T-2 toxin (T-2) and HT-2 toxin (HT-2) are type A trichothecene mycotoxins produced by several Fusarium species, mainly Fusarium sporotrichioides, Fusarium langsethiae and Fusarium poae. Generally, these Fusarium species can grow on cereals and produce T-2 and HT-2 under moist cool conditions already prior to harvesting [1]. Among cereals, oats, wheat, rye and derived products are key dietary sources of T-2 and HT-2 exposure [1]. In order to protect consumer health from the risk of exposure to these toxins, the development of rapid, sensitive and reliable methods for their determination in cereals and cereal-based products is needed. Fluorescence polarization (FP) immunoassay is a homogeneous technique that is getting attention as a screening tool in food safety control due to its simplicity, rapidity, cheapness and reliability. A rapid, sensitive and reliable FP immunoassay has been recently reported for the determination of the sum of T-2 and HT- 2 toxins in wheat [2]. The aim of present work was to evaluate the applicability of the above mentioned FP immunoassay to other unprocessed cereals, such as rye, and cereal-based products, such as oats crispbread, for the quantitative determination of the total content of T-2 and HT-2. The concept of determining the total content of T-2 and HT-2 in cereal samples for both official control purposes and risk assessment studies results in line with the EC Recommendation [3]. Ground samples (25 g) were weighed into a blender jar, added with 2.5 g of NaCl and extracted with 100 mL methanol/water 90:10 (v/v) by blending at high speed for 3 min. The extract was filtered through filter paper. No purification step of extracts was required, although in order to reduce the matrix effect a dilution step with NaCl solution (4 % for rye and 1% for oats crispbread), in a ratio 1:5 (v/v), was necessary to let precipitation of proteins and matrix interfering compounds. The diluted extract was then filtered through a glass microfibre filter and analysed by FP immunoassay. The evaluation of matrix effect in the FP measurements was performed, by comparing the regression line obtained with T-2/ HT-2 standard solutions in the range 0.5-7.5 ng/mL with the regression lines obtained by adding spiked diluted extracts of uncontaminated rye and oats crispbread at 5, 10 and 12 mg of analyzed matrix equivalent by means of parallelism and position statistical tests. No significant differences were observed between slopes (tcalc<2.306; p<0.05) and positions (tcalc<2.262; p<0.05) of the regression lines obtained with T-2/HT-2 standard solutions in buffer and those obtained in the presence of spiked diluted extracts by using amounts of matrix equivalent up to 10 mg for both matrices. In addition for the optimized FP immunoassay, LOD of 0.20 ng/mL (equivalent to 20 ?g/kg in rye and oats crispbread) was calculated. Even though different diluting conditions were required for the tested matrices, LOD obtained in the experimental conditions was lower than the indicative levels suggested by the European Commission for the total content of T-2 and HT-2 toxins in unprocessed rye and cereal-based products. Recovery experiments were performed for rye and oats crispbread in the range 100-400 ?g/kg (sum of toxins). Overall mean recoveries of the optimized FP immunoassay were 105 and 107% for rye and oats crispbread, respectively, with relative standard deviations lower than 4 %, whereas overall mean recoveries for the UHPLC method (after immunoaffinity cleanup) were 94 and 97%, respectively, with relative standard deviations lower than 3 %. The analytical performances of the optimized FP immunoassay in terms of accuracy and precision fulfill the criteria established by the European Commission for the acceptability of analytical method for T-2 and HT-2 determination [4]. In addition, a comparative analysis of the levels of contamination in spiked and blank samples was performed by both FP immunoassay and UHPLC method. In particular, a total of 30 rye and oats crispbread samples, of which 20 spiked samples at levels from 50 to 700 ?g/kg and 10 uncontaminated samples, were analyzed. Good correlations (r=0.998 for rye and r=0.994 for oats crispbread) were found between T-2 and HT-2 concentrations obtained by FP immunoassay and those obtained by the UHPLC method. The linear regression fit was of the form [T-2 +HT-2 by FP] =2.86+1.03 [T-2 + HT-2 by UHPLC] for rye and [T-2 + HT-2 by FP]= 1.51+ 0.90 [T-2 + HT-2 by UHPLC] for oats crispbread, where all data were previously corrected for average recoveries. No false positive result was observed by FP immunoassay for uncontaminated samples. This comparative analysis confirmed the good accuracy of the optimized FP immunoassay. The proposed method coupled performances in terms of sensitivity, accuracy and precision comparable to those of a chromatographic technique with rapidity (20 min), costs and simplicity typical of a high-throughput screening method and can be used as a valid alternative to more expensive and time-consuming LC methods for quantitative determination of T-2 and HT-2 in rye and oats crispbread [5]. ACKNOWLEDGMENTS This work has been supported by the Italian Ministry of Education, University and Research (MIUR) project no. CTN01_00230 CL.A.N. Cluster Tecnologici Nazionali - SAFE&SMART project "New enabling technologies for food safety and food chain integrity within a global scenario". REFERENCES [1] EFSA Panel on Contaminants in the Food Chain Scientific opinion on the risks for animal and public health related to the presence of T-2 and HT-2 toxin in food and feed, vol. 12, pp. 2481-2668, 2011 (available online: www.efsa.europa.eu/ efsajournal). [2] Lippolis V, Pascale M, Valenzano S, Pluchinotta V, Baumgartner S, Krska R, Visconti A, "A rapid fluorescence polarization immunoassay for the determination of T-2 and HT-2 toxins in wheat", Analytical and Bioanalytical Chemistry, vol. 401, pp. 2561-2571, Sept. 2011. [3] European Commission, "Commission recommendation 165/2013/ UE of 27 March 2013 on the presence of T-2 and HT-2 toxin in cereals and cereal products", Official Journal of the European Union L91:12-15, March 2013. [4] European Commission, "Commission Regulation 401/2006/EC 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, L70:12-34, Feb. 2006. [5] Porricelli ACR, Lippolis V, Valenzano S, Cortese M, Suman M, Zanardi S, Pascale M, "Optimization and validation of a fluorescence polarization immunoassay for rapid detection of T-2 and HT-2 toxins in cereals and cereal-based products", Food Analytical Methods, DOI 10.1007/s12161-016-0527-1.
Fluorescence polarization immunoassay for rapid detection of T-2 and HT-2 toxins in cereals and cereal-based products
Vincenzo Lippolis;Stefania Valenzano;Marina Cortese;Michelangelo Pascale
2016
Abstract
T-2 toxin (T-2) and HT-2 toxin (HT-2) are type A trichothecene mycotoxins produced by several Fusarium species, mainly Fusarium sporotrichioides, Fusarium langsethiae and Fusarium poae. Generally, these Fusarium species can grow on cereals and produce T-2 and HT-2 under moist cool conditions already prior to harvesting [1]. Among cereals, oats, wheat, rye and derived products are key dietary sources of T-2 and HT-2 exposure [1]. In order to protect consumer health from the risk of exposure to these toxins, the development of rapid, sensitive and reliable methods for their determination in cereals and cereal-based products is needed. Fluorescence polarization (FP) immunoassay is a homogeneous technique that is getting attention as a screening tool in food safety control due to its simplicity, rapidity, cheapness and reliability. A rapid, sensitive and reliable FP immunoassay has been recently reported for the determination of the sum of T-2 and HT- 2 toxins in wheat [2]. The aim of present work was to evaluate the applicability of the above mentioned FP immunoassay to other unprocessed cereals, such as rye, and cereal-based products, such as oats crispbread, for the quantitative determination of the total content of T-2 and HT-2. The concept of determining the total content of T-2 and HT-2 in cereal samples for both official control purposes and risk assessment studies results in line with the EC Recommendation [3]. Ground samples (25 g) were weighed into a blender jar, added with 2.5 g of NaCl and extracted with 100 mL methanol/water 90:10 (v/v) by blending at high speed for 3 min. The extract was filtered through filter paper. No purification step of extracts was required, although in order to reduce the matrix effect a dilution step with NaCl solution (4 % for rye and 1% for oats crispbread), in a ratio 1:5 (v/v), was necessary to let precipitation of proteins and matrix interfering compounds. The diluted extract was then filtered through a glass microfibre filter and analysed by FP immunoassay. The evaluation of matrix effect in the FP measurements was performed, by comparing the regression line obtained with T-2/ HT-2 standard solutions in the range 0.5-7.5 ng/mL with the regression lines obtained by adding spiked diluted extracts of uncontaminated rye and oats crispbread at 5, 10 and 12 mg of analyzed matrix equivalent by means of parallelism and position statistical tests. No significant differences were observed between slopes (tcalc<2.306; p<0.05) and positions (tcalc<2.262; p<0.05) of the regression lines obtained with T-2/HT-2 standard solutions in buffer and those obtained in the presence of spiked diluted extracts by using amounts of matrix equivalent up to 10 mg for both matrices. In addition for the optimized FP immunoassay, LOD of 0.20 ng/mL (equivalent to 20 ?g/kg in rye and oats crispbread) was calculated. Even though different diluting conditions were required for the tested matrices, LOD obtained in the experimental conditions was lower than the indicative levels suggested by the European Commission for the total content of T-2 and HT-2 toxins in unprocessed rye and cereal-based products. Recovery experiments were performed for rye and oats crispbread in the range 100-400 ?g/kg (sum of toxins). Overall mean recoveries of the optimized FP immunoassay were 105 and 107% for rye and oats crispbread, respectively, with relative standard deviations lower than 4 %, whereas overall mean recoveries for the UHPLC method (after immunoaffinity cleanup) were 94 and 97%, respectively, with relative standard deviations lower than 3 %. The analytical performances of the optimized FP immunoassay in terms of accuracy and precision fulfill the criteria established by the European Commission for the acceptability of analytical method for T-2 and HT-2 determination [4]. In addition, a comparative analysis of the levels of contamination in spiked and blank samples was performed by both FP immunoassay and UHPLC method. In particular, a total of 30 rye and oats crispbread samples, of which 20 spiked samples at levels from 50 to 700 ?g/kg and 10 uncontaminated samples, were analyzed. Good correlations (r=0.998 for rye and r=0.994 for oats crispbread) were found between T-2 and HT-2 concentrations obtained by FP immunoassay and those obtained by the UHPLC method. The linear regression fit was of the form [T-2 +HT-2 by FP] =2.86+1.03 [T-2 + HT-2 by UHPLC] for rye and [T-2 + HT-2 by FP]= 1.51+ 0.90 [T-2 + HT-2 by UHPLC] for oats crispbread, where all data were previously corrected for average recoveries. No false positive result was observed by FP immunoassay for uncontaminated samples. This comparative analysis confirmed the good accuracy of the optimized FP immunoassay. The proposed method coupled performances in terms of sensitivity, accuracy and precision comparable to those of a chromatographic technique with rapidity (20 min), costs and simplicity typical of a high-throughput screening method and can be used as a valid alternative to more expensive and time-consuming LC methods for quantitative determination of T-2 and HT-2 in rye and oats crispbread [5]. ACKNOWLEDGMENTS This work has been supported by the Italian Ministry of Education, University and Research (MIUR) project no. CTN01_00230 CL.A.N. Cluster Tecnologici Nazionali - SAFE&SMART project "New enabling technologies for food safety and food chain integrity within a global scenario". REFERENCES [1] EFSA Panel on Contaminants in the Food Chain Scientific opinion on the risks for animal and public health related to the presence of T-2 and HT-2 toxin in food and feed, vol. 12, pp. 2481-2668, 2011 (available online: www.efsa.europa.eu/ efsajournal). [2] Lippolis V, Pascale M, Valenzano S, Pluchinotta V, Baumgartner S, Krska R, Visconti A, "A rapid fluorescence polarization immunoassay for the determination of T-2 and HT-2 toxins in wheat", Analytical and Bioanalytical Chemistry, vol. 401, pp. 2561-2571, Sept. 2011. [3] European Commission, "Commission recommendation 165/2013/ UE of 27 March 2013 on the presence of T-2 and HT-2 toxin in cereals and cereal products", Official Journal of the European Union L91:12-15, March 2013. [4] European Commission, "Commission Regulation 401/2006/EC 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, L70:12-34, Feb. 2006. [5] Porricelli ACR, Lippolis V, Valenzano S, Cortese M, Suman M, Zanardi S, Pascale M, "Optimization and validation of a fluorescence polarization immunoassay for rapid detection of T-2 and HT-2 toxins in cereals and cereal-based products", Food Analytical Methods, DOI 10.1007/s12161-016-0527-1.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
