共振瑞利散射技术测定氟喹诺酮类抗生素的新方法研究
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摘要
共振瑞利散射(RRS)是二十世纪九十年代发展起来的一种新分析技术,由于其简便性、高灵敏性而受到人们的广泛关注。目前,共振瑞利散射已经越来越来多地应用于生物大分子、无机离子和药物的研究和测定,在纳米微粒的研究方面业取得了积极的进展。近年来,RRS技术在药物分析中的研究和应用日益增多,显示了良好的应用前景,但目前对于氟喹诺酮类抗生素的分析应用研究不多。故本文以临床上广泛使用的氟喹诺酮类抗生素作为研究对象,重点研究了这类抗生素与某些染料、金属离子的相互作用以及金属-药物螯合物与某些小分子(如染料)形成三元配合物对RRS光谱的影响。考察了适宜的反应条件,影响因素和分析化学特性,提出了一系列RRS技术测定氟喹诺酮类药物的方法,并结合吸收光谱、荧光光谱的变化和量子化学计算的方法,对相关的反应机理和RRS增强的原因进行了讨论。
1、以赤藓红作探针三种方法测定氟喹诺酮类抗生素
用分光光度法、荧光光度法和共振瑞利散射(RRS)法研究了环丙沙星(CIP),诺氟沙星(NOR),左氧氟沙星(LEV)和洛美沙星(LOM)四种氟喹诺酮类抗生素与酸性染料赤藓红(Ery)的相互作用,结果表明四种药物与赤藓红形成离子缔合物时,不仅引起吸收光谱的变化和荧光猝灭,也导致RRS的显著增强并产生新的RRS光谱。文中研究了结合产物的吸收、荧光和RRS光谱特征,适宜的反应条件及分析化学性质,据此发展了以赤藓红为光谱探针的灵敏、简便、快速测定氟喹诺酮类抗生素的新方法。CIP-Ery、NOR-Ery、LEV-Ery和LOM-Ery体系的RRS法检出限分别0.0172、0.0179、0.0142和0.0272μg·mL~(-1);荧光猝灭法的检出限分别为0.0219、0.1001、0.0361和0.0380μg·mL~(-1);分光光度法的检出限分别为0.0983、0.2647、0.1050和0.0970μg·mL~(-1)。本文还研究了赤藓红与氟喹诺酮类抗生素相互作用对吸收、荧光和RRS光谱的影响。并采用量子化学AM1法计算了反应前后体系的电荷分布、生成焓和平均极化率的变化,讨论了RRS光谱产生和增强的原因及光吸收、荧光和RRS之间的能量转换关系。
研究了诺氟沙星(NOR)、环丙沙星(CIP)、氧氟沙星(OF)、左氧氟沙星(LEV)、洛美沙星(LOM)和司帕沙星(SPA)等氟喹诺酮类抗生素与酸性呫吨染料赤藓红(Ery)的相互作用对于共振瑞利散射(RRS)的影响。结果表明:在pH4.4～4.6的BR缓冲介质中,上述FLQs与Ery反应,形成结合产物时,能引起RRS的明显增强,并且在一定范围内散射增强(ΔI)与赤藓红的浓度成正比,可用于赤藓红的定量测定。其中以司帕沙星(SPA)体系最灵敏,对于Ery的检出限为7.14ng·mL~(-1)本文研究了反应体系的RRS光谱特征,并以SPA体系为例,研究适宜的反应条件和影响因素,实验了共存物质的影响,表明方法有较好的选择性。基于FLQs与Ery的离子缔合反应,发展了一种用RRS技术测定赤藓红的新方法。方法灵敏、简便、快速,可用于某些糖果和饮料中赤藓红的测定。文中还结合吸收光谱、荧光光谱及量子化学计算方法对反应机理进行了讨论。
2.2、以氟喹诺酮类抗生素作探针荧光猝灭法测定赤藓红
研究了诺氟沙星(NOR)、环丙沙星(CIP)、氧氟沙星(OF)、左氧氟沙星(LEV)、洛美沙星(LOM)和司帕沙星(SPA)等氟喹诺酮类抗生素与赤藓红(Ery)相互作用对荧光光谱的影响,结果表明:在弱酸性介质中,这类药物与赤藓红反应形成离子缔合物时,将引起溶液荧光猝灭。当以氟喹诺酮类抗生素作探针时,可用荧光猝灭法测定赤藓红,不同药物体系对于赤藓红的灵敏度存在差异,其检出限在0.016～0.027μg·mL~(-1)之间,其灵敏度的顺序是LEV＞LOM＞CIP＞NOR＞OF＞＞SPA,即以LEV-Ery体系最好,而以SPA-Ery体系最差。本文以LEV-Ery体系为例,研究了适宜的反应条件和影响因素,考察了共存物质的影响,表明方法有较好的选择性,可用于食品中赤藓红的测定。文章还运用量子化学计算方法对反应机理进行了讨论。
3、铈(Ⅳ)-氟喹诺酮类抗生素体系
在pH为4.0～5.0的HCl-NaAc缓冲溶液中,Ce(Ⅳ)能与环丙沙星(CIP),诺氟沙星(NOR),培氟沙星(PEN),洛美沙星(LOM)和司帕沙星(SPA)等氟喹诺酮类抗生素的反应,形成稳定的螯合物,此时导致共振瑞利散射(RRS)的显著增强,5种药物的RRS光谱特征一致,其最大散射峰位于381nm附近,并在534nm附近出现一个较小的散射峰。在一定范围内散射强度与药物的浓度成线性增强,五种药物的线性范围和检出限分别为0.006～4.000μg.mL~(-1)和0.0019μg·mL~(-1)(PEN)、0.008～4.000μg·mL~(-1)和0.0023μg·mL~(-1)(CIP)、0.009～10.000μg·mL~(-1)和0.0028μg·mL~(-1)(NOR)、0.018～5.000μg·mL~(-1)和0.0053μg·mL~(-1)(LOM)、0.0533～3.200μg·mL~(-1)和0.0160μg·mL~(-1)(SPA),其中培氟沙星体系灵敏度最高,而司帕沙星体系灵敏度较低。文中重点研究了反应体系的RRS光谱特征、适宜反应条件和影响因素,考察了共存物质的影响,表明方法具有较好的选择性。利用此反应发展了一种用RRS技术简单、快速、稳定、高灵敏度测定某些氟喹诺酮类抗生素的新方法,此法可用于药物制剂和鱼肉中药物的测定,结果满意。
4、铜(Ⅱ)-氟喹诺酮类抗生素螯合物与赤藓红体系
在pH4.2-5.0的Britton-Robinson缓冲溶液中,环丙沙星(CIP),诺氟沙星(NOR),氧氟沙星(OF),左氧氟沙星(LEV),洛美沙星(LOM)和司帕沙星(SPA)等氟喹诺酮类抗生素(FLQs)能与铜(Ⅱ)形成螯合阳离子,它们能进一步与赤藓红(Ery)阴离子通过静电引力和疏水作用形成FLQs:Cu(Ⅱ):Ery为1:1:1的离子缔合物。此时,能引起吸收光谱的变化,并发生明显的褪色作用,最大褪色波长均位于526nm处,反应具有较高的灵敏度,除NOR的摩尔吸光系数(ε)较低外,其余5种抗生素的s值均大于1.0×10~5L·mol~(-1)·cm~(-1),而且LOM和OF体系的ε值均大于3×10~5L·mol~(-1)·cm~(-1),而SPA的ε值高达7.22×10~5L·mol~(-1)·cm~(-1),可用于这类药物的分光光度测定。离子缔合反应还导致赤藓红的荧光猝灭,反应也具有高灵敏度,上述6种FLQs药物的检出限在7.1～12.2 ng·mL~(-1)之间,为荧光猝灭法测定ng.mL~(-1)级FLQs创造了条件。离子缔合反应更能导致共振瑞利散射(RRS)的显著增强,并产生新的RRS光谱。六种药物的反应产物具有相似的光谱特征,最大散射波长均位于566 nm处,并在333 nm和287 nm处有2个较小的散射峰。在一定条件下散射增强(ΔI)与药物浓度成正比。RRS法较褪色分光光度法和荧光猝灭法具有更高的灵敏度,对不同的FLQs药物的检出限在1.7 ng·mL~(-1)至3.1ng·mL~(-1)之间,更适于痕量的FLQs测定。本文研究了反应产物的吸收、荧光和RRS光谱特征,适宜的反应条件及分析化学性质,结合量子化学计算方法讨论了离子缔合反应的历程及对光谱特征的影响,并研究了RRS法的选择性及分析应用。
5、氟喹诺酮类抗生素与钴(Ⅱ)和刚果红体系
在pH4.5-6.5的Britton-Robinson缓冲溶液中,钻(Ⅱ)与环丙沙星(CIP)、诺氟沙星(NOR)、氧氟沙星(OF)和左氧氟沙星(LEV)等氟喹诺酮类抗生素能形成螯合阳离子,它们能通过静电引力和疏水作用与刚果红阴离子反应,形成1:2:1的三元离子缔合配合物。此时将引起溶液的共振瑞利散射(RRS)显著增强,并出现新的RRS光谱。不同抗生素具有相似的光谱特征,其最大散射波长均位于560 nm处,并在382 nm和278 nm处有2个较小的散射峰。一定浓度的抗生素与散射增强(ΔI)成正比,对不同氟喹诺酮类药物的线性范围和检出限(3σ)分别是0.026-2.64μg·mL~(-1)和7.68 ng·mL~(-1)(CIP),0.045-3.20μg·mL~(-1)和13.00 ng·mL~(-1)(NOR),0.037-4.00μg·mL~(-1)和11.24 ng·mL~(-1)(OF),0.039-4.00μg·mL~(-1)和11.80 ng·mL~(-1)(LEV),据此提出了一种以RRS技术测定氟喹诺酮抗生素的新方法。方法不仅灵敏度高,而且简单、快速,并有良好的选择性和重复性,可用于片剂、针剂、滴眼液和人尿液中氟喹诺酮类药物的测定。文中还对反应机理和RRS增强的原因作了讨论。
Resonance Rayleigh scattering(RRS)technique is a new analytical technique developed in 1990s.It has attracted more and more attention because of its high sensitivity,simplicity and good selectivity.It has been extensively and successfully unitized to study and determination of biological macromolecules,trace metal ions, nonmetal ions and nanoparticle probe et al.In the last years,there are more and more studies and applications by RRS technique in pharmaceutical field,it indicated RRS technique has a good applied future.But it is rarely applied in fluoroquinolones antibiotics field.Therefore,taking fluoroquinolones antibiotics which are applied widely in clinic as studied objects,we investigated the interaction between fluoroquinolones antibiotics with some dyes,metal ions,and the interaction between metal-fluoroquinolones chelates with some small molecules(dyes)by RRS method.The spectral characteristics,the affecting factors and the properties of analytical chemistry and their analytical applications have been studied,and a series of methods to determinate fluoroquinolones antibiotics were developed.Besides,the reaction mechanism and the reasons for enhancement of RRS were discussed by the absorption spectra,the fluorescence spectra and the quantum chemistry method.
1、Three methods to the determination for fluoroquinolones by the erythrosine as probe
In pH 4.4-4.5 Britton-Robinson(BR)buffer solution,fluoroquinolone antibiotics (FLQs)including ciprofloxacin(CIP),norfloxacin(NOR),levofloxacin(LEV)and lomefloxacin(LOM)could react with erythrosine(Ery)to form 1:1 ion-association complexes,which not only resulted in the changes of the absorption spectra and the quenching of fluorescence,but also resulted in the great enhancement of resonance Rayleigh scattering(RRS).These offered some indications of the determination of fluoroquinolone antibiotics by spectrophotometric,fluorescence and resonance Rayleigh scattering methods.The detection limits for fluoroquinolone antibiotics were in the range of 0.097～0.265μg·mL~(-1)for absorption methods,0.022～0.100μg·mL~(-1)for fluorophotometry and 0.014～0.027μg·mL~(-1)for RRS method,respectively.Among them, the RRS method had the highest sensitivity.In this work,the spectral characteristics of the absorption,fluorescence and RRS,the optimum conditions of the reactions and the properties of the analytical chemistry were investigated.The methods have been successfully applied to determination of some fluoroquinolone antibiotics in human urine samples and tablets.Taking CIP-Ery system as an example,the charge distribution,the enthalpy of formation and the mean polarizability were calculated by density function theory(DFT)method.In addition,the reasons for the enhancement of scattering spectra were discussed by the absorption spectra,the fluorescence spectra and the quantum chemistry method.
2、Two methods to the determination for erythrosine by the as fluoroquinolones probe
2.1、RRS method to the determination for erythrosione
The reaction between erythrosine and fluoroquinolone antibiotics(FLQs)including ciprofloxacin(CIP),norfloxacin(NOR),ofloxacin(OF),levofloxacin(LEV), sparfloxacin(SPA)and lomefloxacin(LOM)was studied by resonance Rayleigh scattering methods.Our experiment conclusion indicated the reaction between erythrosine and fluoroquinolones resulted in the great enhancement of RRS,the scattering intensities are enhanced with the increase of erythrosine concentration,so it can be used to determinate erythrosine.Among of six systems,sparfloxacin-erythrosine system has the highest sensitivity which the detection limits is 7.14 ng·mL~(-1).The optimum conditions of reaction excluding pH,concentration and ionic intensity,and the effects of foreign substances have been investigated.Based on the reaction between flouroquinolones and erythrosine,we developed a new method of determination for erythrosine by RRS.The method is simple,rapid and sensitive,can be applied to determinate erythrosine in some candies and drink.In addition,the reasons for the enhancement of scattering spectra were discussed by the absorption spectra,the fluorescence spectra and the density function theory(DFT)method.
2.1、The determination for erythrosione by the quenching of fluorescence method
The influence on fluorescence spectra of the reaction between erythrosine and fluoroquinolone antibiotics(FLQs)including ciprofloxacin(CIP),norfloxacin(NOR), ofloxacin(OF),levofloxacin(LEV),sparfloxacin(SPA)and lomefloxacin(LOM)was studied.The result indicated the reaction between erythrosine and fluoroquinolones resulted in the quenching of fluorescence.By using flouroquinolones as probes,we develope some quenching of fluorescence methods to determinate erythrosine.There are some differences in their sensitivities of six systems,which the detection limits are from 0.016 to 0.027μg·mL~(-1).Taking LEV-Ery system as an example,the optimum conditions of reaction excluding pH,concentration and ionic intensity,and the effects of foreign substances have been investigated.In addition,the reasons for the quenching of fluorescence were discussed.
3、The system of fluoroquinolone and cerium(Ⅳ)
In pH 4.0-5.0 HCl-NaAc buffer solution,cerium(Ⅳ)can react with fluoroquinolones on oxygen of carbonyl to form very stable chelate complexes which are bidentate ligands.The reactions not only result in the changes of absorption spectra, a new peak appear at 205 nm,and make the characteristic peak of fluoroquinolones to bathochromic shift,but also results in the enhancement of RRS,the studied five medications systems have similar spectra which the maximum scattering peaks locate on 381 nm,and there are the small peaks at 534 nm.The scattering intensities are enhanced with the increase of medication concentration,so we develop a new method to determinate fluoroquinolones.In this paper,the optimum conditions and properties of the analytical chemistry are investigated.The methods are good selective,simple,rapid stable and have high sensitivity,so they can be applied to determinate fluoroquinoloncs on pharmaceutical and fish meat,and the results are satisfactory.
4、The system of Cu(Ⅱ)-fluoroquinolone chelates with erythrosine
In pH 4.2-5.0 Britton-Robinson buffer solution medium,fluoroquinolone antibiotics(FLQs),such as ciprofloxacin(CIP),norfloxacin(NOR),ofloxacin(OF), levofloxacin(LEV),lomefloxacin(LOM),and sparfloxacin(SPA),react with Cu(Ⅱ)to form chelate cations,which further bind with erythrosine to form the ion association complexes.They can result in the changes of the absorption spectra.Simultaneously, erythrosine fades obviously and the maximum fading wavelength is located at 526 nm. The fading reactions have high sensitivities.Thus,new spectrophotometries of determination for these drugs are developed.The ion -association reactions result in the quenching of fluorescence,which also have high sensitivities.The detection limits for six antibiotics are in the range of 7.1～12.2 ng·mL~(-1)Furthermore,the reactions can result in the enhancement of resonance Rayleigh scattering(RRS).The maximum scattering peaks of six ion-association complexes are located at 566 nm,and there are two small RRS peaks at 333 nm and 287 nm.The detection limits for fluoroquinolone antibiotics are in the range of 1.70～3.10 ng·mL~(-1)for RRS method.Among the above three methods, the RRS method has the highest sensitivity.In this work,we investigated the spectral characteristics of the absorption,fuorescence and RRS,the optimum conditions of the reactions,and the properties of the analytical chemistry.In addition,the mechanism of reactions was discussed by density function theory(DFT)and AM1 methods.
5、The system of Fluoroquinolone Antibiotics-Cobalt(Ⅱ)-Congo Red
In pH 4.5-6.5 Britton-Robinson buffer medium,fluoroquinolone antibiotics(FLQs) such as ciprofloxacin(CIP),norfloxacin(NOR),ofloxacin(OF),levofloxacin(LEV) react with Co~(2+)to form chelate cations,which further bind with Congo Red to form the ion association complexes.It results in the great enhancement of resonance Rayleigh scattering(RRS).Four FLQs reaction products have similar spectral characteristics and their maximum RRS wavelengths are all located at 560 nm,and there are two smaller RRS peaks in 382 nm and 278 nm.The linear ranges and the detection limits of four FLQs are 0.026-2.64μg·mL~(-1)and 7.68 ng mL~(-1)for CIP,0.045-3.20μg·mL~(-1)and 13.00 ng mL~(-1)for NOR,0.037-4.00μg·mL~(-1)and 11.24 ng mL~(-1)for OF,0.039-4.00μg·mL~(-1) and 11.80 ng·mL~(-1)for LEV,respectively.A simple,rapid and sensitive RRS method for the determination of FLQs in pharmaceutical formulation and human urine sample was developed.The reaction mechanism has been also investigated.
1、以赤藓红作探针三种方法测定氟喹诺酮类抗生素
用分光光度法、荧光光度法和共振瑞利散射(RRS)法研究了环丙沙星(CIP),诺氟沙星(NOR),左氧氟沙星(LEV)和洛美沙星(LOM)四种氟喹诺酮类抗生素与酸性染料赤藓红(Ery)的相互作用,结果表明四种药物与赤藓红形成离子缔合物时,不仅引起吸收光谱的变化和荧光猝灭,也导致RRS的显著增强并产生新的RRS光谱。文中研究了结合产物的吸收、荧光和RRS光谱特征,适宜的反应条件及分析化学性质,据此发展了以赤藓红为光谱探针的灵敏、简便、快速测定氟喹诺酮类抗生素的新方法。CIP-Ery、NOR-Ery、LEV-Ery和LOM-Ery体系的RRS法检出限分别0.0172、0.0179、0.0142和0.0272μg·mL~(-1);荧光猝灭法的检出限分别为0.0219、0.1001、0.0361和0.0380μg·mL~(-1);分光光度法的检出限分别为0.0983、0.2647、0.1050和0.0970μg·mL~(-1)。本文还研究了赤藓红与氟喹诺酮类抗生素相互作用对吸收、荧光和RRS光谱的影响。并采用量子化学AM1法计算了反应前后体系的电荷分布、生成焓和平均极化率的变化,讨论了RRS光谱产生和增强的原因及光吸收、荧光和RRS之间的能量转换关系。
研究了诺氟沙星(NOR)、环丙沙星(CIP)、氧氟沙星(OF)、左氧氟沙星(LEV)、洛美沙星(LOM)和司帕沙星(SPA)等氟喹诺酮类抗生素与酸性呫吨染料赤藓红(Ery)的相互作用对于共振瑞利散射(RRS)的影响。结果表明:在pH4.4～4.6的BR缓冲介质中,上述FLQs与Ery反应,形成结合产物时,能引起RRS的明显增强,并且在一定范围内散射增强(ΔI)与赤藓红的浓度成正比,可用于赤藓红的定量测定。其中以司帕沙星(SPA)体系最灵敏,对于Ery的检出限为7.14ng·mL~(-1)本文研究了反应体系的RRS光谱特征,并以SPA体系为例,研究适宜的反应条件和影响因素,实验了共存物质的影响,表明方法有较好的选择性。基于FLQs与Ery的离子缔合反应,发展了一种用RRS技术测定赤藓红的新方法。方法灵敏、简便、快速,可用于某些糖果和饮料中赤藓红的测定。文中还结合吸收光谱、荧光光谱及量子化学计算方法对反应机理进行了讨论。
2.2、以氟喹诺酮类抗生素作探针荧光猝灭法测定赤藓红
研究了诺氟沙星(NOR)、环丙沙星(CIP)、氧氟沙星(OF)、左氧氟沙星(LEV)、洛美沙星(LOM)和司帕沙星(SPA)等氟喹诺酮类抗生素与赤藓红(Ery)相互作用对荧光光谱的影响,结果表明:在弱酸性介质中,这类药物与赤藓红反应形成离子缔合物时,将引起溶液荧光猝灭。当以氟喹诺酮类抗生素作探针时,可用荧光猝灭法测定赤藓红,不同药物体系对于赤藓红的灵敏度存在差异,其检出限在0.016～0.027μg·mL~(-1)之间,其灵敏度的顺序是LEV＞LOM＞CIP＞NOR＞OF＞＞SPA,即以LEV-Ery体系最好,而以SPA-Ery体系最差。本文以LEV-Ery体系为例,研究了适宜的反应条件和影响因素,考察了共存物质的影响,表明方法有较好的选择性,可用于食品中赤藓红的测定。文章还运用量子化学计算方法对反应机理进行了讨论。
3、铈(Ⅳ)-氟喹诺酮类抗生素体系
在pH为4.0～5.0的HCl-NaAc缓冲溶液中,Ce(Ⅳ)能与环丙沙星(CIP),诺氟沙星(NOR),培氟沙星(PEN),洛美沙星(LOM)和司帕沙星(SPA)等氟喹诺酮类抗生素的反应,形成稳定的螯合物,此时导致共振瑞利散射(RRS)的显著增强,5种药物的RRS光谱特征一致,其最大散射峰位于381nm附近,并在534nm附近出现一个较小的散射峰。在一定范围内散射强度与药物的浓度成线性增强,五种药物的线性范围和检出限分别为0.006～4.000μg.mL~(-1)和0.0019μg·mL~(-1)(PEN)、0.008～4.000μg·mL~(-1)和0.0023μg·mL~(-1)(CIP)、0.009～10.000μg·mL~(-1)和0.0028μg·mL~(-1)(NOR)、0.018～5.000μg·mL~(-1)和0.0053μg·mL~(-1)(LOM)、0.0533～3.200μg·mL~(-1)和0.0160μg·mL~(-1)(SPA),其中培氟沙星体系灵敏度最高,而司帕沙星体系灵敏度较低。文中重点研究了反应体系的RRS光谱特征、适宜反应条件和影响因素,考察了共存物质的影响,表明方法具有较好的选择性。利用此反应发展了一种用RRS技术简单、快速、稳定、高灵敏度测定某些氟喹诺酮类抗生素的新方法,此法可用于药物制剂和鱼肉中药物的测定,结果满意。
4、铜(Ⅱ)-氟喹诺酮类抗生素螯合物与赤藓红体系
在pH4.2-5.0的Britton-Robinson缓冲溶液中,环丙沙星(CIP),诺氟沙星(NOR),氧氟沙星(OF),左氧氟沙星(LEV),洛美沙星(LOM)和司帕沙星(SPA)等氟喹诺酮类抗生素(FLQs)能与铜(Ⅱ)形成螯合阳离子,它们能进一步与赤藓红(Ery)阴离子通过静电引力和疏水作用形成FLQs:Cu(Ⅱ):Ery为1:1:1的离子缔合物。此时,能引起吸收光谱的变化,并发生明显的褪色作用,最大褪色波长均位于526nm处,反应具有较高的灵敏度,除NOR的摩尔吸光系数(ε)较低外,其余5种抗生素的s值均大于1.0×10~5L·mol~(-1)·cm~(-1),而且LOM和OF体系的ε值均大于3×10~5L·mol~(-1)·cm~(-1),而SPA的ε值高达7.22×10~5L·mol~(-1)·cm~(-1),可用于这类药物的分光光度测定。离子缔合反应还导致赤藓红的荧光猝灭,反应也具有高灵敏度,上述6种FLQs药物的检出限在7.1～12.2 ng·mL~(-1)之间,为荧光猝灭法测定ng.mL~(-1)级FLQs创造了条件。离子缔合反应更能导致共振瑞利散射(RRS)的显著增强,并产生新的RRS光谱。六种药物的反应产物具有相似的光谱特征,最大散射波长均位于566 nm处,并在333 nm和287 nm处有2个较小的散射峰。在一定条件下散射增强(ΔI)与药物浓度成正比。RRS法较褪色分光光度法和荧光猝灭法具有更高的灵敏度,对不同的FLQs药物的检出限在1.7 ng·mL~(-1)至3.1ng·mL~(-1)之间,更适于痕量的FLQs测定。本文研究了反应产物的吸收、荧光和RRS光谱特征,适宜的反应条件及分析化学性质,结合量子化学计算方法讨论了离子缔合反应的历程及对光谱特征的影响,并研究了RRS法的选择性及分析应用。
5、氟喹诺酮类抗生素与钴(Ⅱ)和刚果红体系
在pH4.5-6.5的Britton-Robinson缓冲溶液中,钻(Ⅱ)与环丙沙星(CIP)、诺氟沙星(NOR)、氧氟沙星(OF)和左氧氟沙星(LEV)等氟喹诺酮类抗生素能形成螯合阳离子,它们能通过静电引力和疏水作用与刚果红阴离子反应,形成1:2:1的三元离子缔合配合物。此时将引起溶液的共振瑞利散射(RRS)显著增强,并出现新的RRS光谱。不同抗生素具有相似的光谱特征,其最大散射波长均位于560 nm处,并在382 nm和278 nm处有2个较小的散射峰。一定浓度的抗生素与散射增强(ΔI)成正比,对不同氟喹诺酮类药物的线性范围和检出限(3σ)分别是0.026-2.64μg·mL~(-1)和7.68 ng·mL~(-1)(CIP),0.045-3.20μg·mL~(-1)和13.00 ng·mL~(-1)(NOR),0.037-4.00μg·mL~(-1)和11.24 ng·mL~(-1)(OF),0.039-4.00μg·mL~(-1)和11.80 ng·mL~(-1)(LEV),据此提出了一种以RRS技术测定氟喹诺酮抗生素的新方法。方法不仅灵敏度高,而且简单、快速,并有良好的选择性和重复性,可用于片剂、针剂、滴眼液和人尿液中氟喹诺酮类药物的测定。文中还对反应机理和RRS增强的原因作了讨论。
Resonance Rayleigh scattering(RRS)technique is a new analytical technique developed in 1990s.It has attracted more and more attention because of its high sensitivity,simplicity and good selectivity.It has been extensively and successfully unitized to study and determination of biological macromolecules,trace metal ions, nonmetal ions and nanoparticle probe et al.In the last years,there are more and more studies and applications by RRS technique in pharmaceutical field,it indicated RRS technique has a good applied future.But it is rarely applied in fluoroquinolones antibiotics field.Therefore,taking fluoroquinolones antibiotics which are applied widely in clinic as studied objects,we investigated the interaction between fluoroquinolones antibiotics with some dyes,metal ions,and the interaction between metal-fluoroquinolones chelates with some small molecules(dyes)by RRS method.The spectral characteristics,the affecting factors and the properties of analytical chemistry and their analytical applications have been studied,and a series of methods to determinate fluoroquinolones antibiotics were developed.Besides,the reaction mechanism and the reasons for enhancement of RRS were discussed by the absorption spectra,the fluorescence spectra and the quantum chemistry method.
1、Three methods to the determination for fluoroquinolones by the erythrosine as probe
In pH 4.4-4.5 Britton-Robinson(BR)buffer solution,fluoroquinolone antibiotics (FLQs)including ciprofloxacin(CIP),norfloxacin(NOR),levofloxacin(LEV)and lomefloxacin(LOM)could react with erythrosine(Ery)to form 1:1 ion-association complexes,which not only resulted in the changes of the absorption spectra and the quenching of fluorescence,but also resulted in the great enhancement of resonance Rayleigh scattering(RRS).These offered some indications of the determination of fluoroquinolone antibiotics by spectrophotometric,fluorescence and resonance Rayleigh scattering methods.The detection limits for fluoroquinolone antibiotics were in the range of 0.097～0.265μg·mL~(-1)for absorption methods,0.022～0.100μg·mL~(-1)for fluorophotometry and 0.014～0.027μg·mL~(-1)for RRS method,respectively.Among them, the RRS method had the highest sensitivity.In this work,the spectral characteristics of the absorption,fluorescence and RRS,the optimum conditions of the reactions and the properties of the analytical chemistry were investigated.The methods have been successfully applied to determination of some fluoroquinolone antibiotics in human urine samples and tablets.Taking CIP-Ery system as an example,the charge distribution,the enthalpy of formation and the mean polarizability were calculated by density function theory(DFT)method.In addition,the reasons for the enhancement of scattering spectra were discussed by the absorption spectra,the fluorescence spectra and the quantum chemistry method.
2、Two methods to the determination for erythrosine by the as fluoroquinolones probe
2.1、RRS method to the determination for erythrosione
The reaction between erythrosine and fluoroquinolone antibiotics(FLQs)including ciprofloxacin(CIP),norfloxacin(NOR),ofloxacin(OF),levofloxacin(LEV), sparfloxacin(SPA)and lomefloxacin(LOM)was studied by resonance Rayleigh scattering methods.Our experiment conclusion indicated the reaction between erythrosine and fluoroquinolones resulted in the great enhancement of RRS,the scattering intensities are enhanced with the increase of erythrosine concentration,so it can be used to determinate erythrosine.Among of six systems,sparfloxacin-erythrosine system has the highest sensitivity which the detection limits is 7.14 ng·mL~(-1).The optimum conditions of reaction excluding pH,concentration and ionic intensity,and the effects of foreign substances have been investigated.Based on the reaction between flouroquinolones and erythrosine,we developed a new method of determination for erythrosine by RRS.The method is simple,rapid and sensitive,can be applied to determinate erythrosine in some candies and drink.In addition,the reasons for the enhancement of scattering spectra were discussed by the absorption spectra,the fluorescence spectra and the density function theory(DFT)method.
2.1、The determination for erythrosione by the quenching of fluorescence method
The influence on fluorescence spectra of the reaction between erythrosine and fluoroquinolone antibiotics(FLQs)including ciprofloxacin(CIP),norfloxacin(NOR), ofloxacin(OF),levofloxacin(LEV),sparfloxacin(SPA)and lomefloxacin(LOM)was studied.The result indicated the reaction between erythrosine and fluoroquinolones resulted in the quenching of fluorescence.By using flouroquinolones as probes,we develope some quenching of fluorescence methods to determinate erythrosine.There are some differences in their sensitivities of six systems,which the detection limits are from 0.016 to 0.027μg·mL~(-1).Taking LEV-Ery system as an example,the optimum conditions of reaction excluding pH,concentration and ionic intensity,and the effects of foreign substances have been investigated.In addition,the reasons for the quenching of fluorescence were discussed.
3、The system of fluoroquinolone and cerium(Ⅳ)
In pH 4.0-5.0 HCl-NaAc buffer solution,cerium(Ⅳ)can react with fluoroquinolones on oxygen of carbonyl to form very stable chelate complexes which are bidentate ligands.The reactions not only result in the changes of absorption spectra, a new peak appear at 205 nm,and make the characteristic peak of fluoroquinolones to bathochromic shift,but also results in the enhancement of RRS,the studied five medications systems have similar spectra which the maximum scattering peaks locate on 381 nm,and there are the small peaks at 534 nm.The scattering intensities are enhanced with the increase of medication concentration,so we develop a new method to determinate fluoroquinolones.In this paper,the optimum conditions and properties of the analytical chemistry are investigated.The methods are good selective,simple,rapid stable and have high sensitivity,so they can be applied to determinate fluoroquinoloncs on pharmaceutical and fish meat,and the results are satisfactory.
4、The system of Cu(Ⅱ)-fluoroquinolone chelates with erythrosine
In pH 4.2-5.0 Britton-Robinson buffer solution medium,fluoroquinolone antibiotics(FLQs),such as ciprofloxacin(CIP),norfloxacin(NOR),ofloxacin(OF), levofloxacin(LEV),lomefloxacin(LOM),and sparfloxacin(SPA),react with Cu(Ⅱ)to form chelate cations,which further bind with erythrosine to form the ion association complexes.They can result in the changes of the absorption spectra.Simultaneously, erythrosine fades obviously and the maximum fading wavelength is located at 526 nm. The fading reactions have high sensitivities.Thus,new spectrophotometries of determination for these drugs are developed.The ion -association reactions result in the quenching of fluorescence,which also have high sensitivities.The detection limits for six antibiotics are in the range of 7.1～12.2 ng·mL~(-1)Furthermore,the reactions can result in the enhancement of resonance Rayleigh scattering(RRS).The maximum scattering peaks of six ion-association complexes are located at 566 nm,and there are two small RRS peaks at 333 nm and 287 nm.The detection limits for fluoroquinolone antibiotics are in the range of 1.70～3.10 ng·mL~(-1)for RRS method.Among the above three methods, the RRS method has the highest sensitivity.In this work,we investigated the spectral characteristics of the absorption,fuorescence and RRS,the optimum conditions of the reactions,and the properties of the analytical chemistry.In addition,the mechanism of reactions was discussed by density function theory(DFT)and AM1 methods.
5、The system of Fluoroquinolone Antibiotics-Cobalt(Ⅱ)-Congo Red
In pH 4.5-6.5 Britton-Robinson buffer medium,fluoroquinolone antibiotics(FLQs) such as ciprofloxacin(CIP),norfloxacin(NOR),ofloxacin(OF),levofloxacin(LEV) react with Co~(2+)to form chelate cations,which further bind with Congo Red to form the ion association complexes.It results in the great enhancement of resonance Rayleigh scattering(RRS).Four FLQs reaction products have similar spectral characteristics and their maximum RRS wavelengths are all located at 560 nm,and there are two smaller RRS peaks in 382 nm and 278 nm.The linear ranges and the detection limits of four FLQs are 0.026-2.64μg·mL~(-1)and 7.68 ng mL~(-1)for CIP,0.045-3.20μg·mL~(-1)and 13.00 ng mL~(-1)for NOR,0.037-4.00μg·mL~(-1)and 11.24 ng mL~(-1)for OF,0.039-4.00μg·mL~(-1) and 11.80 ng·mL~(-1)for LEV,respectively.A simple,rapid and sensitive RRS method for the determination of FLQs in pharmaceutical formulation and human urine sample was developed.The reaction mechanism has been also investigated.
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