Identification and Quantification of Dityrosine in Grain Proteins by Isotope Dilution Liquid Chromatography-Tandem Mass
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Identification and Quantification of Dityrosine in Grain Proteins by Isotope Dilution Liquid Chromatography-Tandem Mass Spectrometry D. D. Nguyen 1,2 & S. K. Johnson 1 & M. W. Clarke 3
Received: 13 February 2017 / Accepted: 7 April 2017 # Springer Science+Business Media New York 2017
Abstract A novel liquid chromatography-tandem mass spectrometry method was developed and validated for identification and quantification of dityrosine in zein protein. The zein samples spiked with a dityrosine standard and 3,3-13C6-dityrosine, as the internal standard were hydrolysed in a mixture of 6 N hydrochloric acid and propionic acid followed by cleaning-up using Sep Pak C18 cartridges. Good linear regression (R2 = 0.999) was obtained in the range 1–1000 ng/mL. Method limit of detection and method limit of quantification were 42.1 and 140 ng/g, respectively. Recoveries were from 92 to 95.2%. Precision and inter-day reproducibility expressed as relative standard deviation were from 3.9 to 22.1% and from 4.4 to 16.6%, respectively. The validated method was applied to quantify dityrosine in various grain proteins. The levels of dityrosine ranged from 0.38 to 1.92 ng/mg. This validated method will be of great value for understanding the role of dityrosine in grain food texture.
Keywords Dityrosine . Zein . Kafirin . Marama . Gluten . LC-MS/MS
* M. W. Clarke [email protected] 1
Faculty of Health Science, School of Public Health, Curtin University, Perth 6102, Australia
2
Faculty of Food Science and Technology, Vietnam National University of Agriculture, Hanoi, Vietnam
3
Biological and Molecular Mass Spectrometry Facility, Centre for Microscopy, Characterisation and Analysis, University of Western Australia, Perth 6009, Western Australia, Australia
Introduction Dityrosine (DiTyr, 2-amino-3-[4-[4-(2-amino-2-carboxyethyl) -2-hydroxyphenyl]-3-hydroxyphenyl] propionic acid) (Pubchem 2015) was initially discovered by Gross and Sizer (1959) when oxidising L-tyrosine with a peroxide-peroxidase system in vitro. In addition, the formation of DiTyr was also found as a result of ultraviolet radiation, γ-radiation or exposure to oxygen-free radicals in in vitro studies (Boguta and Dancewicz 1981; Davies 1987; Lehrer and Fasman 1967). Because many human diseases have been considered to be a result of oxidative damage to proteins, DiTyr excretion in the urine has been widely used as a biomarker for oxidative stress and pathogenic changes (Hanft and Koehler 2005; Kato et al. 2009; Orhan et al. 2005; Wu et al. 2015). In food processing on the other hand, DiTyr has gained far less attention. However, it has been proposed as a good marker for milk protein oxidation (Fenaille et al. 2004) and as a protein cross-link possibly contributing to the desired viscoelastic structure of wheat gluten network in baked foods such as bread (Tilley et al. 2001). Due to its characteristic intense fluorescence, highperformance liquid chromatography (HPLC) coupled with fluorescence detection has been previously used for quantification of DiTyr in wheat
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