Method for determining ceftizoxime sodium for injection and impurities of ceftizoxime sodium by liquid chromatography

Abstract

The invention discloses a method for determining ceftizoxime sodium for injection and impurities thereof by liquid chromatography, which comprises the following steps: taking octadecylsilane chemically bonded silica as a filler for the ceftizoxime sodium for injection; the column temperature is 35 DEG C; the flow rate is 0.8 ml / min; the detection wavelength is 254 nm; the sample injection volume is 20 microliters; carrying out gradient elution by using a mobile phase A and a mobile phase B; according to the method, the retention time of the impurity 16 can be stabilized, the impurity 09 and the impurity 12 can be effectively separated, and the method has important significance on quality control of the ceftizoxime sodium for injection.

Description

Technical Field

[0001] The invention relates to the field of drug quality control, and in particular to a method for determining ceftizoxime sodium for injection and its impurities by liquid chromatography. Background Art

[0002] Ceftizoxime sodium is a third-generation cephalosporin developed by Fujisawa Pharmaceutical Co., Ltd. in the 1980s. It is a β-lactam used to treat susceptible bacterial sepsis, lower respiratory tract and urinary tract infections, skin and skin structure infections, bone and joint infections, intra-abdominal infections, and meningitis. This product has obtained a Japanese compound invention patent (patent number: Showa 61-34435) and was approved for marketing in Japan in 1982.

[0003] Cefuroxime Sodium for Injection is the first-line drug for the clinical treatment of moderate to severe infections. It has been used clinically for more than 30 years and has been widely recognized by clinicians. Among similar drugs, it has a low bacterial resistance rate, strong antibacterial activity, a long half-life, and few adverse reactions. Compared with similar third-generation cephalosporins, the incidence of hypoprothrombinemia and disulfiram-like reactions is extremely low. However, related substances and polymers of Cefuroxime Sodium for Injection are the main causes of adverse reactions during clinical use. In order to better control product quality and ensure product safety and effectiveness, it is urgent to analyze and study multiple organic impurities from the API starting materials and synthesis process routes, screen and optimize related substance methods, effectively separate each impurity and accurately detect its impurity amount, and comprehensively improve the quality control level. Summary of the invention

[0004] In view of this, one of the objects of the present invention is to provide a method for analyzing ceftizoxime sodium for injection by high performance liquid chromatography.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] 1. A method for analyzing ceftizoxime sodium for injection by high performance liquid chromatography, comprising the following steps:

[0007] (1) Sample preparation: Dissolve ceftizoxime sodium for injection in pH 7.0 phosphate buffer to prepare a solution containing approximately 1.5 mg of ceftizoxime per 1 mL;

[0008] (2) Detection method:

[0009] Chromatographic column: octadecylsilane bonded silica gel as filler;

[0010] Mobile phase: gradient elution of mobile phase A and mobile phase B;

[0011] Column temperature: 35 °C;

[0012] Flow rate: 0.8ml / min;

[0013] Detection wavelength: 254nm;

[0014] Injection volume: 20 μl;

[0015] The gradient elution is as follows:

[0016] The mobile phase A is a buffer solution with a pH value of 3.4 to 4.0; the mobile phase B is a buffer solution with a pH value of 3.4 to 4.0 and acetonitrile with a volume ratio of 15:85.

[0017] Preferably, the gradient elution is:

[0018] 0-10 min, the volume ratio of mobile phase A:mobile phase B was 97:3;

[0019] 10-20 min, the volume ratio of mobile phase A:mobile phase B is 88:12;

[0020] 20-35 min, the volume ratio of mobile phase A:mobile phase B was 76:24;

[0021] 35-46 min, the volume ratio of mobile phase A:mobile phase B was 0:100;

[0022] 46-55min, the volume ratio of mobile phase A:mobile phase B is 97:3.

[0023] Or the elution gradient is:

[0024] 0-10 min, the volume ratio of mobile phase A:mobile phase B was 97:3;

[0025] 10-20 min, the volume ratio of mobile phase A:mobile phase B is 90:10;

[0026] 20-30 min, the volume ratio of mobile phase A:mobile phase B is 75:25;

[0027] Or the elution gradient is:

[0028] 0-10 min, the volume ratio of mobile phase A:mobile phase B was 97:3;

[0029] 10-20 min, the volume ratio of mobile phase A:mobile phase B is 90:10;

[0030] 20-35 min, the volume ratio of mobile phase A:mobile phase B is 80:20;

[0031] 35-41 min, the volume ratio of mobile phase A:mobile phase B was 15:85;

[0032] 41-50 min, the volume ratio of mobile phase A:mobile phase B is 97:3.

[0033] Preferably, the pH 3.4-4.0 buffer solution is prepared as follows: 1.42 g of citric acid and 2.31 g of disodium hydrogen phosphate are dissolved in water and diluted to 1000 ml, and the pH is adjusted to 3.4-4.0 with phosphoric acid.

[0034] Preferably, the buffer solution has a pH of 3.5.

[0035] The beneficial effects of the present invention are as follows: the present invention provides a method for determining sodium cefuroxime for injection and its impurities by liquid chromatography, wherein sodium cefuroxime for injection is filled with octadecylsilane bonded silica gel; column temperature is 35°C; flow rate is 0.8ml / min; detection wavelength is 254nm; injection volume is 20μl; mobile phase A and mobile phase B are used for gradient elution; the method can stabilize the retention time of impurity 16, and impurity 09 and impurity 12 can be effectively separated, which is of great significance to the quality control of sodium cefuroxime for injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0037] Figure 1 The spectrum of ceftizoxime sodium for injection analyzed by high performance liquid chromatography in Example 1;

[0038] Figure 2 The spectrum of ceftizoxime sodium for injection analyzed by high performance liquid chromatography for comparative example;

[0039] Figure 3 The results are repeatable test results;

[0040] Figure 4 This is the sensitivity test result. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0042] According to the analysis of the synthetic process route and degradation pathway of cefuroxime sodium, several process impurities and degradation impurities that may be produced in cefuroxime sodium are inferred. The original technical method cannot effectively separate impurity 09 and impurity 12. The specific impurities are shown in Table 1.

[0043] Table 1. Impurity analysis of ceftizoxime sodium

[0044]

[0045]

[0046]

[0047]

[0048] Example 1

[0049] The method for analyzing ceftizoxime sodium for injection by high performance liquid chromatography comprises the following specific steps:

[0050] (1) Sample processing:

[0051] Preparation of pH 7.0 phosphate buffer: Take 3.63g of potassium dihydrogen phosphate and 14.33g of disodium hydrogen phosphate, add water to dissolve and dilute to 1000ml.

[0052] The test solution is freshly prepared before use. Take an appropriate amount of the product, dissolve it in pH 7.0 phosphate buffer and quantitatively dilute it to make a solution containing about 1.5 mg of ceftizoxime per 1 mL.

[0053] Reference solution: Take an appropriate amount of ceftizoxime reference substance, weigh accurately, dissolve it in pH 7.0 phosphate buffer and prepare a solution containing approximately 3 μg of ceftizoxime per 1 mL.

[0054] Sensitivity solution: Accurately measure an appropriate amount of the reference solution and dilute it with pH 7.0 phosphate buffer to make a solution containing approximately 0.15 μg of cefuroxime per 1 ml.

[0055] System suitability solution (1): Take about 31.6 mg of this product, accurately weigh it, place it in a 20 ml volumetric flask, add about 10 ml of pH 7.0 phosphate buffer to dissolve it, heat it in a 90°C water bath for 10 minutes, take it out, cool it to room temperature, place it in a light box (5000lx) for 24 hours, take it out, dilute it to the scale with pH 7.0 phosphate buffer, and shake it well.

[0056] System suitability solution (2): Take an appropriate amount of cefuroxime sodium, dissolve it in pH 7.0 phosphate buffer and dilute it to make a solution containing about 0.1 g per 1 ml. After standing for 24 hours, dilute it with pH 7.0 phosphate buffer to make a solution containing about 1.5 mg per 1 ml.

[0057] (2) Detection method:

[0058] Chromatographic column: octadecylsilane bonded silica gel as filler;

[0059] Mobile Phase:

[0060] Mobile phase A: pH 3.5 buffer (1.42 g citric acid and 2.31 g disodium hydrogen phosphate, dissolve in water and dilute to 1000 ml, adjust pH to 3.5 with phosphoric acid);

[0061] Mobile phase B: pH 3.5 buffer: acetonitrile = 15:85. The preparation method of pH 3.5 buffer is as follows: take 1.42 g of citric acid and 2.31 g of disodium hydrogen phosphate, dissolve them in water and dilute to 1000 ml, and adjust the pH to 3.5 with phosphoric acid.

[0062] The gradient elution program is shown in Table 1.

[0063] Table 1. Gradient elution program

[0064]

[0065] Column temperature: 35°C

[0066] Flow rate: 0.8ml / min

[0067] Detection wavelength: 254nm

[0068] Injection volume: 20 μl

[0069] Test results such as Figure 1 The results show that impurity 09 and impurity 12 can be separated.

[0070] In order to analyze the effect of mobile phase composition on the separation effect, mobile phase B was adjusted to a mixed solution of salt solution and acetonitrile in a certain proportion. The specific conditions are as follows:

[0071] Chromatographic column: Octadecylsilane bonded silica gel is used as filler.

[0072] Mobile phase: pH 4.0 buffer (1.42 g of citric acid and 2.31 g of disodium hydrogen phosphate, dissolved in water and diluted to 1000 ml) was used as mobile phase A; acetonitrile was used as mobile phase B, eluted according to the table below, with a flow rate of 0.8 ml / min; the column temperature was 35°C; the detection wavelength was 254 nm.

[0073] The specific gradients are shown in Tables 2 to 4:

[0074] Table 2, Gradient 1

[0075]

[0076] Table 3, Gradient 2

[0077]

[0078] Table 4, Gradient 3

[0079]

[0080] The separation results of different gradients are shown in Table 5.

[0081] Table 5. Separation of different elution gradients

[0082]

[0083]

[0084] The results showed that gradient 3 was used as the mobile phase gradient elution program, and the separation between the component peaks was the best, but under this gradient condition, the chromatographic peak of impurity 16 drifted.

[0085] In order to analyze the effect of different pH on the detection effect, mobile phases of pH 3.4, pH 3.7 and pH 4.0 were used for elution, and the results are shown in Table 6. The results show that impurity 09 and impurity 12 can be effectively separated in the pH range of 3.4 to 3.7.

[0086] Table 6. Separation degree of mobile phase elution at pH 3.4 to 4.0

[0087]

[0088] Comparative Example 1

[0089] Chromatographic column: Octadecylsilane bonded silica gel is used as filler.

[0090] Mobile phase: pH 3.6 buffer (1.42 g of citric acid and 2.31 g of disodium hydrogen phosphate, dissolved in water and diluted to 1000 ml) as mobile phase A; acetonitrile as mobile phase B, eluted according to Table 7, flow rate of 0.8 ml / min; column temperature of 35°C; detection wavelength of 254 nm.

[0091] Table 7. Gradient elution program

[0092] Retention time (minutes) Mobile phase A (%) Mobile phase B (%) 0 97 3 10 85 15 20 40 60 25 40 60 26 97 3 35 97 3

[0093] Test results such as Figure 2 The results show that under this condition, impurity 09 and impurity 12 completely overlap and cannot be separated, and when the pH of the mobile phase is 3.6, the retention time of impurity 16 will drift under this condition.

[0094] The method of Example 1 was repeated 5 times, and the results were as follows. Figure 3 The results show that the impurity 09 and impurity 12 can be separated at different repetition times.

[0095] According to the method of Example 1, the sensitivity solution was tested, and the results were as follows Figure 4The results show that the method of the present invention has good sensitivity and can separate various impurities at low concentrations and detect the peaks.

[0096] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A method for analyzing ceftizoxime sodium for injection by high performance liquid chromatography, characterized in that: The steps include: (1) Sample preparation: Dissolve ceftizoxime sodium for injection in pH 7.0 phosphate buffer to prepare a solution containing approximately 1.5 mg of ceftizoxime per 1 mL; (2) Detection method: Chromatographic column: octadecylsilane bonded silica gel as filler; Mobile phase: gradient elution of mobile phase A and mobile phase B; Column temperature: 35°C; Flow rate: 0.8ml / min; Detection wavelength: 254nm; Injection volume: 20 μl; The gradient elution is as follows: The mobile phase A is a buffer solution with a pH value of 3.4 to 4.0; the mobile phase B is a buffer solution with a pH value of 3.4 to 4.0 and acetonitrile with a volume ratio of 15:

85.

2. The method for analyzing ceftizoxime sodium for injection by high performance liquid chromatography according to claim 1, characterized in that: The gradient elution is: 0-10 min, the volume ratio of mobile phase A:mobile phase B was 97:3; 10-20 min, the volume ratio of mobile phase A:mobile phase B is 88:12; 20-35 min, the volume ratio of mobile phase A:mobile phase B was 76:24; 35-46 min, the volume ratio of mobile phase A:mobile phase B was 0:100; 46-55min, the volume ratio of mobile phase A:mobile phase B is 97:

3. Or the elution gradient is: 0-10 min, the volume ratio of mobile phase A:mobile phase B was 97:3; 10-20 min, the volume ratio of mobile phase A:mobile phase B is 90:10; 20-30 min, the volume ratio of mobile phase A:mobile phase B is 75:25; Or the elution gradient is: 0-10 min, the volume ratio of mobile phase A:mobile phase B was 97:3; 10-20 min, the volume ratio of mobile phase A:mobile phase B is 90:10; 20-35 min, the volume ratio of mobile phase A:mobile phase B is 80:20; 35-41 min, the volume ratio of mobile phase A:mobile phase B was 15:85; 41-50 min, the volume ratio of mobile phase A:mobile phase B is 97:

3.

3. The method for analyzing ceftizoxime sodium for injection by high performance liquid chromatography according to claim 1, characterized in that: The preparation method of the pH 3.4-4.0 buffer solution is as follows: 1.42 g of citric acid and 2.31 g of disodium hydrogen phosphate are dissolved in water and diluted to 1000 ml, and the pH is adjusted to 3.4-4.0 with phosphoric acid.

4. The method for analyzing ceftizoxime sodium for injection by high performance liquid chromatography according to claim 1, characterized in that: The buffer solution is a pH 3.5 buffer.

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