高效液相色谱-高分辨质谱法定性筛查牛奶中49种β-内酰胺类抗生素残留

Qualitative screening of 49 kinds of β-lactam antibiotic residues in milk by high performance liquid chromatography coupled with high resolution mass spectrometry

目的开发和验证定性筛查牛奶中49种β-内酰胺类抗生素残留的高效液相色谱-高分辨质谱法(high performance liquid chromatography coupled with high resolution mass spectrometry,HPLC-HRMS)。方法牛奶经磷酸盐缓冲液及乙腈提取后,加入硫酸钠(Na_(2)SO_(4))和氯化钠(NaCl)进行盐析,离心后取部分上清液经Na2SO4和C18混合的分散式固相萃取填料(dispersive solid phase extraction,d-SPE)净化,净化液浓缩定容后,使用HPLC-HRMS分别以全扫描-数据依赖型二级扫描(full scan-data dependent secondary acquisition,Full MS/dd-MS2)和全扫描-平行反应监测二级扫描(full scan-selected ion monitoring/parallel reaction monitoring,Full MS/PRM)两种模式进行数据采集。结果在Full MS/PRM模式下,45种抗生素通过方法验证,假阳性率和假阴性率都小于5%;在Full MS/dd-MS2模式下,43种抗生素通过方法验证,假阳性率和假阴性率在规定的目标浓度下均低于5%,Full MS/PRM数据采集模式更适用于多兽药残留的定性筛查。结论此方法适用于牛奶样品中β-内酰胺类抗生素的定性筛查,并且高分辨质谱的Full MS/PRM采集模式相较于Full MS/dd-MS2采集模式,抗干扰性更好,更适用于复杂基质中目标物的定性筛查。

Objective To developed and validate a qualitative screening method for the determination of 49 kinds of β-lactam antibiotic residues in milk by high performance liquid chromatography coupled with high resolution mass spectrometry(HPLC-HRMS). Method Milk was extracted with a mixture of a phosphate buffer solution and acetonitrile followed by a salt partition using sodium sulphate(Na_(2)SO_(4)) and sodium chloride(NaCl). After centrifugation, an aliquot of the supernatant was cleaned by dispersive solid phase extraction(d-SPE) which consisted of a mixture of Na2SO4 and octadecyl-silica C18 sorbents. After concentration and constant volume of the purification solution, HPLC-HRMS were used for data acquisition in 2 modes: Full scan-data dependent secondary acquisition(Full MS/dd-MS2) and full scan-selected ion monitoring/parallel reaction monitoring(Full MS/PRM). Result The 45 kinds of antibiotic passed through the validation with false positive and negative rate less than 5% in Full MS/PRM mode;43 kinds of antibiotics passed through the validation with false positive and negative rate less than 5% at the specified target concentration in Full MS/dd-MS2 mode, Full MS/PRM was more suitable for the qualitative screening of multiple veterinary drug residues. Conclusion This method is suitable for the qualitative screening of β-lactam antibiotics residues in milk. Comparing to Full MS/dd-MS2, Full MS/PRM is more robust, and it is more suitable for the qualitative screening of veterinary drug residues in complex matrix.