摘要
喹噁啉类是化学合成的具有抗菌、促生长作用的动物专用药,被广泛用作猪、鸡、鱼等动物的饲料添加剂,主要包括卡巴氧、喹乙醇、喹赛多、喹烯酮和乙酰甲喹等5种药物。因毒副作用,卡巴氧和喹乙醇已被禁止或限制使用。中国相继批准了乙酰甲喹和喹烯酮的使用,并正在开发较安全的喹赛多等同类替代产品。但喹噁啉类的滥用和非法使用现象普遍,药物毒性及残留造成的潜在的食品安全危害不容忽视,开展这类药物毒性作用机制及其在饲料和动物源食品中的分析方法研究具有重大意义。喹噁啉类分析方法研究禁锢于单一基质、单组分,不同基质中多种喹噁啉类分析方法研究未见报道。喹噁啉类毒性研究也仅局限于推测其源于脱氧过程,但未见毒性与脱氧速率关系的阐述。本课题从喹噁啉类分析方法展开研究,深入探索并建立饲料中喹噁啉类和动物可食性组织中喹噁啉类残留标示物的同时分析方法,进而初步探讨喹噁啉类脱氧速率和毒性的关系,具体为以下3个研究体系内容。这些研究为这类药物的残留监控和毒性作用机制等研究提供技术支持和理论依据,对动物源食品安全评价具有指导意义和参考价值。
1.饲料中喹噁啉类药物同时分析方法研究
深入探索样品前处理和检测方法,确定最佳条件,建立猪、鸡、鱼饲料中5种喹噁啉类高效液相色谱同时分析方法。样品经甲醇-水-乙腈(35:35:30,v/v/v)溶剂超声提取,中性氧化铝固相萃取小柱净化,高效液相色谱梯度洗脱分离,380 nm紫外波长测定。不同饲料样品中,5种药物的CCα小于0.45 mg/kg,CCβ小于0.75 mg/kg;在1、5、50和200 mg/kg的添加浓度,除喹赛多(>75%)外,其它4种药物的回收率为92.1%~104.3%,日内、日间相对标准偏差为3.0%~13%。
超声提取和固相萃取净化,可以节省样品前处理时间,避免有毒试剂的使用,减少有机试剂用量,消除饲料基质对喹噁啉类药物测定干扰。仪器测定条件选择过程中,采用流动相梯度洗脱方式,考察了流动相成分和比例对5种喹噁啉类药物同时分析的影响,选用380 nm作为紫外检测器测定波长,进一步对5种喹噁啉类药物进行筛选,对比不同溶剂对药物测定分析结果差异,确定5种喹噁啉类药物同时分离分析的高效液相色谱条件。
本方法操作简便、材料易得、环境污染小,可以广泛应用于猪、鸡、鱼等饲料中多种喹噁啉类的监测,对饲料中其它化合物的多组分分析方法研究具有参考价值。
2.动物可食性组织中喹噁啉类多残留分析方法研究
探讨样品提取、净化和测定溶剂等条件,建立猪、鸡、鱼肌肉及猪、鸡肝脏组织中喹噁啉-2-羧酸和3-甲基喹噁啉-2-羧酸的高效液相色谱多残留分析方法。动物组织样品采用偏磷酸甲醇溶液水解,乙酸乙酯提取,MAX阴离子固相萃取小柱净化,碱性甲醇溶解浓缩残渣,高效液相色谱分离,320 nm紫外波长测定。猪、鸡、鱼肌肉组织中喹噁啉-2-羧酸的CCα为0.7~1.0μg/kg,CCβ为1.3~1.7μg/kg;3-甲基喹噁啉-2-羧酸的CCα为1.2~2.6μg/kg,CCβ为2.0~4.4μg/kg。猪、鸡肝脏组织中喹噁啉-2-羧酸的CCα为2.5~2.6μg/kg,CCβ为4.3~5.6μg/kg;3-甲基喹噁啉-2-羧酸的CCα为2.1~2.4μg/kg,CC∞为4.4~5.6μg/kg。喹噁啉-2-羧酸和3一甲基喹噁啉-2-羧酸在各动物肌肉组织中添加浓度为2.0~16μg/kg,回收率为70.7%~104.3%,相对标准偏差<20%;在猪、鸡肝脏组织添加浓度为10~50μg/kg,回收率为72.3%~80.7%,相对标准偏差<13%。
酸性环境水解样品,结合阴离子固相萃取小柱净化,简化了样品前处理步骤,避免强酸、强碱和有毒试剂的使用,可有效消除基质对药物测定的干扰,扩大了分析样品范围,方法灵敏度高。对比不同样品溶剂对两种分析物紫外吸收的影响,明确其经样品前处理后紫外吸收波长的变化,选用碱性甲醇溶剂,抵消了分析物紫外吸光度变化值,减小了回收率测定误差,提高了分析方法的准确度。
本方法样品前处理简单、基质干扰少、分析范围广、检测灵敏度高,基本达到国际领先水平,可广泛应用于喹噁啉类药物的残留监测,对动物组织中多残留分析方法研究具有指导意义,为动物性食品安全评价提供技术支持。
3.喹噁啉类脱氧速率研究
建立脱二氧喹赛多、脱二氧喹烯酮和脱二氧喹乙醇的高效液相色谱测定方法,为喹噁啉类脱氧产物定性分析提供技术方法。3种化合物经高效液相色谱流动相梯度洗脱分离,二极管阵列检测器测定,检测限为0.01μmol/L,定量限为0.02μmol/L,在0.01~10μmol/L范围内,线性关系良好,相关系数(r)为0.995 9~0.997 1。
化学还原体系中5种喹嗯啉类脱氧速率比较,为这类药物脱氧反应符合一级动力学规律提供判断依据。酸性碘化钾还原体系中,5种药物脱氧还原反应近似符合一级化学动力学规律,半衰期为4.80~19.06 min。碱性连二亚硫酸钠还原体系中喹噁啉类迅速还原,高效液相色谱测定反应体系,由其脱氧还原产物类别推测喹赛多脱氧速率最大,喹烯酮次之。化学还原体系中5种喹噁啉脱氧速率关系:喹赛多>卡巴氧>喹烯酮>喹乙醇>乙酰甲喹。
猪肝微粒体中5种喹噁啉类脱氧速率比较,验证喹噁啉类脱氧速率关系。5种药物在加有还原型辅酶的猪肝微粒体中孵育,高效液相色谱测定不同时间原形药物浓度,发现其脱氧还原反应符合一级化学动力学规律,5种药物的半衰期为11.75~69.31 min。脱氧速率喹赛多>喹烯酮>喹乙醇>卡巴氧>乙酰甲喹。
喹噁啉类脱氧速率及毒理学研究结果,进行脱氧速率与毒性相关性分析,证明脱氧速率为:喹赛多>喹烯酮>喹乙醇>卡巴氧>乙酰甲喹,速率越大毒性越低,脱氧速率与毒性之间存在负相关性,可以作为喹噁啉类毒性高低判断的重要参数,此结论可为喹噁啉类毒性作用机制研究提供理论依据。
本课题开展喹噁啉类分析方法和脱氧速率的研究,在国内外首次建立了饲料中多种喹噁啉类同时分析方法和不同动物可食性组织中喹噁啉类多残留分析方法,比较喹噁啉类脱氧速率快慢,分析脱氧速率与毒性大小之间的关系,这些研究为喹噁啉类残留监控和毒性机理研究提供了先进的检测方法和强有力的理论依据,对动物性食品安全评价有重要参考价值。
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 C_(18) 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 quinoxalines
To 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 Na_2S_2O_4 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.
引文
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