HPLC Analysis of Aflatoxin B1 Contamination of Local and Foreign Brands of Cigarette Tobacco Popularly Consumed in Niger State North- Central Nigeria

Under favorable growth conditions, fungi produced aflatoxins. Aflatoxin B1 (AFB1) categorized as a carcinogen is the most toxic and subject of regulation in foods and feeds in many countries. AFB1 contamination has been reported in a wide range of products including cigarette tobacco due to improper processing and storage conditions. This research aim at determining the concentration load of AFB1 mycotoxin in brands of cigarette tobacco commonly consumed in Lapai and Minna metropolis of Niger State Nigeria. Thirty-six (36) brands of cigarette tobacco were purchased in open markets in Minna and Lapai towns of Niger state, Nigeria. Residual AFB1 was extracted from the sample using standard procedures. HPLC analysis was used for the mycotoxin quantification. The result showed that all cigarette samples produces AFB1 with a concentration of 302 μg/mL and 18 μg/mL as the highest and lowest AFB1 concentration respectively for the local – Nigeria brands compared to 166 μg/mL and 8 μg/mL AFB1 concentration observed for the foreign brands. The findings from this study provide sufficient levels of contamination to pose significant health hazard for the consumer of the tobacco. Careful processing and storage is therefore advocated to eliminate or reduce the mycotoxin contamination of the cigarette brands. Ndagi Mohammed Saba, Musa Achimugu Dickson and Ogbiko Cyril http://www.earthlinepublishers.com 242 Introduction Mycotoxins are secondary metabolites produced by fungi that frequently contaminate agricultural commodities. Aflatoxins, produced by common mold fungi in the genus Aspergillus, are one of the most important groups of mycotoxins. Among the most commonly, occurring aflatoxins are aflatoxin B1, G1, B2, G2) (Figure 1). Aflatoxin B1 (AFB1) is the most toxic and has been the subject of regulation in foods and feeds in many countries, including the United States [1]. AFB1 contamination can occur in a varied range of products due to improper storage conditions and has been shown to be hepatotoxic, carcinogenic, and teratogenic, as well as causing other less specific symptoms such as weight loss and impaired immune systems [2]. As a result of its harmful nature, AFB1 has been included as a carcinogen on the list of Harmful and Potentially Harmful Constituents (HPHCs) in tobacco products as regulated by the Food and Drug Administration (FDA) [3]. Tobacco is one among the 25% of world crops affected by mycotoxins every year [4]. Diverse microorganisms community ranging from bacteria (both Gram positive and Gram negative), bacterial spores and toxins (endotoxins and exotoxins), molds, yeasts, as well as fungal spores and their secondary metabolites such as aflatoxin B1 are involved in the production process of cigarette from tobacco, with the spores of such fungi capable of surviving the entire manufacturing process and the level of these toxins production influenced by the substrate on which the fungi grow as well as the moisture level, temperature, and presence of competitive micro-flora [5,6]. Aflatoxin contamination of tobacco and tobacco related products have long been reported. Aspergillus flavus growth on flue-cured tobacco have been observed [7] with several aspergilli reported in the stored leaves of tobacco [8]. AFB1 has been reported in tobacco products such as cigarettes [9] and chewable tobacco mixtures including other plant material such as betel leaf [10]. Exposure to aflatoxins in cigarettes may be mitigated by their potential destruction in the combustion process [11]. AFB1 has traditionally been detected in commodities by use of HPLC with fluorescence detection [12] but with HPLC-MS/MS and UHPLC-MS/MS methods becoming more common [13-15]. In this study, we screened both local and foreign brands of tobacco cigarettes consumed in Lapai and Minna towns of Niger State Nigeria to ascertain the extent of aflatoxin B1 contamination using HPLC method. HPLC Analysis of Aflatoxin B1 Contamination of Local and Foreign Brands ... Earthline J. Chem. Sci. Vol. 5 No. 1 (2021), 241-248 243 Figure 1. Chemical structures of aflatoxins: aflatoxin B1 (AFB1) (1), aflatoxin G1 (AFG1) (2), aflatoxin B2 (AFB2) (3), aflatoxin G2 (AFG2) (4). Materials and Methods Sample collection Thirty six (36) different cigarette brands were procured from randomized cigarette wholesale and retail outlets in Lapai and Minna towns of Niger State. Extraction of residual AFB1 Twenty five (25g) of the tobacco in each of the cigarettes was weighed into a 250 mL Erlenmeyer flask and extracted for 30 minutes on a wrist action shaker using 125 mL of methanol – water mixture (55:45 v/v), 50 mL of n-hexane and 1g of sodium chloride. The mixture was filtered through Whatmann filter paper and the residue discarded. The filtrate was allowed to settle, and the lower methanol phase was collected into a separating funnel. It was then extracted 3 times with 25ml chloroform. The chloroform extracts were concentrated into the vials and evaporated to dryness. The dry film was dissolved with 400μl of the mobile phase (water: methanol: acetonitrile 60:20:20) [16]. Quantification of AFB1 using HPLC analysis AFB1 was quantified by reversed-phase HPLC methods. Aflatoxin B1 was analysed Ndagi Mohammed Saba, Musa Achimugu Dickson and Ogbiko Cyril http://www.earthlinepublishers.com 244 on a Buck Instruments high performance liquid chromatography (HPLC) with UVdetection wavelength of 365nm. A C18 column type, 250 x 4.6mm with 5 micron particle size was used at an ambient temperature of 25 o C. The mobile phase was water:methanol:acetonitrile in the ratio of 60:20:20 at a flow rate of 1ml/min. the injection volume was 20μl. Results AFB1 quantification of the cigarette samples using HPLC analysis reveals varying concentrations of AFB1 in both local and foreign cigarette brands as represented in Figures 2 and 3. Figure 2. (a) and (b): Aflatoxin B1 concentration of analyzed local (Nigerian) brands of cigarette tobacco. HPLC Analysis of Aflatoxin B1 Contamination of Local and Foreign Brands ... Earthline J. Chem. Sci. Vol. 5 No. 1 (2021), 241-248 245 Figure 3. Aflatoxin B1 concentration of analyzed foreign brands of cigarette tobacco. Key: CG 2, 26, 27, 32, 33 (South Korea); CG 5, 34, 35 (Senegal); CG 6, 7, 8, 11 (UAE); CG 10 (Germany); CG 25 (Britain). Discussion Although fungi may be present without producing any toxin, in an earlier report by Ndagi et al. [17], different colonies of fungi ranging from Apergillus niger, Aspergillus fumigatus, Aspergillus nidulans, Chrysonillia sitophilia and Aspergillus flavus were all isolated from Nigeria and foreign brands of cigarette tobacco [17]. This finding was confirmed by Pauly et al. [18] who isolated diverse microbes and microbial toxins from more than 90% individual tobacco flakes from eight different popular US cigarette brands. They reported that these toxins are released from the cigarette during smoking, and carried into mainstream smoke that is sucked deep into the lung. Several other researchers have reported that tobacco cigarettes harbor a plethora of bacteria (Grampositive and Gram-negative), fungi (mold, yeast), spores, and is rich in endotoxin [1922]. This is expected since microorganisms are involved in the production process of tobacco cigarette as well as mycotoxin producing fungi earlier isolated from these cigarette brands. It becomes imperative therefore to ascertain the concentration of aflatoxin B1 mycotoxin concentration in the brands of cigarettes. The results of the mycotoxin concentration show that the local brands (302 μg/mL and 18 μg/mL as the highest and lowest aflatoxin concentration respectively) compared to their foreign counterpart (166 μg/mL and 8 μg/mL as the highest and lowest aflatoxin concentration respectively). This could however be attributed to better processing of the foreign brands than the local Nigeria brands since processing methods had been reported to reduce fungi contamination and consequently their mycotoxins production [17]. Ndagi Mohammed Saba, Musa Achimugu Dickson and Ogbiko Cyril http://www.earthlinepublishers.com 246 Conclusion The HPLC analysis of the tobacco cigarette brands showed that all the cigarette brands (Nigeria and Foreign) were not only contaminated with fungi but produce AFB1with varying concentrations. The presence of fungi and its associated mycotoxins confirms that cigarettes pose a significant health risk to its consumers although the claim of a 100% aflatoxin B1 transfer to smoke during tobacco combustion had been refuted by several research and factory documents.


Introduction
Mycotoxins are secondary metabolites produced by fungi that frequently contaminate agricultural commodities. Aflatoxins, produced by common mold fungi in the genus Aspergillus, are one of the most important groups of mycotoxins. Among the most commonly, occurring aflatoxins are aflatoxin B 1 , G 1 , B 2 , G 2 ) (Figure 1). Aflatoxin B 1 (AFB 1 ) is the most toxic and has been the subject of regulation in foods and feeds in many countries, including the United States [1]. AFB 1 contamination can occur in a varied range of products due to improper storage conditions and has been shown to be hepatotoxic, carcinogenic, and teratogenic, as well as causing other less specific symptoms such as weight loss and impaired immune systems [2]. As a result of its harmful nature, AFB 1 has been included as a carcinogen on the list of Harmful and Potentially Harmful Constituents (HPHCs) in tobacco products as regulated by the Food and Drug Administration (FDA) [3].
Tobacco is one among the 25% of world crops affected by mycotoxins every year [4]. Diverse microorganisms community ranging from bacteria (both Gram positive and Gram negative), bacterial spores and toxins (endotoxins and exotoxins), molds, yeasts, as well as fungal spores and their secondary metabolites such as aflatoxin B 1 are involved in the production process of cigarette from tobacco, with the spores of such fungi capable of surviving the entire manufacturing process and the level of these toxins production influenced by the substrate on which the fungi grow as well as the moisture level, temperature, and presence of competitive micro-flora [5,6].
Aflatoxin contamination of tobacco and tobacco related products have long been reported. Aspergillus flavus growth on flue-cured tobacco have been observed [7] with several aspergilli reported in the stored leaves of tobacco [8]. AFB 1 has been reported in tobacco products such as cigarettes [9] and chewable tobacco mixtures including other plant material such as betel leaf [10]. Exposure to aflatoxins in cigarettes may be mitigated by their potential destruction in the combustion process [11]. AFB 1 has traditionally been detected in commodities by use of HPLC with fluorescence detection [12] but with HPLC-MS/MS and UHPLC-MS/MS methods becoming more common [13][14][15]. In this study, we screened both local and foreign brands of tobacco cigarettes consumed in Lapai and Minna towns of Niger State Nigeria to ascertain the extent of aflatoxin B 1 contamination using HPLC method.  (1), aflatoxin G 1 (AFG 1 ) (2), aflatoxin B 2 (AFB 2 ) (3), aflatoxin G 2 (AFG 2 ) (4).

Sample collection
Thirty six (36) different cigarette brands were procured from randomized cigarette wholesale and retail outlets in Lapai and Minna towns of Niger State.

Extraction of residual AFB 1
Twenty five (25g) of the tobacco in each of the cigarettes was weighed into a 250 mL Erlenmeyer flask and extracted for 30 minutes on a wrist action shaker using 125 mL of methanol -water mixture (55:45 v/v), 50 mL of n-hexane and 1g of sodium chloride. The mixture was filtered through Whatmann filter paper and the residue discarded. The filtrate was allowed to settle, and the lower methanol phase was collected into a separating funnel. It was then extracted 3 times with 25ml chloroform. The chloroform extracts were concentrated into the vials and evaporated to dryness. The dry film was dissolved with 400μl of the mobile phase (water: methanol: acetonitrile 60:20:20) [16].

Quantification of AFB 1 using HPLC analysis
AFB 1 was quantified by reversed-phase HPLC methods. Aflatoxin B 1 was analysed 244 on a Buck Instruments high performance liquid chromatography (HPLC) with UVdetection wavelength of 365nm. A C18 column type, 250 x 4.6mm with 5 micron particle size was used at an ambient temperature of 25 o C. The mobile phase was water:methanol:acetonitrile in the ratio of 60:20:20 at a flow rate of 1ml/min. the injection volume was 20μl.

Discussion
Although fungi may be present without producing any toxin, in an earlier report by Ndagi et al. [17], different colonies of fungi ranging from Apergillus niger, Aspergillus fumigatus, Aspergillus nidulans, Chrysonillia sitophilia and Aspergillus flavus were all isolated from Nigeria and foreign brands of cigarette tobacco [17]. This finding was confirmed by Pauly et al. [18] who isolated diverse microbes and microbial toxins from more than 90% individual tobacco flakes from eight different popular US cigarette brands. They reported that these toxins are released from the cigarette during smoking, and carried into mainstream smoke that is sucked deep into the lung. Several other researchers have reported that tobacco cigarettes harbor a plethora of bacteria (Grampositive and Gram-negative), fungi (mold, yeast), spores, and is rich in endotoxin [19][20][21][22]. This is expected since microorganisms are involved in the production process of tobacco cigarette as well as mycotoxin producing fungi earlier isolated from these cigarette brands. It becomes imperative therefore to ascertain the concentration of aflatoxin B 1 mycotoxin concentration in the brands of cigarettes. The results of the mycotoxin concentration show that the local brands (302 µg/mL and 18 µg/mL as the highest and lowest aflatoxin concentration respectively) compared to their foreign counterpart (166 µg/mL and 8 µg/mL as the highest and lowest aflatoxin concentration respectively). This could however be attributed to better processing of the foreign brands than the local Nigeria brands since processing methods had been reported to reduce fungi contamination and consequently their mycotoxins production [17].

Conclusion
The HPLC analysis of the tobacco cigarette brands showed that all the cigarette brands (Nigeria and Foreign) were not only contaminated with fungi but produce AFB 1 with varying concentrations. The presence of fungi and its associated mycotoxins confirms that cigarettes pose a significant health risk to its consumers although the claim of a 100% aflatoxin B 1 transfer to smoke during tobacco combustion had been refuted by several research and factory documents.