OBJECTIVE ‌To establish a screening strategy and workflow for the illegal peptide additive retatrutide.

METHODS Tricine-SDS-PAGE was performed as the first rudimentary screening. Subsequently, “bottom-up” and “top-down” analytical strategies were established via HPLC-QTOF for identifying the masses of the peptide fragments and the intact peptide. Separation was achieved on an Agilent EC-C₁₈ column(3.0 mm×150 mm, 2.7 μm) using a mobile phase consisting of 0.1% formic acid in water(A) and 0.1% formic acid in acetonitrile(B) with gradient elution at a flow rate of 0.3 mL·min^–1. Data were acquired using an ESI⁺ ion source.

RESULTS The presence of the polypeptide substance was confirmed by Tricine-SDS-PAGE. The sequence of retatrutide was deduced using the “bottom-up” strategy, which was also useful for identifying the fatty acid chain modification site. Ultimately, the structure was confirmed using a “top-down” analytical strategy with a reference standard.

CONCLUSION A screening method for retatrutide was established. The screening procedure is accurate and reliable, which can be applied for the characterization and analysis of the primary structure of GLP-1 receptor agonists, as well as for the screening of unknown illegal peptide adulterants.