Method for identifying polymer in ceftazidime / ceftazidime for injection

Abstract

The invention provides a method for identifying polymers in ceftazidime / ceftazidime for injection. The method is characterized in that the ceftazidime / ceftazidime for injection is analyzed by adopting an RP-HPLC (Reverse Phase-High Performance Liquid Chromatography) method; the chromatographic conditions of the RP-HPLC are as follows: a 10-20 mmol / L ammonium formate solution with the pH value of 3.4-3.6 is used as a mobile phase A; methanol / acetonitrile in a ratio of 1: (1-2) is taken as a mobile phase B; linear gradient elution is carried out, and the flow velocity is 1.0-2.0 ml / min; the detection wavelength is 254 nm. The method can be used for effectively detecting the polymer in the ceftazidime / the ceftazidime for injection.

Description

Technical Field

[0001] The present invention relates to the field of impurity analysis of drugs, and specifically, to a method for identifying polymers in ceftazidime / ceftazidime for injection. Background Art

[0002] β-lactam antibiotics are prone to polymerization reactions during production, transportation, and storage, generating polymeric impurities, which may induce clinical allergic reactions and pose potential risks to medication safety. Currently, the main methods for analyzing polymeric impurities in β-lactam antibiotics are G-10 gel chromatography, TSK high-performance gel chromatography, and reversed-phase HPLC.

[0003] Ceftazidime is a semi-synthetic third-generation cephalosporin antibiotic. The 2020 edition of the Chinese Pharmacopoeia uses G-10 gel chromatography to control polymeric impurities in ceftazidime and ceftazidime for injection. Due to its separation mechanism, in addition to molecular exclusion, adsorption, hydrophobic, and other effects also occur. In G-10 gel chromatography and TSK high-performance gel chromatography, the impurity peaks before the main peak are not all polymeric impurities, but also small molecule degradation impurities. With the continuous in-depth understanding of the polymerization mechanism and structure of polymers in β-lactam antibiotics, using HPLC to analyze oligomers as pointer impurities is the research direction for polymeric impurities in this type of drug. Summary of the Invention

[0004] The present invention aims to overcome the above defects. After establishing an HPLC system with ammonium formate solution - methanol - acetonitrile as the mobile phase suitable for the detection of ceftazidime polymers, the LC-MS / MS analysis was subsequently performed on sample solution (1), sample solution (2), degradation solution (1), and degradation solution (2) using this system. The structures of the strongly retained impurities with relatively high contents in the sample solutions and degradation solutions were speculated through the first-order and second-order mass spectra, thereby providing a basis for the purity and impurity analysis of the samples.

[0005] The present invention provides a method for identifying polymers in ceftazidime / ceftazidime for injection, characterized in that:

[0006] RP-HPLC analysis is performed on ceftazidime / ceftazidime for injection;

[0007] The chromatographic conditions for the above RP-HPLC are as follows:

[0008] 10 - 20 mmol / L ammonium formate solution with a pH of 3.4 - 3.6 is used as mobile phase A;

[0009] Methanol / acetonitrile = 1:1 - 2 is used as mobile phase B;

[0010] Linear gradient elution is performed at a flow rate of 1.0 - 2.0 ml / min;

[0011] The detection wavelength is 254 nm.

[0012] Furthermore, a method for identifying polymers in ceftazidime / ceftazidime for injection provided by the present invention is further characterized in that:

[0013] The pH of the ammonium formate solution is adjusted with formic acid.

[0014] Furthermore, a method for identifying polymers in ceftazidime / ceftazidime for injection provided by the present invention is further characterized in that:

[0015] The chromatographic column uses octadecylsilyl silica gel as the filler.

[0016] Furthermore, a method for identifying polymers in ceftazidime / ceftazidime for injection provided by the present invention is further characterized in that:

[0017] The above linear gradient elution method is as follows:

[0018] Time (min) Mobile Phase A (%) Mobile Phase B (%) 0 75-85 15-25 10 75-85 15-25 35 40-60 40-60 40 20-30 70-80 45 20-30 70-80 46 80-85 15-20 55 80-85 15-20。

[0019] Furthermore, a method for identifying polymers in ceftazidime / ceftazidime for injection provided by the present invention is further characterized in that:

[0020] The polymer impurities appear after 3 times the relative retention time of the main peak.

[0021] Furthermore, a method for identifying polymers in ceftazidime / ceftazidime for injection provided by the present invention is further characterized in that:

[0022] The limit of ceftazidime polymer is that the peak area of polymer impurities with a relative retention time after 3.0 shall not be greater than 0.6 times the peak area of the main peak of the control solution;

[0023] The limit of ceftazidime for injection polymer is that the peak area of polymer impurities with a relative retention time after 3.0 shall not be greater than 2 times the peak area of the main peak of the control solution.

[0024] Furthermore, a method for identifying polymers in ceftazidime / ceftazidime for injection provided by the present invention is further characterized in that:

[0025] The molecular formulas of the polymers that can be identified by the above method for identifying polymers in ceftazidime / ceftazidime for injection are as follows:

[0026] Polymer impurity I: C39H4N11O15S4;

[0027] Derivative of polymer impurity I: C44H45N11O19S4;

[0028] Polymer impurity II: C39H39N11O14S4;

[0029] Derivative of polymer impurity II: C43H45N1O17S4;

[0030] Polymer impurity III: C61H61N17O21S6.

[0031] Furthermore, a method for identifying polymers in ceftazidime / ceftazidime for injection provided by the present invention is further characterized in that:

[0032] Other impurities that can be identified by the above method for identifying polymers in ceftazidime / ceftazidime for injection further include:

[0033] Impurity II: C27H26N6O9S3;

[0034] Impurity III: C38H43N11O14S4;

[0035] Impurity IV: C26H30N6O7S2.

[0036] In addition, the present invention also provides a method for obtaining polymer impurities in ceftazidime / ceftazidime for injection, which is characterized by comprising the following steps:

[0037] S1. Select a destruction solution to destroy ceftazidime / ceftazidime for injection to strengthen the impurity peaks;

[0038] S2. Separate the solution after destruction in S1 by the RP-HPLC method as described in claim 1 above;

[0039] S3. Analyze the mass spectra of the separated components to determine the structures of each impurity.

[0040] Furthermore, the above method for obtaining polymer impurities in ceftazidime / ceftazidime for injection is further characterized in that:

[0041] The above destruction solution is sodium carbonate;

[0042] The specific conditions for the above destruction are as follows: Take about 0.1 g of ceftazidime raw material, add 10 - 20 mg of sodium carbonate, dissolve in 2 - 4 ml of water, stand at room temperature for at least 3 days, and set aside. Before use, dilute the degradation concentrated solution to prepare a solution containing 1 - 2 mg of ceftazidime per 1 ml. Description of the Drawings

[0043] Figure 1 Typical chromatogram of the system suitability solution;

[0044] Figure 2 Linear test results of ceftazidime;

[0045] Figure 3 and the typical chromatogram of the test solution of ceftazidime raw material;

[0046] Figure 4 and the typical chromatogram of the test solution of ceftazidime for injection;

[0047] Figure 5 and the typical chromatogram of the test solution of blank solution;

[0048] Figure 6 and the chromatogram of the retention behavior of the degradation solution on the Hypersil ODS-2 column;

[0049] Figure 7 and the chromatogram of the retention behavior of the system suitability solution on the Diamonsil plus C18 column;

[0050] Figure 8 and the chromatogram of the retention behavior of the system suitability solution on the Luna C18 column;

[0051] Figure 9 and the chromatogram of the sample solution (1) of ceftazidime raw material;

[0052] Figure 10 and the chromatogram of the sample solution (2) of ceftazidime for injection;

[0053] Figure 11 and the chromatogram of the sample solution (3) of the reference ceftazidime for injection;

[0054] Figure 12 and the chromatogram of the degradation solution (1);

[0055] Figure 13 and the chromatogram of the degradation solution (2);

[0056] Figure 14 and the chromatogram of the blank solution;

[0057] Figure 15 and the first and second mass spectra of ceftazidime;

[0058] Figure 16 and the information of the possible second mass spectrometry fragmentation fragments of ceftazidime;

[0059] Figure 17 and the TIC chromatogram and typical chromatogram of the ceftazidime sample solution;

[0060] Figure 18 and the first and second mass spectra of impurity 1;

[0061] Figure 19 and the first and second mass spectra of impurity 2;

[0062] Figure 20 and the first- and second-order mass spectra of impurity 3;

[0063] Figure 21 and the first- and second-order mass spectra of impurity 4;

[0064] Figure 22 Possible structures of unknown impurity 1;

[0065] Figure 23 Possible mass spectrometry fragmentation fragment information of unknown impurity 1;

[0066] Figure 24 Possible structures of unknown impurity 2;

[0067] Figure 25 Possible mass spectrometry fragmentation fragment information of unknown impurity 2;

[0068] Figure 26 Possible structures of unknown impurity 3;

[0069] Figure 27 Possible mass spectrometry fragmentation fragment information of unknown impurity 3;

[0070] Figure 28 Possible structures of unknown impurity 4;

[0071] Figure 29 Possible mass spectrometry fragmentation fragment information of unknown impurity 4;

[0072] Figure 30 TIC chromatogram and typical chromatogram of the degradation solution;

[0073] Figure 31 The first- and second-order mass spectra of impurity 1

[0074] Figure 32 The first- and second-order mass spectra of impurity 2

[0075] Figure 33 The first- and second-order mass spectra of impurity 3

[0076] Figure 34 The first- and second-order mass spectra of impurity 4

[0077] Figure 35 The first- and second-order mass spectra of impurity 5

[0078] Figure 36 The first- and second-order mass spectra of impurity 6;

[0079] Figure 37 Possible structures of unknown impurity 2 and impurity 3

[0080] Figure 38 Possible mass spectrometry fragmentation fragment information of unknown impurity 2 and impurity 3;

[0081] Figure 39 Possible structures of unknown impurity 4

[0082] Figure 40 Possible mass spectrometry fragmentation information of impurity 4;

[0083] Figure 41 Possible structures of unknown impurity 5

[0084] Figure 42 Possible mass spectrometry fragmentation information of impurity 5;

[0085] Figure 43 Possible structures of unknown impurity 6

[0086] Figure 44 Possible mass spectrometry fragmentation information of unknown impurity 6;

[0087] Figure 45 Typical chromatogram of G-10 system chromatography Detailed implementation method

[0088] I. HPLC method (most preferred conditions)

[0089] HPLC chromatographic conditions: Using octadecylsilane-bonded silica gel as the filler (Thermo Hypersil ODS-2 4.6 mm×250 mm, 5 μm or a chromatographic column with equivalent efficiency); Using 10 mmol / L ammonium formate solution (adjusted to pH 3.4 with formic acid) as mobile phase A, and methanol-acetonitrile (1:1) as mobile phase B, perform linear gradient elution according to the following table, with a flow rate of 1.0 ml / min; The detection wavelength is 254 nm, and the injection volume is 20 μl.

[0090] Table 1 Linear gradient elution

[0091]

[0092] Test solution:

[0093] Sample solution (1): Take an appropriate amount of ceftazidime raw material, accurately weigh it, dissolve it in water and quantitatively dilute it to make a solution containing about 2 mg of ceftazidime per 1 ml.

[0094] Sample solution (2): Take an appropriate amount of ceftazidime for injection, accurately weigh it, dissolve it in water and quantitatively dilute it to make a solution containing about 2 mg of ceftazidime per 1 ml.

[0095] Sample solution (3): Take an appropriate amount of ceftazidime for injection (batch number: 2009E0), accurately weigh it, dissolve it in water and quantitatively dilute it to make a solution containing about 2 mg of ceftazidime per 1 ml.

[0096] Degradation solution (1): Take about 0.1 g of ceftazidime raw material (batch number cdp - 2003001), add 10 mg of sodium carbonate, dissolve in 2 ml of water, let stand at room temperature for 3 days, and set aside. Immediately before use, dilute the concentrated degradation solution to prepare a solution containing about 2 mg of ceftazidime per 1 ml.

[0097] Degradation solution (2): Take about 0.1 g of ceftazidime for injection (batch number 2106301), dissolve in 2 ml of water, let stand at room temperature for 3 days, and set aside. Immediately before use, dilute the concentrated degradation solution to prepare a solution containing about 2 mg of ceftazidime per 1 ml

[0098] Reference solution: Accurately measure 1 ml of the test solution, place it in a 200 - ml volumetric flask, dilute to the mark with water, and shake well.

[0099] System suitability solution: Take about 0.1 g of this product, add 10 mg of sodium carbonate, dissolve in 2 ml of water, let stand at room temperature for 3 days, dissolve with water and quantitatively dilute to a solution containing about 2 mg of ceftazidime per 1 ml as the system suitability solution.

[0100] Limit: The limit of ceftazidime polymer is that the peak area of polymer impurities with a relative retention time after 3.0 shall not be greater than 0.6 times (0.3%) of the peak area of the reference solution main peak;

[0101] The limit of ceftazidime polymer for injection is proposed to be that the peak area of polymer impurities with a relative retention time after 3.0 shall not be greater than 2 times (1.0%) of the peak area of the reference solution main peak.

[0102] The typical chromatogram of the system suitability solution is as Figure 1 shown.

[0103] Methodology verification

[0104] 1. Specificity

[0105] It can be known from mass spectrometry analysis that the RP - HPLC method can detect ceftazidime dimer, dimer derivative, trimer and condensate in the sample solution, and the method has good specificity.

[0106] 2. Linearity and range

[0107] Take about 12 mg of ceftazidime reference substance, accurately weigh it, place it in a 50 - ml volumetric flask, dissolve with water and dilute to the mark, shake well as the linear test stock solution. Accurately measure an appropriate amount and dilute with water to prepare solutions containing 0.1 μg, 0.2 μg, 1 μg, 5 μg, 10 μg, 50 μg per 1 ml, which are respectively equivalent to 0.005%, 0.01%, 0.05%, 0.25%, 0.5% and 2.5% of the concentration of the test solution (2 mg / ml). Determine according to this method, and the results are shown in Table 2 and Figure 2 .

[0108] Table 2 Linear test results of ceftazidime

[0109]

[0110] From the linear test, it can be seen that when the concentration of ceftazidime is in the range of 0.1014 μg / ml to 50.71 μg / ml, there is a good linear relationship between the peak area and the concentration: A = 46339C - 3550.2 (r = 1.0000, n = 6).

[0111] 3. Repeatability

[0112] Take this product (raw material with batch number cdp - 2003001; preparation with batch number 2106301, specification: 1.0 g), a total of 6 portions, dissolve in water and quantitatively dilute to make a solution containing about 2 mg of ceftazidime per 1 ml, and inject for analysis according to the chromatographic method in 3.3.1. The typical chromatogram is shown in Figures 3 - 5 , and calculate the polymer content by the self - control method. The repeatability results are shown in detail in Table 3 and Table 4.

[0113] Table 3 Determination results of repeatability test of ceftazidime raw material

[0114]

[0115] Table 4 Determination results of repeatability test of ceftazidime for injection

[0116]

[0117] From the results, it can be seen that this method has good repeatability.

[0118] 4. Stability of sample solution

[0119] Take this product (raw material with batch number cdp - 2003001; preparation with batch number 2106301, specification: 1.0 g), dissolve in water and quantitatively dilute to make a solution containing about 2 mg of ceftazidime per 1 ml, place at 4℃ for different times respectively, inject for analysis, and calculate the polymer content by the self - control method. The results are shown in detail in Table 5 and Table 6.

[0120] Table 5 Determination of polymers of ceftazidime raw material placed at 4℃ for different times

[0121]

[0122] Table 6 Determination of polymers of ceftazidime for injection placed at 4℃ for different times

[0123]

[0124] As can be seen from the results, the content of polymer impurities in the test solution increased significantly when stored at 4°C. It is recommended to prepare the test solution freshly before use.

[0125] 5. Quantitation Limit and Detection Limit

[0126] When the signal-to-noise ratio was 10:1, the quantitation limit of ceftazidime was 0.6 ng. The concentration of ceftazidime in the test solution was 2 mg / ml, and the quantitation limit concentration was equivalent to 0.0015% of the concentration of the test solution. When the signal-to-noise ratio was 3:1, the detection limit of ceftazidime was 0.1 ng, equivalent to 0.0003% of the concentration of the test solution.

[0127] 6. Method Robustness

[0128] The effects of three different brands of C18 chromatographic columns on the separation were investigated, namely Thermo Hypersil ODS-2 column (4.6×250 mm, 5 μm), Diamonsil plus C18 column (4.6×250 mm, 5 μm), and Luna C18 column (4.6×250 mm, 5 μm). The chromatograms of the system suitability solution are shown respectively as Figures 6 - 8 follows. We also investigated the conditions of different mobile phase buffer pH values (pH 3.2 - 3.6), different column temperatures (30°C ± 5°C), and flow rates (0.9 ml / min - 1.1 ml / min). The chromatograms of the system suitability solution were basically the same, indicating that the method has good robustness.

[0129] II. LC-MS

[0130] In this part, the LC-MS / MS analysis was performed on sample solution (1), sample solution (2), degradation solution (1), and degradation solution (2) using this system. The structures of the strongly retained impurities with relatively large contents in the sample solutions and degradation solutions were speculated based on the first-order and second-order mass spectra.

[0131] The chromatograms of ceftazidime raw material sample solution (1) and sample solution (2) of ceftazidime for injection are shown as Figure 9 and Figure 10 follows. The chromatogram of sample solution (3) of the original research ceftazidime for injection is shown as Figure 11 follows. The chromatograms of degradation solutions (1) - (2) are shown as Figure 12 and Figure 13 follows. The chromatogram of the blank solution is shown as Figure 14 follows.

[0132] The results showed that the impurity peaks after the main peak of the ceftazidime raw material were basically the same as those of the preparation, and the impurity peaks after the main peak of the preparation sample solution were basically the same as those of the reference preparation sample solution. Since ceftazidime raw material has poor solubility in water, sodium carbonate was used to assist dissolution for the degradation test. When placed at room temperature, the strongly retained impurities after the main peak of the injection preparation and the raw material solution with added sodium carbonate increased significantly, and the impurity peaks were basically the same and could be eluted.

[0133] 1. Analysis of the cleavage pattern of ceftazidime

[0134] The first-order mass spectrum ([M+H]+, m / z: 547.11) and the second-order mass spectrum of ceftazidime are as Figure 15 shown. The cleavage pattern of the second-order mass spectrum of ceftazidime is speculated as Figure 16 shown, and the structures of unknown impurities can be inferred through the characteristic fragment ions m / z 468.06, 424.07, 396.08, 277.02, 167.03, and 80.05.

[0135] 2. Structural analysis of unknown strongly retained impurities

[0136] Under the above chromatographic conditions, LC-MS / MS analysis was performed on the impurity peaks with retention times longer than the main peak, and the structures of unknown impurities with relatively large contents and molecular weights greater than the main peak after the main peak in the sample were speculated. The mass spectrometer detector used an electrospray ionization source (ESI), positive ion scanning, scanning range (m / z) 100 - 1700, drying gas temperature 250 °C, drying gas flow rate 16 L / min, nebulizer pressure 45 psi, sheath gas temperature 350 °C, sheath gas flow rate 11 L / min, capillary voltage 3500 V, and collision-induced dissociation (CID) voltage 10 - 30 eV.

[0137] Since the polymer impurities after the main peak in the ceftazidime raw material and the injection ceftazidime sample solution were basically the same, taking the injection ceftazidime as an example, the TIC diagram and typical chromatogram of the sample solution (2) are as Figure 17 shown.

[0138] Through the analysis in the Auto-MS / MS mode, the structures of 4 unknown impurities with retention times of 20.5 minutes, 24.5 minutes, 25.9 minutes, and 34.8 minutes were speculated, and their first-order and second-order mass spectra are respectively as Figures 18 - 21 shown.

[0139] The exact molecular weight of the unknown impurity 1 at the retention time of 20.5 minutes is 1031.1. As can be seen from the first-order mass spectrum, in addition to the protonated molecular ion peak [M+H]+ (m / z 1032.17), plasma peaks such as [M+2H]2+ (516.59) can also be generated. Combining the fragment information of the second-order mass spectrum, characteristic ions 126.01, 167.03, 442.08, 486.08, 547.10, 821.16, 909.14, and 953.13 can be found. It is speculated that impurity 1 is a condensate of ceftazidime and its fragment ([M+H]+, m / z 486), and the molecular formula may be C 39 H 41 N 11 O 15 S4, and its possible structure is as Figure 22 shown. Taking 22a as an example, the fragmentation rule of the second-order mass spectrum is as Figure 23 shown, which is ceftazidime dimer I.

[0140] The exact molecular weight of the unknown impurity 2 at the retention time of 24.5 minutes is 674.1. As can be seen from the first-order mass spectrum, protonated molecular ion peaks [M+H]+ (675.10) and [M+2H]+ (338.05) can be generated. Combining the fragment information of the second-order mass spectrum, characteristic ions 126.01, 167.03, 396.08, 524.07, and 596.06 can be found. The molecular formula may be C27H26N6O9S3, and its possible structure is as Figure 24 shown, and the fragmentation rule of the second-order mass spectrum is as Figure 25 shown.

[0141] The exact molecular weight of the unknown impurity 3 at the retention time of 25.9 minutes is 1005.19. As can be seen from the first-order mass spectrum, protonated molecular ion peaks [M+H]+ (1006.20) and [M+2H]+ (503.60) can be generated. Combining the fragment information of the second-order mass spectrum, characteristic ions 126.01, 677.08, 839.17, 883.16, and 927.15 can be found. The molecular formula may be C 38 H43N11O 14 S4, and its possible structure is as Figure 26 shown, and the fragmentation rule of the second-order mass spectrum is as Figure 27 shown.

[0142] The exact molecular weight of unknown impurity 4 at a retention time of 34.8 minutes is 602.16. From the first-order mass spectrum, it can be seen that a protonated molecular ion peak [M + H]+ (603.17) can be generated. Combining the fragment information of the second-order mass spectrum, characteristic ions 167.03, 277.02, 396.08, 468.06, and 524.13 can be found. The molecular formula may be C26H30N6O7S2. It is speculated that it may be impurity E of cephalothin in the European Pharmacopoeia, which is a process impurity, and its possible structure is as Figure 28 shown, and the fragmentation rule of the second-order mass spectrum is as Figure 29 shown.

[0143] Since the polymer impurities after the main peak in the degradation solution (1) of cephalothin raw material and the degradation solution (2) of cephalothin for injection are basically the same, taking cephalothin for injection as an example, the TIC diagram and typical chromatogram of the degradation solution (2) are as Figure 30 shown.

[0144] By analyzing in the Auto-MS / MS mode, the structures of 6 unknown impurities with significantly increased contents at retention times of 20.5 minutes, 21.8 minutes, 26.5 minutes, 27.3 minutes, 29.5 minutes, and 30.6 minutes were speculated. Their first-order and second-order mass spectra are respectively as Figures 31 - 36 shown.

[0145] The exact molecular weight of unknown impurity 1 at a retention time of 20.5 minutes is 1031.1. From the first-order mass spectrum, it can be seen that ion peaks such as [M + H]+ (m / z 1032.17) and [M + 2H]2+ (516.59) can be generated. Combining the fragment information of the second-order mass spectrum, characteristic ions 126.01, 167.03, 505.07, 486.08, 547.10, 821.16, 909.14, and 953.13 can be found. It is speculated that it is a condensate of cephalothin and its fragment ([M + H]+, m / z 486), which is basically the same as impurity 1 in the sample solution. The molecular formula may be C 39 H41N11O 15 S4, which is cephalothin dimer I.

[0146] The exact molecular weights of unknown impurities 2' and 3 at retention times of 21.8 minutes and 26.5 minutes are 1159.2. Through software analysis, their molecular formula is C44H45N11O19S4. From the first-order mass spectrum, ion peaks [M + 2H]2+ (580.589) and [M + H]+ (1160.170) are generated. Combining the fragment information of the second-order mass spectrum, characteristic ions 126.01, 167.03, 653.05, 793.07, 1009.15, 1037.14, and 1081.13 can be found. The speculated possible structure is as Figure 37As shown, it is a ceftazidime dimer I derivative, and the fragmentation pattern of the secondary mass spectrometry is as follows Figure 38 as shown.

[0147] The exact molecular weight of the unknown impurity 4 with a retention time of 27.3 minutes is 1013.16. Through software analysis, its molecular formula is C39H39N11O14S4. From the primary mass spectrometry diagram, it can be seen that the ion peaks of [M + 2H]2+ (507.59) and [M + H]+ (1014.16) are generated. Combining the fragment information of the secondary mass spectrometry diagram, characteristic ions 126.01, 167.03, 634.08, 847.14, 891.13, and 935.12 can be found. The possible structure is speculated as follows Figure 39 As shown, it is ceftazidime dimer II, and the fragmentation pattern of the secondary mass spectrometry is as follows Figure 40 as shown.

[0148] The exact molecular weight of the unknown impurity 5 with a retention time of 29.5 minutes is 1115.19. Through software analysis, its molecular formula is C43H45N1O17S4. From the primary mass spectrometry diagram, it can be seen that the ion peaks of [M + 2H]2+ (558.59) and [M + H]+ (1116.18) are generated. Combining the fragment information of the secondary mass spectrometry diagram, characteristic ions 126.01, 167.03, 787.07, 890.09, 976.12, 993.16, and 1037.15 can be found. The possible structure is speculated as follows Figure 41 As shown, it is a ceftazidime dimer II derivative, and the fragmentation pattern of the secondary mass spectrometry is as follows Figure 42 as shown.

[0149] The exact molecular weight of the unknown impurity 6 with a retention time of 30.6 minutes is 1559.26. Through software analysis, its molecular formula is C61H61N17O21S6. From the primary mass spectrometry diagram, it can be seen that the ion peaks of [M + 2H]2+ (780.64) and [M + 3H]3+ (520.76) are generated. Combining the fragment information of the secondary mass spectrometry diagram, characteristic ions 126.01, 167.03, 396.08, 468.06, 547.12, 741.11 ([M + 2H]2+), 935.12, and 1014.16, etc. can be found. The possible structure is speculated as follows Figure 43 As shown, it is ceftazidime trimer. Taking 36a as an example, the fragmentation pattern of the secondary mass spectrometry is as follows Figure 44 as shown.

[0150] The above results show that: 1. Ceftazidime for injection is prone to generate polymer impurities when placed at room temperature; 2. The HPLC system with ammonium formate buffer - methanol - acetonitrile as the mobile phase can effectively detect the polymer impurities in ceftazidime raw materials and injection preparations (impurity peaks after a relative retention time of 3.0 (20.5 min - 34.8 min)), and can be used for its quality control.

[0151] III. Analyze 3 batches of raw materials, 3 batches of preparations and 1 batch of original research single-agent preparation by this method. The determination results of polymer content are shown in Table 7

[0152] Table 7 Determination Results of Polymer Content in RP-HPLC Chromatographic System

[0153]

[0154] It can be seen from the typical chromatogram of the sample solution that the polymer impurity content after the relative retention time of 3.0 is about 0.03% - 0.07%. Therefore, the limit of ceftazidime polymer is: the peak area of the polymer impurity after the relative retention time of 3.0 shall not be greater than 0.6 times (0.3%) of the main peak area of the control solution;

[0155] The limit of ceftazidime polymer for injection is proposed as: the peak area of the polymer impurity after the relative retention time of 3.0 shall not be greater than 2 times (1.0%) of the main peak area of the control solution.

[0156] It can be seen from the above table that although the detection results of G-10 for polymers are all less than those of the RP-HPLC of the present invention. This is because when analyzing ceftazidime polymer by the G-10 ( Figure 45 ) system, the components do not elute in order of molecular weight. There are both high molecular polymer impurities and degradation impurities with molecular weights smaller than ceftazidime before the main peak of ceftazidime. This leads to the problem of unclear identification of each component. In practice, some polymer impurities are thus ignored, resulting in the problem that its results are generally small.

Claims

1. A method for identifying polymers in ceftazidime / ceftazidime for injection, characterized in that: Ceftazidime / ceftazidime for injection was analyzed by RP-HPLC method; The chromatographic conditions of the RP-HPLC are as follows: 10 - 20 mmol / L ammonium formate solution with a pH of 3.4 - 3.6 was used as mobile phase A; Methanol / acetonitrile = 1:1 - 2 was used as mobile phase B; Linear gradient elution was carried out at a flow rate of 1.0 - 2.0 ml / min; The detection wavelength was 254 nm.

2. The method for identifying polymers in ceftazidime / ceftazidime for injection according to claim 1, characterized in that: The pH of the ammonium formate solution was adjusted with formic acid.

3. The method for identifying polymers in ceftazidime / ceftazidime for injection according to claim 1, characterized in that: The chromatographic column was packed with octadecylsilane chemically bonded silica gel.

4. The method for identifying polymers in ceftazidime / ceftazidime for injection according to claim 1, characterized in that: The method of the linear gradient elution is as follows:

5. The method for identifying polymers in ceftazidime / ceftazidime for injection according to claim 1, characterized in that: The polymer impurities appeared after 3 times the relative retention time of the main peak.

6. The method for identifying polymers in ceftazidime / ceftazidime for injection according to claim 1, characterized in that: The limit of ceftazidime polymer is that the peak area of the polymer impurities with a relative retention time after 3.0 shall not be greater than 0.6 times the peak area of the main peak of the control solution; The limit of ceftazidime for injection polymer is that the peak area of the polymer impurities with a relative retention time after 3.0 shall not be greater than 2 times the peak area of the main peak of the control solution.

7. The method for identifying polymers in ceftazidime / ceftazidime for injection according to any one of claims 1-6, characterized in that: The molecular formula of the polymer that can be identified by the method for identifying the polymer in ceftazidime / ceftazidime for injection is as follows: Polymer impurity I: C 39 H4N 11 O 15 S4; Polymer impurity I derivative: C 44 H 45 N 11 O 19 S4; Polymer Impurity II: C 39 H 39 N 11 O 14 S4; Polymer impurity II derivative: C 43 H 45 N1O 17 S4; Polymer Impurity III: C 61 H 61 N 17 O 21 S6。 8. The method for identifying polymers in ceftazidime / ceftazidime for injection according to claim 7, characterized in that: Other impurities that can be identified by the method for identifying the polymer in ceftazidime / ceftazidime for injection also include: Impurity II: C 27 H 26 N6O9S3; Impurity III: C 38 H 43 N 11 O 14 S4; Impurity IV: C 26 H 30 N6O7S2。 9. A method for obtaining polymer impurities in ceftazidime / ceftazidime for injection, characterized in that, It includes the following steps: S1. Select a destruction solution to destroy ceftazidime / ceftazidime for injection to enhance the impurity peaks; S2. Separate the solution after destruction in S1 by the RP-HPLC method as described in claim 1; S3. Analyze the mass spectra of the separated components to determine the structures of each impurity.

10. The method for obtaining polymer impurities in ceftazidime / ceftazidime for injection according to claim 9, characterized in that: The destruction solution is sodium carbonate; The specific conditions for the destruction are as follows: Take about 0.1 g of ceftazidime raw material, add 10 - 20 mg of sodium carbonate, dissolve in 2 - 4 ml of water, stand at room temperature for not less than 3 days, and set aside. Before use, dilute the degraded concentrated solution to make a solution containing 1 - 2 mg of ceftazidime per 1 ml.

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