Method for detecting polymers in cefoperazone sodium and sulbactam sodium

Abstract

The invention discloses a method for detecting polymers in cefoperazone sodium and sulbactam sodium, and belongs to the technical field of medicine quality control. The invention discloses a method for detecting polymers in cefoperazone sodium and sulbactam sodium. Respectively injecting the test solution and the reference solution into a liquid chromatograph, and detecting the content of the polymer by adopting a high performance liquid chromatography; the chromatographic column is a reversed-phase liquid chromatographic column; the detection wavelength is 240 to 250 nm; the flow velocity of the mobile phase is 0.4-0.8 ml / min; the column temperature is 20-30 DEG C; according to the method, the content of the polymer can be directly tested in one step under the chromatographic condition through liquid chromatography detection, and compared with the prior art in which impurities and main components are separated through a one-dimensional detection method and then the impurities are subjected to two-dimensional chromatographic analysis, the method is simple and high in operability and has remarkable advantages; the method can meet the requirements of qualitative and quantitative analysis, and is good in reproducibility, high in specificity, strong in durability, high in detection accuracy and good in system adaptability.

Description

Technical Field

[0001] The present application relates to a method for detecting a polymer in cefoperazone sodium and sulbactam sodium, and belongs to the technical field of drug quality control. Background Art

[0002] Cefoperazone sodium and sulbactam sodium are two antibiotics commonly used to treat bacterial infections. Cefoperazone belongs to the third-generation cephalosporin antibiotics, while sulbactam is a β-lactamase inhibitor. The two are often used in combination to enhance the antibacterial effect. In the process of drug production, polymers refer to high molecular weight substances that may be formed during the manufacturing process. These polymers may have a negative impact on the stability, solubility and bioavailability of the drug, and may cause immune reactions or other adverse reactions. Therefore, detecting the presence of polymers is a very important step in drug quality control.

[0003] At present, regarding the detection method of impurities in cefoperazone sodium and sulbactam sodium, the Chinese Pharmacopoeia only records the reverse phase chromatography method for the detection of related substances, and does not record the detection method for its polymer. Cefoperazone sodium and cefoperazone sodium for injection are controlled by analytical exclusion method in the Chinese Pharmacopoeia. However, the analytical exclusion chromatography performs a general total amount control on polymer impurities, and the interference of small molecules leads to insufficient method specificity, long detection time and low separation. Chinese patent 202211255091.4 provides a two-dimensional detection method for impurities in cefoperazone sodium and sulbactam sodium for injection, which first needs to perform a one-dimensional chromatographic analysis step to separate the impurities from the main component cefoperazone sodium and sulbactam sodium, and then collect the separated impurities in the collection ring and switch to inject liquid chromatography and mass spectrometry for two-dimensional chromatographic detection. This detection method requires simultaneous one-dimensional chromatographic analysis and two-dimensional chromatographic analysis. The operation process is complicated and not conducive to improving production efficiency and large-scale application. Summary of the invention

[0004] According to one aspect of the present application, a method for detecting polymers in cefoperazone sodium and sulbactam sodium is provided. By liquid chromatography detection, under the chromatographic conditions of the present application, the polymer content can be directly tested in one step. Compared with the prior art in which impurities and main components are first separated by a one-dimensional detection method and then the impurities are subjected to two-dimensional chromatographic analysis, the method is simple and highly operable, and has significant advantages. The method can meet the requirements of qualitative and quantitative analysis, and has good reproducibility, high specificity, strong durability, high detection accuracy, and good system adaptability.

[0005] The method for detecting polymers in cefoperazone sodium and sulbactam sodium comprises:

[0006] The test solution and the reference solution were respectively injected into a liquid chromatograph, and the polymer content was detected by high performance liquid chromatography;

[0007] The chromatographic column is a reverse phase liquid chromatography column;

[0008] The detection wavelength is 240-250nm;

[0009] The flow rate of the mobile phase is 0.4-0.8 ml / min;

[0010] Column temperature is 20-30°C;

[0011] The polymer includes polymerization hydrolysis impurity 1, polymerization hydrolysis impurity 2, polymerization hydrolysis impurity 3, polymerization hydrolysis impurity 4, and polymerization hydrolysis impurity 5;

[0012] The structure of the polymerized hydrolysis impurity 1 is as follows:

[0013]

[0014] The structure of the polymerized hydrolysis impurity 2 is as follows:

[0015]

[0016] The structure of the polymerization hydrolysis impurity 3 is as follows:

[0017]

[0018] The structure of the polymerized hydrolysis impurity 4 is as follows:

[0019]

[0020] The structure of the polymerized hydrolysis impurity 5 is as follows:

[0021]

[0022] Optionally, the chromatographic conditions are as follows:

[0023] Mobile phase A: ammonia-formic acid mixed solution, pH adjusted to 2-3;

[0024] Mobile phase B: acetonitrile.

[0025] Optionally, the volume ratio of aqueous ammonia to formic acid is (2.5-3.5):1; preferably, the volume ratio of aqueous ammonia to formic acid is 3:1.

[0026] Optionally, formic acid is used to adjust the pH.

[0027] Optionally, the pH is adjusted to 2.3.

[0028] Optionally, the solvent of the ammonia-formic acid mixed solution is water.

[0029] Optionally, the concentration of the ammonia-formic acid mixed solution is 0.02-0.03 Vol %; preferably, the concentration of the ammonia-formic acid mixed solution is 0.017 Vol %.

[0030] Optionally, the mobile phase uses a gradient elution.

[0031] Optionally, the gradient elution gradient is as follows:

[0032] 0min, mobile phase A 85Vol%, mobile phase B 15Vol%;

[0033] 2 min, mobile phase A 85 Vol%, mobile phase B 15 Vol%;

[0034] 3 min, mobile phase A 75 Vol%, mobile phase B 25 Vol%;

[0035] 21 min, mobile phase A 75 Vol%, mobile phase B 25 Vol%;

[0036] 40 min, mobile phase A 60 Vol%, mobile phase B 40 Vol%;

[0037] 50 min, mobile phase A 60 Vol%, mobile phase B 40 Vol%;

[0038] 55min, mobile phase A 85Vol%, mobile phase B 15Vol%;

[0039] 60 min, mobile phase A 85 Vol%, mobile phase B 15 Vol%.

[0040] Optionally, the filler of the chromatographic column is octadecylsilane bonded silica gel.

[0041] Optionally, the column length of the chromatographic column is 100-150 mm; preferably, the column length of the chromatographic column is 150 mm.

[0042] Optionally, the inner diameter of the chromatographic column is 3.0-4.6 mm; preferably, the inner diameter of the chromatographic column is 4.6 mm.

[0043] Optionally, the particle size of the chromatographic column is 3.5-5 μm; preferably, the particle size of the chromatographic column is 3.5 μm.

[0044] Optionally, the detection wavelength is 246 nm.

[0045] Optionally, the column temperature is independently selected from any one of 20°C, 22°C, 25°C, 28°C, 30°C or a range between any two of the above.

[0046] Optionally, the column temperature is 20°C.

[0047] Optionally, the injection volume is 20-40 μl; preferably, the injection volume is 30 μl.

[0048] Optionally, the flow rate of the mobile phase is 0.6 ml / min.

[0049] Optionally, the mass ratio of cefoperazone sodium to sulbactam sodium in the cefoperazone sodium and sulbactam sodium is 1:1 or 2:1.

[0050] Optionally, the testing method for the polymer in cefoperazone sodium and sulbactam sodium comprises:

[0051] (1) Prepare the test solution: weigh cefoperazone sodium and sulbactam sodium according to the required ratio to prepare the test solution;

[0052] (2) Preparation of reference solution: Take cefoperazone reference substance and prepare reference solution;

[0053] (3) Take the test solution and the reference solution separately, inject them into the liquid chromatograph, record the chromatogram, and calculate the peak area according to the external standard method.

[0054] Optionally, the concentration of cefoperazone sodium in the test solution is 1-20 mg / ml; preferably, the concentration of cefoperazone sodium in the test solution is 10 mg / ml.

[0055] Optionally, the concentration of cefoperazone sodium in the reference solution is 0.01-0.05 mg / ml; preferably, the concentration of cefoperazone sodium in the reference solution is 0.02 mg / ml.

[0056] Optionally, the polymer content calculation formula of the present application is as follows:

[0057]

[0058] In the formula:

[0059] A 对 is the main peak area in the chromatogram of the reference solution;

[0060] A 供 It is the sum of the peak areas of polymers in the chromatogram of the test solution;

[0061] C 对 is the concentration of the reference solution (mg / ml);

[0062] C 供 is the concentration of the test solution (mg / ml);

[0063] P is the purity of the reference substance (%);

[0064] Labelled amount: Cefoperazone (C25 H 27 N9O8S2) labeled amount (mg).

[0065] The beneficial effects of this application include:

[0066] 1. The detection method of the polymer of the present application is detected by liquid chromatography. Under the chromatographic conditions of the present application, the polymer content can be directly tested in one step. Compared with the prior art of first separating impurities and main components through a one-dimensional detection method and then performing two-dimensional chromatographic analysis on the impurities, the method is simple, highly operational, and has significant advantages.

[0067] 2. After extensive research, the applicant used ammonia-formic acid mixed solution as mobile phase A and acetonitrile as mobile phase B. The mobile phase of the present application can be directly connected to the MS detector, effectively improving the impurity response, and solving the problem that the impurity response values ​​of the prior art using triethylamine acetic acid solution as mobile phase A are too low and cannot be effectively detected. At the same time, the detection method can detect many types of polymers and has strong pertinence, and can effectively control the polymers, thereby ensuring the quality and efficacy of cefoperazone sodium and sulbactam sodium.

[0068] 3. The inventors found that when an aqueous ammonia-formic acid mixed solution was used as the fluidity A and acetonitrile was used as the fluidity B, a problem of poor separation would occur during polymer detection. Through extensive research and improvement, the inventors unexpectedly found that when the concentration of the aqueous ammonia-formic acid mixed solution was controlled at 0.02-0.03 Vol%, the separation of each impurity met the requirements and the peak shape was good.

[0069] 4. Polymer impurities are components that are difficult to separate and detect. The prior art mostly uses increasing the column temperature to improve the separation of polymer components, so as to better distinguish the target substance and polymer impurities. In the prior art, the column temperature is usually around 30°C to achieve the above effect. However, higher temperatures will affect the performance of the stationary phase and cause uneven viscosity of the mobile phase, thereby resulting in unstable separation effect. The present application can reduce the column temperature to 20°C by adjusting the elution gradient, etc., thereby solving the problem of unstable separation of polymer impurities caused by higher column temperatures in the prior art.

[0070] 5. The inventors have discovered through experiments that the mobile phase, mobile phase concentration, elution gradient, flow rate, and detection wavelength in this application complement each other. The above conditions are controlled within the scope of this application to obtain better separation degree and separation effect. Any condition that is not within the scope of this application will affect the separation effect.

[0071] 6. The detection method of polymers in cefoperazone sodium and sulbactam sodium in the present application can meet the requirements of qualitative and quantitative analysis, and has good reproducibility, high specificity, strong durability, high detection accuracy and good system adaptability. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 This is the blank liquid chromatogram of Example 1;

[0073] Figure 2 This is the liquid chromatogram of the test sample in Example 1;

[0074] Figure 3 The comparative liquid chromatogram of Example 1;

[0075] Figure 4 Liquid chromatography of reference preparations, homemade preparations and destroyed samples. DETAILED DESCRIPTION

[0076] The present application is described in detail below with reference to embodiments, but the present application is not limited to these embodiments.

[0077] Unless otherwise specified, the raw materials in the examples of this application were purchased through commercial channels.

[0078] The polymer content is calculated as follows:

[0079]

[0080] Where:

[0081] A 对 is the main peak area in the chromatogram of the reference solution;

[0082] A 供 It is the sum of the peak areas of polymers in the chromatogram of the test solution;

[0083] C 对 is the concentration of the reference solution (mg / ml)

[0084] C 供 is the concentration of the test solution (mg / ml)

[0085] P is the purity of the reference substance (%)

[0086] Labelled amount: Cefoperazone (C 25 H 27 N9O8S2) labeled amount (mg).

[0087] Example 1

[0088] Phosphate buffer: 0.2 mol / L sodium dihydrogen phosphate solution-0.2 mol / L sodium dihydrogen phosphate solution (volume ratio is 39:61).

[0089] Solvent: Water.

[0090] Test solution:

[0091] Take 212 mg of cefoperazone sodium and sulbactam sodium (mass ratio 1:1), weigh accurately, place in a 10 ml volumetric flask, add water to dissolve and dilute to the scale, shake well. (Cefoperazone sodium 10 mg / ml)

[0092] Reference solution: Accurately weigh 10 mg of cefoperazone reference substance, accurately weigh, place in a 50 ml volumetric flask, add 2 ml of phosphate buffer to dissolve, dilute to the scale with water, shake well, accurately measure 5 ml, place in a 50 ml volumetric flask, dilute to the scale with water, shake well (cefoperazone 0.02 mg / ml).

[0093] Accurately measure 30 μl of the test solution and reference solution, inject them into the liquid chromatograph, record the chromatogram, and calculate the peak area according to the external standard method.

[0094] The chromatographic conditions were as follows:

[0095] Mobile phase A: ammonia-formic acid mixed solution; the volume ratio of ammonia and formic acid is 3:1; the solvent is water, and the pH is adjusted to 2.3 with formic acid; the concentration of ammonia-formic acid mixed solution is 0.017 Vol%;

[0096] Mobile phase B: acetonitrile;

[0097] The gradient elution was as follows:

[0098] 0min, mobile phase A 85Vol%, mobile phase B 15Vol%;

[0099] 2 min, mobile phase A 85 Vol%, mobile phase B 15 Vol%;

[0100] 3 min, mobile phase A 75 Vol%, mobile phase B 25 Vol%;

[0101] 21 min, mobile phase A 75 Vol%, mobile phase B 25 Vol%;

[0102] 40 min, mobile phase A 60 Vol%, mobile phase B 40 Vol%;

[0103] 50 min, mobile phase A 60 Vol%, mobile phase B 40 Vol%;

[0104] 55min, mobile phase A 85Vol%, mobile phase B 15Vol%;

[0105] 60 min, mobile phase A 85 Vol%, mobile phase B 15 Vol%.

[0106] Column: SunFire C18, 4.6×150 mm, 3.5 μm;

[0107] The flow rate is 0.6m1 per minute;

[0108] The detection wavelength is 246nm;

[0109] The column temperature was 25 °C;

[0110] Injection volume: 30 μl.

[0111] Sample test results:

[0112]

[0113] Blank HPLC Figure 1 , the liquid chromatogram of the test sample is shown in Figure 2 , see the reference liquid chromatogram Figure 3 .

[0114] The liquid phase of this method can be directly connected to a MS detector, which can effectively improve the impurity response.

[0115] This method was used to test reference preparations, homemade preparations, and damaged samples.

[0116] The operation of destroying the sample is as follows: use a 36-month-old homemade product, dissolve it in water, and place it at 40°C for 4 hours.

[0117] Liquid chromatography of reference preparations, homemade preparations and destroyed samples is shown in Figure 4 ,In the figure, from top to bottom are the reference preparation, the homemade preparation and the destroyed sample.

[0118] Through LC-MS mass spectrometry analysis, the impurity peaks are as follows:

[0119]

[0120]

[0121] After analysis, the structures of the impurities are as follows:

[0122]

[0123]

[0124]

[0125] The method was validated and the results are as follows:

[0126]

[0127]

[0128]

[0129]

[0130] Comparative Example 1

[0131] Specific implementation method is the same as Example 1, except that the mobile phase A is triethylamine acetic acid solution.

[0132] Polymers were detected in both the reference preparation and the homemade preparation using this method. Since mobile phase A included triethylamine solution with poor volatility, the compatibility was poor when mass spectrometry was used to analyze polymer impurities, requiring column switching analysis. In addition, the response values ​​of each impurity were too low to be effectively detected in the mass spectrometry.

[0133] Comparative Example 2

[0134] The specific implementation is the same as Example 1, except that the mobile phase A is an ammonia acetic acid solution.

[0135] The separation degree of the polymer impurities in this comparative example is <1.0, the peak shape is poor, and the separation effect is not good.

[0136] Comparative Example 3

[0137] The specific implementation is the same as in Example 1, except that the elution gradient is as follows:

[0138] 0min, mobile phase A 90 Vol%, mobile phase B 10 Vol%;

[0139] 2 min, mobile phase A 90 Vol%, mobile phase B 10 Vol%;

[0140] 3 min, mobile phase A 85 Vol%, mobile phase B 15 Vol%;

[0141] 21 min, mobile phase A 85 Vol%, mobile phase B 15 Vol%;

[0142] 40 min, mobile phase A 70 Vol%, mobile phase B 30 Vol%;

[0143] 50 min, mobile phase A 70 Vol%, mobile phase B 30 Vol%;

[0144] 55 min, mobile phase A 90 Vol%, mobile phase B 10 Vol%;

[0145] 60 min, mobile phase A 90 Vol%, mobile phase B 10 Vol%.

[0146] The separation degree of impurities in this comparative example is poor, the separation effect is not good, and the types of polymer impurities detected are missing.

[0147] Comparative Example 4

[0148] Specific implementation method The same as Example 1, except that the flow rate of the mobile phase is 1.2 ml / min.

[0149] The polymer impurity separation degree of this comparative example is poor, and the separation effect cannot meet the requirements.

[0150] Comparative Example 5

[0151] Specific implementation method The same as Example 1, except that the concentration of the ammonia-formic acid mixed solution in the mobile phase A is 0.6 Vol%.

[0152] The polymer impurity separation degree of this comparative example is poor, and the separation effect cannot meet the requirements.

[0153] The above are only a few embodiments of the present application and do not constitute any form of limitation to the present application. Although the present application is disclosed as above with preferred embodiments, it is not intended to limit the present application. Any technician familiar with the profession, without departing from the scope of the technical solution of the present application, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. A method for detecting polymers in cefoperazone sodium and sulbactam sodium, characterized in that: include: The test solution and the reference solution were respectively injected into a liquid chromatograph, and the polymer content was detected by high performance liquid chromatography; The chromatographic column is a reverse phase liquid chromatography column; The detection wavelength is 240-250nm; The flow rate of the mobile phase is 0.4-0.8 ml / min; Column temperature is 20-30°C; The polymer includes polymerization hydrolysis impurity 1, polymerization hydrolysis impurity 2, polymerization hydrolysis impurity 3, polymerization hydrolysis impurity 4, and polymerization hydrolysis impurity 5; The structure of the polymerized hydrolysis impurity 1 is as follows: The structure of the polymerized hydrolysis impurity 2 is as follows: The structure of the polymerization hydrolysis impurity 3 is as follows: The structure of the polymerized hydrolysis impurity 4 is as follows: The structure of the polymerized hydrolysis impurity 5 is as follows:

2. The method for detecting polymers in cefoperazone sodium and sulbactam sodium according to claim 1, characterized in that: The chromatographic conditions were as follows: Mobile phase A: ammonia-formic acid mixed solution, pH adjusted to 2-3; Mobile phase B: acetonitrile.

3. The method for detecting polymers in cefoperazone sodium and sulbactam sodium according to claim 2, characterized in that: The volume ratio of ammonia water and formic acid is (2.5-3.5):

1.

4. The method for detecting polymers in cefoperazone sodium and sulbactam sodium according to claim 2, characterized in that: The concentration of the ammonia-formic acid mixed solution is 0.02-0.03 Vol%.

5. The method for detecting polymers in cefoperazone sodium and sulbactam sodium according to any one of claims 1 to 4, characterized in that: The mobile phase adopts gradient elution, and the gradient of the gradient elution is as follows: 0min, mobile phase A 85Vol%, mobile phase B 15Vol%; 2 min, mobile phase A 85 Vol%, mobile phase B 15 Vol%; 3 min, mobile phase A 75 Vol%, mobile phase B 25 Vol%; 21 min, mobile phase A 75 Vol%, mobile phase B 25 Vol%; 40 min, mobile phase A 60 Vol%, mobile phase B 40 Vol%; 50 min, mobile phase A 60 Vol%, mobile phase B 40 Vol%; 55min, mobile phase A 85Vol%, mobile phase B 15Vol%; 60 min, mobile phase A 85 Vol%, mobile phase B 15 Vol%.

6. The method for detecting polymers in cefoperazone sodium and sulbactam sodium according to claim 5, characterized in that: The filler of the chromatographic column is octadecylsilane bonded silica gel.

7. The method for detecting polymers in cefoperazone sodium and sulbactam sodium according to claim 6, characterized in that: The column length of the chromatographic column is 100-150 mm; Preferably, the inner diameter of the chromatographic column is 3.0-4.6 mm; Preferably, the particle size of the chromatographic column is 3.5-5 μm.

8. The method for detecting polymers in cefoperazone sodium and sulbactam sodium according to any one of claims 1 to 4, characterized in that: In the test solution, the concentration of cefoperazone sodium is 1-20 mg / ml.

9. The method for detecting polymers in cefoperazone sodium and sulbactam sodium according to any one of claims 1 to 4, characterized in that: In the reference solution, the concentration of cefoperazone is 0.01-0.05 mg / ml.

10. The method for detecting polymers in cefoperazone sodium and sulbactam sodium according to any one of claims 1 to 4, characterized in that: The mass ratio of cefoperazone sodium to sulbactam sodium in the cefoperazone sodium and sulbactam sodium is 1:1 or 2:1.

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