Development and Validation of a Liquid Chromatography/Tandem Mass Spectrometry Method for the Quantification of the GLP-1 Analog Semaglutide in Rat Plasma, and Its Application in a Pharmacokinetic Study.

Semaglutide, a long-acting glucagon-like peptide-1 (GLP-1) analog for type 2 diabetes and obesity, requires sensitive and high-throughput bioanalytical methods to support pharmacokinetic studies. However, previously reported liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays have been limited by lengthy run times (~18 min) and suboptimal sensitivity. This study aimed to develop and validate a rapid, sensitive LC-MS/MS method for quantifying semaglutide in plasma. Plasma samples (50 μL) were prepared by acetone-mediated protein precipitation followed by solid-phase extraction. Chromatographic separation was performed on a Cadenza CD-C18 MF column within 9 min, using positive electrospray ionization in multiple reaction monitoring mode with the transitions / 1029.4 → 110.1 for semaglutide and / 938.9 → 109.9 for liraglutide (internal standard). Validation followed the U.S. Food and Drug Administration (FDA) bioanalytical guidelines. The assay showed a lower limit of quantification of 1 ng/mL with linearity across 1-500 ng/mL (R = 0.9999), with sharp peak shape and no carryover. Intra- and inter-day accuracies were 95.69-103.76% and 94.93-100.08%, with precision ≤4.50% and ≤5.88%. Recovery (93.05-107.95%) and matrix effects (96.34-104.12%) were consistent across quality control levels, and the analyte was stable under all tested conditions. The method was successfully applied to a pharmacokinetic study in Sprague-Dawley rats following subcutaneous administration of 50 μg semaglutide. The validated method offers shorter analysis time, improved sensitivity, and reduced sample volume compared with previously reported assays, supporting its application in preclinical pharmacokinetic studies of semaglutide and related GLP-1 analogs.