CN113588817A - Method for simultaneously determining content of atropine sulfate and EDTA-2Na in atropine eye drops - Google Patents
Method for simultaneously determining content of atropine sulfate and EDTA-2Na in atropine eye drops Download PDFInfo
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Abstract
The invention belongs to the technical field of pharmaceutical analysis, and particularly discloses a method for simultaneously determining the content of atropine sulfate and EDTA-2Na in atropine eye drops, which comprises the following steps: s1, preparing EDTA-2Na and atropine sulfate reference substance solution; s2, adding chlorinated metal salt into the atropine eye drops to obtain a sample solution; s3, respectively carrying out HPLC detection on the reference substance solution and the sample solution by adopting the following conditions: the chromatographic column is a reversed phase C18 column; the mobile phase A is acetonitrile-sodium heptanesulfonate aqueous solution, and the mobile phase B is ammonium acetate aqueous solution or tetrabutylammonium hydroxide aqueous solution; controlling the pH of the mobile phase A to be 4.0-5.0; s4, the content of atropine sulfate and EDTA-2Na in the atropine eye drops is obtained by an external standard method. The invention can avoid the mutual influence between EDTA-2Na and atropine sulfate molecules in the atropine eye drops and accurately determine the content of the two.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for simultaneously determining the content of atropine sulfate and EDTA-2Na in atropine eye drops.
Background
The low-concentration atropine sulfate eye drops are the most effective preparation for preventing and treating myopia of teenagers and children at present. In the preparation, production, storage and clinical use processes of the low-concentration atropine sulfate eye drops, the low-concentration atropine sulfate eye drops are influenced by factors such as temperature, illumination, oxygen content, solution pH, storage time, osmotic pressure, viscosity and the like, and unsaturated chemical bonds, ionic bonds, complex coordination bonds and van der Waals force of active pharmaceutical ingredient atropine sulfate, such as small molecular ester bonds, conjugated double bonds, toluene type side chain structures and the like, are easy to be unstable and are degraded or molecular structures are changed, so that the low-concentration atropine eye drops deteriorate or even lose efficacy, and corresponding substances such as alcohol, acid, ketone, ester, alkaloid and the like can be generated.
Aiming at the problems, EDTA-2Na stabilizer is added into the low-concentration atropine sulfate eye drops at present, and EDTA-2Na, also known as disodium edetate, is an important complexing agent and is widely applied as an antiseptic system by combining benzalkonium chloride. The principle of using EDTA-2Na as a stabilizer in liquid preparations such as eye drops and injection is that EDTA-2Na and metal ions form a stable water-soluble chelate, which can prevent autoxidation and is beneficial to improving the stability of the medicament in the processes of preparation, storage and clinical preparation. However, EDTA-2Na can stimulate mucous membrane, upper respiratory tract, eyes and skin, and can combine with calcium ions to form soluble complex, thus reducing blood calcium concentration to cause hypocalcemia, and the long-term use of EDTA-2Na products beyond the standard may have adverse effects on human health, therefore, the content of EDTA-2Na in the eye drops should be strictly controlled, and the accurate quantification of EDTA-2Na must be ensured.
The existing methods for determining the content of EDTA-2Na in the liquid preparation mainly comprise a titration method, a gas chromatography, a high performance liquid chromatography and a liquid chromatography-tandem mass spectrometry. The titration method has large measurement error and complicated operation, and needs a reference substance for calibration; the pretreatment of a gas chromatography sample needs to adopt organic solvent extraction or pre-column derivatization, and the treatment is complex; the liquid chromatography-tandem mass spectrometry has high determination cost, is greatly influenced by the outside and has high difficulty of a detection method; high performance liquid chromatography is relatively low in cost and simple to operate, and is the most commonly used method for detecting EDTA-2Na in a liquid preparation at present.
At present, a great number of researches report the high performance liquid chromatography detection method of EDTA-2Na in eye drops, such as: schachka et al adopts reversed-phase ion-pair high performance liquid chromatography to measure the content of disodium edetate in the suspension eye drops (Schachka, Yanglonhua. reversed-phase ion-pair high performance liquid chromatography measures the content of disodium edetate in the suspension eye drops [ J ]. Chinese medicine, 2012,21(01): 24-25); measuring the content of disodium edetate in the sodium chloride eye drops by adopting HPLC (measuring the content of disodium edetate [ J ] in the sodium chloride eye drops by adopting HPLC (food and medicine 2015,17(04): 270-) -272); patent CN111398443A discloses a method for measuring disodium edetate in tafluprost eye drops. However, the determination of the stabilizing agent EDTA-2Na in the atropine sulfate eye drops is not as easy as that of eye drops of other raw material medicines, and because the molecules of the atropine sulfate are ester-like complexes and interact with EDTA-2Na, the contents of the EDTA-2Na and the atropine sulfate cannot be accurately determined. When the HPLC method provided by the reference document is used for detection, the problems of serious tailing, flat peak or no peak of an EDTA-2Na chromatographic peak and the like can occur, and the problems of serious tailing and the like of the front edge of the atropine sulfate chromatographic peak caused by the influence of EDTA-2Na molecules in a solution can occur; in addition, in the prior art, no report is provided for a method for simultaneously detecting the content of atropine sulfate and EDTA in the low-concentration atropine eye drops.
Disclosure of Invention
In order to solve the problems, the invention provides a method for simultaneously measuring the content of atropine sulfate and EDTA-2Na in the atropine eye drops, which can avoid the mutual influence between the molecules of EDTA-2Na and atropine sulfate and accurately measure the content of the atropine sulfate and the EDTA-2 Na.
A method for simultaneously determining the content of atropine sulfate and EDTA-2Na in atropine eye drops comprises the following steps:
s1, preparing a reference solution: weighing EDTA-2Na standard substance, and dissolving with pure water to obtain EDTA-2Na reference substance solution; weighing atropine sulfate standard substance, and dissolving with pure water to obtain atropine sulfate reference substance solution;
s2, preparing a sample solution: measuring atropine eye drops, and adding metal chloride to obtain a sample solution;
s3, respectively carrying out HPLC detection on the reference substance solution and the sample solution by adopting the following conditions:
a chromatographic column: a reversed phase C18 column;
mobile phase: the mobile phase A is acetonitrile-sodium heptanesulfonate aqueous solution, and the mobile phase B is ammonium acetate aqueous solution or tetrabutylammonium hydroxide aqueous solution;
pH: controlling the initial pH of the mobile phase A to be 4.0-5.0;
s4, recording the concentrations and corresponding peak areas of the EDTA-2Na and the atropine sulfate of the reference solution and the peak areas of the EDTA-2Na and the atropine sulfate of the sample solution; and (4) obtaining the corresponding atropine sulfate and EDTA-2Na content from the peak area of the sample solution by an external standard method.
The method effectively solves the problems that the content of atropine sulfate and EDTA-2Na cannot be accurately determined or the determination result is inaccurate due to mutual influence between the atropine sulfate and EDTA-2Na molecules when the quantitative determination of the atropine sulfate eye drops is carried out in the low-concentration atropine sulfate eye drops. The method can ensure that the peaks of the EDTA-2Na and the atropine sulfate are normal, the separation degree and the peak shape are good, and the content and the quality control result of the EDTA-2Na and the atropine sulfate are accurate and reliable.
Preferably, in step S2, the chlorinated metal salt is FeCl3、CuCl2、MgCl2Or CaCl2。
Further, the metal chloride salt is added into the atropine eye drops in a solid form, and the adding amount of the metal chloride salt in the atropine eye drops is 2-20 mg/mL.
Preferably, in step S3, acetic acid, formic acid or trifluoroacetic acid is added to mobile phase a to adjust pH.
Preferably, in step S3, the volume fraction of acetonitrile in the mobile phase a is 75% to 95%, and the salt concentration (i.e., sodium heptanesulfonate concentration) of the mobile phase a is 0.01mol/L to 0.05 mol/L; the salt concentration of the mobile phase B is 0.01mol/L-0.05 mol/L.
Preferably, in step S3, the flow rate of HPLC detection is 0.3-1.0mL/min, the detection wavelength is 210-290nm, the column temperature is 25-35 ℃, and the sample injection amount is 15-25 μ L.
Preferably, in step S3, the HPLC assay employs a gradient elution process, which is shown in the following table:
preferably, in step S1, the concentration of the EDTA-2Na control solution is 0.10-1.0mg/mL, and the concentration of the atropine sulfate control solution is 0.05-0.50 mg/mL.
The invention has the following beneficial effects:
1. according to the invention, by adding a metal ion complex into a sample solution, and strictly controlling the pH value of the mobile phase A to be 4.0-5.0 on the basis of selecting an acetonitrile-sodium heptanesulfonate aqueous solution as the mobile phase A and an ammonium acetate aqueous solution or a tetrabutylammonium hydroxide aqueous solution as the mobile phase B, the problem that the content determination of EDTA-2Na and atropine sulfate is influenced by the interaction between the EDTA-2Na and atropine sulfate molecules in the solution is effectively solved, the peaks of EDTA-2Na and atropine sulfate are normal, and the separation degree and the peak shape are good.
2. The method can simultaneously detect the content of atropine sulfate and EDTA-2Na in the low-concentration atropine sulfate eye drops, and has the following advantages: the recovery rate is high, and the accuracy is good; the sensitivity is high; the reproducibility is good; the durability is good. Therefore, the method can effectively control the content of the raw material atropine sulfate and the important auxiliary material EDTA-2Na in the atropine sulfate eye drops, further effectively guide the production process and quality control of the atropine sulfate eye drops, judge the quality and use safety of the eye drops, and provide accurate, rapid and efficient data support for the research and development of the eye drops and the stability of the production process.
3. The method has the advantages of low economic cost, simplicity, high efficiency, small influence of external environmental factors on detection, realization of detection under different laboratories, different HPLC instruments, different analysis testers and analysis environmental conditions, and good applicability.
Drawings
FIG. 1: HPLC test patterns of atropine sulfate and EDTA-2Na in example 2;
FIG. 2: HPLC test patterns of atropine sulfate and EDTA-2Na in comparative example 1;
FIG. 3: HPLC test patterns of atropine sulfate and EDTA-2Na in comparative example 2;
in the figure, a is the peak of atropine sulfate, and b is the peak of EDTA-2 Na.
Detailed Description
The invention is further described below with reference to the accompanying drawings and specific embodiments.
Example 1 for methodological studies, examples 2-10 and comparative examples 1-2 were actual sample tests.
Example 1
This example is a methodological study including system applicability, linearity and range, detection and quantitation limits, repeatability, precision, recovery, durability tests.
1. Chromatographic conditions
Adopting a reverse phase C18 column, wherein the mobile phase A is acetonitrile-sodium heptanesulfonate aqueous solution (85% acetonitrile: 0.02mg/mL sodium heptanesulfonate), the pH of the mobile phase A is adjusted to 4.5 by acetic acid, and the mobile phase B is 0.02mg/mL ammonium acetate aqueous solution; the chromatographic flow rate is 1.0 mg/mL; the detection wavelength is 230nm, the column temperature is 35 ℃, the sample volume is 20 mu L, the sample solution adopts self-made atropine sulfate eye drops (containing EDTA-2 Na) and 5mg/ml ferric chloride solid is added, and gradient elution is carried out according to the following process:
2. specificity test
2.1 System applicability
And (3) respectively taking an atropine sulfate reference solution (0.1 mg/ml), an EDTA-2Na reference solution (0.2 mg/ml), a self-made atropine eye drop test sample solution and a blank reference solution (without atropine sulfate and EDTA-2 Na), and detecting according to the chromatographic conditions. In atropine sulfate reference solution, EDTA-2Na reference solution and test solution, the retention time of atropine sulfate and EDTA-2Na is 2.2min and 5.4min respectively, and the base line separation of the base line chromatographic peak of the atropine sulfate and EDTA-2Na and the base line chromatographic peak of the blank auxiliary material eye drops are completely achieved, and the separation degree is 2.61. Meanwhile, no interference peak is found at 2.2min and 5.4min in the corresponding chromatogram of the blank control solution. The results show that the atropine eye drop blank auxiliary material has no interference on the determination of atropine sulfate and EDTA-2Na under the established chromatographic condition, and the method has good specificity.
2.2 Linear Range
Precisely weighing atropine sulfate and EDTA-2Na reference substances, dissolving with pure water, and diluting by times to obtain atropine sulfate solutions (0.01-1 mg/mL) and EDTA-2Na solutions (0.02-2 m) with 8 different concentrationsg/ml), and performing linear regression by taking the peak areas A of the atropine sulfate and the EDTA-2Na as ordinate and the concentrations C of the atropine sulfate and the EDTA-2Na as abscissa respectively according to the detection of the chromatographic conditions. The results show that: the atropine sulfate concentration and the peak area are in good linear relation within the range of 0.01-1 mg/mL, and the EDTA-2Na concentration and the peak area are in good linear relation within the range of 0.02-2 mg/mL, both of which can reach R2The level is more than or equal to 99.95 percent.
2.3 detection and quantitation limits
And precisely measuring atropine sulfate and EDTA-2Na, diluting with pure water in a stepwise manner, detecting according to the chromatographic conditions, recording peak areas, and taking the corresponding amounts of the atropine sulfate and the EDTA-2Na as a detection limit and a quantification limit when the signal to noise ratio is 3: 1 and 10: 1 respectively. The detection limit of atropine sulfate is 4.8ng (S/N = 3) and the quantification limit is 15.4ng (S/N = 10); the detection limit of EDTA-2Na was 3.8ng (S/N = 3), and the quantification limit was 12.4ng (S/N = 10). It can be seen that the sensitivity of the method of the present invention is high.
2.4 repeatability test
6 parts of the same atropine sulfate (0.1 mg/mL) and EDTA-2Na (0.2 mg/mL) control solution were taken, and subjected to detection according to the above chromatographic conditions, and peak areas were recorded, so that RSD of the atropine sulfate and EDTA-2Na concentrations were 0.94% (n = 6) and 0.96% (n = 6), respectively, and the reproducibility was good.
2.5 precision test
Taking the same atropine sulfate (0.1 mg/mL) and EDTA-2Na (0.2 mg/mL) reference substance solution, detecting and recording peak areas according to the chromatographic conditions, continuously sampling for 6 times within 1 day, recording the peak areas of the atropine sulfate and EDTA-2Na, and counting and calculating to obtain the intra-day precision RSD of the concentrations of the atropine sulfate and the EDTA-2Na, which is 0.75% (n = 6) and 0.81% (n = 6). And taking the same atropine sulfate and EDTA-2Na, continuously treating for 6d by the same method every day, and parallelly and continuously injecting samples for 3 times to obtain the daily precision RSD of the concentration of the atropine sulfate and the EDTA-2Na, wherein the daily precision RSD is 1.0 percent (n = 6) and 1.0 percent (n = 6). It can be seen that the method of the present invention is highly accurate.
2.6 recovery test
Precisely weighing 3 parts of atropine sulfate and EDTA-2Na standard substances of 5.5, 5.0 and 4.5mg respectively, placing the atropine sulfate and EDTA-2Na standard substances in 50mL measuring bottles respectively, adding pure water, performing ultrasonic treatment to completely dissolve the atropine sulfate and EDTA-2Na standard substances, adding 2.5 mL of blank auxiliary material solution, quantifying to a scale by using the pure water, shaking up, and centrifuging (12000 g, 4 ℃, 10 min) to obtain test sample solutions with high, medium and low concentrations. Detecting according to the chromatographic conditions, recording peak areas, obtaining atropine sulfate and EDTA-2Na measured values according to an external standard method, comparing with theoretical values, and parallelly measuring each concentration for 3 times. Average recovery rates of atropine sulfate were determined to be 97.1% (RSD =0.90%, n = 3), 97.0% (RSD =0.10%, n = 3), and 98.7% (RSD =0.59%, n = 3); the average recovery of EDTA-2Na was 98.2% (RSD =0.91%, n = 3), 97.2% (RSD =0.11%, n = 3), and 98.9% (RSD =0.63%, n = 3). Therefore, the method has high recovery rate of atropine sulfate and EDTA-2Na and good quantitative detection accuracy of the atropine sulfate and the EDTA-2 Na.
2.7 durability test
As shown in the following tables A-H, factors such as solution pH, detection wavelength, flow rate, added metal ions and the like of a mobile phase are changed, HPLC detection is respectively carried out on a sample solution of the home-made atropine sulfate eye drops (containing EDTA-2 Na), the durability and result reliability of a research method are examined by examining the recovery rate of the atropine sulfate and the recovery rate of the EDTA-2Na, and the specific results are shown in the following tables:
as can be seen from the above table, changing these factors has little effect on the recovery of atropine sulfate and EDTA-2Na, and the method of the present invention has good durability and reliability on the results of the test.
Example 2
A method for simultaneously determining the content of atropine sulfate and EDTA-2Na in atropine eye drops comprises the following steps:
s1, preparing a reference solution: weighing EDTA-2Na standard substance, dissolving with pure water to obtain EDTA-2Na control solution with concentration of 0.2 mg/mL; weighing atropine sulfate standard substance, dissolving with pure water to obtain atropine sulfate control solution with concentration of 0.1mg/mL (the standard substance of EDTA-2Na and the atropine sulfate are solid standard substances sold in Zhongzhou hospital);
s2, preparing a sample solution: measuring low-concentration atropine eye drops, adding FeCl according to the concentration of 10mg/mL3Solid to obtain a sample solution;
s3, carrying out HPLC detection on the reference substance solution and the sample solution by adopting the following conditions:
a chromatographic column: a reversed phase C18 column;
mobile phase: mobile phase a was acetonitrile-sodium heptanesulfonate aqueous solution (85% acetonitrile: 0.02mg/mL sodium heptanesulfonate), mobile phase B was 0.02mg/mL ammonium acetate aqueous solution;
pH: adjusting the pH of the mobile phase A to 4.5 by using acetic acid;
flow rate: 1.0 ml/min;
detection wavelength: 270 nm;
column temperature: 30 ℃;
sample introduction amount: 20 mu L of the solution;
gradient elution, elution procedure is shown in the following table:
s4, recording the concentrations and corresponding peak areas of the EDTA-2Na and the atropine sulfate of the reference solution and the peak areas of the EDTA-2Na and the atropine sulfate of the sample solution; and (4) obtaining the corresponding atropine sulfate and EDTA-2Na content from the peak area of the sample solution by an external standard method.
Example 3
A method for simultaneously measuring the content of atropine sulfate and the content of EDTA-2Na in atropine eye drops is basically the same as that in example 2, and only the difference is that: in step S3, the pH of mobile phase a was adjusted to 4.0 with acetic acid.
Example 4
A method for simultaneously measuring the content of atropine sulfate and the content of EDTA-2Na in atropine eye drops is basically the same as that in example 2, and only the difference is that: in step S3, the pH of mobile phase a was adjusted to 5.0 with acetic acid.
Example 5
A method for simultaneously measuring the content of atropine sulfate and the content of EDTA-2Na in atropine eye drops is basically the same as that in example 2, and only the difference is that: in step S3, the pH of mobile phase a was adjusted to 5.0 with formic acid.
Example 6
A method for simultaneously measuring the content of atropine sulfate and the content of EDTA-2Na in atropine eye drops is basically the same as that in example 2, and only the difference is that: in this example, step S3, mobile phase A was adjusted to pH 4.5 with trifluoroacetic acid and the flow rate by HPLC was 0.5 ml/min.
Example 7
A method for simultaneously measuring the content of atropine sulfate and the content of EDTA-2Na in atropine eye drops is basically the same as that in example 2, and only the difference is that: in step S3 of this example, the flow rate was 0.8ml/min by HPLC.
Example 8
A method for simultaneously measuring the content of atropine sulfate and the content of EDTA-2Na in atropine eye drops is basically the same as that in example 2, and only the difference is that: in step S2 of this example, FeCl was added at a concentration of 20mg/mL3And (3) a solid.
Example 9
A method for simultaneously measuring the content of atropine sulfate and the content of EDTA-2Na in atropine eye drops is basically the same as that in example 2, and only the difference is that: in step S2 of this example, CuCl was added at a concentration of 2mg/mL2And (3) a solid.
Example 10
A method for simultaneously measuring the content of atropine sulfate and the content of EDTA-2Na in atropine eye drops is basically the same as that in example 2, and only the difference is that: in step S2 of this example, MgCl was added at a concentration of 5mg/mL2And (3) a solid.
Comparative example 1
A method for simultaneously measuring the content of atropine sulfate and the content of EDTA-2Na in atropine eye drops is basically the same as that in example 2, and only the difference is that: in step S3, the pH of mobile phase a was adjusted to 3.0 with acetic acid.
Comparative example 2
A method for simultaneously measuring the content of atropine sulfate and the content of EDTA-2Na in atropine eye drops is basically the same as that in example 2, and only the difference is that: in step S3, the pH of mobile phase a was adjusted to 6.0 with acetic acid.
The test results of examples 2 to 10 and comparative examples 1 to 2 were as follows:
first, peak appearance
In examples 2-10, EDTA-2Na and atropine sulfate showed normal peaks, and the separation degree and peak shape of the peaks of EDTA-2Na and atropine sulfate were good as shown by the example 2 test pattern shown in FIG. 1.
The test pattern of the comparative example 1 is shown in fig. 2, and it can be seen that when the initial pH of the mobile phase a is less than 4, atropine sulfate or EDTA-2Na can cause the atropine sulfate drug substance to often fail to peak due to the related influence between the molecules of the atropine sulfate or EDTA-2Na during detection, so that the content of EDTA-2Na in the atropine eye drops can not be normally tested, and the normal development and production quality control process is greatly influenced; and even though the EDTA-2Na and the atropine sulfate can generate peaks occasionally, the damage of the flowing under low pH value to the chromatographic packing stationary phase is very large, the chromatographic column can be damaged greatly after long-term use, and the service life of the chromatographic column is shortened rapidly, so that the pH value of the mobile phase A is controlled to be 4.0-5.0.
The test pattern of the comparative example 2 is shown in fig. 3, when the initial pH of the mobile phase a is greater than 6, the peak of the EDTA-2Na has a certain degree of front edge or tail due to the related influence between the molecules of the atropine sulfate or the EDTA-2Na during the detection, and according to the integration principle of the "half-peak height" method of liquid chromatography, the tail or front edge of the chromatographic peak is seriously unable to accurately perform quantitative detection of the chromatographic peak shape, so that accurate quantification and quality control of the atropine sulfate and the EDTA-2Na in the atropine sulfate eye drops cannot be simultaneously performed.
Secondly, the specific test data are shown in the following table:
as can be seen from the table above, the atropine sulfate eye drops detected by the method have good stability of the content of atropine sulfate and EDTA-2Na, and can accurately control the quality of the atropine sulfate eye drops containing EDTA-2 Na.
This detailed description is to be construed as illustrative only and is not to be taken as limiting the invention, as any changes that may be made by a person skilled in the art after reading the present specification will be protected by the patent laws within the scope of the appended claims.
Claims (8)
1. A method for simultaneously measuring the content of atropine sulfate and EDTA-2Na in atropine eye drops is characterized in that: the method comprises the following steps:
s1, preparing a reference solution: weighing EDTA-2Na standard substance, and dissolving with pure water to obtain EDTA-2Na reference substance solution; weighing atropine sulfate standard substance, and dissolving with pure water to obtain atropine sulfate reference substance solution;
s2, preparing a sample solution: measuring atropine eye drops, and adding metal chloride to obtain a sample solution;
s3, respectively carrying out HPLC detection on the reference substance solution and the sample solution by adopting the following conditions:
a chromatographic column: a reversed phase C18 column;
mobile phase: the mobile phase A is acetonitrile-sodium heptanesulfonate aqueous solution, and the mobile phase B is ammonium acetate aqueous solution or tetrabutylammonium hydroxide aqueous solution;
pH: controlling the initial pH of the mobile phase A to be 4.0-5.0;
s4, recording the concentrations and corresponding peak areas of the EDTA-2Na and the atropine sulfate of the reference solution and the peak areas of the EDTA-2Na and the atropine sulfate of the sample solution; and (4) obtaining the corresponding atropine sulfate and EDTA-2Na content from the peak area of the sample solution by an external standard method.
2. The method for simultaneously determining the content of atropine sulfate and the content of EDTA-2Na in atropine eye drops according to claim 1, wherein the method comprises the following steps: in step S2, the chlorinated metal salt is FeCl3、CuCl2、MgCl2Or CaCl2。
3. The method for simultaneously determining the content of atropine sulfate and EDTA-2Na in atropine eye drops according to claim 2, wherein the method comprises the following steps: the metal chloride salt is added into the atropine eye drops in a solid form, and the addition amount is 2-20 mg/mL.
4. The method for simultaneously determining the content of atropine sulfate and the content of EDTA-2Na in atropine eye drops according to claim 1, wherein the method comprises the following steps: in step S3, acetic acid, formic acid, or trifluoroacetic acid is added to mobile phase a to adjust pH.
5. The method for simultaneously determining the content of atropine sulfate and the content of EDTA-2Na in atropine eye drops according to claim 1, wherein the method comprises the following steps: in the step S3, the volume fraction of acetonitrile in the mobile phase A is 75-95%, and the salt concentration of the mobile phase A is 0.01-0.05 mol/L; the salt concentration of the mobile phase B is 0.01mol/L-0.05 mol/L.
6. The method for simultaneously determining the content of atropine sulfate and the content of EDTA-2Na in atropine eye drops according to claim 1, wherein the method comprises the following steps: in step S3, the flow rate of HPLC detection is 0.3-1.0mL/min, the detection wavelength is 210-290nm, the column temperature is 25-35 ℃, and the sample injection amount is 15-25 μ L.
7. The method for simultaneously determining the content of atropine sulfate and the content of EDTA-2Na in atropine eye drops according to claim 1, wherein the method comprises the following steps: in step S3, the HPLC detection employs gradient elution, and the gradient elution process is as follows:
reducing the volume fraction of the mobile phase A from 95% to 70% and increasing the volume fraction of the mobile phase B from 5% to 30% in 0-5 min;
5-15 min: the volume fraction of the mobile phase A is kept at 70 percent, and the volume fraction of the mobile phase B is kept at 30 percent;
15-20 min: the volume fraction of the mobile phase A is reduced from 70% to 50%, and the volume fraction of the mobile phase B is increased from 30% to 50%;
20-30 min: the volume fraction of the mobile phase A is kept at 50%, and the volume fraction of the mobile phase B is kept at 50%;
30-31 min: the volume fraction of the mobile phase A is increased from 50% to 95%, and the volume fraction of the mobile phase B is decreased from 50% to 5%;
31-40 min: the volume fraction of mobile phase a remained 95%, and the volume fraction of mobile phase B remained 5%.
8. The method for simultaneously determining the content of atropine sulfate and the content of EDTA-2Na in atropine eye drops according to claim 1, wherein the method comprises the following steps: in step S1, the concentration of the EDTA-2Na control solution is 0.10-1.0mg/mL, and the concentration of the atropine sulfate control solution is 0.05-0.50 mg/mL.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114129511A (en) * | 2021-12-07 | 2022-03-04 | 河南润弘制药股份有限公司 | Atropine sulfate injection with improved stability |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110917133A (en) * | 2019-12-23 | 2020-03-27 | 河北科技大学 | Eye drops for treating myopia and preparation method thereof |
| CN111398443A (en) * | 2020-03-09 | 2020-07-10 | 南京华盖制药有限公司 | Method for determining disodium ethylene diamine tetraacetate in tafluprost eye drops |
-
2021
- 2021-07-20 CN CN202110820534.9A patent/CN113588817B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110917133A (en) * | 2019-12-23 | 2020-03-27 | 河北科技大学 | Eye drops for treating myopia and preparation method thereof |
| CN111398443A (en) * | 2020-03-09 | 2020-07-10 | 南京华盖制药有限公司 | Method for determining disodium ethylene diamine tetraacetate in tafluprost eye drops |
Non-Patent Citations (6)
| Title |
|---|
| NAROLA B ET AL.: "A validated reverse phase HPLC method for the determination of disodium EDTA in meropenem drug substance with UV-detection using precolumn derivatization technique", 《 ANALYTICAL CHEMISTRY INSIGHTS》 * |
| 屈蓉等: "反相离子对高效液相色谱法测定盐酸林可霉素注射液中乙二胺四乙酸二钠的含量", 《药学与临床研究》 * |
| 李增礼等: "高效液相色谱法测定重组人干扰素α-2b注射液中乙二胺四乙酸二钠含量", 《生物技术通讯》 * |
| 李志梅等: "离子对色谱法测定硫酸阿托品滴眼液的含量", 《药学实践杂志》 * |
| 林观样等: "高效液相色谱法测定复方阿托品滴鼻剂中硫酸阿托品的含量", 《温州医学院学报》 * |
| 聂磊等: "高效液相色谱法测定化妆品中EDTA的含量", 《香料香精化妆品》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114129511A (en) * | 2021-12-07 | 2022-03-04 | 河南润弘制药股份有限公司 | Atropine sulfate injection with improved stability |
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