Determination of the Antihypertensive Peptide LHLPLP in Fermented Milk by High-Performance Liquid Chromatography–Mass Spectrometry

(A) Analysis of the water-soluble extract of milk fermented with Enterococcus faecalis CECT 5728 obtained by HPLC with UV detection at 214nm; (B) total ion current chromatogram; (C) HPLC–mass spectrometric (MS) analysis, showing the extracted ion chromatogram of the molecular ion of peptide LHLPLP with m/z 689.4; and (D) HPLC–tandem MS (MS/MS) analysis, showing the extracted ion chromatogram of fragment ions with m/z 574.3, 439.2, 364.2, and 251.0. For experimental conditions see the Material and Methods section.

(A) High-performance liquid chromatograph–mass spectrum of the peptides eluting between 44.3 and 47.3min. (B) Tandem mass spectrum of the singly charged ion m/z 689.4. The sequence of this peptide is displayed with the fragment ions observed in the spectrum. Fragment ions are labeled according to the nomenclature proposed by Roepstorff and Fohlman (1984).

Calibration curves of the peptide LHLPLP in water obtained after HPLC–mass spectrometric (MS) analysis and extraction of the ion m/z 689.4 and its corresponding adducts (○) and after HPLC–ion trap tandem MS (MS/MS) analysis and extraction of different fragment ions (□, ♢, ▵).

Concentration of the peptide LHLPLP in milk fermented with Enterococcus faecalis CECT 5727 (□) and E. faecalis CECT 5728 (○) throughout fermentation (0, 3, 6, 9, 24 and 48h), and the mathematical equation chosen to describe the evolution of the concentration with fermentation time.