**11. Advanced analysis of aflatoxins in biological fluids**

#### **11.1. Sampling and sample preparation**

*In vivo*, as soon as a compound is released from its matrix in the chyme, the compound can be transported across the intestinal epithelium into the body thereby keeping the compound

Different analytical approaches can be applied to measure bioaccessibility of nutrients and bioactive compounds: *in vivo* and *in vitro* studies both present strengths and drawbacks. Within *in vivo* studies, balance studies and tissue concentration are two strategies that allow determination of the absorbed amount of nutrients, bioactive compounds, or their metabo‐ lites. Balance studies determine the absorbed amount by measuring the difference between the fed and excreted amounts of the nutrient or bioactive compound. Tissue concentration consists of monitoring the increase in plasma/serum concentration of the nutrient or bioac‐ tive compound. These approaches have been applied these approaches have been used with both animals and humans to determine absorption of carbohydrates, minerals, vitamins, phytochemicals, and different compounds (Benito, Miller, 1998; Hallberg, 1991). *In vivo* hu‐ man studies are the criterion standard approach to determine bioaccessibility of food nu‐ trients or bioactive compounds, although some experimental approaches are ethically and

Digestion and absorption involve several different steps, and each one could cause an effect on the nutrient or bioactive compound so that a detailed picture is not obtained with the bal‐ ance and bioassay studies. *In vitro* studies have been developed to simulate the physiologic conditions and the sequence of events that occur during digestion in the human gastrointes‐ tinal tract. In a first step, an in vitro gastrointestinal method is applied to the food, mirroring the physiochemical conditions that take place during human digestion, considering the

The main features of the in vitro gastrointestinal methods are temperature, shaking or agita‐ tion, and the chemical and enzymatic composition of saliva, gastric juice, duodenal juice, and bile juice (Wittsiepe, Schrey, Hack, Selenka, Wilhelm, 2001). When physical processes that oc‐ cur in vivo are not reproduced (shear, mixing, hydration, changes in conditions over time, peri‐ stalsis), the in vitro gastrointestinal model is defined as a static or biochemical model. The dynamic models mimic the *in vivo* physical processes so that they take into account new varia‐ bles, such as changes on viscosity of the digesta, particle size reduction, diffusion, and parti‐ tioning of nutrients. Several examples of *in vitro* gastrointestinal static and dynamic models have been described (Rotard, Christmann, Knoth, Mailahn, 1995; Arcand, Mainville, Farn‐ worth, 2007). During the application of the in vitro gastrointestinal method, food nutrients or bioactive compounds can be monitored to determine whether they are affected by digestion conditions (pH, enzymes) or if interactions with other food components (fiber, sucrose polyest‐ er, fat replacers) take place, which could affect efficiency of digestion. The final processed ma‐

To analyze the lipophilic content that has been effectively incorporated to mixed micelles, the micellar fraction can be isolated from that processed material by the application of an ultracentrifugation protocol (Hernell, Staggers, Carey, 1999). In the digestion model, the compounds are not removed from the chyme during digestion and therefore, bioaccesibility

three areas of the human digestive system (mouth, stomach, and intestine).

terial of the experimental procedure is a digesta or intestinal preparation.

concentration low in the chyme.

356 Aflatoxins - Recent Advances and Future Prospects

technically unaffordable.

Sampling and sample preparation remain a considerable source of error in the analytical identification of aflatoxins. Thus, systematic approaches to sampling, sample prepara‐ tion, and analysis are absolutely necessary to determine aflatoxins at the parts-per-billion level. In this regard, specific plans have been developed and tested rigorously for some commodities such as corn, peanuts, and tree nuts; sampling plans for some other com‐ modities have been modeled after them. A common feature of all sampling plans is that the entire primary sample must be ground and mixed so that the analytical test portion has the same concentration of toxin as the original sample. Methods of sampling and analysis for the official control of mycotoxins, including aflatoxins, are laid down in Commission Regulation No 401/2006. This ensures that the same sampling criteria in‐ tended for the control of mycotoxin content in food are applied to the same products by the competent authorities throughout the EU and that certain performance criteria, such as recovery and precision, are fulfilled. In 2008, the Codex Alimentarius set a maximum level of 10 µg/kg total aflatoxins in ready-to-eat almonds, hazelnuts, and pistachios at a level higher than that currently in force in the EU (4 µg/kg total aflatoxins).

**12. Aflatoxins identity assessment**

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

Aflatoxins: Risk, Exposure and Remediation http://dx.doi.org/10.5772/52866 359

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‐

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‐

techniques used involve either chemical derivatization or mass spectrometry (MS).

toxins in different matrices (Campàs, Garibo, Prieto-Simón, 2012).

arette smoke (Edinboro, Karnes, 2005).

#### **11.2. Solid-phase extraction**

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 determination of target analytes.

## **11.3. Thin-layer chromatography**

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 screen and corroborate findings by newer, more rapid techniques.

#### **11.4. Liquid chromatograph**

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 with electrochemical detection.

#### **11.5. Immunochemical methods**

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, such as flour and starch products.
