Development of a high-performance liquid chromatography–electrospray ionization-quadrupole-time-of-flight-mass spectrometry methodology for the determination of three highly antihypertensive peptides in maize crops

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.

► Optimized ESI-MS parameters reduced spontaneous fragmentation of peptide ions.
► SPE C18 Sep-Pak method efficiently removed urea from digested extract.
► In-depth characterization of the method was performed observing adequate results.
► Quantification of antihypertensive peptides in six maize lines was achieved.
► The antihypertensive potency of six maize lines was evaluated.