A method for determining betamethasone sodium phosphate injection edetate disodium by liquid chromatography

Abstract

The invention relates to the field of pharmaceutical analysis technology, and provides a method for determining disodium edetate in betamethasone sodium phosphate injection by liquid chromatography, wherein octadecylsilane bonded silica gel is used as a filler, the detection wavelength is 254nm, the flow rate is 0.8-1.2ml / min, the column temperature is 30-50℃, and the injection volume is 5-50µl. The method for detecting the content of disodium edetate by liquid chromatography of the invention makes up for the problem of inaccurate detection in the prior art, has strong specificity, good method precision, high accuracy, and the solution is stable within 48 hours, and can effectively control the content of disodium edetate in the injection.

Description

Technical Field

[0001] The invention relates to the technical field of medical analysis, in particular to a method for determining edetate disodium in betamethasone sodium phosphate injection by liquid chromatography. Background Art

[0002] Betamethasone sodium phosphate injection is mainly used for allergic and autoimmune inflammatory diseases. It is now mostly used for active rheumatism, rheumatoid arthritis, lupus erythematosus, severe bronchial asthma, severe dermatitis, acute leukemia, etc. It is also used for the comprehensive treatment of certain infections. In the prescription of this product, disodium edetate is used as a chelating agent to form stable water-soluble complexes with alkaline earth metals and heavy metal ions. Disodium edetate (Ethylenediaminetetraacetic acid disodiumsalt), chemical name is disodium ethylenediaminetetraacetic acid, its chemical structure is:

[0003]

[0004] The general reaction formula of metal ion (M) and disodium edetate (Y) is:

[0005]

[0006] Studies have shown that a certain dose of edetate disodium, after being absorbed by the body, will have adverse effects on organs such as the liver, kidneys, and heart, and will also irritate the eye mucosa, upper respiratory tract mucosa, etc. Therefore, in order to ensure the safety of medication for patients, a detection method for the content of edetate disodium in betamethasone sodium phosphate injection should be established to effectively control its content.

[0007] So far, there are relatively few reports on the limits and detection methods of disodium edetate. Patent No. CN202311634960.9 discloses a method for analyzing the content of disodium edetate in a budesonide suspension for inhalation, the method comprising: preparing a suspension sample for adjusting the instrument parameters of a high-performance liquid chromatography instrument, constructing a mobile phase in a liquid storage tank, and calculating the peak separation of disodium edetate in the suspension sample based on the mobile phase properties of the mobile phase, the high-pressure pump properties of the high-pressure pump, and the chromatographic column properties of the chromatographic column; based on the peak separation, determining the mobile phase parameters, high-pressure pump parameters, and chromatographic column parameters of the high-performance liquid chromatography instrument from the mobile phase properties, high-pressure pump properties, and chromatographic column properties; in the chromatographic column, constructing a compound band of the compound to be analyzed separated from the budesonide suspension for inhalation; using a detector to analyze the chromatographic peak area in the compound band, and calculating the disodium edetate content of disodium edetate in the compound to be analyzed based on the chromatographic peak area.

[0008] The invention studies a new method for detecting the content of disodium edetate, which has the characteristics of strong specificity, high sensitivity and the like, is suitable for detecting the content of disodium edetate in betamethasone sodium phosphate injection, and has high accuracy. Summary of the invention

[0009] Therefore, in view of the above-mentioned defects, the present invention provides a method for determining edetate disodium in betamethasone sodium phosphate injection by liquid chromatography, which makes up for the problem of inaccurate detection in the prior art and effectively controls the content of edetate disodium in the injection.

[0010] To achieve the above object, the present invention is achieved through the following technical solutions:

[0011] A method for determining edetate disodium in betamethasone sodium phosphate injection by liquid chromatography, using octadecylsilane bonded silica gel as a filler, with a detection wavelength of 254 nm, a flow rate of 0.8-1.2 ml / min, a column temperature of 30-50° C., and an injection volume of 5-50 μl.

[0012] Further improvements are: the flow rate is 1.0 ml / min, the column temperature is 40° C., and the injection volume is 20 μl.

[0013] A further improvement is that the mobile phase is 0.3% tetrabutylammonium hydroxide solution-water-acetonitrile in a volume ratio of 18-25:45:30-37.

[0014] A further improvement is that the 0.3% tetrabutylammonium hydroxide solution is adjusted to a pH of 4.0 with phosphoric acid.

[0015] A further improvement is: a method for determining betamethasone sodium phosphate injection edetate disodium by liquid chromatography comprises the following steps:

[0016] (1) Prepare the test solution: accurately measure 1 ml of the product, add 100 μl of 0.1% copper acetate solution, and dilute with diluent to make a solution containing 0.01 mg of edetate disodium per 1 ml;

[0017] (2) Preparation of reference solution: accurately weigh 5 mg of edetate disodium reference substance into a 50 ml volumetric flask, add water to dissolve and dilute to scale; accurately weigh 1 ml into a 10 ml volumetric flask, add 100 μl of 0.1% copper acetate solution, and dilute to scale with a diluent to prepare a solution containing 0.01 mg per 1 ml;

[0018] (3) Accurately measure 20 μl of each of the above solutions and inject them into a liquid chromatograph. Record the chromatogram and calculate the content of disodium edetate based on the peak area using the external standard method.

[0019] A further improvement is that the diluent is a phosphoric acid aqueous solution with a pH of 2.5.

[0020] By adopting the above technical solution, the beneficial effects of the present invention are:

[0021] The method for detecting the content of disodium edetate by liquid chromatography has strong specificity, good method precision and high accuracy, and the solution is stable within 48 hours, and has practical significance for the quality control of betamethasone sodium phosphate injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a standard curve diagram of Example 1 of the present invention. DETAILED DESCRIPTION

[0023] The following will describe the implementation methods of the present invention in detail in conjunction with specific embodiments, so that the implementation process of how the present invention applies technical means to solve technical problems and achieve technical effects can be fully understood and implemented accordingly.

[0024] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the reagents and products used are all commercially available. The sources, trade names and components of the reagents used are indicated when they first appear.

[0025] Example 1

[0026] Octadecylsilane bonded silica gel was used as a filler; 0.3% tetrabutylammonium hydroxide solution (adjusted to pH 4.0 with phosphoric acid)-water-acetonitrile (18:45:37) was used as a mobile phase, the concentration of which was 0.3% tetrabutylammonium hydroxide solution adjusted to pH 4.0 with phosphoric acid; the flow rate was 1.0 ml / min per minute; the column temperature was 40° C.; the detection wavelength was 254 nm; and the injection volume was 20 μl.

[0027] A method for determining betamethasone sodium phosphate injection disodium edetate by liquid chromatography comprises the following steps:

[0028] (1) Prepare the test solution: accurately measure 1 ml of the product, add 100 μl of 0.1% copper acetate solution, and dilute with a pH 2.5 phosphoric acid aqueous solution to make a solution containing 0.01 mg of edetate disodium per 1 ml;

[0029] (2) Preparation of reference solution: accurately weigh 5 mg of edetate disodium reference substance into a 50 ml volumetric flask, add water to dissolve and dilute to scale; accurately weigh 1 ml into a 10 ml volumetric flask, add 100 μl of 0.1% copper acetate solution, and dilute to scale with pH 2.5 phosphoric acid aqueous solution to make a solution containing 0.01 mg per ml. Prepare two portions in parallel, respectively as reference solution 1 and reference solution 2;

[0030] (3) Blank excipient solution: Take blank excipient (not containing edetate disodium) and prepare it in the same way as the test solution dilution procedure.

[0031] (4) Accurately measure 20 μl of each of the blank solvent, blank excipient solution, test solution and reference solution (reference solution 1 is injected into 5 needles, reference solution 2 is injected into 2 needles) and inject into the liquid chromatograph, record the chromatogram, and calculate the content of disodium edetate by the peak area using the external standard method. The calculation formula is as follows:

[0032]

[0033] In the formula, A s : Average peak area of ​​edetate disodium in reference solution; A i W: peak area of ​​the component to be tested in the test solution; s : Weight of reference substance, mg; W i : Sample volume of test product, ml; V s : Dilution volume of reference substance, ml; V i : dilution volume of test solution, ml; P s : Content of reference substance, %.

[0034] Example 2

[0035] The detection method of Example 1 was validated from several aspects, including specificity, system applicability, solution stability, linearity and range, accuracy, and repeatability, as described in detail below.

[0036] (1) Exclusivity

[0037] The specificity results are shown in Table 1.

[0038] Table 1 Specificity test results

[0039] name RT / min Test results blank solution / No interference Blank excipient solution / No interference Test solution 12.817 / Reference solution 12.968 /

[0040] As can be seen from Table 1, neither the blank solution nor the blank auxiliary material solution interferes with the detection of edetate disodium, indicating that the detection method of the present invention has good specificity.

[0041] (2) System applicability

[0042] The system suitability results are shown in Table 2.

[0043] Table 2 System suitability experimental results

[0044]

[0045]

[0046] As shown in Table 2, the RSD of the peak area of ​​edetate disodium for 5 consecutive reference solution 1 is ≤2.0%, and the recovery rates of the response factors of the two reference solution are between 98.0% and 102.0%, indicating that the system has good applicability and can meet the test requirements.

[0047] (3) Solution stability

[0048] Take the reference solution and the test solution under the specificity item, place them at room temperature, and accurately measure 20μl of the reference solution at 0, 6, 12, 18, 26, 36, and 48 hours, inject them into the liquid chromatograph, and record the chromatogram; place them at room temperature, and accurately measure 20μl of the test solution at 0, 4, 8, 16, 26, 36, and 48 hours, inject them into the liquid chromatograph, and record the chromatogram. The results are shown in Table 3-4.

[0049] Table 3 Stability results of reference solution

[0050]

[0051] Table 4 Stability results of test solution

[0052]

[0053]

[0054] (4) Linearity and range

[0055] Take the reference substance of disodium edetate, dilute it into the solution of 20%~200% of the prescription concentration (0.1mg / ml), accurately measure 20μl of each of the above solutions, inject it into the liquid chromatograph, and record the chromatogram. In the concentration range of 20%~200%, with the concentration as the abscissa (X) and the peak area as the ordinate (Y), perform linear regression analysis, and the results are shown in Table 5.

[0056] Table 5 Linearity results

[0057]

[0058] The test results show that within the range of 2.046 to 20.460 μg / ml, the linear equation of concentration and peak area is y=9.9309x-0.3011, r=1.0000, indicating that the method has a good linear relationship.

[0059] (5) Repeatability

[0060] Accurately measure 1 ml of this product and place it in a 10 ml volumetric flask, add 100 μl of 0.1% copper acetate solution, dilute to the scale with a pH 2.5 phosphoric acid aqueous solution, shake well, and you are done. Prepare 6 portions in parallel. Accurately weigh 5 mg of disodium edetate reference substance and place it in a 50 ml volumetric flask, dissolve it with water and dilute to the scale; accurately measure 1 ml and place it in a 10 ml volumetric flask, add 100 μl of 0.1% copper acetate solution, dilute to the scale with a pH 2.5 phosphoric acid aqueous solution, shake well, and you are done. Prepare 2 portions in parallel, as reference solution 1 and reference solution 2, respectively. Accurately measure 20 μl each of the test solution and reference solution (reference solution 1 is injected into 5 needles, reference solution 2 is injected into 2 needles), inject into the liquid chromatograph, and record the chromatogram. The results are shown in Table 6.

[0061] Table 6 Repeatability test results

[0062]

[0063] The test results show that: the RSD of the content of disodium edetate in 6 samples was less than 2.0% in parallel determination, indicating that the repeatability of this method is good.

[0064] (6) Intermediate precision

[0065] Have another tester accurately measure 1 ml of this product and place it in a 10 ml volumetric flask, add 100 μl of 0.1% copper acetate solution, dilute to the mark with a diluent, shake well, and you are done. Prepare 6 portions in parallel. Accurately weigh 5 mg of disodium edetate reference substance and place it in a 50 ml volumetric flask, dissolve it with water and dilute to the mark; accurately measure 1 ml and place it in a 10 ml volumetric flask, add 100 μl of 0.1% copper acetate solution, dilute to the mark with a diluent, shake well, and you are done. Prepare 2 portions in parallel, as reference solution 1 and reference solution 2, respectively. Accurately measure 20 μl each of the test solution and reference solution (reference solution 1 is injected into 5 needles, reference solution 2 is injected into 2 needles), inject into the liquid chromatograph, and record the chromatogram. The results are shown in Table 7.

[0066] Table 7 Intermediate precision test results

[0067]

[0068] The test results show that: the RSD of the content of disodium edetate in 12 samples was less than 2.0% when the method was used for parallel determination, indicating that the intermediate precision of the method was good.

[0069] (7) Accuracy

[0070] The solution preparation is shown in Table 8. 20 μl of each of the blank solution, blank excipient solution, reference solution (reference solution 1 is injected into 5 needles, reference solution 2 is injected into 2 needles), and spiked test solution are injected into the liquid chromatograph and the chromatogram is recorded. The test results are shown in Table 9.

[0071] Table 8 Accuracy solution preparation

[0072]

[0073] Table 9 Reference solution stability solution results

[0074]

[0075]

[0076] As shown in Table 9, within the range of 50%, 100%, and 150% of the prescription concentration (0.1 mg / ml), the average recovery rate (n=9) was 98.0% to 102.0%; RSD ≤ 2.0%, indicating that the method has good accuracy.

[0077] The above records are only embodiments of the present invention. Any modification or change made by a person familiar with the present technology using the present invention shall fall within the patent scope claimed by the present invention and shall not be limited to those disclosed in the embodiments.

Claims

1. A method for determining betamethasone sodium phosphate injection edetate disodium by liquid chromatography, characterized in that: Octadecylsilane bonded silica gel was used as filler, the detection wavelength was 254 nm, the flow rate was 0.8~1.2 ml / min, the column temperature was 30~50 °C, and the injection volume was 5~50 µl.

2. A method for determining betamethasone sodium phosphate injection disodium edetate by liquid chromatography according to claim 1, characterized in that: The flow rate was 1.0 ml / min, the column temperature was 40° C., and the injection volume was 20 μl.

3. A method for determining betamethasone sodium phosphate injection disodium edetate by liquid chromatography according to claim 1, characterized in that: The mobile phase was 0.3% tetrabutylammonium hydroxide solution-water-acetonitrile in a volume ratio of 18~25:45:30~37.

4. A method for determining betamethasone sodium phosphate injection disodium edetate by liquid chromatography according to claim 3, characterized in that: The concentration of the 0.3% tetrabutylammonium hydroxide solution was adjusted to pH 4.0 with phosphoric acid.

5. A method for determining betamethasone sodium phosphate injection disodium edetate by liquid chromatography according to claim 1, characterized in that: The following steps are involved: (1) Preparation of test solution: Accurately measure 1 ml of the product, add 100 μl of 0.1% copper acetate solution, and dilute with diluent to make a solution containing 0.01 mg of edetate disodium per 1 ml; (2) Preparation of reference solution: accurately weigh 5 mg of edetate disodium reference substance into a 50 ml volumetric flask, add water to dissolve and dilute to scale; accurately weigh 1 ml into a 10 ml volumetric flask, add 100 μl of 0.1% copper acetate solution, and dilute to scale with diluent to make a solution containing 0.01 mg of edetate disodium per 1 ml; (3) Accurately measure 20 µl of the test solution and the reference solution and inject them into the liquid chromatograph. Record the chromatogram and calculate the content of disodium edetate based on the peak area using the external standard method.

6. A method for determining betamethasone sodium phosphate injection edetate disodium by liquid chromatography according to claim 5, characterized in that: The diluent is a phosphoric acid aqueous solution with a pH of 2.5.

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