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| 微波消解-高效液相色谱-串联质谱法定量检测牛乳中糠氨酸 |
| Quantitative detection of furosine in milk by microwave hydrolysis-ultra performance liquid chromatography-tandem mass spectrometry |
| 投稿时间：2025-04-21 修订日期：2025-06-12 |
| DOI： |
| 中文关键词: 微波消解 高效液相色谱-串联质谱法 牛奶 糠氨酸 响应面优化 |
| 英文关键词:microwave hydrolysis Liquid chromatography-tandem mass spectrometry Milk Furosine Response surface optimization |
| 基金项目:国家乳业技术创新中心项目(2023-KFKT-27） |
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| 中文摘要: |
| 建立微波消解-高效液相色谱-串联质谱法（ultra performance liquid chromatography-tandem mass spectrometry, UPLC-MS/MS）检测牛乳中糠氨酸含量的方法。方法 运用单因素试验及响应面法优化微波水解最佳条件。在最佳水解条件下获得的水解液过滤定容后，滤液经6.0 g/L的乙酸铵溶液稀释10倍后进样分析，以0.1%甲酸水溶液和甲醇溶液为流动相进行梯度洗脱，T3液相色谱柱（100 mm×2.1 mm, 1.8 μm）进行分离，在电喷雾离子源正离子模式（the positive ion mode of electrospray ion source, ESI+）下，选用多反应监测（multiple reaction monitoring, MRM）模式检测，外标法定量。结果 牛奶中糠氨酸的最佳微波水解条件为水解温度160 ℃、盐酸浓度8.1 mol/L、水解时间52 min，影响因素主次顺序为盐酸浓度＞水解时间＞水解温度。牛奶基质中糠氨酸在0.005~0.500 μg/mL质量浓度范围内呈良好的线性关系，相关系数（r）为0.9958。定量限（limit of quantification, LOQ）和检出限（limit of detection, LOD）分别为0.20 μg/mL和0.07 μg/mL。平均加标回收率为89.09%~97.33%，相对标准偏差（relative standard deviations, RSDs）为3.37％～7.02％ (n=6)。结论 该方法高效、准确，与传统的水解方法（12~24 h）相比，微波水解将水解时间缩短至52 min，且与NY/T 939—2016《巴氏杀菌乳和UHT灭菌乳中复原乳的鉴定》中常规水解方法对比，检测结果无显著差异（P＞0.05），可为乳制品质量控制及热处理工艺评估提供技术参考。 |
| 英文摘要: |
| ABSTRACT: Objective To establish a method for the detection of furosine in milk by microwave hydrolysis-ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Methods The optimal conditions for microwave hydrolysis were optimized by single factor experiments and response surface methodology. The hydrolysis solution obtained under the optimal hydrolysis conditions was filtered to a constant volume. The hydrolysate was diluted 10-fold with 6.0 g/L ammonium acetate solution and then analyzed. Gradient elution was performed with 0.1% formic acid aqueous solution and methanol solution as mobile phases, and the separation was carried out on a T3 liquid chromatography column (100 mm × 2.1 mm, 1.8 μm). Under the positive ion mode of electrospray ion source（ESI+）, multiple reaction monitoring (MRM) mode was selected for detection and external standard method was used for quantification. Results The optimal microwave hydrolysis conditions for furosine in milk were hydrolysis temperature of 160 ℃, hydrochloric acid concentration of 8.1 mol/L, and hydrolysis time of 52 min. The primary-to-secondary order of influencing factors was hydrochloric acid concentration>hydrolysis time>hydrolysis temperature. Furosine in milk showed a good linear relationship within the range of 0.005–0.500 μg/mL, with a correlation coefficient (r) of 0.9958. The limits of quantitation (LOQ) and limits of detection (LOD) were 0.2 μg/mL and 0.07 μg/mL, respectively. The average recoveries were 89.09%~97.33%, with relative standard deviations (RSDs) of 3.37%–7.02% (n=6). Conclusion This method is efficient and accurate. Compared with the traditional hydrolysis methods (12-24 hours), microwave hydrolysis shortens the hydrolysis time to 52 minutes, and there is no significant difference (P>0.05) in the detection results compared with the conventional hydrolysis methods in the industry standard NY/T 939-2016 identification of reconstituted milk in pasteurized and UHT milk. It can provide technical reference for quality control and heat treatment process evaluation of dairy products. |
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