Enhanced specificity in molecular imprinting: A dual-role chitosan-bentonite substrate coupled with hierarchical monomers for ultrasensitive diclofenac potassium and metabolite enrichment

Diclofenac potassium (DCF-K) is a widely used nonsteroidal anti-inflammatory drug with enhanced therapeutic effects mediated by its bioactive metabolites, particularly 4′-hydroxydiclofenac (4′-OH-DCF) and 5-hydroxydiclofenac (5-OH-DCF). However, the accurate detection of DCF-K and its metabolites in complex biological matrices remains challenging due to trace concentrations and matrix interference. To address this challenge, a chitosan-bentonite molecularly imprinted polymer (CB-MIP) was developed using a dual-role strategy, where chitosan-bentonite (CB) serves as both substrate and functional monomer, combined with a hierarchical functional monomer system (acrylamide as an auxiliary monomer) to enhance binding affinity and selectivity for the specific capture of DCF-K and its metabolites. Comprehensive characterization confirmed the successful synthesis of CB-MIP, revealing improved stability, hydrophilicity, and a mesoporous structure. CB-MIP exhibited good adsorption capacity for DCF-K (17.2× higher than CB), with Langmuir isotherm and pseudo-second-order kinetics indicating monolayer chemisorption. Selectivity studies demonstrated good recognition capability for DCF-K, achieving a selectivity factor up to 77.3 against structural analogs and co-administered drugs. Furthermore, cluster analysis revealed the CB-MIP sensitivity to DCF-K concentration variations as low as 0.3 μg·mL −1 in complex matrices. Mechanistic studies identified electrostatic and hydrogen bonding interactions as the primary binding forces, validated by electrostatic potential simulations, hydrogen-bond modeling, and spectroscopic analyses. Practical application in mouse plasma and liver samples confirmed efficient enrichment and detection of DCF-K and metabolites (4′-OH-DCF, 5-OH-DCF, diclofenac acyl glucuronide) using CB-MIP coupled with HPLC/UPLC-MS/MS, achieving high sensitivity (limit of detection: 0.231 μg·mL −1 in liver) and reproducibility (relative standard deviation: 1.22 %–5.81 %). This work establishes CB-MIP as a robust pretreatment material for targeted drug/metabolite analysis in complex biological systems.