Puchalska Patrycja, García M Concepción, Marina M Luisa
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.
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.
J Chromatogr A. 2014 Apr 18;1338:85-91. doi: 10.1016/j.chroma.2014.02.059. Epub 2014 Mar 1.
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.
大豆肽VLIVP具有非常高的降压活性（IC50值为1.69μM），甚至高于对牛奶中广泛研究的IPP和VPP肽。然而，这种肽并未受到太多关注，并且没有能够在大豆中测定它的方法。这项工作的目的是为此开发一种分析方法。一种由大豆蛋白提取、用蛋白酶P酶消化、使用毛细管高效液相色谱进行色谱分离以及离子阱质谱检测组成的方法得到了优化。蛋白质提取通过使用高强度聚焦超声进行，以缩短提取时间。色谱和质谱参数的优化使得VLIVP肽在短短7分钟内得以分离并实现灵敏检测。通过线性度、检测限、定量限研究、基质干扰存在情况研究、精密度和回收率研究，对毛细管高效液相色谱 - 离子阱质谱法的分析特性进行了评估。该方法能够检测低至3.6纳克的肽，并能测定1克大豆（以干基计）中低至12纳克的肽。最后，将所开发的方法应用于不同大豆品种中降压肽VLIVP的测定。结果表明，来自波兰的Mazowiecka II品种中VLIVP肽的产量最高。
