**1. Introduction**

Aflatoxins are a group of chemically similar poisonous, carcinogenic fungal secondary metabolites produced by *Aspergillus flavus*, *A*. *parasiticus* and *A*. *nomius*, which are abundant in warm and humid regions of the world. They are probably the most intensively researched toxins in the world due to their carcinogenic and mutagenic effects. Aflatoxins have also been identified as a potential biological weapon for food and water contamination. The word aflatoxins is the combination of three words: first letter "A" from genus *Aspergillus*, next three letters "FLA" from species *flavus*, and the noun "TOXIN". Aflatoxins are quite stable and are resistant to degradation [1, 2]. There are about 18 different aflatoxins, and six types have been identified to be more important and they are labeled AFB1, AFB2, AFG1, AFG2, AFM1 and AFM2, and they exhibit different molecular structures. The B-group have cyclopentane ring and exhibits blue fluorescence under UV light, while the G group contains lactone ring and exhibits yellow-green fluorescence under UV light. Aflatoxins M1 and M2 are hydroxylated derivatives of aflatoxins B and were first isolated from milk. The behavior under UV light made them easily identified and quantified using fluorescence spectroscopy [1, 3]. The aim of this work is to review various aspects of QuEChERS techniques including its various modification for the analysis of aflatoxins in food samples.

#### **2. Aflatoxin and its health impacts**

Food industries in developed and developing countries are facing serious challenges with contamination of mycotoxins especially aflatoxin in food and feed products. The Food and Agricultural Organization (FAO) concluded that around 25% of the world's cereals are contaminated by mycotoxins including aflatoxins [4]. The most common food commodities affected by aflatoxins are cereals (corn, wheat, barley, maize, oats and rye), nuts (hazelnut, peanut and pistachio nut), dried fruits (fig), and spices (chili powder) [2]. Thus, aflatoxins are quite chemically stable and are highly resistant to degradation. Among the 18 common groups of aflatoxins, B1, B2, G1, G2, M1 and M2 are the major classes and derivatives of bifuranocoumarins. Health implications of contaminated aflatoxins in humans and animals through consumption, contact or inhalation of foodstuffs in both developed and developing countries cannot be underestimated, where billions of people are chemically exposed to uncontrolled amounts of aflatoxins, which causes disease known as aflatoxicosis [1]. Aflatoxins are toxic and fatal in poultry animals (livestock) and are carcinogenic to humans [5].

The International Agency for Research on Cancer (IARC) classified AFBI as class I human carcinogen and has a positive association between dietary aflatoxins and liver cell cancer (LCC). This was the third leading cancer death globally. Vomiting, abdominal pain, pulmonary edema, convulsions and coma. Enlargement of internal organs such as liver, kidneys and heart are common symptoms of aflatoxicosis. Different regions and countries have set maximum levels (MLs) for different mycotoxins in food. In Europe, limits of 2 ppb (for aflatoxin B1) and 4 ppb (for total aflatoxins (B1 + B2 + G1 + G2), for cereals and cereal products (including maize and maize products) for direct human consumption are in place. Likewise, MLs of 5 ppb for aflatoxin B1 and 10 ppb for total aflatoxins are set for maize to be sorted or otherwise processed physically before human consumption. The European Commission further set a method for sampling cereals and cereals products in view of the prescribed limits. The regulated limits of mycotoxins in the European region are defined in the regulation of the European Community EG-VO 1881/2006. Codex Alimentarius Commission is responsible for setting maximum limits for mycotoxins in food and feed at the global level. The Codex Commission has already adopted MLs for mycotoxins as shown below [6]:


#### **3. Sampling in Aflatoxins**

Aflatoxin is a subclass of mycotoxins which are strains of the fungi *Aspergilllus flavus* and *A. parasiticus* and the less common *A. nomius*. Aflatoxins B1, B2, G1, G2, M1 and M2 are the most common types of Aflatoxins, which can be grouped in two based on their chemical structure, that is difurocoumarocyclopentanone and ifurocoumarolactone [2]. However, many foods and feeds that are prone to mycotoxin contamination cannot be directly analyzed in the absence of extraction and clean-up steps [7]. Researchers have used various extraction and clean-up methods to extract aflatoxins from complex matrices [7]. Dry, wet and cryogenic grindings are common homogenization techniques in cereal-based foods, oil seeds, spices, trees nuts and peanuts, contaminated by aflatoxins. Spanjer et al. [8] successfully used dry milling to process peanut, pistachio, wheat, maize, cornflakes, raising and figs for the analysis of different mycotoxins including aflatoxins. Evaluation of homogenization is always done in terms of analytical results, coefficients of variation for different mills, sample and subsample sizes and particle size distributions [7].

The European Union defined sampling method for mycotoxins in agricultural commodities through Commission Regulation No EC401/2006, to show that sample preparation plays important role in the precision of the determination of mycotoxins. Hydrophobic mycotoxins are extracted in the presence of organic solvents, such as methanol, acetone, chloroform and acetonitrile, while polar mycotoxins are extracted in mixture of organic solvents and water [3, 9]. Studies have shown that near infrared region (NIR) (800–2500 nm) is capable of differentiating kernels containing >100 ppb or <10 ppb levels of total aflatoxins. Research conducted on 168 samples of corn collected from different parts of Italy demonstrates that FT-NIR spectroscopy is better, easier and faster to detect FB1 and FB2 in corn compared to other analytical methods such as HPLC and ELISA [10].

#### **3.1 Sample Preparation**

Sample preparation stage is the most crucial and critical step in the analysis of contaminants in complex food samples [11]. Owing to the complexity and structural nature of mycotoxins in foods and feeds there is an urgent need for simple, effective and environmentally friendly methods of sample preparation for the detection and

quantification of aflatoxins in food samples [3]. The goals of sample treatment step are as follows.


Indeed, sample preparation is of great importance in analytical procedures because its steps account for one-third of the errors generated by analytical [13]. An efficient sample preparation method provides reliable, precise and accurate results, especially when trace or ultra-trace level of analytes in complex matrices (biological and environmental) are analyzed. Low operational cost, adequate removal of matrices interference, use of small amount of solvent, limiting the number of steps and high reproducibility and recovery, high sample throughput are characteristics of good sample preparation [14].

Extraction methods based on QuEChERS (quick, easy, cheap, effective, rugged and safe) developed by Anastassiades and his co-researchers. Anastassiades et al. [15] have been widely used in analysis of mycotoxins (aflatoxin, ochratoxin A, zearalenone, fusarenon X, α and β zearalenone) due to their simplicity and effectiveness for isolating mycotoxins from complex matrices. In contrast, traditional methods of extraction such as liquid–liquid extraction and solid phase extraction use highly toxic solvents, time-consuming and large amount of sample. QuEChERS ensures minimum sample loss by limiting the number of steps, improving sample throughput, low operational cost and effective removal of matrix component interference with high productivity and recovery [14].

The extraction method (**Figure 1**) is based on microscale extraction/partitioning followed by dispersive solid phase extraction (dSPE) for cleanup [17]. The analyte is partitioned between an aqueous and an organic layer by using MgSO4 and NaCl,

**Figure 1.** *Steps in original QuEChERS extraction procedure [16].*

followed by manual mixing and then centrifugation for a period of time and then the supernatant is cleaned up with the combination of primary-secondary amine (PSA) adsorbent and MgSO4 for the removal of interfering substances [18, 19]. The aliquot of the cleaned-up extract can then be analyzed with any of the analytical instruments.
