title: Development of a capillary high performance liquid chromatography-ion trap-mass spectrometry method for the determination of VLIVP antihypertensive peptide in soybean crops.
abstract: Soybean peptide VLIVP presents a very high antihypertensive activity (IC50 value 1.69μM), even higher than extensively studied IPP and VPP peptides from milk. Nevertheless, no much attention has been paid to this peptide and there is no method enabling its determination in soybeans. The aim of this work was the development of an analytical methodology for this purpose. A methodology consisting of the extraction of soybean proteins, their digestion with Protease P enzyme, their chromatographic separation using capillary-HPLC, and IT-MS detection was optimized. Protein extraction was performed by the use of high intensity focused ultrasounds to obtain a reduced extraction time. Optimization of chromatographic and mass spectrometry parameters enabled the separation of VLIVP peptide within just 7min and its sensitive detection. The analytical characteristics of the capillary-HPLC-IT-MS method were evaluated through the study of linearity, LOD, LOQ, study of the presence of matrix interferences, precision, and recovery. The method enabled to detect as low as 3.6ng of peptide and to determine as low as 12ng of peptide in 1g of soybean (as dry basis). Finally, the developed method was applied to the determination of the antihypertensive peptide VLIVP in different soybean varieties. The results showed the highest yield of VLIVP peptide in variety Mazowiecka II from Poland.
authors.affiliation: Department of Analytical Chemistry, Physical Chemistry, and Chemical Engineering, Faculty of Biology, Environmental Sciences and Chemistry, University of Alcalá, Ctra. Madrid-Barcelona Km. 33.600, 28871 Alcalá de Henares, Madrid, Spain. Electronic address: patrycia.puchalska@uah.es.
authors.forename: Patrycja
authors.initials: P
authors.lastname: Puchalska
authors.affiliation: Department of Analytical Chemistry, Physical Chemistry, and Chemical Engineering, Faculty of Biology, Environmental Sciences and Chemistry, University of Alcalá, Ctra. Madrid-Barcelona Km. 33.600, 28871 Alcalá de Henares, Madrid, Spain.
authors.forename: M Concepción
authors.initials: MC
authors.lastname: García
authors.affiliation: Department of Analytical Chemistry, Physical Chemistry, and Chemical Engineering, Faculty of Biology, Environmental Sciences and Chemistry, University of Alcalá, Ctra. Madrid-Barcelona Km. 33.600, 28871 Alcalá de Henares, Madrid, Spain. Electronic address: mluisa.marina@uah.es.
authors.forename: M Luisa
authors.initials: ML
authors.lastname: Marina
mesh_terms: D000959:Antihypertensive Agents / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D010455:Peptides / Q000032:analysis*; D010936:Plant Extracts / Q000737:chemistry; D030262:Soybean Proteins / Q000032:analysis / Q000138:chemical synthesis*; D013025:Glycine max / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000959:Antihypertensive Agents; D010455:Peptides; D010936:Plant Extracts; D030262:Soybean Proteins
keywords: Antihypertensive peptide; Ion trap; Micro-HPLC; Quantification; Soybean