CN110794045A - Method for measuring content of edetate disodium in acetylcysteine liquid preparation - Google Patents
Method for measuring content of edetate disodium in acetylcysteine liquid preparation Download PDFInfo
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Abstract
The invention belongs to the field of medicine detection, and discloses a method for measuring the content of edetate disodium in an acetylcysteine liquid preparation. The method adopts HPLC method, and adopts acetylcysteine solution preparation, cyclodextrin and/or cyclodextrin derivative, and Fe3+And preparing a sample solution by using a mobile phase, using an aminosilane bonded silica gel chromatographic column as a filler, using a mixed solvent of an ion pair buffer solution and an organic phase as the mobile phase, and measuring the content of the edetate disodium in the sample solution. The method is simple to operate and good in repeatability, and can accurately measure the content of edetate disodium in the acetylcysteine liquid preparation, so that the product quality of the acetylcysteine liquid preparation can be better controlled.
Description
Technical Field
The invention belongs to the technical field of medicine detection, relates to a method for detecting a metal chelating agent in a medicine, and particularly relates to a method for measuring the content of edetate disodium in an acetylcysteine liquid preparation.
Background
At present, disodium edetate is taken as a common pharmaceutical adjuvant, is collected in Chinese pharmacopoeia, British pharmacopoeia, European pharmacopoeia and the like, and has a structure shown in the following structural formula (I):
the molecular formula is as follows: cl0H14N2Na2O8·2H2O；
Molecular weight: 372.23.
the conventional determination method of the content of the edetate disodium is a complexometric titration method, wherein the specific determination method of Chinese pharmacopoeia comprises the following steps: taking about 0.4g of the product, accurately weighing, adding 40mL of water for dissolving, adding 10mL of ammonia-ammonium chloride buffer solution (pH10.0), titrating with zinc titration solution (0.05mol/L), adding a small amount of chrome black T indicator near the end point, and continuously titrating until the solution turns from blue to purple red. Each 1mL of zinc titrant (0.05mol/L) corresponded to 18.61mg of Cl0H14N2Na2O8·2H2And O. Because the concentration of edetate disodium in the pharmaceutical preparation is low, the complexometric titration method cannot meet the requirement of content determination.
Acetylcysteine solution for inhalation, which is one of acetylcysteine solution preparations, was developed by zapa, italy and marketed in 1965 in italy, with a specification of 3 mL: 0.3g, and is mainly used for treating respiratory diseases with excessive thick mucus secretion, such as: acute bronchitis, chronic bronchitis and patients with exacerbation of the bronchitis, emphysema, mucoviscidosis and bronchiectasis, the medicament has the action mechanism that molecules contain sulfydryl, and disulfide bonds (-S-S-) of mucoprotein peptide bonds can be broken, so that the mucoprotein chains are changed into small molecular peptide chains, the viscosity of mucoprotein is reduced, and the medicament is a dissolving medicament for mucophlegm, purulent phlegm and respiratory mucus. Acetylcysteine is represented by the following structural formula (II):
because the acetylcysteine contains free sulfydryl on the structural formula, the acetylcysteine is particularly sensitive to metal ions and is easy to degrade under the condition of the existence of the metal ions, and the edetate disodium serving as a pharmaceutic adjuvant has wide coordination performance and can form stable chelate with almost all the metal ions. Therefore, disodium edetate is added into the original acetylcysteine solution for inhalation as a metal ion chelating agent for improving the stability of the product, but the disodium edetate has a certain stimulation effect on mucous membranes and upper respiratory tracts in the prescription, and the content of the disodium edetate needs to be strictly controlled to ensure the safety and effectiveness of the product, and the dosage range is generally 0.005-0.05 percent in terms of mass-to-volume ratio (g/mL).
CN201510638593 discloses a method for detecting edetate disodium in clevidipine butyrate injection emulsion, which adopts high performance chromatography to measure the content of edetate disodium, wherein the preferable chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent, the detection wavelength is 254nm, the column temperature is 35 ℃, mobile phases are respectively an A phase and a B phase which are composed of an organic phase and a water phase, and gradient elution is adopted. When the method is used for measuring the content of edetate disodium in the acetylcysteine solution for inhalation, the active ingredient acetylcysteine in the preparation can cause serious interference on the measurement of edetate disodium, and accurate quantification can not be carried out. In addition, the patent application document also discloses a preparation method of a reference substance solution, but all other components except the edetate disodium in the preparation process of the reference substance solution need to be added, and great inconvenience is brought to daily inspection.
CN201510305734 discloses a quantitative detection method of edetate disodium in the ginseng and astragalus strengthening injection, which is ion chromatography, and the preferred chromatographic conditions are as follows: the chromatographic column was AS11-HC (4X 250mm), the eluent was 11.5mmol/L potassium hydroxide solution, and the detector was an electrical conductivity detector. When the method is used for measuring the content of edetate disodium in the acetylcysteine solution for inhalation, the phenomenon that the effective component acetylcysteine seriously interferes the measurement of edetate disodium is also found, and the related requirements of content measurement cannot be met.
Therefore, the prior art does not satisfy the requirement of accurately quantifying edetate disodium in acetylcysteine solution for inhalation.
Disclosure of Invention
Based on the defects of the prior art, the invention provides the detection method which has strong specificity, good reproducibility and simple operation and is used for measuring the content of the metal ion chelating agent edetate disodium in the acetylcysteine liquid preparation.
The invention provides a method for measuring the content of edetate disodium in an acetylcysteine liquid preparation, which comprises the following steps:
(1) preparing a test solution, wherein the test solution is a preparation comprising the following raw materials: acetylcysteine liquid preparation, cyclodextrin and/or cyclodextrin derivative, and Fe3+A solution and a mobile phase;
(2) and (3) determination: and (3) measuring the content of the edetate disodium in the sample solution by adopting a High Performance Liquid Chromatography (HPLC) method, using a chromatographic column with aminosilane bonded silica gel as a filler, and using a mixed solution of an ion pair buffer solution and an organic phase as a mobile phase.
According to the invention, said edetate disodium is edetate disodium or its hydrate, such as dihydrate, of formula Cl0H14N2Na2O8·2H2O。
According to the present invention, the acetylcysteine solution preparation may be selected from acetylcysteine injection, acetylcysteine oral liquid, acetylcysteine solution for inhalation; illustratively, the acetylcysteine solution formulation is an acetylcysteine solution for inhalation.
Preferably, the inhalation acetylcysteine solution includes acetylcysteine, edetate disodium, and sodium hydroxide. Further, the acetylcysteine solution for inhalation comprises, in mass volume percentage (g/mL): 10-20% of acetylcysteine, 0.005-0.05% of edetate disodium and 2.5-5% of sodium hydroxide. Illustratively, the inhalation acetylcysteine solution is available from American Regent, Inc.
According to the invention, the concentration of the edetate disodium in the test solution is 0.005-0.05 mg/mL; for example, the concentration is 0.01-0.03 mg/mL; illustratively, the concentration is 0.015mg/mL, 0.025mg/mL, 0.04mg/mL, 0.05 mg/mL.
According to the invention, the cyclodextrin comprises α -, β -or gamma-cyclodextrin, the cyclodextrin derivative comprises but is not limited to one, two or more of α -, β -, gamma-cyclodextrin ether derivative, ester derivative, polymer and the like, for example, the ether derivative is selected from one, two or more of glucose derivative, hydroxypropyl derivative, methyl derivative and the like, and exemplarily, the derivative is selected from one, two or more of hydroxyethyl- β -cyclodextrin, hydroxypropyl- β -cyclodextrin, sulfobutyl- β -cyclodextrin and the like.
Preferably, the concentration of the cyclodextrin or the cyclodextrin derivative in the test solution is 2-20 mg/mL; for example, the concentration is 5-15 mg/mL; illustratively, the concentration is 7mg/mL, 10mg/mL, 18 mg/mL.
According to the present invention, the Fe is contained in the test solution3+The mass concentration of the active carbon is 0.02-0.2%; for example, 0.05 to 0.15%; illustratively, the concentration is 0.1%, 0.12%, 0.18%.
According to the invention, the Fe3+The solution can be ferric trichloride solution, ferric sulfate solution, ferric nitrate solution, ferric phosphate solution, etc. Preferably ferric chloride solution; for example, adding a ferric trichloride solution with the mass concentration of 0.1-1% to prepare a test solution; illustratively, a 0.5% by mass solution of ferric chloride is added.
Preferably, the Fe3+The solution is an acidic solution, preferably having a pH of 4 or less, more preferably 1.2 to 2.0, prepared by adding an acid, preferably with Fe3+Those acids having the same salt-forming ion in the solution, such as ferric chloride solution, ferric sulfate solution, ferric nitrate solution, and ferric phosphate solution, are added with hydrochloric acid, sulfuric acid, nitric acid, and phosphoric acid, respectively, preferably ferric chloride hydrochloric acid solution.
Preferably, the ion pair buffer is a buffer comprising tetradecylammonium bromide and tetraheptylammonium bromide. Preferably, the mass concentration of the tetradecyl ammonium bromide in the buffer solution is 0.05-2%; illustratively, the concentration is 0.1%, 0.2%, 1%, 2%. Preferably, the mass concentration of the tetraheptyl ammonium bromide in the buffer solution is 0.025-1%; illustratively, the concentration is 0.05, 0.1%, 0.5%, 1%.
Preferably, the organic phase is methanol or acetonitrile.
Preferably, the volume ratio of the ion pair buffer solution to the organic phase is (70-95): 5-30); for example, the volume ratio is (75-90): (10-25), (80-90): 10-20); illustratively, the volume ratio is 90:10, 85:15, 80:20, 78:22, 87: 13.
Preferably, the pH value of the mobile phase is between 1.0 and 5.0; for example, between 1.0 and 3.0; illustratively, the pH is 1.5, 2.0, 2.5. According to the invention, the pH value of the mobile phase is adjusted by using acid; the acid can be at least one of hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, formic acid, etc.; phosphoric acid is preferred.
According to the invention, the chromatographic conditions of the high performance liquid chromatography are as follows:
column temperature: 20 to 40 ℃, such as 25 to 35 ℃, illustratively 30 ℃;
detection wavelength: 240 to 280nm, such as 250 to 270nm, illustratively 257 nm;
flow rate of mobile phase: 0.5 to 2mL/min, such as 0.8 to 1.6mL/min, illustratively at a flow rate of 0.5mL/min, 1.0mL/min, 2.0 mL/min;
sample introduction amount: 5 to 50. mu.L, for example 10 to 30. mu.L, illustratively 5. mu.L, 20. mu.L, 50. mu.L.
According to the invention, the method also comprises the preparation of a control solution, which is a formulation comprising the following raw materials: edetate disodium control substance, mobile phase, cyclodextrin and/or cyclodextrin derivative and Fe3+And (3) solution.
Preferably, the edetate disodium, the mobile phase, the cyclodextrin and/or cyclodextrin derivative and Fe3+The solution has the meaning as described above.
Preferably, the edetate disodium, the cyclodextrin and/or the cyclodextrin derivative and the Fe in the control solution3+The concentration of (A) is kept consistent with the concentration of each substance in the sample solution.
According to the technical scheme of the invention, the method for measuring the content of edetate disodium in the acetylcysteine liquid preparation specifically comprises the following steps:
(1) preparation of test solution:
preparing a test solution: weighing acetylcysteine liquid preparation, mixing with cyclodextrin and/or cyclodextrin derivative, and Fe3+Uniformly mixing the solution and the mobile phase to obtain a test sample solution;
preparation of a reference solution: mixing edetate disodium water solution with cyclodextrin and/or cyclodextrin derivative, and Fe3+Mixing with mobile phase to obtain reference solution;
(2) and (3) determination: and (3) measuring the content of the edetate disodium in the sample solution by adopting a High Performance Liquid Chromatography (HPLC) method, using a chromatographic column with aminosilane bonded silica gel as a filler, and using a mixed solution of an ion pair buffer solution and an organic phase as a mobile phase.
The invention also provides a test solution used in the determination method, wherein the test solution is a preparation comprising the following raw materials: acetylcysteine liquid preparation, cyclodextrin and/or cyclodextrin derivative, and Fe3+Solution and mobile phase.
Preferably, the acetylcysteine liquid preparation, cyclodextrin and/or cyclodextrin derivative, Fe3+Both the solution and the mobile phase have the meaning as described above.
Further, the present invention also provides a method for preparing the above test solution, comprising the steps of: mixing acetylcysteine solution preparation with cyclodextrin and/or cyclodextrin derivative, and Fe3+Mixing the solution and the mobile phase to obtain a test solution.
Preferably, the acetylcysteine liquid preparation, cyclodextrin and/or cyclodextrin derivative, Fe3+Both the solution and the mobile phase have the meaning as described above.
Preferably, the volume-mass ratio of the acetylcysteine liquid preparation to the cyclodextrin and/or the cyclodextrin derivative is 0.001-0.1 mL/mg; for example, 0.01 to 0.05 mL/mg; illustratively, the volume to mass ratio is 0.01, 0.02, 0.03, 0.04 mL/mg.
Preferably, the Fe3+Can be prepared by adding ferric salt solution, wherein the volume ratio of the medicine solution to the ferric salt solution is 0.05 to5; for example, 0.5 to 3, 1.0 to 2.5; illustratively, the volume ratio is 0.5, 1.5, 2.0.
According to the technical scheme of the invention, the preparation method of the test solution specifically comprises the following steps: precisely measuring 0.2-2 mL of acetylcysteine liquid preparation, mixing with 20-200 mg of cyclodextrin and/or cyclodextrin derivative and 0.1-1% of iron salt solution, finally adding a mobile phase to a constant volume of 10mL, and filtering to obtain the acetylcysteine liquid preparation.
According to an exemplary technical scheme of the invention, the preparation method of the test solution comprises the specific steps of precisely measuring 0.2-2 mL of acetylcysteine solution for inhalation, mixing with 20-200 mg of hydroxypropyl- β -cyclodextrin and 0.1-1% of ferric trichloride solution by mass concentration, finally adding a mobile phase to a constant volume of 10mL, and filtering to obtain the test solution.
The invention also provides a reference substance solution used in the determination method, wherein the reference substance solution is a preparation comprising the following raw materials: edetate disodium, mobile phase, cyclodextrin and/or cyclodextrin derivative and Fe3+And (3) solution.
Preferably, the edetate disodium, the mobile phase, the cyclodextrin and/or cyclodextrin derivative and Fe3+The solution has the meaning as described above.
Further, the present invention also provides a method for preparing the above-mentioned reference solution, which comprises: mixing edetate disodium water solution with cyclodextrin and/or cyclodextrin derivative, and Fe3+The solution and the mobile phase are mixed to obtain a reference solution.
Preferably, the mass concentration of the edetate disodium water solution is 0.1-0.5 mL/mg; for example, 0.15-0.4 mL/mg, 0.2-0.3 mL/mg; illustratively, the concentration is 0.25 mL/mg.
Preferably, the edetate disodium, cyclodextrin and/or cyclodextrin derivative, Fe3+Both the solution and the mobile phase have the meaning as described above.
Preferably, the volume-mass ratio of the edetate disodium aqueous solution to the cyclodextrin and/or the cyclodextrin derivative is 0.001-0.1 mL/mg; for example, 0.01 to 0.05 mL/mg; illustratively, the volume to mass ratio is 0.01, 0.02, 0.03, 0.04 mL/mg.
Preferably, the Fe3+The method can be implemented by adding an iron salt solution, wherein the volume ratio of the edetate disodium water solution to the iron salt solution is 0.05-5; for example, 0.5 to 3, 0.5 to 2.5; illustratively, the volume ratio is 0.5, 1.5, 2.0.
According to the technical scheme of the invention, the preparation method of the reference substance solution specifically comprises the following steps: precisely measuring 0.2-2 mL of edetate disodium aqueous solution, mixing with 20-200 mg of cyclodextrin and/or cyclodextrin derivative and 0.1-1% of iron salt solution, adding a mobile phase to a constant volume of 10mL, and filtering to obtain the edetate disodium salt.
According to an exemplary technical scheme of the invention, the preparation method of the reference solution specifically comprises the steps of precisely measuring 0.2-2 mL of edetate disodium aqueous solution, mixing with 20-200 mg of hydroxypropyl- β -cyclodextrin and 0.1-1% of ferric trichloride solution, adding a mobile phase to a constant volume of 10mL, and filtering to obtain the reference solution.
The invention has the beneficial effects that:
when the content of the edetate disodium in the pharmaceutical composition is determined, the content of the edetate disodium cannot be accurately measured due to interference of the diversity of the pharmaceutical components on the detection of the edetate disodium. For example, in acetylcysteine solution preparations, acetylcysteine interferes with the determination of disodium edetate in the preparation, and particularly, when the concentration of acetylcysteine in the acetylcysteine solution preparation for inhalation is high (about 100mg/mL), the determination of disodium edetate is severely interfered. The HPLC chromatographic detection method can effectively eliminate the interference of acetylcysteine on the determination of edetate disodium.
Furthermore, the method provided by the invention is used for determining the content of edetate disodium in the acetylcysteine liquid preparation, especially the acetylcysteine solution for inhalation, and has the advantages of easy realization of chromatographic conditions, high analysis sensitivity, high stability of a test sample and a reference sample, good reproducibility of the method, accurate and reliable detection results, and capability of better controlling the product quality of the acetylcysteine solution for inhalation.
Drawings
FIG. 1 is an HPLC chromatogram of a sample solution for measuring the content of edetate disodium in acetylcysteine solution for inhalation in example 3.
FIG. 2 is an HPLC chromatogram of a control solution for determining the content of edetate disodium in acetylcysteine solution for inhalation in example 3.
The abscissa in fig. 1 and 2 represents: time (minutes).
Detailed Description
The measurement method of the present invention will be described in further detail with reference to specific examples. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.
Apparatus and materials used in the examples:
liquid chromatograph: waters high performance liquid chromatograph model 2695;
inhalation acetylcysteine solution: available from American Regent, Inc.
Disodium edetate as a control: an EP standard;
disodium edetate is chromatographically pure, water is ultrapure water, and other reagents are analytically pure.
Example 1
1. Chromatographic conditions are as follows:
a chromatographic column: using amino silane bonded silica gel as a filler, wherein the size of the Agilent is 4.6 multiplied by 250mm, and the diameter of filler particles is 5 mu m;
column temperature: 25 ℃;
mobile phase: using a solution containing 0.1% of tetradecyl ammonium bromide and 0.05% of tetraheptyl ammonium bromide (pH is adjusted to 2.0 by phosphoric acid) -methanol (volume ratio is 95:5) as a mobile phase;
flow rate: 2 mL/min;
sample introduction volume: 5 mu L of the solution;
detection wavelength: 240 nm.
2. Preparation of test solution:
(1) the sample solution is prepared by precisely measuring 0.2mL of acetylcysteine solution for inhalation, placing into a 10mL measuring flask, adding 20mg of hydroxypropyl- β -cyclodextrin, adding 0.4mL of 0.5% ferric trichloride solution, adding mobile phase for dilution to scale, shaking, and filtering to obtain the sample solution.
(2) The preparation method of the control solution comprises precisely weighing 25mg disodium edetate, placing in a 100mL measuring flask, adding water to dissolve and dilute to scale, precisely weighing 0.2mL, placing in a 10mL measuring flask, adding 20mg hydroxypropyl- β -cyclodextrin, adding 0.5% ferric trichloride solution 0.4mL, adding mobile phase to dilute to scale, shaking, and filtering to obtain the control solution.
3. And (3) determination:
precisely measuring 5 μ L of each of the test solution and the reference solution, respectively injecting into a liquid chromatograph, recording chromatogram, and calculating edetate disodium content by peak area according to external standard method.
Example 2
1. Chromatographic conditions are as follows:
a chromatographic column: amino alkyl silane bonded silica gel is used as a filling agent, Agilent is 4.6 multiplied by 250mm, and the diameter of filling particles is 5 mu m; column temperature: 35 ℃;
mobile phase: using a solution containing 2% of tetradecyl ammonium bromide and 1% of tetraheptyl ammonium bromide (the pH is adjusted to 3.0 by phosphoric acid) -methanol (the volume ratio is 70:30) as a mobile phase;
flow rate: 0.5 mL/min;
sample introduction volume: 50 mu L of the solution;
detection wavelength: 280 nm.
2. Preparation of test solution:
(1) the sample solution is prepared by precisely measuring 2mL of acetylcysteine solution for inhalation, placing into a 10mL measuring flask, adding 200mg of hydroxypropyl- β -cyclodextrin, adding 4mL of 0.5% ferric trichloride solution, adding mobile phase for dilution to scale, shaking, and filtering to obtain sample solution.
(2) The preparation method of the control solution comprises precisely weighing 25mg of edetate disodium, placing in a 100mL measuring flask, adding water to dissolve and dilute to scale, precisely weighing 2mL, placing in a 10mL measuring flask, adding 200mg of hydroxypropyl- β -cyclodextrin, adding 4mL of 0.5% ferric trichloride solution, adding mobile phase to dilute to scale, shaking, and filtering to obtain the control solution.
3. And (3) determination:
precisely measuring the sample solution and the reference solution by 50 μ L, respectively injecting into a liquid chromatograph, recording chromatogram, and calculating edetate disodium content by peak area according to external standard method.
Example 3
1. Chromatographic conditions are as follows:
a chromatographic column: amino alkyl silane bonded silica gel is used as a filling agent, Agilent is 4.6 multiplied by 250mm, and the diameter of filling particles is 5 mu m;
column temperature: 30 ℃;
mobile phase: using a solution containing 0.2% of tetradecyl ammonium bromide and 0.1% of tetraheptyl ammonium bromide (pH is adjusted to 1.5 by phosphoric acid) -methanol (volume ratio is 90:10) as a mobile phase;
flow rate: 1 mL/min;
sample introduction volume: 20 mu L of the solution;
detection wavelength: 257 nm.
2. Test solution preparation:
(1) the sample solution is prepared by precisely measuring 1mL of acetylcysteine solution for inhalation, placing into a 10mL measuring flask, adding 100mg of hydroxypropyl- β -cyclodextrin, adding 2mL of 0.5% ferric trichloride solution, adding mobile phase for dilution to scale, shaking, and filtering to obtain sample solution.
(2) The preparation method of the control solution comprises precisely weighing 25mg of edetate disodium, placing in a 100mL measuring flask, adding water to dissolve and dilute to scale, precisely weighing 1mL, placing in a 10mL measuring flask, adding 100mg of hydroxypropyl- β -cyclodextrin, adding 2mL of 0.5% ferric trichloride solution, adding mobile phase to dilute to scale, shaking, and filtering to obtain the control solution.
3. And (3) determination:
precisely measuring 20 μ L of each of the test solution and the reference solution, respectively injecting into a liquid chromatograph, recording chromatogram, and calculating edetate disodium content by peak area according to external standard method.
Example 4 methodology examination
4.1, linear relationship
4.1.1 test design
Precisely weighing 25mg of edetate disodium reference substance, placing in a 50mL measuring flask, adding water, shaking to dissolve, adding water to dilute to scale, shaking uniformly, precisely weighing 1mL, 2mL, 3mL, 5mL and 10mL, placing in a 100mL measuring flask, adding water to dilute to scale, shaking uniformly, precisely weighing 1mL of the prepared solution respectively, placing in different 10mL measuring flasks, adding 100mg of hydroxypropyl- β -cyclodextrin and 2mL of 0.5% ferric trichloride solution respectively, adding mobile phase to dilute to scale, and shaking uniformly to obtain the reference substance solution.
Precisely measuring 20 μ L of each reference solution, injecting into a liquid chromatograph, recording chromatogram, and drawing a standard curve with peak area as ordinate and concentration as abscissa.
4.1.2 test results
The test results are shown in table 1.
Table 1.
4.1.3 conclusion of the test
Within the concentration range of 5.024 mu g/mL-50.24 mu g/mL, the peak area of edetate disodium and the concentration have good linear relation.
4.2 accuracy
4.2.1 test design
A control solution was prepared with reference to example 3.
And precisely weighing 20mg, 25mg and 30mg of edetate disodium reference substances, precisely weighing, respectively putting 3 parts of edetate disodium reference substances into 100mL measuring bottles, shaking to dissolve blank auxiliary material solutions (the solution components are acetylcysteine and sodium hydroxide, and the dosage of the acetylcysteine and the sodium hydroxide is the same as the content of the acetylcysteine solution for inhalation), diluting to a scale, shaking uniformly, precisely weighing 1mL of the solution components, putting into 10mL measuring bottles, respectively, adding 100mg of hydroxypropyl- β -cyclodextrin and 2mL of 0.5% ferric trichloride solution, diluting to the scale by adding a mobile phase, and shaking uniformly to obtain a sample solution.
Precisely measuring 20 μ L of each of the reference solution and the sample solution, respectively injecting into a liquid chromatograph, recording chromatogram, and calculating according to external standard method by peak area.
4.2.2 test results
The test results are shown in table 2.
Table 2.
4.2.3 conclusion of the test
The method for measuring the content of the disodium edetate has the average recovery rate of 99.19 percent and the RSD value of 1.33 percent, and meets the relevant requirements.
4.3 solution stability
4.3.1 test design
Reference example 3 a control solution and a test solution were prepared. Precisely measuring the reference solution and the sample solution for 0, 2, 4, 6, 8, 12 and 24h respectively, injecting into a liquid chromatograph, recording chromatogram, and observing the stability of the reference solution and the sample solution according to the peak area change condition.
4.3.2 test results
The test results are shown in table 3.
Table 3.
| Time (h) | 0 | 2 | 4 | 8 | 12 | 24 | RSD |
| Peak area of control | 758824 | 755048 | 753985 | 760037 | 756443 | 762212 | 0.42％ |
| Peak area of test sample | 756445 | 753561 | 751035 | 760085 | 752339 | 759772 | 0.51％ |
4.3.3 conclusion of the test
The reference solution and the test solution have good stability within 24 hours.
4.4 repeatability
In order to further verify the repeatability of the method, the content of edetate disodium in the self-made sample is measured by adopting the chromatographic conditions and the solution preparation method in the example 3, the operation is performed for 6 times in parallel, the test result shows that the RSD of the 6 times of test result is 0.53%, the RSD meets the relevant requirements, and the specific result is detailed in a table 4.
TABLE 4 determination of the content of disodium edetate repeatability test (unit:%)
| Numbering | 1 | 2 | 3 | 4 | 5 | 6 | Mean value | RSD |
| Content (wt.) | 99.55 | 100.3 | 98.9 | 99.2 | 99.6 | 100.1 | 99.61 | 0.53 |
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for measuring the content of edetate disodium in an acetylcysteine liquid preparation comprises the following steps:
(1) preparing a test solution, wherein the test solution is a preparation comprising the following raw materials: acetylcysteine liquid preparation, cyclodextrin and/or cyclodextrin derivative, and Fe3+A solution and a mobile phase;
(2) and (3) determination: and (3) measuring the content of the edetate disodium in the sample solution by adopting a High Performance Liquid Chromatography (HPLC) method, using a chromatographic column with aminosilane bonded silica gel as a filler, and using a mixed solution of an ion pair buffer solution and an organic phase as a mobile phase.
2. The assay method of claim 1, wherein the acetylcysteine solution formulation is selected from the group consisting of acetylcysteine injection, acetylcysteine oral liquid, acetylcysteine solution for inhalation; illustratively, the acetylcysteine solution formulation is an acetylcysteine solution for inhalation.
Preferably, the cyclodextrin comprises α -, β -, or γ -cyclodextrin;
preferably, the cyclodextrin derivatives include, but are not limited to, one, two or more of α -, β -, gamma-cyclodextrin ether derivatives, ester derivatives, polymers, and the like, for example, the ether derivatives are selected from one, two or more of glucose derivatives, hydroxypropyl derivatives, methyl derivatives, and the like, and illustratively, the derivatives are selected from one, two or more of hydroxyethyl- β -cyclodextrin, hydroxypropyl- β -cyclodextrin, sulfobutyl- β -cyclodextrin, and the like.
Preferably, the Fe3+The solution can be ferric trichloride solution, ferric sulfate solution, ferric nitrate solution and ferric phosphate solution.
Preferably, the ion pair buffer is a buffer comprising tetradecylammonium bromide and tetraheptylammonium bromide.
Preferably, the organic phase is methanol or acetonitrile.
3. An assay according to claim 1 or 2, wherein the concentration of edetate disodium in the sample solution is 0.005-0.05 mg/mL; for example, the concentration is 0.01-0.03 mg/mL; illustratively, the concentration is 0.015mg/mL, 0.025mg/mL, 0.04mg/mL, 0.05 mg/mL.
Preferably, the concentration of the cyclodextrin or the cyclodextrin derivative is 2-20 mg/mL; for example, the concentration is 5-15 mg/mL; illustratively, the concentration is 7mg/mL, 10mg/mL, 18 mg/mL.
Preferably, the Fe is present in the test solution3+The mass concentration of the active carbon is 0.02-0.2%; for example, 0.05 to 0.15%; illustratively, the concentration is 0.1%, 0.12%, 0.18%.
Preferably, the mass concentration of the tetradecyl ammonium bromide in the ion pair buffer solution is 0.05-2%; illustratively, the concentration is 0.1%, 0.2%, 1%, 2%. Preferably, the mass concentration of the tetraheptyl ammonium bromide in the buffer solution is 0.025-1%; illustratively, the concentration is 0.05, 0.1%, 0.5%, 1%.
4. The assay method according to any one of claims 1 to 3, wherein,
the volume ratio of the ion pair buffer solution to the organic phase is (70-95): 5-30); for example, the volume ratio is (75-90): (10-25), (80-90): 10-20); illustratively, the volume ratio is 90:10, 85:15, 80:20, 78:22, 87: 13.
Preferably, the pH value of the mobile phase is between 1.0 and 5.0; for example, between 1.0 and 3.0; illustratively, the pH is 1.5, 2.0, 2.5.
Preferably, the pH of the mobile phase is adjusted with an acid; the acid can be at least one of hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, formic acid, etc.; phosphoric acid is preferred.
5. The assay method of claim 4, further comprising preparing a control solution, the control solution being a formulation comprising: edetate disodium control substance, mobile phase, cyclodextrin and/or cyclodextrin derivative and Fe3+And (3) solution.
Preferably, the edetate disodium, the cyclodextrin and/or the cyclodextrin derivative and the Fe in the control solution3+Concentration of (2)The concentration of the test solution is consistent with that of each substance in the test solution.
Preferably, the chromatographic conditions of the high performance liquid chromatography are:
column temperature: 20-40 ℃;
detection wavelength: 240-280 nm;
flow rate of mobile phase: 0.5-2 mL/min;
sample introduction amount: 5-50 μ L.
6. A test solution for use in the assay method of any one of claims 1 to 5, said test solution being a formulation comprising: acetylcysteine liquid preparation, cyclodextrin and/or cyclodextrin derivative, and Fe3+Solution and mobile phase.
Preferably, the acetylcysteine liquid preparation, cyclodextrin and/or cyclodextrin derivative, Fe3+Both the solution and the mobile phase have the meaning as stated in any of claims 1 to 5.
7. A method of preparing a test solution according to claim 6, the method comprising the steps of: mixing acetylcysteine solution preparation with cyclodextrin and/or cyclodextrin derivative, and Fe3+Mixing the solution and the mobile phase to obtain a test solution.
Preferably, the acetylcysteine liquid preparation, cyclodextrin and/or cyclodextrin derivative, Fe3+Both the solution and the mobile phase have the meaning as stated in any of claims 1 to 5.
8. The preparation method according to claim 7, wherein the volume-to-mass ratio of the acetylcysteine liquid preparation to the cyclodextrin and/or the cyclodextrin derivative is 0.001-0.1 mL/mg; for example, 0.01 to 0.05 mL/mg; preferably, the volume-to-mass ratio is 0.01, 0.02, 0.03, 0.04 mL/mg.
Preferably, the Fe3+The method can be realized by adding an iron salt solution, wherein the volume ratio of the medicine solution to the iron salt solution is 0.05-5; for example, 0.5 to 3, 1.0 to 2.5; preferably, the volume ratio is 0.5, 1.5, 2.0.
9. A control solution for use in the assay method of claim 5, said control solution comprising: edetate disodium, mobile phase, cyclodextrin and/or cyclodextrin derivative and Fe3+And (3) solution.
Preferably, the edetate disodium, the mobile phase, the cyclodextrin and/or cyclodextrin derivative and Fe3+The solution has the meaning as claimed in any of claims 1 to 5.
10. A method of preparing a control solution according to claim 9, the method comprising: mixing edetate disodium water solution with cyclodextrin and/or cyclodextrin derivative, and Fe3+The solution and the mobile phase are mixed to obtain a reference solution.
Preferably, the mass concentration of the edetate disodium water solution is 0.1-0.5 mL/mg; for example, 0.15-0.4 mL/mg, 0.2-0.3 mL/mg; illustratively, the concentration is 0.25 mL/mg.
Preferably, the edetate disodium, cyclodextrin and/or cyclodextrin derivative, Fe3+Both the solution and the mobile phase have the meaning as stated in any of claims 1 to 5.
Preferably, the volume-mass ratio of the edetate disodium aqueous solution to the cyclodextrin and/or the cyclodextrin derivative is 0.001-0.1 mL/mg; for example, 0.01 to 0.05 mL/mg; illustratively, the volume to mass ratio is 0.01, 0.02, 0.03, 0.04 mL/mg.
Preferably, the Fe3+The solution can be prepared by adding an iron salt solution, wherein the volume ratio of the edetate disodium water solution to the iron salt solution is 0.05-5; for example, 0.5 to 3, 0.5 to 2.5; illustratively, the volume ratio is 0.5, 1.5, 2.0.
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