Method for detecting content of rifamycin sodium injection by high performance liquid chromatography

Abstract

The invention provides a method for detecting the content of rifamycin sodium injection by high performance liquid chromatography. The content of rifamycin sodium in an analyte is efficiently and accurately measured by adopting a high performance liquid chromatography. The method comprises the following steps: S1, preparing a to-be-detected solution, wherein the to-be-detected solution comprises a reference solution and a test solution; and S2, determining the sample obtained in the step S1 by adopting a high performance liquid chromatography, and analyzing the determination result of the sample solution. According to the high performance liquid chromatography method, octadecyl silane bonded silica gel is used as a filling agent; a 0.025 mol / L sodium dihydrogen phosphate solution (the pH value is adjusted to 7.5 by phosphoric acid) and acetonitrile (55: 45) are used as a mobile phase; the method has the advantages of simplicity and convenience in operation, strong specificity and high sensitivity, is suitable for detection and analysis of the multi-component rifamycin sodium injection, and provides a good method for reverse engineering, component analysis, auxiliary material content determination and the like of pharmaceutical preparations and other products.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method for detecting the content of rifamycin sodium injection by high performance liquid chromatography. Background Technology

[0002] Rifamycin sodium injection is a broad-spectrum antibiotic in the semi-synthetic rifamycin class. It has strong antibacterial activity against Staphylococcus aureus (including penicillin-resistant and neomycin-resistant strains) and Mycobacterium tuberculosis. Its activity against common Gram-negative bacteria is weak. Its mechanism of action is to inhibit the activity of bacterial ribonucleic acid polymerase, thereby affecting ribonucleic acid synthesis and protein metabolism, leading to the cessation of bacterial growth and reproduction, thus achieving a bactericidal effect. Its indications are for the combined treatment of Mycobacterium tuberculosis infection and severe methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, and refractory Legionella infections. According to research, currently only the European Pharmacopoeia (EP) includes rifamycin sodium; no other official pharmacopoeias include it. The method for testing the content of the raw material is the antibiotic microbiological method. Currently, based on the antibiotic microbiological assay method for rifamycin sodium injection published in the draft for comments by the Pharmacopoeia Commission, an optimized method for determining the content of rifamycin sodium injection is being developed and validated.

[0003] To address the current lack of a high-performance liquid chromatography (HPLC) method for detecting the content of rifamycin sodium injection, this invention provides a HPLC method for detecting the content of rifamycin sodium injection. Summary of the Invention

[0004] Objective of the invention: To address the current lack of a method for detecting the content of rifamycin sodium injection using high-performance liquid chromatography (HPLC), this invention provides a method for determining the content of rifamycin sodium injection using HPLC. The content of rifamycin sodium injection is determined by HPLC.

[0005] This invention provides a method for determining the content of rifamycin sodium injection by high performance liquid chromatography, comprising the following steps: S1. Prepare the test solution, wherein the test solution includes a reference solution and a test solution; S2. The sample obtained in step S1 is measured by high performance liquid chromatography, and the measurement results of the sample solution are analyzed. The reference solution is prepared by accurately weighing an appropriate amount of rifamycin sodium reference standard, dissolving it in 0.025 mol / L sodium dihydrogen phosphate solution (adjusted to pH 3.0 with phosphoric acid) - acetonitrile (50:50) solvent, and quantitatively diluting it to prepare a solution containing approximately 0.1 mg per ml.

[0006] The test solution is prepared by quantitatively diluting an appropriate amount of this product with 0.025 mol / L sodium dihydrogen phosphate solution (adjusting the pH to 3.0 with phosphoric acid) and acetonitrile (50:50) to a solution containing approximately 0.1 mg per ml.

[0007] Preferably, the high performance liquid chromatography method uses octadecylsilane-bonded silica gel as the packing material and 0.025 mol / L sodium dihydrogen phosphate solution (pH adjusted to 7.5 with phosphoric acid) - acetonitrile (55:45) as the mobile phase.

[0008] In a preferred embodiment, according to the method of the present invention, the column temperature is 30°C, the detection wavelength is 254 nm, the flow rate is 1.0 ml / min, and the injection volume is 20 μL.

[0009] Beneficial effects: Compared with the prior art, the present invention has the following advantages: (1) The present invention will develop a method for detecting the content of rifamycin sodium injection by high performance liquid chromatography, which will greatly improve the detection efficiency.

[0010] (2) All of the present invention uses C18 chromatographic columns and water as the mobile phase. The preparation process is simple and has the advantages of low cost and easy availability.

[0011] (3) The high performance liquid chromatography method used in this invention is used to determine the content of rifamycin sodium injection and to conduct related methodological research and investigation. It is simple to operate, highly specific and sensitive, and is suitable for the detection and analysis of multi-component rifamycin sodium injection. It provides a good method for reverse engineering, component analysis and content determination of excipients in pharmaceutical preparations and other products (such as food and cosmetics). Attached Figure Description

[0012] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0013] Figure 1 Example 1: Chromatogram of the system adaptability of rifamycin sodium injection.

[0014] Figure 2 Comparative Example 1: Chromatogram of the system adaptability of rifamycin sodium injection.

[0015] Figure 3 Comparative Example 2: Chromatogram of the system adaptability of rifamycin sodium injection.

[0016] Figure 4 Chromatogram of the reference solution in Example 1.

[0017] Figure 5 Chromatogram of the test solution in Example 1.

[0018] Figure 6 Example 1: Linear chromatogram of rifamycin sodium injection. Detailed Implementation

[0019] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0020] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] This invention provides a method for determining the content of rifamycin sodium injection by high performance liquid chromatography, comprising the following steps: S1. Prepare the test solution, wherein the test solution includes a reference solution and a test solution; S2. The sample obtained in step S1 is measured by high performance liquid chromatography, and the measurement results of the sample solution are analyzed. The reference solution is prepared by accurately weighing an appropriate amount of rifamycin sodium reference standard, dissolving it in 0.025 mol / L sodium dihydrogen phosphate solution (adjusted to pH 3.0 with phosphoric acid) - acetonitrile (50:50) solvent, and quantitatively diluting it to prepare a solution containing approximately 0.1 mg per ml.

[0022] The test solution is prepared by quantitatively diluting an appropriate amount of this product with 0.025 mol / L sodium dihydrogen phosphate solution (adjusting the pH to 3.0 with phosphoric acid) and acetonitrile (50:50) to a solution containing approximately 0.1 mg per ml.

[0023] Preferably, the high performance liquid chromatography uses octadecylsilane-bonded silica gel as the packing material and 0.025 mol / L sodium dihydrogen phosphate solution (adjusted to pH 7.5 with phosphoric acid)-acetonitrile as the mobile phase.

[0024] In a preferred embodiment, according to the method of the present invention, the column temperature is 30°C, the detection wavelength is 254 nm, the flow rate is 1.0 ml / min, and the injection volume is 20 μL.

[0025] The present invention will now be described in detail through specific embodiments.

[0026] Example 1: Determination of the content of rifamycin sodium injection.

[0027] Step S1: Prepare the test solution, which includes a reference solution and a test solution. The specific preparation methods for each solution are as follows: S11. Reference solution: Take an appropriate amount of rifamycin sodium reference standard, accurately weigh it, add 0.025mol / L sodium dihydrogen phosphate solution (adjust the pH value to 3.0 with phosphoric acid)-acetonitrile (50:50) solvent to dissolve and quantitatively dilute to prepare a solution containing about 0.1mg per ml.

[0028] S12. Test solution: Take an appropriate amount of this product and dilute it quantitatively with 0.025mol / L sodium dihydrogen phosphate solution (adjust the pH value to 3.0 with phosphoric acid)-acetonitrile (50:50) to prepare a solution containing about 0.1mg per ml.

[0029] Step S2: The reference solution and test solution obtained in step S1 are determined by high performance liquid chromatography, and the results are analyzed. The chromatographic conditions were as follows: octadecylsilane-bonded silica gel was used as the packing material; 0.025 mol / L sodium dihydrogen phosphate solution (pH adjusted to 7.5 with phosphoric acid) - acetonitrile (55:45) was used as the mobile phase; the column temperature was 30℃; the detection wavelength was 254 nm; the flow rate was 1.0 ml / min; and the injection volume was 20 μL.

[0030] The chromatograms of the reference solution and the test solution are shown below. Figures 4-5 As shown.

[0031] Example 2: Detailed verification results of the specificity, precision, linearity and range, and accuracy of the detection method.

[0032] (a) Exclusivity: Test solution: Take an appropriate amount of this product and dilute it quantitatively with a solvent to prepare a solution containing approximately 0.1 mg per 1 ml.

[0033] Blank excipient solution: Take 1 ml of blank excipient, place it in a 50 ml volumetric flask, add solvent to make up to the mark, and shake well.

[0034] Take 20 μL each of the solvent, blank excipient solution and test sample solution, and inject them.

[0035] Results: The solvent and blank excipient had no effect on the determination of the test solution, which met the requirements, and the method showed good specificity.

[0036] (ii) System Applicability: System suitability solution: Take 25 mg of rifamycin sodium and 8 mg of rifamycin S reference standard, place them in a 250 ml volumetric flask, add solvent to dissolve and dilute to the mark, and shake well.

[0037] Assay: Inject the system suitability solution into the liquid chromatograph, record the chromatogram, and examine the resolution between rifamycin and rifamycin S.

[0038] Conclusion: The separation degree between rifamycin and rifamycin S was 3.9, indicating good separation; the theoretical plate number of the rifamycin peak was 6788, which is greater than 2000; and the tailing factor was 1.51, which is less than 2.0, all of which meet the requirements.

[0039] (iii) Precision: Reference solution: Weigh an appropriate amount of rifamycin sodium reference standard accurately, dissolve it in solvent and dilute quantitatively to prepare a solution containing approximately 0.1 mg per ml.

[0040] Assay: Inject the reference solution into the liquid chromatograph, record the chromatogram, inject five times consecutively, and examine the injection precision. The results are as follows:

[0041] Conclusion: The peak area RSD of rifamycin was 0.08%, which is less than 2.0% and meets the requirements.

[0042] (iv) Linearity and Range: Accurately weigh an appropriate amount of rifamycin sodium reference standard, dissolve and dilute it with a solvent to prepare a solution containing approximately 1 mg per ml, which serves as the reference standard stock solution. Accurately transfer 1 mL (to a 50 ml volumetric flask), 1 mL (to a 20 ml volumetric flask), 2 mL (to a 25 ml volumetric flask), 1 mL (to a 10 ml volumetric flask), 3 mL (to a 25 ml volumetric flask), 3 mL (to a 20 ml volumetric flask), and 2 mL (to a 10 ml volumetric flask) of the reference standard stock solution to the mark with solvent, respectively, to prepare linear solutions of 20%, 50%, 80%, 100%, 120%, 150%, and 200%.

[0043] Accurately measure 20 μl of each of the above linear solutions and inject them into the liquid chromatograph. Perform linear regression with concentration as the x-axis and peak area as the y-axis. The results are shown in the table below.

[0044]

[0045] Conclusion: Rifamycin sodium showed a good linear relationship with peak area in the concentration range of 0.018-0.175 mg / ml, with a correlation coefficient r of 1.00.

[0046] (5) Accuracy: 1. Rifamycin sodium stock solution: Take an appropriate amount of rifamycin sodium reference standard, accurately weigh it, and dilute it with a solvent to prepare a solution containing approximately 1 mg per 1 ml.

[0047] 2. Blank excipient stock solution: Accurately transfer 1 ml of blank excipient solution (containing vitamin C, disodium edetate, sodium metabisulfite, etc.) into a 50 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain the stock solution.

[0048] 3. Spiking recovery solution: Accurately transfer 2 ml of rifamycin sodium stock solution and 2 ml of blank excipient solution into a 20 ml volumetric flask, dilute to the mark with solvent, and shake well. Prepare 6 parallel aliquots.

[0049] 4. Reference solution: Accurately transfer 2 ml of rifamycin sodium stock solution into a 20 ml volumetric flask, dilute to the mark with solvent, and shake well. Prepare two parallel solutions.

[0050] Determination: Take the reference solution and the recovery solution and inject them into the high performance liquid chromatograph.

[0051]

[0052] Conclusion: The average recovery rate of rifamycin sodium was 99.8%, which meets the requirements. The RSD of the recovery rate was 0.19%, indicating that the method is accurate and feasible for the determination of rifamycin sodium injection content.

[0053] (6) Solution stability: Take the test solution and the reference solution, and inject them at time points of 0h, 2h, 4h, 6h, 8h and 10h, respectively. Record the chromatograms and investigate the changes in content under room temperature conditions. The results are shown in the table below.

[0054]

[0055] The results showed that the peak area of ​​the test sample solution changed by 98%-102% within 10 hours compared to the peak area at 0 hours, and the test sample solution remained stable at room temperature for 10 hours. The peak area of ​​the reference solution changed by 98%-102% only within 2 hours, and the reference solution remained stable at room temperature for 2 hours.

[0056] Comparative Example 1: The difference between this comparative example and Example 1 is that other chromatographic conditions remain unchanged, and the mobile phase is 0.025 mol / L sodium dihydrogen phosphate solution (pH adjusted to 7.5 with phosphoric acid) - acetonitrile (50:50).

[0057] Specifically, this comparative example provides a method for determining the content of rifamycin sodium injection and a method pre-validation.

[0058] Step S1: Prepare the test solution, which includes a reference solution and a test solution. The specific preparation methods for each solution are as follows: S11. Reference solution: Take an appropriate amount of rifamycin sodium reference standard, accurately weigh it, add 0.025mol / L sodium dihydrogen phosphate solution (adjust the pH value to 3.0 with phosphoric acid)-acetonitrile (50:50) solvent to dissolve and quantitatively dilute to prepare a solution containing about 0.1mg per ml.

[0059] S12. Test solution: Take an appropriate amount of this product and dilute it quantitatively with 0.025mol / L sodium dihydrogen phosphate solution (adjust the pH value to 3.0 with phosphoric acid)-acetonitrile (50:50) to prepare a solution containing about 0.1mg per ml.

[0060] Step S2: The reference solution and test solution obtained in step S1 are determined by high performance liquid chromatography, and the results are analyzed. The chromatographic conditions were as follows: octadecylsilane-bonded silica gel was used as the packing material; 0.025 mol / L sodium dihydrogen phosphate solution (pH adjusted to 7.5 with phosphoric acid) - acetonitrile (50:50) was used as the mobile phase; the column temperature was 30℃; the detection wavelength was 254 nm; the flow rate was 1.0 ml / min; and the injection volume was 20 μL.

[0061] Comparative Example 2: The difference between this comparative example and Example 1 is that other chromatographic conditions remain unchanged, and the mobile phase is 0.025 mol / L sodium dihydrogen phosphate solution (pH adjusted to 7.5 with phosphoric acid) - acetonitrile (60:40).

[0062] Specifically, this comparative example provides a method for determining the content of rifamycin sodium injection and a method pre-validation.

[0063] Step S1: Prepare the test solution, which includes a reference solution and a test solution. The specific preparation methods for each solution are as follows: S11. Reference solution: Take an appropriate amount of rifamycin sodium reference standard, accurately weigh it, add 0.025mol / L sodium dihydrogen phosphate solution (adjust the pH value to 3.0 with phosphoric acid)-acetonitrile (50:50) solvent to dissolve and quantitatively dilute to prepare a solution containing about 0.1mg per ml.

[0064] S12. Test solution: Take an appropriate amount of this product and dilute it quantitatively with 0.025mol / L sodium dihydrogen phosphate solution (adjust the pH value to 3.0 with phosphoric acid)-acetonitrile (50:50) to prepare a solution containing about 0.1mg per ml.

[0065] Step S2: The reference solution and test solution obtained in step S1 are determined by high performance liquid chromatography, and the results are analyzed. The chromatographic conditions were as follows: octadecylsilane-bonded silica gel was used as the packing material; 0.025 mol / L sodium dihydrogen phosphate solution (pH adjusted to 7.5 with phosphoric acid) - acetonitrile (60:40) was used as the mobile phase; the column temperature was 30℃; the detection wavelength was 254 nm; the flow rate was 1.0 ml / min; and the injection volume was 20 μL.

[0066]

[0067] Conclusion: The separation effects of Example 1 and Comparative Examples 1 and 2 are similar. The tailing of Comparative Example 2 is slightly larger than that of Example 1 and Comparative Example 1. In summary, compared with Comparative Example 1, the solubility of phosphate in high proportion of acetonitrile is poor, which poses a risk of precipitation. Therefore, Example 1 was selected as the mobile phase for the content method and the methodology was validated.

[0068] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the concept described herein through the above teachings or related technical or knowledge. Any modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention should be within the protection scope of the appended claims.

Claims

1. A method for determining the content of rifamycin sodium injection by high performance liquid chromatography, characterized in that, The content determination method includes the following steps: S1. Prepare the test solution, wherein the test solution includes a reference solution and a test solution; S2. The sample obtained in step S1 is measured using high performance liquid chromatography, and the measurement results of the sample solution are analyzed.

2. The reference solution according to claim 1, characterized in that... Accurately weigh an appropriate amount of rifamycin sodium reference standard, dissolve it in 0.025 mol / L sodium dihydrogen phosphate solution-acetonitrile solvent, and quantitatively dilute it to prepare a solution containing approximately 0.1 mg per ml. The pH of the 0.025 mol / L sodium dihydrogen phosphate solution is adjusted to 3.0 with phosphoric acid, and the ratio of sodium dihydrogen phosphate solution to acetonitrile is 50:

50.

3. The test solution according to claim 1, characterized in that... Take an appropriate amount of this product and dilute it quantitatively with 0.025 mol / L sodium dihydrogen phosphate solution-acetonitrile solvent to prepare a solution containing approximately 0.1 mg per ml.

4. The high-performance liquid chromatography method according to claim 1, characterized in that... The column temperature was 30℃, the detection wavelength was 254nm, the flow rate was 1.0ml / min, and the injection volume was 20μL.

5. The high-performance liquid chromatography method according to claim 1, characterized in that... Octadecylsilane-bonded silica gel is used as a filler; The mobile phase consisted of 0.025 mol / L sodium dihydrogen phosphate solution and acetonitrile, wherein the pH of the 0.025 mol / L sodium dihydrogen phosphate solution was adjusted to 7.5 with phosphoric acid, and the ratio of sodium dihydrogen phosphate solution to acetonitrile was 55:45.

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