STUDY OF AFLATOXINS IN CASHEW NUTS PRODUCED IN CÔTE D'IVOIRE BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (LCHP)

The objective of this study is to isolate, identify and quantify four types of aflatoxins noted AFB1, AFB2, AFG1, and AFG2 that can be found in cashews grown in Côte d'Ivoire. These carcinogenic mycotoxins (AF) are secondary metabolite toxins produced by Aspergillus molds in plant foods.This work involved eleven (11) samples of 500 g of cashew nuts from eleven (11) cities of Côte d'Ivoire for the 2018-2019 campaign. These cities are: Béoumi, Bondoukou, Dabakala, Daloa, Douékoué, Ferkessédougou, Korhogo, Mignignan, Odienné, Sinématiali, and Touba. The test were carried out by high performance liquid chromatography (HPLC) after extraction of the four (4) mycotoxins on an immunoaffinity column at a flow rate of 3 mL / minute. These aflatoxins were identified and quantified from the following pairs of Retention time (Rt) in minutes and Limit of Detection (LD) in µg / kg: (13.777; 0.00143), (10.583 ; 0.00136), (9.901; 0.00151), and (8.184; 0.00564) respectively for AFB1, AFB2, AFG1, and AFG2. Our results show that all eleven (11) samples from these eleven (11) different cities contain aflatoxin (AFB1, AFB2, AFG2 and AFG1) contents below the national standard (2 µg / kg), that of the CODEX Alimentarius (1.4 µg / kg) and that of the European Union (2 µg / kg) indicating that cashews produced in Côte d'Ivoire comply with international standards and their consumption does not pose any risk to human health caused by the studied aflatoxins.


; Codex Alimentarius Commission, 2004).
In developing countries producers of cashew nuts like Côte d'Ivoire, aflatoxins are problematic for a public health. Indeed, it can happen that the cashew nut is contaminated by these aflatoxins in the fields, after harvesting and during storage. Producers of contaminated crops therefore suffer financial losses due to the rejection of their products, by the reduction of the value of said products on the market, the impossibility of accessing international trade and the trade restrictions imposed by international standards (Eric, 2003).
The various studies carried out on aflatoxins aim to preserve the health of populations threatened by this type of mycotoxin. To find a solution to this public health problem, it is essential to assess the degree of contamination of cashew products and derivatives, to specify the impact of the consumption of cashew products contaminated by aflatoxins on human and animal health.
The objective of this study is to identify and quantify aflatoxins by high performance liquid chromatography (HPLC), in the cashews produced in Côte d'Ivoire in 2019.

Analysis protocol:
The analysis protocol used during this study is similar to that described by Nadia (2011). The whole sample is ground and 20 g are taken and then mixed with 100 mL of extraction solvent composed of water / methanol (V / V). The solution obtained is centrifuged at 5000 rpm and homogenized, protected from light. The supernatant is filtered under vacuum on Wattman glassmicrofiber filters(GF/C circles 45 µm diameter) in an Erlenmeyer flask. 10 mL of this filtrate is diluted in 40 mL of a phosphate buffer solution (PBS) and then filtered on Wattman glass. The phosphate saline solution (PBS) of pH 7.3 is prepared by dissolving 0.2 g, 0.2 g, 1.16 g, and 8 g respectively of KCl, KH 2 PO 4 , Na 2 HPO 4 , and NaCl in one liter of distilled water (Daradimos et al., 2000).
A volume (10mL) of the filtrate is loaded onto the immunoaffinity column at a flow rate of 3 mL / minute. In a hemolysis tube, there (immunoaffinity column) is applied 1.5 ml of methanol (the eluent). The column is dried completely by applying vacuum. The eluate is collected, diluted in 1.5 ml of distilled water and recovered in a 2 ml Vial for chromatographic analysis.

Analytical conditions at HPLC:
The chromatograms were recorded and analyzed using a DELL computer with LC solution software. The analytical conditions at HPLC are presented in Table I. Limit of detection (LD) and quantification (Logeais, 1986; wafa, 2013) The LD was calculated according to (Eq. 1) 557 LD = , × (1) σ is the standard deviation; S the slope of the calibration curve. The limit of quantification (LQ) is defined as being 3.3 time the limit of detection (Jennifer, 2009) (Eq. 2).

Quantification of Aflatoxins by HPLC in Cashews • Aflatoxin content in a contaminated reference sample
The quantitative aflatoxin analysis was performed on a reference sample of contaminated cashews. The chromatogram from the contaminated sample is shown in Figure 2  The parameters resulting from the chromatogram are mentioned in Table II.

Aflatoxins content in cashew samples
Quantitative analysis of total aflatoxin (B1, B2, G1, G2) was performed on 11 cashew samples from 11 production cities. The results are reported in Table III.  ., (2011)), the presence of aflatoxin B1 is explained by the fact that hygienic control measures, adequate harvesting and collection methods, drying, sorting and storage are not well controlled. Aflatoxin B1 is also found in several plant foods.
However, the levels of all the aflatoxins (G2, G1, B1 and B2) obtained in the eleven (11) samples are lower than the national standard (2 µg / kg), CODEX Alimentarius (1.4 µg / kg), and European Union (2 µg / kg) (Julien, 2013). Furthermore, the results from our testings are in the same order than those obtained by José et al. (2015) in cashew nuts from Mexico (0.02 µg / kg). Likewise, the results obtained are consistent with those reported by many authors in other foods. According to Aissata et al., (2017), the analysis of 12 samples of peanut paste taken from the markets of three large municipalities, namely Yopougon, Abobo, and Adjamé, showed aflatoxin B1 contamination at levels as low as 2 µg / kg (authorized contamination limit). All these observations show that all samples analyzed have acceptable levels of aflatoxins for a safe consumption.

Conclusion:-
This work helps to better understand the problem of aflatoxins in Côte d'Ivoire, in particular in the case of cashew nuts from the 2018-2019 campaign. The analyzes carried out by high pressure liquid chromatography (HPLC) on the eleven (11) averages of cashew samples from eleven (11) different cities available to us show that these samples contain aflatoxin contents (AFB1, AFB2; AFG1 and AFG2) lower than the national standard (2 µg / kg), CODEX Alimentarius (1.4 µg / kg), and European Union (2 µg / kg). Therefore, there is no risk for the consumption of cashew-based products from Côte d'Ivoire based on the level of aflatoxins Consequently, the cashew nuts from the 2018-2019 campaign meet international standards and can be exported to European and African markets.