CN113447592A - Method for detecting ethylene diamine tetraacetic acid disodium in metronidazole gel - Google Patents

Method for detecting ethylene diamine tetraacetic acid disodium in metronidazole gel Download PDF

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CN113447592A
CN113447592A CN202110798124.9A CN202110798124A CN113447592A CN 113447592 A CN113447592 A CN 113447592A CN 202110798124 A CN202110798124 A CN 202110798124A CN 113447592 A CN113447592 A CN 113447592A
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ethylene diamine
solution
metronidazole gel
diamine tetraacetate
disodium ethylene
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刘永波
蔡蓓蕾
唐小晴
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Jiangsu Zhiyuan Pharmaceutical Co ltd
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Jiangsu Zhiyuan Pharmaceutical Co ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
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    • G01N2030/047—Standards external

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Abstract

The invention discloses a detection method of disodium ethylene diamine tetraacetate in metronidazole gel, which is characterized in that a high performance liquid chromatograph is adopted to detect the content of disodium ethylene diamine tetraacetate in a sample; the method comprises the following steps: (1) sample pretreatment: mixing metronidazole gel to be detected with phosphoric acid aqueous solution, filtering, and collecting filtrate as test solution; (2) and (3) elution: injecting the test solution and the reference solution into a liquid chromatograph, pumping the eluent by an ultraviolet detector, and eluting by a gradient elution program; (3) and (3) detection: and recording the chromatogram, and calculating according to the peak area by an external standard method to obtain the content of the ethylene diamine tetraacetic acid in the metronidazole gel. The detection method has the advantages of good specificity, high sensitivity, linear range and accuracy meeting the measurement requirements, and good durability.

Description

Method for detecting ethylene diamine tetraacetic acid disodium in metronidazole gel
Technical Field
The invention relates to the technical field of analysis and detection, and particularly relates to a detection method of disodium ethylene diamine tetraacetate in metronidazole gel.
Background
The metronidazole gel is a commonly used external medicine in clinic, is a light yellow transparent gel in appearance, contains effective components of metronidazole, can kill a plurality of anaerobic bacteria and mites, and is a commonly used medicine for treating skin diseases such as facial rosacea, pimple acne, pustule and the like.
MetronidazoleThe British name is Metronidazole, the chemical name is 2-methyl-5-nitroimidazole-1-ethanol, and the molecular formula is C6H9N3O3Molecular weight is 171.16, CAS number is 443-48-1, and the chemical structural formula is as follows:
Figure 442225DEST_PATH_IMAGE001
at present, 5 metronidazole gels approved in China have the specification of 0.75%. The original research is not imported to China. Meanwhile, no related substance and component inspection item exists in the Chinese pharmacopoeia, and a key ring for comprehensively controlling the quality of metronidazole gel is lacked.
Therefore, an analytical method which is simple in analytical process and can effectively control the quality of metronidazole gel is urgently needed at present.
Disclosure of Invention
The invention aims to provide a method for detecting ethylene diamine tetraacetic acid in metronidazole gel, which solves one or more of the problems in the prior art.
The invention provides a method for detecting disodium ethylene diamine tetraacetate in metronidazole gel, which is used for detecting the content of the disodium ethylene diamine tetraacetate in a sample by adopting a high performance liquid chromatograph.
In certain embodiments, a method for detecting edetate disodium in a metronidazole gel, comprising the steps of:
(1) sample pretreatment: mixing metronidazole gel to be detected with phosphoric acid aqueous solution, filtering, and collecting filtrate as test solution;
(2) and (3) elution: injecting the test solution and the reference solution into a liquid chromatograph, pumping the eluent by an ultraviolet detector, and eluting by a gradient elution program;
(3) and (3) detection: and recording the chromatogram, and calculating according to the peak area by an external standard method to obtain the content of the ethylene diamine tetraacetic acid in the metronidazole gel.
In certain embodiments, step (3) is specifically: respectively measuring standard reference substance solutions and test substance solutions with different concentrations by using a high performance liquid chromatograph, respectively recording the concentration of the standard reference substance solution, the corresponding peak area and the peak area of the test substance, and performing linear regression analysis on each mass concentration of the reference substance stock solution and the peak area of a chromatogram to obtain a regression equation and a correlation coefficient to make a standard curve;
and (4) calculating the content of the corresponding ethylene diamine tetraacetic acid in the metronidazole gel from the peak area of the test solution by using an external standard method.
In certain embodiments, a gradient elution is used as measured by hplc.
In certain embodiments, phosphate buffer is used as mobile phase a: the concentration is 0.049-0.051 mol/L, preferably 0.05mol/L, and the pH value is 1.5; the mobile phase B is acetonitrile;
wherein: the preparation steps of the phosphate buffer solution are as follows: sodium dihydrogen phosphate dihydrate 7.8g was taken, 1000mL of water was added, and the pH was adjusted to 1.5 with phosphoric acid.
In certain embodiments, the gradient elution procedure is as set forth in the following table:
Figure 703442DEST_PATH_IMAGE002
。
in certain embodiments, the control solution is prepared by:
taking 20mg of an ethylene diamine tetraacetic acid reference substance, precisely weighing, placing the reference substance in a 100ml measuring flask, adding 0.1% phosphoric acid aqueous solution to dissolve and dilute the reference substance to a constant volume, shaking up, precisely transferring 2ml of the solution, placing the solution in a 20ml measuring flask, adding 0.1% phosphoric acid aqueous solution to dilute the solution to a constant volume, shaking up, taking the solution as a reference substance solution, and preparing two parts in parallel.
In certain embodiments, the test solution is prepared by:
2g of metronidazole gel is precisely weighed, placed in a 50ml measuring flask, added with 0.1% phosphoric acid aqueous solution, ultrasonically dissolved, diluted to scale by 0.1% phosphoric acid aqueous solution, shaken up and filtered to be used as a test solution;
wherein: the concentration of the test solution is 38-40 mg/mL, preferably 40 mg/mL.
In certain embodiments, the limit of detection of impurities is that the peak area of the disodium edetate should not be greater than the major peak area of the control solution.
In certain embodiments, the detection conditions of the hplc are:
octadecylsilane chemically bonded silica is used as a filling agent, and the column temperature is set to be 40 ℃; the detection wavelength is 210 nm; the injection volume is 20 mu L; the flow rate is 0.8-1.2 mL/min, preferably 1.0 mL/min.
Has the advantages that: the invention selects a proper mobile phase system by screening chromatographic column and flow equal conditions in the analysis of the high performance liquid chromatograph. On the aspect of proportion adjustment of a mobile phase, the diluent and the auxiliary materials are ensured not to interfere the detection of the disodium ethylene diamine tetraacetate in the sample, the separation degree between chromatographic peaks of metronidazole and the disodium ethylene diamine tetraacetate is ensured to meet the requirement, and finally, a gradient elution HPLC analysis method is established, so that the detection and control of the disodium ethylene diamine tetraacetate in metronidazole gel are realized. The detection method of the invention has positive effects on strictly controlling the quality of the medicine and ensuring the safety and effectiveness of the medicine.
Drawings
FIG. 1 is a high performance liquid chromatogram for detecting the content of disodium edetate in metronidazole gel in example 1.
Detailed Description
The present invention will be described in further detail below with reference to embodiments.
Example 1 detection of disodium EDTA in Metronidazole gel
Measured according to high performance liquid chromatography (China pharmacopoeia 2020 edition general regulation 0512).
Chromatographic conditions are as follows: octadecylsilane chemically bonded silica was used as a filler (Atlantis T3, 250 × 4.6mm, 5 μm); gradient elution was performed using 0.05mol/l phosphate buffer (sodium dihydrogen phosphate dihydrate 7.8g, water 1000ml was added to dissolve, and phosphoric acid was used to adjust the pH to 1.5) as mobile phase A and acetonitrile as mobile phase B according to the following table; the column temperature was 40 ℃; the detection wavelength is 210 nm; the flow rate is 1.0 ml/min; wherein:
the gradient elution procedure is listed in the table below:
Figure 683905DEST_PATH_IMAGE003
。
the determination method comprises the following steps: precisely weighing about 20mg of disodium ethylene diamine tetraacetate, placing the disodium ethylene diamine tetraacetate into a 100ml measuring flask, adding 0.1% phosphoric acid aqueous solution to dissolve and dilute the disodium ethylene diamine tetraacetate, fixing the volume to a scale, shaking up, precisely transferring 2ml of the disodium ethylene diamine tetraacetate into a 20ml measuring flask, adding 0.1% phosphoric acid aqueous solution to dilute the disodium ethylene diamine tetraacetate to a scale, shaking up to obtain a reference solution. Two portions were prepared in parallel. Taking about 2g of metronidazole gel, precisely weighing, placing in a 50ml measuring flask, adding 0.1% phosphoric acid aqueous solution, ultrasonically dissolving, diluting with 0.1% phosphoric acid aqueous solution to scale, shaking, and filtering to obtain sample solution.
1. Detection limit and quantitative limit test of disodium ethylene diamine tetraacetate
Taking a proper amount of disodium ethylene diamine tetraacetate reference substance, respectively dissolving with 0.1% phosphoric acid aqueous solution, gradually diluting, taking a signal-to-noise ratio of 3:1 as a detection limit and taking a signal-to-noise ratio of 10:1 as a quantification limit, and obtaining the following test results.
Figure 739586DEST_PATH_IMAGE005
。
2. Linear regression of disodium ethylenediaminetetraacetate
Taking a proper amount of an ethylene diamine tetraacetic acid reference substance, dissolving and diluting the disodium ethylene diamine tetraacetate reference substance by using 0.1% phosphoric acid aqueous solution to prepare a series of solutions with gradient concentration, precisely measuring 20 mu l of the solution, injecting the solution into a liquid chromatograph, drawing a standard curve by taking a peak area A as a vertical coordinate and a corresponding concentration C as a horizontal coordinate, and calculating a linear regression equation, wherein the test result is as follows.
Figure 306965DEST_PATH_IMAGE006
。
The test result shows that the solution concentration (mu g/mL) of the disodium ethylene diamine tetraacetate in the range of 0.995 mu g/mL-39.780 mu g/mL has a good linear relation with the peak area, the linear equation is Y =2.514X-0.725, and the linear correlation coefficient R = 0.999.
3. Repeatability of
Precisely weighing about 20mg of disodium ethylene diamine tetraacetate reference substance, placing the disodium ethylene diamine tetraacetate reference substance into a 100ml measuring flask, adding 0.1% phosphoric acid aqueous solution to dissolve and dilute the disodium ethylene diamine tetraacetate reference substance to a constant volume, shaking up, precisely transferring 2ml of the disodium ethylene diamine tetraacetate reference substance into a 20ml measuring flask, adding 0.1% phosphoric acid aqueous solution to dilute the disodium ethylene diamine tetraacetate reference substance to a scale, shaking up to obtain the reference substance solution. Two portions were prepared in parallel. Taking about 2g of metronidazole gel, precisely weighing, placing in a 50ml measuring flask, adding 0.1% phosphoric acid aqueous solution, ultrasonically dissolving, diluting with 0.1% phosphoric acid aqueous solution to scale, shaking, and filtering to obtain sample solution. 6 parts are prepared in parallel. Precisely measuring 20 μ l of each of the reference sample and the sample, injecting into a liquid chromatograph, and calculating the content of disodium edetate in each sample. The test results are as follows:
Figure DEST_PATH_IMAGE007
。
the test result shows that: the repeatability test result RSD of the ethylene diamine tetraacetic acid disodium salt is 5.5 percent, which shows that the method has good repeatability.
4. Recovery rate of disodium ethylene diamine tetraacetate
And (3) adding a proper amount of ethylene diamine tetraacetic acid reference substance into the metronidazole gel blank auxiliary material according to 80-120% of the limit, and calculating the recovery rate according to the ratio of the measured amount to the added amount. The test results are as follows:
recovery test results of disodium ethylenediaminetetraacetate:
Figure DEST_PATH_IMAGE008
。
the test result shows that: the test result of the recovery rate of the disodium ethylene diamine tetraacetate accords with the pharmacopoeia regulations, and the method has better recovery rate and high accuracy.
In summary, the following steps: the detection method has the advantages of good specificity, high sensitivity, linear range and accuracy meeting the measurement requirements, and good durability.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various changes and modifications can be made without departing from the inventive concept of the present invention, and these should be considered as within the scope of the present invention.

Claims (10)

1. A detection method of disodium ethylene diamine tetraacetate in metronidazole gel is characterized in that a high performance liquid chromatograph is adopted to detect the content of disodium ethylene diamine tetraacetate in a sample.
2. The method for detecting the disodium ethylene diamine tetraacetate in the metronidazole gel as claimed in claim 1, characterized by comprising the following steps:
(1) sample pretreatment: mixing metronidazole gel to be detected with phosphoric acid aqueous solution, filtering, and collecting filtrate as test solution;
(2) and (3) elution: injecting the test solution and the reference solution into a liquid chromatograph, pumping the eluent by an ultraviolet detector, and eluting by a gradient elution program;
(3) and (3) detection: and recording the chromatogram, and calculating according to the peak area by an external standard method to obtain the content of the ethylene diamine tetraacetic acid in the metronidazole gel.
3. The method for detecting disodium ethylene diamine tetraacetate in metronidazole gel as claimed in claim 2, wherein step (3) specifically comprises: respectively measuring standard reference substance solutions and test substance solutions with different concentrations by using a high performance liquid chromatograph, respectively recording the concentration of the standard reference substance solution, the corresponding peak area and the peak area of the test substance, and performing linear regression analysis on each mass concentration of the reference substance stock solution and the peak area of a chromatogram to obtain a regression equation and a correlation coefficient to make a standard curve;
and (4) calculating the content of the corresponding ethylene diamine tetraacetic acid in the metronidazole gel from the peak area of the test solution by using an external standard method.
4. The method for detecting disodium edetate in metronidazole gel as claimed in claim 2, characterised in that gradient elution is used for the determination by high performance liquid chromatography.
5. The method for detecting disodium ethylene diamine tetraacetate in metronidazole gel according to claim 4, characterized in that phosphate buffer is used as mobile phase A: the concentration is 0.049-0.051 mol/L, preferably 0.05mol/L, and the pH value is 1.5; the mobile phase B is acetonitrile;
wherein: the preparation steps of the phosphate buffer solution are as follows: sodium dihydrogen phosphate dihydrate 7.8g was taken, 1000mL of water was added, and the pH was adjusted to 1.5 with phosphoric acid.
6. The method for detecting ethylenediaminetetraacetic acid disodium salt in metronidazole gel as claimed in claim 4, characterized in that the gradient elution procedure is as follows:
Figure 291877DEST_PATH_IMAGE001
。
7. the method for detecting disodium ethylene diamine tetraacetate in metronidazole gel as claimed in claim 2, wherein the preparation method of the reference substance solution is as follows:
taking 20mg of an ethylene diamine tetraacetic acid reference substance, precisely weighing, placing the reference substance in a 100ml measuring flask, adding 0.1% phosphoric acid aqueous solution to dissolve and dilute the reference substance to a constant volume, shaking up, precisely transferring 2ml of the solution, placing the solution in a 20ml measuring flask, adding 0.1% phosphoric acid aqueous solution to dilute the solution to a constant volume, shaking up, taking the solution as a reference substance solution, and preparing two parts in parallel.
8. The method for detecting the disodium ethylene diamine tetraacetate in the metronidazole gel as claimed in claim 2, wherein the preparation method of the test solution is as follows:
2g of metronidazole gel is precisely weighed, placed in a 50ml measuring flask, added with 0.1% phosphoric acid aqueous solution, ultrasonically dissolved, diluted to scale by 0.1% phosphoric acid aqueous solution, shaken up and filtered to be used as a test solution;
wherein: the concentration of the test solution is 38-40 mg/mL, preferably 40 mg/mL.
9. The method for detecting the disodium ethylene diamine tetraacetate in the metronidazole gel as claimed in claim 2, characterized in that the detection limit of impurities is that the peak area of the disodium ethylene diamine tetraacetate is not larger than the main peak area of the control solution.
10. The method for detecting the disodium ethylene diamine tetraacetate in the metronidazole gel as claimed in claim 2, wherein the detection conditions of the high performance liquid chromatograph are as follows:
octadecylsilane chemically bonded silica is used as a filling agent, and the column temperature is set to be 40 ℃; the detection wavelength is 210 nm; the injection volume is 20 mu L; the flow rate is 0.8-1.2 mL/min, preferably 1.0 mL/min.
CN202110798124.9A 2021-07-15 2021-07-15 Method for detecting ethylene diamine tetraacetic acid disodium in metronidazole gel Pending CN113447592A (en)

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
周会芹 等: "RP-HPLC法测定甲硝唑氯化钠注射液中乙二胺四乙酸二钠的含量", 《首都医药》 *
赵杰 等: "HPLC法测定注射用泮托拉唑钠中EDTA- 2Na的含量", 《医学信息》 *

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