Animal-Free LC-MS Workflow for Endotoxin and Microbial Differentiation

Endotoxin contamination remains a critical risk in drug products and medical devices. Conventional assays rely on clotting proteins from horseshoe crab blood, raising sustainability and accuracy concerns. This proposal introduces a same-day, animal-free workflow using liquid chromatography–mass spectrometry (LC-MS) to directly measure endotoxin molecular fragments. The approach detects diagnostic biomarkers such as Lipid A and characteristic lipopolysaccharide (LPS) motifs, while also enabling microbial differentiation by targeting peptidoglycan fragments (Gram-positive bacteria) and chitin/β-glucan oligomers (fungi). The result is a rapid, accurate, and environmentally sustainable test that provides both pass/fail assessment and classification of contamination. This conceptual framework reduces reliance on animal resources, minimizes false positives from excipients, and enables reliable endotoxin and microbial assessment in hours.