DETERMINATION OF MYCOTOXINE DEOXYNIVALENOL IN WHEAT FLOUR AND WHEAT BRAN

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1 Revue de Cytologie et Biologie végétales-le Botaniste (s.i.): DETERMINATION OF MYCOTOXINE DEOXYNIVALENOL IN WHEAT FLOUR AND WHEAT BRAN by F. ISRAEL-ROMING (1) M. AVRAM (2) V. GAGIU (2) G. CAMPEANU (1) (1) Center of Research for Applied Biochemistry an Biotechnology, Department of Biotechnologies, 59 Marasti Boulevard, Sector 1, Bucharest, Romania; (2) Institute of Bioresources, 6 Dinu Vintila Street, Sector 2, Bucharest, Romania; Corresponding author: Israel-Roming Florentina, 59 Marasti Boulevard, Sector 1, Bucharest, Romania. tel/fax: , Abstract Deoxynivalenol (DON), also known as vomitoxin, belongs to trichotecenes family and is produced by several species of genus Fusarium. The present study was carried out to find a method for this mycotoxin determination and to evaluate the natural occurrence of DON in wheat flour and wheat bran from southern part of Romania. 25 grams of sample were extracted in distilled water, filtered and then centrifuged at 4000 rpm for 10 minutes. The samples were cleaned-up using DONprep immunoaffinity columns. DON was eluted with methanol, injected in HPLC system and than chromatographed on a reversephase C18 column with water-acetonitrile-methanol (90:5:5 v/v) as mobile phase. Quantification was made using an UV detector at 218 nm. As control we used certified reference material with g/g DON. The assay shoved a high degree of linearity (r 2 = ). The recoveries for the assay were generally higher than 80%. The detection limit was 0.02 g/g DON. Key words Deoxynivalenol (DON), mycotoxin, HPLC, wheat flour, wheat bran 1.- Introduction The mycotoxin deoxynivalenol (DON) is a metabolite produced by several Fusarium genus. Also known as vomitoxin, DON belongs to trichotecenes chemical family: sesquiterpenes with 12, 13-epoxy group. Recent studies demonstrated that DON is commonly found at high levels in cereals intended for human and animal consumption (CAHILL et al. 1999). In addition many studies have shown substantial adverse effects of DON on animal and human health. Even at low levels, deoxynivalenol may cause feeding refuse and at higher levels induce vomiting. Regarding these concerns about DON it is more and more important to determine the level of this mycotoxin in food and feeds. DON determination must take into account the sampling from large quantities, the extraction of the mycotoxin, the cleaning-up and the final analysis. Regarding extraction, this was made in water (CAHILL et al., 1999; MATEO et al., 2001; MARTINS and MARTINS, 2001; KOTAL and RADOVA, 2002), methanolwater in different proportions (50:50, 70:30) [MATEO et al., 2001] and aconitrile:water (84:16) (RUPP, 2002, FAZEHAS et al., 2000; TRUCKNESS et al., 1998). Cleaning-up procedure is performed with: SPE columns (florisil and activated charcoal) (FAZEHAS et al., 2000; TRUCKNESS et al.,1998), MycoSep multifunctional columns (MATEO et al,. 2001; RUPP, 2002) and immunoaffinity columns (KOTAL and RADOVA, 2002; MATEO et al., 2002). DON separation and detection is realised by HPLC and GS methods (MATEO et al., 2001; BETINA, 1989). This paper describes the method developed for DON determination in wheat using immunoaffinity columns for cleaning-up, HPLC for separation and UV for detection. In order to demonstrate the trueness of the method a certified reference material was used. Recovery experiments were realised with spiked samples with 0.1 and 0.3 g/g DON. Finally several samples of wheat flour and wheat bran were analyzed according to the described method.

2 Materials and methods Chemicals and reagents The deoxynivalenol standard was supplied by Sigma Aldrich. Stock solution of DON was prepared in acetonitrile with concentration 1 mg/ml and stored at 20 o C. The working standard solution was freshly prepared in mobile phase, ranging from 1 μg/ml to 10 μg/ml. The solvents (methanol and acetonitrile) were HPLC grade and were supplied by Merck. Ultrapure water was obtained using Simplicity 185 system from Millipore, DONprep immunoaffinity column were supplied by R-Biopharm Rhone. Certified reference material (wheat flour containing DON 0.7±0.2 μg/g) was supplied by R- Biopharm Rhone. Blank cereal samples (wheat flour) and contaminated wheat samples (wheat flour and wheat bran) were obtained from Institute of Bioresources (Bucharest, Romania). Apparatus The Waters 2690 Alliance liquid chromatography system was used with RP 4.6x150 mm, 5.0 μm spherical particle, C 18 Spherisorb column. Detection was made using Waters 2487 UV detector. Data were analysed and processed with Millennium 32 software. Analytical procedure 1. Extraction procedure: 25 g of sample were extracted with 200 ml distilled water by shaking at 200 rpm for 30 minutes. Extracts were filtered through 113 Watman filter paper and then centrifuged at 4000 rpm. 2. Clean-up procedure: 2 ml of extract was applied on a DONprep column. The column was washed with 5 ml ultrapure water and passing 1.5 ml methanol eluted the mycotoxin. The eluate was dried and redissolved in 1 ml mobile phase. 3. HPLC chromatography: the sample is placed in the auto sampler and then injected in the separation system of the HPLC. The used mobile phase consisted of acetonitrile:methanol:water solution (5:5:90, v/v). The elution was performed with 1 ml/min flow rate for 10 minutes. Detection of DON was made at a wavelength of 218 nm. 3.- Results and discussion Calibration curve For realising the calibration curve there were made three replicates injections of five standard levels of DON: μg, 0.01 μg, μg, μg and 0.05 μg. These levels were obtained by injection of different volumes of the diluted standard solution (0.001 μg/μl). The calibration curve obtained by plotting peak areas as a function of amount of DON injected (fig. 1) showed that under the proposed HPLC conditions and within the working range studied deviations from linearity are minimal (r 2 = ) (table 1). The limit of detection LOD (signal-to-noise=3) was 20 ng/g and the limit of quantification LOQ was 60 ng/g. Fig.1. Calibration curve

3 57 Determination of DON using certified reference material The trueness of the method was assessed by analysing a certified reference material (fig. 2). Five replicates were analysed according to the described method. Table 1. Results of the determinations for the calibration levels All results are:summarized in the attached table 2. The mean amount of the five determinations was μg in 100 μl injected that corresponds to 0.73 μg/g ± 0.02 μg/g. The certified value was 0.7±0.2 μg/g. The average of the relative standard deviation (RSD) was 0.13% for the retention time and 2.6% for the amount of DON. Fig. 2.- Overlay chromatograms of five replicates of the certified reference material

4 58 Table 2. Results of the determination of DON from certified reference material Recovery tests using spiked samples Recovery was determined using wheat samples experimentally contaminated with DON at two levels: 0.1 and 0.3 μg/g (fig. 3 and fig. 4). The three replicates made for 0.1 μg/g level conducted to a mean of μg/100 μl (table 3) that corresponds to μg/g. In this case the recovery was 98%. The average of the relative standard deviation (RSD) was 0.07% for the retention time and 10.38% for the amount of DON. Fig. 3.- Overlay chromatogram of three replicates of spiked sample 0.1 μg/g DON

5 59 Table 3. Results of the determination of DON from spiked samples 0.1 g/g The three replicates made for 0.3 μg/g level conducted to a mean of μg/100 μl (table 4) that corresponds to 0.26 μg/g. In this case the recovery was 87%, lower than the first level. The average of the relative standard deviation (RSD) was 0.05% for the retention time and 1.08 % for the amount of DON. Fig. 4.- Overlay chromatogram of three replicates of spiked sample 0.3 μg/g DON Determination of DON in naturally contaminated samples Before analysing by HPLC method, the samples of wheat flour and wheat bran were tested with ELISA kits. From 22 samples of wheat flour and 17 samples of wheat bran we selected only four from each group, the rest of them showing no activity at the screening test. One of the reason the results obtained using ELISA test kits were so pour is that all the samples were harvested in 2004 and the specialists didn t report any considerable attack of Fusarium sp.

6 60 Table 4. Results of the determination of DON from spiked samples 0.3 g/g Table 5. Results of the determination of DON in naturally contaminated samples Wheat flour Wheat bran Sample DON (μg/g) Sample DON (μg/g) F T F T F T F T The results of the DONprep-HPLC determinations are presented in table 5. All the concentrations obtained for wheat flour are very small, lower than LOQ but higher than LOD. The values obtained for wheat bran are higher, probably because the contamination is made at the surface of the kernels. 4.- Conclusion The DONprep-HPLC method described provides a precise instrument for determination of deoxynivalenol in cereals provides. The calibration curve is linear for the studied range, the linearity coefficient being The limit of detection is 0.02 μg/g and the limit of quantification is 0.06 μg/g. The accuracy of the method was studied using certified reference material and spiked samples. The recoveries were higher than 80%. Several samples of wheat flour and wheat bran were analysed according to the method. The values obtained for wheat bran were higher than those obtained for wheat flour, probably because of the localisation of DON in outer layers of the kernels. Acknowledgements Financial support from the Romanian Ministry of Education and Research (project BIOTECH 3366/2003) is acknowledged. 6.- References BETINA (V.), Chromatographic methods as tools in the field of mycotoxin. J. Chromatogr. 477 (2): CAHILL (L.M.), KRUGER (S.C.), MCALICE (B.T.), RAMSEY (C.S.), PRIOLI (R.), KHON (B.), Quantification of deoxynivalenol in wheat using immunoaffinity column and liquid chromatography. J. Cromatogr. 859: FAZEKAS (B.), HAJDU (E.T.), TAR (A.K.), TANYI (J.), Natural deoxynivalenol (DON) contaminants of wheat sam grown in 1998 as determined by HPLC. Acta Vet. Hung, 48 (2): KOTAL (F.), RADOVA (Z.), 2002, A simple method for determination of deoxynivalenol in cereals and flours. Czech. J. Food Sci. 20 (2): MARTINS (M.L.), MARTINS (H.M.), Determination of deoxynivalenol in wheat-based breakfast cereals marketed in Portugal. J. of Food Protection, 64: MATEO (J.J.), LLORENS (A.), MATEO (R.), JIMENEZ (M.), Critical study of and improvements in chromatographic methods for analysis of type B trichotecenes. J. Cromatogr. A 918: MATEO (J.J.), MATEO (R.), HINJO (M.J.), JIMENEZ (M.), Liquid chromatographic determination of toxigenic secondary metabolites produced by Fusarium strains. J. Chromatogr. A, 955 (2): RUPP (H.), 2002, Determination of deoxynivalenol in whole wheat flour and wheat bran. J. AOAC, 85 (6): TRUCKNESS (M.W.), PAGE (S.W.), WOOD (G.E.), CHO (T.H.), Determination of deoxynivalenol in white flour, whole wheat flour, and bran by solid-phase extraction/liquid chromatography: interlaboratory study. J. AOAC, 81(4):

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