Parada-Suárez David Alexander, Mendoza-Mendoza Jose David, Huertas-Ortiz Kevin Andrey, Reyes-Calderón Juan Esteban, Rivera-Monroy Jhon Erick, Fierro-Medina Ricardo, García-Castañeda Javier Eduardo, Rivera-Monroy Zuly Jenny
Departamento de Farmacia, Facultad de Ciencias, Universidad Nacional de Colombia, Carrera 45 # 26-85, Bogotá 111321, Colombia.
Instituto de Biotecnología, Facultad de Ciencias, Universidad Nacional de Colombia, Carrera 45 # 26-85, Bogotá 111321, Colombia.
ACS Omega. 2025 Jun 6;10(23):24169-24180. doi: 10.1021/acsomega.4c10923. eCollection 2025 Jun 17.
Whey adulteration in milk is a widespread issue that often escapes detection by conventional methods due to its similar composition and minimal impact on milk's sensory characteristics. This study addresses this challenge by evaluating synthetic peptides derived from caseinomacropeptide (CMP) as potential standards for detecting whey adulteration in milk using LC-MS. Two principal peptides, MAIPPKKNQDKTEIPTINT (CMP-1), derived from pepsin digestion of CMP, and TEIPTINT (CMP-3), obtained through a double digestion with pepsin followed by trypsin, were synthesized, purified, and comprehensively characterized to assess their viability as biomarkers. Isomerism in CMP-1 was initially observed through LC-MS and confirmed as proline-proline isomerism by 2D-NMR analyses (TOCSY and ROESY), which complicates its application as a standard. Consequently, CMP-3, an isomerism-free derivative, was synthesized and demonstrated stability in both pepsin and trypsin digestions. The use of three surrogates for quantifying CMP was evaluated: CMP, CMP-1, and CMP-3, for both nonadulterated and adulterated milk samples, which allowed for effective detection and quantification of whey adulteration. Calibration curves for CMP-1 and CMP-3 were generated on high-resolution (LC-HRMS) and low-resolution (LC-LRMS) mass spectrometers, yielding values between 0.91 and 0.99. The calibration curves displayed adequate linearity, and variability in ionization efficiency underscored the need for an internal standard to ensure greater reproducibility. CMP-3 is proposed as a promising biomarker and/or standard for quantifying whey adulteration in milk, offering a reliable and reproducible approach for routine LC-MS analysis.
牛奶中的乳清掺假是一个普遍存在的问题，由于其成分相似且对牛奶感官特性影响极小，传统方法往往难以检测到。本研究通过评估源自酪蛋白巨肽（CMP）的合成肽作为使用液相色谱 - 质谱法检测牛奶中乳清掺假的潜在标准，来应对这一挑战。合成、纯化并全面表征了两种主要肽段，即通过胃蛋白酶消化CMP得到的MAIPPKKNQDKTEIPTINT（CMP - 1），以及先经胃蛋白酶消化再经胰蛋白酶消化得到的TEIPTINT（CMP - 3），以评估它们作为生物标志物的可行性。最初通过液相色谱 - 质谱法观察到CMP - 1存在异构现象，并通过二维核磁共振分析（TOCSY和ROESY）确认为脯氨酸 - 脯氨酸异构，这使其作为标准的应用变得复杂。因此，合成了无异构的衍生物CMP - 3，并证明其在胃蛋白酶和胰蛋白酶消化中均具有稳定性。评估了使用三种替代物（CMP、CMP - 1和CMP - 3）对非掺假和掺假牛奶样品中CMP进行定量，从而能够有效检测和定量乳清掺假。在高分辨率（液相色谱 - 高分辨质谱）和低分辨率（液相色谱 - 低分辨质谱）质谱仪上生成了CMP - 1和CMP - 3的校准曲线，相关系数值在0.91至0.99之间。校准曲线显示出足够的线性，电离效率的变化突出了需要内标以确保更高的重现性。CMP - 3被提议作为一种有前景的生物标志物和/或用于定量牛奶中乳清掺假的标准，为常规液相色谱 - 质谱分析提供了一种可靠且可重现的方法。
