Introduction
Food crops and feed materials can be easily infected by fungal species which may produce mycotoxins during growth, harvest, and storage (
Monbaliu et al., 2010). When considering the heat stability of mycotoxins, these substances constitute a potential risk for human and animal health (
Zinedine and Mañes, 2009).
Currently, more than 400 mycotoxins have been identified in the world (
Salem and Ahmad, 2010). Among the many known toxins, aflatoxins (AFs) are highly toxic and carcinogenic compounds that can cause diseases in livestock and humans (
Groopman and Donahue, 1988;
Simon et al., 1998). AFs are a group of mycotoxins produced as secondary metabolites by the spoilage of fungi
Aspergillus, particularly
A. flavus and
A. parasiticus (
Kurtzman et al., 1987). The most important members of AFs are AFB1, AFB2, AFG1 and AFG2. The International Agency for Research on Cancer (IARC) has classified AFB1, AFB2, AFG1 and AFG2 as Group I human carcinogens (
IARC, 2002).
Deoxynivalenol (DON), zearalenone (ZEN) and fumonisins (FBs) are mycotoxins produced by several species of the grain pathogen ascomycete genus
Fusarium spp. (
Gelderblom et al., 1988;
Schaafsma et al., 1998;
Bennett and Klich, 2003). The toxicity of DON and ZEN is relatively lower compared to that of AFs. However, acute exposure to high levels of DON doses causes vomiting, diarrhea and even gastrointestinal hemorrhage; chronic exposure may lead to reduced weight gain and anorexia in animals (
Pestka, 2007). ZEN has been associated with early puberty, hyperplasic and neoplastic endometrium, and cervical cancer in humans (
Thongrussamee et al., 2008). Additionally, ingestion of foods highly contaminated with FBs results in increased risk of esophageal cancer and neural tube defects in humans (
Chu and Li, 1994;
Missmer et al., 2006). In pigs, high levels of FB1 in feeds can be hepatotoxic, inducing pulmonary edema and cardiovascular changes (
Haschek et al., 2001;
Marin et al., 2007). Mycotoxins can cause serious problems on human and animal health. The maximum tolerating levels to mycotoxin in foodstuffs and feedstuffs have been established in many countries (
Wang et al., 2008).
Mycotoxins can occur both in temperate and tropical regions of the world, depending on the species of fungi (
Salem and Ahmad, 2010). Maize is a very good substrate for fungal growth and toxinogenesis (
Trung et al., 2008). The natural occurrence of mycotoxins in maize, maize-based food and feeds has been steadily reported from all over the world (
Doko and Visconti, 1994;
Fandohan et al., 2005;
Lino et al., 2007;
Martins et al., 2007;
Ok et al., 2007;
Silva et al., 2007;
Jajić et al., 2008;
Marinamartins et al., 2008;
Gong et al., 2009;
Kimanya et al., 2009). However, there is no information available on the natural occurrence of some mycotoxins in maize throughout China. The purpose of this study was to investigate AFs (mostly including AFB1, AFB2, AFG1 and AFG2), DON, ZEN, FB1 and FB2 contamination incidence and their levels in maize collected in Hebei Province of northern China.
Materials and methods
Chemicals and apparatus
HPLC-grade acetonitrile, water and methanol were purchased from Dikma (Lake Forest, USA), AFB1, AFB2, AFG1, AFG2, DON, ZEN, FB1 and FB2 standards were obtained from Sigma Chemicals Co. (St. Louis, USA). The multifunctional columns, PuriToxSR column TC-M160 and PuriToxSR column TC-T200, were obtained from Trilogy Analytical Laboratory (Washington, USA). Strong anion-exchange columns (SAX) (500 mg, 3 mL) were obtained from Thermo Electron Corporation (Boston, USA). O-Phthaldialdehyde (OPA), 2-mercaptoethanol and potassium borate buffer (o-phtalaldehyde diluent, OD104) were supplied by Pickering Laboratories (Mountain View, USA).
A 20 mg/L stock solution of DON was prepared in methanol and stored at 4°C. A 20 mg/L stock solution of ZEN was prepared in acetonitrile and stored at -20°C. A 20 mg/L stock solutions of AFB1, AFB2, AFG1 and AFG2 were prepared in benzene:acetonitrile (98∶2, v:v). Solutions for AFB1, AFG1 and AFG2 were stored at 4°C and at -20°C for AFB2. Stock solutions of FB1 and FB2 (20 mg/L) were prepared in acetonitrile:water (50∶50, v:v) and stored at 4°C. The HPLC (Agilent Technologies 1200 Series) apparatus used in this study consisted of a quaternary pump (model G1311A), a vacuum degasser (model G1322A), a thermostated autosampler (model G1329A), a column compartment (model G1316A), a DAD detector (model G1315D) and a fluorescence detector (model G1321A). Online post-column chemical derivatization was performed with a Pinnacle PCX reactor supplied by Pickering Laboratories (Mountain View, USA). The ZORBAX SB-C18 column (250 mm × 4.6 mm i.d., particle size 5 µm), was supplied by Agilent Technologies.
Chromatographic conditions
For AFs analysis
The injection volume was 10 µL and the column temperature was set at 42°C. The mobile phase was methanol-acetonitrile-water (22∶22∶56, v:v:v) and pumped at a flow rate of 1 mL/min. Fluorescence detection was performed at the optimized excitation and emission wavelengths of 365 and 430 nm, respectively. The post-column derivatization conditions were set as the following: runtime of 20 min; equilibration time for 1 min; reactor temperature at 95°C; reactor volume of 1.4 mL; and pump1 (100 mg/L, iodine solution) flow rate of 0.3 mL/min.
For DON analysis
The injection volume was 10 µL and the column temperature was set at 30°C. The mobile phase was methanol-water (20∶80, v:v) pumped at a flow rate of 1 mL/min. DON was detected at 220 nm with a DAD detection.
For ZEN analysis
The injection volume was 10L and the column temperature was set at 30°C. The mobile phase was methanol: water (80∶20, v:v) and pumped at a rate of 1 mL/min. Fluorescence detection excitation and emission wavelengths were 274 and 440 nm, respectively.
For FBs analysis
The injection volume was 100 µL and the column temperature was set at 40°C. The mobile phase consisted of methanol (A) and 0.05 M citric acid buffer (pH 4) (B) at a flow rate of 1.0 mL/min. The optimized elution gradient was 7 min isocratic step at 62% A and 38% B; 3 min linear gradient to 66% (A) and 34% (B) and held constantly for 5 min, followed by the initial mobile phase composition, whose system was re-equilibrated for 10 min. Fluorescence detection was performed at the optimized excitation and emission wavelengths of 335 and 440 nm, respectively. The OPA derivatization reagent was prepared fresh with 2.0 g OPA dissolved in 10 mL methanol and diluted with OD104, then added with 6 mL 2-mercaptoethanol, and finally diluted with OD104 to 500 mL. The post-column derivatization solution was under a 25 min runtime; 1 min equilibration time; reactor at 40°C; 1.4 mL reactor volume; at pump1 (2% sodium hydroxide) flow rate of 0.4 mL/min and pump2 (OPA) flow rate of 0.8 mL/min.
Sampling and sample preparation
A total of 25 samples of corn harvested in 2009 were collected from the main corn growing areas of Hebei Province in the northern part of China. The location selected for sampling was divided into plots of 80 m2 each. Ten ears of corn were sampled from each plot, taken as one sample, from which a 1 kg subsample was taken. The samples were kept at 4°C until analysis showed they came from the same maize breed, i.e., Zhengdan 958. All the samples were finely milled in the switching apparatus. A ground sample (10 g) was placed into a blender jar and mixed with 40 mL acetonitrile:water at 84∶16 (v:v). The mixture was blended at a high speed for 3 min, filtered through Neutral filter paper, and collected in a 50-mL centrifuge tube.
A 4 mL aliquot of the filtrate was loaded onto a multifunctional cleanup column TC-M160 without pre-conditioning and was pushed completely through the column using a plunger while being collected directly in a 15 mm × 125 mm test tube. A 2 mL sample of the filtrate was evaporated in a 60°C water bath under a stream of nitrogen. The residue was reconstituted in 0.5 mL of acetonitrile. After filtration (0.45 µm pore size), the samples were subjected to HPLC analysis for their AFs and ZEN contents.
A 2 mL sample of the filtrate from the initial processing was loaded to a multifunctional cleanup column TC-T200, and pushed through with a plunger while the filtrate was being collected in a 15 mm × 125 mm test tube. The plunger was removed, 4 mL acetonitrile:water (84∶16, v:v) was added into the top of the column, pushed all through the column and collected in the same test tube. The rinsing process was repeated. All the filtrate was evaporated in a 60°C water bath under a stream of nitrogen. The residue was redissolved in 0.5 mL acetonitrile. After filtration (0.45 µm pore size), the sample was subjected to HPLC analysis for DON content.
Eight mL of the filtrate was loaded onto a SAX cartridge preconditioned with 5 mL of methanol followed by 5 mL of acetonitrile /water (3∶1, v:v) at a flow rate of approximately 1 mL/min. The cartridge was washed with 5 mL of acetonitrile/water (3∶1, v:v), followed by 3 mL of methanol. Finally, fumonisins were eluted with 10 mL of 1% methanolic acetic acid (v:v). The eluate was evaporated in a 60°C water bath under a stream of nitrogen, and redissolved in 1 mL of acetonitrile/water (50∶50, v:v). After filtration (0.45 µm pore size), the sample was subjected to HPLC analysis for FB content.
Results
Method validation
The analytical method was within the laboratory validated with respect to linearity, recovery, precision, limits of detection (LODs) and limits of quantification (LOQs).
The standard curves for AFs, DON, ZEN and FBs at two determinations of eight concentration levels, were linear, with correlation coefficients (R2) from 0.9990 to 0.9999 (Table 1). Precision was evaluated in terms of intraday repeatability and inter-day reproducibility as RSD%. The intraday variability of the assay was tested by six consecutive injections on the same day. The interday reproducibility of the assay was determined by six injections on five different days. The results are shown in Table 1. The accuracy of the method was confirmed by determining the recovery. Average recovery rates of AFs, DON, ZEN and FBs, which varied from 74.9% to 104.8% with a relative standard deviation from 2.4% to 8.9%, by adding different spiking levels to analyte-free corn samples are presented in Table 2. The limits of detection (LODs) for AFB1, AFB2, AFG1, AFG2, DON, ZEN, FB1 and FB2 were 0.05, 0.1, 0.1, 0.2, 11, 12, 10 and 9 µg/kg, respectively at a signal-to-noise ratio of 3∶1, and the limits of quantitation (LOQs) for AFB1, AFB2, AFG1, AFG2, DON, ZEN, FB1 and FB2 were 0.2, 0.4, 0.4, 0.7, 35, 40, 65 and 62 µg/kg, respectively at 10∶1 (Table 2).
Determination of mycotoxins in maize
In this investigation, a total of 25 samples were analyzed for mycotoxins. The occurrences of AFs, DON, ZEN and FBs in the analyzed samples are presented in Table 3. 68% of the 25 samples analyzed were contaminated with DON at levels ranging from 28 to 2533 µg/kg. The average level of DON in all samples analyzed in the present study was 605 µg/kg.
For ZEN analysis, 15 samples (60%) were found to be contaminated with ZEN at levels ranging from 60 to 1239 µg/kg, and the average level of ZEN in all samples was 238 µg/kg.
The presence of FB1 and FB2 was verified in 8 samples (32%) and 3 (12%) samples, respectively. In positive samples, the concentration of FB1 ranged from 150 to 3480 µg/kg and the concentration of FB2 ranged from 170 to 1000 µg/kg. The average concentration in this study for the totality of maize samples was 360 µg/kg for FB1 and 58 µg/kg for FB2.
AFs were not detected in maize in our study.
Discussion
The study was undertaken to evaluate the natural occurrence of some mycotoxins (AFB1, AFB2, AFG1, AFG2, DON, ZEN, FB1 and FB2) in maize from Hebei Province. To the best of our knowledge, there have been no previous reports on the occurrence of multiple mycotoxins in corn throughout China.
As reported in the available literature, the incidence and levels of DON in maize varied widely. The average contamination level of DON was found to be 605 µg/kg, much higher than the 0-338 µg/kg reported in Argentina, France and Serbia (
González et al., 1999;
Jajić et al., 2008;
Scudamore and Patel, 2009). However, higher values were also reported in maize samples from Austria, with an average contamination level of 753 µg/kg (
Berthiller et al., 2009). Among all the samples, 7 samples were found to have a higher level of DON than the maximum tolerable limit of 1000 µg/kg set as the Chinese standard in maize, although the average of DON did not exceed the Standard (
GB, 2005).
Low levels of ZEN were reported in Argentina (7 µg/kg), France (26 µg/kg) and Italy (42 µg/kg) according to some reports (
Visconti and Pascale, 1998;
González et al., 1999;
Scudamore and Patel, 2009), whereas higher levels of ZEN were found in previous papers (
Silva and Vargas, 2001;
Shim et al., 2009). Thongrussamee et al. (
2008) reported that ZEN was found at a maximum level of 686 µg/kg by HPLC in maize. The similar level was found by Silva and Vargas (
2001) in Brazilian corn. In our work, 60% samples contaminated with ZEN were found and all the positive samples were above the maximum level (60 µg/kg in maize) adopted by China. The average concentration of ZEN (238 µg/kg) in our samples was almost four times as high as the maximum tolerable limit of 60 µg/kg set as the Chinese standard in maize (
GB, 2005).
In this study, we found that the mean concentration of FB2 (58 µg/kg) was lower than that of FB1 (360 µg/kg), which was usual in other studies (
Scudamore and Patel, 2000;
Domijan et al., 2005;
Lino et al., 2006). The mean concentration in our study for the totality of maize samples was 360 µg/kg for FB1 and 58 µg/kg for FB2, which was similar to that reported in Croatia (459.8 µg/kg for FB1) and Portugal (329 µg/kg for FB1 and 131 µg/kg for FB2) (
Domijan et al., 2005;
Lino et al., 2006), but lower than that reported in Spain (4800 µg/kg for FB1 and 1900 µg/kg for FB2), Morocco (1930 µg/kg for FB1) and China (6662 µg/kg for FB1) (
Castellá et al., 1999;
Zinedine et al., 2006;
Gong et al., 2009). The maximum permitted level of FBs in feed and groceries has not yet been set in China. No samples, except one (Maize-01), had levels of FBs exceeding the maximum level of 4000 µg/kg in unprocessed corn for direct human consumption established by the European Union (EU) (
European Commission, 2007).
AFs were not detected in maize in our study, although
A. flavus, A. niger and
Fusarium were found in maize samples (further study is on going). Aflatoxin contamination may be influenced by many factors including the nature of the substrate and the presence of other competing fungi (
Betina, 1989). Although fungi, particularly
A. flavus, was detected at very high levels in the market melon seeds, the incidence and level of aflatoxin were low (
Bankole et al., 2004). This agrees with an earlier report in which
A. flavus constituted 60% of total fungi isolated from melon ball snacks, but the aflatoxin contamination was actually detected in only 11% of samples (
Bankole, 1995). Similar results were reported in spices from Egypt by Oman. Abou-Arab et al. (
1999) and Elshafie et al. (
2002) did not detect aflatoxins, but isolated aflatoxigenic strains of
A. flavus. ElShafie et al. (
2002) concluded that spices and other condiments are unsuitable substrates for aflatoxin production because of their essential oils, which may inhibit toxin production. Horn and Wicklow (
1983) have reported that each fungus carries a factor that determines its growth and ability to compete with other fungal species. Thus, it has been pointed out that fungal interactions from competition could lead to a decrease in mycotoxin contamination.
Although the number of samples analyzed was very limited, the investigation showed that the maize in Hebei Province frequently contained DON, ZEN and FBs. As AFs were not detected in maize in this study, continuing detection of AFs is still an essential part of monitoring. Further monitoring of mycotoxins in China is necessary. Furthermore, the producing fungi and their competition need to be thoroughly investigated.
Higher Education Press and Springer-Verlag Berlin Heidelberg
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