Development and validation of a sensitive HPLC-MS/MS method for the analysis of semaglutide in human plasma.

BACKGROUND: Semaglutide is a human glucagon-like peptide-1 (GLP-1) peptide analog. To date, the literature does not report a validated HPLC-MS/MS assay using a triple-quadrupole mass spectrometer for the quantitation of semaglutide in human plasma in support of pharmacokinetics. A robust, sensitive HPLC--MS/MS assay supporting this class of compounds, is provided and full validation demonstrated. METHODS AND RESULTS: A triple-quadrupole HPLC-MS/MS method has been developed and fully validated for the quantitation of semaglutide in human plasma, using a stable isotope labeled internal standard and an effective, efficient protein precipitation sample extraction. The method has a sensitive analytical range of 5 ng/mL to 200 ng/mL (and to 1000 ng/mL with a dilution QC) and exhibits excellent accuracy (92.7% to 109.4%) and precision of <10.6%. This highly selective method provided high recovery of the analyte, with minimal matrix effects. CONCLUSION: A robust and simple HPLC-MS/MS method to quantify semaglutide in human plasma has been fully validated in accordance with the 2022 FDA M10 guidance. This method is suitable for implementation in clinical studies within the validated analytical range and stability conditions and can be extended to the bioanalysis of other GLP-1 peptide-based therapeutics with similar structural and physiochemical properties.