Exploring New Biomarkers for Detecting Whey Adulteration in Milk: Synthesis and Characterization of Caseinomacropeptide-Derived Peptides

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 <i>in-house</i> 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 <i>cis-trans</i> 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 <i>R</i> ² 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 <i>in-house</i> standard for quantifying whey adulteration in milk, offering a reliable and reproducible approach for routine LC-MS analysis.