Impurity separation and detection method and application of ceftazidime avibactam sodium for injection

Abstract

The invention provides an impurity separation and detection method of ceftazidime avibactam sodium for injection and application of the ceftazidime avibactam sodium. According to the impurity separation and detection method, high performance liquid chromatography is adopted, a C18 chromatographic column is used as a chromatographic column of a stationary phase, a mixed solution of a phosphoric acid aqueous solution and a phosphate buffer solution-acetonitrile is used as a mobile phase, and the impurities in the ceftazidime avibactam sodium for injection are separated and detected. The impurity separation and detection method disclosed by the invention is good in specificity, high in sensitivity, high in accuracy, good in repeatability, convenient and rapid, and capable of greatly shortening the analysis time, and has extremely important significance for realizing quality control of the ceftazidime avibactam sodium for injection.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to an impurity separation and detection method for ceftazidime avibactam sodium for injection and an application thereof. Background Art

[0002] Ceftazidime avibactam sodium for injection is a mixture of ceftazidime pentahydrate (ceftazidime pentahydrate / sodium carbonate mixture) and avibactam sodium [ceftazidime (C 22 H 22 N 6 O 7 S 2 ) and avibactam (C 7 H 11 N 3 O 6 S) is a sterile powder composed of the ratio of 4:1] indicated on the label.

[0003] Ceftazidime is (6R, 7R)-7-[[(2-amino-4-thiazolyl)-[(1-carboxy-1-methylethoxy)imino]acetyl]amino]-2-carboxy-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-3-methylpyridinium inner salt pentahydrate, and its chemical structure is as follows:

[0004]

[0005] Avibactam sodium is [(2S,5R)-2-carbamoyl-7-oxo-1,6-diazabicyclo[3.2.1]-octane-6-yl] sodium sulfate, and its chemical structure is as follows:

[0006]

[0007] Ceftazidime avibactam sodium for injection is an antibiotic drug used to treat complicated intra-abdominal infections, hospital-acquired pneumonia and ventilator-associated pneumonia, and to treat infections caused by the following Gram-negative bacteria that are sensitive to ceftazidime avibactam sodium in adult patients with limited treatment options: Klebsiella pneumoniae, Enterobacter cloacae, Escherichia coli, Proteus mirabilis and Pseudomonas aeruginosa.

[0008] Some existing technologies have not studied the separation and detection of impurities such as ceftazidime process impurities and degradation impurities (such as ceftazidime impurity G, ceftazidime impurity A, ceftazidime impurity B), and because the impurities related to avibactam sodium are mainly terminal absorption (the maximum absorption wavelength is about 190nm~200nm), the sensitivity of avibactam and its related impurities at the detection wavelength of the chromatographic method is poor, and avibactam related impurities (such as decarbonyl avibactam) cannot be studied and controlled. Some existing technologies have not conducted targeted research on ceftazidime related impurities and avibactam sodium related impurities. Some other existing technologies only conduct single component research on avibactam sodium and ceftazidime main components, or ceftazidime polymer impurities, or ceftazidime related impurities, or avibactam sodium related impurities, and multiple detection methods need to be established to measure the samples.

[0009] The synthesis of ceftazidime avibactam sodium for injection uses ceftazidime, avibactam sodium and sodium carbonate as starting materials. In theory, all starting materials and intermediates may be introduced into the final product as impurities. At present, the pharmacopoeia does not include the detection method of the above-mentioned impurities in ceftazidime avibactam sodium for injection. Therefore, it is urgent to develop a method that is fast, simple and can accurately detect ceftazidime, avibactam sodium and their related impurities, which is of great significance to the quality control of ceftazidime avibactam sodium for injection. Summary of the invention

[0010] Therefore, the purpose of the present invention is to overcome the defects in the prior art and provide an impurity separation and detection method and application of ceftazidime avibactam sodium for injection. Thereby achieving the purpose of further improving the specificity, accuracy and sensitivity of the existing detection method, the impurity separation and detection method of the present invention can simultaneously control multiple impurities in the two components of ceftazidime and avibactam sodium, each impurity has good separation and high sensitivity, the method is simple to operate, and the results are stable and reliable.

[0011] Before describing the present invention, the terms used in this article are defined as follows:

[0012] The term "C18 chromatographic column" refers to a chromatographic column with octadecylsilane bonded silica gel as the stationary phase.

[0013] The term "RP-HPLC" means: High Performance Liquid Chromatography.

[0014] To achieve the above-mentioned object, the first aspect of the present invention provides a method for separating and detecting impurities of ceftazidime avibactam sodium for injection, wherein the impurity separation and detection method adopts high performance liquid chromatography, a chromatographic column with a C18 chromatographic column as a stationary phase, and a mixed solution of an aqueous phosphoric acid solution and a phosphate buffer solution-acetonitrile as a mobile phase, to separate and detect impurities in the ceftazidime avibactam sodium for injection;

[0015] Wherein, the phosphate buffer solution-acetonitrile mixed solution is prepared by mixing a phosphate buffer solution and acetonitrile; preferably, in the phosphate buffer solution-acetonitrile mixed solution:

[0016] The concentration of the phosphate buffer solution is 0.01 mol / L to 0.5 mol / L, preferably 0.02 mol / L to 0.3 mol / L, more preferably 0.02 to 0.22 mol / L;

[0017] The concentration of acetonitrile is 20% to 100%, preferably 50% to 100%, and most preferably 100%; and

[0018] The volume ratio of the phosphate buffer solution to the acetonitrile is 30-60:40-80, preferably 40-50:50-70, more preferably 50:50-60, and most preferably 50:50.

[0019] According to the impurity separation and detection method of the first aspect of the present invention, wherein:

[0020] The pH value of the phosphoric acid aqueous solution is adjusted to 2.0 to 4.0 with sodium hydroxide, preferably 2.5 to 3.5, and most preferably 3.5;

[0021] The pH value of the phosphate buffer solution is 5.0 to 8.0, preferably 6.0 to 7.5, most preferably 6.8; and / or

[0022] The volume ratio of phosphoric acid to water in the phosphoric acid aqueous solution is 0.01:100 to 0.05:100, preferably 0.01:100 to 0.03:100, and most preferably 0.01:100.

[0023] According to the impurity separation and detection method of the first aspect of the present invention, the phosphate buffer solution is selected from one or more of the following: sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, anhydrous disodium hydrogen phosphate, anhydrous potassium dihydrogen phosphate, diammonium hydrogen phosphate, ammonium dihydrogen phosphate, sodium phosphate, preferably selected from one or more of the following: sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and most preferably potassium dihydrogen phosphate.

[0024] According to the impurity separation and detection method of the first aspect of the present invention, the separation and determination method comprises the following steps:

[0025] 1) Prepare the test solution, reference solution, system suitability solution and sensitivity solution of ceftazidime avibactam sodium for injection to be tested respectively;

[0026] 2) setting chromatographic conditions, injecting the test solution, reference solution, system suitability solution and sensitivity solution prepared in step 1) into a liquid chromatograph, recording a chromatogram, and separating and determining impurities in the ceftazidime avibactam sodium for injection.

[0027] According to the impurity separation and detection method of the first aspect of the present invention, wherein in the step 1):

[0028] The preparation method of the test solution comprises: taking ceftazidime avibactam sodium for injection to be tested, dissolving it in water to prepare the test solution;

[0029] The preparation method of the reference substance solution comprises: taking avibactam sodium reference substance and ceftazidime reference substance respectively, dissolving them in water to prepare the reference substance solution;

[0030] The method for preparing the system suitability solution comprises: taking the test solution and preparing the system suitability solution in a water bath; and / or

[0031] The preparation method of the sensitivity solution comprises: taking the reference solution and diluting it with water to prepare the sensitivity solution;

[0032] Preferably, in the test solution: the concentration of ceftazidime is 1 to 6 mg / mL, preferably 3 to 5 mg / mL, and most preferably 4 mg / mL; the concentration of avibactam is 0.5 to 3 mg / mL, preferably 0.5 to 2 mg / mL, and most preferably 1 mg / mL;

[0033] Preferably, in the reference solution: the concentration of ceftazidime is 5 to 20 μg / mL, preferably 6 to 15 μg / mL, most preferably 8 μg / mL, and the concentration of avibactam is 0.5 to 10 μg / mL, preferably 2 to 5 μg / mL, most preferably 2 μg / mL;

[0034] Preferably, in the system suitability solution: the amount of the test solution is 5 mL to 25 mL, more preferably 8 mL to 20 mL, and further preferably 10 mL to 15 mL; the temperature of the water bath is 50 to 80° C., more preferably 55 to 70° C., and most preferably 60° C.; the time of the water bath is 20 to 50 min, more preferably 30 to 40 min, and most preferably 30 min; and / or

[0035] Preferably, in the sensitivity solution: the amount of the reference solution is 1 mL to 10 mL, more preferably 2 mL to 8 mL, and further preferably 3 mL to 5 mL.

[0036] According to the impurity separation and detection method of the first aspect of the present invention, the step 2) further comprises: eluting by gradient elution;

[0037] Preferably, the flow rate of the gradient elution is 0.7 to 1.1 mL / min, more preferably 0.8 to 1.0 mL / min, and most preferably 0.9 mL / min;

[0038] Preferably, in the gradient elution, the initial volume ratio of the phosphoric acid aqueous solution to the mixed solution of the phosphate buffer solution-acetonitrile is 10-98:2-90, more preferably 20-75:25-80, further preferably 30-60:40-70; and / or

[0039] Preferably, the chromatographic conditions include:

[0040] The column temperature is 25°C to 50°C, preferably 30°C to 45°C, and most preferably 40°C;

[0041] The injection volume is 10 to 30 μL, preferably 15 to 25 μL, and most preferably 20 μL;

[0042] The sample pan temperature is 4°C to 30°C, preferably 15°C to 30°C, and most preferably 25°C;

[0043] The detector is an ultraviolet detector;

[0044] The wavelength is 190 nm to 210 nm, preferably 193 nm to 205 nm, and most preferably 195 nm.

[0045] According to the impurity separation and detection method of the first aspect of the present invention, the impurities in the ceftazidime avibactam sodium for injection include one or more of the following: ceftazidime impurity A, ceftazidime impurity B, ceftazidime impurity G, pyridine, decarbonyl avibactam sodium, avibactam and ceftazidime; wherein,

[0046] The chemical structural formula of the ceftazidime impurity A is shown in formula (A):

[0047]

[0048] The chemical structural formula of the ceftazidime impurity B is shown in formula (B):

[0049]

[0050] The chemical structural formula of the ceftazidime impurity G is shown in formula (C):

[0051]

[0052] The chemical structural formula of the pyridine is shown in formula (D):

[0053]

[0054] The chemical structural formula of the decarbonyl avibactam sodium is shown in formula (E):

[0055]

[0056] The chemical structural formula of avibactam is shown in formula (F):

[0057]

[0058] The chemical structural formula of ceftazidime is shown in formula (G):

[0059]

[0060] The second aspect of the present invention provides a method for preparing ceftazidime avibactam sodium for injection, the method comprising: separating and detecting impurities in the ceftazidime avibactam sodium for injection by high performance liquid chromatography.

[0061] The third aspect of the present invention provides a ceftazidime avibactam sodium for injection, wherein the ceftazidime avibactam sodium for injection is:

[0062] The method for impurity separation and detection described in the first aspect is carried out to obtain the product by performing impurity separation and determination; or

[0063] The method is described in the second aspect.

[0064] The fourth aspect of the present invention provides the use of the ceftazidime avibactam sodium for injection described in the third aspect in the preparation of an antibiotic drug for treating infections caused by Gram-negative bacteria;

[0065] Preferably, the Gram-negative bacteria are selected from one or more of the following: Klebsiella pneumoniae, Enterobacter cloacae, Escherichia coli, Proteus mirabilis, and Pseudomonas aeruginosa.

[0066] According to a preferred embodiment of the present invention, the high performance liquid chromatography (RP-HPLC) of the related substances of ceftazidime avibactam sodium for injection of the present invention, the test solution and the reference solution are respectively injected into the liquid chromatograph, and the high performance liquid chromatography is used for determination, and the high performance liquid chromatography uses a C18 chromatographic column, the mobile phase A is a phosphoric acid aqueous solution, and the mobile phase B is a phosphate buffer solution-acetonitrile (50:50). The high performance liquid chromatography is eluted by gradient elution, and the elution gradient conditions are as follows:

[0067] Table 1 Gradient elution of HPLC

[0068] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 98 2 15 75 25 35 10 90 40 10 90 40.1 98 2 50 98 2

[0069] The volume ratio of the phosphoric acid aqueous solution is 0.01% to 0.05%, preferably 0.01% phosphoric acid aqueous solution (v / v).

[0070] The concentration of the phosphate buffer solution is 0.1 mol / L to 0.5 mol / L, and preferably the concentration of the phosphate buffer solution is 0.2 mol / L.

[0071] The phosphate buffer solution is selected from one or more of sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate, and the phosphate buffer solution is preferably potassium dihydrogen phosphate.

[0072] The specifications of the C18 chromatographic column used were 4.6 mm inner diameter, 250 mm length, and 5 μm filler particle size.

[0073] The high performance liquid chromatography method uses an ultraviolet detector for detection, and the detection wavelength is 195 nm.

[0074] The flow rate of the gradient elution is 0.7-1.1 mL / min, preferably 0.9 mL / min.

[0075] The injection volume was 20 μL.

[0076] The temperature of the injector was room temperature.

[0077] The concentration of ceftazidime in the test solution was 4 mg / mL and the concentration of avibactam was 1 mg / mL.

[0078] The concentration of ceftazidime in the reference solution was 8 μg / mL, and the concentration of avibactam was 2 μg / mL.

[0079] The high performance liquid chromatography method specifically comprises the following steps:

[0080] Step S1, taking ceftazidime avibactam sodium for injection, and dissolving it in water to prepare a test solution;

[0081] Step S2, taking appropriate amounts of avibactam sodium reference substance and ceftazidime reference substance respectively, and dissolving them in water to prepare reference substance solutions;

[0082] Step S3, place the test solution in a 60°C water bath for 30 minutes, take it out and cool it to make a system suitability solution;

[0083] Step S4, diluting the reference solution in step S2 with water to prepare a sensitivity solution;

[0084] Step S5: inject the test solution, reference solution, system suitability solution and sensitivity solution prepared in the above steps S1 to S4 into a liquid chromatograph and record the chromatogram.

[0085] Among the related substances of ceftazidime avibactam sodium for injection, the structural formulas of ceftazidime impurity A, ceftazidime impurity B, ceftazidime impurity G, pyridine, decarbonyl avibactam sodium, avibactam and ceftazidime are shown in Table 1.

[0086] Table 2 Structural formula of related substances of ceftazidime avibactam sodium for injection

[0087]

[0088]

[0089] The present invention also provides an application of the above-mentioned high-performance liquid chromatography, that is, a method for separating and determining ceftazidime, avibactam sodium and related substances thereof by using the above-mentioned high-performance liquid chromatography. The ceftazidime impurity A, ceftazidime impurity B, ceftazidime impurity G, pyridine, decarbonyl avibactam sodium, avibactam and ceftazidime have structural formulas as shown in Table 2. The present invention can quickly detect whether the finished product contains impurities, and detect the impurity content by high-performance liquid chromatography analysis to achieve the purpose of drug quality control.

[0090] The present invention relates to the field of medical technology, and discloses a high performance liquid chromatography (RP-HPLC) method for related substances of ceftazidime avibactam sodium for injection. A test solution and a reference solution are respectively injected into a liquid chromatograph, and the high performance liquid chromatography method is used for determination. The high performance liquid chromatography method uses a C18 chromatographic column, and the mobile phase A is a phosphoric acid aqueous solution, and the mobile phase B is a phosphate buffer solution-acetonitrile (50:50). The present invention adopts a determination method that can separate ceftazidime and avibactam sodium from their impurities, so as to realize the qualitative and quantitative determination of impurities in ceftazidime avibactam sodium for injection, and the detection method of the present invention has good specificity, high sensitivity, high accuracy, good repeatability, and is convenient and fast, greatly shortening the analysis time, which is of great significance for realizing the quality control of ceftazidime avibactam sodium for injection.

[0091] The present invention establishes an HPLC method capable of simultaneously controlling multiple impurities in two components of ceftazidime and avibactam sodium, and can overcome the defect that only a single component impurity can be controlled in the prior art.

[0092] Compared with the prior art, the impurity separation and detection method of ceftazidime avibactam sodium for injection of the present invention can have but is not limited to the following beneficial effects:

[0093] 1. The present invention uses high performance liquid chromatography to separate the main components of ceftazidime and avibactam sodium from their impurities, so as to achieve the qualitative and quantitative purpose of impurities in ceftazidime avibactam sodium for injection, thereby achieving the purpose of drug quality control.

[0094] 2. The test solution of the present invention has good stability, the separation degree of the main components of ceftazidime and avibactam sodium and their related impurities is good, the detection method has good specificity, high sensitivity, high accuracy, good repeatability, and is convenient and fast, greatly shortening the analysis time, which is of great significance for achieving quality control of ceftazidime avibactam sodium for injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0096] Figure 1 The chromatograms of the blank solution, the system suitability solution and the reference solution in Example 1 of the present invention are shown.

[0097] Figure 2 The chromatogram of the test solution in Example 1 of the present invention is shown.

[0098] Figure 3 The chromatogram of the test solution in Example 2 of the present invention is shown.

[0099] Figure 4 The chromatogram of the test solution in Example 3 of the present invention is shown.

[0100] Figure 5 The chromatogram of the test solution in Example 4 of the present invention is shown.

[0101] Figure 6 The chromatogram of the spiked test solution in Example 5 of the present invention is shown.

[0102] Figure 7 The chromatograms of the sample destroyed by acid, alkali, oxidation, strong light and high temperature in Example 6 of the present invention are shown. DETAILED DESCRIPTION

[0103] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed and specific description and should not be understood as limiting the present invention in any form.

[0104] This section generally describes the materials and test methods used in the experiments of the present invention. Although many materials and operating methods used to achieve the purpose of the present invention are well known in the art, the present invention is still described as detailed as possible. It is clear to those skilled in the art that in the context, if not specifically stated, the materials and operating methods used in the present invention are well known in the art.

[0105] The reagents and instruments used in the following examples are as follows:

[0106] Instrument: High performance liquid chromatograph (model Waters 2695 / 2996, purchased from Waters Company);

[0107] Reagents: Phosphoric acid (chromatographic grade) and acetonitrile (chromatographic grade) were purchased from Merck; sodium hydroxide (analytical grade), potassium dihydrogen phosphate (analytical grade), and potassium hydroxide (analytical grade) were purchased from Guangzhou Chemical Reagent Factory;

[0108] Chromatographic column, C18 column, Inertsil ODS-3, 4.6 mm×250 mm, 5 μm, purchased from GL Sciences.

[0109] Example 1

[0110] This example is an exemplary description of the impurity separation and detection method of ceftazidime avibactam sodium for injection of the present invention.

[0111] Prepare the test solution: Take 3±0.001g of ceftazidime avibactam sodium for injection to be tested, place it in a 100mL volumetric flask, dissolve it with water and dilute it to the scale, shake well, then accurately measure 5mL, place it in a 25mL volumetric flask, dilute it to the scale with 0.01% phosphoric acid aqueous solution, shake well, and use it as the test solution.

[0112] Preparation of reference solution: Accurately weigh 32 mg of ceftazidime reference substance and 8 mg of avibactam sodium reference substance, and quantitatively dilute with water to prepare a solution containing approximately 2 μg of avibactam and 8 μg of ceftazidime per 1 mL as the reference solution, wherein the concentration of ceftazidime is 8 μg / mL and the concentration of avibactam is 2 μg / mL.

[0113] Preparation of system suitability solution: Take 15 mL of the test solution, place it in a 60°C water bath for 30 minutes, take it out, and cool it to prepare the system suitability solution, in which the concentration of ceftazidime is 4 mg / mL and the concentration of avibactam is 1 mg / mL.

[0114] Preparation of sensitivity solution: Take 5 mL of the reference solution and dilute it with water to prepare a sensitivity solution, wherein the concentration of ceftazidime is 1.6 μg / mL and the concentration of avibactam is 0.4 μg / mL.

[0115] The conditions for high performance liquid chromatography were:

[0116] Chromatographic column: Octadecylsilane bonded silica gel is used as filler;

[0117] Mobile phase: Mobile phase A is phosphoric acid aqueous solution (volume ratio of 0.01:100 (ml / ml), pH value adjusted to 3.5 with 0.05 mol / L sodium hydroxide solution), mobile phase B is a mixture of phosphate buffer solution and acetonitrile (volume ratio of 50:50; 0.02 mol / L potassium dihydrogen phosphate solution, pH value adjusted to 6.8 with 0.05 mol / L potassium hydroxide solution);

[0118] Linear gradient elution was performed according to Table 3 below; the flow rate was 0.9 mL per minute;

[0119] Column temperature: 40°C;

[0120] Detection wavelength: 195nm;

[0121] The injection volume was 20 μL.

[0122] Table 3 Gradient elution program of Example 1

[0123] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 98 2 15 75 25 35 10 90 40 10 90 40.1 98 2 50 98 2

[0124] Take the test solution, reference solution, system suitability solution and sensitivity solution prepared above and inject them into the liquid chromatograph, and record the chromatogram. Calculate the content of each impurity in the test solution, and the determination results are shown in Figure 1 and Figure 2 .like Figure 1 and 2 As shown, pyridine, avibactam and its related impurities, ceftazidime and its related impurities eluted in sequence, and baseline separation was achieved between the chromatographic peaks.

[0125] Example 2

[0126] This example is another exemplary description of the impurity separation and detection method of ceftazidime avibactam sodium for injection of the present invention.

[0127] The flow rate was 1.1 mL / min, and other chromatographic conditions, elution procedure and sample preparation were the same as in Example 1.

[0128] Calculate the content of each impurity in the test solution. The results are shown in Figure 3 .

[0129] like Figure 3 As shown, when the flow rate was changed, the elution order of pyridine, avibactam and its related impurities, ceftazidime and its related impurities remained unchanged, the retention time of each chromatographic peak was shortened by 0.2 minutes, and baseline separation was achieved between each chromatographic peak.

[0130] Example 3

[0131] This example is another exemplary description of the impurity separation and detection method of ceftazidime avibactam sodium for injection of the present invention.

[0132] The column temperature was 35°C, and other chromatographic conditions, elution procedure and sample preparation were the same as in Example 1.

[0133] Calculate the content of each impurity in the test solution. The results are shown in Figure 4 .

[0134] like Figure 4 As shown, when the column temperature was changed, the elution order of pyridine, avibactam and its related impurities, ceftazidime and its related impurities remained unchanged, and the baseline separation was achieved between the chromatographic peaks.

[0135] Example 4

[0136] This example is another exemplary description of the impurity separation and detection method of ceftazidime avibactam sodium for injection of the present invention.

[0137] Mobile phase A was phosphoric acid aqueous solution (volume ratio of 0.05:100 (ml / ml), pH adjusted to 3.5 with 0.05 mol / L sodium hydroxide solution), and other chromatographic conditions, elution procedure and sample preparation were the same as in Example 1.

[0138] Calculate the content of each impurity in the test solution. The results are shown in Figure 5 .

[0139] like Figure 5 As shown, when the concentration of mobile phase A changed, the elution order of pyridine, avibactam and its related impurities, ceftazidime and its related impurities remained unchanged, and baseline separation was achieved between the chromatographic peaks.

[0140] Example 5

[0141] This example is another exemplary description of the impurity separation and detection method of ceftazidime avibactam sodium for injection of the present invention.

[0142] Accurately weigh 12.5 mg of decarbonyl avibactam sodium reference substance, 10 mg of ceftazidime impurity A reference substance, 10 mg of ceftazidime impurity B reference substance and 10 mg of ceftazidime impurity G reference substance, dissolve in water and dilute to the scale to make a solution containing 0.125 mg of decarbonyl avibactam sodium and 0.1 mg each of ceftazidime impurity A, ceftazidime impurity B and ceftazidime impurity G per mL as the impurity reference substance solution.

[0143] Take 3±0.001 g of ceftazidime avibactam sodium for injection to be tested, place it in a 100 mL volumetric flask, dissolve it with water and dilute it to the scale, shake it well, then accurately measure 5 mL, place it in a 25 mL volumetric flask, accurately measure 2 mL of impurity reference solution, dilute it to the scale with 0.01% phosphoric acid aqueous solution, shake it well, and use it as the spiked test solution, wherein the concentration of ceftazidime is 4 mg / mL, the concentration of avibactam is 1 mg / mL, the concentration of decarbonyl avibactam sodium is 10 μg / mL, and the concentration of ceftazidime impurity A is 8 μg / mL.

[0144] Other chromatographic conditions, elution procedures and sample preparation were the same as in Example 1.

[0145] Calculate the content of each impurity in the spiked test solution. The results are shown in Figure 6 .

[0146] like Figure 6 As shown, pyridine, decarbonyl avibactam, avibactam, ceftazidime impurity A, ceftazidime impurity G, ceftazidime, and ceftazidime impurity B are eluted in sequence, and baseline separation can be achieved between each chromatographic peak.

[0147] The determination results of Examples 1 to 5 are shown in Table 4. Each impurity can be accurately located and detected. The determination method of the present invention can better improve and control the quality of ceftazidime avibactam sodium for injection.

[0148] Table 4 Measurement results of Examples 1 to 5

[0149] Impurity Name Example 1 Example 2 Example 3 Example 4 Example 5 Pyridine 0.09％ 0.09％ 0.09％ 0.11％ 0.09％ Descarbonyl avibactam Not detected Not detected Not detected Not detected 0.97％ Ceftazidime Impurity A 0.20％ 0.18％ 0.19％ 0.19％ 0.40％ Ceftazidime Impurity B 0.02％ 0.01％ 0.01％ Not detected 0.23％ Ceftazidime Impurity G 0.05％ 0.05％ 0.05％ 0.06％ 0.25％ Other individual impurities of Avibactam Not detected Not detected Not detected Not detected Not detected Other single impurities of ceftazidime 0.07％ 0.07％ 0.06％ 0.06％ 0.08％ Total impurities 0.50％ 0.48％ 0.45％ 0.53％ 2.12％

[0150] Example 6

[0151] This example is yet another exemplary description of the impurity separation and detection method of ceftazidime avibactam sodium for injection of the present invention.

[0152] Mobile phase B is a mixture of phosphate buffer solution and acetonitrile (volume ratio of 50:50; 0.2 mol / L potassium dihydrogen phosphate solution or 0.15 mol / L potassium dihydrogen phosphate solution or 0.18 mol / L potassium dihydrogen phosphate solution or 0.22 mol / L potassium dihydrogen phosphate solution, pH adjusted to 6.8 with 0.05 mol / L potassium hydroxide solution; acetonitrile), and other chromatographic conditions, elution procedure and system suitability solution are the same as in Example 1.

[0153] As shown in the table below, when the concentration of phosphate buffer in mobile phase B changes, the peak elution order of specific impurities and main components in system suitability is consistent, and the retention time of the chromatographic peaks changes slightly, but baseline separation can be achieved between the chromatographic peaks.

[0154] Table 5 Effect of changes in phosphate buffer concentration in mobile phase B

[0155]

[0156] Example 7

[0157] This example is an illustrative description of the impurity separation and detection method of ceftazidime avibactam sodium for injection after being destroyed by acid, alkali, oxidation, strong light and high temperature.

[0158] Acid destruction: Take about 3±0.001g of the ceftazidime avibactam sodium for injection to be tested, place it in a 100mL volumetric flask, dissolve it with water and dilute it to the scale, shake it well, then accurately measure 5mL, place it in a 25mL volumetric flask, add 2mL of 0.01mol / L HCI, let it stand for 2 hours, then add 2mL of 0.01mol / L NaOH to neutralize it, let it cool, dilute it to the scale with 0.01% phosphoric acid aqueous solution, and shake it well.

[0159] Alkali destruction: Take 3±0.001g of ceftazidime avibactam sodium for injection to be tested, place it in a 100mL volumetric flask, dissolve it in water and dilute it to the scale, shake it well, then accurately measure 5mL, place it in a 25mL volumetric flask, add 2mL of 0.01mol / L NaOH, let it stand for 60 minutes, then add 2mL of 0.01mol / L HCI to neutralize it, let it cool, dilute it to the scale with 0.01% phosphoric acid aqueous solution, and shake it well.

[0160] Oxidative damage: Take 3 ± 0.001 g of ceftazidime avibactam sodium for injection to be tested, place it in a 100 mL volumetric flask, dissolve it in water and dilute it to the mark, shake it well, then accurately measure 5 mL, place it in a 25 mL volumetric flask, and add 2 mL of 0.05% H 2 O 2 , let stand for 30 minutes, dilute to scale with 0.01% phosphoric acid aqueous solution, and shake well.

[0161] Strong light destruction: Take 3±0.001g of the ceftazidime avibactam sodium for injection to be tested, place it in a 100mL volumetric flask, dissolve it with water and dilute it to the scale, shake it well, then accurately measure 5mL, place it in a 25mL volumetric flask, place it under 4500lx±500lx for 24 hours, dilute it to the scale with 0.01% phosphoric acid aqueous solution, and shake it well.

[0162] High temperature destruction: Take 3±0.001g of ceftazidime avibactam sodium for injection to be tested, place it in a 100mL volumetric flask, dissolve and dilute to the scale with water, shake well, then accurately measure 5mL, place it in a 25mL volumetric flask, place it in a 60℃ water bath, leave it for 30 minutes, take it out, cool it, dilute it to the scale with 0.01% phosphoric acid aqueous solution, and shake well.

[0163] The test solutions prepared by the above acid, alkali, oxidation, strong light and high temperature destruction were all measured according to the chromatographic conditions of Example 1. The results are shown in Figure 7 .

[0164] like Figure 7 It is shown that ceftazidime avibactam sodium for injection is unstable under the above conditions, and corresponding impurities are generated respectively. However, under the determination method provided by the present invention, these impurities can be well separated by baseline.

[0165] Although the effects of some embodiments are shown above, those skilled in the art should understand that according to the concept of the present invention, the aforementioned other embodiments without specific effects or other technical solutions of the present invention not shown in the embodiments can also achieve the following technical effects stated in the invention content section that are equivalent to the embodiments:

[0166] 1. The present invention uses high performance liquid chromatography to separate the main components of ceftazidime and avibactam sodium from their impurities, so as to achieve the qualitative and quantitative purpose of impurities in ceftazidime avibactam sodium for injection, thereby achieving the purpose of drug quality control.

[0167] 2. The test solution of the present invention has good stability, the separation degree of the main components of ceftazidime and avibactam sodium and their related impurities is good, the detection method has good specificity, high sensitivity, high accuracy, good repeatability, and is convenient and fast, greatly shortening the analysis time, which is of great significance for achieving quality control of ceftazidime avibactam sodium for injection.

[0168] Although the present invention has been described to a certain extent, it is obvious that appropriate changes in various conditions can be made without departing from the spirit and scope of the present invention. It is understood that the present invention is not limited to the embodiments described, but belongs to the scope of the claims, which includes equivalent replacements of each factor described.

Claims

1. A method for separating and detecting impurities of ceftazidime avibactam sodium for injection, characterized in that: The impurity separation and detection method adopts high performance liquid chromatography, with a C18 chromatographic column as a chromatographic column with a stationary phase, and a mixed solution of an aqueous phosphoric acid solution and a phosphate buffer solution-acetonitrile as a mobile phase, to separate and detect impurities in the ceftazidime avibactam sodium for injection; Wherein, the phosphate buffer solution-acetonitrile mixed solution is prepared by mixing a phosphate buffer solution and acetonitrile; preferably, in the phosphate buffer solution-acetonitrile mixed solution: The concentration of the phosphate buffer solution is 0.01 mol / L to 0.5 mol / L, preferably 0.02 mol / L to 0.3 mol / L, more preferably 0.02 to 0.22 mol / L; The concentration of acetonitrile is 20% to 100%, preferably 50% to 100%, and most preferably 100%; and The volume ratio of the phosphate buffer solution to the acetonitrile is 30-60:40-80, preferably 40-50:50-70, more preferably 50:50-60, and most preferably 50:

50.

2. The impurity separation and detection method according to claim 1, characterized in that: The pH value of the phosphoric acid aqueous solution is adjusted to 2.0 to 4.0 with sodium hydroxide, preferably 2.5 to 3.5, and most preferably 3.5; The pH value of the phosphate buffer solution is 5.0 to 8.0, preferably 6.0 to 7.5, most preferably 6.8; and / or The volume ratio of phosphoric acid to water in the phosphoric acid aqueous solution is 0.01:100 to 0.05:100, preferably 0.01:100 to 0.03:100, and most preferably 0.01:

100.

3. The impurity separation and detection method according to claim 1 or 2, characterized in that: The phosphate buffer solution is selected from one or more of the following: sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, anhydrous disodium hydrogen phosphate, anhydrous potassium dihydrogen phosphate, diammonium hydrogen phosphate, ammonium dihydrogen phosphate, sodium phosphate, preferably selected from one or more of the following: sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and most preferably potassium dihydrogen phosphate.

4. The impurity separation and detection method according to any one of claims 1 to 3, characterized in that: The separation assay method comprises the following steps: 1) Prepare the test solution, reference solution, system suitability solution and sensitivity solution of ceftazidime avibactam sodium for injection to be tested respectively; 2) setting chromatographic conditions, injecting the test solution, reference solution, system suitability solution and sensitivity solution prepared in step 1) into a liquid chromatograph, recording a chromatogram, and separating and determining impurities in the ceftazidime avibactam sodium for injection.

5. The impurity separation and detection method according to claim 4, characterized in that: In the step 1): The preparation method of the test solution comprises: taking ceftazidime avibactam sodium for injection to be tested, dissolving it in water to prepare the test solution; The preparation method of the reference substance solution comprises: taking avibactam sodium reference substance and ceftazidime reference substance respectively, dissolving them in water to prepare the reference substance solution; The method for preparing the system suitability solution comprises: taking the test solution and preparing the system suitability solution in a water bath; and / or The preparation method of the sensitivity solution comprises: taking the reference solution and diluting it with water to prepare the sensitivity solution; Preferably, in the test solution: the concentration of ceftazidime is 1 to 6 mg / mL, preferably 3 to 5 mg / mL, and most preferably 4 mg / mL; the concentration of avibactam is 0.5 to 3 mg / mL, preferably 0.5 to 2 mg / mL, and most preferably 1 mg / mL; Preferably, in the reference solution: the concentration of ceftazidime is 5 to 20 μg / mL, preferably 6 to 15 μg / mL, most preferably 8 μg / mL, and the concentration of avibactam is 0.5 to 10 μg / mL, preferably 2 to 5 μg / mL, most preferably 2 μg / mL; Preferably, in the system suitability solution: the amount of the test solution is 5 mL to 25 mL, more preferably 8 mL to 20 mL, and further preferably 10 mL to 15 mL; the temperature of the water bath is 50 to 80° C., more preferably 55 to 70° C., and most preferably 60° C.; the time of the water bath is 20 to 50 min, more preferably 30 to 40 min, and most preferably 30 min; and / or Preferably, in the sensitivity solution: the amount of the reference solution is 1 mL to 10 mL, more preferably 2 mL to 8 mL, and further preferably 3 mL to 5 mL.

6. The impurity separation and detection method according to claim 4 or 5, characterized in that: The step 2) further comprises: eluting by gradient elution; Preferably, the flow rate of the gradient elution is 0.7 to 1.1 mL / min, more preferably 0.8 to 1.0 mL / min, and most preferably 0.9 mL / min; Preferably, in the gradient elution, the initial volume ratio of the phosphoric acid aqueous solution to the mixed solution of the phosphate buffer solution-acetonitrile is 10-98:2-90, more preferably 20-75:25-80, further preferably 30-60:40-70; and / or Preferably, the chromatographic conditions include: The column temperature is 25°C to 50°C, preferably 30°C to 45°C, and most preferably 40°C; The injection volume is 10 to 30 μL, preferably 15 to 25 μL, and most preferably 20 μL; The sample pan temperature is 4°C to 30°C, preferably 15°C to 30°C, and most preferably 25°C; The detector is an ultraviolet detector; The wavelength is 190 nm to 210 nm, preferably 193 nm to 205 nm, and most preferably 195 nm.

7. The impurity separation and detection method according to any one of claims 1 to 6, characterized in that: The impurities in the ceftazidime avibactam sodium for injection include one or more of the following: ceftazidime impurity A, ceftazidime impurity B, ceftazidime impurity G, pyridine, decarbonyl avibactam sodium, avibactam and ceftazidime; wherein, The chemical structural formula of the ceftazidime impurity A is shown in formula (A): The chemical structural formula of the ceftazidime impurity B is shown in formula (B): The chemical structural formula of the ceftazidime impurity G is shown in formula (C): The chemical structural formula of the pyridine is shown in formula (D): The chemical structural formula of the decarbonyl avibactam sodium is shown in formula (E): The chemical structural formula of avibactam is shown in formula (F): The chemical structural formula of ceftazidime is shown in formula (G):

8. A method for preparing ceftazidime avibactam sodium for injection, characterized in that: The preparation method comprises: separating and detecting impurities in the ceftazidime avibactam sodium for injection by high performance liquid chromatography.

9. A ceftazidime avibactam sodium for injection, characterized in that: The ceftazidime avibactam sodium for injection adopts: The method for impurity separation and detection according to any one of claims 1 to 7 is carried out to obtain the product after the impurity separation and determination is performed; or Prepared by the preparation method according to claim 8.

10. Use of the ceftazidime avibactam sodium for injection according to claim 9 in the preparation of an antibiotic drug for treating infections caused by Gram-negative bacteria; Preferably, the Gram-negative bacteria are selected from one or more of the following: Klebsiella pneumoniae, Enterobacter cloacae, Escherichia coli, Proteus mirabilis, and Pseudomonas aeruginosa.

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