Monitoring the large-scale production of the antihypertensive peptides RYLGY and AYFYPEL by HPLC-MS.

This work reports the quantitative analysis of two novel antihypertensive peptides alpha(s1)-CN f(90-94), with sequence RYLGY, and alpha(s1)-CN f(143-149), with sequence AYFYPEL, by high-performance liquid chromatography-mass spectrometry in food-grade hydrolysates of milk proteins. The method was validated and showed sufficient specificity, reproducibility, linearity and recovery. Linear calibrations of the molecular ions m/z 671.2 and 902.3 were selected for the determination of the peptides RYLGY and AYFYPEL, respectively, and showed good statistical results (R(2) > or = 0.995 and with no significant lack-of-fit). The simplicity of RP-HPLC-MS method allowed the automated quantification of both antihypertensive peptides without any sample pretreatment. The application of this method permitted the evaluation of some hydrolysis variables, i.e., substrate, temperature, hydrolysis time or enzyme/substrate ratio, on the formation of antihypertensive peptides. The quantitative analysis of RYLGY and AYFYPEL showed that ultrafiltration was not effective to improve the content in active peptides, containing the hydrolysates and their respective permeates similar peptide amounts.