畜禽粪便和污水中金霉素及其代谢物的液质联用(LC-MS/MS)检测方法研究

An innovated analytical method for determine the CTC and its metabolites in animal excreta and sewage by LC-MS/MS

建立了畜禽粪便和污水中金霉素(CTC)及其两种代谢物——差向金霉素(ECTC)和异金霉素(ICTC)的LC-MS/MS检测方法。粪便样品经Na_2EDTA-McIlvaine缓冲液(pH=4)分别提取3次,离心,收集上清液,过Waters的Oasis HLB固相萃取小柱净化、浓缩。用6 mL0.01 mol·L^(-1)草酸甲醇溶液洗脱,收集洗脱液,40 ℃水浴氮气吹至近干,甲醇定容至1 mL,过0.22 μm滤膜,用LC-MS/MS测定。污水取100 mL经滤纸和0.65μm纤维膜过滤后,加1 mL 5%Na_2EDTA溶液,调节pH值至7.0,然后过预先用5 mL甲醇和10 mL Na_2EDTA-McIlvaine缓冲液(pH=4)活化过的Waters Oasis HLB固相萃取小柱,净化、浓缩,后用6mL 0.01 mol·L^(-1)草酸甲醇溶液洗脱,收集洗脱液,40℃水浴氮气吹至近干,甲醇定容至1 mL,过0.22 μm滤膜,上机测定。该方法在10～1 000μg·L^(-1)之间呈线性相关,金霉素、差向金霉素和异金霉素的平均回收率分别为79.64%～97.23%、75.34%～98.37%和73.67%～95.08%,日间偏差在1.78%～6.18%,日内偏差在1.07%～6.28%。粪便样品和污水样品中的检测限分别为20μg·kg^(-1)和10μg·L^(-1),符合作为检测方法的要求。

This paper aims to introduce a sensitive LC -MS/MS analytical method for determining CTC, ECTC and ICTC in animal excreta and sewage developed by the present author. As we know, for the time being, the most frequently used antibacterial for treating animal disease and yotmg animal growth-promotor is Chlortetracyeline （CTC）. However,CTC eann＇t be fully absorbed and a considerable amount of CTC and its main metabolites were excreted directly into the excreta in reality. For this reason, it has become a great concern for the potential risk of the manure-derived CTC and its main metabolites that may permeate into the surfaee water. In order to find effective resistant microorganism to deal with the problem, we have done experiments by extracting animal excreta samples three times by using 6 mL 0.1 mol· L^- 1 Na2EDTA-Mellvaine buffer （pH = 4） and then cleaned the samples up by using Oasis HLB solid phase extraction（SPE） column. Later, the sewage was let to be filtrated by 0.65 μm film firstly and then mixed with 5% Na2EDTA. In doing so, the solution was then treated oll the Oasis HLB column after their pH value was adjusted to 7. The analytes were then separated by means of liquid chromatography and determined with liquid chromatography coupled to tandem mass spectrometry under the electro-sprayed ion and multiple reaction monitor mode. The results indicate that the calibration curves for CTC, ECTC and ICTC turned to be linear at a concentration range of 0.01 - 1.0 mg· L^-1, with the correlation coefficients being from 0.998 7 to 0.999 7, respectively. The limits of detection （LOD） for CTC, ECTC and ICTC prove to be 20 μg· kg^-1 in animal excretion and 10 μg· kg^-1 in the sewage. The average recovery rate ofCTC, ECTC and ICTC spiked samples was made to reach 79.64 % - 88.73% , 75.34% -87.34% and 73.67 -84.21% in animal excretion, respectively, and 93.27% - 97.23% , 91.34% - 98.37% and 93.15% -95.08% in the sewage. The inter-day standard deviations （RSD） and intra-day RSD deviations tend to range from 1.78% to 6.18% and from 1.07% to6.28%, respectively. Thus, that the method developed in this study is simple in operation, sensitive and repeatable, as well as favorable for the simultaneous determination of CTC, ECTC and ICTC either in animal excretion or in the sewage.