Development and validation of a multiplexed LC-HRMS method for nine GLP-1 receptor agonists and its pharmaceutical application.
This study presents a validated LC-HRMS method that can accurately quantify GLP-1 receptor agonists, including retatrutide, with high sensitivity and specificity.
Where it sits
this study against the rest of the retatrutide (ly3437943) corpusSummary and findings
A multiplexed liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method was developed for the identification and quantitation of nine GLP-1 receptor agonists, including retatrutide. The method demonstrated good linearity and was successfully applied to marketed formulations. Mean recoveries for the marketed formulations ranged from 113.7% to 118.4%.
Abstract
A rapid, sensitive, and selective multiplexed liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method was developed and validated for the identification and quantitation of nine structurally diverse peptide-based glucagon-like peptide-1 receptor agonists (GLP-1 RAs): bofanglutide, ecnoglutide, exenatide, liraglutide, mazdutide, retatrutide, semaglutide, survodutide, and tirzepatide. These peptides represent single, dual, and triple receptor agonists, including four globally approved medications, two recently approved by China's National Medical Products Administration (NMPA), and three investigational drugs in Phase III clinical trials. Chromatographic separation was achieved on an ACQUITY Premier BEH C18 column (2.1 × 150 mm, 1.7 µm) at 50 °C using a multistep linear gradient with water and acetonitrile containing 0.1% difluoroacetic acid (DFA) and 0.5% dimethyl sulfoxide (DMSO) as mobile phases (flow rate 0.3 mL/min). The optimised method provided excellent resolution with baseline separation for most peptides within a 20-minute runtime. A key advantage of this work is the implementation of a dual strategy that combines high-resolution full-scan MS (HRMS) for accurate mass measurement with data-dependent HRMS/MS fragmentation for structural confirmation. Peptide identification adopted a top-down approach, achieving mass accuracy of 0.0-0.5 ppm and fragment ion coverage of 76-100%. The method showed good linearity (r² ≥ 0.995) over 4-235 ng/mL, with LODs of 0.44-2.06 ng/mL and LOQs of 8.29-11.74 ng/mL. It was successfully applied to marketed formulations of liraglutide, semaglutide, and tirzepatide using a simple dilute-and-shoot procedure, yielding mean recoveries of 113.7-118.4%. This LC-HRMS method offers superior selectivity and flexibility for analysing current and next-generation GLP-1 RAs.
Background
Not reported in abstract.
Methods
Not reported in abstract.
Results
Not reported in abstract.
Interpretation
Not reported in abstract.
Limitations
Not reported in abstract.