| Quinoxalines are widely applicated as feed additives in animal husbandry of china. They product many metabolites in animal and these metabolites also have certain pharmacodynamics and toxicity. For the purpose of affording useful technology for drug safety evaluation, as same as for pharmacology and toxicology research of quinoxalines, rapid screening methods and conformation methods for analysis of these drugs and their metabolites were developed in this paper.Immuno hapten was synthesized by transformating chemical structure of olaquindox and then combined to bovine serum albumin (BSA) by ester activation method. Polyclonal antibody was prepared by immunizing newzealand rabbits with the hapten-protein conjugates. Based on the polyclonal antibody, an indirect ELISA method for rapid screening eight kinds of quinoxalines (olaquindox, carbadox, mequindox, quinocetone,1-desoxy-cyadox, bidesoxy-quinocetone, bidesoxy-mequindox and quinoxaline-2-carboxylic acid) in swine liver was established.2-Acrylic acid-1,4-bidesoxyquinoxaline was selected as coatantigen. IC50of all target compounds to the Pab were from O.10μg·L-1to2.50μg·L-1. Samples were extracted by2mol-L-1hydrochloric acid-acetonitrile (3+5, v/v). The extraction was concentrated for ELISA detection. When spiked with1-100μg·kg-1of target compounds in swine liver, the recoveries were ranged from80.14%to96.90%, with intra-day variation coefficient of5.67-13.82%and inter-day variation coefficient of6.22-14.19%. The detection limit of this method was0.03-0.79μg·kg-1.Methyl-3-quinoxaline-2-carboxylic acid-BSA conjugates was synthesized by ester activation method and immuned to Balb/c mouse for preparing antiserum. Then monoclonal antibody was produced by spleen cells fusion method and used for establish an ELISA method for detecting methyl-3-quinoxaline-2-carboxylic acid in swine liver. IC50of5.72ng-mL-1was obtained for methyl-3-quinoxaline-2-carboxylic acid. Sample was treated with2mol·L-1of hydrochloricacid, followly extracted by ethyl acetate-hexane-isopropanol(8+1+1,v/v/v) and detected by ELISA. At the spiking range of1-100μg-kg-1,the average recovery of methyl-3-quinoxaline-2-carboxylic acid was85.44-100.02%, the intra-day coefficient of variation was6.64-10.57%and the inter-day coefficient of variation was7.29-10.88%. The limit of detection was0.27μg·kg-1.An UPLC/Q-TOF-MS method was established for determination and confirmation of quinocetone and its four metabolites (1-desoxy quinocetone,4-disoxy quinocetone, bidesoxy quinocetone and methyl-3-quinoxaline-2-carboxylic acid) in swine liver. Firstly, extraction was performed by ethyl acetate. Secondly, the remained sample was extracted by andhydrochloric acid solution.The target compounds in acid extract was transferred to ethyl acetate by liquid to liquid extraction procedure.All ethyl acetate extractions were combined together to a heart-shaped bottle. After phosphate buffer was added in the bottle, a rotary evaporation was done to evaporate ethyl acetate. The residual liquid was passed through an MAX cartridge for purification. When spiked at20-100μg·kg-1of methyl-3-quinoxaline-2-carboxylic acid in blank liver, average recovery of the five compounds was77.99-86.54%, with intra-day variation coefficient of2.00-6.62%and inter-day variation coefficient of2.13-5.14%. Limit of detection was1.4-4.8μg·kg-1and limit of quantification was4.6-15.9μg·kg-1An UPLC-MS/MS method was established for determination and confirmation of cyadox and its metabolites (quinoxaline-2-carboxylic acid,1-desoxycyadox and bidesoxycyadox) in pork and shrimp. Samples were extracted by acetonitrile-0.5mol·L-1hydrochloric acid (9+1, v/v) and purified by MAX cartridge. When spiked at2-50μg·kg-1of mixed compounds, the average recovery of four compounds was80.0-94.7%, with intra-day variation coefficient of’1.5-14.6%and inter-day variation coefficient of3.1-13.5%. Limit of detection was0.38-1.16μg·kg-1and limit of quantification was1.73-3.87μg·kg-1.Determination and confirmation of thirteen kinds of quinoxalinesand their metabolite in liver tissues was performed by an UPLC-MS/MS method, including carbadox, quinoxaline-2-carboxylic acid, olaquindox,4-desoxycyadox, cyadox, bidesoxycyadox,4-desquinoceton,1-desoxymequindox, bidesoxyquincetone, bidesoxymequindox, quinocet one, methyl-3-quinoxaline-2-carboxylic acid and mequindox) After acidified samples with3mol-L-1of0.1%hydrochloric acid, target compounds were extracted with formic acid-acetonitrile solution(4+6) and purified with HLB cartridge. When spiked at10-500μg·kg-1of mixed compounds in blank liver, the average recovery was79.8-93.7%, with intra-day variation coefficient of3.7-14.0%and inter-day variation coefficient of5.3-15.6%. For these thirteen compounds, limit of detection was0.33-2.51μg·kg-1and limit of quantification was1.10-8.37μg·kg-1. |
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