Objective To develop an immunomagnetic separation-enzyme-linked immunosorbent assay (IMS-ELISA) for the determination of 5-hydroxymethylfurfural (5-HMF) and to evaluate its applicability for near-line monitoring and process optimization of the honey-refining process in traditional Chinese medicine (TCM) preparations. Methods Anti-5-HMF antibodies were immobilized on magnetic beads to enable selective capture and magnetic enrichment of 5-HMF. Biotinylated 5-HMF-BSA served as the competitive antigen, while horseradish peroxidase-streptavidin (HRP-SA) and 3,3′,5,5′-tetramethylbenzidine (TMB) were used for signal generation. Immunomagnetic separation was employed to reduce matrix interference and enhance analytical sensitivity. The method was systematically validated in terms of linearity, limit of detection (LOD), specificity, precision, and accuracy. Subsequently, the established IMS-ELISA was applied to near-line analysis by monitoring 5-HMF levels at key stages of the honey-processing workflow, including variations in temperature, pressure, processing time, storage duration, and reheating conditions. Results The IMS-ELISA exhibited good linearity over the concentration range of 0.4—160 ng/mL (R² = 0.997 3), with a detection limit of 0.4 ng/mL. No significant cross-reactivity was observed with structurally related compounds such as furfural or 5-methylfurfural. The intra-assay relative standard deviation was 4.29%, and the results obtained for real samples showed no significant difference from those measured by HPLC (P > 0.05). Near-line application demonstrated that honey-processing conditions of 75—85 ℃, 20.0 kPa, and 10—15 min were associated with relatively lower 5-HMF formation, whereas storage for ≥ 48 h and reheating at ≥ 80 ℃ markedly promoted 5-HMF accumulation. Conclusion The proposed IMS-ELISA method offers high sensitivity, good specificity, and low operational cost. It is suitable for near-line monitoring of 5-HMF during honey processing and pill manufacturing in TCM production, providing methodological support for process optimization and contributing to process visualization and intelligent manufacturing of TCM products.

5-hydroxymethylfurfural  /  immunomagnetic separation  /  enzyme-linked immunosorbent assay  /  traditional Chinese medicine honeyed pill preparation  /  honey-refining process