| Illegal use of banned drugs, irrational use of veterinary drugs and mycotoxin contamination are the three main factors affecting feed safety. Feed safety issues caused by the three main factors affect the quality of meat, eggs, milk and other animal products, and also harm to animal safety and human health. Therefore, it has important significance to establish analysis method of above three types of drugs and monitoring and control of feed quality based on these analysis mehtods. However, there are many difficulties and problems in the feed safety testing due to the complexity of the feed sample matrix, time-comsuing and laborious of single component determination, low analytical efficiency, high cost of pretreatment, large dosage of organic solvents, and so on. The purpose of the study is to develop fast, efficient and multicomponent determination technology to effectively ensuring feed quality safety.The study thoroughly study the feed safety multicomponent analytical sample pretreatment technique and liquid chromatography tandem mass spectrometry (LC-MS/MS) analytical methodology with β-receptor agonists, resorcylic acid lactones, sulfonamides and quinolones antibiotics as research objects in this study. Carbon nanotubes materials and dispersive solid-phase extraction technology, which had aroused wide concern, have been used during development of sample pretreatment method. Therefore, the established pretreatment method effectively improves the efficiency of sample pretreatment. UPLC has been used for rapid chromatographic separation and tandem mass spectrometry has been used for qualitative and quantitative determination. Moreover, isotope standard dilution has been used to improve the accuracy and precision of the method. The main research contents and results are as follows:1. Study on mass spectral characteristics and fragmentation mechanism of β-receptor agonistsTo clarify pyrolysis mechanism of β-receptor agonists under postitive mode, representative drugs including clenbuterol, salbutamol and ractopamine had been chosen to study with AB SCIEX5500QTRAP triple quadrupole mass spectrometry and IDA-EPI technology. The results showed that there are some similarities and differences for ESI-MS/MS behavior of β-receptor agonists due to them belong to phenylethanolamine compounds, which have the same basic skeleton and similar structure features and different connection for the benzene ring and amino functional groups. Under ESI postivie mode, β-receptor agonists are easy to form [M+1]+. The strcture is easy to loss neutral molecules, such as H2O. Moreover, the C-N bond connected with amino group is easy to broken and produce conjugated ion structure with benzene. This study has important theory research significance and practical value, such as providing reliable basis for other β-receptor agonists structural analysis and providing powerful means to track newp-receptor agonists.2. Study on the analysis110-receptor agonists in feeds by LC-MS/MS coupled with dispersive solid phase extraction using multiwalled carbon nanotubes as absorbentA simple, sensitive and reliable analytical method was developed for determination of (3-receptor agonists (clenbuterol, ractopamine, salbutamol, phenylethanolamine A, clorprenaline, tulobuterol, cimaterol, terbutaline, mabuterol, bambuterol, penbuterol) in feeds by ultra high performance liquid chromatography-positive electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) using dispersive solid phase extraction with multi-walled carbon nanotubes. To improve sample throughout and sensitivity of analytical method, the systematic optimization of sample pretreatment method had been carried out including the type and amount of MWCNTs, the pH value of the sample, the sample extraction time, and the volume of eluent. Feed samples were extracted with formic acid solution. The eluate was directly analyzed by LC-MS/MS on an Acquity UPLC BEH C18column with a mixture of methanol and0.1%formic acid solution as the mobile phase. The samples were quantified with the internal standard calibration curve method. The results showed that good linearities were obtained for the β-receptor agonists at the concentration of0.1-20μg/L with the linear relative coefficient more than0.997. The recoveries for the β-receptor agonists in formular feed, concentrated feed and premix feed of swine were89.6%-112.9%at the fortified levels of0.5-100μg/kg. The limits of quantitation of11β-receptor agonists were0.05-0.48μg/kg, and the relative standard deviations were less than15%. Compared with the method of Announcement No.1486of the Ministry of Agriculture in positive sample analysis, results which obtained with the two methods, were consistent.3. Simultaneous determination of zearalenone and analogues in feed using LC-MS/MS and multiwalled carbon nanotubes as dispersive solid phase extraction sorbent A simple and cost-effective pre-treatment procedure was developed for six zearalenone and analogues (α-zearalanol, β-zearalanol, a-zearalenol, β-zearalenol, Zearalanone, Zearalenone) in feed using dispersive solid phase extraction (dSPE) with multi-walled carbon nanotubes (MWCNTs). After extraction with acetonitriel/water (80:20, v/v) and dilution with water, a dSPE procedure was carried out with MWCNTs. The pH value of the extract, extraction time with MWCNTs, type and amount of MWCNTs and type of eluent were optimized to increase the sample throughput and sensitivity. The eluate was directly analyzed by LC-MS/MS on an Acquity UPLC BEH C18column with a mixture of water and acetonitrile solution as the mobile phase. The samples were quantified using zearalenone-D6as internal standard. Mean recoveries from fortified swine mixed feed samples ranged from94.6%to107.2%, with relative standard deviations2.6%-8.5%, the limits of detection and quantification for RALs were in the ranges of0.12-0.27μg/kg and0.40-0.90μg/kg, respectively. At the same time, compared the positive results of ZEN with agriculture standard NY/T2071-2011method, relative standard deviation obtained with two analytical methods is within15%.4. Using carbon nanotubes as dispersion solid phase extraction and LC-MS/MS determination of22sulfonamides and quinolones antibiotics in feedA simple and cost-effective pre-treatment procedure was developed for determination of22sulfonamides and quinolones (sulfacetamide, sulfadiazine, sulfamethoxazole, sulfathiazole, sulfadimethoxine, sulfadoxine, sulfamethazine, sulfamethoxydiazine, sulfamethoxypyridazine, sulfamonomethoxine, sulfaquinoxaline, sulfachloropyridazine, flumequine, norfloxacin, ciprofloxacin, fleroxacin, orbifloxacin, lomefloxacin, enrofloxacin, ofloxacin, sarafloxacin, difluoxacin) in feed using dispersive solid phase extraction with multiwalled carbon nanotubes. The type and amount of MWCNTs, pH value of the extract, extraction time with MWCNTs, and type of eluent were optimized to increase the sample throughput and sensitivity. The eluate was analyzed by LC-MS/MS on a Waters Atlantis Cis column with0.1%formic acid solution and acetonitrile as the mobile phase with gradient elution, and determined in electrospray ionization positive mode (ESI+) with multiple reaction monitoring (MRM). The samples were quantified using internal standard calibration. The results showed that22sulfonamides and quinolones were linear in the concentration range of1.0-1000μg/L, the linear correlation coefficient(r2) were more than0.992. The limits of detection were3.0-10μg/kg, and the limits of quantification wei.;10-25μg/kg. When spiked level at0.05-50mg/kg in formular feed, concentrated feed and premix feed of swine and chicken, the recoveries of sulfonamides and quinolones were71.5%-114.7%, and the relative standard deviation were in the range of1.5%to12.5%. One positive feed samples with sulfaquinoxaline were tested with the method, and compared with the method of GB/T19542. The results showed that the result was (58.9±5.75) mg/kg (n=5) with our developed method, and was (61.2±6.25)mg/kg (n=5) with GB/T19542method. |
