Polymer analysis method of cefazolin sodium for injection

Abstract

The invention discloses a polymer analysis method of cefazolin sodium for injection, relates to the field of medicine detection, and aims to provide a polymer analysis method of cefazolin sodium for injection, which is high in sensitivity and good in stability. Chromatographic conditions are as follows: an MG series C18 chromatographic column is adopted as a chromatographic column; a mobile phase A is 10 mmol / L-50 mmol / L acetate aqueous solution, a mobile phase B is methanol, and gradient elution is carried out; the column temperature is 25-40 DEG C; the flow velocity is 0.5 mL / min to 1.5 mL / min; the sample size is 20 microliters; the detection wavelength is 254 nm; the sample concentration is 1mg / mL to 5mg / mL. The method has the advantages of high detection sensitivity, good reproducibility, high stability and the like, and is suitable for detecting the cefazolin sodium for injection and a polymer of the cefazolin sodium serving as a raw material medicine of the cefazolin sodium.

Description

Technical Field

[0001] The invention relates to the field of medical detection, and in particular to a polymer analysis method for cefazolin sodium for injection. Background Art

[0002] Cefazolin is a first-generation semi-synthetic cephalosporin with the common characteristics of β-lactam antibiotics, that is, it is easy to self-polymerize and generate polymer impurities, thereby inducing allergic reactions. Therefore, in order to further ensure the quality and safety of cefazolin sodium, it is of great significance to strictly control the polymer content in the raw materials and preparations of this product.

[0003] The 2020 edition of the "Chinese Pharmacopoeia" still uses molecular exclusion chromatography-dextran gel G-10 column to control the polymers of this product. The current editions of the pharmacopoeias of other countries do not conduct polymer quality control on cefazolin sodium raw materials and preparations. The detection methods that can be found in most literature are the TSK chromatography system to analyze cephalosporin polymer impurities.

[0004] Considering that the G-10 gel system cannot effectively separate and control polymer impurities exclusively, it can only control the polymer in total. The TSK chromatography system also has obvious disadvantages when analyzing polymer impurities, that is, the polymer chromatographic peak is easily interfered by the co-emission peak of other small molecular impurities with greater polarity in the drug, thereby affecting the quantitative accuracy of polymer impurities. However, there are literature reports that cefazolin sodium may contain a polymer with a molecular weight of 794. The commercial impurity manufacturers and impurity preparation manufacturers cannot obtain the polymer impurity reference substance. Therefore, developing a polymer analysis method for cefazolin sodium for injection to study the type and quantity of polymers in cefazolin sodium and the control limit is of great significance to the clinical application and safety of this variety. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a polymer analysis method for cefazolin sodium for injection.

[0006] The present invention provides a polymer analysis method for cefazolin sodium for injection, wherein the polymer analysis method for cefazolin sodium for injection adopts reverse phase C18 chromatography to analyze polymer impurities in cefazolin sodium for injection and its raw material cefazolin sodium, as follows:

[0007] Step 1: Preparation of test solution

[0008] Take Cefazolin Sodium for Injection or its raw material Cefazolin Sodium, weigh accurately, dissolve in water and quantitatively dilute to make a solution containing about 1-5 mg of Cefazolin per 1 mL, and shake well;

[0009] Step 2: Preparation of control solution

[0010] Take the test solution prepared in step 1, place it in a volumetric flask, quantitatively dilute it to the scale with mobile phase A, shake well, and obtain a control solution with a concentration of 0.001-0.005 mg / mL;

[0011] Step 3: HPLC detection

[0012] Chromatographic conditions: MG series C18 chromatographic column, octadecylsilane bonded silica gel as filler; 10mmol / L~50mmol / L acetate aqueous solution as mobile phase A, methanol as mobile phase B; flow rate 0.5mL / min~1.5mL / min, gradient elution; column temperature 25℃~40℃; detection wavelength 254nm; injection volume 20μl; sample concentration 1mg / mL~5mg / mL;

[0013] The gradient elution program was as follows: 0→10 min, the amount of mobile phase A was 95%, and the amount of mobile phase B was 5%; 10→20 min, the amount of mobile phase A was 90%, and the amount of mobile phase B was 10%; 20→35 min, the amount of mobile phase A was 85%, and the amount of mobile phase B was 15%; 35→45 min, the amount of mobile phase A was 78%, and the amount of mobile phase B was 22%; 45→50 min, the amount of mobile phase A was 60%, and the amount of mobile phase B was 40%; 50→55 min, the amount of mobile phase A was 95%, and the amount of mobile phase B was 5%; 55→65 min, the amount of mobile phase A was 95%, and the amount of mobile phase B was 5%.

[0014] The method of the present invention utilizes a column switching 2D-LC-MS method to further identify polymer impurities through high-resolution mass spectrum information and fragment information, and a total of 2 polymer impurities are identified.

[0015] Furthermore, the MG series C18 chromatographic column is CAPCELL PAK C18 MG, CAPCELL PAK C18 MGⅡ, CAPCELL PAK C18 MGⅢ or a chromatographic column with equivalent performance.

[0016] Furthermore, the chromatographic column model is CAPCELL PAK C18 MGⅡ, the specification is 250mm×4.6mm, 5μm, the mobile phase A is 20mmol / L ammonium acetate solution, the mobile phase B is methanol, and the gradient elution is performed; the column temperature is 35°C; the flow rate is 1.1mL / min; the injection volume is 20μl; the detection wavelength is 254nm; the sample concentration is 2mg / mL; the gradient elution program is: 0→10min, the amount of mobile phase A is 95%, the amount of mobile phase B is 5%; 10→20min, the amount of mobile phase A is 90%, the amount of mobile phase B is 5%; The amount of mobile phase B is 10%; 20→35min, the amount of mobile phase A is 85%, and the amount of mobile phase B is 15%; 35→45min, the amount of mobile phase A is 78%, and the amount of mobile phase B is 22%; 45→50min, the amount of mobile phase A is 60%, and the amount of mobile phase B is 40%; 50→55min, the amount of mobile phase A is 95%, and the amount of mobile phase B is 5%; 55→65min, the amount of mobile phase A is 95%, and the amount of mobile phase B is 5%.

[0017] Further, the preparation of HPLC test system suitability solution:

[0018] Take cefazolin sodium for injection, weigh it accurately, put it in a volumetric flask, add mobile phase A to dissolve it, add hydrochloric acid solution, shake until the test sample precipitates, seal the bottle and place it at 60℃ for 1 to 2 hours, take it out, put it at room temperature, add sodium hydroxide solution, dilute it to the scale with water, and shake well.

[0019] Furthermore, the mass volume ratio of the cefazolin sodium for injection to the mobile phase A is 6-8 mg:1 mL.

[0020] Furthermore, the mass volume ratio of the cefazolin sodium for injection to the hydrochloric acid solution is 20-23 mg:1 mL; wherein the molar concentration of the hydrochloric acid solution is 1 mol / L.

[0021] Furthermore, the mass volume ratio of the cefazolin sodium for injection to the sodium hydroxide solution is 20-23 mg:1 mL; wherein the molar concentration of the sodium hydroxide solution is 1 mol / L.

[0022] Furthermore, the polymers of the injection of cefazolin sodium are polymer 1 and polymer 2, the molecular formulas of which are respectively C 25 H 27 N 14 O9S4,C 28 H 32 N 16 O 10 S6.

[0023] The structural formula, molecular formula and molecular weight information of polymer 1 and polymer 2 are shown in the following table:

[0024]

[0025] Furthermore, after the system suitability solution is injected into a liquid chromatograph, polymer 1 is located at a relative retention time of about 1.06 to 1.11 in the chromatogram, and polymer 2 is located at a relative retention time of about 1.37 to 1.43.

[0026] Furthermore, a system sensitivity solution is used for sensitivity detection in HPLC detection, and the system sensitivity solution is prepared as follows:

[0027] Take the control solution, place it in a volumetric flask, quantitatively dilute it to the scale with mobile phase A, shake well, and obtain a sensitivity solution with a concentration of 0.00025-0.00125 mg / mL.

[0028] After the sensitivity solution was injected into the liquid chromatograph, the signal-to-noise ratio of the cefazolin peak in the chromatogram was greater than 10.

[0029] The present invention has the following beneficial effects:

[0030] In order to meet the requirements of separation and detection methods for polymer impurities of injection cefazolin sodium and its raw material cefazolin sodium, the present invention develops and establishes a reverse phase C18 chromatography method, investigates and optimizes degradation conditions, conducts comparative analysis on potential polymer impurities, adopts a column switching 2D-LC-MS method, further identifies polymer impurities through high-resolution mass spectrometry information and fragment information, and identifies a total of 2 polymer impurities. The present invention has the advantages of high detection sensitivity, strong specificity, good reproducibility, high stability, etc., and is suitable for polymer inspection of injection cefazolin sodium and its raw material cefazolin sodium. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a typical spectrum of system suitability;

[0032] Figure 2 This is the detection spectrum at a flow rate of 1.0 mL / min;

[0033] Figure 3 This is the detection spectrum at a flow rate of 1.2 mL / min;

[0034] Figure 4 This is the detection spectrum at a column temperature of 33°C;

[0035] Figure 5 This is the detection spectrum at a column temperature of 37°C;

[0036] Figure 6 This is the detection spectrum of the Agilent instrument;

[0037] Figure 7 The detection spectra of different chromatographic columns. DETAILED DESCRIPTION

[0038] In order to make the objectives, technical solutions and advantages of the embodiments of the present invention more clearly understood, the spirit of the contents disclosed by the present invention will be described in detail below. After understanding the embodiments of the contents of the present invention, any technician in the relevant technical field can change and modify them according to the techniques taught by the contents of the present invention without departing from the spirit and scope of the contents of the present invention.

[0039] The exemplary embodiments of the present invention and the description thereof are used to explain the present invention, but are not intended to limit the present invention.

[0040] Example 1

[0041] A polymer analysis method of cefazolin sodium for injection in this embodiment is as follows:

[0042] 1) Preparation of system suitability solution

[0043] Take about 21 mg of this product (equivalent to about 20 mg of cefazolin), accurately weigh it, put it in a 10 mL volumetric flask, add 3 mL of mobile phase A to dissolve it, add 0.8 mL of 1 mol / L hydrochloric acid solution, shake for 60 seconds until the test product precipitates, seal the bottle cap and place it at 60 ° C for 1.5 hours, take it out, put it at room temperature, add 0.8 mL of 1 mol / L sodium hydroxide solution to dissolve it, dilute it to the scale with water, shake well, and prepare it fresh before use.

[0044] 2) Preparation of test solution

[0045] Take an appropriate amount of cefazolin sodium for injection or its raw material cefazolin sodium, weigh accurately, add water to dissolve and quantitatively dilute to make a solution containing about 2 mg of cefazolin per 1 mL, and shake well.

[0046] 3) Preparation of control solution

[0047] Take an appropriate amount of cefazolin reference substance, weigh it accurately, add an appropriate amount of mobile phase A to dissolve it, and dilute it with water to make a solution containing about 2 μg of cefazolin per 1 mL, and shake well.

[0048] 4) Preparation of sensitivity solution

[0049] Take 2.5 mL of the above control solution, place it in a 10 mL volumetric flask, quantitatively dilute it to the scale with mobile phase A, and shake well.

[0050] Example 2

[0051] This example is the optimization and determination of the chromatographic conditions used in the analysis method of Cefazolin Sodium for Injection polymer

[0052] (1) Determination of detection wavelength

[0053] The maximum absorption wavelengths of the main component and each impurity are close to 254nm, and the signal-to-noise ratio of the sensitivity solution at this concentration is greater than 10, which meets the requirements.

[0054] (2) Optimal chromatographic conditions

[0055] Chromatographic column: CAPCELL PAK C18 MGⅡ, specification: 4.6mm×250mm, 5μm; mobile phase A is 20mmol / L ammonium acetate solution, mobile phase B is methanol, gradient elution; column temperature is 35℃; flow rate is 1.1mL / min; injection volume is 20μl; detection wavelength is 254nm; sample concentration is 2mg / mL. The elution program was: 0→10 min, mobile phase A~mobile phase B (95:5); 10→20 min, mobile phase A~mobile phase B (90:10); 20→35 min, mobile phase A~mobile phase B (85:15); 35→45 min, mobile phase A~mobile phase B (78:22); 45→50 min, mobile phase A~mobile phase B (60:40); 50→55 min, mobile phase A~mobile phase B (95:5); 55→65 min, mobile phase A~mobile phase B (95:5).

[0056] pass Figures 1 to 7 The above-mentioned optimal detection wavelength and optimal chromatographic conditions can be determined.

[0057] (3) Methodological validation

[0058] The above-mentioned optimal chromatographic conditions were used to verify the test product of Example 1.

[0059] 1) Detection limit and quantification limit Take 50 mg of cefazolin sodium for injection, place it in a 25 mL volumetric flask, and quantitatively dilute it with mobile phase A until the signal-to-noise ratio of the sample solution is greater than 10, which is the quantification limit and the result is 0.1015 μg / mL. The signal-to-noise ratio is greater than 3, which is the detection limit and the result is 0.0304 μg / mL.

[0060] 2) Linearity: Take 5 mg of cefazolin sodium for injection, put it in a 25 mL volumetric flask, and dilute it to the scale with mobile phase A. Accurately measure 3.0 mL, 2.0 mL, 1.0 mL, 0.5 mL, 0.2 mL, and 0.1 mL, and put them in 100 mL volumetric flasks, respectively, and dilute them to the scale with mobile phase. As the linear solution, the chromatographic conditions described in the present invention are adopted for determination, and linear regression is performed with concentration C as the abscissa and peak area A as the ordinate. The regression equation is A=26394.339C-65.7265, and the correlation coefficient r=1.0000.

[0061] 3) Precision The precision was measured on different dates, by different analysts, using different equipment under the above test conditions. Six measurements were performed and the relative standard deviations of the single and total impurities of the test solution were calculated 12 times. The results are shown in Table 1. The precision was less than 5.0%.

[0062] 4) Separation Degree According to the system suitability test requirements, the system suitability solution is measured under the chromatographic conditions of the present invention, and the separation degree between the main peak and the peaks before and after it is greater than 1.5.

[0063] 5) Room temperature solution stability Under the chromatographic conditions of the present invention, a test solution of cefazolin sodium for injection was prepared with mobile phase A, placed at room temperature, and sampled and checked at 0, 2, 4, 8, 12, and 24 hours. The results are shown in Table 2. It can be seen from Table 2 that after the cefazolin sodium solution for injection was placed at room temperature for 24 hours, the RSD of polymer 1 was 2.02%, indicating that the cefazolin sodium for injection was stable when placed at room temperature for 24 hours.

[0064] 6) 4°C Solution Stability Under the chromatographic conditions of the present invention, a test solution of cefazolin sodium for injection was prepared with mobile phase A, placed at 4°C, and sampled and checked at 0, 2, 4, 8, 12, and 24 hours. The results are shown in Table 3. It can be seen from Table 3 that after the cefazolin sodium for injection was placed at 4°C for 24 hours, the RSD of polymer 1 was 1.45%, indicating that the cefazolin sodium for injection was stable when placed at 4°C for 24 hours.

[0065] 7) Durability The test solution of cefazolin sodium for injection was taken, and the durability of the chromatographic conditions described in the present invention was investigated by making small changes in the column temperature, flow rate, different instruments and chromatographic column brands. The results showed that slight changes in the above chromatographic conditions did not affect the polymer detection of cefazolin sodium for injection. The results are shown in Tables 4 to 6.

[0066] Table 1 Precision results

[0067]

[0068] Table 2 Room temperature solution stability results

[0069]

[0070] Table 3 4℃ solution stability results

[0071]

[0072]

[0073] Table 4 Results of the suitability investigation of the durability test system

[0074]

[0075] Table 5 Durability test results of the test solution

[0076]

[0077] Table 6 Durability test results of reference solution

[0078]

Claims

1. A polymer analysis method for cefazolin sodium for injection, characterized in that: The polymer analysis method of Cefazolin Sodium for Injection is to analyze the polymer impurities in Cefazolin Sodium for Injection and its raw material Cefazolin Sodium by reverse phase C18 chromatography, as follows: Step 1: Preparation of test solution Take Cefazolin Sodium for Injection or its raw material Cefazolin Sodium, weigh accurately, dissolve in water and quantitatively dilute to make a solution containing about 1-5 mg of Cefazolin per 1 mL, and shake well; Step 2: Preparation of control solution Take the test solution prepared in step 1, place it in a volumetric flask, quantitatively dilute it to the scale with mobile phase A, shake well, and obtain a control solution with a concentration of 0.001-0.005 mg / mL; Step 3: HPLC detection Chromatographic conditions: MG series C18 chromatographic column, octadecylsilane bonded silica gel as filler; 10mmol / L~50mmol / L acetate aqueous solution as mobile phase A, methanol as mobile phase B; flow rate 0.5mL / min~1.5mL / min, gradient elution; column temperature 25℃~40℃; detection wavelength 254nm; injection volume 20μl; sample concentration 1mg / mL~5mg / mL; The gradient elution program was as follows: 0→10 min, the amount of mobile phase A was 95%, and the amount of mobile phase B was 5%; 10→20 min, the amount of mobile phase A was 90%, and the amount of mobile phase B was 10%; 20→35 min, the amount of mobile phase A was 85%, and the amount of mobile phase B was 15%; 35→45 min, the amount of mobile phase A was 78%, and the amount of mobile phase B was 22%; 45→50 min, the amount of mobile phase A was 60%, and the amount of mobile phase B was 40%; 50→55 min, the amount of mobile phase A was 95%, and the amount of mobile phase B was 5%; 55→65 min, the amount of mobile phase A was 95%, and the amount of mobile phase B was 5%.

2. The polymer analysis method of cefazolin sodium for injection according to claim 1, characterized in that The MG series C18 chromatographic column is CAPCELL PAK C18 MG, CAPCELL PAK C18 MGⅡ, CAPCELL PAK C18 MGⅢ or a chromatographic column with equivalent performance.

3. A polymer analysis method for cefazolin sodium for injection according to claim 1 or 2, characterized in that The chromatographic column model is CAPCELL PAK C18 MGⅡ, the specification is 250mm×4.6mm, 5μm, the mobile phase A is 20mmol / L ammonium acetate solution, the mobile phase B is methanol, and the gradient elution is performed; the column temperature is 35°C; the flow rate is 1.1mL / min; the injection volume is 20μl; the detection wavelength is 254nm; the sample concentration is 2mg / mL; the gradient elution program is: 0→10min, the amount of mobile phase A is 95%, the amount of mobile phase B is 5%; 10→20min, the amount of mobile phase A is 90%, the amount of mobile phase B is 5%; The amount of mobile phase B is 10%; 20→35min, the amount of mobile phase A is 85%, and the amount of mobile phase B is 15%; 35→45min, the amount of mobile phase A is 78%, and the amount of mobile phase B is 22%; 45→50min, the amount of mobile phase A is 60%, and the amount of mobile phase B is 40%; 50→55min, the amount of mobile phase A is 95%, and the amount of mobile phase B is 5%; 55→65min, the amount of mobile phase A is 95%, and the amount of mobile phase B is 5%.

4. The polymer analysis method of cefazolin sodium for injection according to claim 1, characterized in that Preparation of HPLC test system suitability solution: Take cefazolin sodium for injection, weigh it accurately, put it in a volumetric flask, add mobile phase A to dissolve it, add hydrochloric acid solution, shake until the test sample precipitates, seal the bottle and place it at 60℃ for 1 to 2 hours, take it out, put it at room temperature, add sodium hydroxide solution, dilute it to the scale with water, and shake well.

5. The polymer analysis method of cefazolin sodium for injection according to claim 4, characterized in that The mass volume ratio of the cefazolin sodium for injection to the mobile phase A is 6-8 mg:1 mL.

6. The polymer analysis method of cefazolin sodium for injection according to claim 4, characterized in that The mass volume ratio of the cefazolin sodium for injection to the hydrochloric acid solution is 20-23 mg:1 mL; wherein the molar concentration of the hydrochloric acid solution is 1 mol / L.

7. The polymer analysis method of cefazolin sodium for injection according to claim 4, characterized in that The mass volume ratio of the cefazolin sodium for injection to the sodium hydroxide solution is 20-23 mg:1 mL; wherein the molar concentration of the sodium hydroxide solution is 1 mol / L.

8. The polymer analysis method of cefazolin sodium for injection according to claim 1, characterized in that The polymers of Cefazolin Sodium for injection are polymer 1 and polymer 2, the molecular formulas of which are respectively C 25 H 27 N 14 O9S4,C 28 H 32 N 16 O 10 S6.

9. The polymer analysis method of cefazolin sodium for injection according to claim 8, characterized in that After the system suitability solution is injected into the liquid chromatograph, polymer 1 is located at a relative retention time of about 1.06 to 1.11 in the chromatogram, and polymer 2 is located at a relative retention time of about 1.37 to 1.

43.

10. The polymer analysis method of cefazolin sodium for injection according to claim 1, characterized in that The system sensitivity solution is used for sensitivity detection in HPLC detection. The system sensitivity solution is prepared as follows: Take the control solution, place it in a volumetric flask, quantitatively dilute it to the scale with mobile phase A, shake well, and obtain a sensitivity solution with a concentration of 0.00025-0.00125 mg / mL.

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