In the present work a high performance liquid-chromatography UV/Vis method was developed to quantify gliadins in foods. Gliadins are prolamins and are one of the constituents of wheat gluten (approximately 50%). These compounds have particular importance in food analysis since they are responsible for the celiac disease, which is an intolerance ar hypersensitivity to ingested prolamins.info:eu-repo/semantics/publishedVersio

Dias, L. G.

Machado, A. A. S. C.

Meirinho, Sofia G.

Morais, Jorge Sá

Peres, António M.

Veloso, Ana C. A.

English

Universidade do Minho: RepositoriUM

6° Encontro Nacional de Cromatografia Universidade da Madeira 2009 - ~. - -.,;::::::: --- --·- ............. UNMRSID.-\Dl da MADURA 6° encontro nacional de cromatografia comun\cay6es em poster - P.60 P.60 LIQUID CHROMATOGRAPHY ASSAY FOR GLIADINS QUANTIFICATION: APPLICATION TO GLUTEN-CONTAINING AND "GLUTEN-FREE" FOOD PRODUCTS J. Sa Morais' . S. Meirinho', A. M. Peres' ·', A. C. Veloso'·', L. G. Dias ' , A. A. S. C. Machado5 1 CIMO- Mountain Research Centre, Escola Superior Agraria de Braganga, Braganga, Portugal 2 LSRE- Laboratory of Separation and Reaction Eng ineering, Escola Superior Agraria, Braganga, Portugal 3 Departamento de Engenharia Qui mica e Biol6gica, lnstituto Superior de Engenharia de Coimbra, Coimbra, Portugal 4 IBB - Center of Biological Engineering, Univerdidade do Minho, Braga, Portugal 5 LAOUIPAI - Departamento de Ouimica, Faculdade de Ciencias da Universidade do Porto, Porto, Portugal In the present work a high performance liquid-chromatography UV!Vis method was developed to quantify gliadins in foods. Gliadins are prolamins and are one of the constituents of wheat gluten (approximately 50%). These compounds have particular importance in food analysis since they are responsible for the celiac disease, which is an intolerance or hypersensitivity to ingested prolamins. The only known treatment for celiac disease is a gluten free diet. Therefore , the development of analytical methods to assess the gliadins contents in food products is crucial , namely in those labeled as "gluten-free". A "gluten-free" alimentary product cannot contain more than 1 OOppm of gluten, which corresponds to 50ppm of gliadins. Fifteen wheat based foods (7 "gluten-free" and 8 gluten-containing food samples, according to the label) were purchased in commercial supermarkets and analyzed using the HPLC method developed. The samples studied included flours, biscuits, breakfast cereals and bread. The HPLC equipment consisted of a Varian chromatographic system, equipped with a Varian Prostar 220 pump and a 7725i Rheodyne manual injector with a 1 Of-IL loop. A Varian Pro Star 330 Photodiode Array detector was used. The equipment was controlled using the Star Chromatography Workstation software (version 4.5) , which also controlled the solvent gradient, the data acquisition and data processing. The chromatographic separation was performed with a chromatographic column PLRP-S (polystyrene divinylbenzene stationary phase, particle size 8 microns, pore 300A and 150x4.6mm id). The column was placed inside an oven (Jones, Model 7981) and kept at 40±0.1 °C. The best chromatographic resolution for the gliadins was achieved using a gradient elution with acetonitrilerrFA/water. The elution was performed at a constant flow of 0.6mllmin and at a temperature of 40±0.1 °C. The detection was made at a wave-lenght of 21 On m. Each sample analysis took 30min. The gliadins concentrations of the calibration standard solutions (different standard gliadin masses dissolved in ethanol/water mixed in the proportion of volume 7/3) were confirmed using a Pierce Coomassie Plus kit test. The gliadins were extracted from the food samples using an ethanol/water solution (70:30, v/v) solution and, after vortex homogenization and centrifugation , the solutions were filtered through a Whatman 0.2f-1m nylon filter , before HPLC analysis. Calibration results showed a linear relationship between peak areas related to gliadins and concentrations with a correlation coefficient of 0.9993. The calibration curve was established by the external standard calibration method. The dynamic range of gliadins concentrations studied was between 50 and 1309ppm. The detection and quantification limits obtained were 46.5 and 140.91 pp m of gliadins, respectively. Data refer to the overall area of all peaks. The values obtained for repeatabil ity and intermediate precision were acceptable since the relative standard deviation percentage values were lower than 4%. The gliadins peaks in the food samples were identified based on the retention times obtained for the standard solutions of gliadins and by analysis of the UV spectrum associated to each peak. Results confirmed the presence of gliadins in all food samples indicating gluten-containing label. Moreover, gliad ins were also detected in two "gluten-free" food samples. Furthermore, in one of 199 6° encontro nacional de cromatografia comunica~6es em poster - P. 60 them it was also possible to quantify the gliadin level, which was greater than 50ppm, indicating that the "gluten-free" label was incorrect. The results obtained showed that the HPLC method developed in this work can be a useful tool for assessing gliadin contents in "gluten-free" products, although some additional effort must be made to decrease the detection and quantification limits of the proposed method. Acknowledgements Financial support received from FCT, Lisbon, under research projects POCI/QUI/58706/2004 and PPCDT/QUI/58076/2004 is gratefully acknowledged. 200 Liquid chromatography assay for gliadins quantification: Application to gluten-containing and "gluten-free" food products J. Sa Morais1· , S. Meirinho1, A. M. Peres1·2 , A. C. A. Veloso3.4, L. G . Dias1,, A. A. S. C. Machado5 1CIMO - Mountain Research Centre, Escola Superior Agraria de Braganqa, Braganqa, Portugal 2LSRE - Laboratory of Separation and Reaction Engineering, Escola Superior Agraria de Bragam;;a, Braganqa, Portugal 3Departamento de Engenharia Qui mica e Biol6gica, lnstituto Superior de Engenharia de Coimbra, Coimbra, Portugal 4188 - Center of Biological Engineering, Universidade do Minho, Braga, Portugal 5LAQU/PAI - Departamento de Quimica, Faculdade de Ciencias da Universidade do Porta, Porta, Portugal jsamorais@ipb.pt, www.esa.ipb.pt OBJECTIVES HPLC analytical method implementation for Gliadin analysis in foodstuffs, especially those declared as "gluten-free" INTRODUCTION •Celiac disease (CLD) is an autoimmune-mediated disorder, which is triggered by the ingestion of prolamins, namely gliadins • The gluten wheat proteins contain about 50% of gliadins • CLD prevalence is in the range of0.5 to 1.0% in Europe and U.S.A. • CLD may revert if a strict "gluten-free" lifelong diet is established • "Gluten-free" and "low gluten content" products must not contain more than 20 and I 00 ppm of gluten, respectively • "Gluten-free" foods must be analytically controlled SAMPLES 15 Food samples Label indicating Label indicating Food Food Gluten Containina Food Gluten Free Flour 2 1 Bread 1 1 Babv food formula 2 2 Wafer 2 2 Breakfast cereals 1 1 GLIADIN SOLUTION STANDARDIZATION Coomassie Plus - The Better BradfordTM Assay JUt BioTek Instrument ELx800 Microplates Software GeneS Detection at 595nm Albumine standard solutions HPLC SYSTEM Varian Prostar 220 Pump Varian ProS tar 330 Photodiode Array Rheodyne 7725i manual injector with Loop of lOf!L Software Star Chromatography Workstation, version 6.4 PLRP-S Polymer Laboratories Column {8f!m, 300A, 150x4.6mm i.d.) Jones 7981 Chromatography Column Oven HPLC CONDITIONS Column temperature at 40°C A) 99% ofwater/1% of acetonitrile (0.01% ofTFA) B) 99% of acetonitrile/1% of water (0.01% ofTFA) Eluent gradient: 20% to 80% ofB in 30min Flux of0.6mL/min Eluent temperature at 40°C Detection at 2 1 Onm SAMPLE GLIADIN EXTRACTION Gliadin was extracted with 70% ethanolic solution 15min of Vortex homogenization Centrifugation at 5000rpm for lOmin at 25°C Filtration with 0.21llJl Whatrnan Nylon Filters GLIADIN IDENTIFICATION IN SAMPLES Based on the retention times of the gliadins standard solution Confirmation based on the UV spectra of each peak detected Acknowledgement!!: Financia1 support received from FCf, Lisbon, under research projects POCUQUI/58706/2004 and PPCDT/QUI/58076/2004 is gratefully acknowledged. STANDARD GLIADIN IDENTIFICATION Signal 1 o.a 0.6 D.4 0.2 Zone if Gliadinspaaks o t-A..,..--;:=.::!._-""'==== -0.2 10 15 20 25 30 t(min) 5 Figure 2. Typical chromatograph of a gliadins Standard solution - Peak A - PeakB PaakC 225 250 275 300 325 nm Figure 3. Gliadins UV spectra of HPLC peaks SAMPLE GLIADIN IDENTIFICATION Signal 0.2 -H Gliadin UV spectra 0.15 -0.1 -0.05 • 0 5 10 15 20 25 30 -0.05 - t (min) Figure 4. Typical chromatograph of a gliadins sample solution Abs 0,30.,-----------., 0,25 0,20 0,15 0,10 0,05 o.oo -1-o~---~--~--1 0,00 0,50 1,00 1,50 2,00 Concentration (g/L) Figure 1. Albumin protein calibration (BSA kit Pierce) at 595nm RESULTS -9- Linearity: correlation coefficient of 0.9993 -9- LD ~ 46.5 g/L and LQ ~ 140.9 g/L of gliadins -9- Acceptable repeatability (CV%< 5%) ?Acceptable intermediate precision (CV% < 5%) -9- Acceptable accuracy (in general, CV% < 5%) -9- Gliadins detected in Flour 3 ("gluten-free") Figure 5. UV spectra of HPLC peaks GLIADIN HPLC QUANTIFICATION 3.so~:8T----------~ 3,00E+08 2,50E+08 2,00E+08 1,50E+08 1,00E+08 5,00E+07 O,OOE+OO -1--':...._~- ---~~_J 300 600 900 1200 Concentration (ppm) Figure 6. HPLC gliadins calibration measured at 210nm. Table1 . Results of gliadins analysis in the food samples ood samoles "with luten" Name C m Madln sam le Flour 1 11297 Flour2 10562 Bread1 3111 Babvfood 1 1090 Bab food 2 2982 Waffer1 d. -9- Gliadins quantified in Baby food 3 ("gluten-free") Waffer 2 d. CONCLUSION The results showed that "gluten-free" foods must be analysed to determine the gliadins content to ensure that the product label information is correct and, therefore, can be consumed by celiac patients. LSRE Breakfeast cereals 1 d. ood samoles "aluten free" Name Flour 3 Bread 2 Babv food 3 Babvfood 4 Waffer3 Waffer4 Breakfeast cereals 2 C l~ Nadlnslk sam le d. n.d. 930 n.d. n.d. n.d. n.d. 6°ENCONTRO NACIONAL DE CROMATOGRAFIA 

Liquid chromatography assay for gliadins quantification: application to gluten-containing and gluten-free food products

In the present work a high performance liquid-chromatography UV/Vis method was developed to quantify gliadins in foods. Gliadins are prolamins and are one of the constituents of wheat gluten (approximately 50%). These compounds have particular importance in food analysis since they are responsible for the celiac disease, which is an intolerance ar hypersensitivity to ingested prolamins

Veloso, Ana C.A.

Dias, L.G.

Machado, Adélio A.S.C.

Biblioteca Digital do IPB

Liquid chromatography assay for gliadins quantification: application to gluten-containing and “gluten-free” food products

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Liquid chromatography assay for gliadins quantification: application to gluten-containing and gluten-free food products

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