【作者】 孙国仁；

【导师】 祁克宗； 朱良强；

【作者基本信息】 安徽农业大学 ， 基础兽医学， 2006， 硕士

【摘要】 为了灵敏、快速、高效的检测氟喹诺酮类药物残留,本研究基于铽离子对氟喹诺酮类药物有很强的荧光增强作用,详细讨论该体系的反应条件及影响因素,以肉仔鸡为实验模型,建立高灵敏度的检测鸡肉中氟喹诺酮类药物残留的方法。1.铽增敏荧光分光光度法的初步建立样品经过预处理,通过荧光分光光度计进行扫描,发现铽离子对FQs有明显的增敏作用。分析了铽-环丙沙星、铽-诺氟沙星、铽-恩诺沙星衍生体系,摸索铽与氟喹诺酮类药物反应条件。同时,详细分析了铽-环丙沙星体系,以环丙沙星浓度(C)为纵坐标,以荧光强度(F)为横坐标,进行线性回归,并作空白对照,初步建立铽增敏荧光分光光度法,回归方程为: C = 3.0031F - 48.198 r=0.981环丙沙星浓度在1～1000ng·ml-1范围内呈线性关系,方法回收率98.92%,日内RSD=8.2%。日间RSD=10.9%。由CL=KSm/S,计算其最低检测限为1ng·ml-(1g-1)。实验表明,铽-环丙沙星、铽-诺氟沙星、铽-恩诺沙星衍生体系的最大发射波长都在545nm,最大激发波长也均在330nm左右;又因为铽增敏荧光分光光度法的检测限大大降低,为此,本方法可以作为环丙沙星、诺氟沙星、恩诺沙星药物残留的初筛方法。2.铽增敏HPLC柱后衍生法的确立用磷酸盐缓冲液提取出鸡肉组织中的氟喹诺酮类药物,过C18固相萃取柱净化,铽增敏HPLC柱后衍生法检测。色谱条件:柱温40℃;流速0.8mL·min-1;进样量20μL;衍生泵温度40℃;衍生泵流速0.5mL·min-1;荧光检测器,λEx=330nm,λEm=545nm。环丙沙星、诺氟沙星、恩诺沙星三种药物的保留时间分别为8.16 min、7.25 min和11.49min,当药物浓度在1-200ng?ml-1(g-1)时与峰面积(S)呈良好的线性关系,其线性方程分别为:环丙沙星:S环丙=59.81C+6.38 r=0.9981诺氟沙星: S诺氟=87.33C+3.81 r=0.9896恩诺沙星: S恩诺=108.47C+9.39 r=0.9823三种氟喹诺酮类药物的平均回收率及其相对标准偏差(RSD)分别为:环丙沙星:78.64%, 7.45%;诺氟沙星:80.92%,11.73%;恩诺沙星:77.70%,13.17%。其在鸡肉组织中的最低检测限分别为:环丙沙星:0.05ng·g-1;诺氟沙星:0.05ng·g-1;恩诺沙星:0.08ng·g-1。比现在报道的最低检测限降低了50倍,即灵敏度提高了50倍。3.应用铽增敏HPLC柱后衍生法检测三种氟喹诺酮类药物在鸡肉中的残留用铽增敏HPLC柱后衍生法检测肉仔鸡连续4d内服环丙沙星、诺氟沙星、恩诺沙星混合药物后的残留。停药第2d,肌肉组织中均可检测到高残留的三种药物;停药第5d,鸡肉组织中药物残留量也较高;停药第16d,诺氟沙星和恩诺沙星在肌肉组织中残留药物浓度低于我国规定的MRLs,环丙沙星在肌肉组织中仍有较高残留;停药第22d,肌肉中也检测不到诺氟沙星和恩诺沙星,但可以检测到环丙沙星;停药第29d,肌肉中三种药物均未检测到。本研究首次建立了检测鸡肉中三种氟喹诺酮类药物残留的Tb3+增敏HPLC柱后衍生法,提高了氟喹诺酮类药物的检测灵敏度,为氟喹诺酮类药物在鸡肉组织中的残留分析和研究提供了实验手段。同时,在条件摸索的过程中,建立了Tb3+增敏荧光分光光度法检测鸡肉组织中的环丙沙星的方法,样品前处理简单、快速,整个实验操作方便快捷,经济,灵敏度高,为本类药物残留检测的快速筛选提供了手段。更多还原

【Abstract】 In order to dertermine residue of fluoroquinolones high-sensitively, this research is based on that the terbium ion has strong fluorescence enhancement function against fluoroquinolones, the systematic reaction condition and infection factor was discussed detailly, a high-sensitivity determination method of the residue of fluoroquinolines in chicken was established according to the experimental model.Ⅰ. Preliminary establishment of terbium sensitized fluorescence spectrophotometryAfter pretreatment of the sample, which scanned by fluorescence spectrophotometer. Terbium ion has strong fluorescence enhancement function against fluoroquinolones was found. Tb3+-Ciprofloxacin, Tb3+-Norfloxacin and Tb3+-Enrofloxacin derivation systems was analyzed and the reaction condition of terbium and fluoroquinolones was groped. The same time Tb3+-Ciprofloxacin derivation systems was analyzed detailly, took ciprofloxacin density (C) as the y-coordinate and fluorescence intensity (F) as the x-coordinate, carried out linear regression, and made the blank comparison. We can get the regression equation as followed: C = 3.0031F - 48.198 r=0.981When the density of ciprofloxacin is in the range of 1～1000ng/ml that indicate linear relation, and the recovery of method is 98.92% with the within-day RSD of 8.2% and between-day RSD of 10.9%. As CL equals KSm/S, the lowest detection limit is 1ng?ml-1(g-1).The experiment indicated the biggest transmitted wave length of Tb3+-Ciprofloxacin, Tb3+-Norfloxacin and Tb3+-Enrofloxacin derivation systems are all about 545nm, and their biggest excitation wave length are all about 330nm. Because the detection limit of terbium sensitized fluorescence spectrophotometry has reduced greatly, we can conjecture that this method can be the primary screening method of Ciprofloxacin, Norfloxacin and Enrofoxacin.Ⅱ. Establishment of terbium sensitized fluorescence and post-column derivation HPLCExtract fluoroquinolones from chicken tissue with phosphate buffer, and purify with C18 solid phase extract column, and then detect drug density with terbium sensitized fluorescence and post-column derivation HPLC. Chromatographic condition: column temperature is 40℃; float rate 0.8mL?min-1; sample size is 20μL; derivation pump temperature is 40℃; derivation pump float rate is 0.5mL?min-1;λEx=330nm,λEm=545nm. The retention time of Ciprofloxacin, Norfloxacin and Enrofoxacin are respectively 7.25min, 8.16min and 11.49min. The calibration curves for the area (S) is linear in the range of 1～200ng?ml-1(g-1), and the linear equations are respectively are: Ciprofloxacin: SCIP=59.81C+6.38 r=0.9981 Norfloxacin: SNOR=87.33C+3.81 r=0.9896 Enrofloxacin:SENR=108.47C+9.39 r=0.9823The average recoveries and relative standard deviations of these 3 kinds of fluoroquinolones are respectively: Ciprofloxacin: 78.64% 7.45% Norfloxacin: 80.92% 11.73% Enrofloxacin: 77.70% 13.17%The lowest detection limits of these 3 kinds of fluoroquinolones in chicken tissue are as followed: Ciprofloxacin: 0.05ng?g-1, Norfloxacin:0.05ng?g-1, Enrofloxacin:0.08ng?g-1.They are 50 times lower than the current reported lowest detection limit. That is to say, the sensitivity has been increased 50 times.Ⅲ. The terbium increase sensitivity to HPLC post-column derivation method that was used to determine the residues after continuously in-take 3 kinds of fluoroquinolone drugs in chickenThe terbium increase sensitivity to HPLC post-column derivation method that was used to determine the residues after meat chicken had continuously in-taken the mixed drugs of Ciprofloxacin, Norfloxacin and Enrofloxacin for 4 days. After stopped taking drugs, on the second day, the high residue of these 3 kinds of drugs could be all detected in chicken tissue; on the sixteenth day, the residues were still high; on the tenth day, the density of drug residue of Norfloxacin and Enrofloxacin was lower than MRLs in chicken tissue, and Ciprofloxacin still had high residue; on the twenty-second day, Norfloxacin and Enrofloxacin couldn’t be found in chicken tissue, but Ciprofloxacin could be found; on the twenty-nine day, these 3 kinds of drugs couldn’t be detected in chicken tissue. This research has established the HPLC post-column derivation method of detecting 3 kinds of fluoroquinolones residues in chicken for the first time, and it has increased the determination sensitivity of fluoroquinolones. It also provides experiment method for drug residue analysis and research. In the process of groping conditions, the terbium sensitized fluorescence spectrophotometry of detecting Ciprofloxacin in chicken tissue was established. The preliminary treatment of sample is simple and rapid. The operation is convenient and quick. This method is economical and highly sensitive, and it provides a means for fast screening of drug residue analysis.更多还原