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.
Bibliographical noteFunding Information:
Authors thank the Spanish Ministry of Science and Innovation (project CTQ2009-09022), the Spanish Ministry of Economy and Competitiveness (project AGL2012-36362), 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 Amano Enzyme Inc., Nagoyo, Japan and Centro de Recursos Fitogenéticos del Instituto Nacional de Investigaciones Agrarias, Madrid, Spain for the kind donation of enzyme and soybean varieties, respectively.
- Antihypertensive peptide
- Ion trap