Study On Non-Targeted Screening Technology And Application Of Feed And Urine

Melamine,clenbuterol and other safety incidents had significant damages to China’s milk and meat industry,and remaining influential so far.The main reason is that the tests for several original specifically targeted compounds are unable to realize the rapid screening for non-targeted risky compounds,and cannot achieve effective monitoring and early warning.At the same time,the veterinary drugs have the characteristics of various types,fast update and hidden adding means,which resulted in enormous potential security risks in aquaculture industry.It is beneficial to the study of related researches on early detection of new unknown additives in the aquaculture industry,to change the after event active regulation.The early detection and early treatment could avoid significant impacts and ensure the quality and safety of feed and meat products.This study is aim to establish a method for non-targeted screening of chemical additives in feed and urine samples using a high-resolution instrument and a metabolomics approach,and provide compound specific signals which can be used for further investigations of the identity of the possible contaminants detected.The main contents and results are as follows:(1)Through the optimization of the instrument parameters and sample pretreatment conditions,the LC-Q-TOF/MS method was developed for ?-agonists in feed samples.The method involves 0.1% formic acid in aqueous solutions of EDTA 0.1%(w/v)-acetonitrile-methanol(1:1:1,v/v)extraction,full scan mode of data collection.TOF full scan mode and MS mode were used to collect data of product ions.IDA mode(automatic CE,ions that greater than 100 cps)was used to collect data of daughter ions in feed samples and Targeted MS/MS(Fragmentor 125 V,15-50 V automatic CE)for urine samples.(2)The non-targeted screening method was developed using LC-Q-TOF/MS in combination with PCA and OPLS-DA.The results indicated that all these two statictical models could separate the abnormal samples from the control samples using the information of product ions.Through the study of the discriminant ability of these two models under the condition of different number of drugs,different kind of feeds and different concentration of drugs,the non-targeted screening method was established.This method of LC-Q-TOF/MS coupled with PCA and OPLS-DA could be used to identify new or unexpected chemical hazards in feed ignoring the influence of substrate and concentrate.(3)Based on the results of ions in PCA and OPLS-DA,the method of finding abnormal ions in samples was established.The results showed that the ions which PC1 weights of loading plot in PCA was greater than 0.18 and the content of abnormal ions was four times bigger than that in control group of profile plot could be judged as the abnormal ions in feed samples.The ions that P-Cov was greater than 150 of S-Plot in PCA and the content minus was bigger than 10 in Box-whisker Plot could be judged as the abnormal ions in urine samples.In the model of OPLS-DA,ions that P value was greater than 0.1 in S-Plot and VIP value was greater than 1 could be judged as the abnormal ions.The determination method could used to identify the abnoemal ions ignoring the influence of substrate and concentrate.(4)The non-targeted screening can completely separate between control group and spiked group using the model of PCA and OPLS-DA.The abnormal ions leading to the differences between the two groups were accurately identified.The structures of these ions were comfirmed by comparing with self-built library and Chemispider website using the information of accurate molecular weight,fragment ions and isotopic abundance combined with the matching score greater than 85 and the information of molecular formula.(5)On the basis of the established screening methods of non-targeted compounds in feed and urine,an actual sample test was carried out of non-targeted screeing.The results showed that: different kind of veterinary drugs could be identified using LC-Q-TOF/MS in combination with PCA and OPLS-DA.And the LC-MS/MS method was carried out to verify the exactness of the results.The antibiotic drugs were sieved out in the highest rate and the antiviral drugs and agnoists had a relatively low rate.The method of LC-Q-TOF/MS together with chemometrics can be applied to the non-targeted screening work in the actual samples.