The Simultaneous Voltammetric Determination of Aflatoxins В1 and М1 on a Glassy-Carbon Electrode

For the first time, the possibility of using stripping voltammetry for the simultaneous determination of aflatoxins В1 and М1 on a glassy-carbon electrode has been shown. The influence of various factors (Еэ, τэ, w, and the nature of the background electrolyte) on the potential and magnitude of the oxidation current of mycotoxins has been estimated. Working conditions for voltammetric determination and reproducibility of analytical signals for two mycotoxins have been selected. The mutual influence of aflatoxins В1 and М1 on the value of analytical signals in their simultaneous presence has been studied. It has been found that, in the range of their detectable contents, the presence of aflatoxin В1 reduces the analytical signal of aflatoxin М1 by 45–50%, but the linearity of the calibration dependence is preserved. The content of aflatoxin М1 in determination of aflatoxin B1 does not exert a significant effect in the range of 10–15%. Based on the results obtained, a procedure has been proposed for determining the content of aflatoxins В1 and М1 in their joint presence in milk by voltammetry in the concentration ranges 2 × 10−3 ÷ 2 × 10−1 mg/dm3 and 2 × 10−4 ÷ 2 × 10−2 mg/dm3, respectively (Sr not more than 18%).


Introduction
Aflatoxin M 1 is a metabolite of aflatoxin B 1 , a product of life activity of Aspergillus microscopic fungi. In natural conditions, aflatoxin B 1 contaminates cereals, legumes, various nuts, oil seeds, cocoa and coffee, animal feed, and other food products. It can be converted into aflatoxin M 1 in the body of animals and is present in meat [1]. In [2], the information on admissible levels of contents of the specified kinds of mycotoxins in flour obtained from various kinds of grains used for cooking human food, as well as in the composition of feed of various animal species, is presented. With contaminated feed, aflatoxins enter the body of animals, and their residual quantities are found in meat, milk, and eggs. Aflatoxins are the only mycotoxins that are strictly regulated in markets such as the EU and US [3]. e information on tolerable levels of mycotoxins taken in Australia, China, Guatemala, India, Ireland, Kenya, and Taiwan is reported [4]. Aflatoxin M 1 is a hydroxylated metabolite present in human milk and animals exposed to aflatoxin B 1 . Like its precursor, aflatoxin B 1 , aflatoxin M 1 already in low concentrations poses a serious threat to the health of animals and humans. Aflatoxin B 1 is found not only in whole milk (including reconstituted milk) but also in cottage cheese, cheese, and yoghurt. Dairy products contaminated with aflatoxin M 1 are environmentally hazardous to humans. In adult food and baby food, aflatoxins are not allowed. Figure 1 shows the structural formula of aflatoxins B 1 and M 1 .
Aflatoxin B 1 is (6aR-cis) ( ere are known methods of simultaneous quantitative determination of aflatoxins B 1 and M 1 , aflatoxins B 1 , C 2 , G 1 , G 2 , and M 1 , and ochratoxin A by the method of highperformance liquid chromatography with fluorescent detection in breast milk [5][6][7][8] with detection limits between 0.5 and 0.25 μg/L [7], of 5 ng/L [7], and from 0.005 to 0.03 ng/mL [8]. e method of high-performance liquid chromatography was used to determine aflatoxin M 1 in eggs [9], and together with tandem mass-spectrometry, it was used to determine aflatoxins B 1 and M 1 in fresh and dry milk after ultrasonic extraction; the detection limit was 0.05 μg/kg, and the limit of quantification was 0.1 μg/kg [10]. e detection limits of aflatoxins B 1 , C 2 , G 1 , G 2 , and M 1 and ochratoxin A in food products of animal origin were in the range of 0.07-0.59 μg/kg [11]. In case of simultaneous determination of six aflatoxins (B 1 , C 2 , G 1 , G 2 , M 1, and M 2 ) by this method in peanut, the detection limits are in the range of 0.03 to 0.26 ng/g and 0.1 to 0.88 ng/g [12]. e use of chromatography methods is complicated by the duration and the need to use expensive equipment and highly toxic solvents as a mobile phase. e possibility of indirect competitive enzyme-linked immunosorbent assay for the determination of aflatoxin M 1 in various objects was demonstrated in [13]. At present, highly sensitive, inexpensive, and easy-to-use electrochemical methods, in particular voltammetry, are becoming increasingly popular for the determination of a number of organic substances including aflatoxins. In literature, there is a rather large number of works on the individual determination of aflatoxins B 1 [14][15][16][17][18][19] and M 1 [20][21][22][23] using amperometric and voltammetric immunosensors. e use of enzymes and nanomaterials to design sensors provides high sensitivity and selectivity for detection. At the same time, the analysis of numerous publications in the databases of Science Direct, Scopus, Web of Science, and so on shows that, at the moment, there is no research work on the simultaneous quantification of aflatoxins C 1 and N 1 by the method of voltammetry.
e purpose of the work consists of studying the possibility of simultaneous voltammetric determination of aflatoxins C 1 and N 1 on a glassy-carbon electrode (GCE), selecting working conditions for measurements and developing a method for their determination in whole milk.

Methods and Materials
In this study, the voltammetric analyzer "STA" (Russia) consisting of electronic and measuring units and an IBMcompatible personal computer with the installed software package "STA" was used. As the indicator electrode, a glassycarbon electrode (GCE) was used, and the conventional silver chloride electrode (CSE) served as an auxiliary and reference electrode.
e measurements were carried out in a constantcurrent sweep mode with the speed w � 30 mV/s in the potential range from 0.0 to +1.1 V. To mix the analyzed solution, vibration of the electrodes without removal of dissolved oxygen was used. e working solutions of aflatoxins C 1 and N 1 were prepared from standard samples of aflatoxin

Preparation of the Sample of Whole Milk
When preparing for analysis, a sample of whole milk 25.00 g is taken in a conical flask with the capacity of 100 cm 3 , and 1.0 cm 3 of hydrochloric acid with a concentration of 6-7 mol/dm 3 is added in portions of 0.2 cm 3 . e mixture is slightly stirred and left for 15 minutes, poured into centrifuge tubes, and then centrifuged at 15,000 rpm within 15 minutes. e centrifugate is poured into a conical flask, and 5-6 g of ammonium sulfate ((NH) 2 SO 4 ) is added in portions of 2 to 3 grams, each time stirring the contents of the flask with a glass rod until the salt dissolves.
e flask is left for 20 minutes, after which the contents of the flask are poured into centrifuge tubes and centrifuged within 15 minutes at the speed of 6000 rpm. e centrifugate is filtered into a clean cup with the capacity of 30 cm 3 through the doublelayered filter paper (blue tape). e resulting filtrate is a prepared sample. For analysis, an aliquot of the prepared sample of 5.0 cm 3 is taken.

Results and Discussion
Studies on the effect of the background electrolyte composition on the analytical signals of aflatoxins C 1 and N 1 on a glassy-carbon electrode under working conditions previously developed for the determination of aflatoxin C 1 were conducted [24]. Experiments on the choice of the background electrolyte showed that the value of the analytical signal aflatoxin M 1 on background electrolytes: 0.1 M Na 3 PO 4 , 0.1 M Na 2 HPO 4 , 0.1 M K 2 HPO 4 , and 0.1 M ZnSO 4 , was found to be low, and it was high on background electrolytes: 0.1 M (NH 4 ) 2 SO 4 and 0.1 M Li 2 CO 3 ; the maximum current of its electric oxidation was obtained against the background of 0.1 M C 6 H 5 O 7 (NH 4 ) 3 . Changing the cation-anion composition and pH of the background electrolyte may negatively shift the peak potential of the electric oxidation peak of aflatoxin N 1 in the range (0.6 ± 0.08) V. e effect of the background electrolyte pH on the analytical signals of these aflatoxins was studied, and it was shown that it was preferable to use neutral or weak acidic solutions as background ones, since mycotoxins decompose into nontoxic or low-toxic compounds in the alkaline medium, and the use of background electrolytes with pH > 6.5 is impractical. In Figure 2, calibration curves of the peak current of electric oxidation of aflatoxins C 1 and N 1 in various background electrolytes are presented. According to the calibration curves, two background electrolytes were selected: 0.1 M 3-substituted ammonium citrate and 0.1 M ammonium sulfate solution, providing a high detection sensitivity coefficient in the range of determined contents 2 × 10 −4 ÷ 0.6 mg/dm 3 . In Figure 3, voltammograms of the electric oxidation of aflatoxins C 1 and N 1 on the GCE in the selected background electrolyte are shown. Analytical signals are well separated and reproduced.
Both electrolytes can be used for the joint quantification of mycotoxins, but 0.1 N S 6 O 5 P 7 (NH 4 ) 3 was selected as the working background electrolyte providing sufficient resolution and satisfactory reproducibility of the analytical signal. Figure 4 shows the dependences of the current derivatives of the peak of aflatoxins C 1 (curve 1) and N 1 (curve 2) on the accumulation potential of the GCE in the selected background electrolyte. It can be seen in Figure 4 that the maximum values of the electric oxidation currents of aflatoxins are observed at the potential of 0.0 V that was selected as the accumulation potential for further studies. e mutual influence of aflatoxins C 1 and N 1 in their simultaneous determination on a glassy-carbon electrode was studied. For this purpose, the current derivative of the peak of aflatoxin C 1 electric oxidation was obtained as a function of the concentration of aflatoxin N 1 in the solution ( Figure 5) and the calibration curves of aflatoxin N 1 in the presence of aflatoxin C 1 (Figure 6).
In Figure 5, it can be seen that, in the concentration range studied, the effect of aflatoxin M 1 on the aflatoxin B 1 current is practically negligible at the ratio C aΦB 1 : C aΦM 1 � 1 : 1 in the presence of a two-or threefold excess of aflatoxin M 1 , the peak current of aflatoxin B 1 decreases by 10-15% (curve 1), and the potential of the peak remains unchanged. It is shown that the systematic error in determination of aflatoxin B 1 in the presence of aflatoxin M 1 at the ratio C B 1 /C M 1 ≤ 1 : 40 does not exceed 20%.
In Figure 6, it is seen that, in the presence of aflatoxin B 1 , the derivative of the peak current of aflatoxin M 1 decreases almost 1.5 times and the peak potential shifts to the anode region from +0.85 V to +0.95 V, but the linearity of the calibration dependence remains in a wide range which proves the possibility of their simultaneous determination.
Based on the conducted studies, the working conditions of the simultaneous voltammetric determination of aflatoxins B 1 and N 1 were proposed (Table 1).
On the basis of the obtained data of the electrochemical behavior of aflatoxins, an algorithm for quantifying these toxic substances in order to effectively control the detection of their minimum acceptable amounts in whole milk was developed. e algorithm for quantification of mycotoxins in whole milk includes the following steps: (1) Taking a sample (2) Acid hydrolysis with concentrated HCl and centrifugation (3) Precipitation of proteins with ammonium salts of sulfate (NH 4 ) 2 SO 4 and centrifugation (4) Filtration of the obtained precipitate (5) Quantitative determination of the aflatoxins content by the method of differential voltammetry Verification of the correctness of the proposed procedure was carried out by the "introduced-found" method ( Table 2). e data in Table 2 show that the voltammetric joint for determination of quantities of aflatoxins B 1 and M 1 is possible with the measurement error of 15-20% in the concentration ranges 2 × 10 −3 ÷ 2 × 10 −1 mg/dm 3 and 2 × 10 −4 ÷ 2 × 10 −2 mg/dm 3 , respectively. e proposed method is simple, and it does not require a lot of reagents and labor. e range of detectable (2) C afB 1 � 2 × 10 −3 mg/dm 3 and C afM 1 � 0; (3) C afB 1 � 2 × 10 −3 mg/dm 3 and C afM 1 � 2 × 10 −4 mg/dm 3 .
concentrations is from 0.001 to 0.12 mg/dm 3 . e relative standard deviation (Sr) is not more than 30%.

Conclusion
us, the possibility of the simultaneous voltammetric determination of aflatoxins B 1 and M 1 on the GCE in the background electrolyte 0.1 N S 6 O 5 P 7 (NH 4 ) 3 has been shown. (1) C afB 1 � 0; (2) C afB 1 � 2 × 10 −3 mg/dm 3 .  Figure 4: Dependences of the current derivatives of the aflatoxins C 1 and N 1 peak on the accumulation potential of the GCE. e background electrolyte is 0.1 N S 6 O 5 P 7 (NH 4 ) 3 ; τ ; � 30 s; w � 30 mV/s; (1) C afM 1 � 2 × 10 −3 mg/dm 3 ; (2) C afM 1 � 2 × 10 −4 mg/dm 3 . When determining aflatoxins, the method of "soft" sample preparation has been used for separating the matrix by hydrolysis and salting out proteins followed by their separation by centrifugation or filtration which reduces the analysis time to less than one hour as compared with thinlayer and high-performance chromatography (GOST 30711-2001). e developed technique has a number of advantages in comparison with the already known methods of analysis. e algorithm of the technique is characterized by the express analysis (the analysis time does not exceed 1 hour), sensitivity (the range of the determined contents is not inferior, and in the case of aflatoxin M 1 , it exceeds the capabilities of chromatographic methods), and the equipment cheapness. e technique is characterized by simplicity of execution, minimal consumption of reagents, and improved metrological characteristics.

Conflicts of Interest
e authors declare that they have no conflicts of interest for this research.