**12. Aflatoxins identity assessment**

level of 10 µg/kg total aflatoxins in ready-to-eat almonds, hazelnuts, and pistachios at a

All analytical procedures include three steps: extraction, purification, and determination. The most significant recent improvement in the purification step is the use of solid-phase ex‐ traction. Extracts are extracts are cleaned up before instrumental analysis (thin layer or liq‐ uid chromatography) to remove coextracted materials that often interfere with the

Thin layer chromatography (TLC), also known as flat bed chromatography or planar chro‐ matography is one of the most widely used separation techniques in aflatoxin analysis. Since 1990, it has been considered the AOAC official method and the method of choice to identify and quantitate aflatoxins at levels as low as 1 ng/g. The TLC method is also used to

Liquid chromatography (LC) is similar to TLC in many respects, including analyte applica‐ tion, stationary phase, and mobile phase. Liquid chromatography and TLC complement each other. For an analyst to use TLC for preliminary work to optimize LC separation condi‐ tions is not unusual. Liquid chromatography methods for the determination of aflatoxins in foods include normal-phase LC (NPLC), reversed-phase LC (RPLC) with pre- or before-col‐ umn derivatization (BCD), RPLC followed by postcolumn derivatization (PCD), and RPLC

Thin layer chromatography and LC methods for determining aflatoxins in food are labo‐ rious and time consuming. Often, these techniques require knowledge and experience of chromatographic techniques to solve separation and and interference problems. Through advances in biotechnology, highly specific antibody-based tests are now commercially available that can identify and measure aflatoxins in food in less than 10 minutes. These tests are based on the affinities of the monoclonal or polyclonal antibodies for aflatoxins. The three types of immunochemical methods are radioimmunoassay (RIA), enzymelinked immunosorbent assay (ELISA), and immunoaffinity column assay (ICA). These are mostly chemical methods of detection but still provide an insight into the immuno‐ chemical methods such as ELISA and RIA which can used to detect aflatoxins in foods,

screen and corroborate findings by newer, more rapid techniques.

level higher than that currently in force in the EU (4 µg/kg total aflatoxins).

**11.2. Solid-phase extraction**

358 Aflatoxins - Recent Advances and Future Prospects

determination of target analytes.

**11.3. Thin-layer chromatography**

**11.4. Liquid chromatograph**

with electrochemical detection.

**11.5. Immunochemical methods**

such as flour and starch products.

Although analytical methods might consist of different extraction, clean-up, and quantita‐ tion steps, the results of the analyses by such methods should be similar when the methods are applied properly. Since the reliability of the quantitative data is not in question, the problem still to be solved is the confirmation of identity of the aflatoxins. The confirmation techniques used involve either chemical derivatization or mass spectrometry (MS).

Different analytical methods have been reported in the literature in order to facilitate the in‐ vestigation of the role of ingested aflatoxins in small volumes of human sera (Grio, Jose, Fre‐ nich, Martinez Vidal, Luis, Romero-Gonzalez, 2010; Yuanjing, Yi, Huiming, Bingnan, Haicheng, Fanli, Miaomiao, Wei, Wendong, 2010). Aflatoxin B1 has been extracted from 1 mL or less of human sera spiked with a known concentration of aflatoxin B1 and analyzed using high-performance liquid chromatography (HPLC) as the detection system. Several methods have been used to analyze feed, foods and bodyfluids, human and animal plasma, serum, milk, etc. (Santini, Ferracane, Meca, Ritieni, 2009; Rampone, Piccinelli, Aliberti, Ras‐ trelli, 2009; Monbaliu, Van Poucke, Detavernier, Dumoulin, Van De Velde, Schoeters, Van Dyck, Averkieva, Van Peteghem, De Saeger, 2010). The ELISA (Zhu, Zhang, Hu, Xiao, Chen, Xu, Fremy, Chu, 1987) or radioimmunoassay (RIA) methods (Groopman, Donahue, 1988; Tang, Pang, 2009; Li, Zhang, Zang, 2009) allow the quantification of the total amount of afla‐ toxins, and results are expressed in term of aflatoxin B1 equivalents. Both methods however involve the use of specific antibodies not commercially available. Recently immunoensors (Sun, Yan, Tang, Zhang, 2012) and biosensor have been proposed for the analyses of myco‐ toxins in different matrices (Campàs, Garibo, Prieto-Simón, 2012).

The International Agency for Research on Cancer has classified aflatoxin B1 as a human car‐ cinogen and aflatoxins B2, G1 and G2 as possible nephrotoxic natural compounds and carci‐ nogenic to humans (IARC, 1993; Commission Regulation (EC) No. 1525/98, 1998). Due to carryover in food and feed they are considered nowadays to have the most severe impact of all mycotoxins on human health. Maximum residue levels have been set down to the g/mL range in a wide variety of agricultural commodities, food, feed and milk, e.g. 0.01 mg/kg of aflatoxin M1 in milk for infants (Groopman, Donahue, Zhu, Chen, Wogan,1985).

Methods like liquid chromatography-mass spectroscopy (LC/MS) have been repeatedly used for structural elucidation in metabolism on aflatoxin containing analytes and specific matrices but only a limited number of quantitative methods have been published to deter‐ mine the more common aflatoxins present in food (Papp, Otta, Zaray, Mincsovics, 2002; Bi‐ selli, Hartig, Wegener, Hummert, 2004; Biselli, Hartig, Wegener, Hummert, 2005; Sorensen, Elbaek, 2005; Kokkonen, Jestoi, Rizzo, 2005) milk, (Sorensen, Elbaek, 2005) cheese, (Cava‐ liere, Foglia, Pastorini, Samperi, Lagana, 2006) herbs, (Ventura, Gomez, Anaya, Diaz, Broto, Agut, Comellas, 2004) urine, (Scholl, Musser, Groopman, 1997; Walton, Egner, Scholl, Walk‐ er, Kensler, Groopman, 2001; Egner, Yu, Johnson, Nathasingh, Groopman, Kensler, Roe‐ buck, 2003; Wang-Buhler, Lee, Chung, Stevens, Tseng, Hseu, Hu, Westerfield, Yang, Miranda, Buhler, 2005) airborne dust (Kussak, Nilsson, Andersso, Langridge, 1995) and cig‐ arette smoke (Edinboro, Karnes, 2005).

LC/MS has been used as a confirmation technique for the already well established, reliable and robust LC-FL methodology (Kussak, Nilsson, Andersson, Langridge, 1995; Abbas, Williams, Windham, Pringle, Xie, Shier, 2002; Blesa, Soriano, Molto, Marin, Manes, 2003; Abbas, Cart‐ wright, Xie, Shier, 2006) and has also been used to confirm positive results of TLC and ELISA based screening analyses. All the aflatoxins exhibit good ESI ionisation efficiency in the posi‐ tive ion mode with abundant protonated molecules [MH]+ and sodium adduct ions (Blesa, Sor‐ iano, Molto, Marin, Manes, 2003; Ventura, Gomez, Anaya, Diaz, Broto, Agut, Comellas, 2004; Kussak, Nilsson, Andersson, Langridge, 1995) and typically, for aflatoxins B1, B2, G1 and G2, the formation of sodium adduct ions can easily be suppressed by the addition of ammonium ions to the mobile phase leading to a better mass spectroscopy (MS) sensitivity (Cavaliere, Foglia, Pastorini, Samperi, Lagana, 2006). Reports about the utility of atmospheric pressure chemical ionization (APCI) interfaces and ionization efficiencies in this mode seem to be highly depend‐ ent on the aflatoxin studied and the APCI interface geometry (Abbas, Williams, Windham, Pringle, Xie, Shier, 2002; Abbas, Cartwright, Xie, Shier, 2006).

point. However, in the presence of moisture and at elevated temperatures there is destruc‐ tion of aflatoxin and this can occur with aflatoxin in oilseed meals, roasted peanuts or in aqueous solution at pH 7. Although the reaction products have not been examined in detail it seems likely that such treatment leads to opening of the lactone ring with the possibility of decarboxylation at elevated temperatures. At a temperature of about 100°C, ring opening followed by decarboxylation occurs, and reaction may proceed further, leading to the loss of

Aflatoxins: Risk, Exposure and Remediation http://dx.doi.org/10.5772/52866 361

In alkali solution reversible hydrolysis of the lactone moiety occurs. Recyclization has been

In the presence of acids, aflatoxin B1 and G1 are converted in to aflatoxin B2A and G2A due to acid-catalyzed addition of water to the double bond in the furan ring. In the presence of ace‐ tic anhydride and hydrochloric acid the reation proceeds further to give the acetoxy deriva‐ tive. Similar adducts of aflatoxin B1 and G1 are formed with formic acid-thionyl chloride,

Many oxidizing agents, e.g. sodium hypochlorite, potassium permanganate, chlorine, hy‐ drogen peroxide, ozone and sodium perborate react with aflatoxin and change the aflatoxin molecule in some way as indicated by the loss of fluorescence. The mechanisms of these re‐ actions are uncertain and the reaction products remain unidentified in most cases. Reduc‐ tion of aflatoxin B1 and B2 with sodium borohydride yielded aflatoxin RB1 and RB2, respectively. These arise as a result of opening of the lactone ring followed by reduction of the acid group and reduction of the keto group in the cyclopentene ring. Hydrogenation of aflatoxin B1 and G1 yields aflatoxin B2 and G2 respectively. Further reduction of aflatoxin B1

Food and feed contaminated with mycotoxins pose a severe health risk to animals and they may cause big economical losses due to the lower efficacy of animal husbandry and crop

In addition, directly or indirectly (carry on through animal products) contaminated foods may also pose a health risk to humans. For this reason it is understandable that many re‐ search has been addressed in an attempt to salvage mycotoxin contaminated commodities

Relevant basic criteria to be followed when a decontamination strategy is assessed have

**•** the mycotoxin must be inactivated (destroyed) by transformation to non-toxic com‐

**•** the food or feed material should retain its nutritive value and remain palatable for con‐

**•** fungal spores and mycelia should be destroyed, so that new toxins are not produced;

observed after acidification of a basic aflatoxin containing solution.

the methoxy group from the aromatic ring.

acetic acid-thionyl chloride and trifluoroacetic acid.

using 3 moles of hydrogen yields tetrahydroxyaflatoxin.

and to avert health risks associated with the toxins.

been suggested (Scott, 1990; Pomeranz, Bechtel, Sauer, Seitz, 1990):

**•** the physical properties of raw material should not change significantly;

performances.

pounds;

sumers;

This method has been proved to be more sensitive for the simultaneous determination of aflatoxins B1, B2, G1, G2, M1, M2, and moreover smaller sample volumes of serum can be used for the analysis. Aflatoxins are in free equilibrium with the albumin combined form and it is reported in the literature the effect of pH and/or serum concentration of fatty acids on the formation of the adducts. Moreover, a recent study showed that green tea polyphenols might modulate the formation of the adducts between aflatoxin B1 and albumin (Tang, Tang, Xu, Luo, Huang, Yu, Zhang, Gao, Cox, Wang, 2008).

Advanced spectrometric methods, such as LC-MS/MS, permit quantification and recogni‐ tion of the free aflatoxins in the sera with fewer problems on recovery, sensitivity and chem‐ ical identification (Santini, Ferracane, Meca, Ritieni, 2009; Huang, Zheng, Zengxuan, Yongjiang, Yiping, 2010) evaluating the aflatoxin exposure directly from their free forms.
