Development and optimisation of a generic micro LC-ESI-MS method for the qualitative and quantitative determination of 30-mer toxic gliadin peptides in wheat flour for food analysis.

Abstract

We sometimes see manufactured bakery products on the market which are labelled as being gluten free. Why is the content of such gluten proteins of importance for the fabrication of bakery industry and for the products? The gluten proteins represent up to 80% of wheat proteins, and they are conventionally subdivided into gliadins and glutenins. Gliadins belong to the proline and glutamine-rich prolamin family. Its role in human gluten intolerance,as a consequence of its harmful effects, is well documented in the scientific literature. The only known therapy so far is a gluten-free diet, and hence, it is important to develop robust and reliable analytical methods to quantitatively assess the presence of the identified peptides causing the so-called coeliac disease. This work describes the development of a new, fast and robust micro ion pair-LC-MS analytical method for the qualitative and quantitative determination of 30-mer toxic gliadin peptides in wheat flour. The use of RapiGest™ SF as a denaturation reagent prior to the enzymatic digestion showed to shorten the measuring time. During the optimisation of the enzymatic digestion step, the best 30-mer toxic peptide was identified from the maximum recovery after 3h of digestion time. The lower limit of quantification was determined to be 0.25ng/μL. The method has shown to be linear for the selected concentration range of 0.25-3.0ng/μL. The uncertainty related to reproducibility of measurement procedure, excluding the extraction step, has shown to be 5.0% (N = 12). Finally, this method was successfully applied to the quantification of 30-mer toxic peptides from commercial wheat flour with an overall uncertainty under reproducibility conditions of 6.4% including the extraction of the gliadin fraction. The results were always expressed as the average of the values from all standard concentrations. Subsequently, the final concentration of the 30-mer toxic peptide in the flour was calculated and expressed in milligrams per gram unit. The determined, calculated concentration of the 30-mer toxic peptide in the flour was found to be 1.29 ± 0.37μg/g in flour (N = 25, s y = 545,075, f = 25 - 2 (t = 2.069), P = 95%, two-sided).