Accurate antibiotic residue detection in honey is crucial for food safety and veterinary drug regulation in apiculture. Honey is a particularly challenging matrix. The high sugar content， strong viscosity， and pronounced matrix effects of the sample make it difficult to extract and detect target analytes effectively. Moreover， multiple antibiotics are used in beekeeping， while existing residue standards often provide incomplete coverage. These limitations highlight the urgent need for a robust and environmentally friendly multi-residue analytical method. In this study， a green and sensitive method was developed for the simultaneous determination of 38 antibiotic residues， including chloramphenicol， quinolones， and sulfonamides， in honey. Sample preparation was based on magnetic dispersive solid-phase extraction （MDSPE） using a mixed-mode hydrophilic-lipophilic balance （mHLB） magnetic sorbent. This sorbent combines hydrophilic and hydrophobic interactions， enabling efficient extraction of chemically diverse antibiotics. Method optimization was performed systematically. A Plackett-Burman design was used to screen seven factors influencing extraction. The sorbent dosage， sample volume， and extraction time were identified as the most significant. These variables were further optimized by response surface methodology， which minimized the number of experimental runs while clarifying interactions among factors. The final protocol required only 10 mg of sorbent， 5 mL of sample extract， and 1 mL of methanol for elution. This miniaturized approach represents an environmentally sustainable method. Chromatographic separation was achieved on a Poroshell 120 SB-C18 column （100 mm×2.1 mm，2.7 μm） using a gradient of acidified methanol and water. Detection was carried out by high performance liquid chromatography-tandem mass spectrometry （HPLC-MS/MS） with electrospray ionization operated in both positive and negative modes， and multiple reaction monitoring ensured high sensitivity and selectivity. The method was fully validated. Excellent linearity was obtained for all analytes in the range of 0.02-200 μg/L， with correlation coefficients above 0.99. Limits of detection ranged from 0.01 to 0.12 μg/kg， while limits of quantification ranged from 0.02 to 0.39 μg/kg. Recoveries at three spiked levels ranged from 70.5% to 109.3%， with relative standard deviations （RSDs） below 10.8%. Matrix effects ranged from 0.82 to 1.19， indicating effective suppression of interference and reliable quantification in complex honey samples. The method was successfully applied to 42 commercial honey batches from different geographical regions. Several antibiotics were detected， including chloramphenicol， as well as other compounds without established maximum residue limits. These findings indicate potential misuse of antibiotics in beekeeping and reveal important gaps in current regulatory frameworks， underscoring the need for continuous monitoring and timely updates to residue standards. In addition to high sensitivity and wide applicability， the method demonstrated strong environmental advantages. Green performance was assessed using the AGREEprep tool， which evaluates twelve principles of sustainable sample preparation. The method achieved a high score of 0.73， reflecting minimal solvent consumption， low sorbent use， low energy demand， and negligible hazardous waste. These features align with the principles of green analytical chemistry and significantly reduce the environmental impact of residue analysis. In conclusion， this work establishes an MDSPE-HPLC-MS/MS for multi-class antibiotic residue detection in honey. The method combines simplicity， sensitivity， robustness， and environmental compatibility. It provides reliable technical support for risk assessment and regulatory improvement of antibiotic residues in honey， and it offers a valuable reference for the development of green analytical methods for trace contaminants in other complex food and environmental matrices.
蜂蜜中抗生素残留的精准检测是保障蜂产品质量与规范用药监管的关键环节。针对蜂蜜中糖类含量高、黏度大、基质干扰强以及抗生素种类多样、残留标准覆盖有限等问题，本工作构建了一种基于混合模式亲水-疏水平衡（mHLB）磁性材料的磁分散固相萃取-高效液相色谱-串联质谱（MDSPE-HPLC-MS/MS）方法，用于蜂蜜中氯霉素、喹诺酮类和磺胺类共38种抗生素残留的定量分析。通过Plackett-Burman设计筛选影响mHLB萃取效率的关键因素，并结合响应面法系统优化MDSPE的前处理步骤，在确保分析性能的同时显著简化操作流程。在优化的MDSPE条件下（10 mg mHLB吸附5 mL样品提取液，1 mL甲醇洗脱），38种抗生素在0.02~200 μg/L范围内线性关系良好（R²>0.99），检出限为0.01~0.12 μg/kg，定量限为0.02~0.39 μg/kg，回收率为70.5%~109.3%，相对标准偏差低于10.8%。该方法可有效控制基质干扰，适用于痕量抗生素的准确定量。在42批次实际样品的应用中，检出部分禁用或尚未纳入限量目录的抗生素，提示蜂产品中潜在的用药风险与监管局限性。此外，与已有方法比较，本方法操作简便，灵敏度高，适用性强，绿色评分达0.73，具备良好的环境友好性和方法学先进性。本研究为蜂蜜中抗生素残留的风险监测和标准完善提供了可靠的技术支持，并可为复杂基质中痕量污染物的绿色分析方法的开发提供参考。
Keywords: antibiotic residues; green analytical chemistry; honey; magnetic dispersive solid-phase extraction （MDSPE）; mixed-mode sorbent.