Studies On Quantification Analysis Methods And Deoxidation Rates Of Quinoxalines

Quinoxalines are a group of synthetic antibacterial agents which are widely used as medicinal feed additives as antibacterial growth promoters.In some countries,carbadox and olaquindox have been banned or limited to be used in food animal due to their potential toxicity.Mequindox and quinocetone have been successively registered and the safer medicine of cyadox which belongs to quinoxalines family is being developed in China.However,it can not be neglected of the hazards from the residues on food safety for some abuse and illegal use of the drugs.So it is more important to do deep studies on toxicity mechanism of quinoxalines and analysis methods for determination of these drugs in feeds and animal foods.Though some methods have been reported for determination of the individual quinoxaline in simple matrix,no method has yet been developed for simultaneous determination of the five compounds in different matrices. And there is only suppose that toxicity is relative to desoxidation of the quinoxalines. However,no report is for clarifying relativity between deoxidation rates and toxicity of quinoxalines.Based on the two aspects,this study aims to explore analysis methods for determination of multi quinoxalines in feeds and animal edible tissues deeply,and investigate the relativity between deoxidation rates and toxicity of quinoxalines primarily, to provide technical support for monitoring and reasonable evidence for studying on toxicity mechanism of quinoxalines.The study also has a great value for evaluation on animal food safety.1.Development of a method for simultaneous determination of the quinoxalines in feeds.An HPLC method with UV detection has been established for simultaneous quantitative determination of the 5 quinoxalines(carbadox,olaquindox,cyadox, mequindox,quinocetone) in porcine,chicken and fish feeds.Feed samples were extracted with methanol-acetonitrile-water(35:35:30,v/v/v) in an ultrasonic bath,and purified by solid phase extraction on Alumina N cartridges.The samples were analyzed on an Eclipse XDB C₁₈ liquid chromatography column using a gradient program with methanol and water.Except for cyadox(＞75%),recoveries of the drugs from feed samples spiked at 1, 5,50 and 200 mg/kg ranged from 92.1%to 104.3%.Coefficients of variation were 3.0%～13%.The decision limits(CCα) for the five compounds were＜0.45 mg/kg,and the detection capabilities(CCβ) were＜0.75 mg/kg.The sample pretreatment has been simplified for using the ultrasonic extraction and solid phase extraction in the method.The volume of the organic solvent was decreased using the sample pretreatment method,and avoiding the toxicity solvent using in the method.In the same time,the interference peaks were deleted.The gradient elute of the mobile phase was used to separate five quinoxalines in a liquid column.The wavelength was set at 380 nm.In the experiment,the conditions for mobile phase and the wavelength have been investigated.This simply and little contaminative method with the familiar materials can be applied on monitoring the use of multi-quinoxalines in porcine,chicken,fish feed,etc.This research can also provide reference for developing methods for determination of muti-ingredient in feeds.2.Development of a method for simultaneous quantification of marker residues of quinoxalines in animal edible tissues.A method of high-performance liquid chromatography with UV detection has been established for simultaneous quantitative determination of quinoxaline-2-carb- oxylic acid (QCA) and methyl-3-quinoxaline-2-carboxylic acid(MQCA),the marker residues for quinoxalines,respectively,in the muscles and livers of porcine and chicken and in the muscle of fish.And some conditions such as sample extraction,purification and sample solvent have been deeply investigated in this experiment.Tissue samples were subject to acid hydrolysis followed by liquid-liquid extraction and Oasis MAX solid-phase extraction clean-up.Residues of concentration were solved with alkaline methanol and determined by high performance liquid chromatography with UV detection at wavelength of 320 nm.CCαwere 0.7～1.0μg/kg,CCβwere 1.3～1.7μg/kg for QCA and CCαwere 1.2～2.6μg/kg and CCβwere 2.0～4.4μg/kg for MQCA in muscle tissues.CCαwere 2.5～5.6μg/kg,CCβwere 4.3～5.6μg/kg for QCA and CCαwere 2.1～2.4μg/kg and CCβwere 4.4～5.6μg/kg for MQCA in liver tissues. Recoveries of QCA and MQCA,spiked in muscle tissues at levels of 2～16μg/kg,were from 70.7%to 104.3%,the relative standard deviation values were＜20%.Recoveries of QCA and MQCA,spiked in liver tissues at levels of 10～50μg/kg,were from 72.3%to 80.7%,the relative standard deviation values were＜13%.This simple and sensitive method with little interference for the drugs can be applied to monitoring for the possible misuse of quinoxalines.The experiment can be taken as an instruction to do studies on analysis methods for determination of multi-residue in animal tissues and provide technical support for evaluation on animal food safety.3.Comparative study on deoxidation rates of quinoxalinesTo provide evidence for qualitative analysis of the productions from deoxidation of quinoxalines,HPLC with DAD detector was used to determine three desoxy compounds including desoxy cyadox,desoxy olaquindox and desoxy quinocetone.The detection limit was 0.01μmol/L and the quantitation limit was 0.02μmol/L.Within the range of 0.01～10μmol/L,the linearity relations between response and concentrations of three desoxy quinoxalines are good.The coefficient correlation(r) is 0.995 9 to 0.997 1.Deoxidation rates of quinoxalines in chemical reductive systems were compared. The reaction in acid KI system was according with the first order kinetic equation.And half-lives of the five drugs were 4.80～19.06.In alkaline Na₂S₂O₄ system,deoxidation reaction was occurred immediately and the productions were determined by HPLC.From the results,it could be inferred that deoxidation rate of cyadox was the fastest among,and quinocetone is the second.In the chemical systems,it can be reduced that deoxidation rates of quinoxaliens are:cyadox＞carbadox＞quinocetone＞olaquindox＞mequindox.Deoxidation rates of quinoxalines in porcine liver microsome reductive system were compared.The 5 drugs were incubated in porcine liver microsome with reducing type coenzyme.It is found that the reaction is also according to the first order rule and the half-lives were 11.75～69.31 min for the drugs.Deoxidation rates of quinoxaline drugs are:cyadox＞quinocetone＞olaquindox＞carbadox＞mequindox.Above results are confirmed with toxicology experimental result of quinoxalines.It is proved that deoxidation rates of quinoxaline drugs are:cyadox＞quinocetone＞olaquindox＞carbadox＞mequindox.Toxic intensity of quinoxalines could be judged by their deoxidation rates.In a word,faster the rates,stronger the toxicity.The experiments could provide some reasonable evidences for study on toxicity mechanism of quinoxalines.From above,analysis methods for determination of quinoxalines and toxicity mechanism of the drugs were studied in this paper circled on animal food safety.Analysis methods for determination of quinoxlaines in feeds and multi-residue of quinoxalines in animal edible tissues have been firstly established in this study.The relativity between deoxidation rates and toxicity of the drugs was illustrated,which could be one of judgments for toxicity level of the drugs.All the results from the study can provide advanced technologies and reasonable evidence for monitoring or residue surveillance for quinoxlaines and studying on toxicity mechanism of quinoxalines.And all of them have great vales for animal food safety.