From Targeted LC-MS/MS to High-Resolution Mass Spectrometry: Emerging Analytical Strategies for the Characterization of Antiviral and Antibacterial Drugs, Impurities and Degradation Products: A Critical Review
TL;DR
Overall, the available evidence supports an integrated analytical strategy in which HRMS is primarily employed to elucidate chemical identity and targeted LC-MS/MS is applied when sensitive, selective and robust routine quantification is required.
Abstract
Antiviral and antibacterial medicines present a particularly broad analytical challenge. The same laboratory may need to quantify a known impurity at trace levels, investigate an unexpected peak arising during stability studies and distinguish a genuine degradation product from an adduct, isomer, or source-generated fragment. Targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS), particularly triple-quadrupole multiple-reaction monitoring (MRM), remains highly effective for the sensitive and selective quantification of predefined analytes. In contrast, high-resolution mass spectrometry (HRMS) provides complementary capabilities through accurate-mass full-scan measurements and product-ion data, which can be interrogated when the composition of an impurity is not known in advance. This review examines how these complementary capabilities can be integrated into the analysis of antiviral and antibacterial drug substances and pharmaceutical products. Recent studies published between 2022 and 2026 are considered alongside established analytical principles, with particular emphasis on forced degradation, impurity origin, chromatographic selectivity, ionization behaviour, fragmentation evidence, isomeric ambiguity, matrix effects and the level of evidence required to support structural assignments. Recent examples in antiviral drug analysis include Maillard-reaction impurities of acyclic nucleoside analogues, forced-degradation products of darunavir, impurities of peramivir, bictegravir-related substances and degradation products of umifenovir. An example from antibacterial drug analysis is the LC-HRMS/MS characterization of dalbavancin degradation products. Collectively, these studies demonstrate that accurate-mass measurements are most informative when interpreted in conjunction with retention behaviour, dependence on stress conditions, isotope patterns, product-ion shifts and , where necessary, reference standards or orthogonal structural characterization techniques. Accordingly, this review considers targeted LC-MS/MS and LC-HRMS as complementary components of a fit-for-purpose analytical workflow rather than as competing platforms. The current principles outlined in ICH Q2(R2) and Q14 are also considered in the context of analytical procedure performance and lifecycle development. Emerging approaches, including data-independent acquisition, ion mobility spectrometry and computational spectral annotation, are discussed cautiously as complementary tools that may strengthen, rather than replace, expert interpretation. Overall, the available evidence supports an integrated analytical strategy in which HRMS is primarily employed to elucidate chemical identity and targeted LC-MS/MS is applied when sensitive, selective and robust routine quantification is required.