超高效液相色谱串联质谱法同时检测复杂基质中四环素类抗生素及其代谢产物

Simultaneous Determination of Tetracyclines and Their Degradation Products in Complicated Matrix by Ultra Performance Liquid Chromatography with Tandem Mass Spectrometry

为同时检测复杂基质中（由猪粪和蘑菇渣混合而成的堆肥原料）3种四环素类抗生素（四环素TC、土霉素OTC和金霉素CTC）及其代谢产物，建立了超高效液相色谱串联质谱检测方法（UPLC—MS／MS）。该方法同时采用pH值=4的Na2EDTA-Mellvaine缓冲溶液和乙腈为提取溶液，经过固相萃取净化后，以乙腈和0．1％的甲酸水溶液为流动相，采用超高效液相色谱柱进行分离，在电喷雾正离子模式下，用四极杆串联质谱仪进行定性和定量分析。3种四环素类抗生素及其代谢产物均在7min内完成分离，总共分析时间为12min。在0-6mg·kg^-1DW（Dryweight）浓度范围内，3种四环素类抗生素及其代谢产物的标准曲线线性良好，线性相关系数R2均大于0．9960，重现l生也较好（n=11，相对标准偏差均小于15％）。在3个加标水平0．2mg·kg^-1DW、1mg·kg^-1DW和4mg·kg^-1DW下，TC、OTC、CTC的回收率分别为71％-89％、66％～94％和66％～84％；去甲基金霉素（DMCTC）的回收率为52％-64％；差向异构产物的回收率在32％～51％之间；脱水产物以及差向脱水产物的回收率均低于30％。3种四环素类抗生素及其代谢产物的检出限和定量限分别在1．668～17．270μg·kg^-1和5．561—45．918μg·kg^-1范围内，表明该方法具有较高的灵敏度。对北京市某露天堆肥场中的样品进行测定发现，TC、OTC、CTC的浓度分别为0．4、1．6、2．9mg·kg^-1，检测到的代谢产物主要为相应的差向异构体，其中差向金霉素（ECTC）的浓度最高，达到2．7mg·kg^-1，和母体的含量水平比较接近，其他代谢产物也有不同程度的检出。

An analytical method was developed to simultaneously determine the three tetracyclinesltetracycline （TC）, oxytetracycline （OTC） and chlrotetracycline（CTC ）] and their degradation products in complicated matrix, which consisted of swine manure and mushroom residues used for composting, using ultra performance liquid chromatography with tandem mass spectrometry （UPLC-MS/MS）. Samples were extract- ed by Na2EDTA-McIlvaine buffer at pH 4 in combination with acetonitrile and then purified through solid phase extraction. The separation was performed on UPLC column using acetonitrile and 0.1% formic acid as mobile phase. Qualitative and quantitative analysis were carried out by a triple quadrupole mass spectrometer equipped with an electrospray ionization source that operated in the positive ionization mode （ESI＋）. All the three tetracyclines and their degradation products achieved complete separation within 7 min and the total running time was 12 min. Accompanying with a pleasant reproducibility（n= 11, RSD〈 15% ）, the linearity of calibration curve was rather good in the range of 0~6 mg· kg-1 DW（dry weight）, with the R2 all above 0.996 0. The recoveries of TC, OTC and CTC were 71%~89%, 66%-94% and 66%N 84%, respectively, at the three spiked levels of 0.2 mg· kg-1 DW, 1 mg· kg-1 DW and 4 mg· kg-1 DW. The recovery of demeclocycline was in the range of 52%~64%, and for the epimers, the recovery varied between 32% and 51% while the anhydro-tetracyclines and their corre-sponding epimers showed poor recoveries below 30%. The limit of detection and quantitation for the three tetracyclines and their degradation products were in the range of 1.668~17.270 μg·kg^-1 and 5.561-45.918 μg·kg^-1, indicating a high sensitivity for this method. Samples taken from an open windrow composting plant located in Beijing were determined by this method and the results presented that the concentrations of TC, OTC and CTC were 0.4 mg· kg-1, 1.6 mg-kg-1 and 2.9 mg· kg-1, respectively. The detected degradation products were predominately their epimers, of which epi-chlortetracycline（ECTC ） showed a highest concentration of 2.7 mg· kg-1, closing to its parent compound CTC. Other degradation products were also found at different levels.