The simultaneous quantification of three highly antihypertensive peptides (LRP, LSP, and LQP) in six maize crops using novel HPLC-ESI-Q-ToF methodology is presented. The method included the extraction of α-zein proteins from maize, their purification by acetone precipitation, digestion with thermolysin and HPLC separation in a fused-core column. Several MS parameters were optimized to increase sensitivity and reduce spontaneous fragmentation of peptide ions into the ESI source. The ions with m/. z 193.1315, 385.2558 (for LRP), 316.1867 (for LSP), and 357.2132 (for LQP) were monitored in the optimization and characterization of the method. In order to improve the repeatability, sensitivity, and the stability of peptides in the sample, the removal of urea was required. The use of two solid-phase extraction methods to remove urea from digested extract was evaluated. For the first time filter-aided sample preparation approach for the study of bioactive peptides in foodstuffs has been proposed. The optimized HPLC-ESI-Q-ToF method was characterized by the evaluation of linearity, LOD, LOQ, precision, and recovery. A study on the existence of matrix interferences was also performed. The developed method was applied to the quantification of LRP, LQP, and LSP peptides in maize lines using the standard addition method. The results showed the highest yield of LSP peptide in EZ11A line and LRP and LQP peptides in A632 line.
Bibliographical noteFunding Information:
Authors thank the Spanish Ministry of Science and Innovation (project CTQ2009-09022) and the Comunidad Autónoma of Madrid (Spain) and European funding from FEDER program (project S2009/AGR-1464, ANALISYC-II). Patrycja Puchalska thanks the University of Alcalá for her pre-doctoral contract. Authors gratefully acknowledge Ángel Álvarez from Experimental Station of Aula Dei, CSIC, Zaragoza, Spain for the kind donation of the maize crops.
- Antihypertensive peptides
- Fused-core column