Analytical Considerations and Often-Overlooked Aspects in the Therapeutic Drug Monitoring of Polymyxins in Human Biological Matrices Using LC-MS/MS

liquid chromatography-tandem mass spectrometry (LC-MS/MS) is compromised by a series of often-overlooked yet critical analytical challenges. This comprehensive review identifies and addresses three major pre-analytical and analytical pitfalls: (1) the inherent variability in compositional heterogeneity and the salt form of commercial standards, which we propose to correct using a batch-specific weighted average molecular weight formula;(2) significant nonspecific adsorption（NSA） to laboratory surfaces, requiring proactive mitigation strategies such as silanization and additive use; (3) the pronounced and pH-dependent instability of colistimethate sodium (CMS), which necessitates strict non-acidic sample preparation protocols to prevent artificial hydrolysis; Furthermore, we clarify the clinical contexts for measuring active versus total colistin concentrations. By synthesizing current evidence and offering practical methodological refinements, this work aims to enhance the accuracy, reproducibility, and clinical utility of polymyxins TDM, ultimately supporting more precise dosing and improved patient.