Design of Experiment (DoE) for Optimization of HPLC Conditions for the Simultaneous Fractionation of Seven α-Amylase/Trypsin Inhibitors from Wheat (Triticum aestivum L.)

Wheat alpha-amylase/trypsin inhibitors remain a subject of interest considering the latest findings showing their implication in wheat-related non-celiac sensitivity (NCWS). Understanding functions such disorder is still unclear and for further study, need pure ATI molecules one limiting problems. In this work, simplified approach based on successive fractionation extracts by reverse phase ion exchange chromatography was developed. ATIs were first extracted from wheat flour using combination Tris buffer chloroform/methanol methods. The separation C18 column generated two main fractions F1 F2. response surface methodology with Doehlert design allowed optimizing operating parameters strong anion chromatography. Finally, seven major namely P01083, P17314, P16850, P01085, P16851, P16159, P83207 recovered purity levels (according to targeted LC-MS/MS analysis) 98.2 ± 0.7; 98.1 0.8; 97.9 0.5; 95.1 98.3 0.4; 96.9 0.5, 96.2 0.4%, respectively. MALDI-TOF-MS analysis revealed single peaks each mass yielded deviations 0.4, 1.9, 0.1, 0.2, 0.9, 0.1% between theoretical determined masses P83207, Overall, study establishing an efficient purification process most important ATIs. This paves way in-depth investigation gain more knowledge related involvement NCWS disease allow absolute quantification samples.