Food Additives and Contaminants: Part B Vol. 5, No. 3, September 2012, 208–211
Occurrence of fumonisins and aflatoxins in cereals from markets of Hebei province of China Y.P. Liua, L.X. Yanga, N.J. Yanga, B. Donga, L.L. Caob*, K. Wangb and L.X. Yangb a
Hebei Provincial Center for Disease Control and Prevention, Shijiazhuang, Hebei Province, China; bShijiazhuang Center for Disease Control and Prevention, Shijiazhuang, Hebei Province, China (Received 1 November 2011; final version received 23 May 2012) Fumonisins B1, B2 and B3 (FB1, FB2 and FB3) and aflatoxins B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2) are both major mycotoxins of food concern, because of their wide range of concentration and possible co-occurrence. Therefore, a contamination survey in corn and wheat flour by liquid chromatography–tandem mass spectrometry was carried out. Quantification of fumonisins and aflatoxins was based on internal calibration (by the use of 13C34-fumonisin) and external calibration, respectively. Fumonisins were detected in 95% of corn samples and in 7% of wheat flour samples, with the mean level (FB1 þ FB2 þ FB3) of 441 mg kg1 and 0.09 mg kg1, respectively. Low levels of aflatoxins were detected in 37% of the samples with a mean level (B1 þ B2 þ G1 þ G2) of 0.12 mg kg1. Fumonisins and aflatoxins were not detected in 29% of the samples analysed. Simultaneous occurrence of fumonisins and aflatoxins was observed in 12% of samples. Keywords: fumonisin; aflatoxin; corn; wheat flour; liquid chromatography–tandem mass spectrometry
Introduction As major groups of mycotoxins, fumonisin and aflatoxins are commonly present in different kinds of foodstuffs, especially in cereals and grains. Fumonisin has three major types: B1 (FB1), B2 (FB2) and B3 (FB3). Aflatoxin has four major types: B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2). Some studies have demonstrated that they are regarded a potential risk to human and animal health, including genotoxic (AFB1), carcinogenic, hepatotoxic (FB1) and immunosuppressive (Shephard et al. 1999; Gelderblom et al. 2001; Voss et al. 2001). Furthermore, corn and wheat flour are two of the most important cereal crops in China and are used primarily for direct human and animal consumption. Hence, the presence of fumonisin and aflatoxins in cereals must be paid close attention. The natural occurrence of fumonisins, together with aflatoxins, has been reported in different foods and feeds from many countries (Kedera et al. 1999; Gutema et al. 2000; Omurtag 2001; Ferracane et al. 2007; Idris et al. 2010). The maximum admissible levels of fumonisins and aflatoxins vary from one country to another. In the United States, the FDA has set a maximum permissible level of 2 mg kg1 for fumonisins in human food and 20 mg kg1 for total aflatoxins in all foodstuffs (Wu 2004). European Union (EU) has set a fumonisin level of 1 mg kg1 and AFB1 level of 2 mg kg1 for human consumption. In China, the
*Corresponding author. Email: [email protected] ISSN 1939–3210 print/ISSN 1939–3229 online ß 2012 Taylor & Francis http://dx.doi.org/10.1080/19393210.2012.697486 http://www.tandfonline.com
legal limit of AFB1 in corn is set at 20 mg kg1 (National food safety standard GB2761-2011). The aim of the present study was to survey the contamination levels of fumonisins and aflatoxins in cereals and prepare for further assessing their potential risks to human health.
Materials and methods Samples In this paper, a total of 51 cereals (30 wheat flour and 21 corn samples) were purchased from supermarkets in Shijiazhuang, Hebei Province, of China.
Chemicals All solvents were high-performance liquid chromatography (HPLC)-grade and purchased from J T Baker (Philipsburg, MT, USA). The single standard solutions of FB1, FB2 and FB3 (50 mg mL1) were obtained from Sigma-Aldrich Laborchemikalien GmbH (Steinheim, Germany). The isotope internal standards 13C34-FB1 (25 mg mL1), 13C34-FB2 (10 mg mL1), 13C34-FB3 (10 mg mL1) were also obtained from Sigma-Aldrich Laborchemikalien GmbH. MultiSepÕ 211 FUM columns were obtained from Romer Labs (Tulln, Austria).
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The parameters used for MS under ESIþ mode were set as follows: spray voltage 3500 V, Sheath Gas Pressure 40, Aux Gas Pressure 6, capillary temperature 320 C, capillary offset 114. Table 1 lists the parameters and collision energies of parent, primary and secondary daughter ions, limits of detection (LOD, S/N ¼ 3) and of quantification (LOQ, S/N ¼ 10) and linearity (R2) for FBs. Quantification was carried out by isotope internal calibration with standards in solvent.
The standards of AFB1, AFB2, AFG1 and AFG2 were purchased from Romer Labs. Immunoaffinity columns (AflaStarTM R, IAC1004 column), used for rapid and simple purification of aflatoxin B1, B2, G1 and G2 were purchased from Romer Labs, Inc. (Union, MO, USA). Milli-Q quality water (Millipore, Bedford, MA, USA) was used for all analyses.
Sample extraction and analysis for fumonisins Sample portions of 2 g were accurately weighed, spiked with a known amount of internal standards (13C-FB1, 13 C-FB2 and 13C-FB3) and extracted with 10 mL acetonitrile–water (50/50) by shaking for 3 min. After that, extracts were centrifuged at 4000 rpm for 10 min. Then 1 mL supernatant was diluted into 10 mL methanol–water (3/1). The solutions were flown through multifunction cartridges, previously conditioned by the successive passage of 5 mL methanol and 5 mL methanol–water (3/1). The cartridges were washed with 5 mL methanol–water (3/1) followed by 5 mL methanol. The fumonisins were eluted with 10 mL methanol– acetic acid (99/1). The eluates were dried and reconstituted with 1 mL of acetonitrile–water (50/50). Before LC-MS/MS, the solution was passed through a 0.22 -mm nylon filter. The analysis was performed by using an LC-MS/ MS system consisting of a Finnigan Surveyor LC in combination with an ultra-TSQ tandem mass spectrometer (Thermo Fisher Scientific, Barrington, Illinois, USA). The chromatographic column was a 15 mm 2.1 mm i.d. Atlantis T3 (Waters Corporation, Wexford, Ireland, USA). The mobile phase, with a flow rate of 0.2 mL min1, consisted of (A) 0.1% formic acid–water solution and (B) acetonitrile–methanol (50/50, v/v). Organic solvent percentage in the mobile phase linearly varied as follows: 0 min, 30%; 8 min, 70%; 13 min, 70%; 17 min, 100%; 19 min, 100%; 20 min, 30%; 30 min, 30%. The injection volume was 20 mL and the column temperature was maintained at 25 C.
Sample extraction and analysis for aflatoxins Samples were accurately weighed (5 g, precision 0.1 mg) and 5 g NaCl and methanol–water (7/3, v/v) were added up to 20 mL final volume. The whole mixture was extracted on a mechanical shaker for 2 min. After filtration, 15 mL extract was collected and diluted with 30 mL HPLC-grade water. Then 15-mL sample extracts were passed through immunoaffinity columns at a rate of about 6 mL min1. The column was washed with 10 mL HPLC water for two times. Aflatoxins were eluted from the column with 1 mL methanol. Finally, the methanolic elutes were dried and reconstituted with 1 mL of 0.1% formic acid solution before LC-MS/MS analysis. The same LC-MS/MS system was used for aflatoxins analysis, with a 15 mm 2.1 mm i.d. X Terra chromatographic column (Waters). The mobile phase consisted of (A) water containing 0.1% formic acid and (B) acetonitrile–methanol (50/50, v/v) with a flow rate of 0.2 mL min1. Organic solvent percentage in the mobile phase linearly varied as follows: 0 min, 22%; 8 min, 45%; 11 min, 100%; 13 min, 100%; 17 min, 22% and 24 min, 22%. The injection volume was 20 mL and the column temperature was maintained at 25 C. The parameters used for MS, also in ESIþ mode, were spray voltage of 3500 V, Sheath Gas Pressure of 40, Aux Gas Pressure of 10, capillary temperature of 300 C and capillary offset of 35. Table 2 lists the parameters and collision energies of parent, primary daughter and secondary daughter ions, limits of detection (LOD, S/N ¼ 3) and of quantification
Table 1. Collision energies of parent, primary and secondary daughter ions, LOD (S/N ¼ 3), LOQ (S/N ¼ 10) and linearity (R2) for fumonisins.
FBs
Parent ion (m/z)
Primary daughter ion (m/z)
Collision energy (eV)
Secondary daughter ion (m/z)
Collision energy (eV)
R2
LOD (mg kg1)
LOQ (mg kg1)
FB1 FB2 FB3 13 C34-FB1 13 C34-FB2 13 C34-FB3
723.0 707.0 707.0 756.2 740.3 740.3
353.5 337.2 337.2 374.2 358.2 358.2
42 36 36 36 39 39
705.3 354.2 354.2 356.3 376.2 376.2
28 26 26 37 45 45
0.9920 0.9971 0.9975 – – –
0.6 0.5 0.4 – – –
1.9 1.6 1.4 – – –
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Table 2. Collision energies of parent, primary and secondary daughter ions, LOD (S/N ¼ 3), LOQ (S/N ¼ 10) and linearity (R2) for aflatoxins.
AFs
Parent ions (m/z)
Primary daughter ion (m/z)
Collision energy (eV)
Secondary daughter ion (m/z)
Collision energy (eV)
R2
LOD (mg kg1)
LOQ (mg kg1)
AFB1 AFB2 AFG1 AFG2
313.0 315.0 328.9 331.1
241.0 259.1 242.9 189.0
44 34 33 40
285.0 287.1 283.0 256.9
24 18 42 30
0.9991 0.9943 0.9981 0.9960
0.05 0.02 0.09 0.04
0.16 0.06 0.26 0.11
(LOQ, S/N ¼ 10) and linearity (R2) for AFs. Quantification was carried out by external calibration with standards in solvent.
Evaluation of method performance Accuracy was estimated by spiking blank samples in recovery experiments (n ¼ 3). Method reproducibility studies were done by injecting three replicates of the same standard solution in three different days and in the same day. Both the intraday and interday precision showed relative standard deviations below 15%. The spiked concentrations of FB1, FB2 and FB3 were evaluated at levels of 2, 10, 100 and 500 mg kg1. All recoveries were higher than 75% (75.9%–111.2%), indicating good accuracy of the method. RSDs (n ¼ 6) ranged from 2.1% to 9.4% and LODs from 0.4 to 0.6 mg kg1. The spiked concentrations of AFB1, AFB2, AFG1 and AFG2 were evaluated with high (500 mg kg1), intermediate (10 and 100 mg kg1) and low (2 mg kg1) levels for each AF standard. All recoveries were higher than 80%. RSDs ranged from 6.8% to 12.4% and LODs from 0.02 to 0.09 mg kg1 for aflatoxins. RSDs (n ¼ 6) ranged from 2.9% to 12.9%.
Results and discussion Fumonisins contamination in corn and wheat flour In total, 51 samples (30 wheat flour and 21 corn samples) intended for human consumption were analysed for the incidence and levels of fumonisins B1, B2 and B3. Forty-one percent of the samples contained FB1 with concentrations ranging from 1 to 1997 mg kg1. FB2 was detected in 33% of the samples with concentrations ranging from 0.6 to 190 mg kg1. Forty-three percent of the samples contained FB3, with levels ranging from 0.6 to 541 mg kg1. The simultaneous occurrence of FB1, FB2 and FB3 was observed in 31% of the samples. According to different sample types, fumonisins were detected in most of corn samples (95% of occurrence), whereas only low concentration of fumonisins was found in a small part of wheat flour (7% of
occurrence). The results indicated that corn samples in the present study showed high levels of fumonisin contamination, and the majority of wheat samples in this study were safe. The average levels of the fumonisin-positive corn samples were 325, 34 and 82 mg kg1, for FB1, FB2 and FB3, respectively. The incidence of fumonisin in our work was close to those reported in corn-based human food products in the United States (Sydenham et al. 1991; Stack and Eppley 1992). In the United States, 94% of corn meal for human consumption analysed in 1991 was contaminated, with mean levels of 1048 and 298 mg kg1 for FB1 and FB2, respectively. Although the mean levels of fumonisins were lower than the cited US levels, the levels of fumonisin B1 in three corn samples were found to be high (1219, 1997 and 1578 mg kg1), which exceeded the proposed fumonisin level of 1 mg kg1 by the European Union. Fumonisin levels obtained from our work were also lower than those found by Yoshizawa et al. (1994), who found mean levels of 872 mg kg1 for FB1 and 448 mg kg1 for FB2 in Linxian corn.
Aflatoxins contamination in corn and wheat flour In total, aflatoxin contamination in corn and wheat flour was at low levels in this study. The highest aflatoxin B1 contamination was found in one corn sample (2.1 mg kg1), which was slightly higher than the proposed B1 levels of 2 mg kg1 for human consumption as set by EU. Positive aflatoxin levels in the other samples were lower than 0.5 mg kg1. The levels of AFB1, AFB2, AFG1 and AFG2 in all samples ranged from 0.10 to 2.1, 0.02 to 0.2, 0.1 to 0.5 and 0.1 to 0.5 mg kg1, respectively. In most of the samples, no aflatoxins were detected. This result was similar to the report by Moreno et al. (2009), where aflatoxins were not detected in 92% of the analysed corn samples. AFB1 contamination existed in four corn samples and four wheat flour samples, whereas AFB2 was found in two corn samples and two wheat flour samples. No corns were determined with AFG1 and AFG2. However, AFG1 was detected in 4 out of 30 wheat flour samples and AFG2 was found in another 5
Food Additives and Contaminants: Part B wheat flour samples. Hence, most of the samples analysed were below the legal limits.
Comparison of fumonisin and aflatoxin levels in cereals In this study, determination of both fumonisin and aflatoxin levels allowed comparison between these two classes of contaminants. Overall, the average concentration of fumonisins (FB1, FB2 and FB3 were 134, 14 and 34 mg kg1, respectively) in these cereals were much higher than that of aflatoxins (0.1 mg kg1, for AFB1 þ AFB2 þ AFG1 þ AFG2). This indicates that corn is more susceptible to fumonisin contamination. No correlation between fumonisin and aflatoxin contamination was obtained. However, Abbas et al. (2006) reported a positive correlation between aflatoxin and fumonisins and concluded that natural infection with Fusarium spp. apparently did not protect against aflatoxin production.
Conclusions In this study, co-occurrence of fumonisins and aflatoxins was evaluated in 51 cereals samples. Fumonisins were detected in 95% of corn samples and in 7% of wheat flour samples, with a mean level (FB1 þ FB2 þ FB3) of 444 and 0.1 mg kg1, respectively. The results indicated that corn was more susceptible to fumonisin contamination. Most of the samples analysed had aflatoxin contamination below the EU limit (37% of occurrence). Further studies should investigate the fumonisin and aflatoxins co-occurrence in cereal samples from other districts of China.
Acknowledgements The authors wish to thank Dr Shang in Chinese CDC and Dr Li in Tianjin Entry-Exit Inspection and Quarantine Bureau for providing some literature documents.
References Abbas HK, Cartwright RD, Xie WP, Shier WT. 2006. Aflatoxin and fumonisin contamination of corn (maize, Zea mays) hybrids in Arkansas. Crop Prot. 25:1–9.
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Ferracane R, Tafuri A, Logieco A, Galvano F, Balzano D, Ritieni A. 2007. Simultaneous determination of aflatoxin B1 and ochratoxin A and natural occurrence in Mediterranean virgin olive oil. Food Addit Contam. 24:173–180. Gelderblom WC, Abel S, Smuts CM, Marnewick J, Marasas WF, Lemmer ER, Ramljak D. 2001. Fumonisin-induced hepatocarcinogenesis: mechanisms related to cancer initiation and promotion. Environ Health Persp. 109:291–300. Gutema T, Munimbazi C, Bullerman LB. 2000. Occurrence of fumonisins and moniliformin in corn and corn-based food products of U.S. origin. J Food Prot. 63:1732–1737. Idris YMA, Mariod AA, Elnour IA, Mohamed AA. 2010. Determination of aflatoxin levels in Sudanese edible oils. Food Chem Toxicol. 48:2539–2541. Kedera CJ, Plattner RD, Desjardins AE. 1999. Incidence of Fusarium spp and levels of fumonisins in maize in Western Kenya. Appl Environ Microbiol. 65:41–44. Moreno EC, Garcia GT, Augusto Ono M, Vizoni E, Kawamura O, Hirooka EY, Sataque Ono EY. 2009. Cooccurrence of mycotoxins in corn samples from the northern region of Parana State, Brazil. Food Chem. 116:220–226. National food safety standard: standards for limits of mycotoxins in food GB2761-2011. Omurtag GZ. 2001. Determination of fumonisin B1 and B2 in corn and corn-based products in Turkey by HPLC. J Food Prot. 64:1072–1075. Shephard GS, Thiel PG, Stocienstrom S, Sydenham E. 1999. Worldwide survey of fumonisin contamination of corn and corn based products. J Assoc Off Anal Chem Int. 79:671–686. Stack ME, Eppley RM. 1992. Liquid chromatographic determination of fumonisins B1 and B2 in corn and corn products. J Assoc Off Anal Chem. 75:834–837. Sydenham EW, Shephard GS, Thiel PG, Marasas WFO, Stockenstrom S. 1991. Fumonisin contamination of commercial corn-based human foodstuffs. J Agric Food Chem. 39:2014–2018. Voss KA, Riley RT, Norred WP, Bacon CW, Meredith FI, Howard PC, Plattner RD, Collins TF, Hansen DK, Porter JK. 2001. An overview of rodent toxicities: liver and kidney effects of fumonisins and Fusarium moniliforme. Environ Health Persp. 109:259–266. Wu F. 2004. Mycotoxin risk assessment for the purpose of setting international regulatory standards. Environ Sci Technol. 38:4049–4055. Yoshizawa T, Yamashita A, Luo Y. 1994. Fumonisin occurrence in corn from high- and low-risk areas for human esophageal cancer in China. Appl Environ Microbiol. 60:1626–1629.
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Occurrence of fumonisins and aflatoxins in cereals from markets of Hebei province of China Y.P. Liua, L.X. Yanga, N.J. Yanga, B. Donga, L.L. Caob*, K. Wangb and L.X. Yangb a
Hebei Provincial Center for Disease Control and Prevention, Shijiazhuang, Hebei Province, China; bShijiazhuang Center for Disease Control and Prevention, Shijiazhuang, Hebei Province, China (Received 1 November 2011; final version received 23 May 2012) Fumonisins B1, B2 and B3 (FB1, FB2 and FB3) and aflatoxins B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2) are both major mycotoxins of food concern, because of their wide range of concentration and possible co-occurrence. Therefore, a contamination survey in corn and wheat flour by liquid chromatography–tandem mass spectrometry was carried out. Quantification of fumonisins and aflatoxins was based on internal calibration (by the use of 13C34-fumonisin) and external calibration, respectively. Fumonisins were detected in 95% of corn samples and in 7% of wheat flour samples, with the mean level (FB1 þ FB2 þ FB3) of 441 mg kg1 and 0.09 mg kg1, respectively. Low levels of aflatoxins were detected in 37% of the samples with a mean level (B1 þ B2 þ G1 þ G2) of 0.12 mg kg1. Fumonisins and aflatoxins were not detected in 29% of the samples analysed. Simultaneous occurrence of fumonisins and aflatoxins was observed in 12% of samples. Keywords: fumonisin; aflatoxin; corn; wheat flour; liquid chromatography–tandem mass spectrometry
Introduction As major groups of mycotoxins, fumonisin and aflatoxins are commonly present in different kinds of foodstuffs, especially in cereals and grains. Fumonisin has three major types: B1 (FB1), B2 (FB2) and B3 (FB3). Aflatoxin has four major types: B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2). Some studies have demonstrated that they are regarded a potential risk to human and animal health, including genotoxic (AFB1), carcinogenic, hepatotoxic (FB1) and immunosuppressive (Shephard et al. 1999; Gelderblom et al. 2001; Voss et al. 2001). Furthermore, corn and wheat flour are two of the most important cereal crops in China and are used primarily for direct human and animal consumption. Hence, the presence of fumonisin and aflatoxins in cereals must be paid close attention. The natural occurrence of fumonisins, together with aflatoxins, has been reported in different foods and feeds from many countries (Kedera et al. 1999; Gutema et al. 2000; Omurtag 2001; Ferracane et al. 2007; Idris et al. 2010). The maximum admissible levels of fumonisins and aflatoxins vary from one country to another. In the United States, the FDA has set a maximum permissible level of 2 mg kg1 for fumonisins in human food and 20 mg kg1 for total aflatoxins in all foodstuffs (Wu 2004). European Union (EU) has set a fumonisin level of 1 mg kg1 and AFB1 level of 2 mg kg1 for human consumption. In China, the
*Corresponding author. Email: [email protected] ISSN 1939–3210 print/ISSN 1939–3229 online ß 2012 Taylor & Francis http://dx.doi.org/10.1080/19393210.2012.697486 http://www.tandfonline.com
legal limit of AFB1 in corn is set at 20 mg kg1 (National food safety standard GB2761-2011). The aim of the present study was to survey the contamination levels of fumonisins and aflatoxins in cereals and prepare for further assessing their potential risks to human health.
Materials and methods Samples In this paper, a total of 51 cereals (30 wheat flour and 21 corn samples) were purchased from supermarkets in Shijiazhuang, Hebei Province, of China.
Chemicals All solvents were high-performance liquid chromatography (HPLC)-grade and purchased from J T Baker (Philipsburg, MT, USA). The single standard solutions of FB1, FB2 and FB3 (50 mg mL1) were obtained from Sigma-Aldrich Laborchemikalien GmbH (Steinheim, Germany). The isotope internal standards 13C34-FB1 (25 mg mL1), 13C34-FB2 (10 mg mL1), 13C34-FB3 (10 mg mL1) were also obtained from Sigma-Aldrich Laborchemikalien GmbH. MultiSepÕ 211 FUM columns were obtained from Romer Labs (Tulln, Austria).
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The parameters used for MS under ESIþ mode were set as follows: spray voltage 3500 V, Sheath Gas Pressure 40, Aux Gas Pressure 6, capillary temperature 320 C, capillary offset 114. Table 1 lists the parameters and collision energies of parent, primary and secondary daughter ions, limits of detection (LOD, S/N ¼ 3) and of quantification (LOQ, S/N ¼ 10) and linearity (R2) for FBs. Quantification was carried out by isotope internal calibration with standards in solvent.
The standards of AFB1, AFB2, AFG1 and AFG2 were purchased from Romer Labs. Immunoaffinity columns (AflaStarTM R, IAC1004 column), used for rapid and simple purification of aflatoxin B1, B2, G1 and G2 were purchased from Romer Labs, Inc. (Union, MO, USA). Milli-Q quality water (Millipore, Bedford, MA, USA) was used for all analyses.
Sample extraction and analysis for fumonisins Sample portions of 2 g were accurately weighed, spiked with a known amount of internal standards (13C-FB1, 13 C-FB2 and 13C-FB3) and extracted with 10 mL acetonitrile–water (50/50) by shaking for 3 min. After that, extracts were centrifuged at 4000 rpm for 10 min. Then 1 mL supernatant was diluted into 10 mL methanol–water (3/1). The solutions were flown through multifunction cartridges, previously conditioned by the successive passage of 5 mL methanol and 5 mL methanol–water (3/1). The cartridges were washed with 5 mL methanol–water (3/1) followed by 5 mL methanol. The fumonisins were eluted with 10 mL methanol– acetic acid (99/1). The eluates were dried and reconstituted with 1 mL of acetonitrile–water (50/50). Before LC-MS/MS, the solution was passed through a 0.22 -mm nylon filter. The analysis was performed by using an LC-MS/ MS system consisting of a Finnigan Surveyor LC in combination with an ultra-TSQ tandem mass spectrometer (Thermo Fisher Scientific, Barrington, Illinois, USA). The chromatographic column was a 15 mm 2.1 mm i.d. Atlantis T3 (Waters Corporation, Wexford, Ireland, USA). The mobile phase, with a flow rate of 0.2 mL min1, consisted of (A) 0.1% formic acid–water solution and (B) acetonitrile–methanol (50/50, v/v). Organic solvent percentage in the mobile phase linearly varied as follows: 0 min, 30%; 8 min, 70%; 13 min, 70%; 17 min, 100%; 19 min, 100%; 20 min, 30%; 30 min, 30%. The injection volume was 20 mL and the column temperature was maintained at 25 C.
Sample extraction and analysis for aflatoxins Samples were accurately weighed (5 g, precision 0.1 mg) and 5 g NaCl and methanol–water (7/3, v/v) were added up to 20 mL final volume. The whole mixture was extracted on a mechanical shaker for 2 min. After filtration, 15 mL extract was collected and diluted with 30 mL HPLC-grade water. Then 15-mL sample extracts were passed through immunoaffinity columns at a rate of about 6 mL min1. The column was washed with 10 mL HPLC water for two times. Aflatoxins were eluted from the column with 1 mL methanol. Finally, the methanolic elutes were dried and reconstituted with 1 mL of 0.1% formic acid solution before LC-MS/MS analysis. The same LC-MS/MS system was used for aflatoxins analysis, with a 15 mm 2.1 mm i.d. X Terra chromatographic column (Waters). The mobile phase consisted of (A) water containing 0.1% formic acid and (B) acetonitrile–methanol (50/50, v/v) with a flow rate of 0.2 mL min1. Organic solvent percentage in the mobile phase linearly varied as follows: 0 min, 22%; 8 min, 45%; 11 min, 100%; 13 min, 100%; 17 min, 22% and 24 min, 22%. The injection volume was 20 mL and the column temperature was maintained at 25 C. The parameters used for MS, also in ESIþ mode, were spray voltage of 3500 V, Sheath Gas Pressure of 40, Aux Gas Pressure of 10, capillary temperature of 300 C and capillary offset of 35. Table 2 lists the parameters and collision energies of parent, primary daughter and secondary daughter ions, limits of detection (LOD, S/N ¼ 3) and of quantification
Table 1. Collision energies of parent, primary and secondary daughter ions, LOD (S/N ¼ 3), LOQ (S/N ¼ 10) and linearity (R2) for fumonisins.
FBs
Parent ion (m/z)
Primary daughter ion (m/z)
Collision energy (eV)
Secondary daughter ion (m/z)
Collision energy (eV)
R2
LOD (mg kg1)
LOQ (mg kg1)
FB1 FB2 FB3 13 C34-FB1 13 C34-FB2 13 C34-FB3
723.0 707.0 707.0 756.2 740.3 740.3
353.5 337.2 337.2 374.2 358.2 358.2
42 36 36 36 39 39
705.3 354.2 354.2 356.3 376.2 376.2
28 26 26 37 45 45
0.9920 0.9971 0.9975 – – –
0.6 0.5 0.4 – – –
1.9 1.6 1.4 – – –
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Table 2. Collision energies of parent, primary and secondary daughter ions, LOD (S/N ¼ 3), LOQ (S/N ¼ 10) and linearity (R2) for aflatoxins.
AFs
Parent ions (m/z)
Primary daughter ion (m/z)
Collision energy (eV)
Secondary daughter ion (m/z)
Collision energy (eV)
R2
LOD (mg kg1)
LOQ (mg kg1)
AFB1 AFB2 AFG1 AFG2
313.0 315.0 328.9 331.1
241.0 259.1 242.9 189.0
44 34 33 40
285.0 287.1 283.0 256.9
24 18 42 30
0.9991 0.9943 0.9981 0.9960
0.05 0.02 0.09 0.04
0.16 0.06 0.26 0.11
(LOQ, S/N ¼ 10) and linearity (R2) for AFs. Quantification was carried out by external calibration with standards in solvent.
Evaluation of method performance Accuracy was estimated by spiking blank samples in recovery experiments (n ¼ 3). Method reproducibility studies were done by injecting three replicates of the same standard solution in three different days and in the same day. Both the intraday and interday precision showed relative standard deviations below 15%. The spiked concentrations of FB1, FB2 and FB3 were evaluated at levels of 2, 10, 100 and 500 mg kg1. All recoveries were higher than 75% (75.9%–111.2%), indicating good accuracy of the method. RSDs (n ¼ 6) ranged from 2.1% to 9.4% and LODs from 0.4 to 0.6 mg kg1. The spiked concentrations of AFB1, AFB2, AFG1 and AFG2 were evaluated with high (500 mg kg1), intermediate (10 and 100 mg kg1) and low (2 mg kg1) levels for each AF standard. All recoveries were higher than 80%. RSDs ranged from 6.8% to 12.4% and LODs from 0.02 to 0.09 mg kg1 for aflatoxins. RSDs (n ¼ 6) ranged from 2.9% to 12.9%.
Results and discussion Fumonisins contamination in corn and wheat flour In total, 51 samples (30 wheat flour and 21 corn samples) intended for human consumption were analysed for the incidence and levels of fumonisins B1, B2 and B3. Forty-one percent of the samples contained FB1 with concentrations ranging from 1 to 1997 mg kg1. FB2 was detected in 33% of the samples with concentrations ranging from 0.6 to 190 mg kg1. Forty-three percent of the samples contained FB3, with levels ranging from 0.6 to 541 mg kg1. The simultaneous occurrence of FB1, FB2 and FB3 was observed in 31% of the samples. According to different sample types, fumonisins were detected in most of corn samples (95% of occurrence), whereas only low concentration of fumonisins was found in a small part of wheat flour (7% of
occurrence). The results indicated that corn samples in the present study showed high levels of fumonisin contamination, and the majority of wheat samples in this study were safe. The average levels of the fumonisin-positive corn samples were 325, 34 and 82 mg kg1, for FB1, FB2 and FB3, respectively. The incidence of fumonisin in our work was close to those reported in corn-based human food products in the United States (Sydenham et al. 1991; Stack and Eppley 1992). In the United States, 94% of corn meal for human consumption analysed in 1991 was contaminated, with mean levels of 1048 and 298 mg kg1 for FB1 and FB2, respectively. Although the mean levels of fumonisins were lower than the cited US levels, the levels of fumonisin B1 in three corn samples were found to be high (1219, 1997 and 1578 mg kg1), which exceeded the proposed fumonisin level of 1 mg kg1 by the European Union. Fumonisin levels obtained from our work were also lower than those found by Yoshizawa et al. (1994), who found mean levels of 872 mg kg1 for FB1 and 448 mg kg1 for FB2 in Linxian corn.
Aflatoxins contamination in corn and wheat flour In total, aflatoxin contamination in corn and wheat flour was at low levels in this study. The highest aflatoxin B1 contamination was found in one corn sample (2.1 mg kg1), which was slightly higher than the proposed B1 levels of 2 mg kg1 for human consumption as set by EU. Positive aflatoxin levels in the other samples were lower than 0.5 mg kg1. The levels of AFB1, AFB2, AFG1 and AFG2 in all samples ranged from 0.10 to 2.1, 0.02 to 0.2, 0.1 to 0.5 and 0.1 to 0.5 mg kg1, respectively. In most of the samples, no aflatoxins were detected. This result was similar to the report by Moreno et al. (2009), where aflatoxins were not detected in 92% of the analysed corn samples. AFB1 contamination existed in four corn samples and four wheat flour samples, whereas AFB2 was found in two corn samples and two wheat flour samples. No corns were determined with AFG1 and AFG2. However, AFG1 was detected in 4 out of 30 wheat flour samples and AFG2 was found in another 5
Food Additives and Contaminants: Part B wheat flour samples. Hence, most of the samples analysed were below the legal limits.
Comparison of fumonisin and aflatoxin levels in cereals In this study, determination of both fumonisin and aflatoxin levels allowed comparison between these two classes of contaminants. Overall, the average concentration of fumonisins (FB1, FB2 and FB3 were 134, 14 and 34 mg kg1, respectively) in these cereals were much higher than that of aflatoxins (0.1 mg kg1, for AFB1 þ AFB2 þ AFG1 þ AFG2). This indicates that corn is more susceptible to fumonisin contamination. No correlation between fumonisin and aflatoxin contamination was obtained. However, Abbas et al. (2006) reported a positive correlation between aflatoxin and fumonisins and concluded that natural infection with Fusarium spp. apparently did not protect against aflatoxin production.
Conclusions In this study, co-occurrence of fumonisins and aflatoxins was evaluated in 51 cereals samples. Fumonisins were detected in 95% of corn samples and in 7% of wheat flour samples, with a mean level (FB1 þ FB2 þ FB3) of 444 and 0.1 mg kg1, respectively. The results indicated that corn was more susceptible to fumonisin contamination. Most of the samples analysed had aflatoxin contamination below the EU limit (37% of occurrence). Further studies should investigate the fumonisin and aflatoxins co-occurrence in cereal samples from other districts of China.
Acknowledgements The authors wish to thank Dr Shang in Chinese CDC and Dr Li in Tianjin Entry-Exit Inspection and Quarantine Bureau for providing some literature documents.
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