays techniques [3], Thin layer chromatography (TLC) [4]. High-Performance liquid chromatography (HPLC) with fluorescence detection [5]. Not long ago, analytical methods based on clean-up with immunoaffinity column and HPLC with postcolumn derivatization and fluorescence detection have gained much popu‐ larity. Even though, several works have been reported to determine AFs in foods by using these methods, only few validation studies are available which comply with certain regula‐ tions. There are immunochemical methods which are based principally on enzyme-linked immunosorbent assay (ELISA) that has a good sensitivity; speed and simplicity; however these kinds of instruments are expensive. An alternative of improving the disadvantages of the previous methods are trying to be solved by biosensors which are devices that enable identification and quantification of aflatoxins. Exists a variety of biosensors that base their performance in several principles, those are: optical, optoelectronic, electrochemical, piezo‐ electric, DNA and combined. In the same way, there are other methods not as common as the previous methods but they have a wide utility as well. The most important are those that

The chapter has two main proposes. First, to give general description of the most common methods used for quantifying aflatoxin concentrations. And second, to give a perspective about the tendencies in the development of systems, based on the so far used methods,

Chromatography is one of the most common methods for quantifying aflatoxins. This meth‐ od started with Gas chromatography (GC). However, technology advancements allow the development of new chromatography-based techniques. Examples of these improvements are Liquid Chromatography (LC), Thin Layer Chromatography (TLC) and High-Perform‐ ance Liquid Chromatography (HPLC). The quantification of aflatoxins using chromatogra‐ phy relies principally on fluorescence detection depending of the compounds under analysis. So that, nowadays there are several works employing a variety of fluorescence de‐ tection in order to improve the sensibility of these techniques. In the same manner, UV visi‐ ble (Vis) wavelength spectrum has been used to improve the detection and quantification of aflatoxins. Others methods employed to accomplish the chromatographic quantification of

Before the chromatographic analysis, the toxic compounds must be extracted to remove the interfering particles; such extraction is commonly done by a solvent in a clean-up step that regularly uses an immunoaffinity column (IAC). This procedure increases the sensitivity and diminishes the necessary sample quantity in the analysis. Other system used to quantify single and multiple aflatoxins is the mass spectrometer which is coupled commonly with a HPLC system. This section explains the most common methods based on chromatographic princi‐ ple and the steps before and after for accomplishing the analysis with better assessments.

which could be employed in the near future to detect and quantify aflatoxins in food.

base their principle on electrochemistry, spectroscopy and fluorescence.

**2. Chromatography methods**

290 Aflatoxins - Recent Advances and Future Prospects

AFs are array of diodes and refraction index.

The instrumentation of gas chromatography comprises well defined components that ac‐ complish specific functions of the overall process. GC almost reaches the complete develop‐ ment of technological level in 50 years. The Figure 1 shows the principal components that constitute a Gas Chromatography System.

**Figure 1.** Block diagram of a gas chromatograph.

Gas supply means to move the sample through the column; the possible gases to choice are restricted and the most commonly used are nitrogen and helium. It is also necessary to con‐ trol the gas flow because it can have impact on the separating performance. Tramps can be purchased to reduce or remove hydrocarbons and oxygen in the carrier gas. The chromato‐ graphic process starts when the sample is introduced into the column, ideally without dis‐ rupting flows in the column. Therefore, the deliberation of the sample into the column should be controlled, reproducible and rapid. The GC include an oven which is an impor‐ tant component in this process, because the vapor state must be maintained thought the GC separation, therefore, a good control of temperature must be kept. Another important com‐ ponent of the gas chromatography is the detector which has been evolved through the years. Nowadays, the mass spectrometer (MS) promises to be the most suitable method to be cou‐ pled with GC.

#### **2.2. Liquid Chromatography (LC)**

The principle of liquid chromatography is the separation process which is based on the dis‐ tribution between two phases. The sample is propelled by a liquid which percolates a solid stationary phase. Thus a variety of stationary phases can be used in liquid chromatographic systems. The liquid chromatographic process and the separation of the sample may be ach‐ ieved, both, in low and high pressure systems. And the correct selection of the separation mode stationary phase and mobile phase may be straight (normal) phase, reversed phase and size-exclusion (SEC) or ion-exchange (IEC) liquid chromatography respectively.

#### **2.3. Thin-Layer Chromatography (TLC)**

Thin-layer chromatography is a very commonly used technique in syntactic chemistry. This technique identifies compounds by determining the purity and progress of a reaction. Such reaction is fast and only requires a small quantity of the compounds. In TLC the mobile phase is liquid and the stationary process is a solid adsorbent. Several factors determine the efficiency of a chromatographic separation. The adsorbent should show a maximum of se‐ lectivity toward the substances that are being separated so that the differences in rate of elu‐ tion will be large. For the separation of any mixture, some adsorbents may be too strongly adsorbing or too weakly adsorbing.

In the work presented by [14], aflatoxin B1 was detected in animal liver (pig, chicken, turkey, beef, calf) and chicken eggs by a process consisting of sample immunoaffinity column cleanup and liquid chromatography with post column bromination in kobra cell and florescence detection, which was introduced and validated. The validation process was done based on the decision 2002/657/EC established by European commission. Figure 2 shows an exempli‐ fication of the common steps included in the detection and quantification of aflatoxins used nowadays. These steps include the processing of the sample before introduced it into the HPLC system. The most commonly pre-process technique is the IAC which permits to puri‐ fy and decreases the quantity of sample needed in the experiment. The other problem that IAC tries to diminish is the non homogenization of the sample. HPLC is the most common based-chromatography technique used at this time; nevertheless, it needs of the steps shown in Figure 2 to work better. The principal advantages of IAC are the effectiveness and specif‐ icity in the purification of the extraction that provide the economic use of solvents and the improved chromatographic performance achieved with samples [15]. Also IAC can be used to analyze commodities that contain different aflatoxins. The fluorescence of aflatoxins make possible the use of a fluorometers to detect and quantify the concentration of aflatoxins in a sample, however, sometimes it is necessary to improve this property. Derivatization proc‐ esses have been employed to improve the fluorescence of aflatoxins and by consequence the sensitivity of the system. Years ago, the quantification of the concentration of aflatoxins was accomplished by comparison of the sample and authentic standards using visual estimation of fluorescence of the separated spot long wavelength UV radiation [15]. A great advance in the detection and quantification of aflatoxins is the introduction of mass spectrometry as a viable detector system. The advantage of coupling mass spectrometer to LC is that, it allows improving the detection limits. For aflatoxins quantification, a number of instruments have been used including single quadrapole, triple quadrapole and lines ion single quadrapole

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and linear ion trap instrument.

**Figure 2.** Common steps followed actually to quantify aflatoxins using HPLC.

Chromatography has been one of the most common methods used for the detection and quantification of aflatoxins. This technique has been evolving through the years from GC to HPLC which nowadays is the most used chromatography-based technique employed for aflatoxin detection. TLC and LC are methods that have been going displaced by HPLC be‐ cause of its sensitivity, specificity and facility of operation. At the present time, HPLC by it‐ self is not enough and it is necessary to employ pre-process and derivatization techniques that, jointly with detectors, improve the purity of the sample and the fluorescence property of the aflatoxins. In consequence, the detectors, which generally take into account the fluo‐ rescence of the aflatoxins, accomplish better quantification and offer more sensitivity. The most common method for pre-processing the sample is IAC that allow having high specifici‐

## **2.4. High-Performance Liquid Chromatography (HPLC)**

HPLC is now the most common used chromatographic technique for a detection of a wide diversity of mycotoxins, especially for aflatoxins [6]. The analysis sample cleanup can be performed by liquid-liquid partitioning, solid phase extraction (SPE), column chromatogra‐ phy, immunoaffinity clean-up (IAC) columns, and multifunctional clean columns [7]. Re‐ cently the utility of the IAC columns has become very popular because of its high selectivity. IAC columns can be used for sample preparation before HPLC analysis either in off-line or in-line mode [8]. While in the off-line immunoaffinity cleanup the purification step is done separately by an expert, the IAC column is directly coupled to the HPLC system in the inline immnunoaffinity cleanup. A chromatographic process can be defined as separation technique which involves mass-transfer between stationary and mobile phase. HPLC uti‐ lizes a liquid mobile phase to separate the components of a mixture. The stationary phase can be a liquid or a solid.

#### **2.5. Combined methods**

Nowadays there are combinations of the aforementioned methods with pre-process techni‐ ques. Such methods are able to detect in a better way, the concentration of aflatoxins in a solution. Immunoaffinity column sample clean-up followed by a normal or reverse phase of HPLC separation with fluorometric detection is mostly used for quantitative determination of AFM1 because of the characteristics of specificity, high sensitivity and simplicity of operation [9].

There are several works that employ IAC combined with HPLC and fluorometric detection for detecting and quantifying precisely concentrations of AFM1 [10]. In the reference [11] the authors employ IAC, HPLC and an optimized photo-derivatization to assess the concentra‐ tion of mycotoxins of airborne from a house in Dalian, china. In [12] is employed, in the same manner, IAC and an HPLC equipped with fluorescence detector to determine the quantity of aflatoxins AFM1 in milk used for preparing market milk, yogurt and infant for‐ mula products in South Korea. As can be seen, the HPLC process commonly needs a clean‐ up process with immunoaffinity columns before detection. Such sample preparations are multistage, expensive and time-consuming. The combination of GC with MS (GC-MS) for the analysis of aflatoxins can provide definitive, qualitative and quantitative results, but it requires a derivatization step, which lengthens the analysis time and may compromise ana‐ lyte recoveries [13]. Post-column derivatization is a version of chromatography where the components that were separated eluting from the column are derivatized prior to entering the detector. The derivatization process is generally carried out during the process, during the transfer of the sample components from the column to the detector. The derivatization may also be accomplished before the sample enters into the column or the planar medium, thus it is called pre-column derivatization.

In the work presented by [14], aflatoxin B1 was detected in animal liver (pig, chicken, turkey, beef, calf) and chicken eggs by a process consisting of sample immunoaffinity column cleanup and liquid chromatography with post column bromination in kobra cell and florescence detection, which was introduced and validated. The validation process was done based on the decision 2002/657/EC established by European commission. Figure 2 shows an exempli‐ fication of the common steps included in the detection and quantification of aflatoxins used nowadays. These steps include the processing of the sample before introduced it into the HPLC system. The most commonly pre-process technique is the IAC which permits to puri‐ fy and decreases the quantity of sample needed in the experiment. The other problem that IAC tries to diminish is the non homogenization of the sample. HPLC is the most common based-chromatography technique used at this time; nevertheless, it needs of the steps shown in Figure 2 to work better. The principal advantages of IAC are the effectiveness and specif‐ icity in the purification of the extraction that provide the economic use of solvents and the improved chromatographic performance achieved with samples [15]. Also IAC can be used to analyze commodities that contain different aflatoxins. The fluorescence of aflatoxins make possible the use of a fluorometers to detect and quantify the concentration of aflatoxins in a sample, however, sometimes it is necessary to improve this property. Derivatization proc‐ esses have been employed to improve the fluorescence of aflatoxins and by consequence the sensitivity of the system. Years ago, the quantification of the concentration of aflatoxins was accomplished by comparison of the sample and authentic standards using visual estimation of fluorescence of the separated spot long wavelength UV radiation [15]. A great advance in the detection and quantification of aflatoxins is the introduction of mass spectrometry as a viable detector system. The advantage of coupling mass spectrometer to LC is that, it allows improving the detection limits. For aflatoxins quantification, a number of instruments have been used including single quadrapole, triple quadrapole and lines ion single quadrapole and linear ion trap instrument.

**Figure 2.** Common steps followed actually to quantify aflatoxins using HPLC.

efficiency of a chromatographic separation. The adsorbent should show a maximum of se‐ lectivity toward the substances that are being separated so that the differences in rate of elu‐ tion will be large. For the separation of any mixture, some adsorbents may be too strongly

HPLC is now the most common used chromatographic technique for a detection of a wide diversity of mycotoxins, especially for aflatoxins [6]. The analysis sample cleanup can be performed by liquid-liquid partitioning, solid phase extraction (SPE), column chromatogra‐ phy, immunoaffinity clean-up (IAC) columns, and multifunctional clean columns [7]. Re‐ cently the utility of the IAC columns has become very popular because of its high selectivity. IAC columns can be used for sample preparation before HPLC analysis either in off-line or in-line mode [8]. While in the off-line immunoaffinity cleanup the purification step is done separately by an expert, the IAC column is directly coupled to the HPLC system in the inline immnunoaffinity cleanup. A chromatographic process can be defined as separation technique which involves mass-transfer between stationary and mobile phase. HPLC uti‐ lizes a liquid mobile phase to separate the components of a mixture. The stationary phase

Nowadays there are combinations of the aforementioned methods with pre-process techni‐ ques. Such methods are able to detect in a better way, the concentration of aflatoxins in a solution. Immunoaffinity column sample clean-up followed by a normal or reverse phase of HPLC separation with fluorometric detection is mostly used for quantitative determination of AFM1 because of the characteristics of specificity, high sensitivity and simplicity of operation [9].

There are several works that employ IAC combined with HPLC and fluorometric detection for detecting and quantifying precisely concentrations of AFM1 [10]. In the reference [11] the authors employ IAC, HPLC and an optimized photo-derivatization to assess the concentra‐ tion of mycotoxins of airborne from a house in Dalian, china. In [12] is employed, in the same manner, IAC and an HPLC equipped with fluorescence detector to determine the quantity of aflatoxins AFM1 in milk used for preparing market milk, yogurt and infant for‐ mula products in South Korea. As can be seen, the HPLC process commonly needs a clean‐ up process with immunoaffinity columns before detection. Such sample preparations are multistage, expensive and time-consuming. The combination of GC with MS (GC-MS) for the analysis of aflatoxins can provide definitive, qualitative and quantitative results, but it requires a derivatization step, which lengthens the analysis time and may compromise ana‐ lyte recoveries [13]. Post-column derivatization is a version of chromatography where the components that were separated eluting from the column are derivatized prior to entering the detector. The derivatization process is generally carried out during the process, during the transfer of the sample components from the column to the detector. The derivatization may also be accomplished before the sample enters into the column or the planar medium,

adsorbing or too weakly adsorbing.

292 Aflatoxins - Recent Advances and Future Prospects

can be a liquid or a solid.

**2.5. Combined methods**

thus it is called pre-column derivatization.

**2.4. High-Performance Liquid Chromatography (HPLC)**

Chromatography has been one of the most common methods used for the detection and quantification of aflatoxins. This technique has been evolving through the years from GC to HPLC which nowadays is the most used chromatography-based technique employed for aflatoxin detection. TLC and LC are methods that have been going displaced by HPLC be‐ cause of its sensitivity, specificity and facility of operation. At the present time, HPLC by it‐ self is not enough and it is necessary to employ pre-process and derivatization techniques that, jointly with detectors, improve the purity of the sample and the fluorescence property of the aflatoxins. In consequence, the detectors, which generally take into account the fluo‐ rescence of the aflatoxins, accomplish better quantification and offer more sensitivity. The most common method for pre-processing the sample is IAC that allow having high specifici‐ ty in the selection of aflatoxin in samples contaminated with more compounds. So that, it is clear that the tendency of the technology is principally to improve the stage of pre-processes and derivatizations in order to achieve a more precise quantification. The more sophisticat‐ ed the technology of pre-process and derivatization the more high specificity and sensitivity the method gets.

exposed to a buffer with the tested sample. Antibodies are released into the buffer; some of them will bind to the immobilized conjugate, while the remainder will join the analytes in the buffer. After a while, it is released a second group of enzyme-labeled antibodies, or other fluorescent-core kind of signal material; these are joined to the first antibodies, which in turn

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The process of a direct competitive assay does not require a second labeled antibody. For this type of analysis, either aflatoxins (aflatoxin protein conjugate) or specific antibodies can be immobilized. The complementary component to that immobilized is marked and added to the sample. A competitive reaction occurs between the Ab-Ag, so that some marked components remain adhered to the immobilized one, while others adhere to those

For non-competitive assays, Abs are immobilized. When immobilized Abs make contact with the sample molecules and AF bind to them because Abs are attached to a sensitive sur‐ face, the amount of analyte bounded by Abs will result in an electrical or optical variation. However, sandwich format is preferred for this kind of assay, in which the sample is mixed previously with Bovine Serum Albumine (BSA) because AF molecules are small. Previously it was carried out a treating of the sample with Bovine Serum Albumine (BSA) (carrier pro‐

Any type of assay involving Ab-Ag reaction, where one of the reactants is conjugated with an enzyme, is considered as an ELISA. Amplification and visualization of Ab-Ag interaction are achieved by this enzyme conjugation. ELISA is the most used immunoassays used in

Antibodies or antigens are immobilized on a solid-phase matrix by linking them, either through adsorption or covalently. Reactants are usually adsorbed on to the wells of 96- or 384- microtitre plate of polystyrene, where adsorption is characterized by a strong hydro‐ phobic binding and slow dissociation rate. After this coating process, the residual proteinbinding capacity of solid matrix is blocked by exposing it to an excess of unrelated protein (e.g. gelatin or bovine serum albumin "BSA"). The next step is the addition of a test solution, which may be serum with an unknown concentration of antibodies against the immobilized antigen. After incubation and washing, binding of specific antibodies is visualized by the addition of antiimmunoglobulin-enzyme conjugate followed by a substrate, generating a colored product when hydrolysed. This change of color is proportional to the amount of an‐ tibodies bounded and may be recorded visually or spectrophotometrically. In case of an an‐ tigen measurement, the process is the same but may be done by using competitive- or sandwich-type assays. When using microarray format, ELISA may detect other toxins, such

tein). BSA binds to the AFs and the conjugate is captured by immobilized Abs.

**3.3. Enzyme-Linked Immunoabsorbent Assays (ELISAs)**

are attached to the protein-conjugate immobilized aflatoxins.

present in the sample.

food-aflatoxin detection.

as AFs in a sample [18].
