Recombinant somatotropin (rbST) is a synthetic hormone that mimics endogenous bovine somatotropin (GH), differing by a single N-terminal amino acid (ala-bST vs. met-rbST). While rbST is approved in some countries to boost milk production, it is banned in Europe. Given the risk of illicit use, its detection in milk and dairy products is essential. This study aimed to develop an LC-MS/MS method for rbST identification.

Stock solutions (1000 µg/mL in water) of endogenous and recombinant somatotropin peptides were prepared and stored at 4 °C. Daily dilutions were used to assess linearity and repeatability of the LC-MS/MS method using synthetic peptides at various concentrations. Analyses were performed with an Exion LC Sciex UHPLC system coupled with a 6500 QTRAP mass spectrometer (SCIEX, Milan, Italy).

Serum samples (n=48) from six Holstein cows in farms in Piedmont were subjected to extraction, purification, and digestion prior to LC-MS analysis. Method optimization included LC separation and MS condition setup, with MS acquisition parameters refined using 1 mg/mL standard solutions. The HPLC-MS/MS method showed excellent performance: linearity (R² = 0.999, 1–250 ppb), repeatability (RSD <10% at 1, 5, and 50 ppb), and recovery (95–106%). Analysis of real samples is still ongoing. The ability to determine illicit treatments is of special interest because of regulations aimed at ensuring that consumer and farmer rights are protected. Development and application of valuable analytical tools suitable to identify various hormones are matters of interest in order to protect consumers

In this study we have developed an LC- method for the endogenous (ala-bst) and recombinant protein (met-rbst) identification in serum.