CN112945878A - Method for measuring dopamine by indirect photometry - Google Patents

Method for measuring dopamine by indirect photometry Download PDF

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CN112945878A
CN112945878A CN202110150585.5A CN202110150585A CN112945878A CN 112945878 A CN112945878 A CN 112945878A CN 202110150585 A CN202110150585 A CN 202110150585A CN 112945878 A CN112945878 A CN 112945878A
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dopamine
solution
absorbance
dopamine hydrochloride
concentration
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刘威
安慧萍
穆艳超
王宝凤
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Anyang Maternal And Child Health Hospital Anyang Children's Hospital
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    • G—PHYSICS
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    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light

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Abstract

The invention discloses a method for measuring dopamine by an indirect photometry method, which reduces Fe (III) into Fe (II) by using dopamine hydrochloride, and reduces the generated Fe (II) and K3[Fe(CN)6]The concentration of the dopamine hydrochloride and the absorbance of the soluble prussian blue generated by the reaction form a linear relation, and the maximum absorption wavelength of the soluble prussian blue is 735nm according to the absorbance by indirectly measuring the content of the dopamine hydrochloride. The method has the advantages of simplicity, rapidness, wide linear range and high sensitivity, the concentration of the dopamine hydrochloride is in a good linear relation with the absorbance within the range of 0.32-8.96 mu g/mL, and the detection limit is 0.025 mu g/mL.

Description

Method for measuring dopamine by indirect photometry
Technical Field
The invention relates to a method for measuring dopamine, in particular to a method for measuring dopamine by an indirect photometry.
Background
Dopamine (DA) is one of the most important catecholamine neurotransmitters in the central nervous system of mammals, and plays a very important role in the functional regulation of the central nervous system, cardiovascular system, endocrine system and renal system. The abnormal DA content in the biological fluid is closely related to some nervous system diseases, such as Parkinson's disease, schizophrenia, autism, Huntington's chorea and Alzheimer's disease. Therefore, accurate detection of DA is crucial for neurophysiology, pathology, and clinical disease diagnosis.
At present, the main methods for detecting the content of dopamine comprise: electrochemical method, high performance liquid chromatography, fluorescence spectroscopy, capillary electrophoresis, etc. Although these methods have high sensitivity and strong specificity, they have the disadvantages of complexity, long time consumption, expensive instruments and the like.
Potassium ferricyanide has been used as a chemiluminescent reagent and is widely used in chemiluminescent analysis. The research shows that: under certain experimental conditions, hydroxyl contained in dopamine has certain reducibility, Fe (III) can be reduced into Fe (II), and Fe (II) and K generated by reduction3[Fe(CN)6]Reacting to obtain soluble Prussian blue KFe III [ Fe II (CN)6]Therefore, the inventors have conducted intensive studies on the determination of dopamine content using potassium ferricyanide as a chemiluminescent reagent.
Disclosure of Invention
In view of the above, the present invention provides a method for spectrophotometrically detecting dopamine by using potassium ferricyanide as a probe reagent, aiming at the defects of the prior art. The method has the characteristics of low detection limit, large linear range, simple operation, low cost and the like, and has a satisfactory result when being used for measuring the content of the dopamine in the medicine.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for measuring dopamine by indirect photometry comprises reducing Fe (III) to Fe (II) with dopamine hydrochloride, and reducing the generated Fe (II) and K3[Fe(CN)6]The concentration of dopamine hydrochloride and the absorbance of the soluble prussian blue generated by the reaction are in a linear relation, and the maximum absorption wavelength of the soluble prussian blue is 735nm according to the absorbance by indirectly measuring the content of dopamine hydrochloride; preferably, the concentration of the dopamine hydrochloride is 0.32-8.96 mu g/mL.
Preferably, the detection limit of the method for measuring dopamine by the indirect photometry on dopamine hydrochloride is 0.025 mu g/mL.
More preferably, the pH of hydrochloric acid in the dopamine hydrochloride is 3.
As a preferable embodiment of the present invention, the method for measuring dopamine by indirect photometry comprises the following steps:
1) measuring the absorbance: taking K of corresponding volume and concentration3[Fe(CN)6]Solution and FeCl3Diluting the solution and the dopamine hydrochloride solution in a colorimetric tube to a scale, shaking up, reacting, taking a reagent blank at 735nm as a reference, and measuring absorbance;
2) drawing a standard curve to obtain a linear regression equation;
3) and (3) calculating the concentration of dopamine hydrochloride: and (4) calculating the concentration of the dopamine hydrochloride according to the correlation between the concentration and the absorbance.
Preferably, the method for measuring the absorbance in the step 1) is to take K3[Fe(CN)6]And FeCl3And (3) putting the solution and 1.00mL of 80 mu g/mL dopamine hydrochloride solution in a 25-mL ground colorimetric tube, adjusting the pH value, diluting to a scale, shaking up, reacting, taking a blank reagent at 735nm as a reference, and determining the absorbance.
More preferably, K3[Fe(CN)6]The molar concentration of the solution was 1.5X 10-2mol/L and/or dosage is 0.50mL-2.00 mL; and/or FeCl3Has a molar concentration of 1.5X 10-2mol/L and/or dosage is 0.50mL-2.00 mL;
preferably, K3[Fe(CN)6]The molar concentration of the solution was 1.5X 10-2mol/L and/or the dosage is 0.80 mL; and/or FeCl3The molar concentration of the solution was 1.5X 10-2mol/L and/or the amount is 0.80 mL.
Preferably, the pH is adjusted to 4 or less.
Preferably, the temperature of the reaction is 4-50 ℃; preferably, the temperature of the reaction is 25-50 ℃; more preferably, the temperature of the reaction is 25 ℃.
Preferably, the reaction time is 5-10 min; preferably, the reaction time is 5 min.
An application of a method for measuring dopamine by an indirect photometry is used for quickly measuring the content of dopamine hydrochloride in a medicament.
Preferably, the dosage forms of the medicament comprise injections, tablets, pills, capsules and other all dosage forms which can contain dopamine hydrochloride.
The invention has the beneficial effects that:
the experiment establishes the method based on that dopamine hydrochloride can reduce Fe (III)3[Fe(CN)6]A novel method for measuring the content of dopamine hydrochloride in a medicament by a visible spectrophotometry of a-Fe (III) system. The method has the advantages of simplicity, rapidness, wide linear range and high sensitivity, the concentration of the dopamine hydrochloride is in a good linear relation with the absorbance within the range of 0.32-8.96 mu g/mL, and the detection limit is 0.025 mu g/mL. And the used reagent is simple and easy to obtain, and is suitable for rapidly determining the content of the dopamine hydrochloride in the medicaments of various formulations.
Drawings
FIG. 1: absorption spectrum chart
a: a product absorption spectrum; b: FeCl3And K3[Fe(CN)6]Mixed solution absorption spectrum; c: dopamine hydrochloride absorption spectrum; dopamine hydrochloride (80. mu.g/mL) solution: 1.00 mL; FeCl3(1.5×10-2mol/L) solution: 0.80 mL; k3[Fe(CN)6](1.5×10-2mol/L) solution: 1.00 mL;
FIG. 2: FeCl3Influence of dosage
Dopamine hydrochloride (80. mu.g/mL) solution: 1.00 mL; k3[Fe(CN)6](1.5×10-2mol/L) solution: 0.80 mL; reaction temperature: 25 ℃; reaction time: 5 min; total volume: 25 mL;
FIG. 3K3[Fe(CN)6]Influence of dosage
Dopamine hydrochloride (80. mu.g/mL) solution: 1.00 mL; fe Cl3(1.5×10-2mol/L) solution: 0.80 mL; reaction temperature: 25 ℃; reaction time: 5 min; total volume: 25 mL;
FIG. 4 Effect of reaction time
Dopamine hydrochloride (80. mu.g/mL) solution:1.00mL；Fe Cl3(1.5×10-2mol/L) solution: 0.80 mL; k3[Fe(CN)6](1.5×10-2mol/L) solution: 0.80 mL; reaction temperature: 25 ℃; total volume: 25 mL;
FIG. 5 influence of pH
Dopamine hydrochloride (80. mu.g/mL) solution: 1.00 mL; FeCl3(1.5×10-2mol/L) solution: 0.80 mL; k3[Fe(CN)6](1.5×10-2mol/L) solution: 0.80 mL; reaction temperature: 25 ℃; reaction time: 5 min; total volume: 25 mL; FIG. 6 Standard Curve
The absorbance is taken as the ordinate, and the concentration of dopamine hydrochloride is taken as the abscissa.
Detailed Description
The invention is further described below with reference to the figures and examples.
First, experimental part
1.1 Experimental instruments
Cary 60 uv-visible spectrophotometer (agilent technologies, ltd.);
FD-101S heat-collecting type constant temperature heating magnetic stirrer (Zhengzhou prefecture instruments, Inc.).
1.2 Experimental drugs and reagents
Dopamine (A.R., Shanghai Alatin Biotechnology, Inc.) standard stock solution 1.00mg/mL, stored at 4 deg.C in dark place, and diluted to 80 μ g/mL standard solution when used;
potassium ferricyanide (a.r., beijing chemical plant) standard solution:
1.5×10-2mol/L; standard solution of ferric chloride (a.r., tianjin chemical industry, three factories): 1.5X 10-2mol/L。
All other reagents used in the experiment are analytically pure, and the water used in the experiment is secondary distilled water.
1.3 Experimental methods
Pipette accurately 0.80mL of 1.5X 10-2mol/L of K3[Fe(CN)6]And 0.80mL of 1.5X 10- 2FeCl in mol/L3The solution and 1.00mL of 80. mu.g/mL dopamine hydrochloride solution are put into a 25-mL ground colorimetric tube to ensure thatDiluting with secondary distilled water to scale, shaking, reacting at 25 deg.C for 5min, and measuring absorbance with 735nm reagent blank as reference.
Second, result and discussion
2.1 reaction mechanism
Dopamine has a catechol structure, catechol has strong reducibility, ferric iron can be reduced into ferrous iron, and ferrous iron ions and potassium ferricyanide can react to generate soluble Prussian blue KFe [ Fe (CN)6]The solution has strong absorption to visible light. The stoichiometric ratio of dopamine to Fe (iii) reaction is assumed to be 1:2 from electron gain and loss, and the possible reaction mechanism is scheme 1:
Ⅰ.
Figure BDA0002932562280000031
Ⅱ.
Figure BDA0002932562280000032
2.2 absorption Spectrum
The absorption spectra of the reagent blank and dopamine hydrochloride were measured separately using water as reference, and the prussian blue reaction product was measured using the reagent blank as reference, and are shown in fig. 1. FIG. 1 shows that the maximum absorption wavelength of the reaction product is 735nm, dopamine hydrochloride is not absorbed in the range of 500-800 nm, and the reagent blank has smaller absorption near 735 nm. In order to eliminate reagent interference, the product Prussian blue KFe is measured at 735nm by taking reagent blank as referenceⅢ[FeⅡ(CN)6]Absorbance of (b).
2.3 Effect of the amount of ferric trichloride
Fixed at 1.5X 10-2mol/L K3[Fe(CN)6]The amount of (B) is 0.80mL, the amount of 80. mu.g/mL dopamine hydrochloride is 1.00mL, FeCl is examined3Influence of dosage on absorbance. The results are shown in FIG. 2 when FeCl is used3When the amount of the compound added was 0.80mL, the absorbance reached the maximum. Continued addition of FeCl3When the amount reaches 2.00mL, the absorbance is still unchanged. This indicates dopa hydrochloride in solutionThe amine has been completely oxidized by Fe (III), the amount of Fe (II) produced is maximized, and the concentration of soluble Prussian blue formed therewith is also maximized. Therefore, in the subsequent experiment, FeCl3The amount was selected to be 0.80 mL.
2.4 Effect of Potassium ferricyanide dosage
Fixed at 1.5X 10-2mol/L FeCl3The dosage is 0.80mL, the dosage of 80 mu g/mL dopamine hydrochloride is 1.00mL, other conditions are not changed, and K is considered3[Fe(CN)6]Influence of dosage on absorbance. The results are shown in FIG. 3, when K is3[Fe(CN)6]When the addition amount is 0.80mL, the absorbance reaches the maximum and does not follow K any more3[Fe(CN)6]The increase in the amount of the solution indicated that the concentration of prussian blue produced in the solution was maximized. To complete the reaction of dopamine hydrochloride with Fe (III), K is selected3[Fe(CN)6]The amount of (B) was 0.80 mL.
2.5 Effect of reaction time
Fixed at 1.5X 10-2mol/L K3[Fe(CN)6]The dosage of (B) is 0.80mL, 1.5X 10-2mol/L FeCl3The dosage is 0.80mL, the dosage of 80 mug/mL dopamine hydrochloride is 1.00mL, the reaction temperature is 25 ℃, and the influence of different reaction time on the absorbance is examined. The result is shown in FIG. 4, when the reaction time is 5min, the absorbance reaches the maximum, and can be kept unchanged for 45min, thereby meeting the experimental determination requirements. Therefore, the optimal reaction time in the subsequent experiments is selected to be 5 min.
2.6 Effect of pH
Fixed FeCl3And K3[Fe(CN)6]The dosage is 0.80mL, the dosage of 80 mug/mL dopamine hydrochloride is 1.00mL, the reaction temperature is 25 ℃, and the reaction time is 5 min. The acidity of the solution was adjusted with 1mol/L HCl and 0.5mol/L NaOH, and the effect of different pH values on the absorbance was examined, and the results are shown in FIG. 5. When the pH value is less than 4, the absorbance of the solution is stable, and when the pH value is more than 4, the absorbance of the solution is obviously reduced. This is because Fe (III) is easily hydrolyzed to Fe (OH) under higher pH conditions3Precipitation, which affects the reaction of Fe (III) with dopamine hydrochloride, reduces the absorbance of the solution. This is also in agreement with the experimental observation that pH values above 4.0 are observedThe reaction solution became cloudy. In order to complete the reaction, the pH value of the solution is controlled below 4.0 in the experiment.
3.7 Effect of coexisting Components
According to the experimental method, the fixed dopamine dosage is 3.20 mu g/mL, the relative error is controlled within 5 percent, and interference tests are carried out on common pharmaceutical excipients (starch, glucose and arginine), amino acid and mineral ions. The results show that: 320 ug/mL glucose, 160 ug/mL starch, 20 ug/mL arginine, cystine, 40 ug/mL glutamic acid, 10 ug/mL Co2+、Cd2+，0.1μg/mL Cu2+、Zn2+A large amount of K+、Na+、Ca2+、Mg2+、Ba2+、Al3+、Mn2+、NO3 －、SO4 2－、Br－And Cl－The measuring result is not influenced.
3.8 drawing of Standard Curve
Fixed FeCl3(1.5×10-2mol/L) is 0.80mL, K3[Fe(CN)6](1.5×10-2mol/L) is 0.80mL, the reaction temperature is 25 ℃, and the reaction time is 5 min. 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.80, 1.00, 1.20, 1.40, 1.60, 1.80, 2.00, 2.20, 2.40, 2.60, 2.80mL of 80. mu.g/mL dopamine hydrochloride standard solution was accurately transferred to a 25mL colorimetric tube, diluted to the scale (pH 3.0 in this case), shaken, reacted at room temperature for 5min, and the absorbance of the solution at 735nm was measured. The absorbance was plotted as the ordinate and the concentration of dopamine hydrochloride as the abscissa to draw a standard curve, and the result is shown in fig. 6. The result shows that the concentration of the dopamine hydrochloride is in a good linear relation with the absorbance within the range of 0.32-8.96 mu g/mL, the linear regression equation A is 0.0089+0.0879c (mu g/mL), the correlation coefficient R is 0.9997, and the molar absorption coefficient epsilon is 2.3 multiplied by 104L/mol. cm. The absorbance of 15 blank solutions was measured in parallel, and the detection limit was found to be 0.025. mu.g/mL by dividing 3 times the standard deviation by the slope of the linear regression equation.
3.9 sample analysis
2mL of dopamine hydrochloride injection (2 mL: 20mg, far-reaching medicine (China) Co., Ltd.) is taken out of a 250mL volumetric flask, the flask is washed 3-5 times by secondary distilled water, the volume is determined to 250mL, and the concentration of the obtained solution is 80 mug/mL. The dopamine content of the injection is measured according to the experimental method, and the result is shown in table 1.
TABLE 1 determination of samples and recovery
Figure BDA0002932562280000051
Third, conclusion
The experiment establishes the method based on that dopamine hydrochloride can reduce Fe (III)3[Fe(CN)6]A novel method for measuring the content of dopamine hydrochloride in a medicament by a visible spectrophotometry of a-Fe (III) system. The research shows that: fe (III) can be reduced to Fe (II) by dopamine hydrochloride, and Fe (II) and K generated by reduction3[Fe(CN)6]Reaction to form soluble Prussian blue KFeⅢ[FeⅡ(CN)6]And the dopamine hydrochloride content can be indirectly measured according to the absorbance. The method has the characteristics of simplicity, rapidness, wide linear range, high sensitivity and the like, and the used reagent is simple and easy to obtain, so that the method is suitable for rapidly determining the content of the dopamine hydrochloride in the medicament.
Finally, the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting, and other modifications or equivalent substitutions made by the technical solutions of the present invention by those of ordinary skill in the art should be covered within the scope of the claims of the present invention as long as they do not depart from the spirit and scope of the technical solutions of the present invention.

Claims (10)

1. The method for measuring dopamine by indirect photometry is characterized in that Fe (III) is reduced into Fe (II) by hydrochloric acid dopamine, and the reduced Fe (II) and K are generated3[Fe(CN)6]The concentration of dopamine hydrochloride and the absorbance of the soluble Prussian blue are in a linear relation, and the maximum absorption wavelength of the soluble Prussian blue is indirectly measured according to the absorbanceIs 735 nm; preferably, the concentration of the dopamine hydrochloride is 0.32-8.96 mu g/mL.
2. The method of indirect photometric dopamine determination according to claim 1 wherein the method has a detection limit for dopamine hydrochloride of 0.025 μ g/mL.
3. The method of indirect photometric determination of dopamine according to claim 1 or 2, characterized by the following steps:
1) measuring the absorbance: taking K of corresponding volume and concentration3[Fe(CN)6]Solution and FeCl3Diluting the solution and the dopamine hydrochloride solution in a colorimetric tube to a scale, shaking up, reacting, taking a reagent blank at 735nm as a reference, and measuring absorbance;
2) drawing a standard curve to obtain a linear regression equation;
3) and (3) calculating the concentration of dopamine hydrochloride: and (4) calculating the concentration of the dopamine hydrochloride according to the correlation between the concentration and the absorbance.
4. The method for indirect photometry of dopamine according to any one of claims 1 to 3, wherein the method for measuring absorbance in step 1) is to take K3[Fe(CN)6]And FeCl3And (3) putting the solution and 1.00mL of 80 mu g/mL dopamine hydrochloride solution in a 25-mL ground colorimetric tube, adjusting the pH value, diluting to a scale, shaking up, reacting, taking a blank reagent at 735nm as a reference, and determining the absorbance.
5. The method of indirect photometry of dopamine according to any one of claims 1-4, where K is3[Fe(CN)6]The molar concentration of the solution was 1.5X 10-2mol/L and/or dosage is 0.50mL-2.00 mL; and/or FeCl3Has a molar concentration of 1.5X 10-2mol/L and/or dosage is 0.50mL-2.00 mL;
preferably, K3[Fe(CN)6]The molar concentration of the solution was 1.5X 10-2mol/L and/or the dosage is 0.80 mL; and/or FeCl3The molar concentration of the solution was 1.5X 10-2mol/L and/or the amount is 0.80 mL.
6. The method of indirect photometric dopamine determination according to claim 4 wherein the pH adjustment is such that the pH is adjusted to a value below pH 4.
7. The method of indirect photometric determination of dopamine according to any one of claims 1 to 4, characterized in that the temperature of the reaction is 4-50 ℃; preferably, the reaction temperature is 25-50 ℃; more preferably, the temperature of the reaction is 25 ℃.
8. The method of indirect photometric dopamine according to any one of claims 1 to 4 wherein the reaction time is 5-10 min; preferably, the reaction time is 5 min.
9. Use of a method according to any one of claims 1 to 8 for the rapid determination of the dopamine hydrochloride content of a medicament.
10. The use according to claim 9, wherein the pharmaceutical dosage forms comprise injections, tablets, pills, capsules and all other dosage forms that may contain dopamine hydrochloride.
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