Strain-promoted azide-alkyne cycloaddition-based fluorometric detection of azvudine using DBCO-functionalized carbon dots.

Azvudine, a nucleoside analog antiviral agent approved for COVID-19 and HIV treatment, requires precise analytical methods for therapeutic drug monitoring and pharmacokinetic studies. This study presents the first strain-promoted azide-alkyne cycloaddition (SPAAC)-based fluorometric method for azvudine determination. The innovative methodology exploits the unique structural feature of azvudine containing an azide functional group, enabling highly selective covalent binding with dibenzocyclooctyne (DBCO)-functionalized carbon dots through copper-free click chemistry, resulting in concentration-dependent aggregation-induced fluorescence quenching. The method demonstrates excellent analytical performance with linear response in the range 2.0-150.0 ng/mL (R² = 0.9987), and achieves a limit of detection of 0.87 ng/mL. The bioorthogonal SPAAC reaction ensures exceptional selectivity, with no significant interference observed from endogenous plasma components, co-administered medications, or structurally related compounds. The method’s successful validation in complex biological matrices demonstrates robust extraction recoveries of 96.50-99.25% across clinically relevant concentrations, with relative standard deviations below 4%. Comprehensive application to real plasma samples from drug-treated human volunteers confirms the method’s practical utility for pharmacokinetic studies. This SPAAC-based fluorometric approach offers significant advantages over existing LC-MS/MS methods, including simplified sample preparation, reduced instrumentation costs, enhanced accessibility, and suitability for high-throughput therapeutic drug monitoring applications while maintaining comparable analytical performance.