超高效液相色谱-三重四级杆质谱联用-内标法对豆芽中抗生素的测定

Determination of antibiotics in bean sprouts by UHPLC-MS/ MS-internal standard method

  • 摘要:
    目的 建立超高效液相色谱⁃三重四级杆质谱联用(UHPLC⁃MS/MS)⁃内标法同时测定豆芽中喹诺酮类、磺胺类、四环素类、大环内酯类和硝基咪唑类抗生素等59种抗生素。
    方法 在5.0 g样品中加入内标物、5.0 mL含0.1%甲酸的乙腈,超声提取,提取液过0.22 μm滤膜后直接进样1.0 μL,以含0.1%甲酸的甲醇溶液和含0.1%甲酸的2.0 mmol·L-1甲酸铵水溶液作为流动相进行梯度洗脱,在ACQUITY PREMIER BEH C18色谱柱上实现分离,采用电喷雾离子源正离子多反应监测模式检测,内标法定量。
    结果 豆芽中59种抗生素在10~1 000 μg·kg-1范围内线性关系良好,黄豆芽的方法检出限为0.1~3.0 μg·kg-1,定量限为0.4~10.0 μg·kg-1;绿豆芽的方法检出限为0.1~ 2.0 μg·kg-1,定量限为0.4~7.0 μg·kg-1。59种抗生素在10、50、200、800 μg·kg-1 4个添加水平下,黄豆芽的平均回收率为82.1%~122.3%,相对标准偏差为1.2%~15.4%(n=6);绿豆芽的平均回收率为87.3%~119.1%,相对标准偏差为0.9%~15.0%(n=6)。
    结论 该方法快速、灵敏和准确,可用于大批量豆芽使用喹诺酮类、磺胺类、四环素类、大环内酯类和硝基咪唑类抗生素的快速筛查。

     

    Abstract:
    Objective To establish an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS)-internal standard method for the simultaneous determination of 59 antibiotics, including quinolones, sulfonamides, tetracyclines, macrolides and nitroimidazole in bean sprouts.
    Methods The internal standard compounds and 5.0 mL of acetonitrile containing 0.1% formic acid were added into 5.0 g of the sample, and then extracted by ultrasonication. The extracted supernatant was filtered by 0.22 µm membrane and then injected into 1.0 µL of the sample directly. Methanol containing 0.1% formic acid and 2.0 mmol·L-1 ammonium formate containing 0.1% formic acid were taken as the mobile phases to conduct gradient elution. The separation was achieved on an ACQUITY PREMIER BEH C18 column. The separated analytes were detected by the electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode, and quantified by internal standard method.
    Results The 59 kinds of antibiotics in bean sprouts had a good linear relationship in the range of 10 μg·kg-1 to 1 000 μg·kg-1. The limits of detection (LODs) and the limits of quantification (LOQs) of the method for soybean sprouts ranged from 0.1 μg·kg-1 to 3.0 μg·kg-1 and 0.4 μg·kg-1 to 10.0 μg·kg-1, respectively. While, the LODs and LOQs of the method for mung bean sprouts ranged from 0.1 μg·kg-1 to 2.0 μg·kg-1 and 0.4 μg·kg-1 to 7.0 μg·kg-1, respectively. The average recoveries of the 59 kinds of antibiotics at four levels (10、50、200、800 μg·kg-1) in soybean sprouts were 82.1%‒122.3%, with the relative standard deviations (RSDs) of 1.2%‒15.4% (n=6). However, the average recoveries of the 59 kinds of antibiotics in mung bean sprouts were 87.3%‒119.1%, with the RSDs of 0.9%‒15.0% (n=6).
    Conclusion The method is rapid, highly sensitive and accurate, which can be used for rapid screening of quinolones, sulfonamides, tetracyclines, macrolides, and nitroimidazole antibiotics in large quantities of bean sprouts.

     

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