- Email: [email protected]
Presence of trichothecenes and co-occurrence in cereal-based food from Catalonia (Spain)
Food Control 22 (2011) 490e495
Contents lists available at ScienceDirect
Food Control journal homepage: www.elsevier.com/locate/foodcont
Presence of trichothecenes and co-occurrence in cereal-based food from Catalonia (Spain) G. Cano-Sancho a, *, F.M. Valle-Algarra b, M. Jiménez b, P. Burdaspal c, T.M. Legarda c, A.J. Ramos a, V. Sanchis a, S. Marín a a b c
Applied Mycology Unit, Food Technology Department, University of Lleida, XaRTA-UTPV, Rovira Roure 191, 25198 Lleida, Spain Microbiology and Ecology Department, University of Valencia, Dr. Moliner 50, E-46100 Burjassot, Valencia, Spain National Centre for Food (Spanish Food Safety and Nutrition Agency), 28220 Majadahonda, Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 July 2010 Received in revised form 20 September 2010 Accepted 28 September 2010
The most important trichothecenes are HT-2 toxin (HT2) and T-2 toxin (T2) from type A and deoxynivalenol (DON) from type B. Thus, the aim of the current study was to assess the occurrence of these trichothecenes in the Catalonian market. 479 food samples were taken from the most susceptible to trichothecenes contamination and most commonly consumed in Catalonia commodities. DON, T2 and HT2 toxin were determined in breakfast cereals, snacks and pasta samples following extraction, clean-up, derivatization and finally analysis by GCeECD. Moreover, these mycotoxins were determined in sliced bread, sweet corn and beer by LCeDAD. Our results showed that DON was the main trichothecene present in the cereal-based food from Catalonian market with percentages of positive samples ranging from 1.4 to 100.0%. Despite the high incidence of DON, only five samples were above EU limits. Concerning HT2 toxin, it was present in a low percentage of samples of sliced bread (15.3%), wheat flakes (14.8%), pasta (10.0%), corn snacks (8.5%), sweet corn (6.9%) and corn flakes (6.2%), while the T2 toxin only was quantified in 5 samples out of the total 479. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Cereal-based food Deoxynivalenol T2 HT2 Mycotoxins Occurrence
1. Introduction Trichothecenes are a family of related cyclic sesquiterpenoids, which are divided into four groups (types AeD) according to their characteristic functional groups, being the type A and B, the most common. Type A is represented by HT-2 toxin (HT2) and T-2 toxin (T2) and type B is most frequently represented by deoxynivalenol (DON). Trichothecenes are produced in several cereals by species of Fusarium, thus, a wide range of cereal-based food have been confirmed to be contaminated by these toxins (JECFA, 2001; pp. 281e320). T2 is a potent inhibitor of protein synthesis and mitochondrial function both in vivo and in vitro, and shows immunosuppressive and cytotoxic effects. Moreover, it has been reported that the toxin has extremely toxic effects on skin and mucous (Eriksen & Pettersson, 2004, Sudakin, 2003; Visconti, 2001; Visconti, Minervini, Lucivero, & Gambatesa, 1991). It has been shown that through deacetylation of T2, it is obtained HT2 as the major metabolite; however, little
* Corresponding author. Tel.: þ34 973702670; fax: þ34 973702596. E-mail address: [email protected] (G. Cano-Sancho). 0956-7135/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2010.09.033
information is available regarding toxicity of HT2 alone (Sudakin, 2003; Visconti, 2001). Although DON is not as toxic as other trichothecenes such as T2 or HT2, this mycotoxin is one of the most common contaminants of cereals worldwide (Jelinek, Pohland, & Wood, 1989; Scott, 1989). Acute effects of food poisoning in humans are abdominal pains, dizziness, headache, throat irritation, nausea, vomiting, diarrhoea, and blood in the stool (Rotter, Prelusky, & Pestka, 1996). Maximum level of DON permitted in Europe is 0.750 mg/g in pasta and 0.500 mg/g in bread, pastries, biscuits, cereal snacks and breakfast cereals (European Commission, 2006b). The Joint FAO/ WHO Expert Committee on Food Additives (JECFA), after assessing the toxic effect of T2 and HT2, concluded that the toxic effects of these mycotoxins could not be differentiated. Thus, the provisional maximum tolerable daily intake (PMTDI) for the combination of these toxins or alone was set at 0.06 mg/kg body weight/day (JECFA, 2001; pp. 281e320). Concerning DON, a tolerable daily intake (TDI) of 1 mg/kg body weight based on a reduction of body weight gain was established by the EC SCF (SCF, 2002). Occurrence of DON, T2 and HT2 toxin has been widely reported in raw food and foodstuff, around of the European region (JECFA, 2001; pp. 281e320), confirming that food processing methods do not completely remove the mycotoxins in the matrix (Hazel & Patel, 2004). Nevertheless, the occurrence of these trichothecenes in
G. Cano-Sancho et al. / Food Control 22 (2011) 490e495
491
corn-based food from Spain, has been partially addressed in two previous studies, where the breakfast cereals and corn snacks, were the main foodstuffs analyzed (Castillo et al., 2008; Cervero, Castillo, Montes & Hernandez, 2007), thereby, currently there is a lack of raw contamination data of foodstuffs for human consumption, in order to be used in accurate exposure assessment studies, specially concerning T2 and HT2. The aim of the present study was to study the occurrence of trichothecenes DON, T2 and HT2 toxin in Catalonian market.
Each standard of DON, HT2 and T2 was dissolved in acetonitrile at a concentration of 1.0 mg/ml and stored at 20 C in a sealed vial until use. Working standards (10.0, 2.0, 1.0, 0.5, 0.25, 0.1, 0.05 and 0.02 mg/ml) were prepared by appropriate dilution of known volumes of the stock solution with acetonitrile and used to obtain calibration curves after derivatization (when it was necessary) and injection in the appropriated chromatographic system.
2. Materials and methods
2.4. Equipment
2.1. Samples
The GC system was composed of an HP-6890 plus gas chromatograph, equipped with a 63Ni ECD (HewlettePackard, Avondale, PA, USA) and an Agilent 7683 Series injector (Agilent Technologies, Waldbronn, Germany). Signals were processed by HP GC ChemStation software Version A.10:02(1757) (HewlettePackard). To analyze trichothecenes in bread a GC system, 6890N-5973 equipped with a mass spectrometer was used. An HP-5MS fused silica capillary column (30 m 0.25 mm, film thickness 0.25 mm) from Agilent Technologies was used. The LC system consisted of a Waters 600 pump, a Waters 717 automatic injector and a Waters 996 UV diode-array detector (DAD) (Waters Corporation, Manchester, UK). Millennium 32 software, version 3.05.01 (Waters Co., Milford, MA, USA) was used to control the system.
Food samples were taken from the most susceptible to trichothecenes contamination and most commonly consumed in Catalonia commodities (Serra-Majem et al., 2003). During the months of June and July 2008, corn flakes (n ¼ 168), wheat flakes (n ¼ 27), sweet corn (n ¼ 185), corn snacks (n ¼ 213), pasta (n ¼ 201), beer (n ¼ 213), sliced bread (n ¼ 147) and bread (n ¼ 31) were obtained in six hypermarkets and supermarkets from twelve main cities (Tortosa, Tarragona, Reus, Vilanova i la Geltrú, l’Hospitalet de Llobregat, Barcelona, Terrassa, Sabadell, Mataró, Girona, Manresa and Lleida) of Catalonia, Spain, representative of 72% of the population. From each supermarket or hypermarket, 3 items (if present) of each product were randomly taken. The level of trichothecenes was determined in a total of 72 composite samples obtained by pooling the 3 items taken from each ́ store if were available (12 cities 6 stores/city ¼ 72 samples/category). ́ However, in some cases, 3 items were not available in the same store, thus, less than 72 composites could be obtained. Regarding brands, we finally obtained 62 of corn flakes, 29 of wheat flakes, 31 of sweet corn, 79 of corn snacks,105 of pasta, 64 of beer, 43 of sliced bread, which can be considered the majority of market share in Catalonia of these products, as well as in the rest of Spanish market. The samples were transported and stored under suitable conditions until analysis. 2.2. Chemicals and reagents Trichothecene standards, including DON, HT2 and T2 were supplied by Sigma (SigmaeAldrich, Alcobendas, Spain). Toluene was purchased from Sigma. Acetonitrile and methanol were purchased from J.T. Baker (Deventer, The Netherlands). Bencene and n-hexane were purchased from Merck (Darmstadt, Germany). All solvents were LC grade. Standardized 70e230 mesh aluminium oxide 90 (0.063e0.2 mm particle size) and ammonium hydroxide solution (32%, v/v) were purchased from Merck. C18 silica was purchased from Waters (Milford, MA, USA). Activated charcoal (Norit) was purchased from Fluka (SigmaeAldrich, Alcobendas, Spain). Glass microfibre filters (GF/C) and filter papers (Whatman No 4) were purchased from Whatman (Maidstone, UK). Pentafluoropropionic anhydride (PFPA), 4-dimethylaminopyridine (DMAP), sodium hydrogen carbonate, zinc acetate dihydrate (33% w/v) and potassium hexacyanoferrate (II) were also purchased from Sigma. T2/HT2 HPLC and DONTEST HPLC monoclonal antibodybased immunoaffinity columns were purchased from Vicam (Waters Business, Milford, MA, USA). Mycosep # 225 and Multisep #216 columns were purchased from Romer Laboratories. Imidazole and sodium sulfate anhydrous for analysis, were purchased from Merck. Pure water was obtained from a Milli-Q apparatus (Millipore, Billerica, MA, USA) and was used when water was required. Phosphate buffer saline (PBS) was prepared with potassium chloride (0.2 g) (Panreac, Castellar del Vallès, Spain), potassium dihydrogen phosphate (0.2 g) (Sigma), disodium phosphate anhydrous (1.16 g) (Panreac), sodium chloride (8.0 g) (J.T Baker) in 1 l of pure water; the pH was brought to 7.4.
2.3. Preparation of standard solutions
2.5. Trichothecene determination in the studied samples Development and optimization of the analytical method for trichothecenes determination in breakfast cereals, corn snacks and pasta samples, was carried out by means of various steps: firstly, a mycotoxin extraction phase, then a clean-up procedure, after a derivatization step and finally analysis by GCeECD, while the bread samples were analyzed by GCeMS. Concerning sliced bread, sweet corn and beer samples, the procedure for the determination of trichothecenes was carried out by means of extraction, clean-up and LCeDAD steps, because, this method showed the best accuracy and recovery rates for these matrices. 2.5.1. Extraction procedure Bread, sliced bread and sweet corn samples were previously dried for 48 h at 50 C. Fifty grams of composites of breakfast cereals, corn snacks, pasta, sweet corn and sliced bread were finely ground with a laboratory mill and 2 g of flour was poured into a 50 ml screw top tube. After adding 15 ml of acetonitrileewater (84:16, v/v), the mixture was blended in an orbital blender for 90 min. The procedure was slightly modified for bread, 5 g of dried sample was extracted with 20 ml of acetonitrileewater (84:16, v/v) by shaking with an ultraturrax for 3 min. After filtering through Whatman No. 4 filter, the extraction mixture was stored in a tightly closed glass bottle at 20 C until use. 2.5.2. Clean-up procedures 2.5.2.1. Breakfast cereals, corn snacks and pasta samples. ‘Made-inlaboratory’ cartridges were prepared using 5 ml sterile plastic syringes. A glass microfibre filter was placed at the bottom. Then, a mixture of packing bed was poured on it. The packed material consisted of 1.16 g of aluminaecharcoaleC18 silica (75:1:40, w/w/w). Another glass microfibre filter was placed on the bed top. Then, it was pressed tightly but carefully with a plunger. Three milliliters of sample extract was passed through the prepared cartridge and collected in a vial. The cartridge was rinsed with 2 ml of acetonitrileewater (84:16, v/v). The eluate was collected in the same vial and the purified extract was concentrated to dryness at 45 C
492
G. Cano-Sancho et al. / Food Control 22 (2011) 490e495
under gentle stream of nitrogen. The samples of breakfast cereals, corn snacks and pasta continued the process with the derivatization of the extracts. 2.5.2.2. Sliced bread and sweet corn samples. Firstly, the clean-up procedure is the same that for breakfast cereals, corn snacks and pasta samples. Then, 10 ml of PBS was added to the dried extract and were shaken for 30 s with the help of a Vortex. Diluted solution was loaded into the immunoaffinity columns (both columns were consecutively coupled) and passed at a flow rate at one to two drops per second. The columns were washed with 5 ml of pure water. Then, it was eluted with 2 ml of methanol at a rate of about one drop per second in a 4 ml vial. The purified extract was concentrated to dryness at 45 C under gentle stream of nitrogen and solved in 0.25 ml of initial LC mobile phase. 2.5.2.3. Bread. A 5 ml aliquot of the extract was transferred to the tube that follows the Mycosep 225 columns. The Mycosep column was put on the top of the tube (as a cap) and mix carefully. The extract was pressed slowly through the Mycosep column (about 25e30 s per column). 3 ml of the purified extract was loaded into the Mycosep 216 column previously conditioned with 10 ml acetonitrile/water (84:16). Then it was eluted with 10 ml acetonitrile/water (9:1). The purified extract was evaporated to dryness under a gentle stream of nitrogen at 60 C. 2.5.2.4. Beer. In the case of beer samples, the process of clean-up was as following: about 50 ml of cool beer was thoroughly degassed in ultrasonic bath for 1 h in 500 ml Erlenmeyer flask. 10 ml of degassed beer was transferred to a screw top tube. Then, 0.1 ml of ammonium hydroxide solution (32%, v/v) was added to alkalinize the sample and hence to precipitate proteins and other matrix components. The mixture was shaken and let stand for 10 min. Then, 0.4 ml of a 25% aqueous solution of zinc acetate dihydrate was added to eliminate dyes without affecting trichothecene levels. Zn2þ excess was controlled with 0.4 ml of potassium hexacyanoferrate (II) (10%, w/v) which provided a voluminous precipitate of zinc hexacyanoferrate (II). The mixture was vigorously shaken for 1 min, centrifuged at 6840 g for 10 min and the supernatant was collected to another tube. The supernatant was loaded into the immunoaffinity columns and passed at a flow rate at oneetwo drops per second. The columns were washed with 5 ml of pure water. Then, it was eluted with 2 ml of methanol at a rate of about one drop per second in a 4 ml vial. The purified extract was concentrated to dryness at 45 C under a gentle stream of nitrogen and solved in 0.25 ml of initial LC mobile phase. 2.5.3. Trichothecene derivatization for GCeECD determination in samples of corn snacks, pasta and breakfast cereals One hundred microliters of a 2 mg/l solution of DMAP in tolueneeacetonitrile (80:20, v/v) and 50 ml of pentafluoropropionic anhydride (PFPA) were added to each dry extract in a screw cap vial. After capping tightly, the reaction mixture was heated at 60 C for 60 min in an aluminium heater block. After the mixture had cooled, 1 ml of a 3% (w/v) aqueous solution of NaHCO3 was added and the vial was vortexed for 15 s. The two layers were allowed to separate. The top (organic phase) layer was transferred to a GC autoinjector vial and analyzed by GCeECD as pointed out below. 2.5.4. Trichothecene derivatization for GCeMS determination in samples of bread One milliliter benzene was added to the evaporated extract and mixed for 1 min. The extract was evaporated once again to dryness and 500 ml of 0.4 M imidazole (as catalyst) in tolueneeacetonitrile (85 þ 15) and 100 ml PFPA were added to each derivatization vial
and mixed for 1 min. The reaction was left for derivatization at 60 C for 1 h and cooled afterwards for about 10 min and washed (to remove the excess reagent) by adding 500 ml hexane and 1 ml 5% sodium hydrogen carbonate solution. The derivatives were mixed carefully and the vials were opened carefully to reduce the pressure and mixed 1 min more. The aqueous phase was removed and 1 ml water was added and mixed 1 min. Then the aqueous phase was removed completely. A small amount of sodium sulfate was added to remove any water left. Finally, about 200 ml of the derivatized extract were transferred to a GC vial. 2.5.5. GCeECD analysis in the samples of corn snacks, pasta and breakfast cereals The GCeECD determination was carried out using the following chromatographic conditions. One microliter of solution was injected in splitless mode. The temperatures of the injection port and the detector were 250 and 300 C, respectively. The procedure used a fused silica capillary column HP-5 [5% methyl phenyl siloxane (30 m 0.32 mm, 0.25 mm film thickness, Agilent Technologies)]. The oven temperature program was: 90 C held for 1 min, 40 C/min to 160 C, 1.5 C/min to 182 C, 5 C/min to 240 C, and then 40 C/min to 275 C, held for 8 min. Helium at a constant pressure of 42.1 kPa was used as carrier gas. 2.5.6. GCeMS analysis in bread samples The GCeMS conditions were as follows: 1 ml of extract was injected in splitless mode. The temperature of the injection port was 250 C and the temperatures of the detector were 230 C (ion source) and 150 C (quadrupole). The oven temperature program was: 120 C held for 2 min, 30 C/min to 175 C (held for 3 min), 1 C/ min to 180 C and finally 25 C/min to 265 C (held for 15 min). Helium at a constant flow of 0.7 ml/min was used as carrier gas. The transfer line temperature was held at 260 C and selected ion monitoring at m/z 309 and 543 for DON, 377 and 555 for HT2 and 377, 451 for T2 were scanned for GCeMS analysis. 2.5.7. LCeDAD analysis in the samples of sweet corn, sliced bread and beer Fifty microliter dissolved extract were injected in the chromatographic system. The elution was carried out at a flow of 1 ml/ min with a mixture of acetonitrile (solvent A) and water (solvent B) applying the following gradient program: (i) initial solvent: 94% B, maintained for 6 min, (ii) lineal variation of B from 94% to 70% for 4 min, (iii) lineal variation of B from 70% to 50% for 12 min, (iv) 50% of B for 3 min, and (v) return to the initial conditions in 0.2 min. Separation was performed on a Zorbax Eclipse Plus C18 column (150 mm 4.6 mm, 3.5 mm particle size) connected to an Eclipse Plus C18 guard column (12.5 mm 4.6 mm, 3.5 mm particle size) (Agilent Technologies Inc, Santa Clara, USA) filled with the same phase. The column was kept at 35 C. Quantification of trichothecenes was performed by measuring its peak area with the help of a calibration curve calculated by regression from standard solutions. Detection wavelengths were 200 nm for HT2 and T2, and 220 nm for DON. The UV spectrum of compounds in the sample was compared with the standard UV spectrum for the mycotoxin identification. 2.6. Validation of analytical methods The analytical methods used for DON, HT2 and T2 were assessed for selectivity, linearity, and precision. Selectivity was checked by injecting three times 50 ml of mycotoxin standard solutions before injecting extracted samples and comparing the peak retention times and the UV spectra of the substances that produce these peaks.
G. Cano-Sancho et al. / Food Control 22 (2011) 490e495
493
Table 1 Method performance characteristics for DON, HT2 and T2. Sample ¼ 3
Pasta
Corn snacks
Sweet corn
Sliced bread
Beer
Corn flakes
Wheat flakes
Bread
Spiking level
a
HT2
LOQ
Recovery
RSDr
LOQ
(mg/g)
(mg/g)
(%)
(%)
0.05 0.2 1 0.05 0.2 1 0.05 0.2 1 0.05 0.2 1 0.05 0.2 1 0.05 0.2 1 0.05 0.2 1
0.042
143.1 78.1 77 105.2 91.6 77.8 110.5 89.3 94.7 109.6 109.9 86.4 110.2 101.5 93.6 95.6 99.8 70.9 89.3 84.5 85.8 80.3 76.6
19.2 17.9 17.9 17.4 16.5 8.9 16.8 5.1 12.3 10.0 12.2 5.7 10.0 6.1 5.4 19.8 7.6 8.1 14.0 11.9 18.4 5.1 6.4
a b
b
DON
0.066
0.027
0.030
0.012
0.045
0.041
0.018
T2 Recovery
RSDr
LOQ
Recovery
RSDr
(mg/g)
(%)
(%)
(mg/g)
(%)
(%)
0.030
113.2 99.3 88.4 112.7 93.9 82.4 88.8 74 83.4 98.9 107.1 104.8 125 121.7 73.9 129.7 87.3 79.8 109.5 93.6 86 88.2 87.0
12.3 11.2 18.9 24.2 10.1 14.7 6.7 14.9 9.2 8.2 6.6 10.3 16.9 7.7 14.4 26.7 5.2 10.5 17.5 5.5 8.2 5.9 3.9
0.063
90.9 80.9 86.2 115.6 94.7 92.4 e 69.8 101.1 e 90.0 94.4 e 90.3 83.3 119.6 102.8 78.3 66.6 96.5 85.5 95.0 90.7
7.8 24.7 13.5 8.8 21.5 11.7 e 4.0 5.2 e 14.0 8.6 e 5.8 5.9 14.4 18.9 7.9 19.7 6.0 3.1 4.6 5.4
0.061
0.024
0.030
0.009
0.030
0.036
0.050
0.054
0.135
0.108
0.057
0.057
0.042
0.070
Spiking level DON: 0.018 mg/g, HT2: 0.050 mg/g, T2: 0.070 mg/g. Spiking level DON: 0.473 mg/g, HT2: 0.437 mg/g, T2: 0.612 mg/g.
Linearity was assessed by performing triplicate injections of standard solutions whose concentrations were 0.02, 0.05, 0.1, 0.25, 0.5 and 1.0 mg each of mycotoxin/ml. Standard curves were generated by linear regression of peak areas against concentrations. Accuracy and recovery were established for each method by determination of DON, HT2 and T2 in samples of pasta, corn snack, sliced bread, beer, breakfast cereal and sweet corn, covering the range of the method (range between 0.05 and 1.0 mg/g). Recovery was determined by comparing the absolute responses of trichothecenes obtained from the studied samples with the absolute responses of calibration standards. The limit of detection (LOD) was considered as the mycotoxin concentration that provides a signal equal to b þ 3Sb, where b is the intercept of the calibration curve and Sb is the standard error of the estimate assuming to be the blank, and the limit of quantification (LOQ) was considered equal to 3LOD. Samples of pasta, corn snacks, sweet corn, sliced bread, beer, corn flakes and wheat flakes were spiked at levels of 1, 0.2 and 0.05 mg/g of DON, HT2 and T2 in triplicate. Concerning bread, these samples were spiked with each mycotoxin at LOQ level and the concentrations 0.473, 0.437 and 0.612 mg/g for DON, HT2 and T2, respectively. Analytical procedures were carried out three times. Calibration curves showed good linearity with correlations coefficients R2 ranging from 0.9799 to 0.9994. Percentage of recovery and relative standard deviation (RSDr) for each food matrix and spiking level are shown in Table 1. Recovery ranges between 77e143.1, 78.3e115.6 and 74e129.7% were found for DON, T2 and HT2, respectively, according to performance criteria established by Commission Regulation (EC) No 401/2006 (European Commission, 2006a), with the exception of recovery value of 143.1%, found in pasta samples spiked at 0.05 mg/ g. Maximum relative standard deviation (RSDr) levels for DON, T2 and HT2 were 19.8, 24.7 and 26.7%. Quantification limits of DON, T2 and HT2 toxin ranged from 0.012 mg/g (beer) to 0.066 mg/g (corn snacks), 0.042 mg/g (wheat flakes) to 0.135 mg/g (sweet corn) and 0.009 mg/g (beer) to 0.061 mg/g (corn snacks), respectively.
3. Results and discussion 3.1. Occurrence of DON In this study 479 “composite” samples pooled from 1147 individual samples from Catalonian market, were analyzed to detect and quantify DON, T2 and HT2. Previous studies conducted in Spain were carried out with fewer samples than the current work, in the order of 25 (Cerveró et al., 2007) and 175 (Castillo et al., 2008), thus, this work, represents the widest survey conducted in Spain to assess the occurrence of trichothecenes in foodstuffs for human consumption, to date. As shown in Table 2, the mycotoxin DON was found in all cerealbased food items analyzed in this study. The lowest percentage of positive samples (quantified) was found in beer (1.4%), sweet corn (2.8%) and sliced bread (16.7%). In contrast, high percentage of positive samples was found in breakfast cereals (74.1 and 73.4%), corn snacks (78.9%), pasta (74.3%) and specially in bread, where DON was found in all samples. Mean values of positive samples ranged from 0.012 mg/g, found in beer, to 0.246 0.158 mg/g found in bread. Despite the high percentage of positive samples in foodstuffs from Catalonian market, only two samples were above the
Table 2 Occurrence of DON in foodstuff from Catalonian market. N N Samples Mean sd individual composite > LOQ mg/g Wheat flakes Corn flakes Beer Sweet corn Corn snacks Pasta Sliced bread Bread
27 168 213 185 213 201 147 31
27 65 71 72 71 70 72 31
20/27 49/65 1/70 2/72 56/71 52/70 12/72 31/31
0.190 0.109 0.012 0.114 0.153 0.226 0.068 0.246
Median Max
mg/g
0.117 0.157 0.078 0.093 0.012 0.036 0.114 0.058 0.143 0.177 0.199 0.018 0.068 0.158 0.242
mg/g 0.437 0.580 0.012 0.139 0.304 0.946 0.098 0.739
494
G. Cano-Sancho et al. / Food Control 22 (2011) 490e495
Table 3 Occurrence of T2 and HT2 toxin in food from Catalonian market. N N Samples Mean sd individual composite > LOQ mg/g T2 Wheat flakes Corn flakes Beer Sweet corn Corn snacks Pasta Sliced bread Bread HT-2 Wheat flakes Corn flakes Beer Sweet corn Corn snacks Pasta Sliced bread Bread
27 156 216 185 213 201 147 31
27 65 71 72 71 70 72 31
2/27 0/65 0/71 2/72 1/71 0/70 0/72 0/31
27 156 216 185 213 201 147 31
27 65 71 72 71 70 72 31
4/27 4/65 0/71 5/72 6/71 7/70 11/72 0/31
Table 4 Co-occurrence of trichothecenes in foodstuff from Catalonian market. Median Max
mg/g
mg/g
0.072 0.005 0.072 e e e e 0.215 0.058 0.215 0.070 0.70 e e e e e e
0.075 e e 0.256 0.070 e e e
0.066 0.061 0.017 0.034 e 0.023 0.033 0.334 0.078 0.022 0.046 0.012 0.042 e
0.183 0.065 e 0.084 0.895 0.080 0.075 e
0.087 0.041 e 0.043 0.214 0.051 0.047 e
limit of 0.750 mg/g in pasta (0.946 mg/g), while one sample of corn flakes (0.580 mg/g) and two of bread (0.523 and 0.739 mg/g), were above the limit of 0.500 mg/g, established by European Commission (European Commission, 2006a). The previous studies, carried out to assess the occurrence of DON in foodstuff from Spanish market, commonly found lower levels of positive samples, mean and maximum values than our study. For example, in the earliest study the range of 0.038e0.195 mg/g was reported for corn flakes, 0.035e0.061 mg/g for sweet corn and 0.028e0.109 mg/g for fried corn snacks (Cerveró et al., 2007). Moreover, in other recent study, concentrations of 0.030e0.121 and 0.026e0.080 mg/g, were found in breakfast cereals and fried snacks, respectively (Castillo et al., 2008). However, we found a wide range of contamination for these foodstuffs, with maximum values of 0.580, 0.139 and 0.304 mg/g in corn flakes, sweet corn and corn snacks, respectively. In the line with our results, there is a study conducted in Canada with breakfast cereals where the authors showed a maximum level and mean of positive samples of 0.420 and 0.070 mg/g, respectively (Roscoe et al., 2008), while in Italy, a lowest range, between 0.012 and 0.047 mg/g was found (Cirillo, Ritieni, Galvano, & Cocchieri, 2003). Highest levels were stated in other study performed in Portugal, where the range 0.103e6.040 mg/g and the mean 0.754 mg/g were found in wheat-based breakfast cereals (Martins & Martins, 2001). Regarding baked products, in the The First French Total Diet Study the mean of the positive samples of bread and rusk was estimated to be 0.109 mg/g (Leblanc, Tard, Volatier, & Verger, 2005). While in a German study, the authors reported a high percentage of positive samples in conventional and organic bread, with ranges of 0.015e0.690 mg/g and 0.015e0.224 mg/g, and means of 0.184 and 0.062 mg/g, respectively (Schollenberger et al., 2005). In Italy, the level of DON in bread and related food was observed between 0.007 and 0.270 mg/g (Cirillo et al., 2003), all these values being much lower than our range of 0.027e0.739 mg/g. Few data is available about the levels of DON in pasta. This mycotoxin was not detected in France (Leblanc et al., 2005) and the levels ranged from 0.009 to 0.077 mg/g in Italy (Cirillo et al., 2003), while, the level in noodles from Germany was between 0.015 and 1.670 mg/g, with a mean of 0.158 0.334 mg/g (Schollenberger, Suchy, Jara, Drochner, & Müller, 1999), closest to our study with a respective mean and maximum value of 0.226 and 0.946 mg/g. Concerning beer, to our knowledge, few studies have been conducted to assess the level of DON in this alcoholic beverage from
Wheat flakes Corn flakes Beer Pan loaf Sweet-corn Corn snacks Pasta Bread Total
DON-T2
DON-HT2
T2-HT2
DON-T2-HT2
2 1 0 1 0 2 2 0 8
8 17 0 4 0 20 16 0 55
0 0 0 0 3 0 0 0 3
4 4 0 0 0 4 12 0 24
Note: Co-occurrence was determined considering samples above LOD, and expressed as the number of samples with simultaneous contamination the mycotoxins.
European markets. In an early study that reported the occurrence of 176 beer samples collected in European and North American markets, the authors found that 64% of samples were above the LOQ of 0.003 mg/g, and the mean ranged from 0.002 to 0.011 mg/g; the maximum value was 0.036 mg/g (Kostelanska et al., 2009). The higher occurrence in this previous study could be explained by the lower LOQ of this study (0.012 mg/g). 3.2. Occurrence of T2 and HT2 toxin Percentage of positive samples of T2 and HT2 toxin and contamination levels in food marketed in Catalonia, are shown in Table 3. Low percentage of positive samples was found for these type-A trichothecenes, specially for T2, which was quantified only in 5 samples of 479 (wheat flakes, sweet corn and corn flakes). HT2 was present in a low percentage of sliced bread samples (15.3%), wheat flakes (14.8%), pasta (10%), corn snacks (8.4%), sweet corn (6.9%) and corn flakes (6.2%). Mean levels of HT2 in positive samples were between 0.041 0.017 mg/g (corn flakes) and 0.214 0.334 mg/g (corn snacks), being the maximum level 0.895 mg/g in a corn snack sample. In a previous study conducted in Spain, the authors did not find T2 in any sample out of the 25 samples analyzed, above the LOD of 0.030 mg/g neither in corn flakes, sweet corn nor in corn snacks (Cerveró et al., 2007). Leblanc et al. (2005) quantified HT2 in one sample out of 238 composite samples with a level of 0.270 mg/g. In Germany, HT2 was the main type-A trichothecene found in cereal-based food, with ranges of 0.012e0.032 mg/g in bread and related products, 0.012e0.025 mg/g in noodles and 0.012e0.022 mg/ g in breakfast cereals, slightly lower values than our results (Schollenberger et al., 1999). 3.3. Co-occurrence of trichothecenes in the samples Several studies have suggested that the mycotoxins could have synergistic effect in vivo (Bacon, Porter, Norred, & Leslie, 1996; Javed et al., 1993), therefore the knowledge of the occurrence of all mycotoxins should be considered a challenge in exposure assessment studies. In our work, the co-occurrence of different mycotoxins in the same sample, was studied considering positive those samples above LOD (Table 4). The results suggest that the most common case of co-occurrence with trichothecenes can be drawn by the simultaneous occurrence of DON and HT2 toxin, specially in corn snacks, corn flakes and pasta, as was elucidated previously by Schollenberger et al. (1999). 4. Conclusions With 479 analytical samples, this is the widest study to assess the occurrence of trichothecenes in food for human consumption
G. Cano-Sancho et al. / Food Control 22 (2011) 490e495
until now in Spain. We have found that the occurrence of DON was high in cereal-based foodstuff from Catalonian market, specially in wheat flakes, corn flakes, corn snacks, pasta and bread. Despite the high percentage of positive samples in foodstuff from Catalonian market, only five samples were above the limit established by European Commission (European Commission, 2006a). In the other hand, T2 toxin was only quantified in 5 samples. HT2 was present in a low percentage of the samples and it was not detected in samples of bread and beer. Although no worrying values have been found in this study for DON, T2 and HT2 with samples collected in 2008, the annual variability of the levels of trichothecenes in the crops makes necessary the monitoring of these mycotoxins.
Acknowledgements The authors would like to acknowledge Exposure Assessment of Spanish Population to Fusarium Toxins Project, National Plan of Spanish Government (AGL2008-05030-C02-01), Catalonian Food Safety Agency of ‘Generalitat de Catalunya’ Health Department and University of Lleida for their financial support. Also, the authors thank to M. J. Nogueiras and A. Dieguez for their technical assistance in the analysis carried out in the National Centre for Food.
References Bacon, C. W., Porter, J. K., Norred, W. P., & Leslie, J. F. (1996). Production of fusaric acid by Fusarium species. Applied and Environmental Microbiology, 62(11), 4039e4043. Castillo, M., Montes, R., Navarro, A., Segarra, R., Cuesta, G., & Hernández, E. (2008). Occurrence of deoxynivalenol and nivalenol in Spanish corn-based food products. Journal of Food Composition and Analysis, 21(5), 423e427. Cerveró, M. C., Castillo, M. A., Montes, R., & Hernández, E. (2007). Determination of trichothecenes, zearalenone and zearalenols in commercially available cornbased foods in Spain. Revista Iberoamericana de Micología, 24(1), 52e55. Cirillo, T., Ritieni, A., Galvano, F., & Cocchieri, R. A. (2003). Natural co-occurrence of deoxynivalenol and fumonisins B-1 and B-2 in Italian marketed foodstuffs. Food Additives and Contaminants Part A-Chemistry Analysis Control Exposure & Risk Assessment, 20(6), 566e571. Eriksen, G. S., & Pettersson, H. (2004). Toxicological evaluation of trichothecenes in animal feed. Animal Feed Science and Technology, 114(1e4), 205e239. European Commission. (2006a). Commission Regulation (EC) No. 401/2006 of 23 February 2006. Laying down the methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs. Official Journal of the European Union L, 70, 12e34.
495
European Commission. (2006b). Commission Regulation (EC) No. 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. Official Journal of the European Union L, 364, 5e24. Hazel, C. M., & Patel, S. (2004). Influence of processing on trichothecene levels. Toxicology Letters, 153(1), 51e59. Javed, T., Bennett, G. A., Richard, J. L., Dombrink-Kurtzman, M. A., Côté, L. M., & Buck, W. B. (1993). Mortality in broiler chicks on feed amended with Fusarium proliferatum culture material or with purified fumonisin B1 and moniliformin. Mycopathologia, 123(3), 171e184. Jelinek, C. F., Pohland, A. E., & Wood, G. E. (1989). Worldwide occurrence of mycotoxins in foods and feeds-an update. Journal of the Association of Official Analytical Chemists, 72(2), 223e230. Joint FAO/WHO Expert Committee on Food Additives (JECFA). (2001). Safety evaluation of certain mycotoxins in food. Rome, Italy: Food and Agriculture Organization. Kostelanska, M., Hajslova, J., Zachariasova, M., Malachova, A., Kalachova, K., Poustka, J., et al. (2009). Occurrence of deoxynivalenol and its major conjugate, deoxynivalenol-3-glucoside, in beer and some brewing intermediates. Journal of Agricultural and Food Chemistry, 57(8), 3187e3194. Leblanc, J., Tard, A., Volatier, J., & Verger, P. (2005). Estimated dietary exposure to principal food mycotoxins from The First French Total Diet Study. Food Additives and Contaminants, 22(7), 652e672. Martins, M. L., & Martins, H. M. (2001). Determination of deoxynivalenol in wheatbased breakfast cereals marketed in Portugal. Journal of Food Protection, 64(11), 1848e1850. Roscoe, V., Lombaert, G. A., Huzel, V., Neumann, G., Melietio, J., Kitchen, D., et al. (2008). Mycotoxins in breakfast cereals from the Canadian retail market: a 3-year survey. Food Additives and Contaminants Part A, 25(3), 347e355. Rotter, B. A., Prelusky, D. B., & Pestka, J. J. (1996). Toxicology of deoxynivalenol (vomitoxin). Journal of Toxicology and Environmental Health - Part A, 48(1), 1e34. SCF. (2002). Opinion on Fusarium TOXINSdPart 6: Group evaluation of T-2 toxin, HT-2 toxin, nivalenol and deoxynivalenol (adopted on 26 February 2002). Available in: http://europa.eu.int/comm/food/fs/sc/scf/out123 en.pdf Accessed 15.02.10. Schollenberger, M., Müller, H., Rüfle, M., Suchy, S., Planck, S., & Drochner, W. (2005). Survey of Fusarium toxins in foodstuffs of plant origin marketed in Germany. International Journal of Food Microbiology, 97(3), 317e326. Schollenberger, M., Suchy, S., Jara, H. T., Drochner, W., & Müller, H. (1999). A survey of Fusarium toxins in cereal-based foods marketed in an area of southwest Germany. Mycopathologia, 147(1), 49e57. Scott, P. M. (1989). The natural occurrence of trichothecenes. In V. R. Beasley (Ed.), Trichothecene mycotoxicosis: Pathophysiologic effects (pp. 1e26). Boca Raton: CRC Press. Serra-Majem, L., Ribas, L., Salvador, G., Castells, C., Serra, J., & Jover, L. (2003). caAvaluació de l’estat nutricional de la població catalana 2002e2003. Evolució dels hàbits alimentaris i del consum d’aliments i nutrients a Catalunya (1992e2003). Barcelona: Direcció General de Salut Pública, Departament de Sanitat i Seguretat Social, Generalitat de Catalunya. Sudakin, D. L. (2003). Trichothecenes in the environment: relevance to human health. Toxicology Letters, 143(2), 97e107. Visconti, A. (2001). Problems associated with Fusarium mycotoxins in cereals. Bulletin of the Institute for Comprehensive Agricultural Sciences Kinki University, 9, 39e55. Visconti, A., Minervini, F., Lucivero, G., & Gambatesa, V. (1991). Cytotoxic and immunotoxic effects of Fusarium mycotoxins using a rapid colorimetric bioassay. Mycopathologia, 113(3), 181e186.
Contents lists available at ScienceDirect
Food Control journal homepage: www.elsevier.com/locate/foodcont
Presence of trichothecenes and co-occurrence in cereal-based food from Catalonia (Spain) G. Cano-Sancho a, *, F.M. Valle-Algarra b, M. Jiménez b, P. Burdaspal c, T.M. Legarda c, A.J. Ramos a, V. Sanchis a, S. Marín a a b c
Applied Mycology Unit, Food Technology Department, University of Lleida, XaRTA-UTPV, Rovira Roure 191, 25198 Lleida, Spain Microbiology and Ecology Department, University of Valencia, Dr. Moliner 50, E-46100 Burjassot, Valencia, Spain National Centre for Food (Spanish Food Safety and Nutrition Agency), 28220 Majadahonda, Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 July 2010 Received in revised form 20 September 2010 Accepted 28 September 2010
The most important trichothecenes are HT-2 toxin (HT2) and T-2 toxin (T2) from type A and deoxynivalenol (DON) from type B. Thus, the aim of the current study was to assess the occurrence of these trichothecenes in the Catalonian market. 479 food samples were taken from the most susceptible to trichothecenes contamination and most commonly consumed in Catalonia commodities. DON, T2 and HT2 toxin were determined in breakfast cereals, snacks and pasta samples following extraction, clean-up, derivatization and finally analysis by GCeECD. Moreover, these mycotoxins were determined in sliced bread, sweet corn and beer by LCeDAD. Our results showed that DON was the main trichothecene present in the cereal-based food from Catalonian market with percentages of positive samples ranging from 1.4 to 100.0%. Despite the high incidence of DON, only five samples were above EU limits. Concerning HT2 toxin, it was present in a low percentage of samples of sliced bread (15.3%), wheat flakes (14.8%), pasta (10.0%), corn snacks (8.5%), sweet corn (6.9%) and corn flakes (6.2%), while the T2 toxin only was quantified in 5 samples out of the total 479. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Cereal-based food Deoxynivalenol T2 HT2 Mycotoxins Occurrence
1. Introduction Trichothecenes are a family of related cyclic sesquiterpenoids, which are divided into four groups (types AeD) according to their characteristic functional groups, being the type A and B, the most common. Type A is represented by HT-2 toxin (HT2) and T-2 toxin (T2) and type B is most frequently represented by deoxynivalenol (DON). Trichothecenes are produced in several cereals by species of Fusarium, thus, a wide range of cereal-based food have been confirmed to be contaminated by these toxins (JECFA, 2001; pp. 281e320). T2 is a potent inhibitor of protein synthesis and mitochondrial function both in vivo and in vitro, and shows immunosuppressive and cytotoxic effects. Moreover, it has been reported that the toxin has extremely toxic effects on skin and mucous (Eriksen & Pettersson, 2004, Sudakin, 2003; Visconti, 2001; Visconti, Minervini, Lucivero, & Gambatesa, 1991). It has been shown that through deacetylation of T2, it is obtained HT2 as the major metabolite; however, little
* Corresponding author. Tel.: þ34 973702670; fax: þ34 973702596. E-mail address: [email protected] (G. Cano-Sancho). 0956-7135/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2010.09.033
information is available regarding toxicity of HT2 alone (Sudakin, 2003; Visconti, 2001). Although DON is not as toxic as other trichothecenes such as T2 or HT2, this mycotoxin is one of the most common contaminants of cereals worldwide (Jelinek, Pohland, & Wood, 1989; Scott, 1989). Acute effects of food poisoning in humans are abdominal pains, dizziness, headache, throat irritation, nausea, vomiting, diarrhoea, and blood in the stool (Rotter, Prelusky, & Pestka, 1996). Maximum level of DON permitted in Europe is 0.750 mg/g in pasta and 0.500 mg/g in bread, pastries, biscuits, cereal snacks and breakfast cereals (European Commission, 2006b). The Joint FAO/ WHO Expert Committee on Food Additives (JECFA), after assessing the toxic effect of T2 and HT2, concluded that the toxic effects of these mycotoxins could not be differentiated. Thus, the provisional maximum tolerable daily intake (PMTDI) for the combination of these toxins or alone was set at 0.06 mg/kg body weight/day (JECFA, 2001; pp. 281e320). Concerning DON, a tolerable daily intake (TDI) of 1 mg/kg body weight based on a reduction of body weight gain was established by the EC SCF (SCF, 2002). Occurrence of DON, T2 and HT2 toxin has been widely reported in raw food and foodstuff, around of the European region (JECFA, 2001; pp. 281e320), confirming that food processing methods do not completely remove the mycotoxins in the matrix (Hazel & Patel, 2004). Nevertheless, the occurrence of these trichothecenes in
G. Cano-Sancho et al. / Food Control 22 (2011) 490e495
491
corn-based food from Spain, has been partially addressed in two previous studies, where the breakfast cereals and corn snacks, were the main foodstuffs analyzed (Castillo et al., 2008; Cervero, Castillo, Montes & Hernandez, 2007), thereby, currently there is a lack of raw contamination data of foodstuffs for human consumption, in order to be used in accurate exposure assessment studies, specially concerning T2 and HT2. The aim of the present study was to study the occurrence of trichothecenes DON, T2 and HT2 toxin in Catalonian market.
Each standard of DON, HT2 and T2 was dissolved in acetonitrile at a concentration of 1.0 mg/ml and stored at 20 C in a sealed vial until use. Working standards (10.0, 2.0, 1.0, 0.5, 0.25, 0.1, 0.05 and 0.02 mg/ml) were prepared by appropriate dilution of known volumes of the stock solution with acetonitrile and used to obtain calibration curves after derivatization (when it was necessary) and injection in the appropriated chromatographic system.
2. Materials and methods
2.4. Equipment
2.1. Samples
The GC system was composed of an HP-6890 plus gas chromatograph, equipped with a 63Ni ECD (HewlettePackard, Avondale, PA, USA) and an Agilent 7683 Series injector (Agilent Technologies, Waldbronn, Germany). Signals were processed by HP GC ChemStation software Version A.10:02(1757) (HewlettePackard). To analyze trichothecenes in bread a GC system, 6890N-5973 equipped with a mass spectrometer was used. An HP-5MS fused silica capillary column (30 m 0.25 mm, film thickness 0.25 mm) from Agilent Technologies was used. The LC system consisted of a Waters 600 pump, a Waters 717 automatic injector and a Waters 996 UV diode-array detector (DAD) (Waters Corporation, Manchester, UK). Millennium 32 software, version 3.05.01 (Waters Co., Milford, MA, USA) was used to control the system.
Food samples were taken from the most susceptible to trichothecenes contamination and most commonly consumed in Catalonia commodities (Serra-Majem et al., 2003). During the months of June and July 2008, corn flakes (n ¼ 168), wheat flakes (n ¼ 27), sweet corn (n ¼ 185), corn snacks (n ¼ 213), pasta (n ¼ 201), beer (n ¼ 213), sliced bread (n ¼ 147) and bread (n ¼ 31) were obtained in six hypermarkets and supermarkets from twelve main cities (Tortosa, Tarragona, Reus, Vilanova i la Geltrú, l’Hospitalet de Llobregat, Barcelona, Terrassa, Sabadell, Mataró, Girona, Manresa and Lleida) of Catalonia, Spain, representative of 72% of the population. From each supermarket or hypermarket, 3 items (if present) of each product were randomly taken. The level of trichothecenes was determined in a total of 72 composite samples obtained by pooling the 3 items taken from each ́ store if were available (12 cities 6 stores/city ¼ 72 samples/category). ́ However, in some cases, 3 items were not available in the same store, thus, less than 72 composites could be obtained. Regarding brands, we finally obtained 62 of corn flakes, 29 of wheat flakes, 31 of sweet corn, 79 of corn snacks,105 of pasta, 64 of beer, 43 of sliced bread, which can be considered the majority of market share in Catalonia of these products, as well as in the rest of Spanish market. The samples were transported and stored under suitable conditions until analysis. 2.2. Chemicals and reagents Trichothecene standards, including DON, HT2 and T2 were supplied by Sigma (SigmaeAldrich, Alcobendas, Spain). Toluene was purchased from Sigma. Acetonitrile and methanol were purchased from J.T. Baker (Deventer, The Netherlands). Bencene and n-hexane were purchased from Merck (Darmstadt, Germany). All solvents were LC grade. Standardized 70e230 mesh aluminium oxide 90 (0.063e0.2 mm particle size) and ammonium hydroxide solution (32%, v/v) were purchased from Merck. C18 silica was purchased from Waters (Milford, MA, USA). Activated charcoal (Norit) was purchased from Fluka (SigmaeAldrich, Alcobendas, Spain). Glass microfibre filters (GF/C) and filter papers (Whatman No 4) were purchased from Whatman (Maidstone, UK). Pentafluoropropionic anhydride (PFPA), 4-dimethylaminopyridine (DMAP), sodium hydrogen carbonate, zinc acetate dihydrate (33% w/v) and potassium hexacyanoferrate (II) were also purchased from Sigma. T2/HT2 HPLC and DONTEST HPLC monoclonal antibodybased immunoaffinity columns were purchased from Vicam (Waters Business, Milford, MA, USA). Mycosep # 225 and Multisep #216 columns were purchased from Romer Laboratories. Imidazole and sodium sulfate anhydrous for analysis, were purchased from Merck. Pure water was obtained from a Milli-Q apparatus (Millipore, Billerica, MA, USA) and was used when water was required. Phosphate buffer saline (PBS) was prepared with potassium chloride (0.2 g) (Panreac, Castellar del Vallès, Spain), potassium dihydrogen phosphate (0.2 g) (Sigma), disodium phosphate anhydrous (1.16 g) (Panreac), sodium chloride (8.0 g) (J.T Baker) in 1 l of pure water; the pH was brought to 7.4.
2.3. Preparation of standard solutions
2.5. Trichothecene determination in the studied samples Development and optimization of the analytical method for trichothecenes determination in breakfast cereals, corn snacks and pasta samples, was carried out by means of various steps: firstly, a mycotoxin extraction phase, then a clean-up procedure, after a derivatization step and finally analysis by GCeECD, while the bread samples were analyzed by GCeMS. Concerning sliced bread, sweet corn and beer samples, the procedure for the determination of trichothecenes was carried out by means of extraction, clean-up and LCeDAD steps, because, this method showed the best accuracy and recovery rates for these matrices. 2.5.1. Extraction procedure Bread, sliced bread and sweet corn samples were previously dried for 48 h at 50 C. Fifty grams of composites of breakfast cereals, corn snacks, pasta, sweet corn and sliced bread were finely ground with a laboratory mill and 2 g of flour was poured into a 50 ml screw top tube. After adding 15 ml of acetonitrileewater (84:16, v/v), the mixture was blended in an orbital blender for 90 min. The procedure was slightly modified for bread, 5 g of dried sample was extracted with 20 ml of acetonitrileewater (84:16, v/v) by shaking with an ultraturrax for 3 min. After filtering through Whatman No. 4 filter, the extraction mixture was stored in a tightly closed glass bottle at 20 C until use. 2.5.2. Clean-up procedures 2.5.2.1. Breakfast cereals, corn snacks and pasta samples. ‘Made-inlaboratory’ cartridges were prepared using 5 ml sterile plastic syringes. A glass microfibre filter was placed at the bottom. Then, a mixture of packing bed was poured on it. The packed material consisted of 1.16 g of aluminaecharcoaleC18 silica (75:1:40, w/w/w). Another glass microfibre filter was placed on the bed top. Then, it was pressed tightly but carefully with a plunger. Three milliliters of sample extract was passed through the prepared cartridge and collected in a vial. The cartridge was rinsed with 2 ml of acetonitrileewater (84:16, v/v). The eluate was collected in the same vial and the purified extract was concentrated to dryness at 45 C
492
G. Cano-Sancho et al. / Food Control 22 (2011) 490e495
under gentle stream of nitrogen. The samples of breakfast cereals, corn snacks and pasta continued the process with the derivatization of the extracts. 2.5.2.2. Sliced bread and sweet corn samples. Firstly, the clean-up procedure is the same that for breakfast cereals, corn snacks and pasta samples. Then, 10 ml of PBS was added to the dried extract and were shaken for 30 s with the help of a Vortex. Diluted solution was loaded into the immunoaffinity columns (both columns were consecutively coupled) and passed at a flow rate at one to two drops per second. The columns were washed with 5 ml of pure water. Then, it was eluted with 2 ml of methanol at a rate of about one drop per second in a 4 ml vial. The purified extract was concentrated to dryness at 45 C under gentle stream of nitrogen and solved in 0.25 ml of initial LC mobile phase. 2.5.2.3. Bread. A 5 ml aliquot of the extract was transferred to the tube that follows the Mycosep 225 columns. The Mycosep column was put on the top of the tube (as a cap) and mix carefully. The extract was pressed slowly through the Mycosep column (about 25e30 s per column). 3 ml of the purified extract was loaded into the Mycosep 216 column previously conditioned with 10 ml acetonitrile/water (84:16). Then it was eluted with 10 ml acetonitrile/water (9:1). The purified extract was evaporated to dryness under a gentle stream of nitrogen at 60 C. 2.5.2.4. Beer. In the case of beer samples, the process of clean-up was as following: about 50 ml of cool beer was thoroughly degassed in ultrasonic bath for 1 h in 500 ml Erlenmeyer flask. 10 ml of degassed beer was transferred to a screw top tube. Then, 0.1 ml of ammonium hydroxide solution (32%, v/v) was added to alkalinize the sample and hence to precipitate proteins and other matrix components. The mixture was shaken and let stand for 10 min. Then, 0.4 ml of a 25% aqueous solution of zinc acetate dihydrate was added to eliminate dyes without affecting trichothecene levels. Zn2þ excess was controlled with 0.4 ml of potassium hexacyanoferrate (II) (10%, w/v) which provided a voluminous precipitate of zinc hexacyanoferrate (II). The mixture was vigorously shaken for 1 min, centrifuged at 6840 g for 10 min and the supernatant was collected to another tube. The supernatant was loaded into the immunoaffinity columns and passed at a flow rate at oneetwo drops per second. The columns were washed with 5 ml of pure water. Then, it was eluted with 2 ml of methanol at a rate of about one drop per second in a 4 ml vial. The purified extract was concentrated to dryness at 45 C under a gentle stream of nitrogen and solved in 0.25 ml of initial LC mobile phase. 2.5.3. Trichothecene derivatization for GCeECD determination in samples of corn snacks, pasta and breakfast cereals One hundred microliters of a 2 mg/l solution of DMAP in tolueneeacetonitrile (80:20, v/v) and 50 ml of pentafluoropropionic anhydride (PFPA) were added to each dry extract in a screw cap vial. After capping tightly, the reaction mixture was heated at 60 C for 60 min in an aluminium heater block. After the mixture had cooled, 1 ml of a 3% (w/v) aqueous solution of NaHCO3 was added and the vial was vortexed for 15 s. The two layers were allowed to separate. The top (organic phase) layer was transferred to a GC autoinjector vial and analyzed by GCeECD as pointed out below. 2.5.4. Trichothecene derivatization for GCeMS determination in samples of bread One milliliter benzene was added to the evaporated extract and mixed for 1 min. The extract was evaporated once again to dryness and 500 ml of 0.4 M imidazole (as catalyst) in tolueneeacetonitrile (85 þ 15) and 100 ml PFPA were added to each derivatization vial
and mixed for 1 min. The reaction was left for derivatization at 60 C for 1 h and cooled afterwards for about 10 min and washed (to remove the excess reagent) by adding 500 ml hexane and 1 ml 5% sodium hydrogen carbonate solution. The derivatives were mixed carefully and the vials were opened carefully to reduce the pressure and mixed 1 min more. The aqueous phase was removed and 1 ml water was added and mixed 1 min. Then the aqueous phase was removed completely. A small amount of sodium sulfate was added to remove any water left. Finally, about 200 ml of the derivatized extract were transferred to a GC vial. 2.5.5. GCeECD analysis in the samples of corn snacks, pasta and breakfast cereals The GCeECD determination was carried out using the following chromatographic conditions. One microliter of solution was injected in splitless mode. The temperatures of the injection port and the detector were 250 and 300 C, respectively. The procedure used a fused silica capillary column HP-5 [5% methyl phenyl siloxane (30 m 0.32 mm, 0.25 mm film thickness, Agilent Technologies)]. The oven temperature program was: 90 C held for 1 min, 40 C/min to 160 C, 1.5 C/min to 182 C, 5 C/min to 240 C, and then 40 C/min to 275 C, held for 8 min. Helium at a constant pressure of 42.1 kPa was used as carrier gas. 2.5.6. GCeMS analysis in bread samples The GCeMS conditions were as follows: 1 ml of extract was injected in splitless mode. The temperature of the injection port was 250 C and the temperatures of the detector were 230 C (ion source) and 150 C (quadrupole). The oven temperature program was: 120 C held for 2 min, 30 C/min to 175 C (held for 3 min), 1 C/ min to 180 C and finally 25 C/min to 265 C (held for 15 min). Helium at a constant flow of 0.7 ml/min was used as carrier gas. The transfer line temperature was held at 260 C and selected ion monitoring at m/z 309 and 543 for DON, 377 and 555 for HT2 and 377, 451 for T2 were scanned for GCeMS analysis. 2.5.7. LCeDAD analysis in the samples of sweet corn, sliced bread and beer Fifty microliter dissolved extract were injected in the chromatographic system. The elution was carried out at a flow of 1 ml/ min with a mixture of acetonitrile (solvent A) and water (solvent B) applying the following gradient program: (i) initial solvent: 94% B, maintained for 6 min, (ii) lineal variation of B from 94% to 70% for 4 min, (iii) lineal variation of B from 70% to 50% for 12 min, (iv) 50% of B for 3 min, and (v) return to the initial conditions in 0.2 min. Separation was performed on a Zorbax Eclipse Plus C18 column (150 mm 4.6 mm, 3.5 mm particle size) connected to an Eclipse Plus C18 guard column (12.5 mm 4.6 mm, 3.5 mm particle size) (Agilent Technologies Inc, Santa Clara, USA) filled with the same phase. The column was kept at 35 C. Quantification of trichothecenes was performed by measuring its peak area with the help of a calibration curve calculated by regression from standard solutions. Detection wavelengths were 200 nm for HT2 and T2, and 220 nm for DON. The UV spectrum of compounds in the sample was compared with the standard UV spectrum for the mycotoxin identification. 2.6. Validation of analytical methods The analytical methods used for DON, HT2 and T2 were assessed for selectivity, linearity, and precision. Selectivity was checked by injecting three times 50 ml of mycotoxin standard solutions before injecting extracted samples and comparing the peak retention times and the UV spectra of the substances that produce these peaks.
G. Cano-Sancho et al. / Food Control 22 (2011) 490e495
493
Table 1 Method performance characteristics for DON, HT2 and T2. Sample ¼ 3
Pasta
Corn snacks
Sweet corn
Sliced bread
Beer
Corn flakes
Wheat flakes
Bread
Spiking level
a
HT2
LOQ
Recovery
RSDr
LOQ
(mg/g)
(mg/g)
(%)
(%)
0.05 0.2 1 0.05 0.2 1 0.05 0.2 1 0.05 0.2 1 0.05 0.2 1 0.05 0.2 1 0.05 0.2 1
0.042
143.1 78.1 77 105.2 91.6 77.8 110.5 89.3 94.7 109.6 109.9 86.4 110.2 101.5 93.6 95.6 99.8 70.9 89.3 84.5 85.8 80.3 76.6
19.2 17.9 17.9 17.4 16.5 8.9 16.8 5.1 12.3 10.0 12.2 5.7 10.0 6.1 5.4 19.8 7.6 8.1 14.0 11.9 18.4 5.1 6.4
a b
b
DON
0.066
0.027
0.030
0.012
0.045
0.041
0.018
T2 Recovery
RSDr
LOQ
Recovery
RSDr
(mg/g)
(%)
(%)
(mg/g)
(%)
(%)
0.030
113.2 99.3 88.4 112.7 93.9 82.4 88.8 74 83.4 98.9 107.1 104.8 125 121.7 73.9 129.7 87.3 79.8 109.5 93.6 86 88.2 87.0
12.3 11.2 18.9 24.2 10.1 14.7 6.7 14.9 9.2 8.2 6.6 10.3 16.9 7.7 14.4 26.7 5.2 10.5 17.5 5.5 8.2 5.9 3.9
0.063
90.9 80.9 86.2 115.6 94.7 92.4 e 69.8 101.1 e 90.0 94.4 e 90.3 83.3 119.6 102.8 78.3 66.6 96.5 85.5 95.0 90.7
7.8 24.7 13.5 8.8 21.5 11.7 e 4.0 5.2 e 14.0 8.6 e 5.8 5.9 14.4 18.9 7.9 19.7 6.0 3.1 4.6 5.4
0.061
0.024
0.030
0.009
0.030
0.036
0.050
0.054
0.135
0.108
0.057
0.057
0.042
0.070
Spiking level DON: 0.018 mg/g, HT2: 0.050 mg/g, T2: 0.070 mg/g. Spiking level DON: 0.473 mg/g, HT2: 0.437 mg/g, T2: 0.612 mg/g.
Linearity was assessed by performing triplicate injections of standard solutions whose concentrations were 0.02, 0.05, 0.1, 0.25, 0.5 and 1.0 mg each of mycotoxin/ml. Standard curves were generated by linear regression of peak areas against concentrations. Accuracy and recovery were established for each method by determination of DON, HT2 and T2 in samples of pasta, corn snack, sliced bread, beer, breakfast cereal and sweet corn, covering the range of the method (range between 0.05 and 1.0 mg/g). Recovery was determined by comparing the absolute responses of trichothecenes obtained from the studied samples with the absolute responses of calibration standards. The limit of detection (LOD) was considered as the mycotoxin concentration that provides a signal equal to b þ 3Sb, where b is the intercept of the calibration curve and Sb is the standard error of the estimate assuming to be the blank, and the limit of quantification (LOQ) was considered equal to 3LOD. Samples of pasta, corn snacks, sweet corn, sliced bread, beer, corn flakes and wheat flakes were spiked at levels of 1, 0.2 and 0.05 mg/g of DON, HT2 and T2 in triplicate. Concerning bread, these samples were spiked with each mycotoxin at LOQ level and the concentrations 0.473, 0.437 and 0.612 mg/g for DON, HT2 and T2, respectively. Analytical procedures were carried out three times. Calibration curves showed good linearity with correlations coefficients R2 ranging from 0.9799 to 0.9994. Percentage of recovery and relative standard deviation (RSDr) for each food matrix and spiking level are shown in Table 1. Recovery ranges between 77e143.1, 78.3e115.6 and 74e129.7% were found for DON, T2 and HT2, respectively, according to performance criteria established by Commission Regulation (EC) No 401/2006 (European Commission, 2006a), with the exception of recovery value of 143.1%, found in pasta samples spiked at 0.05 mg/ g. Maximum relative standard deviation (RSDr) levels for DON, T2 and HT2 were 19.8, 24.7 and 26.7%. Quantification limits of DON, T2 and HT2 toxin ranged from 0.012 mg/g (beer) to 0.066 mg/g (corn snacks), 0.042 mg/g (wheat flakes) to 0.135 mg/g (sweet corn) and 0.009 mg/g (beer) to 0.061 mg/g (corn snacks), respectively.
3. Results and discussion 3.1. Occurrence of DON In this study 479 “composite” samples pooled from 1147 individual samples from Catalonian market, were analyzed to detect and quantify DON, T2 and HT2. Previous studies conducted in Spain were carried out with fewer samples than the current work, in the order of 25 (Cerveró et al., 2007) and 175 (Castillo et al., 2008), thus, this work, represents the widest survey conducted in Spain to assess the occurrence of trichothecenes in foodstuffs for human consumption, to date. As shown in Table 2, the mycotoxin DON was found in all cerealbased food items analyzed in this study. The lowest percentage of positive samples (quantified) was found in beer (1.4%), sweet corn (2.8%) and sliced bread (16.7%). In contrast, high percentage of positive samples was found in breakfast cereals (74.1 and 73.4%), corn snacks (78.9%), pasta (74.3%) and specially in bread, where DON was found in all samples. Mean values of positive samples ranged from 0.012 mg/g, found in beer, to 0.246 0.158 mg/g found in bread. Despite the high percentage of positive samples in foodstuffs from Catalonian market, only two samples were above the
Table 2 Occurrence of DON in foodstuff from Catalonian market. N N Samples Mean sd individual composite > LOQ mg/g Wheat flakes Corn flakes Beer Sweet corn Corn snacks Pasta Sliced bread Bread
27 168 213 185 213 201 147 31
27 65 71 72 71 70 72 31
20/27 49/65 1/70 2/72 56/71 52/70 12/72 31/31
0.190 0.109 0.012 0.114 0.153 0.226 0.068 0.246
Median Max
mg/g
0.117 0.157 0.078 0.093 0.012 0.036 0.114 0.058 0.143 0.177 0.199 0.018 0.068 0.158 0.242
mg/g 0.437 0.580 0.012 0.139 0.304 0.946 0.098 0.739
494
G. Cano-Sancho et al. / Food Control 22 (2011) 490e495
Table 3 Occurrence of T2 and HT2 toxin in food from Catalonian market. N N Samples Mean sd individual composite > LOQ mg/g T2 Wheat flakes Corn flakes Beer Sweet corn Corn snacks Pasta Sliced bread Bread HT-2 Wheat flakes Corn flakes Beer Sweet corn Corn snacks Pasta Sliced bread Bread
27 156 216 185 213 201 147 31
27 65 71 72 71 70 72 31
2/27 0/65 0/71 2/72 1/71 0/70 0/72 0/31
27 156 216 185 213 201 147 31
27 65 71 72 71 70 72 31
4/27 4/65 0/71 5/72 6/71 7/70 11/72 0/31
Table 4 Co-occurrence of trichothecenes in foodstuff from Catalonian market. Median Max
mg/g
mg/g
0.072 0.005 0.072 e e e e 0.215 0.058 0.215 0.070 0.70 e e e e e e
0.075 e e 0.256 0.070 e e e
0.066 0.061 0.017 0.034 e 0.023 0.033 0.334 0.078 0.022 0.046 0.012 0.042 e
0.183 0.065 e 0.084 0.895 0.080 0.075 e
0.087 0.041 e 0.043 0.214 0.051 0.047 e
limit of 0.750 mg/g in pasta (0.946 mg/g), while one sample of corn flakes (0.580 mg/g) and two of bread (0.523 and 0.739 mg/g), were above the limit of 0.500 mg/g, established by European Commission (European Commission, 2006a). The previous studies, carried out to assess the occurrence of DON in foodstuff from Spanish market, commonly found lower levels of positive samples, mean and maximum values than our study. For example, in the earliest study the range of 0.038e0.195 mg/g was reported for corn flakes, 0.035e0.061 mg/g for sweet corn and 0.028e0.109 mg/g for fried corn snacks (Cerveró et al., 2007). Moreover, in other recent study, concentrations of 0.030e0.121 and 0.026e0.080 mg/g, were found in breakfast cereals and fried snacks, respectively (Castillo et al., 2008). However, we found a wide range of contamination for these foodstuffs, with maximum values of 0.580, 0.139 and 0.304 mg/g in corn flakes, sweet corn and corn snacks, respectively. In the line with our results, there is a study conducted in Canada with breakfast cereals where the authors showed a maximum level and mean of positive samples of 0.420 and 0.070 mg/g, respectively (Roscoe et al., 2008), while in Italy, a lowest range, between 0.012 and 0.047 mg/g was found (Cirillo, Ritieni, Galvano, & Cocchieri, 2003). Highest levels were stated in other study performed in Portugal, where the range 0.103e6.040 mg/g and the mean 0.754 mg/g were found in wheat-based breakfast cereals (Martins & Martins, 2001). Regarding baked products, in the The First French Total Diet Study the mean of the positive samples of bread and rusk was estimated to be 0.109 mg/g (Leblanc, Tard, Volatier, & Verger, 2005). While in a German study, the authors reported a high percentage of positive samples in conventional and organic bread, with ranges of 0.015e0.690 mg/g and 0.015e0.224 mg/g, and means of 0.184 and 0.062 mg/g, respectively (Schollenberger et al., 2005). In Italy, the level of DON in bread and related food was observed between 0.007 and 0.270 mg/g (Cirillo et al., 2003), all these values being much lower than our range of 0.027e0.739 mg/g. Few data is available about the levels of DON in pasta. This mycotoxin was not detected in France (Leblanc et al., 2005) and the levels ranged from 0.009 to 0.077 mg/g in Italy (Cirillo et al., 2003), while, the level in noodles from Germany was between 0.015 and 1.670 mg/g, with a mean of 0.158 0.334 mg/g (Schollenberger, Suchy, Jara, Drochner, & Müller, 1999), closest to our study with a respective mean and maximum value of 0.226 and 0.946 mg/g. Concerning beer, to our knowledge, few studies have been conducted to assess the level of DON in this alcoholic beverage from
Wheat flakes Corn flakes Beer Pan loaf Sweet-corn Corn snacks Pasta Bread Total
DON-T2
DON-HT2
T2-HT2
DON-T2-HT2
2 1 0 1 0 2 2 0 8
8 17 0 4 0 20 16 0 55
0 0 0 0 3 0 0 0 3
4 4 0 0 0 4 12 0 24
Note: Co-occurrence was determined considering samples above LOD, and expressed as the number of samples with simultaneous contamination the mycotoxins.
European markets. In an early study that reported the occurrence of 176 beer samples collected in European and North American markets, the authors found that 64% of samples were above the LOQ of 0.003 mg/g, and the mean ranged from 0.002 to 0.011 mg/g; the maximum value was 0.036 mg/g (Kostelanska et al., 2009). The higher occurrence in this previous study could be explained by the lower LOQ of this study (0.012 mg/g). 3.2. Occurrence of T2 and HT2 toxin Percentage of positive samples of T2 and HT2 toxin and contamination levels in food marketed in Catalonia, are shown in Table 3. Low percentage of positive samples was found for these type-A trichothecenes, specially for T2, which was quantified only in 5 samples of 479 (wheat flakes, sweet corn and corn flakes). HT2 was present in a low percentage of sliced bread samples (15.3%), wheat flakes (14.8%), pasta (10%), corn snacks (8.4%), sweet corn (6.9%) and corn flakes (6.2%). Mean levels of HT2 in positive samples were between 0.041 0.017 mg/g (corn flakes) and 0.214 0.334 mg/g (corn snacks), being the maximum level 0.895 mg/g in a corn snack sample. In a previous study conducted in Spain, the authors did not find T2 in any sample out of the 25 samples analyzed, above the LOD of 0.030 mg/g neither in corn flakes, sweet corn nor in corn snacks (Cerveró et al., 2007). Leblanc et al. (2005) quantified HT2 in one sample out of 238 composite samples with a level of 0.270 mg/g. In Germany, HT2 was the main type-A trichothecene found in cereal-based food, with ranges of 0.012e0.032 mg/g in bread and related products, 0.012e0.025 mg/g in noodles and 0.012e0.022 mg/ g in breakfast cereals, slightly lower values than our results (Schollenberger et al., 1999). 3.3. Co-occurrence of trichothecenes in the samples Several studies have suggested that the mycotoxins could have synergistic effect in vivo (Bacon, Porter, Norred, & Leslie, 1996; Javed et al., 1993), therefore the knowledge of the occurrence of all mycotoxins should be considered a challenge in exposure assessment studies. In our work, the co-occurrence of different mycotoxins in the same sample, was studied considering positive those samples above LOD (Table 4). The results suggest that the most common case of co-occurrence with trichothecenes can be drawn by the simultaneous occurrence of DON and HT2 toxin, specially in corn snacks, corn flakes and pasta, as was elucidated previously by Schollenberger et al. (1999). 4. Conclusions With 479 analytical samples, this is the widest study to assess the occurrence of trichothecenes in food for human consumption
G. Cano-Sancho et al. / Food Control 22 (2011) 490e495
until now in Spain. We have found that the occurrence of DON was high in cereal-based foodstuff from Catalonian market, specially in wheat flakes, corn flakes, corn snacks, pasta and bread. Despite the high percentage of positive samples in foodstuff from Catalonian market, only five samples were above the limit established by European Commission (European Commission, 2006a). In the other hand, T2 toxin was only quantified in 5 samples. HT2 was present in a low percentage of the samples and it was not detected in samples of bread and beer. Although no worrying values have been found in this study for DON, T2 and HT2 with samples collected in 2008, the annual variability of the levels of trichothecenes in the crops makes necessary the monitoring of these mycotoxins.
Acknowledgements The authors would like to acknowledge Exposure Assessment of Spanish Population to Fusarium Toxins Project, National Plan of Spanish Government (AGL2008-05030-C02-01), Catalonian Food Safety Agency of ‘Generalitat de Catalunya’ Health Department and University of Lleida for their financial support. Also, the authors thank to M. J. Nogueiras and A. Dieguez for their technical assistance in the analysis carried out in the National Centre for Food.
References Bacon, C. W., Porter, J. K., Norred, W. P., & Leslie, J. F. (1996). Production of fusaric acid by Fusarium species. Applied and Environmental Microbiology, 62(11), 4039e4043. Castillo, M., Montes, R., Navarro, A., Segarra, R., Cuesta, G., & Hernández, E. (2008). Occurrence of deoxynivalenol and nivalenol in Spanish corn-based food products. Journal of Food Composition and Analysis, 21(5), 423e427. Cerveró, M. C., Castillo, M. A., Montes, R., & Hernández, E. (2007). Determination of trichothecenes, zearalenone and zearalenols in commercially available cornbased foods in Spain. Revista Iberoamericana de Micología, 24(1), 52e55. Cirillo, T., Ritieni, A., Galvano, F., & Cocchieri, R. A. (2003). Natural co-occurrence of deoxynivalenol and fumonisins B-1 and B-2 in Italian marketed foodstuffs. Food Additives and Contaminants Part A-Chemistry Analysis Control Exposure & Risk Assessment, 20(6), 566e571. Eriksen, G. S., & Pettersson, H. (2004). Toxicological evaluation of trichothecenes in animal feed. Animal Feed Science and Technology, 114(1e4), 205e239. European Commission. (2006a). Commission Regulation (EC) No. 401/2006 of 23 February 2006. Laying down the methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs. Official Journal of the European Union L, 70, 12e34.
495
European Commission. (2006b). Commission Regulation (EC) No. 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. Official Journal of the European Union L, 364, 5e24. Hazel, C. M., & Patel, S. (2004). Influence of processing on trichothecene levels. Toxicology Letters, 153(1), 51e59. Javed, T., Bennett, G. A., Richard, J. L., Dombrink-Kurtzman, M. A., Côté, L. M., & Buck, W. B. (1993). Mortality in broiler chicks on feed amended with Fusarium proliferatum culture material or with purified fumonisin B1 and moniliformin. Mycopathologia, 123(3), 171e184. Jelinek, C. F., Pohland, A. E., & Wood, G. E. (1989). Worldwide occurrence of mycotoxins in foods and feeds-an update. Journal of the Association of Official Analytical Chemists, 72(2), 223e230. Joint FAO/WHO Expert Committee on Food Additives (JECFA). (2001). Safety evaluation of certain mycotoxins in food. Rome, Italy: Food and Agriculture Organization. Kostelanska, M., Hajslova, J., Zachariasova, M., Malachova, A., Kalachova, K., Poustka, J., et al. (2009). Occurrence of deoxynivalenol and its major conjugate, deoxynivalenol-3-glucoside, in beer and some brewing intermediates. Journal of Agricultural and Food Chemistry, 57(8), 3187e3194. Leblanc, J., Tard, A., Volatier, J., & Verger, P. (2005). Estimated dietary exposure to principal food mycotoxins from The First French Total Diet Study. Food Additives and Contaminants, 22(7), 652e672. Martins, M. L., & Martins, H. M. (2001). Determination of deoxynivalenol in wheatbased breakfast cereals marketed in Portugal. Journal of Food Protection, 64(11), 1848e1850. Roscoe, V., Lombaert, G. A., Huzel, V., Neumann, G., Melietio, J., Kitchen, D., et al. (2008). Mycotoxins in breakfast cereals from the Canadian retail market: a 3-year survey. Food Additives and Contaminants Part A, 25(3), 347e355. Rotter, B. A., Prelusky, D. B., & Pestka, J. J. (1996). Toxicology of deoxynivalenol (vomitoxin). Journal of Toxicology and Environmental Health - Part A, 48(1), 1e34. SCF. (2002). Opinion on Fusarium TOXINSdPart 6: Group evaluation of T-2 toxin, HT-2 toxin, nivalenol and deoxynivalenol (adopted on 26 February 2002). Available in: http://europa.eu.int/comm/food/fs/sc/scf/out123 en.pdf Accessed 15.02.10. Schollenberger, M., Müller, H., Rüfle, M., Suchy, S., Planck, S., & Drochner, W. (2005). Survey of Fusarium toxins in foodstuffs of plant origin marketed in Germany. International Journal of Food Microbiology, 97(3), 317e326. Schollenberger, M., Suchy, S., Jara, H. T., Drochner, W., & Müller, H. (1999). A survey of Fusarium toxins in cereal-based foods marketed in an area of southwest Germany. Mycopathologia, 147(1), 49e57. Scott, P. M. (1989). The natural occurrence of trichothecenes. In V. R. Beasley (Ed.), Trichothecene mycotoxicosis: Pathophysiologic effects (pp. 1e26). Boca Raton: CRC Press. Serra-Majem, L., Ribas, L., Salvador, G., Castells, C., Serra, J., & Jover, L. (2003). caAvaluació de l’estat nutricional de la població catalana 2002e2003. Evolució dels hàbits alimentaris i del consum d’aliments i nutrients a Catalunya (1992e2003). Barcelona: Direcció General de Salut Pública, Departament de Sanitat i Seguretat Social, Generalitat de Catalunya. Sudakin, D. L. (2003). Trichothecenes in the environment: relevance to human health. Toxicology Letters, 143(2), 97e107. Visconti, A. (2001). Problems associated with Fusarium mycotoxins in cereals. Bulletin of the Institute for Comprehensive Agricultural Sciences Kinki University, 9, 39e55. Visconti, A., Minervini, F., Lucivero, G., & Gambatesa, V. (1991). Cytotoxic and immunotoxic effects of Fusarium mycotoxins using a rapid colorimetric bioassay. Mycopathologia, 113(3), 181e186.