The objective of this study was to quantitatively evaluate mycotoxins in samples of maize and poultry feed produced in Brazil. A multimycotoxin method based on HPLC-MS/MS was applied to investigate the occurrence of toxical fungal metabolites in 119 samples collected from poultry feed factory integrated poultry farms: maize grain (74), poultry feed (36), and feed factory residue (9). Twenty of 101 fungal metabolites investigated were detected and quantified in the samples: aflatoxins B1, B2, G1, and G2, fumonisins B1, B2, and B3, hydrolyzed fumonisin B1, zearalenone, agroclavine, chanoclavine, deoxynivalenol, and nivalenol, and enniatin A, A1, B, B1, beauvericin, kojic acid, and moniliformin. Most samples were contaminated with more than one mycotoxin. All samples were contaminated with fumonisins, with medians values of 1,840
Brazil is the third major maize producer country of the world after United States and China. In particular, in 2012, it produced 71.5 million tons [
The infection of cereal crops by phytopathogenic
Research efforts to establish the magnitude of the mycotoxin occurrence in Latin America were initiated in the late 1960s after the outbreak of Turkey X disease. The bulk of mycotoxin research in Latin America has been conducted on maize and specifically on aflatoxins, although other toxins such as zearalenone, T-2 toxin, DON, penicillic acid, kojic acid, and ochratoxin have been detected in that cereal [
According to Salay and Zerlotti Mercadante [
In a review dealing with mycotoxin research in Brazil between 1991 and 2000, Rodriguez-Amaya and Sabino [
The results from another study indicate a low occurrence of trichothecenes mycotoxins in maize-based products commercialized in the city of São Paulo in spite of high levels of T-2 and HT-2 toxins found in one sample and show no immediate cause of concern. Nonetheless, more extensive surveys conducted for several years are advisable in order to furnish a more complete picture of the incidence of these toxins as well as other eventual (emergent) toxins in Brazilian products [
Maize is the major crop frequently exposed to the risk of contamination by all these mycotoxins. In particular, for maize, the European Commission has established maximum permitted levels for aflatoxins (AFB1, 2
While most screening methods for mycotoxins addressed by legislation are based on immunoassays, unambiguous analytes confirmation can be easily achieved with mass spectrometric methods, such as gas chromatography/mass spectrometry (GC/MS) or liquid chromatography/mass spectrometry (LC/MS). During the last few years, this technical and instrumental progress had also an increasing impact on the expanding field of mycotoxin analysis [
The present work aimed to investigate mycotoxin contamination in a poultry maize-based feed chain in Brazil by using a HPLC-MS/MS multimycotoxin method.
Methanol and acetonitrile (both LC gradient grade) were purchased from J.T. Baker (Deventer, The Netherlands) and ammonium acetate (MS grade) and glacial acetic acid (p.a.) from Sigma-Aldrich (Vienna, Austria). Water was purified successively by reverse osmosis and a Milli-Q plus system from Millipore (Molsheim, France). Details concerning standards of the investigated mycotoxins (which include trichothecenes, zearalenone derivatives, fumonisins, ergot alkaloids, aflatoxins, ochratoxins, and some other metabolites produced by
A total of 119 samples of maize grains, subproducts, and poultry feeds were collected from a poultry feed factory and integrated poultry farms in Paraná State, in Brazil, from 2005 to 2006. The samples obtained were as follows: (i) 74 samples of maize grains were randomly withdrawn from trucks (from each truck one sample of 10 kg) in the poultry feed factory reception and factory processing steps (3 kg); (ii) 36 samples of poultry feeds (3 kg) in the integrated poultry farms; and (iii) 9 samples of maize factory residues (10 kg each) collected in the discarding of first cleaning (after sieving).
All samples were ground in a TREU mill (7.5 CV, 1720 rpm) with a 20 mesh sieved at Embrapa Food Technology, homogenized during 15 min (Chopin MR10L), packed under vacuum, and frozen stored until analyzed.
To 5 g of milled sample, 20 mL of extraction solvent (acetonitrile/water/acetic acid 79 : 20 : 1, v/v/v) was added. Extraction, dilution, and analysis were performed as described by Sulyok et al. [
Ionization (ESI) source and an 1100 Series HPLC System were brought from Agilent, Waldbronn, Germany. Chromatographic separation was performed at 25°C on a Gemini C18 column, 150 × 4.6-mmi.d., 5-
The recovery was determined in duplicate by spiking in three different maize and feed sample. It spiked 0.5 g of sample in an open vial with appropriate amounts of a multianalyte working solution. The samples were subsequently stored for one day at room temperature to allow solvent evaporation. After this period, 2 mL of extraction solvent (acetonitrile/water/acetic acid 79 : 20 : 1, v/v/v) was added, and the same analytical procedure used as for the investigated samples was followed. Because all the investigated samples were naturally contaminated by fumonisins, the samples with the lowest levels were used for spiked experiments.
Limits of detection were calculated from the signal to noise ratios (LOD = 3 × S/N) of the respective multiple reaction monitoring (MRM) chromatograms deriving from the analysis of spiked samples.
The significance of mycotoxin contamination in food gained much attention over the past four decades. The cooccurrence of mycotoxins had been already described in maize and others foods [
Quantitative analysis of raw extracts by LC-MS/MS can be disturbed by signal suppression due to matrix effects. As these were investigated in maize only for a smaller set of 39 analytes [
Spiking levels (SL), limit of detection (LOD), and average apparent recoveries of spiked maize and feed.
Toxins | SL | LOD | Recovery (%) | |
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Maize | Feed | |
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Fumonisin B1 (FB1) | 504 | 8 |
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Fumonisin B2 (FB2) | 505 | 7 |
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Fumonisin B3 (FB3) | 50.0 | 4 |
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Hydrolysed fumonisin B1 (HFB1) | 54.9 | 17 |
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Aflatoxin B1 (AFL B1) | 25 | 0.8 |
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Aflatoxin G1 (AFL G1) | 25 | 0.5 |
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Aflatoxin B2 (AFL B2) | 25 | 0.7 |
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Aflatoxin G2 (AFL G2) | 25 | 1 |
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Ochratoxin A (OTA) | 20 | 1 |
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Ochratoxin B (OTB) | 20 | 1 |
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Ochratoxin |
11 | 3 | 77 | 84 |
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Zearalenone (ZON) | 100 | 0.4 |
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Zearalenone-4-sulfate | 0.4 | 0.3 | 86 | 95 |
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20 | 3 |
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20 | 4 |
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120 | 0.8 |
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120 | 1 | 110 | 99 |
Zearalenone-4-glucoside | 20 | 5 |
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Beauvericin (BEA) | 10 | 2 | 66 | 86 |
Enniatin A (EA) | 0.8 | 0.1 |
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Enniatin A1 (EA1) | 0.56 | 0.15 |
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Enniatin B (EB) | 0.53 | 0.3 |
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Enniatin B1 (EB1) | 1.51 | 0.2 |
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Enniatin B3 (EB3) | 0.63 | 0.04 | 93 | 87 |
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Agroclavine | 3.4 | 0.2 |
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Chanoclavine | 50 | 0.4 |
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Festuclavine | 50 | 0.15 |
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Elymoclavine | 50 | 1 |
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Elymoclavine fructoside | 50 | 4 |
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Oxidized elymoclavine | 50 | 3 |
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Ergine | 1.08 | 0.1 |
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Ergotamine | 1.08 | 0.7 | 24 | 37 |
Ergocornine | 1.08 | 1 | 36 | 30 |
Ergocorninine | 0.692 | 0.15 | 52 | 62 |
Ergocristine | 1.08 | 0.3 | 23 | 33 |
Ergocristinine | 0.692 | 0.2 | 59 | 61 |
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1.08 | 0.2 | 30 | 36 |
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0.692 | 0.1 | 67 | 69 |
Ergometrine | 2.17 | 0.1 | 90 | 80 |
Ergometrinine | 0.432 | 0.07 |
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Ergosine | 1.08 | 0.13 | 37 | 31 |
Ergosinine | 0.692 | 0.02 | 82 | 48 |
Dihydroergotamine | 1.08 | 0.5 | 49 | 43 |
Oxidized luol | 50 | 0.3 | 72 | 75 |
Dihydrolysergol | 50 | 0.2 |
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Lysergol | 50 | 1 |
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Deoxynivalenol (DON) | 100 | 20 |
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15-Acetyl-deoxynivalenol | 50.4 | 50 |
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3-Acetyl-deoxynivalenol | 100 | 20 |
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Deoxynivalenol-3-glucoside | 20 | 15 |
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Deepoxydeoxynivalenol | 25.5 | 15 | 127 | 114 |
Nivalenol (NIV) | 100 | 50 |
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Fusarenon X (F-X) | 101 | 50 |
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Toxin HT-2 (HT2) | 100 | 20 |
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Toxin T-2 (T2) | 100 | 20 |
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Neosolaniol (NEO) | 27 | 3 |
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Monoacetoxyscirpenol | 10 | 2 |
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Diacetoxyscirpenol | 100 | 1 |
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Verrucarol | 200 | 180 |
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Verrucarin A | 10.7 | 5 | 95 | 91 |
Roridin A | 13.7 | 1 | 89 | 87 |
T2-Tetraol | 42.7 | 20 | 76 | 89 |
T2-Triol | 42.7 | 20 | 79 | 77 |
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Moniliformin (MON) | 204 | 81 | 87 | 112 |
Kojic acid. | 300 | 160 |
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Emodin | 8.5 | 4 | 89 | 65 |
Penicillic acid | 62.5 | 20 |
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Brefeldin A | 62.5 | 60 |
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Roquefortin C | 62.5 | 4 |
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Gibberellic acid | 85.4 | 20 | 102 | 101 |
Patulin (PAT) | 64.2 | 100 | 16 | 22 |
Gliotoxin | 42.7 | 12 | 58 | 12 |
Fumitremorgin C | 6.4 | 4 | 90 | 79 |
Altenuene | 8.5 | 6 | 89 | 102 |
Alternariol | 17.1 | 2 | 91 | 82 |
Alternariol monomethyl ether | 8.5 | 0.1 | 99 | 81 |
Sterigmatocystin | 8.5 | 0.4 | 78 | 84 |
Citrinin (CTN) | 25.6 | 30 | 90 | 122 |
Cytochalasin A | 62.5 | 30 |
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Cytochalasin B | 62.5 | 10 |
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Cytochalasin C | 62.5 | 2 |
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Cytochalasin D | 62.5 | 4 |
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Cytochalasin H | 62.5 | 30 |
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Cytochalasin J | 62.5 | 5 |
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Mevinolin | 42.7 | 7 | 109 | 65 |
Mycophenolic acid | 23.9 | 10 | 106 | 103 |
Paxilline | 42.7 | 25 | 99 | 66 |
Penitrem A | 12.8 | 5 | 133 | 121 |
Sulochrin | 21.3 | 4 | 84 | 83 |
Tentoxin | 3.39 | 0.5 | 148 | 152 |
Chaetoglobosin A | 21.3 | 9 | 10 | 50 |
Chetomin | 64.0 | 100 | 18 | 17 |
Meleagrin | 21.3 | 2 | 92 | 99 |
Verruculogen | 24.4 | 50 | 78 | 86 |
Griseofulvin | 21.3 | 10 | 90 | 90 |
Methysergide | 0.70 | 0.4 | 75 | 83 |
Alamethicin-F30 | 40 | 3 | 99 | 85 |
HC toxin | 43.5 | 20 |
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Figure
Total ions chromatogram (TIC) of 80 mycotoxins standards in positive mode (a) and of 21 mycotoxins standards in negative mode (b) obtained by HPLC-MS/MS (ESI).
Table
Mycotoxins and metabolites detected in maize, poultry feed, and factory residue by liquid chromatography-tandem mass spectrometry (LC-MS/MS). It shows the contamination range, median, and percentage of contaminated samples.
Toxin | Maize | Poultry feed | Factory residue | |||||||||
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Min. ( |
Median ( |
Max. ( |
% | Min. ( |
Median ( |
Max. ( |
% | Min. ( |
Median ( |
Max. ( |
% | |
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Fumonisin B1 (FB1) | 32 | 1,300 | 6,000 | 100 | 50 | 185 | 1,118 | 100 | 14,085 | 17,153 | 27,145 | 100 |
Fumonisin B2 (FB2) | 9 | 540 | 2,760 | 99 | 8 | 54 | 474 | 100 | 5,927 | 7,412 | 10,867 | 100 |
Fumonisin B3 (FB3) | 7 | 190 | 820 | 99 | nd | 27 | 142 | 92 | 1,422 | 1,853 | 3,090 | 100 |
Fumonisin total (FB1 + FB2) | 41 | 1,840 | 8,760 | 100 | 58 | 239 | 1,592 | 100 | 20,012 | 23,676 | 36,040 | 100 |
Hydrolysed fumonisin B1 (HFB1) | nd* | 6.0 | 170 | 9 | nd | nd | nd | 0 | 168 | 366 | 909 | 100 |
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Aflatoxin B1 (AFL B1) | nd | nd | 3.0 | 16 | nd | nd | nd | 0 | nd | nd | 5.96 | 44 |
Aflatoxin B2 (AFL B2) | nd | nd | nd | 0 | nd | nd | nd | 0 | nd | nd | 1.10 | 22 |
Aflatoxin G1 (AFL G1) | nd | nd | 0.6 | 1 | nd | nd | nd | 0 | nd | nd | 0.52 | 11 |
Aflatoxin G2 (AFL G2) | nd | nd | 1.8 | 4 | nd | nd | 1,43 | 14 | 1.0 | 1.73 | 2.51 | 100 |
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Deoxynivalenol (DON) | nd | nd | 30.0 | 4 | nd | nd | 20 | 3 | nd | nd | nd | 0 |
Nivalenol (NIV) | nd | nd | 120.0 | 5 | nd | nd | 67 | 17 | nd | nd | nd | 0 |
Zearalenone (ZON) | nd | nd | 9.8 | 12 | nd | nd | 6.5 | 39 | nd | nd | nd | 0 |
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Beauvericin (BEA) | nd | 12 | 160 | 96 | nd | 3.6 | 16.7 | 92 | 59 | 116 | 211 | 100 |
Enniatin A (EA) | nd | nd | 0.1 | 1 | nd | nd | nd | 0 | nd | nd | nd | 0 |
Enniatin A1 (EA1) | nd | nd | 0.3 | 12 | nd | nd | 0.72 | 17 | nd | nd | 0.27 | 22 |
Enniatin B (EB) | nd | nd | 5.0 | 34 | nd | nd | 4.6 | 78 | nd | nd | 3.21 | 44 |
Enniatin B1 (EB1) | nd | 0.1 | 1.3 | 12 | nd | nd | 12 | 67 | nd | nd | 1.12 | 33 |
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Kojic acid | nd | 12 | 230 | 65 | nd | 12 | 84 | 67 | 2.9 | 28 | 344 | 100 |
Agroclavine | nd | nd | 7.2 | 1 | nd | nd | nd | 0 | nd | nd | nd | 0 |
Chanoclavine | nd | nd | nd | 0 | nd | nd | nd | 0 | nd | nd | 3.95 | 33 |
Moniliformin (MON) | nd | nd | 170 | 8 | nd | nd | 120 | 3 | nd | 220 | 336 | 89 |
In addition, fully hydrolyzed fumonisin B1 (HFB1), also named aminopentol (AP1), was found in 9% of maize samples. Although numerous fumonisins have been characterized, FB1 is usually the most abundant in contaminated foods, except when maize has been treated with base to produce maize flour for tortillas, which hydrolyzes FB1 to AP1. AP1 also appears to have the same liver cancer promoting activity as FB1. Heretofore, these in vivo effects of AP1 have been somewhat puzzling because AP1 is less potent than FB1 as an inhibitor of ceramide synthase
Considering the maximum limits established in Brazil or even by the EC for aflatoxins, these toxins were detected in very low levels and only in four samples. The maximum value found for AFB1 in maize was 3.0
The trichothecenes DON and NIV were also found, however, in few samples and in low concentrations, considering the limits established by Brazilian Regulation in cereals of 2,000
Among the mycotoxins most frequently found in the samples, there was also beauvericin (BEA) which was detected in 96% of maize samples with a media of 12
Despite a relatively low amount of agroclavine (7.20
Unfortunately, there is only a limited number of surveys concerning
Last column of Table
Kojic acid was also detected in 100% of sample reaching concentrations of 344
The HPLC-MS/MS method used in this study constituted an alternative to conventional techniques for mycotoxin analysis showing an ultralarge mycotoxin spectra, good sensitivity, rapidness, and applicability to complex matrices such as maize and maize-based feed. It could therefore be applied as routine method for different types of food as well as food production testing. The recovery was between 70 and 120% for 73 mycotoxins in maize while 65 mycotoxins in feed.
Concerning fumonisins, all samples were contaminated, and in some samples, contamination levels exceeded the maximum levels established by the EC. This would lead to increased risk to the consumer health from mycotoxins and emphasizes the urgency for establishing regular monitoring programs for mycotoxins in staple grains in developing countries. The results claim for an urgent regulation for fumonisins in Brazil.
This is the first study dealing with agroclavine, chanoclavine, enniatin A, A1, B, beauvericin, and kojic acid contamination of maize and poultry feeds from Brazil. Although some mycotoxin content in maize was low, most samples were contaminated with more than one mycotoxin analyzed. This study suggests that more investigations are needed in this commodity since this survey only covers 2005/2006, and the occurrence may change from year to year implying that further monitoring of mycotoxin in Brazil is justified.
This result reinforces the need to know other mycotoxins in food products to verify the real extension of the mycotoxins in food and feed to protect public health.
The authors thank the European Commission for funding this work through the MYCOTOX project (contract no. ICA4-CT-2002-10043; INCODEV Program); the EMBRAPA, Brazil, for financial support and fellowship of PhD student Maria L. M. Souza; Center for Analytical Chemistry, Department for Agrobiotechnology, IFA-Tulln (University of Natural Resources and Applied Life Sciences) where the samples were carried out, the Lower Austrian Government for the financial support, and Biopure and Romers Labs for providing some mycotoxin standard solutions.