Determination of Shrimp Allergens in Meat Products by Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry
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摘要: 目的：建立超高效液相色谱-串联质谱（ultra performance liquid chromatography-tandem mass spectrometry，UPLC-MS/MS）法检测肉制品中虾过敏原的定量方法。方法：选取基质较为复杂的肉制品（西式火腿、香肠和肉丸）作为研究对象。样品经磷酸盐缓冲液（Phosphate buffer solution，PBS）超声提取30 min，离心（10000 r/min，15 ℃，10 min）后加入乙腈去除脂肪，取样液加入内标肽段（2.5 μmol/L，40 μL）和胰蛋白酶（1 mg/mL，10 μL）在37 ℃下酶解16 h后上液质进行分析，样品经T3柱进行分离，0.1%甲酸-水溶液和乙腈梯度洗脱，多反应监测（multiple reaction monitoring，MRM）正离子模式采集数据，内标法定量。结果：采用该方法测定肉制品中虾过敏原蛋白含量，其中定量肽段在0.001~2.0 μmol/L范围内，线性关系良好，决定系数R2为1.0000，检出限为0.67 mg/kg，定量限为2.00 mg/kg；在三个加标浓度水平下，回收率为83.2%~104.5%，精密度为2.63%~7.92%。结论：该方法具有特异性强、灵敏度高和回收率好等优势，适用于肉制品中虾过敏原的筛查定量。
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关键词:
- 超高效液相色谱-串联质谱法 /
- 肉制品 /
- 虾过敏原 /
- 特征肽段 /
- 原肌球蛋白
Abstract: Objective: To establish a method for the determination of shrimp allergens in meat products by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Methods: Meat products (cooked ham, sausages and meatballs) with complex matrix components were selected as the detection target. The samples were extracted by ultrasonic in phosphate buffer solution (PBS) for 30 min, and acetonitrile was added to remove fat after centrifugation (10000 r/min, 15 ℃, 10 min). Finally, trypsin (1 mg/mL, 10 μL) was added to the sample diluents which had been mixed with internal peptide (2.5 μmol/L, 40 μL), and the enzymolysis process lasted 16 h at 37 ℃ before UPLC-MS/MS analysis. The samples were separated on a T3 column, and eluted by gradient elution with 0.1% formic acid aqueous solution and acetonitrile. The data was collected by multiple reaction monitoring (MRM) in positive ion mode, and quantified by internal standard method. Results: The method was used to determine the content of shrimp allergen in meat products, and the quantitative peptide had a good linear relationship with the determination coefficients 1.0000 in the range of 0.001~2.0 μmol/L. The limits of detection was 0.67 mg/kg, and the limits of quantification was 2.00 mg/kg. The recovery was 83.2%~104.5% and the precision was 2.63%~7.92% at three spiked levels. Conclusion: The method has the advantages of strong specificity, high sensitivity and good recovery. It was suitable for the screening and quantification of shrimp allergens in meat products. -
图 1 Q-Extractive plus Orbitrap高分辨质谱中特征肽段IQLLEEDLER和IVELEEER的碎片离子
Figure 1. Fragment ions of signature peptides IQLLEEDLER and IVELEEER by Q-Extractive plus Orbitrap HRMS
表 1 质谱检测和定量过敏原的设置
Table 1. Analytical settings used for detection and quantification of allergens by mass spectrometry (MS)
肽段名称简写 肽段类型 序列 分子量（g/mol） 母离子（m/z） 子离子（m/z） 锥孔电压（V） 碰撞能量（eV） IR-10 定性 IQLLEEDLER 1257.42 629.6 1016.8# 2 21 242.2 2 21 IR-10* 内标 IQL*LEEDLER 1264.42 632.9 1023.7# 2 21 242.2 8 21 IR-9 定量 IVELEEER 1129.29 565.5 917.6# 4 17 213.3 4 17 IR-9* 内标 IVEL*EEER 1136.29 569.0 924.7# 38 22 213.3 38 22 注：#表示定量离子；IR-10*为IR-10的内标；IR-9*为IR-9的内标。 
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表 2 与其他检测虾过敏原技术对比
Table 2. Comparison with other techniques for detecting shrimp allergens
检测方法 样品基质 目标物 定量限
（mg/kg）对比数据来源 UPLC-MS/MS 西式火腿、
肉丸等原肌球蛋白
特征肽段2.00 本方法 LC-HRMS/MSa 鱼棒、蟹爪等 原肌球蛋白
特征肽段3.5 Stella等[29] HPLC-MSb 鸡肉肠、曲奇等 原肌球蛋白
特征肽段1.6 Wang等[30] qPCR 虾馅模型 16S rRNA 1 Fernandes等[33] ELISA 鱼糜、鳕鱼皮等 原肌球蛋白 1 Werner等[34] 电化学免疫
传感器细胞 原肌球蛋白
Pen a 10.15 μg/mL Jiang等[35] 注：a液相色谱-高分辨率串联质谱（liquid chromatography high-resolution tandem mass spectrometry，LC-HRMS/MS）；b高效液相色谱-质谱（ high performance liquid chromatography mass spectrometry，HPLC-MS）。 
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表 3 加标回收率和精密度（n=6）
Table 3. Recoveries and relative standard deviations (n=6)
添加浓度（mg/kg） 测定浓度（mg/kg） 回收率（%） 精密度（%） 2.00 1.67 83.2 6.44 5.25 4.54 86.5 7.92 20.00 20.90 104.5 2.63 注：mg/kg指过敏原蛋白质量/称样量。 
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表 4 样品测定结果（n=3）
Table 4. Determination of allergens in samples (n=3)
类别 样品编号 测定含量（mg/kg） 标签 香肠 1 46.7±4.2 √ 2 2.3±0.1 √ 3 2.7±0.2 √ 4 ND √ 西式火腿 5 ND √ 6 NQ — 7 ND √ 8 ND — 9 ND √ 10 ND — 11 ND — 12 ND √ 13 ND √ 14 ND — 15 ND √ 16 ND √ 17 ND — 肉丸 18 448.1±31.4 √ 19 ND — 20 ND — 注：—表示未标出；ND表示小于方法检出限；NQ表示小于方法定量限；编号1~4为香肠，5~17为西式火腿，18~20为肉丸。 
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