Polypeptide LC-MS Bioanalysis Strategies in Biological Matrices: Glucagon and Plasma Analogs

Abstract

Glucagon and its analogs are polypeptide pharmaceuticals designed for the management of metabolic disorders, including hypoglycemia, diabetes, and obesity. Owing to their significant potency, these medicines are often found at low quantities in plasma during pharmacokinetic (PK) investigations, necessitating a very sensitive bioanalytical technique. This study delineates a fast and sensitive LC-MS/MS technique using protein precipitation (PPT) with acidified acetonitrile for sample preparation. Analytes were separated on an ACE C18 column using a linear gradient of water and acetonitrile containing 0.1% acetic acid. Detection was conducted on an AB Sciex mass spectrometer using positive electrospray ionization mode, employing the 5+ charge state as the precursor ion. Selective MS/MS fragment ion monitoring enhanced the signal-to-noise ratio and reduced endogenous interference. The test exhibited a linear range of 0.5–500 ng/mL, characterized by great accuracy, precision, and durability. It has increased sensitivity, selectivity, minimal matrix effects, and is appropriate for regular pharmacokinetic analysis with high throughput and low expense.

Keywords: LC-MS/MS, Polypeptide bioanalysis, Glucagon, GLP-1, GLP-2, Plasma matrix.