Simultaneous determination of antibiotics in tap water with automatic solid phase extraction-UHPLC-MS/MS
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摘要:
目的 建立一种同时检测自来水中四环素类、大环内酯类、氟喹诺酮类、酚类和磺胺类27种抗生素残留的全自动固相萃取 – 超高效液相色谱 – 串联质谱分析方法。 方法 采集200 mL自来水样本在24 h内加入内标混合溶液,经玻璃纤维滤纸过滤后,调节pH到7.5~8.5;在全自动固相萃取仪上使用固相萃取柱完成样本富集,用甲醇溶液洗脱后氮吹至干,20 %甲醇水溶液复容,经T3色谱柱在40 %乙腈甲醇 – 0.2 %甲酸水溶液的流动相下梯度分离,高效液相色谱串联质谱检测,内标法定量分析;并采用该方法对浙江省某农村地区的70份自来水样本进行检测。 结果 实验结果显示,27种抗生素在1~100 ng/mL范围内线性关系良好,r值为0.9963~1.0000;该方法检出限(MDL)为0.004~0.296 ng/L,方法定量限(MQL)为0.013~0.988 ng/L,平均回收率为70.0 %~120.8 %;通过对浙江省某农村70份自来水样本进行检测,检测到4种抗生素残留,皆为大环内酯类抗生素,分别为罗红霉素[(0.162~2.858)ng/L]、克拉霉素[(0.063~0.849)ng/L]、红霉素[(0.100~1.804)ng/L]和替米考星[(0.167~1.418)ng/L],其余23种抗生素均未检出。 结论 本研究建立的全自动固相萃取 – 超高效液相色谱 – 串联质谱法操作简单、省时省力、灵敏度高、准确可靠,可适用于自来水中27种抗生素残留的快速检测。 -
关键词:
- 超高效液相色谱 – 串联质谱法 /
- 全自动固相萃取 /
- 抗生素 /
- 自来水
Abstract:Objective To establish a rapid method for simultaneous determination of 27 antibiotics in tap water with ultra high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) combined with automatic solid phase extraction. Methods After added internal standard solutions of 27 antibiotics within 24 hours, the tap water sample (200 mL) was filtered with glass fiber filters and adjusted for pH value of 7.5 – 8.5. The reference antibiotics in the tap water samples were concentrated with extraction column in the automated solid phase extraction instrument and then eluted with methanol solution. The reference antibiotics in the eluate was reconstituted into constant solution with 20% methanol and separated gradiently with T3 chromatography column using 40% acetonitrile methanol and 0.2% formic acid solution as mobile phase; and finally detected with UHPLC-MS/MS and quantified with internal standard method. Thereafter, 70 tap water samples were collected in rural areas of Zhejiang province and 27 antibiotics in the samples were detected simultaneously using the established method. Results A good linearity was observed for detections of the 27 antibiotics at the concentration of 1 to 100 ng/mL, with the correlation coefficient ranging from 0.9963 to 1.0000. For the established method, the detection limits were between 0.004 – 0.296 ng/L; the quantification limits were from 0.013 to 0.998 ng/L; and the mean recoveries ranged from 70.0% – 120.8%. For the 70 rural tap water samples, only four macrolide antibiotics were detected, including roxithromycin, clarithromycin, erythromycin, and tilmicosin, with the concentration ranges (ng/L) of 0.162 – 2.858, 0.063 – 0.849, 0.100 – 1.804, and 0.100 – 1.804, respectively. Conclusion The established method is simple to operate, time- and labor-saving; the method is also of high sensitivity, accuracy and reliability; and the method could be applied to rapid determination of 27 antibiotics in tap water. -
Key words:
- UHPLC-MS/MS /
- automatic solid phase extraction /
- antibiotics /
- tap water
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表 1 27种抗生素药物及其6种内标物质的质谱参数
化合物名称 保留时间(min) 母离子 定量子离子 定性子离子 去簇电压(V) 碰撞电压(定量/定性,V) 环丙沙星 4.97 332.1 288.1 245.1 80 25/33 氧氟沙星 4.89 362.2 318.1 261.1 80 26/38 诺氟沙星 4.91 320.1 276.1 233.1 80 26/35 培氟沙星 4.95 334.1 316.1 290.2 80 27/25 恩诺沙星 5.10 360.1 316.1 245.1 80 28/36 达氟沙星 5.04 358.1 340.1 314.1 77 30/24 双氟沙星 5.35 400.1 356.1 299.1 80 28/41 洛美沙星 5.07 352.0 265.0 308.1 80 33/28 氟苯尼考 6.11 356.0 219.2 119.0 – 70 – 16/– 23 氯霉素 6.41 321.0 152.1 256.9 – 75 – 24/– 17 甲砜霉素 5.31 353.9 289.9 184.9 – 75 – 18/– 28 强力霉素 5.87 445.0 428.1 154.1 80 24/38 四环素 5.09 445.1 410.2 427.1 80 24/29 金霉素 5.66 479.1 462.0 444.0 80 24/28 土霉素 4.90 461.2 426.2 443.2 80 25/17 阿奇霉素 5.32 749.6 591.5 158.0 100 29/34 罗红霉素 6.85 837.6 679.5 158.1 50 30/37 克拉霉素 6.82 748.6 590.4 158.0 100 27/33 脱水红霉素 6.31 734.5 576.4 158.0 30 26/36 替米考星 5.63 869.5 696.5 174.2 100 52/52 螺旋霉素 5.22 843.5 142.1 174.2 60 45/50 甲氧苄氨嘧啶 4.75 291.1 230.1 123.1 95 33/34 磺胺嘧啶 4.64 251.1 156.0 92.0 40 22/38 磺胺甲恶唑 6.03 254.1 156.0 108.0 65 22/36 磺胺二甲嘧啶 5.36 279.1 186.1 156.0 60 23/27 乙酰化磺胺甲嘧啶 5.39 321.1 134.0 186.0 50 35/25 乙酰化磺胺甲恶唑 6.20 296.0 198.0 134.0 70 24/32 阿奇霉素 – D3 5.30 752.4 594.5 158.0 100 38/44 磺胺甲恶唑 – D4 6.01 258.1 160.0 112.0 65 22/36 氯霉素 – D5 6.39 326.0 157.0 262.0 – 75 – 23/– 16 四环素 – D6 5.08 451.2 416.2 433.3 80 26/18 罗红霉素 – D7 6.83 844.5 686.4 158.2 50 30/37 环丙沙星 – D8 4.95 340.0 296.0 249.0 80 24/33 表 2 27种抗生素药物的线性参数及仪器性能参数
类别 名称 线性方程 r 值 IDL(µg/L) IQL(µg/L) 日内精密度(%) 日间精密度(%) 氟喹诺酮类 环丙沙星 y = 1.99762x + 0.00030 0.9994 0.0344 0.1147 1.36 1.64 氧氟沙星 y = 11.55295x – 0.01254 0.9997 0.0057 0.0191 1.43 1.83 诺氟沙星 y = 1.71629x + 0.00866 0.9993 0.0364 0.1213 1.98 1.79 培氟沙星 y = 9.11526x – 0.05177 0.9994 0.0417 0.1392 1.75 3.60 恩诺沙星 y = 7.11977x + 0.04373 0.9991 0.0074 0.0246 1.65 1.79 达氟沙星 y = 15.90120x – 0.12187 0.9995 0.0196 0.0652 1.84 2.81 双氟沙星 y = 6.39833x – 0.01570 0.9997 0.0178 0.0592 2.64 3.69 洛美沙星 y = 6.64743x + 0.03654 0.9992 0.0095 0.0318 2.36 2.67 酚类 氯霉素 y = 1.17566x + 0.00355 1.0000 0.0141 0.0470 3.53 3.21 甲砜霉素 y = 0.61883x + 0.01184 0.9991 0.0256 0.0854 2.96 3.14 氟苯尼考 y = 0.16734x + 0.00389 0.9993 0.0341 0.1137 3.16 3.65 四环素类 四环素 y = 0.84604x + 0.00306 0.9992 0.0587 0.1958 1.28 1.35 土霉素 y = 0.98315x – 0.00257 0.9997 0.0640 0.2134 1.02 3.04 金霉素 y = 0.33857x + 0.00400 0.9973 0.0830 0.2767 1.29 1.76 强力霉素 y = 1.57520x + 0.00748 0.9995 0.0378 0.1259 1.54 1.72 大环内酯类 阿奇霉素 y = 0.48366x + 0.00832 1.0000 0.0294 0.0979 1.57 1.99 罗红霉素 y = 1.39340x + 0.02052 0.9995 0.0037 0.0124 2.30 1.89 克拉霉素 y = 1.31656x + 0.02119 0.9996 0.0026 0.0085 3.06 2.46 红霉素 y = 1.59802x + 0.03415 0.9990 0.0058 0.0192 1.65 2.16 替米考星 y = 0.51372x + 0.00475 0.9995 0.0114 0.0380 3.13 2.93 螺旋霉素 y = 0.01871x + 0.00008 0.9993 0.0829 0.2763 3.95 6.63 磺胺类 甲氧苄氨嘧啶 y = 5.58975x + 0.12082 0.9963 0.0015 0.0049 3.44 2.98 磺胺嘧啶 y = 1.56764x + 0.01207 0.9998 0.0042 0.0140 2.34 2.08 磺胺甲恶唑 y = 1.35660x + 0.00257 1.0000 0.0125 0.0415 1.98 2.00 磺胺二甲嘧啶 y = 3.23837x + 0.04558 0.9993 0.0032 0.0108 3.03 3.53 乙酰化磺胺甲恶唑 y = 0.72177x – 0.00201 0.9999 0.0283 0.0943 2.88 2.39 乙酰化磺胺甲嘧啶 y = 0.77589x – 0.00262 0.9999 0.0095 0.0315 3.26 2.95 表 3 方法回收率及MDL和MQL
类别 化合物 加标回收率(%) MDL(ng/L) MQL(ng/L) 低浓度 中浓度 高浓度 平均值 氟喹诺酮类 环丙沙星 88.7 88.5 103.1 93.4 0.092 0.307 氧氟沙星 73.5 72.8 73.5 73.3 0.020 0.065 诺氟沙星 103.8 96.9 111.1 103.9 0.088 0.292 培氟沙星 87.0 96.2 97.4 93.5 0.112 0.372 恩诺沙星 86.1 104.9 108.3 99.8 0.019 0.062 达氟沙星 85.8 90.5 90.9 89.1 0.055 0.183 双氟沙星 83.6 96.3 99.5 93.1 0.048 0.159 洛美沙星 111.8 105.5 106.6 108.0 0.022 0.074 酚类 氯霉素 101.1 89.5 87.6 92.7 0.038 0.127 甲砜霉素 117.0 107.3 126.3 116.9 0.055 0.183 氟苯尼考 118.2 101.8 101.8 107.2 0.080 0.265 四环素类 四环素 77.6 84.9 88.8 83.8 0.175 0.584 土霉素 93.6 114.3 135.8 114.6 0.140 0.465 金霉素 89.6 65.2 55.2 70.0 0.296 0.988 强力霉素 84.9 63.2 64.8 70.9 0.133 0.444 大环内酯类 阿奇霉素 87.3 95.0 109.9 97.4 0.075 0.251 罗红霉素 86.3 102.1 107.7 98.7 0.009 0.031 克拉霉素 115.0 129.1 103.9 116.0 0.006 0.018 红霉素-水 79.6 101.1 115.9 98.9 0.015 0.049 替米考星 73.8 97.1 98.0 89.6 0.032 0.106 螺旋霉素 101.6 65.2 64.4 77.1 0.269 0.896 磺胺类 甲氧苄氨嘧啶 101.1 99.1 94.2 98.1 0.004 0.013 磺胺嘧啶 66.9 68.4 74.8 70.0 0.015 0.050 磺胺甲恶唑 114.0 104.6 108.6 109.0 0.029 0.095 磺胺二甲嘧啶 120.1 116.2 126.2 120.8 0.007 0.022 乙酰化磺胺甲恶唑 121.5 114.8 115.8 117.4 0.060 0.201 乙酰化磺胺甲嘧啶 119.0 108.7 133.0 120.2 0.020 0.065 -
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