Identification and Quantification of Dityrosine in Grain Proteins by Isotope Dilution Liquid Chromatography-Tandem Mass Spectrometry
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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 (R 2 = 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.
KeywordsDityrosine Zein Kafirin Marama Gluten LC-MS/MS
The authors would like to acknowledge Dr. Francesco Busetti, School of Science, Edith Cowan University, and Ms. Dori Hahne, Centre for Microscopy, Characterisation and Analysis, University of Western Australia, for their valuable suggestions on liquid chromatography and mass spectrometry conditions. We thank Dr. Prashant Kumar, Institute of Chemical Technology (ICT), Mumbai, India, and Professor John Taylor, University of Pretoria, for kindly providing the kafirin and marama bean protein, respectively. We are grateful to the Australia-India Strategic Research Fund (BF040035) for the financial support. The authors also wish to acknowledge federal funding from the National Collaborative Research Infrastructure Strategy (NCRIS) provided through Metabolomics Australia and Bioplatforms Australia.
Compliance with Ethical Standards
This study was funded by the Australia-India Strategic Research Fund (BF040035).
Conflict of Interest
Duc Doan Nguyen declares that he has no conflict of interest. Michael Clarke declares that he has no conflict of interest. Stuart Johnson declares that he has no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
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