EJBR2018v8i3art121 ISSN 2449-8955 European Journal of Biological Research Research Article European Journal of Biological Research 2018; 8 (3): 121-130 Profile of major and emerging mycotoxins in sesame and soybean grains in the Federal Capital Territory, Abuja, Nigeria Stephen O. Fapohunda 1 , Toba S. Anjorin* 2 , Michael Sulyok 3 , Rudolf Krska 3 1 Department of Microbiology, Babcock University, Ilishan-Remo, Ogun State, Nigeria 2 Department of Crop Protection, Faculty of Agriculture, University of Abuja, PMB117, Abuja, Nigeria 3 Center for Analytical Chemistry, Department of Agrobiotechnology (IFA-Tulln), University of Natural Resources and Life Sciences Vienna (BOKU), Konrad Lorenzstr, 20, A-3430 Tulln, Austria *Corresponding author: Toba S. Anjorin; E-mail: oyindamola35@gmail.com ABSTRACT The spectrum of major and emerging mycotoxins in sesame and soybean grains from the six zones of the Federal Capital Territory (FCT), Abuja, Nigeria was determined using Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS). A total of 47 samples (24 sesame and 23 soybean were collected from farmers’ stores. Seven regulated mycotoxins in sesame and five in soybean including aflatoxin B1 (AFB1), aflatoxin B2 (AFB2) and fumonisin B1 (FB1) were detected. However, concentrations were generally lower than regulatory limits set in the EU for raw grains with the exception of ochratoxin A (OTA) exhibiting a maximum concentration level of 23.1 µ g kg-1 in one of the soybean samples. This is the first report concerning the contamination of sesame and soybean in Abuja, FCT-Nigeria with the emerging mycotoxins addressed by recent European Food Safety Authority (EFSA) opinion papers totalling 10 in number. These include beauvericin (BEA), moniliformin (MON), sterigmatocystin (STE), altertoxin-I (ATX-I), alternariol (AOH), alternariol methylether (AME) though at relatively low µ g kg-1 range. This preliminary data indicate that sesame and soybean might be relatively safe commodities in view of the profile of mycotoxins. Keywords: Emerging mycotoxins; Nigeria; Liquid chromatography/tandem mass spectrometry; Regu- lated mycotoxins; Sesame; Soybean. 1. INTRODUCTION Sesame (Sesamum indicum L.) and soybean (Glycine max L. Merril) are two very nutritious food items in Nigeria. Just like other crops produce, the occurrence of mycotoxins in these two crops is hardly avoidable. Unimpressive agricultural prac- tices and lack of well-organized and effective regulations in Nigeria and most of sub Saharan Africa make control gloomy. Even where regulatory measures exist, they have little impact in rural areas and subsistence farming communities [1]. Post- harvest mishandling of the grains especially before and during storage is one of the major causes of mycotoxin invasion of the crops produce [2]. Sesame stored or marketed in some north central States of Nigeria has been shown over many seasons to be contaminated with agriculturally Received: 22 May 2018; Revised submission: 25 June 2018; Accepted: 06 July 2018 Copyright: © The Author(s) 2018. European Journal of Biological Research © T.M.Karpiński 2018. This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial 4.0 International License, which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited. DOI: http://dx.doi.org/10.5281/zenodo.1307184 122 | Fapohunda et al. Profile of major and emerging mycotoxins in sesame and soybean grains European Journal of Biological Research 2018; 8 (3): 121-130 important toxins [3]. This might be linked to favourable climatic and crop storage conditions which are frequently conducive to fungal growth and mycotoxin production, as much of the popu- lation relies on subsistence farming and unregulated local markets. Mycotoxins in soybean have a wide geographical distribution from Argentina [4, 5] to Croatia [6] and their production can be due to the effect of temperature [7]. Health challenges from the synergistic effect of many mycotoxins [8, 9] or through immunomodulation by a variety of co- occurring mycotoxins [10]. Emerging mycotoxins attract an increasing interest among the scientific community due to their high occurrence in feed and food commodities, sometimes at relatively high concentrations, and potential toxicity towards animals and humans. There is not yet existing regulation given to these metabolites despite their established toxicity. The main producers of emerging Alternaria metabolites are A. alternata, A. dauci, A. cucumerina, A. solani, and A. tenuissima, A. citri. These mycotoxins are found in wheat, rice, rye, olives, sorghum, tobacco, apples, peppers, sunflower seeds, oilseed rape, pecan nuts, tomatoes and mandarins [11, 12]. The incidence of emerging Fusarium myco- toxins has been reported in different products such as soybean [13], wheat and barley [14], rice [15], grain-based products [16] and infant cereals [17]. Jestoi [18] reported that limited data on emerging, less reported mycotoxins might be due to their late recognition especially because of the late understanding of their role as mycotoxins. Studies focusing on this class of mycotoxins are still quite low in number an extensive review published in 2015 showed that among all mycotoxin-related studies, only 7% were directed toward emerging mycotoxins. Some metabolites such as fusaric acid and fusaproliferin referred to as “emerging mycotoxins” by certain authors such as Jestoi [18] are not yet addressed by EFSA. In Nigeria, there has been difficulty in determining the actual pecuniary value of food and feed ingredient losses due to mycotoxin load. This was due to paucity of detailed analysis information as well as intervention steps. There has been lack of attention on the probable additive effect and risks posed by emerging mycotoxins. The objective of the present study was to determine the major mycotoxins and emerging fungal metabolite profile of sesame and soybean in the FCT, Abuja Nigeria. This surveillance could be a critical step forward in the cocktail of intervention strategies against mycotoxin contamination in food and feed in Nigeria. 2. MATERIALS AND METHODS 2.1. Sampling Surveys were conducted the FCT, Abuja Nigeria (between Lat. 9o 40’ N, Long. 7o 29’ E and Lat. 8º 83’N, Long. 7º 17’ E, 388-566 m above the sea level between January and February, 2015. The farmers’ stores were located in the six zones of the territory (Table 1). A total of 47 samples (24 sesame and 23 soybean grains) were collected from farmers in the six zones of the FCT namely Abuja Municipal Area Council (AMAC), Abaji, Bwari, Gwagwalada (GWA), Kuje and Kwali. Simple random sampling plan was adopted in the collection of the seed samples. The sampled locations and number of sample types collected from each district were uneven but the quantity were the same. Only samples shelled and stored for less than 30 days after harvest were collected from the farmers. Each sample was collected as a bulk sample (1.8-2 kg) and comprised of four subsamples of 0.5 ± 0.05 kg each. The subsamples were obtained from random points in farmer’s basins or other storage containers and mixed to form the bulk. The samples were comminute and quartered such that 100-150 g of representative samples was obtained from each bulk as described by Ezekiel et al. [19]. Representative samples were stored at 4ºC until they were analyzed for multiple mycotoxins and microbial metabolites. 2.2. Sampling area The sampling location in the six zones of the FCT, Abuja is as shown in Table 1. 2.3. Mycotoxin analysis 2.3.1. Chemicals and reagents Methanol (LC gradient grade) and glacial acetic acid (p.a.) were purchased from Merck 123 | Fapohunda et al. Profile of major and emerging mycotoxins in sesame and soybean grains European Journal of Biological Research 2018; 8 (3): 121-130 (Darmstadt, Germany), acetonitrile (LC gradient grade) from VWR (Leuven, Belgium), and ammo- nium acetate (MS grade) from Sigma-Aldrich (Vienna, Austria). Water was purified successively by reverse osmosis and an Elga Purelab ultra analytic system from Veolia Water (Bucks, UK) to 18.2 MΩ. Standards of fungal and bacterial metabolites were obtained either as gifts from various research groups or from the following commercial sources: Romer Labs® Inc. (Tulln, Austria), Sigma-Aldrich (Vienna, Austria), Iris Biotech GmbH (Marktred- witz, Germany), Axxora Europe (Lausanne, Swit- zerland) and LGC Promochem GmbH (Wesel, Germany). Stock solutions of each analyte were prepared by dissolving the solid substance in acetonitrile (preferably), acetonitrile/water 1:1 (v/v), methanol, methanol/water 1:1 (v/v) or water. Thirty- four combined working solutions were prepared by mixing the stock solutions of the corresponding analytes for easier handling, and were stored at -20°C. All solutions were however brought to room temperature before use. The final working solution was freshly prepared prior to spiking experiments by mixing the combined working solutions. Table 1. Number of samples and sample collection points in the FCT Abuja, Nigeria. Zone Total no. of samples/per zone Community Sesame Soybean Abaji 3 3 , Abaji Centra Pandagi, Sabongari AMAC 3 2 Gwagwa, Kabusa, Karshi, Karu and Orozo Bwari 3 2 Bwari Central, Byazhin, Ushafa Gwagwalada 6 4 Gwako, Paiko and Tungan Maje Kuje 4 6 Chibiri, Gaube, Saaji, Chukuku, Kuje Central, and Kwaku Kwali 5 6 Kilankwa, Kwali Central and Yangoji Total 24 23 2.3.2. Extraction procedures Five grams of each homogenized grain sample, previously pulverized in a mill with a 1 mm2 mesh (Cyclotech, Foss Tecator, Höganäs, Sweden), were weighed into a 50 ml polypropylene centrifuge tube (Sarstedt, Nümbrecht, Germany). Ten millilitres of water was added and briefly vortex and allowed to hydrate for ≥15 min. 20 ml of the extraction solvent (acetonitrile/water/acetic acid 79:20:1, v/v/v) were added before being vortex for 5-10 min to extract the mycotoxins. The extracts were filtered and centrifuged for 5 min at ≥3000 x g (4oC) in order to remove any interfering particles before further clean-up of the supernatant. For spiking experiments, 0.25 g sample was used for extraction. Samples were extracted for 90 min on a GFL 3017 rotary shaker (GFL, Burgwedel, Germany) and diluted (1 + 1) with dilution solvent (acetonitrile/water/acetic acid 20:79:1, v/v/v). Five microliters of the diluted extracts were subsequently injected [20]. 2.3.3. LC-MS/MS parameters The LC-MS/MS has been previously descri- bed by Malachova et al. [21]. Analysis was per- formed with a QTrap 5500 LC-MS/MS System (Applied Biosystems, Foster City, CA, USA) equipped with TurboIon Spray electrospray ioniza- tion (ESI) source and a 1290 Series HPLC System (Agilent, Waldbronn, Germany). Chromatographic separation was performed at 25°C on a Gemini C18-column, 150 × 4.6 mm i.d., 5 μm particle size, equipped with a C18 4 × 3 mm i.d. security guard cartridge (Phenomenex, Torrance, CA, USA). ESI-MS/MS based Spectrum 380® program was performed in the time-scheduled multiple reaction monitoring (MRM) mode both in positive and negative polarities in two separate chroma- 124 | Fapohunda et al. Profile of major and emerging mycotoxins in sesame and soybean grains European Journal of Biological Research 2018; 8 (3): 121-130 tographic runs per sample by scanning two fragmentation reactions per analyte. The MRM detection window of each analyte was set to its expected retention time ±27 s and ±48 s in the positive and the negative modes, respectively. Confirmation of positive analyte identification was obtained by the acquisition of two MRMs per analyte (with the exception of moniliformin that exhibited only one fragment ion). This yielded 4.0 identification points according to the European Union Commission decision 2002/657. In addition, the LC retention time and the intensity ratio of the two MRM transitions agreed with the related values of an authentic standard within 0.1 min and 30%, respectively. Quantification was performed during external calibration based on serial dilution of a multi-analyte stock solution which was the liquid standards. Results were corrected by apparent recoveries that had been determined by spiking five different blank samples at two concentration levels. 2.3.4. Statistical analysis Median, mean and standard deviation (SD) were calculated for concentrations of all toxins and metabolites. Pictorial representation of prevalence level was carried out using Excel package 2010. 3. RESULTS 3.1. Occurrence and contamination level of mycotoxins in sesame and soybean grains from the FCT, Abuja, Nigeria The percentage of contaminated samples, the mean, median and the range of contamination level of the regulated and emerging mycotoxins, in sesame and soybean from the FCT, Abuja are presented Table 2. Table 2. Occurrence and contamination level of mycotoxins in sesame and soybean grains from the FCT, Abuja, Nigeria. Mycotoxin Concentration in sesame (µg kg-1)* Concentration in soybean (µg kg-1) Positive (n=24) (%) Median Mean Range Positive (n= 23) (%) Median Mean Range Aflatoxin B1 (AFB1) 3 (13) 1.6 3.6 0.4-7.2 5 (22) 1.2 1.88 0.5-4.02 Aflatoxin B2 (AFB2) 2 (8) 1.5 1.5 0.8-1.6 0 - - - Aflatoxin G1 (AFG1) 0 - - - 1 (4) 21.1 21.1 21.1 Ochratoxin A (OTA) 0 - - - 2 (9) 16.8 16.8 10.5-23.1 Fumonisin B1 (FB1) 5 (21) 17.1 17.3 7.3-26.7 2 (9) 11.84 11.84 10.3-13.4 Fumonisin B2 (FB2) 2 (8) 8.6 8.6 6.1-11.2 0 - - - Fumonisin B4 (FB4) 1 (4) 5.72 5.72 5.72 0 - - - Deoxynivalenol (DON) 14 (58) 63.7 78.3 28-171 0 - - - Zearalenone (ZEN) 11 (46) 3.2 3.6 0.1-18.3 3 (13) 0.7 0.73 0.3-1.4 *Indicated by LC-MS/MS analysis. The major toxins and emerging metabolites common to both grains were three and seven respectively. Sesame samples had relatively higher occurrence of contamination with regulated mycotoxins such as DON (58.33%), ZEN (45.83%) and FB1 (20.88%) while 21.7% and 13.4% of the soybean were contaminated with AFB1 and ZEN respectively. AFG1 and OTA were detected in soyabean but not in sesame, while FB2 and DON were detected in the sesame but not in soyabean. The highest contamination level was from DON in the sesame samples with median of 63.7 µ g kg-1 while soybean had the highest contamination level from OTA with a median contamination level of 16.8 µ g kg-1. 125 | Fapohunda et al. Profile of major and emerging mycotoxins in sesame and soybean grains European Journal of Biological Research 2018; 8 (3): 121-130 3.2. Occurrence and contamination level of emerging mycotoxins in sesame and soybean grains Among the emerging toxins recognized by European Food Safety Authority (EFSA) and the Food and Agriculture Organisation of the United Nations (FAO) and detected in the samples were beauvericin (BEA), moniliformin (MON), steri- gmatocystin (STER), alternariolmethylether (AME), alternariol (AOH) and altertoxin-I (ATX-I). Beauvericin (BEA) had the highest occurrence of emerging mycotoxin both in the sesame (66.67%) and in the soybean samples (56.52%) at a respective maximum concentration of 4.2 and 23.04 µ g kg-1 respectively (Table 3). The next higher occurrence (29.16%) was obtained for Alternariol methyl ether (AME), with concentration range of 0.22-11.3 μg kg-1 in sesame samples. Moniliformin (MON) occurred in the 30.43% of the soybean and with a range concentration of 0.12-33.34 µ g kg-1, while it was detected in 16.67% of sesame with a range of 4.1-17.4 µ g kg-1. Other emerging mycotoxins such as citrinin (CTN) and tenuazonic acid (TeA) were below detectable level. Table 3. Occurrence of emerging mycotoxins in the samples from the FCT, Abuja, Nigeria, detected by LC-MS/MS. Class of emerging mycotoxin Emerging mycotoxin Sesame concentration* (µg kg-1) Soybean concentration (µg kg-1) Positive (n=24) (%) Median Mean Range Positive (n=23) (%) Median Mean Range Fusarium metabolites Beauvericin (BEA) 16 (67) 0.91 0.94 0.08-4.2 13 (57) 0.31 3.69 0.018- 23.04 Moniliformin (MON) 4 (17) 12.77 11.8 4.1-17.4 7 (30) 2.4 9.4 0.12- 33.34 Enniatin B (ENN-B) 0 -** - - 2 (9) 0.00525 0.005 25 0.005- 0.0055 Aflatoxin precursor Sterigmatocystin (STER) 4 (17) 0.45 0.43 0.27-0.55 1 (4.3) 0.6 0.6 0.6 Alternaria metabolites Chanoclavine (CNV) 3 (13) 0.14 0.14 0.06-0.2 0 - - - Festuclavine (FCV) 2 (9) 1.7 3.21 1.12-8.3 0 - - - Elymoclavine (ECV) 0 - - - 1 (4.3) 1.4 1.4 1.4 Altertoxin-I (ATX-I) 2 (9) 3.9 4.26 2.82-6.5 1 (4.3) 0.91 0.91 0.91 Alternariol (AOH) 5 (21) 1.96 8.55 1.14-3.5 1 (4.3) 0.85 0.85 0.85 Alternariol- methylether (AME) 7 (29) 6.54 5.23 0.22-11.3 1 (4.3) 0.6 0.6 0.6 *Indicated by LC-MS/MS analysis; **= below limit of detection; The co-occurrence of toxins and metabolites in the ten highest contaminated samples of sesame and in six soybean samples were pictorially presented as scatter plots in Figures 1 and 2. As shown in Figure 1, only in the 5 out of the 10 most contaminated sesame samples (S1-S5) that DON and ZEN co-occurred with other mycotoxins.As shown in Figure 2, only in the 3 out of the 6 most contaminated soyabean samples (S1-S3),that AFB1 co-occur with other mycotoxins, though at relatively low concentrations below maximum regulatory limits. 126 | Fapohunda et al. Profile of major and emerging mycotoxins in sesame and soybean grains European Journal of Biological Research 2018; 8 (3): 121-130 Figure 1. Scatter plots visualizing co-occurrence of major mycotoxins in the ten highest contaminated samples of sesame from the FCT, Abuja Nigeria. Different colour pattern represents each type of metabolite. Figure 2. Scatter plots visualizing co-occurrence of major mycotoxins in the six highest contaminated samples of soybean from the FCT, Abuja Nigeria. Different colour pattern represents each type of metabolite. 4. DISCUSSION The development of LC-MS/MS methods for the simultaneous detection and quantification of a broad spectrum of mycotoxins has facilitated the screening of a larger number of samples for contamination with a wide array of major and less well-known “emerging” mycotoxins [22, 23] There- fore, this analysis was applied in this investigation. 4.1. Occurrence of regulated mycotoxins In the present study, AFB1 was observed more frequently in soybean than in sesame. In countries having similar climate, like Burkina Faso and Mozambique, AFB1 was observed more frequently in soybean-based food and feeds (Burkina Faso, 50% incidence, median = 23.6 μg kg-1; Mozam- bique, 46% incidence, median = 69.9 μg kg-1) than in groundnuts (Burkina Faso, 22% incidence, median = 10.5 μg kg-1; Mozambique, 14% inciden- ce, median = 3.4 μg kg-1) [24]. Ezekiel et al. [19] observed that mycotoxin levels were higher in the Nigerian soybean-based kunu-zaki (