共振瑞利散射光谱法在某些抗生素类药物分析中的应用研究
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摘要
共振瑞利散射法(Resonance Rayleigh Scattering, RRS)是二十世纪九十年代发展起来的一种新分析技术,它以灵敏度高(检出限可达ng·mL-1级)、仪器廉价、操作简便以及分析快速等优点而引起了人们的广泛兴趣和关注。目前主要利用染料生色团在生物大分子上的聚集作用产生强烈的共振散射而用于核酸、蛋白质和肝素等生物大分子的研究和测定。此外,研究表明,具有相反电荷的两种离子,借助静电引力、疏水作用力和电荷转移作用形成离子缔合物时,也能引起共振瑞利散射的增强,并改变其光谱特征。这种方法已在痕量金属、非金属、有机物、纳米微粒和药物分析中得到越来越多的应用。
本研究论文主要分为两个部分：综述和研究报告。
第一部分综述共振瑞利散射光谱法在药物分析方面的应用进展。
本文对共振瑞利散射光谱法的发展历程和近五年来在药物分析中的应用做了简要综述,并对共振瑞利散射光谱法在药物分析中的应用前景做了展望。
第二部分主要研究共振瑞利散射光谱法在氟喹诺酮类抗生素药物分析中的应用。本文具体研究体系包括铽-氟喹诺酮类抗生素-茜素红相互作用的反应体系；铕-氟喹诺酮类抗生素-铬天青S相互作用的反应体系；钴-氟喹诺酮类抗生素-刚果红相互作用的反应体系。探讨了它们的可见吸收光谱,荧光光谱和RRS光谱特征,适宜反应条件,影响因素及其分析应用；建立和发展了共振瑞利散射光谱法测定上述药物的新方法；并对反应机理,RRS增强的原因,RRS光谱与吸收光谱的关系以及离子缔合物的结构做了初步探讨。内容如下：
1.铽-氟喹诺酮类抗生素-茜素红体系的共振瑞利散射光谱研究及其应用
在弱酸性的HAc-NaAc缓冲溶液中,氟罗沙星(Fleroxacin, FLE),培氟沙星(Pefloxacin, PEFX),环丙沙星(Ciprofloxacin, CIP),洛美沙星(Lomefloxacin, LOM)和帕珠沙星(Pazufloxacin, PAZ)氟喹诺酮类抗生素(fluoroquinolone antibiotics, FLQs)分别与铽(Ⅲ)能形成螯合阳离子,它们可进一步与茜素红(Alizarin Red, AR)反应形成2：1：1(FLQs：Tb3+：AR)三元离子缔合物,导致共振瑞利散射(RRS)显著增强并出现新的RRS光谱,不同的抗生素具有相似的光谱特征,其最大散射波长位于373 nm左右,一定浓度的抗生素与散射增强(△I)成正比。在最大散射波长处,对不同氟喹诺酮类抗生素药物的浓度的线性范围和检出限(3σ)分别是氟罗沙星0.04～3.69μg·mL-1,22.7 ng·mL-1；培氟沙星0.03～3.33μg·mL-1,13.3 ng·mL-1；环丙沙星0.03～3.31μg·mL-1,22.6 ng·mL-1；洛美沙星0.004～3.52μg·mL-1,14.3 ng·mL-1；帕珠沙星0.16～3.18μg·mL-1,28.6 ng·mL-1。方法简单、快速、灵敏,并有良好的选择性,用于片剂、注射液、粉针剂、尿液和人血清中氟喹诺酮类抗生素药物的测定,结果满意。本文还对反应机理作了简要探讨。
2.铕-氟喹诺酮类抗生素-铬天青S体系的共振瑞利散射光谱研究及其应用
在弱酸性的HAc-NaAc缓冲溶液中,氟罗沙星(Fleroxacin, FLE),洛美沙星(Lomefloxacin, LOM)和环丙沙星(Ciprofloxacin, CIP)分别与铕(Ⅲ)能形成螯合阳离子,进而与铬天青S(Chromeazurol S, CAS)阴离子借助静电引力和疏水作用形成三元离子缔合物,使共振瑞利散射(RRS)显著增强并出现新的RRS光谱,基于此,建立了测定氟喹诺酮类抗生素药物的RRS法,并对其光谱特征,影响因素,适宜的反应条件和共存物质的影响进行了探讨。不同的抗生素具有相似的光谱特征,两个散射峰分别位于361 nm和560 nm左右,在361 nm处,对不同氟喹诺酮类抗生素药物的浓度的线性范围和检出限(3σ)分别是氟罗沙星0.037～3.69μg·mL-1,55.8 ng·mL-1；洛美沙星0.035～3.52μg·mL-1,14.5 ng·mL-1；环丙沙星0.033～3.31μg·mL-1,12.4 ng·mL-1。方法简单、快速、灵敏,并有良好的选择性,可用于片剂、注射液、尿液和人血清中氟喹诺酮类抗生素药物的测定。
3.钴-氟喹诺酮类抗生素-刚果红体系的共振瑞利散射光谱研究及其应用
在弱酸性的Britton-Robinson缓冲溶液中,氟罗沙星(Fleroxacin, FLE),加替沙星(Gatifloxacin, GTFX)和培氟沙星(Pefloxacin, PEFX)分别与钴(Ⅱ)能形成螯合阳离子,其共振瑞利散射(RRS)十分微弱,但当该配阳离子与刚果红(Congo Red, CR)阴离子反应形成2：1：1(FLQs：Co2+：CR)三元离子缔合物时,RRS显著增强并出现新的RRS光谱,散射峰分别位于372和560 nm附近。对不同氟喹诺酮类抗生素药物的浓度的线性范围和检出限(3σ)分别是：372 nm处,氟罗沙星0.03～3.69 gg·mL-1,6.0 ng·mL-1；382nm处,加替沙星0.03～5.26μg·mL-1,7.5 ng·mL-1；380 nm处,培氟沙星0.033～3.31μg·mL-1,26.4 ng·mL-1。方法简单、快速、灵敏,并有良好的选择性,用于片剂、尿液和人血清中氟喹诺酮类抗生素药物的测定,结果满意。
Resonance Rayleigh Scattering (RRS) is a new analytical technique developed in 1990s. For its excellence of high sensitivity (detection limit of ng·mL-1 level), cheap instrument, simple operation and rapid analysis speed, the method has attracted to much attention and interest. At present, as the aggregation of dye chromophore on biological macromolecule leading to enhancement of RRS intensity, the method is mainly used to study biological macromolecules such as nucleic acids, proteins and heparin ect. Furthemrore, when two ions of different charges form ion association complexes by electrostatic force, hydrophobic force or charge transferring effect, RRS can be enhanced and the spectra charaeteristic can be changed. This method has been widely applied to the detemrination of trace metal ions, nonmetal ions, ogranic compounds, nanoparticles and pharmaceutical analysis.
The present thesis consisted of two parts:overview and research reports.
The overview involves the history, the basic principles, characteristics and the main systems of RRS, and the applications of RRS systems in pharmaceutical analysis in last five years are intensively reviewed.
The searcher part of the reports mainly involves three aspects:Terbium-fluoroquinolones-alizarin red (AR) systems; Europium--fluoroquinolones--chromeazurol S (CAS) systems; Cobalt-fluoroquinolone--congo red (CR) systems. Their absorption (A), fluorescence and RRS spectral characteristies, optimum reaction conditions, influening factors and analytical applications have been studied. Furthermore, the reaction mechanism, the reason of A or RRS enhanced, and the relationships between RRS spectra and absorption spectra and the structures of the ion-association complexes are discussed. The main research contents are as follows:
1. Study on Terbium--fluoroquinolones antibiotics--alizarin red systems by Resonance Rayleigh Scattering Spectra and their application
In the weak acid of HAc-NaAc buffer solution, fluoroquinolone antibiotics (FLQs) such as: fleroxacin (FLE), pefloxacin (PEFX), ciprofloxacin (CIP), lomefloxacin (LOM) and pazufloxacin (PAZ) can react with Tb (Ⅲ) to form chelated cations, which further bound with alizarin red (AR) to form the 2:1:1 (FLQs:Tb3+:AR) ternary ion-association complexes, resulting in the great enhancement of resonance Rayleigh scattering (RRS). The five reaction products have similar spectral characteristics and the maximum scattering wavelengths are located near 373 nm respectively. The linear ranges and the detection limits (3σ) of the FLQs are 0.04～3.69μg·mL-1, 22.7 ng·mL-1 for FLE at 373 nm; 0.03～3.33μg·mL-1,13.3 ng·mL-1 for PEFX at 373 nm; 0.03～ 3.31μg·mL-1,22.6 ng·mL-1 for CIP at 373 nm; 0.004～3.52μg·mL-1,14.3 ng·mL-1 for LOM at 370 nm; 0.16～3.18μg·mL-1,28.6ng·mL-1 for PAZ at 373 nm, respectively. The method, which is simple, rapid and selective, has been successfully applied to the determination of FLQs in tablet, injection, urine and human serum with satisfactory results. The mechanism of reaction has also been investeinged.
2. Study on Europium--fluoroquinolones--chromeazurol S systems by Resonance Rayleigh Scattering Spectrum and their Application
In the weak acid of HAc-NaAc buffer solution, fleroxacin (FLE), lomefloxacin (LOM) and ciprofloxacin (CIP) can react with Eu (Ⅲ) to form chelated cations, which further bound with chromeazurol S (CAS) by virtue of electrostatic attraction and hydrophobic force to form ternary ion-association complexes, resulting in the great enhancement of resonance Rayleigh scattering (RRS). A new method for the determination of FLQs based on RRS has been developed. The FLQs reaction products have similar spectral characteristics and the maximum scattering wavelengths are located at 361 nm and 560 nm respectively. The linear ranges and the detection limits (3a) of the FLQs are 0.037-3.69μg·mL-1,55.8 ng·mL-1 for FLE; 0.035～3.52μg·mL-1,14.5 ng·mL-1 for LOM; 0.033～3.31μg·mL-1,12.4 ng·mL-1 for CIP at 361 nm, respectively. The method, which is simple, rapid and selective, has been successfully applied to the determination of FLQs in tablet, injection, urine and human serum with satisfactory results.
3. Study on Cobalt--fluoroquinolone antibiotics--congo red systems by Resonance Rayleigh Scattering Spectrum and their Application
In the weak acid of Britton-Robinson buffer solution,fleroxacin (FLE), gatifloxacin (GTFX), and pefloxacin (PEFX) can react with Co(Ⅱ)to form a chelated cation, but its resonance Rayleigh scattering(RRS) was very weak. Significant enhancement of RRS intensity and new RRS spectrum was observed when the chelated cation further reacts with congo red to form an ion association complexe showing two scattering peaks near 372 nm and 560 nm respectively. The linear ranges and the detection limits (3σ) of the FLQs are 0.03～3.69μg·mL-1,6.0 ng·mL-1 for FLE at 372 nm; 0.03～5.26μg·mL-1,7.5 ng·mL-1 for GTFX at 382 nm; 0.033～3.31μg·mL-1,26.4 ng·mL-1 for PEFX at 380 nm, respectively. The proposed method has been applied to the determination of fluoroquinolone antibiotics in tablet, human urine and plasma with satisfactory results.
本研究论文主要分为两个部分：综述和研究报告。
第一部分综述共振瑞利散射光谱法在药物分析方面的应用进展。
本文对共振瑞利散射光谱法的发展历程和近五年来在药物分析中的应用做了简要综述,并对共振瑞利散射光谱法在药物分析中的应用前景做了展望。
第二部分主要研究共振瑞利散射光谱法在氟喹诺酮类抗生素药物分析中的应用。本文具体研究体系包括铽-氟喹诺酮类抗生素-茜素红相互作用的反应体系；铕-氟喹诺酮类抗生素-铬天青S相互作用的反应体系；钴-氟喹诺酮类抗生素-刚果红相互作用的反应体系。探讨了它们的可见吸收光谱,荧光光谱和RRS光谱特征,适宜反应条件,影响因素及其分析应用；建立和发展了共振瑞利散射光谱法测定上述药物的新方法；并对反应机理,RRS增强的原因,RRS光谱与吸收光谱的关系以及离子缔合物的结构做了初步探讨。内容如下：
1.铽-氟喹诺酮类抗生素-茜素红体系的共振瑞利散射光谱研究及其应用
在弱酸性的HAc-NaAc缓冲溶液中,氟罗沙星(Fleroxacin, FLE),培氟沙星(Pefloxacin, PEFX),环丙沙星(Ciprofloxacin, CIP),洛美沙星(Lomefloxacin, LOM)和帕珠沙星(Pazufloxacin, PAZ)氟喹诺酮类抗生素(fluoroquinolone antibiotics, FLQs)分别与铽(Ⅲ)能形成螯合阳离子,它们可进一步与茜素红(Alizarin Red, AR)反应形成2：1：1(FLQs：Tb3+：AR)三元离子缔合物,导致共振瑞利散射(RRS)显著增强并出现新的RRS光谱,不同的抗生素具有相似的光谱特征,其最大散射波长位于373 nm左右,一定浓度的抗生素与散射增强(△I)成正比。在最大散射波长处,对不同氟喹诺酮类抗生素药物的浓度的线性范围和检出限(3σ)分别是氟罗沙星0.04～3.69μg·mL-1,22.7 ng·mL-1；培氟沙星0.03～3.33μg·mL-1,13.3 ng·mL-1；环丙沙星0.03～3.31μg·mL-1,22.6 ng·mL-1；洛美沙星0.004～3.52μg·mL-1,14.3 ng·mL-1；帕珠沙星0.16～3.18μg·mL-1,28.6 ng·mL-1。方法简单、快速、灵敏,并有良好的选择性,用于片剂、注射液、粉针剂、尿液和人血清中氟喹诺酮类抗生素药物的测定,结果满意。本文还对反应机理作了简要探讨。
2.铕-氟喹诺酮类抗生素-铬天青S体系的共振瑞利散射光谱研究及其应用
在弱酸性的HAc-NaAc缓冲溶液中,氟罗沙星(Fleroxacin, FLE),洛美沙星(Lomefloxacin, LOM)和环丙沙星(Ciprofloxacin, CIP)分别与铕(Ⅲ)能形成螯合阳离子,进而与铬天青S(Chromeazurol S, CAS)阴离子借助静电引力和疏水作用形成三元离子缔合物,使共振瑞利散射(RRS)显著增强并出现新的RRS光谱,基于此,建立了测定氟喹诺酮类抗生素药物的RRS法,并对其光谱特征,影响因素,适宜的反应条件和共存物质的影响进行了探讨。不同的抗生素具有相似的光谱特征,两个散射峰分别位于361 nm和560 nm左右,在361 nm处,对不同氟喹诺酮类抗生素药物的浓度的线性范围和检出限(3σ)分别是氟罗沙星0.037～3.69μg·mL-1,55.8 ng·mL-1；洛美沙星0.035～3.52μg·mL-1,14.5 ng·mL-1；环丙沙星0.033～3.31μg·mL-1,12.4 ng·mL-1。方法简单、快速、灵敏,并有良好的选择性,可用于片剂、注射液、尿液和人血清中氟喹诺酮类抗生素药物的测定。
3.钴-氟喹诺酮类抗生素-刚果红体系的共振瑞利散射光谱研究及其应用
在弱酸性的Britton-Robinson缓冲溶液中,氟罗沙星(Fleroxacin, FLE),加替沙星(Gatifloxacin, GTFX)和培氟沙星(Pefloxacin, PEFX)分别与钴(Ⅱ)能形成螯合阳离子,其共振瑞利散射(RRS)十分微弱,但当该配阳离子与刚果红(Congo Red, CR)阴离子反应形成2：1：1(FLQs：Co2+：CR)三元离子缔合物时,RRS显著增强并出现新的RRS光谱,散射峰分别位于372和560 nm附近。对不同氟喹诺酮类抗生素药物的浓度的线性范围和检出限(3σ)分别是：372 nm处,氟罗沙星0.03～3.69 gg·mL-1,6.0 ng·mL-1；382nm处,加替沙星0.03～5.26μg·mL-1,7.5 ng·mL-1；380 nm处,培氟沙星0.033～3.31μg·mL-1,26.4 ng·mL-1。方法简单、快速、灵敏,并有良好的选择性,用于片剂、尿液和人血清中氟喹诺酮类抗生素药物的测定,结果满意。
Resonance Rayleigh Scattering (RRS) is a new analytical technique developed in 1990s. For its excellence of high sensitivity (detection limit of ng·mL-1 level), cheap instrument, simple operation and rapid analysis speed, the method has attracted to much attention and interest. At present, as the aggregation of dye chromophore on biological macromolecule leading to enhancement of RRS intensity, the method is mainly used to study biological macromolecules such as nucleic acids, proteins and heparin ect. Furthemrore, when two ions of different charges form ion association complexes by electrostatic force, hydrophobic force or charge transferring effect, RRS can be enhanced and the spectra charaeteristic can be changed. This method has been widely applied to the detemrination of trace metal ions, nonmetal ions, ogranic compounds, nanoparticles and pharmaceutical analysis.
The present thesis consisted of two parts:overview and research reports.
The overview involves the history, the basic principles, characteristics and the main systems of RRS, and the applications of RRS systems in pharmaceutical analysis in last five years are intensively reviewed.
The searcher part of the reports mainly involves three aspects:Terbium-fluoroquinolones-alizarin red (AR) systems; Europium--fluoroquinolones--chromeazurol S (CAS) systems; Cobalt-fluoroquinolone--congo red (CR) systems. Their absorption (A), fluorescence and RRS spectral characteristies, optimum reaction conditions, influening factors and analytical applications have been studied. Furthermore, the reaction mechanism, the reason of A or RRS enhanced, and the relationships between RRS spectra and absorption spectra and the structures of the ion-association complexes are discussed. The main research contents are as follows:
1. Study on Terbium--fluoroquinolones antibiotics--alizarin red systems by Resonance Rayleigh Scattering Spectra and their application
In the weak acid of HAc-NaAc buffer solution, fluoroquinolone antibiotics (FLQs) such as: fleroxacin (FLE), pefloxacin (PEFX), ciprofloxacin (CIP), lomefloxacin (LOM) and pazufloxacin (PAZ) can react with Tb (Ⅲ) to form chelated cations, which further bound with alizarin red (AR) to form the 2:1:1 (FLQs:Tb3+:AR) ternary ion-association complexes, resulting in the great enhancement of resonance Rayleigh scattering (RRS). The five reaction products have similar spectral characteristics and the maximum scattering wavelengths are located near 373 nm respectively. The linear ranges and the detection limits (3σ) of the FLQs are 0.04～3.69μg·mL-1, 22.7 ng·mL-1 for FLE at 373 nm; 0.03～3.33μg·mL-1,13.3 ng·mL-1 for PEFX at 373 nm; 0.03～ 3.31μg·mL-1,22.6 ng·mL-1 for CIP at 373 nm; 0.004～3.52μg·mL-1,14.3 ng·mL-1 for LOM at 370 nm; 0.16～3.18μg·mL-1,28.6ng·mL-1 for PAZ at 373 nm, respectively. The method, which is simple, rapid and selective, has been successfully applied to the determination of FLQs in tablet, injection, urine and human serum with satisfactory results. The mechanism of reaction has also been investeinged.
2. Study on Europium--fluoroquinolones--chromeazurol S systems by Resonance Rayleigh Scattering Spectrum and their Application
In the weak acid of HAc-NaAc buffer solution, fleroxacin (FLE), lomefloxacin (LOM) and ciprofloxacin (CIP) can react with Eu (Ⅲ) to form chelated cations, which further bound with chromeazurol S (CAS) by virtue of electrostatic attraction and hydrophobic force to form ternary ion-association complexes, resulting in the great enhancement of resonance Rayleigh scattering (RRS). A new method for the determination of FLQs based on RRS has been developed. The FLQs reaction products have similar spectral characteristics and the maximum scattering wavelengths are located at 361 nm and 560 nm respectively. The linear ranges and the detection limits (3a) of the FLQs are 0.037-3.69μg·mL-1,55.8 ng·mL-1 for FLE; 0.035～3.52μg·mL-1,14.5 ng·mL-1 for LOM; 0.033～3.31μg·mL-1,12.4 ng·mL-1 for CIP at 361 nm, respectively. The method, which is simple, rapid and selective, has been successfully applied to the determination of FLQs in tablet, injection, urine and human serum with satisfactory results.
3. Study on Cobalt--fluoroquinolone antibiotics--congo red systems by Resonance Rayleigh Scattering Spectrum and their Application
In the weak acid of Britton-Robinson buffer solution,fleroxacin (FLE), gatifloxacin (GTFX), and pefloxacin (PEFX) can react with Co(Ⅱ)to form a chelated cation, but its resonance Rayleigh scattering(RRS) was very weak. Significant enhancement of RRS intensity and new RRS spectrum was observed when the chelated cation further reacts with congo red to form an ion association complexe showing two scattering peaks near 372 nm and 560 nm respectively. The linear ranges and the detection limits (3σ) of the FLQs are 0.03～3.69μg·mL-1,6.0 ng·mL-1 for FLE at 372 nm; 0.03～5.26μg·mL-1,7.5 ng·mL-1 for GTFX at 382 nm; 0.033～3.31μg·mL-1,26.4 ng·mL-1 for PEFX at 380 nm, respectively. The proposed method has been applied to the determination of fluoroquinolone antibiotics in tablet, human urine and plasma with satisfactory results.
引文
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