目的 建立免疫磁分离-酶联免疫分析（immunomagnetic separation-enzyme-linked immunoassay，IMS-ELISA）法测定5-羟甲基糠醛（5-hydroxymethylfurfural，5-HMF），并用于炼蜜工艺参数优化。方法 将抗5-HMF抗体修饰在磁珠表面，以实现对5-HMF的特异捕获与分离富集。采用生物素化5-HMF-BSA偶联物（BSA为牛血清白蛋白）作为竞争配体，配合辣根过氧化物酶-链霉亲和素（HRP-SA），以3,3′,5,5′-四甲基联苯胺（tetramethylbenzidine，TMB）为底物进行比色显色。通过磁分离实现5-HMF的富集，有效去除基质干扰并提高检测灵敏度。并开展线性关系考察、检出限、专属性、精密度与准确度等方法学验证。随后开展近线检测应用：选取中药蜜丸制剂炼蜜过程中的关键影响因素（温度、压力、时间、储存时长与复热温度），以IMS-ELISA检测5-HMF含量。结果 建立的IMS-ELISA方法在0.4～160.0 ng/mL内线性良好（R²＝0.997 3），检出限0.4 ng/mL；对糠醛及5-HMF无明显交叉反应；批内RSD为4.29%；实际样品含量检测结果与HPLC法无显著性差异（P＞0.05）。在IMS-ELISA的应用中，成功对5-HMF含量进行了监测，且适用性良好。试验结果表明，推荐75～85 ℃、20.0 kPa、10～15 min作为炼蜜的参考参数；储存时长≥48 h及复热温度≥80 ℃均促进5-HMF生成。结论 基于IMS-ELISA的5-HMF检测方法具有高灵敏、强专属性和低成本的特点，可用于现代中药蜜丸制剂炼蜜、合坨等生产单元中的5-HMF近线监测，为中药蜜丸制剂的工艺优化提供方法学依据。

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.

R283.6

国家重点研发计划（2023YFC3504505）；国家自然科学基金资助（82404857）；国家自然科学基金资助（82274110）；北京科技新星交叉项目（20230484458）；中华中医药学会立项项目（CACM-2023-QNRC2-B10）；京津冀科技创新协同（Z251100005225010）