A method for determining ceftazidime and its formulation polymers

Abstract

This invention discloses a method for determining ceftazidime and its formulation polymers, comprising the following steps: 1) preparing a system suitability solution; 2) preparing a reference solution; 3) preparing a test solution for ceftazidime for injection; 4) determination: injecting the system suitability solution, reference solution, and test solution into a liquid chromatograph, respectively, and performing determination using reversed-phase chromatography, wherein the mobile phase of the reversed-phase chromatography contains mobile phase A and mobile phase B, mobile phase A being an aqueous formic acid solution and mobile phase B being methanol. The chromatogram obtained by the method of this invention has a stable baseline, can detect multiple polymeric impurities of ceftazidime, and exhibits good separation of each impurity; the method of this invention has good specificity and system suitability, and its linearity, sensitivity, repeatability, intermediate precision, and robustness are all good; furthermore, the test solution and reference solution of this invention have high stability.

Description

Technical Field

[0001] This invention relates to a method for determining ceftazidime and its formulation polymers, belonging to the field of pharmaceutical analysis. Background Technology

[0002] Ceftazidime has the molecular formula C 12 H 22 N6O7S2•5H2O, chemically named (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-en-3-methylpyridinium inner salt pentahydrate, has the following structural formula:

[0003]

[0004] Ceftazidime is a third-generation cephalosporin antibiotic with strong antibacterial activity and a broad antibacterial spectrum, exhibiting potent activity against both Gram-positive and Gram-negative bacteria. It demonstrates high stability against β-lactamases produced by Gram-positive and Gram-negative bacteria. This product exhibits strong antibacterial activity against Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterococcus, Salmonella, Shigella, Neisseria gonorrhoeae, Neisseria meningitidis, Staphylococcus aureus, hemolytic streptococci, Streptococcus pneumoniae, and Aerobacterium aerogenes. Ceftazidime is indicated for respiratory tract infections, urinary tract infections, skin and soft tissue infections, gastrointestinal, biliary tract and abdominal infections, bone and joint infections, infections associated with dialysis, infection prevention and treatment during various surgeries, as well as sepsis, bacteremia, peritonitis, meningitis, infections in immunocompromised patients, and infected burns.

[0005] Ceftazidime polymers are major degradation products of antibiotic compounds. Currently, the quality detection method for ceftazidime polymers is the G-10 size exclusion method (Chinese Pharmacopoeia 2025, Part IV, General Chapter 0514). However, the specificity of the G-10 size exclusion method for polymer verification has been questioned in recent years. The polymer composition determined by this method cannot represent the true content of ceftazidime polymers in the product and may contain other small molecule impurities.

[0006] Regarding the detection method of ceftazidime polymer content for injection, Chinese patent document with publication number CN115598235A discloses a method for determining ceftazidime polymers. This method uses an ultra-high performance liquid chromatograph, which has high requirements for the instrument and can only resolve 5 polymer impurity peaks.

[0007] Chinese patent application CN 120177699 A discloses a method for identifying polymers in ceftazidime / ceftazidime for injection, using RP-HPLC for analysis. The RP-HPLC chromatographic conditions are as follows: mobile phase A is 10-20 mmol / L ammonium formate solution with pH 3.4-3.6; mobile phase B is methanol / acetonitrile = 1:1-2; linear gradient elution is used at a flow rate of 1.0-2.0 ml / min; and the detection wavelength is 254 nm. This method can detect polymers in ceftazidime / ceftazidime for injection, but its detection efficiency is still unsatisfactory. The applicability chromatogram only shows five peaks, and the resolution and peak shape are both poor. Furthermore, the limits of quantitation and detection need to be reduced.

[0008] Based on the above, this invention is proposed. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a method for detecting the polymer content of ceftazidime and its preparations, which is applicable to general-purpose liquid chromatographs, so as to improve the universality, specificity, accuracy and sensitivity of the detection method; the detection method of the present invention can detect multiple polymer impurities of ceftazidime, and the separation of each impurity is good.

[0010] The technical solution adopted in this invention is as follows:

[0011] A method for determining ceftazidime and its formulation polymers, the method comprising the following steps:

[0012] 1) Prepare test solutions of ceftazidime or ceftazidime for injection;

[0013] 2) Prepare the reference solution;

[0014] 3) Measurement

[0015] The test solution and the reference solution were injected into the liquid chromatograph and determined by reversed-phase chromatography. The qualitative and / or quantitative results of polymer impurities in ceftazidime were obtained based on the chromatographic data.

[0016] The mobile phase in the reversed-phase chromatography method contains mobile phase A and mobile phase B. Mobile phase A is an aqueous solution of formic acid, and mobile phase B is methanol.

[0017] The volume ratio of mobile phase A to mobile phase B is 20~70:80~30.

[0018] Further, the concentration of the formic acid aqueous solution is 0.05~0.2%, more preferably 0.1%.

[0019] Furthermore, the reversed-phase chromatography method used a shim-pack Scepter C18-120 column (250*4.6mm, 5μm); when using the shim-pack Scepter C18-120 column (250*4.6mm, 5μm) to separate ceftazidime impurities, the obtained chromatogram baseline was stable, and the separation of ceftazidime impurities was good.

[0020] Furthermore, a small collection column can be added before the pump.

[0021] Furthermore, the preferred trapping column is a Welch Ghost-Buster Column (4.6*50mm).

[0022] In this invention, reversed-phase chromatography employs gradient elution, with the following elution gradient:

[0023]

[0024] Furthermore, the determination conditions for reversed-phase chromatography are as follows:

[0025] Flow rate: 1.0 ml / min

[0026] Column temperature: 40℃

[0027] Wavelength: 254nm

[0028] Injection volume: 20 μl.

[0029] In this invention, 20 μl of the test solution is injected into the liquid chromatograph, and the chromatogram is recorded. The retention time of the ceftazidime main peak is approximately 4 ± 1 min, and the relative retention time of the polymer impurities is approximately 1.9–9.5, with 6 distinct polymer impurity peaks. The concentration of a single polymer impurity must not exceed 0.2%, and the total polymer concentration must not exceed 0.5%.

[0030] Further, in step 1), the preparation of the test solution of ceftazidime or ceftazidime for injection includes: weighing ceftazidime or ceftazidime for injection, dissolving it in a solvent to prepare the test solution.

[0031] Furthermore, the concentration of the test solution is 2 mg / mL.

[0032] Further, in step 2), the preparation of the reference solution includes: taking ceftazidime reference standard, dissolving and diluting it with a solvent to prepare the reference solution.

[0033] Furthermore, in step 2), the concentration of the reference solution is 10 μg / mL.

[0034] Further, in steps 1) and 2), the solvent is a 3-8% acetonitrile aqueous solution, more preferably a 5% acetonitrile aqueous solution.

[0035] Compared with the prior art, the beneficial effects of the present invention are reflected in:

[0036] The method of the present invention can effectively separate impurity peaks in ceftazidime and its formulation polymers. The method has the advantages of good separation, good stability, good precision and good repeatability. Attached Figure Description

[0037] Figure 1 Chromatogram of the solution for system suitability;

[0038] Figure 2 This is a linear relationship diagram obtained in Example 2. Detailed Implementation

[0039] The present invention will be further described in detail below through embodiments, but it should not be construed that the scope of the present invention is limited to the following embodiments. Any modifications or substitutions made based on ordinary technical knowledge and common practices in the art without departing from the above-described technical premises are included within the scope of the present invention.

[0040] Unless otherwise specified, the percentages or proportions of solvents mentioned in this invention are volume ratios.

[0041] Example 1: Specificity of the Method

[0042] Chromatographic conditions: Detection wavelength: 254 nm

[0043] Column temperature: 40℃

[0044] Injection volume: 20 μl

[0045] Flow rate: 1.0 ml / min

[0046] Chromatographic column: Octadecylsilane-bonded silica gel as packing material: Shimadzu Shim-pack Scepter C18-120 (250*4.6mm, 5μm), with a Welch Ghost-Buster Column (4.6*50mm) connected before the pump.

[0047] Mobile phase A: 0.1% formic acid aqueous solution

[0048] Mobile phase B: Methanol

[0049] Solvent: 5% acetonitrile aqueous solution

[0050] Elution gradient:

[0051]

[0052] Accurately weigh approximately 25 mg of this product (containing approximately 20 mg of ceftazidime) and place it in a 25 ml stoppered test tube. After placing the tube in an oven at 105°C for 3 hours, remove it and allow it to cool to room temperature. Add approximately 4 ml of solvent, sonicate to dissolve, dilute to 10 ml with solvent, shake well, and filter. This solution is intended for system suitability.

[0053] Accurately weigh appropriate amounts of ceftazidime impurities A, B, C, E, G, and H (the above impurities are recorded in the EP pharmacopoeia) and pyridine, dissolve and dilute them separately with diluent to prepare solutions with a concentration of approximately 20 μl / mg, which are used as impurity localization solutions.

[0054] Accurately measure 20 μl each of blank solvent, system suitability solution, and each test solution, and inject them into the liquid chromatograph, recording the chromatograms. The retention times of each solution peak are shown in Table 1.

[0055] Table 1 Retention times of each solution

[0056]

[0057] ① In the blank solvent spectrum, there is no interference at the retention times of each polymer;

[0058] ② The main component of ceftazidime does not interfere with the detection of this product;

[0059] ③ Under the related substances test methods, known impurities such as impurities A, B, C, G, H and pyridine do not interfere with the detection of the polymer of this product under these chromatographic conditions; the polymer peak positions are all between 40 min after impurity H (relative retention time 1.4). Impurity E can be detected within this range, but under the related substances test methods, impurity E is not detected in the sample. Therefore, it is proposed that the chromatographic peaks with retention times between 40 min after impurity H (relative retention time 1.4) be used as polymer controls.

[0060] Example 2: Examination of Linear Relationships

[0061] The chromatographic conditions were the same as in Example 1.

[0062] Preparation of linear solutions: Take an appropriate amount of ceftazidime reference standard, accurately weigh it, dissolve it in 5% acetonitrile aqueous solution and dilute it quantitatively to prepare a series of linearity test solutions with limits of quantitation and test concentrations (calculated as 2 mg / ml of ceftazidime) of 0.05%, 0.1%, 0.2%, 0.5% and 1%.

[0063] Accurately measure 20 μl of each linearity test solution and inject it into the liquid chromatograph, recording the chromatograms. Plot a standard curve with the concentration of the linearity test solution on the x-axis and the peak area on the y-axis, as shown in the table.

[0064] Table 2 Data and Standard Curve

[0065]

[0066] The regression equation for ceftazidime is y = 51971.8339x + 2351.8716, R0 2 =1.0000

[0067] Conclusion: The above results indicate that ceftazidime concentration exhibits a good linear relationship with the corresponding peak area within the range of limit of quantitation to 200% limit.

[0068] Example 3: Limit of Quantification and Limit of Detection

[0069] The chromatographic conditions were the same as in Example 1.

[0070] Accurately weigh an appropriate amount of ceftazidime reference standard and quantitatively dilute it stepwise with 5% acetonitrile aqueous solution. Inject an appropriate amount into the liquid chromatograph and record the chromatogram. The sample injected is considered the limit of detection when the peak height is approximately 2–8 times the baseline noise; the sample injected is considered the limit of quantitation when the peak height is approximately 10–30 times the baseline noise. The results are shown in Table 3.

[0071] Table 3 Measurement Results

[0072]

[0073] Conclusion: The percentage of the limit of quantitation concentration of this product is less than 0.05%, indicating that the sensitivity of this method meets the requirements.

[0074] Example 4 Instrument Precision

[0075] Chromatographic conditions are the same as in Example 1.

[0076] Take the limit of quantitation solution and perform 6 consecutive measurements. Calculate the RSD of retention time and peak area. The results are shown in Table 4.

[0077]

[0078] The quantitation limit solution was measured six times consecutively, and the RSD of the peak area was less than 5.0%, the RSD of the retention time was less than 2.0%, and the instrument precision met the requirements.

[0079] Example 5 Precision Test - Repeatability Test

[0080] The chromatographic conditions were the same as in Example 1.

[0081] Preparation of the test solution (ceftazidime for injection): Accurately weigh approximately 25 mg of this product (containing approximately 20 mg of ceftazidime), place it in a 10 ml volumetric flask, dissolve and dilute to the mark with solvent, shake well, filter, and collect the filtrate. Prepare 6 parallel solutions. The results are shown in Table 5.

[0082] Preparation of the test solution (ceftazidime): Accurately weigh approximately 23 mg of this product (containing approximately 20 mg of ceftazidime), place it in a 10 ml volumetric flask, dissolve and dilute to the mark with solvent, shake well, filter, and collect the filtrate. Prepare 6 parallel solutions. The results are shown in Table 6.

[0083] Table 5 Repeatability Test Results - Test Solution (Cephotirium for Injection)

[0084]

[0085] Table 6 Repeatability Test Results - Test Solution (Cephotirium)

[0086]

[0087] The standard stipulates that: if the result is <0.01%, no comparison is made; if 0.01% ≤ result <0.05%, the range ≤0.02%;

[0088] 0.05% ≤ result ≤ 0.1%, range ≤ 0.05%; 0.1% < result ≤ 0.2%, range ≤ 0.10%;

[0089] 0.2% < result ≤ 0.5%, range ≤ 0.10%; 0.5% < result ≤ 2.0%, range ≤ 0.20%.

[0090] Conclusion: The experimental results show that the method has good repeatability.

[0091] Example 6 Precision Test - Intermediate Precision Test

[0092] The chromatographic conditions were the same as in Example 1.

[0093] Preparation of the test solution (ceftazidime for injection): Accurately weigh approximately 25 mg of this product (containing approximately 20 mg of ceftazidime), place it in a 10 ml volumetric flask, dissolve and dilute to the mark with solvent, shake well, filter, and collect the filtrate. Prepare 6 parallel solutions. The results are shown in Table 7.

[0094] Preparation of the test solution (ceftazidime): Accurately weigh approximately 23 mg of this product (containing approximately 20 mg of ceftazidime), place it in a 10 ml volumetric flask, dissolve and dilute to the mark with solvent, shake well, filter, and collect the filtrate. Prepare 6 parallel solutions. The results are shown in Table 8.

[0095] Table 7 Intermediate Precision Test Results - Test Solution (Cephotirium for Injection)

[0096]

[0097] Table 8 Repeatability Test Results - Test Solution (Cephalexin)

[0098]

[0099] The standard stipulates that: if the result is <0.01%, no comparison is made; if 0.01% ≤ result <0.05%, the range ≤0.02%;

[0100] 0.05% ≤ result ≤ 0.1%, range ≤ 0.05%; 0.1% < result ≤ 0.2%, range ≤ 0.10%;

[0101] 0.2% < result ≤ 0.5%, range ≤ 0.10%; 0.5% < result ≤ 2.0%, range ≤ 0.20%.

[0102] Conclusion: The experimental results show that the intermediate precision of this method is good.

[0103] The results of the precision test are shown in Tables 9 and 10.

[0104] Table 9 shows the results of 12 samples of test solution (ceftazidime for injection) from different personnel at different time points.

[0105]

[0106] Table 10 shows the results of 12 samples of test solution (ceftazidime) from different personnel at different time points.

[0107]

[0108] The standard stipulates that: if the result is <0.01%, no comparison is made; if 0.01% ≤ result <0.05%, the range ≤0.02%;

[0109] 0.05% ≤ result ≤ 0.1%, range ≤ 0.05%; 0.1% < result ≤ 0.2%, range ≤ 0.10%;

[0110] 0.2% < result ≤ 0.5%, range ≤ 0.10%; 0.5% < result ≤ 2.0%, range ≤ 0.20%.

[0111] Conclusion: The experimental results show that the method has good precision.

[0112] Example 7 Solution Stability

[0113] The chromatographic conditions were the same as in Example 1, and the test solution and reference solution were prepared separately.

[0114] Preparation of test solution (ceftazidime for injection): Accurately measure about 25 mg of this product (containing about 20 mg of ceftazidime), place it in a 10 ml volumetric flask, add solvent to dissolve and dilute to the mark, shake well and filter, and take the filtrate.

[0115] Preparation of test solution (ceftazidime): Accurately weigh about 23 mg of this product (containing about 20 mg of ceftazidime), place it in a 10 ml volumetric flask, add solvent to dissolve and dilute to the mark, shake well and filter, and take the filtrate.

[0116] Reference solution: Accurately weigh approximately 20 mg of ceftazidime reference standard and place it in a 20 ml volumetric flask. Add approximately 8 ml of solvent, sonicate to dissolve, and dilute to the mark with solvent. Shake well. Accurately measure 1 ml of the solution and place it in a 100 ml volumetric flask. Dilute to the mark with solvent and shake well.

[0117] The stability of the reference solution was investigated at 4℃, with injections at 0h, 1h, 2h, 3h, 4h, 10h, 17h, 23h, and 30h. The results are shown in Table 11.

[0118] The stability of the test solution (ceftazidime) was investigated at 4℃, with injections at 0h, 2h, 4h, 7h, 9h, and 11h. The results are shown in Table 12.

[0119] The stability of the test solution (ceftazidime for injection) was investigated at 4℃, with injections at 0h, 3h, 5h, 8h, 10h, and 12h. The results are shown in Table 13.

[0120] Table 11 Stability of Reference Solution

[0121]

[0122] Table 12 Stability of the test solution (ceftazidime)

[0123]

[0124] Table 13 Stability of the test solution (ceftazidime for injection)

[0125]

[0126] The standard stipulates: 1. Reference solution: The peak area measured at each time point shall not exceed 2.0% compared with 0h.

[0127] 2. Test solution: The difference between the content of each impurity measured at each time point and the content at 0 hours:

[0128] If the result is <0.01%, no comparison is made; if 0.01% ≤ result <0.05%, the difference is ≤0.02%.

[0129] If 0.05% ≤ result ≤ 0.1%, the difference ≤ 0.05%; if 0.1% < result ≤ 0.2%, the difference ≤ 0.10%.

[0130] 0.2% < result ≤ 0.5%, difference ≤ 0.10%; 0.5% < result ≤ 2.0%, difference ≤ 0.20%, and whether new chromatographic peaks are generated or degraded.

[0131] Conclusion: The experimental results show that the solution obtained by this method has good stability.

[0132] Example 8 Durability - Flow Rate Variation

[0133] Chromatographic conditions are as described in Example 1. Flow rate variations were: Flow rate variation 1: 0.8 ml / min, Flow rate variation 2: 1.2 ml / min, and initial flow rate: 1.0 ml / min. Test results are shown in Tables 14 and 15.

[0134] Table 14 Results of the durability test system suitability assessment

[0135]

[0136] Table 15 Results of the Durability Test of the Test Sample Solution

[0137]

[0138] Conclusion: When the polymers of this product are determined using the above method, the system suitability solution chromatograms can accurately guide the polymer localization. There are no significant differences in the detection results of polymers in the test samples, and the method has good robustness.

[0139] Example 9 Durability - Column Temperature Variation

[0140] The chromatographic conditions are as described in Example 1. The column temperature variations were: Column temperature variation 1: 35℃, Column temperature variation 2: 45℃, Initial column temperature: 40℃. The test results are shown in Tables 16 and 17.

[0141] Table 16 Results of the Durability Test System Suitability Examination

[0142]

[0143] Table 17 Results of the Durability Test of the Test Sample Solution

[0144]

[0145] Conclusion: When the polymers of this product are determined using the above method, the system suitability solution chromatograms can accurately guide the polymer localization. There are no significant differences in the detection results of polymers in the test samples, and the method has good robustness.

[0146] Example 10 Durability - Instrument Brand Change

[0147] The chromatographic conditions are as described in Example 1, with the instrument brand changed to Shimadzu HPLC and the original instrument brand being Agilent HPLC. The test results are shown in Tables 18 and 19.

[0148] Table 18 Results of the Durability Test System Suitability Examination

[0149]

[0150] Table 19 Results of the Durability Test of the Test Sample Solution

[0151]

[0152] Conclusion: When the polymers of this product are determined using the above method, the system suitability solution chromatograms can accurately guide the polymer localization. There are no significant differences in the detection results of polymers in the test samples, and the method has good robustness.

[0153] Example 11: Multiple Batch Testing

[0154] The chromatographic conditions were the same as in Example 1.

[0155] Blank solution: 5% acetonitrile aqueous solution

[0156] Preparation of test solution (ceftazidime for injection): Accurately weigh the powder equivalent to 20 mg of ceftazidime, place it in a 10 ml volumetric flask, add solvent to dissolve and dilute to the mark, shake well, filter, and take the filtrate as the test solution;

[0157] Preparation of test solution (ceftazidime): Accurately weigh the powder equivalent to 20 mg of ceftazidime, place it in a 10 ml volumetric flask, add solvent to dissolve and dilute to the mark, shake well, filter, and take the filtrate as the test solution;

[0158] Preparation of reference solution: Accurately dissolve and dilute ceftazidime reference standard in solvent to prepare a solution containing approximately 10 μg of ceftazidime per 1 ml to the mark, shake well, and use as the reference solution.

[0159] Preparation of system suitability solution: Accurately weigh this product (containing approximately 20 mg of ceftazidime), place it in a 25 ml stoppered test tube, place it in an oven at 105 °C for 3 hours, remove it, cool it to room temperature, add approximately 4 ml of solvent, sonicate to dissolve, dilute with solvent to 10 ml, shake well, and filter.

[0160] Injection: No less than 2 injections of blank solution, 1 injection of system suitability solution, blank solution (n≥1 injection, run until no interference), 5 injections of reference solution, and 1 injection each of test solution.

[0161] Table 20 Multiple batch test results

[0162]

[0163] Conclusion: The method yielded good results.

Claims

1. A method for determining ceftazidime and its formulation polymers, characterized in that: Includes the following steps: 1) Preparation of test solutions of ceftazidime and its formulation, ceftazidime for injection; 2) Prepare the reference solution; 3) Inject the test solution and reference solution into the liquid chromatograph and perform the determination by reversed-phase chromatography. Based on the chromatographic data, obtain the qualitative and / or quantitative results of polymer impurities in ceftazidime. The mobile phase in the reversed-phase chromatography method contains mobile phase A and mobile phase B. Mobile phase A is an aqueous solution of formic acid, and mobile phase B is methanol. The volume ratio of mobile phase A to mobile phase B is 20~70:80~30.

2. The method for determining ceftazidime and its formulation polymers according to claim 1, characterized in that: The concentration of the formic acid aqueous solution is 0.05~0.2%.

3. The method for determining ceftazidime and its formulation polymers according to claim 1, characterized in that: The chromatographic column used in the reversed-phase chromatography was a Shim-pack Scepter C18-120 (4.6mm*250mm, 5μm). During analysis, a trapping column is added before the pump, preferably a Welch Ghost-Buster Column (4.6*50mm).

4. The method for determining ceftazidime and its formulation polymers according to any one of claims 1-3, characterized in that: Reversed-phase chromatography uses gradient elution, with the following elution gradient: 。 5. The method for determining ceftazidime and its formulation polymers according to claim 4, characterized in that: The determination conditions for reversed-phase chromatography also include: Flow rate: 0.5~1.5ml / min Column temperature: 30~50℃ Wavelength: 254nm Injection volume: 20 μl.

6. The method for determining ceftazidime and its formulation polymers according to claim 1, characterized in that: In step 1), the preparation of the test solution of ceftazidime or ceftazidime for injection includes: weighing ceftazidime or ceftazidime for injection, dissolving it in a solvent to prepare the test solution.

7. The method for determining ceftazidime and its formulation polymers according to claim 6, characterized in that: The solvent is a 3-8% acetonitrile aqueous solution; The concentration of the test solution is 1~3 mg / mL.

8. The method for determining ceftazidime and its formulation polymers according to claim 1, characterized in that: In step 2), the preparation of the reference solution includes: taking ceftazidime reference standard, dissolving and diluting it with a solvent to prepare the reference solution.

9. The method for determining ceftazidime and its formulation polymers according to claim 8, characterized in that: In step 2), the solvent is a 3-8% acetonitrile aqueous solution; The concentration of the reference solution is 8~12 μg / mL.

10. The method for determining ceftazidime and its formulation polymers according to claim 1, characterized in that: The polymer impurities are ceftazidime impurity A, impurity B, impurity C, impurity E, impurity G and impurity H.

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