| Quinoxolines are a class of synthetic growth-promoting drugs.Because of their cheap price and good antibacterial effect,such drugs are often added to feeds in livestock,poultry and aquaculture to treat diseases,promote development and improve feed utilization rate.In recent years,due to the improvement of the consumption capacity of the sea cucumber market in China,the scale of sea cucumber cultivation has also expanded rapidly.In order to pursue maximum benefits,some farmers illegally and excessively use the feed containing quinoxoline drugs in the process of sea cucumber farming,and sea cucumber poisoning deaths also occur from time to time.It is reported that quinoxoline drugs have acute toxicity,short-term toxicity,long-term toxicity,genetic toxicity and other toxicities.The quinoxalines gradually accumulate in the animal body along with the food chain,and humans at the top of the food chain are also at risk of poisoning.Therefore,it is necessary to develop a set of methods for the detection of quinoxolines in feed and sea cucumbers to monitor and control the safety of marine food.Current detection methods mostly focus on terrestrial animals and can only detect one or a class of quinoxoline drugs and the pre-treatment requires complex acidolysis,alkaline hydrolysis or enzymatic hydrolysis.In this study,the pre-treatment method was optimized,and a simple and rapid method was established,which could simultaneously detect a variety of quinoxalines in sea cucumber and feed.This method was used to investigate the residues of quinoxoline drugs in sea cucumber and the use of quinoxoline drugs in feed.The main research contents are as follows:1.Simple and fast extraction coupled to UHPLC-MS/MS for the determination of Qd NOs in feed and applied to the detection of actual samples.Four kinds of quinoxaline prototype drugs,including carbadox,mequindox,olaquindox,and quinocetone,were used as the research objects.In this study,the pre-treatment methods of four quinoxoline drugs in sea cucumber were investigated and optimized,without complex acidolysis,alkalohydrolysis or enzymatic hydrolysis feed,four quinoxolines in the feed were extracted twice by acetonitrile-ethyl acetate(50:50,v:v),and then the extract was purified by C18 solid phase extraction column,Waters ACQUITY UPLC BEH C18 column was used for chromatography.The mobile phases were 0.1%formic acid-water(A)and 0.1%formic acid-acetonitrile(B).The flow rate was 0.25 m L/min,and the injection volume was5?L.The column temperature was 30?.Then,the gradient elution was performed.The mass spectrometry was performed by using positive ion mode scanning and samples were accurately quantified by using external standard method.The 4 drugs were well separated and showed good linear relationship,with a determination coefficient of R~2 above 0.99.The LODs in this research ranged from 0.35 to 0.78?g/kg and the LOQs ranged from 1.15 to2.65?g/kg respectively.Spiked feed samples were performed for validation at 20,50,and100?g/kg,the average recoveries of the 4 quinoxolines were all above 80%,with the intra-day RSD<10.5%,and the inter-day RSD<13.5%.The results showed that 35 of the 50feed samples were positive for MEQ and 10 were positive for QCT,with the detection amounts ranging from 1.25 to 12.33?g/kg and 3.05 to 12.30?g/kg.No illegal drugs were detected,such as CBX and OLA.MEQ and QCT were allowed to be added in the veterinary pharmacopoeia.MEQ and QCT both have lower toxicity and better effect,they have been gradually replacing CBX and OLA,and have been widely used in feed.In this study,a simple and fast pretreatment method coupled UHPLC-MS/MS was proposed,which could simultaneously monitor the use of quinoxalines in feed.2.Simple and fast extraction coupled to UHPLC-MS/MS for the determination of Qd NOs and their major metabolites in sea cucumber and applied to the detection of actual samples.Eight kinds of quinoxaline drugs,including carbadox,mequindox,olaquindox,quinocetone,1,4-bisdesoxy-mequindox,4-desoxy-mequindox,quinoxaline-2-carboxylic acid and 3-methyl-quinoxaline-2-carboxylic acid,were used as the research objects.In this study,the pre-treatment methods of 8 quinoxolines and their metabolites in sea cucumberia were investigated and optimized.Samples were di-extracted with acetonitrile and ethyl acetate containing 0.1%formic acid in turn without complicated acidolysis,alkalolysis,and enzymolysis procedures and purified with HLB cartridge before LC-MS/MS analysis.Waters ACQUITY UPLC TSS T3 column was used for chromatography.The mobile phases were 0.1%formic acid-water(A)and 0.1%formic acid-acetonitrile(B).The flow rate was0.25 m L/min,and the injection volume was 2?L.The column temperature was 40?.Then,the gradient elution was performed.The mass spectrometry was performed by using positive ion mode scanning and samples were accurately quantified by using external standard method.The 8 drugs were well separated and showed good linear relationship,with a determination coefficient of R~2 above 0.99.The LODs in this research ranged from 0.17 to1.24?g/kg and the LOQs ranged from 0.55 to 4.53?g/kg respectively.Spiked sea cucumbers samples were performed for validation at 20,50,and 100?g/kg,the average recoveries of the 8 quinoxolines were all above 80%,with intra-day RSD<12.4%and inter-day RSD<16.6%.The results showed that 16 of 20 sea cucumbers were positive for MEQ,accounting for 80%of the total samples,4 and 6 of the sea cucumbers were positive for 4-DMEQ and MQCA,which were consistent with the detection results of quinoxolines in feed.No illegal drugs were detected,such as CBX and OLA.It was speculated that most of the quinoxoline residues in sea cucumber were MEQ and its metabolites.In this study,a simple and fast pretreatment method was proposed,which could simultaneously detect the residual content of quinoxalines and their metabolites in sea cucumber. |
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