Determination of Multiple Receptor Agonist Drug Residues in Pork by Pass-type SPE and Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry
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摘要: 建立了猪肉中多种受体激动剂药物残留同时检测的超高效液相色谱-串联质谱方法。样品经酶解后，以酸化乙腈为提取液，PRIME HLB通过式净化的方式，超高效液相色谱-串联质谱法（ultra performance liquid chromatography-tandem mass spectrometry，UPLC-MS/MS），电喷雾正离子模式，多反应监测采集（MRM），外标法定量。结果表明，11种受体激动剂在0.1～100 μg/L范围内线性关系良好，相关系数在0.9992～0.9999之间，检出限为0.5 μg/kg，得到的平均回收率为81.3%～117.6%，相对标准偏差RSD值在0.7%～6.5%之间。该方法过程简单，适用于猪肉中多种受体激动剂药物残留的同时检测。
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关键词:
- 猪肉 /
- 通过式固相萃取 /
- 受体激动剂 /
- 超高效液相色谱-串联质谱法
Abstract: A method was established for the simultaneous determination of multiple receptor agonist drug residues in pork by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). After enzymatic hydrolysis, the samples were extracted by acidic acetonitrile, and cleaned up by pass-type PRIME HLB, then detected by UPLC-MS/MS in the positive mode and multiple reaction monitoring (MRM) mode, and quantified by the external standard method. Results showed that, good linear relationship were obtained for the 11 multiple receptor agonist drugs in the range of 0.1~100 μg/L, with coefficients of determination between 0.9992 and 0.9999. The limits of detection (LOD) for the method were 0.5 μg/kg, the average recoveries of all compouds were between 81.3% and 117.6%, with the relative standard deviations varying from 0.7% to 6.5%. The method was simple and would be suitable for simultaneous determination of multiple receptor agonist drug residues in pork. -
图 1 11种物质标准溶液的总离子流图（1.0 μg/L）
Figure 1. TIC chromatograms of standard solution of eleven substances (1.0 μg/L)
注：1：西马特罗；2：赛拉嗪；3：可乐定；4：沙丁胺醇；5：安普乐定；6：替扎尼定；7：克伦特罗；8：赛庚啶；9：溴莫尼定；10：莱克多巴胺；11：特布他林。
图 2 11种物质标准溶液的MＲM图（0.5 μg/L）
Figure 2. MＲM chromatograms of standard solution of eleven substances (0.5 μg/L)
注：A：西马特罗；B：赛拉嗪；C：可乐定；D：沙丁胺醇；E：安普乐定；F：替扎尼定；G：克伦特罗；H：赛庚啶；I：溴莫尼定；J：莱克多巴胺；K：特布他林；谱图中响应值高的为定量离子，响应值低的为定性离子。
图 3 不同乙腈浓度下各化合物回收率对比
Figure 3. Comparison of recoveries using different acetonitrile concentrations
图 4 不同甲醇浓度比例下各化合物回收率对比
Figure 4. Comparison of recoveries using different Methanol concentrations
图 5 不同甲酸浓度下各化合物回收率对比
Figure 5. Comparison of analyte recoveries using different concentrations of formic acid
图 6 不同净化条件下各化合物回收率对比
Figure 6. Comparison of recoveries using different clean-up methods
表 1 流动相梯度洗脱程序
Table 1. Condition of gradient elution
时间（min） A（%） B（%） 流速（mL/min） 0.00 95.00 5.00 0.4 1.80 70.00 30.00 0.4 2.50 30.00 70.00 0.4 3.00 10.00 90.00 0.4 5.00 10.00 90.00 0.4 5.20 95.00 5.00 0.4 7.50 95.00 5.00 0.4 
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表 2 11种物质质谱参数
Table 2. Chromatography-mass spectrometry parameters of eleven substances
化合物名称 母离子质荷比 碰撞电压（V） 子离子质荷比 碰撞能量（eV） 莱克多巴胺 302.2 100 164* 17 302.2 100 284 9 溴莫尼定 292.2 152 211.9* 33 292.2 152 44.1 37 赛庚啶 288.0 152 191* 37 288.0 152 96 33 克仑特罗 277.1 100 202.9* 17 277.1 100 258.9 9 替扎尼定 254.1 112 44.1* 33 254.1 112 210 33 安普乐定 245.2 154 44.2* 33 245.2 154 208.9 25 沙丁胺醇 240.2 100 148* 21 240.2 100 222 9 可乐定 230.0 152 44.1* 29 230.0 152 212.8 29 赛拉嗪 221.1 132 90* 25 221.1 132 163.9 29 西马特罗 220.0 92 201.9* 9 220.0 92 159.9 17 特布他林 226.1 92 151.9* 21 226.1 92 106.9 41 注：*定量离子。
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表 3 酶解对α2型与H1型回收率的影响
Table 3. Effect of enzymatic hydrolysis on recovery of α2 and H1
化合物名称 回收率（%） 样品1 样品2 样品3 样品4 安普乐定 89.0 80.2 90.1 86.7 溴莫尼定 73.6 78.4 76.8 80.1 替扎尼定 82.2 79.4 83.3 78.4 可乐定 84.6 82.9 80.4 79.6 赛拉嗪 90.2 89.6 87.6 88.3 克伦特罗 89.6 91.2 92.5 90.7 赛庚啶 95.4 93.8 90.6 91.5
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表 4 11种物质标准溶液在空白基质提取液和初始流动相中的峰面积
Table 4. Peak area of 11 substances in blank matrix extracting solution and initial mobile phase
化合物名称 空白基质提取液 初始流动相 ME(%) 莱克多巴胺 17705.11 20053.88 88.3 溴莫尼定 1863.58 3217.69 57.9 赛庚啶 13750 14972.84 91.8 克伦特罗 27028.87 30935.23 87.4 替扎尼定 13219.79 13899.8 95.1 安普乐定 7241.31 7678.38 94.3 沙丁胺醇 19672.31 48998.62 40.1 可乐定 7567.76 8726.5 86.7 赛拉嗪 11383.45 12631.64 90.1 西马特罗 80843.8 68749.25 117.6 特布他林 15022.77 35916.55 41.8
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表 5 11种物质的线性范围、回归方程、相关系数和检出限
Table 5. Linear ranges, regression equation, R2 and limits of detection (LODs) of 11 substances
化合物名称 线性范围（μg/L） 回归方程 相关系数 检出限（μg/kg） 沙丁胺醇 0.1~100 y=36850.99x+11877.47 0.9999 0.5 安普乐定 0.1~100 y=6296.34x+251.75 0.9999 0.5 溴莫尼定 0.1~100 y=2600.91x+1326.03 0.9997 0.5 替扎尼定 0.1~100 y=19281.71x+3161.48 0.9998 0.5 可乐定 0.1~100 y=12103.94x−2670.20 0.9997 0.5 莱克多巴胺 0.1~100 y=10678.45x+10338.17 0.9994 0.5 赛拉嗪 0.1~100 y=12523.58x−1830.73 0.9998 0.5 克伦特罗 0.1~100 y=26359.43x+2519.20 0.9998 0.5 赛庚啶 0.1~100 y=10204.60x+7333.19 0.9992 0.5 西马特罗 0.1~100 y=49285.47x+3753.23 0.9995 0.5 特布他林 0.1~100 y=25443.57x+795.51 0.9996 0.5
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表 6 11种物质的加标回收试验及精密度（n=6）
Table 6. Recovery and precision for 11 substances (n=6)
化合物名称 添加水平（μg/kg） 平均回收率（%） RSD（%） 沙丁胺醇 0.50
1.00
10.081.3
88.2
109.85.9
2.2
0.7安普乐定 0.50
1.00
10.0105.7
98.2
108.23.1
1.7
2.8溴莫尼定 0.50
1.00
10.098.9
98.5
103.26.1
4.2
3.4替扎尼定 0.50
1.00
10.099.6
97.1
109.03.7
2.0
1.2可乐定 0.50
1.00
10.098.9
96.4
109.15.4
3.6
1.1莱克多巴胺 0.50
1.00
10.0114.0
116.0
108.64.1
2.8
1.1赛拉嗪 0.50
1.00
10.085.5
93.0
107.93.2
3.0
1.8克伦特罗 0.50
1.00
10.085.5
89.4
107.85.8
6.5
1.2赛庚啶 0.50
1.00
10.0115.9
117.6
114.42.0
3.5
1.6西马特罗 0.50
1.00
10.087.3
83.1
89.44.1
6.1
6.4特布他林 0.50
1.00
10.089.3
93.1
99.43.5
4.0
2.4
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