A method for the quantitation of dityrosine in wheat flour and dough by high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) using an isotope dilution assay with the internal standard 3,3'-(13)C(2)-dityrosine in the single-reaction monitoring mode was developed. The method consisted of the release of protein-bound dityrosine by hydrolysis in 4 mol/L hydrochloric acid/8.9 mol/L propionic acid for 24 h at 110 degrees C after addition of the internal standard, cleanup by C(18) solid-phase extraction, and HPLC-MS/MS. The limit of detection of dityrosine was 80 ng/g of sample (0.22 nmol/g), and the limit of quantitation was 270 ng/g of sample (0.75 nmol/g). The method was sensitive enough to analyze wheat flour and dough and to study the effect of flour improvers on the dityrosine content. Furthermore, the effect of the mixing time was studied. The dityrosine concentration in the flour was 0.66 nmol/g. After we mixed a dough to peak consistency, the dityrosine concentration doubled and remained constant on further mixing. Overdoses of hydrogen peroxide and hexose oxidase (HOX, E.C. 1.1.3.5) resulted in a strongly increased dityrosine content, whereas no increase of the dityrosine concentration was found after the addition of ascorbic acid and potassium bromate. Calculation of the percentage of dimeric tyrosine showed that less than 0.1% of the tyrosine residues of wheat protein were cross-linked. Therefore, dityrosine residues seem to play only a very minor role in the structure of wheat gluten.