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Food Additives & Contaminants: Part B: Surveillance Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfab20
Fumonisins in corn (Zea mays L.) from Southern Brazil a
a
a
Vildes M. Scussel , Geovana D. Savi , Lea Luzia Freitas Costas , José Junior Mendonça a
a
a
a
Xavier , Daniel Manfio , Karoline O. Bittencourt , Kin Aguiar & Stephanie M. Stein
a
a
Laboratory of Mycotoxicology and Food Contaminants, Food Science and Technology Department, Center of Agricultural Sciences, Federal University of Santa Catarina, Florianopolis, Santa Catarina, Brazil Accepted author version posted online: 07 Nov 2013.Published online: 13 Jan 2014.
To cite this article: Vildes M. Scussel, Geovana D. Savi, Lea Luzia Freitas Costas, José Junior Mendonça Xavier, Daniel Manfio, Karoline O. Bittencourt, Kin Aguiar & Stephanie M. Stein (2014) Fumonisins in corn (Zea mays L.) from Southern Brazil, Food Additives & Contaminants: Part B: Surveillance, 7:2, 151-155, DOI: 10.1080/19393210.2013.862745 To link to this article: http://dx.doi.org/10.1080/19393210.2013.862745
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Food Additives & Contaminants: Part B, 2014 Vol. 7, No. 2, 151–155, http://dx.doi.org/10.1080/19393210.2013.862745
Fumonisins in corn (Zea mays L.) from Southern Brazil Vildes M. Scussel*, Geovana D. Savi, Lea Luzia Freitas Costas, José Junior Mendonça Xavier, Daniel Manfio, Karoline O. Bittencourt, Kin Aguiar and Stephanie M. Stein Laboratory of Mycotoxicology and Food Contaminants, Food Science and Technology Department, Center of Agricultural Sciences, Federal University of Santa Catarina, Florianopolis, Santa Catarina, Brazil
Downloaded by [Florida International University] at 09:51 29 August 2014
(Received 7 August 2013; accepted 2 November 2013) A total of 232 samples of corn commercialised in Santa Catarina state, Southern Brazil (temperate zone climate), were evaluated from 2007 to 2012 for fumonisins (FBs: FB1 and FB2). Before performing this study, a FBs method with liquid chromatography and fluorescence detection (ex. 335; em. 440 nm) was validated first. FBs were detected in 46.6% (108 samples), with values ranging from 66 to 7832 µg kg−1 for FB1 and 110 to 1201 µg kg−1 for FB2. The number of contaminated corn samples for FB1 and FB2 varied and often presented contamination of FB1 only. Per year of analysis, the numbers were: n = 22/8(FB1/FB2), 44/5(FB1/FB2), 25/12(FB1/FB2), 4(FB1), 6(FB1) and 7(FB1) in 2007, 2008, 2009, 2010, 2011 and 2012, respectively. The contamination percentage was 42.3/15.4, 59.5/6.8, 43.8/21.1, 36.4, 35.3 and 33.3%, respectively, during these years. Consumers can be exposed to these mycotoxins and their health can be at risk through the consumption of contaminated corn. Keywords: corn; mycotoxin; fumonisins; occurrence; HPLC
Introduction Corn (Zea mays L.) is an important worldwide food crop, utilised in several products manufactured by the industry. Brazil is the third largest world producer of corn, after China and the United States of America, with 75 million ton in 2012, of which 50 million are destined for its own consumption, the animal feed industry being the main destination. Corn is quite prone to the fungal attack and mycotoxins contamination. Some species of toxigenic fungi can cause plant pathologies leading to loss of germination, discolouration and reduction of nutritional values. Fusarium verticillioides and Fusarium proliferatum well known as field fungiproducing fumonisins (FBs) are often associated with economical loss and can lead to severe animal and human health problems (Waskiewicz et al. 2012). FBs are associated with several mycotoxicoses, including equine leukoencephalomalacia (Marasas et al. 1988; Kellerman et al. 1990), swine pulmonary oedema (Harrison et al. 1990) and kidney and liver cancers in experiments in rats (Lemmer et al. 1999; Howard et al. 2001). Among FBs, FB1, FB2 and FB3 are the most detected in corn. Generally FB1 makes up 70% of the total FBs produced, followed by FB2 and FB3 (Nelson et al. 1993). The International Agency for Research on Cancer (IARC) classified FB1 as possible carcinogen to humans (IARC 1993). The presence of FBs has been reported in several foods, especially corn, as well in Brazil (Bittencourt et al. 2005; Moreno et al. 2009; Queiroz et al. 2012) as worldwide (Li et al. 2001; Tseng & Liu 2001; Abbas et al. 2006; *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
Martins et al. 2008; Wang et al. 2008; Van der Westhuizen et al. 2010; Garrido et al. 2012). Since 2011, Brazilian regulations proposed maximum levels (MLs) for mycotoxins in foods. The limit will be decreased over time to allow producers and industry to adapt to the legislation without causing shortage of food. From January 2014, FBs limits for corn for further processing will be set at 5000 µg kg−1 (Brasil 2011). Currently, lower limits are fixed by the European Commission for FBs in foodstuffs, based on the sum of FB1 and FB2 with a ML of 1000 µg kg−1 for corn used for direct human consumption, with the exception of corn-based breakfast cereals and snack foods (800 µg kg−1) and processed corn-based foods and baby foods for infants and young children (200 µg kg−1) (EC 2007). Considering that corn and corn-based products are highly consumed by the population and are an important component of animal feed and taking into account that FB contamination is difficult to avoid and to be removed by industrial processing, there is a need of knowing FBs contamination in the corn production chain in order to assess its safety. The aim of this study was to determine the FBs (FB1 and FB2) levels of contamination in corn commercialised in Southern Brazil in the years from 2007 to 2012. Materials and methods Reagents and materials FB standards were supplied by Sigma Aldrich Chemicals (St Louis, MO, USA). FBs solutions were prepared in
Downloaded by [Florida International University] at 09:51 29 August 2014
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V.M. Scussel et al.
methanol at a concentration of 1 mg/ml and stored at – 20ºC until use. Working standard solutions, ranging from 0.005 to 0.025 µg/ml, were prepared from suitable dilutions of the stock solution in the mobile phase 0.1 M methanol: NaH2PO4 (77:23, v/v), adjusted to pH 3.3 with H3PO4 and stored at 4ºC. Solvents, including methanol, were obtained from Panreac (Castellar del Valles, Barcelona, Spain) with LC grade. Other reagents utilised for the analysis were acetic acid, monobasic sodium phosphate (NaH2PO4) and methanol (analar) from Vetec (Rio de Janeiro, RJ, Brazil) and o-phthaldialdeyde (OPA) from Merck (Darmstadt, Germany). Water was obtained from a Milli-Q system on 18.2 MΩ/cm (Millipore, Bedford, MA, USA). For the clean-up step, solid phase extraction (SPE) cartridges of Quaternary Amino N + C18 (Applied Separations, Allentown, PA, USA) were used.
Instruments Determination of FBs was carried out by a high-performance liquid chromatography (HPLC) system, equipped with an isocratic pump (model 805), auto-injector and fluorescence detector (model 321), set at excitation and emission wavelengths of 335 and 440 nm, respectively (Gilson, Middleton, WI, USA) equipped with a C18 reversed-phase column (particle size 5 µ, with 150 and 4.60 mm, length and diameter, respectively), model Luna (Phenomenex, Torrance, USA). Other equipment: a mill (model 1301, Romer Labs, Union, MO, USA), blender (model Lar.2, Metvisa Brusque, SC, Brazil), heating block (model TE-006, Sarge, Piracicaba, SP, Brazil), micropipette 100 µL (Biohit Proline (Helsinki, Finland) and tube shaker (model AP-56, Phoenix, Araquara, SP, Brazil).
FBs determination Each corn sample was ground in a Romer mill with automatic quartering. Portions of 50 g were taken for FBs analysis. The method applied for FBs determination was AOAC Official Methods of Analysis 995.15 (AOAC 2005). Briefly, 50 g of each sample was added into a blender jar with 100 ml of methanol: water (80:20, v/v). The mixtures were blended for 2 min, followed by filtration and extraction with cleaning up through an amino N + C18 SPE cartridge (conditioned with methanol, followed by methanol:water, then sample filtrate added and extract impurities washed off with methanol:water followed by methanol). The FBs were eluted with methanol:acetic acid (99:1, v/v). The eluate was evaporated to dryness with nitrogen at 60ºC and the dry residue was redissolved with 200 µL methanol, mixed and 25 µL was derivatised with 225 µL of OPA. Then, 20 µL of the extract was injected in the LC System. The mobile phase was delivered at a constant flow rate of 1 ml/min. Quantification of FBs levels was performed by measurement of peak area at FBs retention time compared with the standard solutions used for the calibration curve. For quality control of the routine analytical process, samples were analysed on five different days. Proficiency testing was performed in an interlaboratory study with matrix reference materials of Romer Labs, following ISO/IEC 17043:2010 (Conformity Assessment General Requirements for Proficiency Testing) with satisfactory z-scores in the range from –1 to 1 for FB1 and FB2. Laboratory (LABMICO) is accredited by MAPA (Ministry of Agriculture and Food Supplies), following ISO/IEC 17.025.2005. Measurement uncertainty of the sample values (data shown in the database) was performed according to European Commission Regulation No. 401/2006 (EC 2006).
Sampling
Results and discussion Method validation
Corn samples were collected randomly during five years (2007–2012) in a total of 232 samples commercialised in Santa Catarina state, Southern Brazil. The number of samples collected per year was 52, 74, 57, 11, 17 and 21, respectively. No particular preference was used in selecting samples. The samples were collected from bulk batches after filth removal and drying (up to a maximum of 80ºC) in the storage units. Collection was performed using a grains auger from different points of the bulk batches, with a minimum final weight of approximately 10 kg – depending on the batch size. Each sample was homogenised and reduced in portions from 2.0 kg. Samples were packed in polyethylene bags and sent to the Food Science and Technology Department, Laboratory of Mycotoxicology and Food Contaminants of the Federal University of Santa Catarina for FBs analysis.
The method applied was successfully validated under laboratory conditions. The validation criteria were linearity, selectivity, reproducibility, limits of detection and quantification (LOD and LOQ, respectively) and recovery. The retention times (rt) of FBs were 6.5 and 16.5 minutes for FB1 and FB2, respectively. Linearity was confirmed by constructing a calibration curve for each FB in the range of 0.005–0.025 µg/ml, which showed coefficients of correlation r2 = 0.990 and 0.991 for FB1 and FB2, respectively. In corn the LOD (signal-to-noise ratio = 3) was 6.6 and 14.4 µg kg−1, and the LOQ (signal-to-noise ratio = 10) was 21.0 and 44.4 µg kg−1 for FB1 and FB2, respectively. Recovery experiments were conducted by spiking blank corn with FBs at concentrations of 0.005, 0.025 and 0.05 µg kg−1 and analyse them in triplicate. Recoveries ranged from of 70% to 85%, with an overall mean of 80%.
Food Additives & Contaminants: Part B
Downloaded by [Florida International University] at 09:51 29 August 2014
Occurrence of FBs in corn Out of the total corn samples (232), less than a half (46.6% –108) were contaminated with FBs, ranging from 66 to 7832 and from 11 to 1201 µg kg−1 for FB1 and FB2, respectively (Table 1). The contamination in almost all positive (46.6%) samples did not exceed the ML of 5000 µg kg−1 as set by the Brazilian regulation of 2014 in corn (Brasil 2011), with only 2 (3.5%) samples in the year 2009, exceeding the ML. On the other hand, regarding the EC regulation, all years surveyed presented some samples contaminated above the FBs total ML set at 1000 µg kg−1 in corn used for direct human consumption (EC 2007). In the 6 years surveyed (2007–2012), some positive samples were found above that ML for FBs, as follows: 5 (9.6%), 2 (2.7%), 16 (28.1%), 2 (18.2%), 2 (11.8%) and 6 (28.6%) for FB1, respectively, and 3 (5.3%) positives samples, only in the year of 2009, for FB2. On the other hand, most positive samples were below 1000 µg kg−1 for FBs in each year with 17 (32.7%), 42 (56.8%), 9 (15.8%), 2 (18.2%), 4 (23.5%) and 1 (4.8%) for FB1, respectively, and 8 (15.4%), 5 (6.8%) and 9 (15.8%) positive samples in the years of 2007, 2008 and 2009 for FB2, respectively. The results of this study indicate that the incidence of FB1 (46.6%) was higher than of FB2 (10.8%) in the contaminated samples. In the year 2009, the highest
153
contamination levels were detected, in 16 (28.1%) samples (above the EC ML). Despite the lower percentage of FBs contamination detected in the current study, these findings corroborate those of Bittencourt et al. (2005) who analysed 60 corn meal samples in Sao Paulo, in which authors also detected more FB1 contamination, ranging from 1100 to 15300 µg kg−1 (mean 5200 µg kg−1), than FB2, ranging from 200 to 3900 µg kg−1 (mean 1000 µg kg−1). Authors also reported corn flour with lower levels of FB1 from 500 to 7200 µg kg−1 (mean 2100 µg kg−1) and FB2 from 100 to 1800 µg kg−1 (mean 700 µg kg−1). Queiroz et al. (2012) described in 40 samples of corn stored in a family farm in Minas Gerais, Brazil, 100% contamination by FBs with values ranging from 230 to 6450 µg kg−1. In Parana State 100% and 98.9% of freshly-harvested corn samples collected in the storage reception were contaminated in 2003 and 2004, with mean levels of 2.54 and 1.31 µg kg−1, respectively (Moreno et al. 2009). Similarly, Garrido et al. (2012) detected high contamination levels of FBs in corn from 1999 to 2010 in Argentina, in a total of 3246 freshly harvested (1655) and stored (1591) samples. For the percentage of corn samples contaminated by FBs, the authors reported between 90% and 100% for all years studied, with mean levels varying from 1773 to 9093 µg kg−1 and 2525 to 11528 µg kg−1, for freshly harvested and stored corn,
Table 1. Frequency and levels of FBs contamination in corn (Zea mays L.) samples from 2007 to 2012 in Southern Brazil.
Samples Year
Number
2007
52
2008
74
2009
57
2010
11
2011
17
2012
21
Distribution levels FB1/FB2 (µg kg−1) 200–500 500–1000 1000–2000 2000–3000 3000–5000 100–500 500–1000 1000–2000 200–500 500–1000 1000–2000 2000–3000 3000–5000 5000–7000 50–300 1000–2000 2000–3000 100–400 2000–3000 700–1000 1000–2000
Total: 232 a
Note: mean contamination of positive samples.
FBs levels (µg kg−1)
Number of contaminated samples (%) FB1 12 5 2 1 2 34 8 2 4 5 3 4 7 2 2 1 1 4 2 1 6 108
(23.2) (9.6) (3.8) (1.9) (3.8) (45.9) (10.8) (2.7) (7.1) (8.8) (5.3) (7.0) (12.2) (3.5) (18.2) (9.1) (9.1) (23.5) (11.8) (4.8) (28.6) (46.6)
FB2 8 0 0 0 0 5 0 0 4 5 3 0 0 0 0 0 0 0 0 0 0 25
(15.4) (0) (0) (0) (0) (6.8) (0) (0) (7.0) (8.8) (5.3) (0) (0) (0) (0) (0) (0) (0) (0) (0) (0) (10.8)
FB1 min
FB2 max
min
max
FB1
a total
FB2
a total
220–4305
260–400
977
318
137–1510
110–498
432
285
143–7832
287–1201
2397
581
66–3000
Food Additives & Contaminants: Part B: Surveillance Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfab20
Fumonisins in corn (Zea mays L.) from Southern Brazil a
a
a
Vildes M. Scussel , Geovana D. Savi , Lea Luzia Freitas Costas , José Junior Mendonça a
a
a
a
Xavier , Daniel Manfio , Karoline O. Bittencourt , Kin Aguiar & Stephanie M. Stein
a
a
Laboratory of Mycotoxicology and Food Contaminants, Food Science and Technology Department, Center of Agricultural Sciences, Federal University of Santa Catarina, Florianopolis, Santa Catarina, Brazil Accepted author version posted online: 07 Nov 2013.Published online: 13 Jan 2014.
To cite this article: Vildes M. Scussel, Geovana D. Savi, Lea Luzia Freitas Costas, José Junior Mendonça Xavier, Daniel Manfio, Karoline O. Bittencourt, Kin Aguiar & Stephanie M. Stein (2014) Fumonisins in corn (Zea mays L.) from Southern Brazil, Food Additives & Contaminants: Part B: Surveillance, 7:2, 151-155, DOI: 10.1080/19393210.2013.862745 To link to this article: http://dx.doi.org/10.1080/19393210.2013.862745
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Food Additives & Contaminants: Part B, 2014 Vol. 7, No. 2, 151–155, http://dx.doi.org/10.1080/19393210.2013.862745
Fumonisins in corn (Zea mays L.) from Southern Brazil Vildes M. Scussel*, Geovana D. Savi, Lea Luzia Freitas Costas, José Junior Mendonça Xavier, Daniel Manfio, Karoline O. Bittencourt, Kin Aguiar and Stephanie M. Stein Laboratory of Mycotoxicology and Food Contaminants, Food Science and Technology Department, Center of Agricultural Sciences, Federal University of Santa Catarina, Florianopolis, Santa Catarina, Brazil
Downloaded by [Florida International University] at 09:51 29 August 2014
(Received 7 August 2013; accepted 2 November 2013) A total of 232 samples of corn commercialised in Santa Catarina state, Southern Brazil (temperate zone climate), were evaluated from 2007 to 2012 for fumonisins (FBs: FB1 and FB2). Before performing this study, a FBs method with liquid chromatography and fluorescence detection (ex. 335; em. 440 nm) was validated first. FBs were detected in 46.6% (108 samples), with values ranging from 66 to 7832 µg kg−1 for FB1 and 110 to 1201 µg kg−1 for FB2. The number of contaminated corn samples for FB1 and FB2 varied and often presented contamination of FB1 only. Per year of analysis, the numbers were: n = 22/8(FB1/FB2), 44/5(FB1/FB2), 25/12(FB1/FB2), 4(FB1), 6(FB1) and 7(FB1) in 2007, 2008, 2009, 2010, 2011 and 2012, respectively. The contamination percentage was 42.3/15.4, 59.5/6.8, 43.8/21.1, 36.4, 35.3 and 33.3%, respectively, during these years. Consumers can be exposed to these mycotoxins and their health can be at risk through the consumption of contaminated corn. Keywords: corn; mycotoxin; fumonisins; occurrence; HPLC
Introduction Corn (Zea mays L.) is an important worldwide food crop, utilised in several products manufactured by the industry. Brazil is the third largest world producer of corn, after China and the United States of America, with 75 million ton in 2012, of which 50 million are destined for its own consumption, the animal feed industry being the main destination. Corn is quite prone to the fungal attack and mycotoxins contamination. Some species of toxigenic fungi can cause plant pathologies leading to loss of germination, discolouration and reduction of nutritional values. Fusarium verticillioides and Fusarium proliferatum well known as field fungiproducing fumonisins (FBs) are often associated with economical loss and can lead to severe animal and human health problems (Waskiewicz et al. 2012). FBs are associated with several mycotoxicoses, including equine leukoencephalomalacia (Marasas et al. 1988; Kellerman et al. 1990), swine pulmonary oedema (Harrison et al. 1990) and kidney and liver cancers in experiments in rats (Lemmer et al. 1999; Howard et al. 2001). Among FBs, FB1, FB2 and FB3 are the most detected in corn. Generally FB1 makes up 70% of the total FBs produced, followed by FB2 and FB3 (Nelson et al. 1993). The International Agency for Research on Cancer (IARC) classified FB1 as possible carcinogen to humans (IARC 1993). The presence of FBs has been reported in several foods, especially corn, as well in Brazil (Bittencourt et al. 2005; Moreno et al. 2009; Queiroz et al. 2012) as worldwide (Li et al. 2001; Tseng & Liu 2001; Abbas et al. 2006; *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
Martins et al. 2008; Wang et al. 2008; Van der Westhuizen et al. 2010; Garrido et al. 2012). Since 2011, Brazilian regulations proposed maximum levels (MLs) for mycotoxins in foods. The limit will be decreased over time to allow producers and industry to adapt to the legislation without causing shortage of food. From January 2014, FBs limits for corn for further processing will be set at 5000 µg kg−1 (Brasil 2011). Currently, lower limits are fixed by the European Commission for FBs in foodstuffs, based on the sum of FB1 and FB2 with a ML of 1000 µg kg−1 for corn used for direct human consumption, with the exception of corn-based breakfast cereals and snack foods (800 µg kg−1) and processed corn-based foods and baby foods for infants and young children (200 µg kg−1) (EC 2007). Considering that corn and corn-based products are highly consumed by the population and are an important component of animal feed and taking into account that FB contamination is difficult to avoid and to be removed by industrial processing, there is a need of knowing FBs contamination in the corn production chain in order to assess its safety. The aim of this study was to determine the FBs (FB1 and FB2) levels of contamination in corn commercialised in Southern Brazil in the years from 2007 to 2012. Materials and methods Reagents and materials FB standards were supplied by Sigma Aldrich Chemicals (St Louis, MO, USA). FBs solutions were prepared in
Downloaded by [Florida International University] at 09:51 29 August 2014
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V.M. Scussel et al.
methanol at a concentration of 1 mg/ml and stored at – 20ºC until use. Working standard solutions, ranging from 0.005 to 0.025 µg/ml, were prepared from suitable dilutions of the stock solution in the mobile phase 0.1 M methanol: NaH2PO4 (77:23, v/v), adjusted to pH 3.3 with H3PO4 and stored at 4ºC. Solvents, including methanol, were obtained from Panreac (Castellar del Valles, Barcelona, Spain) with LC grade. Other reagents utilised for the analysis were acetic acid, monobasic sodium phosphate (NaH2PO4) and methanol (analar) from Vetec (Rio de Janeiro, RJ, Brazil) and o-phthaldialdeyde (OPA) from Merck (Darmstadt, Germany). Water was obtained from a Milli-Q system on 18.2 MΩ/cm (Millipore, Bedford, MA, USA). For the clean-up step, solid phase extraction (SPE) cartridges of Quaternary Amino N + C18 (Applied Separations, Allentown, PA, USA) were used.
Instruments Determination of FBs was carried out by a high-performance liquid chromatography (HPLC) system, equipped with an isocratic pump (model 805), auto-injector and fluorescence detector (model 321), set at excitation and emission wavelengths of 335 and 440 nm, respectively (Gilson, Middleton, WI, USA) equipped with a C18 reversed-phase column (particle size 5 µ, with 150 and 4.60 mm, length and diameter, respectively), model Luna (Phenomenex, Torrance, USA). Other equipment: a mill (model 1301, Romer Labs, Union, MO, USA), blender (model Lar.2, Metvisa Brusque, SC, Brazil), heating block (model TE-006, Sarge, Piracicaba, SP, Brazil), micropipette 100 µL (Biohit Proline (Helsinki, Finland) and tube shaker (model AP-56, Phoenix, Araquara, SP, Brazil).
FBs determination Each corn sample was ground in a Romer mill with automatic quartering. Portions of 50 g were taken for FBs analysis. The method applied for FBs determination was AOAC Official Methods of Analysis 995.15 (AOAC 2005). Briefly, 50 g of each sample was added into a blender jar with 100 ml of methanol: water (80:20, v/v). The mixtures were blended for 2 min, followed by filtration and extraction with cleaning up through an amino N + C18 SPE cartridge (conditioned with methanol, followed by methanol:water, then sample filtrate added and extract impurities washed off with methanol:water followed by methanol). The FBs were eluted with methanol:acetic acid (99:1, v/v). The eluate was evaporated to dryness with nitrogen at 60ºC and the dry residue was redissolved with 200 µL methanol, mixed and 25 µL was derivatised with 225 µL of OPA. Then, 20 µL of the extract was injected in the LC System. The mobile phase was delivered at a constant flow rate of 1 ml/min. Quantification of FBs levels was performed by measurement of peak area at FBs retention time compared with the standard solutions used for the calibration curve. For quality control of the routine analytical process, samples were analysed on five different days. Proficiency testing was performed in an interlaboratory study with matrix reference materials of Romer Labs, following ISO/IEC 17043:2010 (Conformity Assessment General Requirements for Proficiency Testing) with satisfactory z-scores in the range from –1 to 1 for FB1 and FB2. Laboratory (LABMICO) is accredited by MAPA (Ministry of Agriculture and Food Supplies), following ISO/IEC 17.025.2005. Measurement uncertainty of the sample values (data shown in the database) was performed according to European Commission Regulation No. 401/2006 (EC 2006).
Sampling
Results and discussion Method validation
Corn samples were collected randomly during five years (2007–2012) in a total of 232 samples commercialised in Santa Catarina state, Southern Brazil. The number of samples collected per year was 52, 74, 57, 11, 17 and 21, respectively. No particular preference was used in selecting samples. The samples were collected from bulk batches after filth removal and drying (up to a maximum of 80ºC) in the storage units. Collection was performed using a grains auger from different points of the bulk batches, with a minimum final weight of approximately 10 kg – depending on the batch size. Each sample was homogenised and reduced in portions from 2.0 kg. Samples were packed in polyethylene bags and sent to the Food Science and Technology Department, Laboratory of Mycotoxicology and Food Contaminants of the Federal University of Santa Catarina for FBs analysis.
The method applied was successfully validated under laboratory conditions. The validation criteria were linearity, selectivity, reproducibility, limits of detection and quantification (LOD and LOQ, respectively) and recovery. The retention times (rt) of FBs were 6.5 and 16.5 minutes for FB1 and FB2, respectively. Linearity was confirmed by constructing a calibration curve for each FB in the range of 0.005–0.025 µg/ml, which showed coefficients of correlation r2 = 0.990 and 0.991 for FB1 and FB2, respectively. In corn the LOD (signal-to-noise ratio = 3) was 6.6 and 14.4 µg kg−1, and the LOQ (signal-to-noise ratio = 10) was 21.0 and 44.4 µg kg−1 for FB1 and FB2, respectively. Recovery experiments were conducted by spiking blank corn with FBs at concentrations of 0.005, 0.025 and 0.05 µg kg−1 and analyse them in triplicate. Recoveries ranged from of 70% to 85%, with an overall mean of 80%.
Food Additives & Contaminants: Part B
Downloaded by [Florida International University] at 09:51 29 August 2014
Occurrence of FBs in corn Out of the total corn samples (232), less than a half (46.6% –108) were contaminated with FBs, ranging from 66 to 7832 and from 11 to 1201 µg kg−1 for FB1 and FB2, respectively (Table 1). The contamination in almost all positive (46.6%) samples did not exceed the ML of 5000 µg kg−1 as set by the Brazilian regulation of 2014 in corn (Brasil 2011), with only 2 (3.5%) samples in the year 2009, exceeding the ML. On the other hand, regarding the EC regulation, all years surveyed presented some samples contaminated above the FBs total ML set at 1000 µg kg−1 in corn used for direct human consumption (EC 2007). In the 6 years surveyed (2007–2012), some positive samples were found above that ML for FBs, as follows: 5 (9.6%), 2 (2.7%), 16 (28.1%), 2 (18.2%), 2 (11.8%) and 6 (28.6%) for FB1, respectively, and 3 (5.3%) positives samples, only in the year of 2009, for FB2. On the other hand, most positive samples were below 1000 µg kg−1 for FBs in each year with 17 (32.7%), 42 (56.8%), 9 (15.8%), 2 (18.2%), 4 (23.5%) and 1 (4.8%) for FB1, respectively, and 8 (15.4%), 5 (6.8%) and 9 (15.8%) positive samples in the years of 2007, 2008 and 2009 for FB2, respectively. The results of this study indicate that the incidence of FB1 (46.6%) was higher than of FB2 (10.8%) in the contaminated samples. In the year 2009, the highest
153
contamination levels were detected, in 16 (28.1%) samples (above the EC ML). Despite the lower percentage of FBs contamination detected in the current study, these findings corroborate those of Bittencourt et al. (2005) who analysed 60 corn meal samples in Sao Paulo, in which authors also detected more FB1 contamination, ranging from 1100 to 15300 µg kg−1 (mean 5200 µg kg−1), than FB2, ranging from 200 to 3900 µg kg−1 (mean 1000 µg kg−1). Authors also reported corn flour with lower levels of FB1 from 500 to 7200 µg kg−1 (mean 2100 µg kg−1) and FB2 from 100 to 1800 µg kg−1 (mean 700 µg kg−1). Queiroz et al. (2012) described in 40 samples of corn stored in a family farm in Minas Gerais, Brazil, 100% contamination by FBs with values ranging from 230 to 6450 µg kg−1. In Parana State 100% and 98.9% of freshly-harvested corn samples collected in the storage reception were contaminated in 2003 and 2004, with mean levels of 2.54 and 1.31 µg kg−1, respectively (Moreno et al. 2009). Similarly, Garrido et al. (2012) detected high contamination levels of FBs in corn from 1999 to 2010 in Argentina, in a total of 3246 freshly harvested (1655) and stored (1591) samples. For the percentage of corn samples contaminated by FBs, the authors reported between 90% and 100% for all years studied, with mean levels varying from 1773 to 9093 µg kg−1 and 2525 to 11528 µg kg−1, for freshly harvested and stored corn,
Table 1. Frequency and levels of FBs contamination in corn (Zea mays L.) samples from 2007 to 2012 in Southern Brazil.
Samples Year
Number
2007
52
2008
74
2009
57
2010
11
2011
17
2012
21
Distribution levels FB1/FB2 (µg kg−1) 200–500 500–1000 1000–2000 2000–3000 3000–5000 100–500 500–1000 1000–2000 200–500 500–1000 1000–2000 2000–3000 3000–5000 5000–7000 50–300 1000–2000 2000–3000 100–400 2000–3000 700–1000 1000–2000
Total: 232 a
Note: mean contamination of positive samples.
FBs levels (µg kg−1)
Number of contaminated samples (%) FB1 12 5 2 1 2 34 8 2 4 5 3 4 7 2 2 1 1 4 2 1 6 108
(23.2) (9.6) (3.8) (1.9) (3.8) (45.9) (10.8) (2.7) (7.1) (8.8) (5.3) (7.0) (12.2) (3.5) (18.2) (9.1) (9.1) (23.5) (11.8) (4.8) (28.6) (46.6)
FB2 8 0 0 0 0 5 0 0 4 5 3 0 0 0 0 0 0 0 0 0 0 25
(15.4) (0) (0) (0) (0) (6.8) (0) (0) (7.0) (8.8) (5.3) (0) (0) (0) (0) (0) (0) (0) (0) (0) (0) (10.8)
FB1 min
FB2 max
min
max
FB1
a total
FB2
a total
220–4305
260–400
977
318
137–1510
110–498
432
285
143–7832
287–1201
2397
581
66–3000
