Method for measuring content of metronidazole by using asymmetric porphyrin as fluorescent probe

Abstract

The invention belongs to the field of drug analysis and research, and particularly relates to a method for researching the interaction between cinnamyl amino porphyrin and metronidazole by adopting a micelle sensitization fluorescence quenching analysis method, and by taking the fluorescence quenching effect of metronidazole on a porphyrin compound which is a fluorescent probe molecule as well as the ratio of the reduction degree of fluorescence intensity to the concentration of the substance as an example, the detection of the interaction between cinnamyl amino porphyrin and metronidazole is realized. A novel fluorescence analysis method which is high in sensitivity, good in selectivity and simple and convenient to operate and can be used for measuring the content of a daily drug metronidazole is established; the novel fluorescence analysis method for measuring the content of metronidazole is not reported in literatures; the structure of the cinnamyl amino porphyrin (I) is shown in the specification.

Description

Technical Field

[0001] This invention relates to a novel fluorescence analysis method for studying the interaction between cinnamylaminoporphyrin and metronidazole using micellar-sensitized fluorescence quenching analysis. It utilizes the fluorescence quenching effect of metronidazole on the porphyrin compound, a fluorescent probe molecule, with the decrease in fluorescence intensity proportional to the concentration of the substance. This method establishes a highly sensitive, selective, and easy-to-operate fluorescence analysis method suitable for the determination of metronidazole content in the commonly used drug. Background Technology

[0002] Metronidazole has been used clinically for decades, primarily for treating trichomoniasis and amoebae, and in recent years has been widely used to treat anaerobic bacterial infections. Currently, methods for determining metronidazole content include ultraviolet spectrophotometry, multivariate correction-absorbance spectrophotometry, high-performance liquid chromatography, conversion curve spectrophotometry, dual-wavelength method, coefficient ratio method, novel solutions to simultaneous equations, and non-aqueous titration. Some of these methods are time-consuming, labor-intensive, and cumbersome, while others have high measurement requirements. Therefore, there is a need to explore a new method for determining metronidazole content that is highly sensitive, easy to operate, and highly selective.

[0003] Literature studies have shown that with the deepening research on the photophysical properties of porphyrins, porphyrins have been widely used in the field of drug analysis. The Rayleigh scattering spectroscopy of tetra(benzo-15-crown-5)porphyrin can be used to determine the content of L-glutamine. Tetra(4-aminophenyl)porphyrin was synthesized using the design principle of fluorescent molecular probes, and its fluorescence properties in water were studied. It was also applied to the identification of cobalt and nickel ions in water.

[0004] Therefore, in order to further study the photophysical properties of porphyrins and their applications in drug analysis, a micellar-sensitized fluorescence quenching assay was used to investigate the interaction between cinnamylaminoporphyrin and metronidazole. By utilizing the fluorescence quenching effect of metronidazole on porphyrin compounds as fluorescent probe molecules, the degree of fluorescence intensity decrease is proportional to the concentration of the substance. A new fluorescence analysis method with high sensitivity, good selectivity, and simple operation was established for the determination of metronidazole content in routine drugs.

[0005] There is a current need in this field to establish a fluorescence analysis method for determining metronidazole content that is highly sensitive, easy to operate, and highly selective. Summary of the Invention

[0006] The purpose of this invention is to provide a new fluorescence analysis method for studying the interaction between cinnamylaminoporphyrin and metronidazole using micellar-sensitized fluorescence quenching analysis. This method utilizes the fluorescence quenching effect of metronidazole on porphyrin compounds, a fluorescent probe molecule, with the degree of fluorescence intensity decrease being proportional to the concentration of the substance. The method is highly sensitive, selective, and easy to operate, and can be used for the determination of metronidazole content in routine pharmaceuticals. The method described has not been reported in the literature.

[0007] The structure of the cinnamylaminoporphyrin (I) is as follows:

[0008]

[0009] Wherein, R represents a hydrogen atom or a halogen atom or a hydroxyl group or an amino group or a cyano group or a carboxyl group or selected from one or more of the following: alkyl group (C1-C15), substituted alkyl group (C1-C15), alkoxy group (C1-C15), substituted alkoxy group (C1-C15), alkenyl group (C1-C15), alkynyl group (C6-C15), and heterocyclic group (C1-C15).

[0010] This invention provides a novel fluorescence analysis method for studying the interaction between cinnamylaminoporphyrin and metronidazole using micellar-sensitized fluorescence quenching analysis. Utilizing the fluorescence quenching effect of metronidazole on the porphyrin compound (a fluorescent probe molecule), the decrease in fluorescence intensity is proportional to the concentration of the substance. This method establishes a highly sensitive, selective, and simple fluorescence analysis method applicable to the determination of metronidazole content in routine pharmaceutical products. The method includes the following steps:

[0011] a) Selection of spectral measurement conditions

[0012] A solution of cinnamylaminoporphyrin of a certain concentration was prepared, and UV-Vis spectra were scanned to preliminarily determine the excitation wavelength range. The fluorescence intensity was measured at different excitation wavelengths, and the excitation and emission slit widths were adjusted to make the fluorescence intensity (F value) as close to 1000 as possible. The position of the peak was observed to determine the excitation wavelength range for fluorescence scanning.

[0013] b) Selection of Microemulsions

[0014] The main role of surfactants in microemulsions is to reduce interfacial tension and form adsorption films, thereby promoting microemulsion formation. Microemulsions composed primarily of cationic surfactants are used in studies of the interaction between porphyrins and metronidazole.

[0015] The cationic surfactant used is a quaternary ammonium salt, preferably hexadecyltrimethylammonium bromide (CTAB);

[0016] c) Selection of buffer system

[0017] A buffer solution system was used as the medium for the interaction between porphyrin and metronidazole. The buffer systems used included PBS buffer solution, Britton-Robinson buffer solution, acetate-sodium acetate buffer solution, KH2PO4-Na2B4O7 buffer solution, with Britton-Robinson buffer solution being preferred.

[0018] d) Order of component addition

[0019] The order in which the components are added can cause a large variation in ΔF, which has a significant impact on the experimental results. The added components include: porphyrin, metronidazole, microemulsion, and buffer. The preferred order of addition is: porphyrin + metronidazole + microemulsion + BR buffer.

[0020] e) Interference experiments with coexisting substances

[0021] This study investigated the influence of commonly used excipients in metronidazole tablets on measurement error. The concentrations of these excipients in the system were 40, 500, 500, and 500 times the concentration of metronidazole, respectively. However, the largest measurement error was only 3.1%, and the relative errors were all less than 5%. This demonstrates that the presence of these excipients does not affect the determination of metronidazole content.

[0022] The excipients used include common excipients: starch, dextrin, gum arabic, and glucose;

[0023] f) Determination of the standard curve

[0024] Under optimal experimental conditions, the fluorescence intensity of the interaction between metronidazole solution of different concentrations and cinnamylaminoporphyrin was measured, and a standard curve of the interaction between metronidazole solution and cinnamylaminoporphyrin was established.

[0025] Optimal assay conditions: 0.3 mL of 1×10 -5 A solution of cinnamylaminoporphyrin (mol / L), a series of different volumes of 10 μg / mL metronidazole solution, 0.5 mL of CTAB microemulsion, and 1.5 mL of pH 4.10 BR buffer solution were diluted with water and shaken well. The solution was allowed to stand at room temperature for 15 min, and the fluorescence intensity was measured under optimal conditions.

[0026] g) Determination of detection limit

[0027] According to the formula: LOD = 3σ / K, where σ is the standard deviation of more than 10 measurements in the blank experiment, and K is the slope of the standard curve. The fluorescence intensity of the system was measured under optimal experimental conditions.

[0028] Optimal determination conditions: Add the following substances in sequence to a 10 mL volumetric flask: 0.3 mL of 1×10⁻⁶ -5 A solution of cinnamylaminoporphyrin (mol / L), 0.5 mL of CTAB microemulsion, and 1.5 mL of BR buffer solution at pH 4.10 were diluted with water, shaken well, and allowed to stand at room temperature for 15 min. The reaction was repeated 11 times under the same conditions, and the fluorescence intensity was measured.

[0029] This invention provides a novel fluorescence analysis method for studying the interaction between cinnamylaminoporphyrin and metronidazole using micellar-sensitized fluorescence quenching analysis. By utilizing the fluorescence quenching effect of metronidazole on porphyrin compounds, a fluorescent probe molecule, a new fluorescence analysis method with high sensitivity, good selectivity, and simple operation can be used for the determination of metronidazole content in routine drugs. Detailed Implementation

[0030] To further understand the present invention, preferred embodiments are described below with reference to examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and not for limiting the scope of the claims. The specific structure of the cinnamylaminoporphyrin in the examples is as follows:

[0031]

[0032] Example 1

[0033] Selection of Spectral Measurement Conditions

[0034] Prepare 2.8×10 -6 A mol / L cinnamylaminoporphyrin solution was used to perform UV-vis spectral scanning to preliminarily determine the excitation wavelength range.

[0035] Add the following ingredients in sequence to a 10 mL volumetric flask: 1.0 × 10⁻⁶ -5 0.25 mL of cinnamylaminoporphyrin (mol / L), 0.5 mL of CTAB microemulsion, and 1.5 mL of BR buffer solution at pH 4.10 were diluted to the mark with water, shaken well, and allowed to stand at room temperature for 5 minutes. The fluorescence intensity was then measured at different excitation wavelengths. The excitation and emission slit widths were adjusted to keep the fluorescence intensity (F value) as close to 1000 as possible, and the position of the peak was observed to determine the wavelength range for fluorescence scanning.

[0036] The UV-Vis spectrum of cinnamylaminoporphyrin showed a maximum absorption peak at 420 nm, preliminarily determining that its maximum excitation wavelength should be around 420 nm. Experimental studies showed that the optimal excitation wavelength for the reaction system of cinnamylaminoporphyrin and metronidazole was 410 nm when measuring fluorescence intensity, with emission peaks at 648 nm and 710 nm, and excitation and emission slit widths of 5 nm and 10 nm, respectively.

[0037] Example 2 Selection of Microemulsion

[0038] Different microemulsions formed with different surfactants were used as reaction media. Experimental results showed that the fluorescence quenching was most pronounced (ΔF was the largest) when CTAB microemulsion was used as the reaction media. Therefore, CTAB microemulsion is the optimal reaction media for the interaction between porphyrin and metronidazole, and the optimal volume in a 10 mL volumetric flask is 0.5 mL.

[0039] Example 3: Order of Component Addition

[0040] All components, including porphyrin, metronidazole, microemulsion, and buffer, were added in different orders, and the degree of fluorescence quenching was measured. The study showed that the first group (added in the order of porphyrin + metronidazole + microemulsion + BR buffer) had a fluorescence quenching degree of 110.809, which is the highest value, indicating that this addition order is the most sensitive. Therefore, the preferred order of adding components is porphyrin + metronidazole + microemulsion + BR buffer.

[0041] The above description is only a partial embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention shall fall within the scope of the technical solution of the present invention.

Claims

1. A class of cinnamylaminoporphyrins for fluorescent analysis that can be used to determine metronidazole content, characterized by: It has the following general formula (I): Wherein, R represents a hydrogen atom or a halogen atom or a hydroxyl group or an amino group or a cyano group or a carboxyl group or one or more selected from C1 to C15 alkyl groups, substituted C1 to C15 alkyl groups, C1 to C15 alkoxy groups, substituted C1 to C15 alkoxy groups, C1 to C15 alkenyl groups, C1 to C15 alkynyl groups, C6 to C15 aryl groups and C1 to C15 heterocyclic groups.

2. The interaction between cinnamylaminoporphyrin (I) and metronidazole as described in claim 1 was studied using micellar-sensitized fluorescence quenching analysis. Utilizing the fluorescence quenching effect of metronidazole on the porphyrin compound, a fluorescent probe molecule, a novel fluorescence analysis method with high sensitivity, good selectivity, and simple operation was established for the determination of metronidazole content in routine pharmaceutical products. Its characteristics are as follows: The steps and conditions are as follows: a) Order of component addition All components, including cinnamylaminoporphyrin (I), metronidazole, microemulsion, and buffer, were added in different orders, and the degree of fluorescence quenching was determined. The preferred order of component addition was: porphyrin + metronidazole + microemulsion + BR buffer. b) Selection of Microemulsions Different microemulsions formed by different surfactants were used as reaction media. Microemulsions composed of cationic surfactants were preferred for the study of the interaction between cinnamylaminoporphyrin (I) and metronidazole. The cationic surfactants were quaternary ammonium salts, preferably hexadecyltrimethylammonium bromide (CTAB). c) Selection of buffer system A buffer solution system is used as the medium for the interaction between the cinnamylaminoporphyrin (I) and metronidazole. The buffer system used includes PBS buffer solution, Britton-Robinson buffer solution, acetate-sodium acetate buffer solution, KH2PO4-Na2B4O7 buffer solution, preferably Britton-Robinson buffer solution. d) Interference experiment of coexisting substances This study investigated the effects of commonly used excipients in metronidazole tablets on measurement error. The concentrations of these excipients in the system were 40, 500, 500, and 500 times the concentration of metronidazole, respectively. However, the largest measurement error was only 3.1%, and the relative error values ​​were all less than 5%, which proves that the presence of these excipients does not affect the determination of metronidazole content. The excipients used include common excipients: starch, dextrin, gum arabic, and glucose; e) Determination of the standard curve Under optimal experimental conditions, the fluorescence intensity of the interaction between metronidazole solution of different concentrations and cinnamylaminoporphyrin was measured, and a standard curve of the interaction between metronidazole solution and cinnamylaminoporphyrin was established. Optimal assay conditions: 0.3 mL of 1×10 -5 A solution of cinnamylaminoporphyrin (mol / L), a series of different volumes of 10 μg / mL metronidazole solution, 0.5 mL of CTAB microemulsion, and 1.5 mL of pH 4.10 BR buffer solution were diluted with water and shaken well. The solution was allowed to stand at room temperature for 15 min, and the fluorescence intensity was measured under optimal conditions. g) Determination of detection limit According to the formula: LOD = 3σ / K, where σ is the standard deviation of more than 10 measurements in the blank experiment, and K is the slope of the standard curve. The fluorescence intensity of the system was measured under optimal experimental conditions. Optimal determination conditions: Add the following substances in sequence to a 10 mL volumetric flask: 0.3 mL of 1×10⁻⁶ -5 A solution of cinnamylaminoporphyrin (mol / L), 0.5 mL of CTAB microemulsion, and 1.5 mL of BR buffer (pH 4.10) were diluted to volume with water, shaken well, and allowed to stand at room temperature for 15 min. This process was repeated 11 times under the same conditions, and the fluorescence intensity was measured.

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