Method for detecting content of zoledronic acid concentrated solution for injection

Abstract

The invention provides a method for detecting the content of a concentrated zoledronic acid solution for injection, which comprises the following steps: S1, preparing a test solution: taking the concentrated zoledronic acid solution for injection, diluting with a mobile phase, and uniformly shaking; s2, preparation of a reference substance solution: taking a zoledronic acid reference substance, diluting the zoledronic acid reference substance with a mobile phase, and uniformly shaking; and S3, respectively detecting the test solution and the reference solution by adopting a high performance liquid chromatography, wherein the chromatographic conditions are as follows: a stationary phase is a chromatographic column taking octadecylsilane chemically bonded silica as a filler; and the mobile phase is composed of a phosphate buffer solution, acetonitrile and sodium pentanesulfonate. The detection method has the advantages of good separation effect, high detection sensitivity, high accuracy, improved intermediate precision, simple detection method, strong specificity, good repeatability and the like, can effectively guarantee the safety and effectiveness of the concentrated zoledronic acid solution for injection, and can be used for quality control of the concentrated zoledronic acid solution for injection.

Description

Technical Field

[0001] The invention relates to the technical field of drug detection, and in particular to a method for detecting the content of a concentrated zoledronic acid solution for injection. Background Art

[0002] Zoledronic acid, chemical name 1-hydroxy-2-(imidazol-1-yl)-ethylidene-1,1-diphosphonic acid, molecular formula C5H 10 N2O7P2 is a bisphosphonate drug. Zoledronic acid is a bisphosphonate compound that acts specifically on bones. It can inhibit bone resorption caused by increased osteoclast activity, reduce the risk of bone metastasis and fractures, and is widely used clinically. Zoledronic acid is usually administered as an intravenous injection once a year or every six months. The specific dosage depends on the patient's condition and the doctor's advice. Compared with oral medications, intravenous administration can ensure that the drug quickly reaches an effective concentration in a short period of time.

[0003] Zoledronic acid concentrated solution for injection plays an important role in the treatment of osteoporosis and tumor-related bone diseases. Its unique pharmacological mechanism and clinical effects make it an indispensable drug in the field of bone metabolism. The quality control of zoledronic acid concentrated solution for injection is inseparable from the detection of zoledronic acid content. Through accurate content detection, the safety and effectiveness of the drug can be effectively guaranteed, the patient's medication safety can be maintained, and regulatory requirements can be met. It is an indispensable and important link in drug production.

[0004] HPLC is currently the main method for detecting the content of zoledronic acid. It has high sensitivity and good separation effect. It uses a reversed-phase column, an appropriate mobile phase, and monitors a specific wavelength through a UV detector. During sample preparation and analysis, matrix components may interfere with the test results. Therefore, it is of great significance to continuously optimize the HPLC method, including the improvement of mobile phase ratio, column temperature, flow rate and other conditions, to improve separation efficiency and detection sensitivity. Summary of the invention

[0005] In view of this, the present invention provides a method for detecting the content of a concentrated solution of zoledronic acid for injection to solve the above problems.

[0006] The technical solution of the present invention is achieved in this way:

[0007] The method for detecting the content of zoledronic acid concentrated solution for injection comprises the following steps:

[0008] S1. Preparation of test solution: Take concentrated zoledronic acid solution for injection, dilute it with mobile phase, and shake well;

[0009] S2. Preparation of reference solution: Take zoledronic acid reference solution, dilute it with mobile phase, and shake well;

[0010] S3. Use high performance liquid chromatography to test the test sample and reference solution respectively. The chromatographic conditions are as follows:

[0011] Stationary phase: chromatographic column with octadecylsilane bonded silica gel as filler;

[0012] Mobile phase: composed of phosphate buffer, acetonitrile and sodium pentane sulfonate.

[0013] Preferably, the volume ratio of the phosphate buffer, acetonitrile and sodium pentane sulfonate is 90:9:1.

[0014] Preferably, the detection wavelength is 215 nm.

[0015] Preferably, the column temperature is 25-30°C.

[0016] Preferably, the injection volume is 20-40 μL.

[0017] Preferably, the flow rate of the mobile phase is 0.8 to 1.0 mL / min.

[0018] Preferably, the model specifications of the chromatographic column are: Express C18 chromatographic column, column length 150mm, inner diameter 4.6mm, filler particle size 2.7μm.

[0019] Preferably, the pH of the phosphate buffer is 7.9.

[0020] Preferably, the phosphate buffer solution is prepared by taking anhydrous disodium hydrogen phosphate or disodium hydrogen phosphate dodecahydrate and tetrahexylammonium hydrogen sulfate, dissolving them in water, adding disodium ethylenediaminetetraacetic acid solution, mixing, adjusting the pH value to 7.9 with sodium hydroxide solution, and mixing.

[0021] Preferably, the concentration of the reference solution is 0.16 mg / mL.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention uses a mobile phase as a diluent to prepare a reference solution and a test solution of a certain concentration, uses high performance liquid chromatography, optimizes chromatographic conditions and the mobile phase component ratio, and determines the content of a concentrated solution of zoledronic acid for injection. The detection method has the advantages of good separation effect, high detection sensitivity, high accuracy, improved intermediate precision, etc. The method for detecting the content of a concentrated solution of zoledronic acid for injection provided by the present invention is simple, has strong specificity, and has good repeatability, and can effectively ensure the safety and effectiveness of a concentrated solution of zoledronic acid for injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1This is the HPLC spectrum of the solvent in the content determination of the concentrated zoledronic acid solution for injection in Example 1 of the present invention.

[0025] Figure 2 This is the HPLC spectrum of the blank auxiliary material in the content determination of the concentrated solution of zoledronic acid for injection in Example 1 of the present invention.

[0026] Figure 3 The HPLC spectrum of the test solution in the content determination of the concentrated solution of zoledronic acid for injection in Example 1 of the present invention.

[0027] Figure 4 The HPLC spectrum of the reference substance solution in the content determination of the concentrated zoledronic acid solution for injection in Example 1 of the present invention. DETAILED DESCRIPTION

[0028] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0029] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0030] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0031] The concentrated zoledronic acid solution for injection used in the present invention is produced by Hainan Huluwa Pharmaceutical Group Co., Ltd., and the specific prescription is:

[0032] Table 1 Prescription of Zoledronic Acid Concentrated Solution for Injection

[0033]

[0034]

[0035] Example 1

[0036] Determination of the content of zoledronic acid concentrated solution for injection:

[0037] Inspection method: High performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0512)

[0038] Chromatographic conditions:

[0039] Chromatographic column: Octadecylsilane bonded silica gel is used as filler ( Express C18, 150mm×4.6mm, 2.7μm);

[0040] Detection wavelength: 215nm;

[0041] Column temperature: 25°C;

[0042] Injection volume: 40 μl;

[0043] Flow rate: 1.0ml / min

[0044] Mobile phase: phosphate buffer (pH 7.9) 注 - acetonitrile-sodium pentane sulfonate (volume ratio 90:9:1);

[0045] Note: Phosphate buffer (pH 7.9): Take 5.7 g of anhydrous disodium hydrogen phosphate and 4.5 g of tetrahexyl ammonium hydrogen sulfate, add 900 ml of water to dissolve, add 2 ml of disodium ethylenediaminetetraacetic acid solution, mix well, adjust the pH to 7.9 with 2 mol / L sodium hydroxide solution, and mix well.

[0046] Disodium ethylenediaminetetraacetate solution: Take 0.365 g of disodium ethylenediaminetetraacetate dihydrate, add 5 ml of 2 mol / L sodium hydroxide solution to dissolve it, add water to 100 ml, mix well, and you have the solution.

[0047] Solution preparation:

[0048] Solvent: Mobile phase

[0049] Complete excipient solution: Take 240 mg of sodium citrate and 2.2 g of mannitol, place them in a 50 ml volumetric flask, add water to dissolve and dilute to the scale, and shake well.

[0050] Excipient solution: Accurately measure 2 ml of the entire excipient solution, place it in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0051] Reference solution: Take about 16 mg of zoledronic acid reference substance, weigh accurately, place in a 100 ml volumetric flask, dissolve and dilute to the scale with solvent, and shake well.

[0052] Test solution: Take 2 ml of concentrated zoledronic acid solution for injection, place it in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0053] Determination method: Accurately measure 40 μL of reference solution and test solution, respectively, and inject them into the liquid chromatograph to record the chromatogram. The test solution is collected until the excipient peak is completely discharged, and the reference solution is collected until the zoledronic acid peak is completely collected. Calculation formula:

[0054] K=(W 对 ×P 对 ) / (A 对 × dilution factor of reference substance)

[0055] Content (%) = [(A 样 ×K×dilution factor of the test sample) / (V 样 ×Specification)]×100%

[0056] Where: A 样 - Zoledronic acid peak area of ​​the test solution;

[0057] A 对 - Zoledronic acid peak area of ​​reference solution;

[0058] V 样 - Volume of the test sample, mL;

[0059] W 对 -weight of the reference substance, g;

[0060] P 对 -Content of reference substance, %;

[0061] Specification: 5ml: 4mg.

[0062] Result determination: Calculated by peak area according to external standard method, the content of zoledronic acid (C5H 10 N2O7P2·H2O) to anhydrous zoledronic acid (C5H 10 N2O7P2), it should be 95.0% to 105.0% of the labeled amount.

[0063] HPLC chromatogram results are as follows Figures 1 to 4 As shown, the retention time of the main peak of the reference solution is 24.984min, the peak area is 4851502, and the retention time of the main peak of the test solution is 25.063min, the peak area is 5235466. Under this chromatographic condition, the blank solvent and excipients do not interfere with the main component elution.

[0064] According to the 2020 Chinese Pharmacopoeia Part IV 9101 Analytical Method Validation Guidelines, a systematic method validation was conducted on the content determination method of zoledronic acid concentrated solution for injection. The method validation content includes: specificity, linearity and range, accuracy test, precision test (repeatability and intermediate precision), durability test and solution stability test. The validation results are summarized in the following table:

[0065] Table 2 Results of content determination method validation

[0066]

[0067]

[0068] The validation results showed that this method was suitable for the determination of the content of zoledronic acid concentrated solution for injection.

[0069] Example 2

[0070] The difference between this example and Example 1 is that the chromatographic conditions are different, column temperature: 30°C; injection volume: 40 μL; flow rate: 1.0 mL / min. The results show that the peak shapes of the blank solvent and the main component zoledronic acid are good and there is no mutual interference.

[0071] Example 3

[0072] The difference between this example and Example 1 is that the chromatographic conditions are different, column temperature: 30°C; injection volume: 40 μL; flow rate: 0.8 mL / min. The results show that the peak shapes of the blank solvent and the main component zoledronic acid are good and there is no mutual interference.

[0073] Comparative Example 1

[0074] The difference between this comparative example and Example 1 is that the solvent is replaced by water from the mobile phase when preparing the solution. The accuracy test results show that the reference solution was continuously injected for 5 injections, the RSD of the main peak area was 2.1%, and the RSD of the recovery rate of 9 sampled test solutions was 1.85%. Compared with the mobile phase as solvent, the accuracy is reduced. The mobile phase as solvent can provide a more stable baseline signal, thereby improving the accuracy of the determination and reducing the system error.

[0075] Comparative Example 2

[0076] The difference between this comparative example and Example 1 is that the chromatographic conditions are different, column temperature: 30°C; injection volume: 40 μL; flow rate: 1.2 mL / min. The results show that the blank solvent peaks at the main peak position, interfering with the main component.

[0077] Comparative Example 3

[0078] The difference between this comparative example and Example 1 is that the stationary phase is different. The stationary phase in this example is Ascentis Express C8 chromatographic column, the chromatographic column length is 150 mm, the inner diameter is 4.6 mm, and the particle size of the filler is 2.7 μm. The results show that the retention time of zoledronic acid is shortened, and the blank solvent peaks at the main peak position, which will interfere with the main component.

[0079] Comparative Example 4

[0080] The difference between this comparative example and Example 1 is that the stationary phase is different. The stationary phase in this example is a Sepax GP-C18 liquid chromatography column with a specification of 4.6 mm×250 mm and a filler particle size of 5 μm. The results show that the peak shape of zoledronic acid is severely tailed, affecting the accuracy of the quantitative results.

[0081] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting the content of a concentrated solution of zoledronic acid for injection, characterized in that: The following steps are involved: S1. Preparation of test solution: Take concentrated zoledronic acid solution for injection, dilute it with mobile phase, and shake well; S2. Preparation of reference solution: Take zoledronic acid reference solution, dilute it with mobile phase, and shake well; S3. Use high performance liquid chromatography to test the test sample and reference solution respectively. The chromatographic conditions are as follows: Stationary phase: chromatographic column with octadecylsilane bonded silica gel as filler; Mobile phase: composed of phosphate buffer, acetonitrile and sodium pentane sulfonate.

2. The method for detecting the content of zoledronic acid concentrated solution for injection according to claim 1, characterized in that: The volume ratio of the phosphate buffer, acetonitrile and sodium pentane sulfonate is 90:9:

1.

3. The method for detecting the content of zoledronic acid concentrated solution for injection according to claim 1, characterized in that: The detection wavelength is 215 nm.

4. The method for detecting the content of zoledronic acid concentrated solution for injection according to claim 1, characterized in that: The column temperature is 25-30℃.

5. The method for detecting the content of zoledronic acid concentrated solution for injection according to claim 1, characterized in that: The injection volume is 20-40 μL.

6. The method for detecting the content of zoledronic acid concentrated solution for injection according to claim 1, characterized in that: The flow rate of the mobile phase is 0.8~1.0mL / min.

7. The method for detecting the content of zoledronic acid concentrated solution for injection according to claim 1, characterized in that: The model specifications of the chromatographic column are: Express C18 chromatographic column, column length 150mm, inner diameter 4.6mm, filler particle size 2.7μm.

8. The method for detecting the content of zoledronic acid concentrated solution for injection according to claim 1, characterized in that: The pH of the phosphate buffer is 7.

9.

9. The method for detecting the content of zoledronic acid concentrated solution for injection according to claim 8, characterized in that: The preparation method of the phosphate buffer is as follows: taking anhydrous disodium hydrogen phosphate or disodium hydrogen phosphate dodecahydrate and tetrahexylammonium hydrogen sulfate, adding water to dissolve, adding disodium ethylenediaminetetraacetic acid solution, mixing, adjusting the pH value to 7.9 with sodium hydroxide solution, and mixing.

10. The method for detecting the content of zoledronic acid concentrated solution for injection according to claim 1, characterized in that: The concentration of the reference solution is 0.16 mg / mL.

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