A new method for the detection of ceftriaxone sodium polymers

Abstract

This invention belongs to the field of pharmaceutical analysis technology and discloses a novel method for detecting ceftriaxone sodium polymers. The method employs reversed-phase high-performance liquid chromatography (RP-HPLC) to detect ceftriaxone sodium samples. The chromatographic conditions include: a column using either octadecylsilane-bonded silica gel or octylsilane-bonded silica gel as the packing material; mobile phase A comprising a mixed solution of an aqueous phase and an organic phase at a volume ratio of 78:22 to 68:32, wherein the aqueous phase can be dipotassium hydrogen phosphate or n-octylamine solution, and the organic phase can be methanol or acetonitrile; mobile phase B is methanol or acetonitrile, with gradient elution. This novel detection method can be used for detecting polymeric impurities in ceftriaxone sodium and its preparations, and has advantages such as good specificity, high sensitivity, and good robustness, enabling precise separation of different polymeric impurities.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical analysis, and particularly relates to a new detection method of ceftriaxone sodium polymers. BACKGROUND

[0002] Ceftriaxone sodium is a third-generation cephalosporin antibiotic, which belongs to the beta-lactam antibiotic. Its antibacterial spectrum is similar to that of cefotaxime, but the antibacterial spectrum of ceftriaxone sodium is more biased towards gram-positive bacteria, and it has the largest coverage of coccus among the third-generation cephalosporins. Therefore, ceftriaxone sodium has a wide range of clinical applications.

[0003] Polymer research is an important part of drug quality research. Especially, cephalosporin antibiotics are prone to polymerization to produce polymer impurities. Polymer impurities are the main allergens of cephalosporin antibiotics and can induce allergic reactions. Therefore, it is necessary to analyze and control polymers.

[0004] Ceftriaxone sodium raw materials are included in the Chinese Pharmacopoeia (2020 edition) (ChP2020), the United States Pharmacopoeia (43) (USP43) and the European Pharmacopoeia 10.0 (EP10.0). However, only ChP2015 controls polymers, and the analysis method used is the Sephadex gel chromatography method (G-10 method). The known impurities listed in other national pharmacopoeias (such as USP43 and EP10.0) do not include polymer impurities. However, when the G-10 method is used to separate polymer impurities in ceftriaxone sodium, some small molecule impurities co-elute with polymers, which cannot be distinguished, resulting in poor specificity and poor quantitative accuracy of the method.

[0005] In addition to the G-10 method, the commonly used polymer analysis method is the TSK method, which uses a TSK chromatographic column for the chromatographic system. However, in practical application, it is found that the chromatogram obtained by the TSK method has the problem of co-elution of different polymer impurities, which cannot achieve fine separation of different polymer impurities, and thus is not conducive to the control of individual polymer impurities in the quality monitoring of ceftriaxone sodium.

[0006] Therefore, the present application is proposed. SUMMARY

[0007] The technical problem to be solved by the present application is to overcome the shortcomings of the prior art and provide a new detection method of ceftriaxone sodium polymers, which has good specificity, high sensitivity and good durability, and can achieve fine separation of polymer impurities in ceftriaxone sodium.

[0008] To solve the above technical problems, the basic idea of the technical solution of the present application is:

[0009] A novel method for detecting ceftriaxone sodium polymers employs reversed-phase high-performance liquid chromatography (RP-HPLC) to detect ceftriaxone sodium samples. The chromatographic conditions include:

[0010] The chromatographic column can be packed with either octadecylsilane-bonded silica gel or octylsilane-bonded silica gel.

[0011] Mobile phase A consists of a mixed solution of an aqueous phase and an organic phase in a volume ratio of 78:22 to 68:32. The aqueous phase can be dipotassium hydrogen phosphate or n-octylamine solution, and the organic phase can be methanol or acetonitrile. Mobile phase B is methanol or acetonitrile, and gradient elution is performed.

[0012] Specifically, in this invention, the organic phase composition of mobile phase A is preferably the same as that of mobile phase B. That is, if the organic phase in mobile phase A is methanol, then the corresponding mobile phase B is methanol. And if the organic phase in mobile phase A is acetonitrile, then the corresponding mobile phase B is acetonitrile.

[0013] In the above scheme, reversed-phase high-performance liquid chromatography (RPLC) combined with a gradient elution process can achieve fine separation of polymer impurities (i.e., ceftriaxone sodium polymers) in ceftriaxone sodium. Different polymer impurities can be separated into peaks in the obtained chromatogram, and the separation between each polymer impurity peak is good, which is conducive to achieving the purpose of controlling individual polymer impurities in the quality monitoring of ceftriaxone sodium.

[0014] Experiments have shown that the mobile phase component selected in this invention has higher sensitivity to polymers that may be present in ceftriaxone sodium compared to other commonly used mobile phase components, and can achieve effective separation of ceftriaxone sodium polymers.

[0015] In this invention, the specificity of the detection method was investigated using a system suitability solution. The chromatogram of the obtained system suitability solution showed good separation of the polymer impurity peaks. Further mass spectrometry analysis was used to study the components corresponding to each polymer impurity peak, confirming that they were ceftriaxone sodium polymers. This demonstrates that the detection method of this invention can effectively analyze ceftriaxone sodium polymers and exhibits good specificity.

[0016] Furthermore, after the aqueous phase and the organic phase are mixed, phosphoric acid is added to adjust the pH value to 6.3-6.7, preferably to 6.5, as the mobile phase A;

[0017] Preferably, the aqueous phase and the organic phase are mixed at a volume ratio of 73:27;

[0018] Preferably, the concentration of the aqueous phase is 0.018 mol / L to 0.022 mol / L, and more preferably 0.020 mol / L.

[0019] Further, the gradient elution includes:

[0020] The elution time is 0–5 min, the volume percentage of mobile phase A is maintained at 100%, and the volume percentage of mobile phase B is maintained at 0%.

[0021] The elution time was 5–45 min, during which the volume percentage of mobile phase A decreased from 100% to 70%, and the volume percentage of mobile phase B increased from 0% to 30%.

[0022] The elution time was 45–55 min, during which the volume percentage of mobile phase A decreased from 70% to 60%, and the volume percentage of mobile phase B increased from 30% to 40%.

[0023] Gradient elution offers advantages such as shorter elution time, improved separation efficiency, and better peak shape. In this invention, the chromatogram obtained from the detection also shows peaks corresponding to four polymeric impurities (denoted as polymeric impurities 1-4), in addition to the ceftriaxone peak. These peaks are designated as polymer peak 1 (corresponding to polymeric impurity 1), polymer peak 2 (corresponding to polymeric impurity 2), polymer peak 3 (corresponding to polymeric impurity 3), and polymer peak 4 (corresponding to polymeric impurity 4). Initially, maintaining 100% mobile phase A allows for the elution of non-polymeric impurities, preventing interference from other non-polymeric impurities or their degradation, thus improving the specificity of the detection method. During the elution period of 5–45 min, as mobile phase A decreases from 100% to 70%, different polymeric impurities are eluted in descending order of polarity, resulting in sharp peaks and good separation.

[0024] In this invention, the gradient elution process using the controlled mobile phase described above can effectively separate the polymer peaks 1-4, and the separation degree between adjacent peaks meets the requirements. Further experiments confirm that if equal gradient elution is used during the detection process, the aforementioned polymer impurity peaks cannot be separated, and if a gradient elution process different from the above is used, the separation effect on polymer impurities also decreases.

[0025] Furthermore, the chromatographic conditions also include one or more of the following:

[0026] a) The flow rate is 0.5–1.5 mL / min, preferably 1.0 mL / min;

[0027] b) The detection wavelength is 249–261 nm, preferably 254 nm;

[0028] c) The column temperature of the chromatographic column is 28–32°C, preferably 30°C;

[0029] d) The injection volume is 10–30 μL, preferably 20 μL.

[0030] Furthermore, the chromatographic column is a Kromasil 100-C18, Capcell Pak C18 MG II, Innoval C18 column, or Kromasil 100-5-C8 column;

[0031] The length of the chromatographic column is 150-300 mm, preferably 250 mm;

[0032] The inner diameter of the chromatographic column is 3.0–4.6 mm, preferably 4.6 mm;

[0033] The filler has a particle size of 3–10 μm, preferably 5 μm.

[0034] A system suitability solution containing polymer impurities was injected into a liquid chromatograph, and the separation effect of the solution under different chromatographic conditions was tested. Experiments showed that different ceftriaxone sodium polymers could be effectively separated when the pH of mobile phase A was controlled at 6.3–6.7; different ceftriaxone sodium polymers could be effectively separated when the column temperature was controlled at 28–32°C; and different ceftriaxone sodium polymers could be effectively separated when the column was a Kromasil 100-C18, Capcell Pak C18 MG II, or Innovation C18 column. Therefore, the detection method of this invention has good robustness.

[0035] Furthermore, the new detection method includes the following steps:

[0036] (1) Take ceftriaxone sodium sample, dissolve it with methanol-water solution or acetonitrile-water solution, and prepare test solution and control solution;

[0037] (2) Measure equal amounts of the test solution and the control solution and inject them into the liquid chromatograph for determination.

[0038] Further, the preparation of the test solution includes: taking an appropriate amount of ceftriaxone sodium sample, accurately weighing it, adding a methanol-water solution or acetonitrile-water solution with an organic phase to aqueous phase volume ratio of 22:78 to 32:68 to dissolve it, and preparing a test solution containing 0.10 to 0.50 mg of ceftriaxone sodium sample per 1 mL;

[0039] Preferably, a solution with a volume ratio of organic phase to aqueous phase of 27:73 is added for dissolution;

[0040] Preferably, each 1 mL of the test solution contains 0.22 mg of ceftriaxone sodium sample.

[0041] Further, the preparation of the control solution includes: accurately measuring x mL of the test solution, transferring it to a 100x mL volumetric flask, adding a solution with a volume ratio of organic phase to aqueous phase of 22:78 to 32:68 and diluting to the mark to obtain the control solution;

[0042] Preferably, a solution with a volume ratio of organic phase to aqueous phase of 27:73 is added and the volume is adjusted to the mark.

[0043] In the above scheme, the corresponding organic phase refers to the organic phase used in preparing the test solution and the control solution that has the same composition as the organic phase used in mobile phase A under the chromatographic conditions. Specifically, if the organic phase used in mobile phase A under the chromatographic conditions is methanol, then methanol-water solution is used for dissolution and volume adjustment when preparing the test solution and the control solution. If the organic phase used in mobile phase A under the chromatographic conditions is acetonitrile, then acetonitrile-water solution is used for dissolution and volume adjustment when preparing the test solution and the control solution.

[0044] In this invention, both the test solution and the control solution must be freshly prepared before use. Preferably, when preparing the control solution, 1 mL of the test solution is accurately measured and transferred to a 100 mL volumetric flask for dilution to volume. After the test solution and control solution are prepared, 20 μL of each solution are accurately measured and injected into the liquid chromatograph, and the chromatograms are recorded.

[0045] After detection, chromatograms of the test solution and the control solution were obtained. In this invention, the main component self-comparison method without correction factors was used to quantitatively analyze the content of polymer impurities. Specifically, if a polymer impurity peak is present in the chromatogram of the test solution, it is compared with the main peak (i.e., the ceftriaxone peak) in the chromatogram of the control solution to calculate the content of polymer impurities.

[0046] Furthermore, it also includes system suitability testing:

[0047] Take ceftriaxone reference standard, dissolve it in methanol-water solution or acetonitrile-water solution, heat in a water bath and then dilute to obtain degradation solution;

[0048] The degradation solution was injected into a liquid chromatograph, and the resulting chromatogram was recorded.

[0049] In the above scheme, the degradation solution is injected into the liquid chromatograph as a system suitability solution to examine whether the chromatographic system used is effective and suitable for the detection of ceftriaxone sodium polymers. After the ceftriaxone reference standard solution is left to stand at a certain temperature for a period of time, polymer impurities will be generated. These impurities are diluted and injected into the liquid chromatograph. If the various polymer impurity peaks can be observed in the resulting chromatogram, it indicates that the current chromatographic system can effectively detect ceftriaxone sodium polymers.

[0050] Further, the preparation of the degradation solution specifically includes:

[0051] Take an appropriate amount of ceftriaxone reference standard and dissolve it in a solution with a volume ratio of organic phase to aqueous phase of 22:78 to 32:68 to prepare a solution containing 5 to 15 mg of ceftriaxone reference standard per 1 mL.

[0052] After heating in a water bath at 40–60°C for 4–8 hours, measure y mL and transfer it to a 10y mL volumetric flask. Add a solution with an organic phase to aqueous phase volume ratio of 22:78–32:68 and bring the volume to the mark to obtain the degradation solution.

[0053] Preferably, a solution with a volume ratio of organic phase to aqueous phase of 27:73 is added during dissolution and volume adjustment.

[0054] Preferably, each 1 mL of the prepared solution contains 10 mg of ceftriaxone reference standard;

[0055] Preferably, a 60°C water bath is used for heating for 8 hours.

[0056] In the above scheme, the corresponding organic phase is similar to that described above; that is, the organic phase used to prepare the degradation solution is the same as the organic phase used in mobile phase A under chromatographic conditions. When the organic phase used in mobile phase A under chromatographic conditions is methanol, the degradation solution is prepared by dissolving and adjusting the volume using a methanol-water solution. If the organic phase used in mobile phase A under chromatographic conditions is acetonitrile, the degradation solution is prepared by dissolving and adjusting the volume using an acetonitrile-water solution.

[0057] In this invention, preferably, 1 mL of the solution heated in a water bath is transferred to a 10 mL volumetric flask and diluted to volume to prepare the degradation solution. During the system suitability test, 20 μL of the degradation solution is injected into a liquid chromatograph and the chromatogram is recorded.

[0058] Ceftriaxone sodium belongs to the β-lactam antibiotic class. Studies have found that high-concentration solutions of β-lactam antibiotics easily generate polymer impurities when left to stand at room temperature. However, the time required for polymer formation at room temperature is relatively long, generally requiring 72 hours of standing before the polymers can be clearly detected. To reduce the preparation time of the degradation solution, repeated experiments revealed that heating a high-concentration solution of ceftriaxone reference standard in a water bath at 40–60°C for 4–8 hours, compared to leaving it to stand at room temperature for 72 hours, resulted in a diluted solution that produced the same polymer impurity profile, meaning that polymer impurities of the same composition were obtained.

[0059] Furthermore, in the chromatogram of the degradation solution, the retention time of the ceftriaxone peak is 12-15 min, and after the ceftriaxone peak, polymer peak 1, polymer peak 2, polymer peak 3 and polymer peak 4 emerge sequentially.

[0060] Preferably, the retention time of the ceftriaxone peak is approximately 13 minutes;

[0061] Preferably, the resolution between each polymer peak is greater than 1.5.

[0062] Specifically, in this invention, polymer peak 1 corresponds to polymer impurity 1, the structural formula of which is shown in Formula I; polymer peak 2 corresponds to polymer impurity 2, the structural formula of which is shown in Formula II; polymer peak 3 corresponds to polymer impurity 3; and polymer peak 4 corresponds to polymer impurity 4, wherein polymer impurity 3 and polymer impurity 4 are a pair of isomers with the structural formula shown in Formula III.

[0063]

[0064] By adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art.

[0065] This invention employs reversed-phase high-performance liquid chromatography (RP-HPLC) combined with a gradient elution process to achieve effective separation of polymeric impurities in ceftriaxone sodium. The novel detection method of this invention offers advantages such as good specificity, high sensitivity, and robustness. Compared to the G-10 and TSK methods, it can achieve more precise separation of polymeric impurities, thereby enabling control over individual polymeric impurities and facilitating the quality monitoring of ceftriaxone sodium.

[0066] The specific embodiments of the present invention will be described in further detail below. Attached Figure Description

[0067] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings:

[0068] Figure 1 These are the RPLC chromatograms of the test solution / degradation solution in Examples 1-14 of this invention;

[0069] Figure 2 This is a comparison RPLC chromatogram of the test solution and the degradation solution in Example 15 of the present invention;

[0070] Figure 3 This is the RPLC chromatogram of the degradation solution in Example 16 of the present invention;

[0071] Figure 4This is the UV chromatogram analyzed by LC-MS in Experimental Example 1 of this invention;

[0072] Figure 5 This is a possible polymerization mode of ceftriaxone dimer;

[0073] Figure 6 This is the reaction formula for the secondary degradation reaction of ceftriaxone fragments;

[0074] Figure 7 These are the primary and secondary mass spectra of chromatographic peak 1 in Experimental Example 1 of this invention;

[0075] Figure 8 These are the primary and secondary mass spectra of chromatographic peak 2 in Experimental Example 1 of this invention;

[0076] Figure 9 These are the primary and secondary mass spectra of chromatographic peak 3 in Experimental Example 1 of this invention;

[0077] Figure 10 These are the primary and secondary mass spectra of chromatographic peak 4 in Experimental Example 1 of this invention;

[0078] Figure 11 This is the fitting result of the linear experiment in Experiment Example 3 of this invention;

[0079] Figure 12 This is the RPLC chromatogram obtained when mobile phase A has different pH values ​​in Experimental Example 5 of this invention;

[0080] Figure 13 These are the RPLC chromatograms obtained in Experimental Example 6 of this invention when the chromatographic column has different column temperatures;

[0081] Figure 14 These are the RPLC chromatograms obtained using different chromatographic columns in Experiment Example 7 of this invention;

[0082] Figure 15 This is a chromatographic comparison of degradation solutions obtained under different standing conditions in Experiment Example 8 of this invention;

[0083] Figure 16 This is the chromatogram obtained by analyzing the degradation solution using the G-10 method in Comparative Example 1 of this invention;

[0084] Figure 17 These are separation chromatograms of the effluent from the G-10 method at different time periods in the RPLC system in Comparative Example 1 of this invention;

[0085] Figure 18 This is the chromatogram obtained by analyzing the degradation solution using the TSK method in Comparative Example 2 of this invention;

[0086] Figure 19This is a separation chromatogram of the effluent from the TSK method at different time periods in the RPLC system in Comparative Example 2 of this invention;

[0087] Figure 20 This is a schematic diagram of the column-switched LC-MS system used in Experimental Example 1 of the present invention;

[0088] Figure 21 This is the RPLC chromatogram of the degradation solution in Example 17 of the present invention.

[0089] It should be noted that these accompanying drawings and textual descriptions are not intended to limit the scope of the invention in any way, but rather to illustrate the concept of the invention to those skilled in the art by referring to specific embodiments. Detailed Implementation

[0090] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0091] The instruments used in the embodiments and test examples of this invention include:

[0092] LC-20A liquid chromatography system, equipped with a diode array detector (Shimadzu Corporation, Japan);

[0093] Two-dimensional liquid chromatography system, Summit 100 (including P680 dual ternary low-pressure gradient pump, ACI-100 autosampler, Tcc-100 column oven and PDA100 diode array detector, workstation is Chromeleon 7.2SR5 (Thermo Scientific, USA);

[0094] The column-switching LC-MS system consists of a Thermo HPLC system (including a U3000 quaternary low-pressure gradient pump, autosampler, column oven, switching valve, and four-wavelength detector) and a Q Exactive Focus mass spectrometer (Therm Corporation, USA), with an Xcalibur 2.3 workstation.

[0095] Examples 1-13

[0096] In Examples 1-13 of the present invention, the specific information of the ceftriaxone sodium samples used is shown in Table 1 below. The manufacturer of all samples is Jinhong Pharmaceutical Co., Ltd.

[0097] Table 1 Sample Information Table

[0098]

[0099] The polymer impurity content of different samples in Table 1 was determined using the following detection method.

[0100] Preparation of the test solution: Take an appropriate amount of ceftriaxone sodium sample, accurately weigh it, add acetonitrile-water solution with a volume ratio of 27:73 to dissolve it, and prepare a test solution containing 0.22 mg of ceftriaxone sodium sample per 1 mL.

[0101] Preparation of the control solution: Accurately measure 1 mL of the above test solution and transfer it to a 100 mL volumetric flask. Add an acetonitrile-water solution with a volume ratio of 27:73 (acetonitrile to water) and dilute to the mark. Shake well to obtain the control solution.

[0102] Chromatographic analysis: Accurately measure 20 μL each of the test solution and the control solution, inject them into the liquid chromatograph, and record the resulting chromatograms.

[0103] Chromatographic conditions for chromatographic analysis include:

[0104] (1) The chromatographic column was a Capcell Pak C18 MG II column with a specification of 250×4.6mm and 5μm;

[0105] (2) Mobile phase A is a 0.02 mol / L n-octylamine solution-acetonitrile mixed solution (volume ratio 73:27), and the pH value is adjusted to 6.5 with phosphoric acid; Mobile phase B is acetonitrile;

[0106] (3) The flow rate is 1.0 mL / min;

[0107] (4) The detection wavelength is 254nm;

[0108] (5) The column temperature of the chromatographic column is 30℃;

[0109] (6) Perform gradient elution according to the following procedure:

[0110]

[0111] In the above embodiments, the chromatograms of the test solution are as follows: Figure 1 As shown. Among them, Figure 1 In the chromatograms shown, starting with the second curve from the bottom, the chromatograms are of the test solutions from Examples 1-13.

[0112] Example 14

[0113] This embodiment is a system suitability test of the chromatographic system used in embodiments 1-13 above. A degradation solution of ceftriaxone reference standard was prepared as a system suitability solution and injected into the liquid chromatograph for chromatographic analysis.

[0114] In this embodiment, the ceftriaxone reference standard used had batch number 130480-201504 and a content of 83.9%. System suitability testing was conducted according to the following steps:

[0115] (1) Take an appropriate amount of ceftriaxone reference standard, add acetonitrile-water solution with a volume ratio of 27:73 to dissolve it, and prepare a solution containing 10 mg of ceftriaxone reference standard per 1 mL.

[0116] (2) After heating in a water bath at 60℃ for 8 hours, measure 1 mL and transfer it to a 10 mL volumetric flask. Add an acetonitrile-water solution with a volume ratio of 27:73 to the water and bring it to the mark. Shake well to obtain the degradation solution.

[0117] (3) Take 20 μL of the degradation solution and inject it into a liquid chromatograph for chromatographic analysis. The chromatographic conditions are the same as those in Examples 1-13 above. Record the obtained chromatogram.

[0118] In this embodiment, the chromatogram of the degradation solution is as follows: Figure 1 The bottom curve is shown in the figure. It can be seen that four polymer impurity peaks were detected in the chromatogram, namely the four peaks marked with asterisks * in the figure, which are labeled as polymer peak 1, polymer peak 2, polymer peak 3 and polymer peak 4 from left to right.

[0119] In the chromatogram obtained in this embodiment, the retention time of the ceftriaxone peak, i.e., the main peak, is approximately 13 minutes, and polymer peaks 1-4 eluted sequentially after the ceftriaxone peak. It can be seen that the peak shapes and separation of each impurity peak are good, achieving fine separation of polymer impurities of different components.

[0120] Example 15

[0121] The chromatograms obtained in Example 14 above can be used to determine the retention times of different polymer impurities in the chromatograms. Based on this, this example uses the principal component self-comparison method without correction factors to calculate the content of polymer impurities in the ceftriaxone sodium samples in Examples 1-13.

[0122] Specifically, such as Figure 2 As shown, curve B is the chromatogram of the degradation solution in Example 14, and curve A is the chromatogram of the test solution in Example 1. It can be seen that curve A contains polymer impurity peaks corresponding to the retention times of peaks 3 and 4 in curve B, indicating the presence of polymer impurities 3 and 4 in the tested ceftriaxone sodium sample. However, the polymer impurity peaks corresponding to the retention times of peaks 1 and 2 in curve B are very low in curve A, almost indistinguishable upon direct observation, indicating that the content of polymer impurities 1 and 2 in the sample is relatively low.

[0123] The chromatograms of the test solutions obtained in other embodiments are similar to those obtained in other embodiments. Figure 2 The shape of curve B is also quite similar (see Figure 1 In other words, the polymer impurity peaks corresponding to the retention times of peaks 3 and 4 in curve A can be clearly observed, but the polymer impurity peaks corresponding to the retention times of peaks 1 and 2 in curve A are more difficult to distinguish.

[0124] Further, the areas of polymer peaks 1-4 in the chromatograms of each test solution obtained in Examples 1-13 were calculated, and the area of ​​the ceftriaxone peak in each control solution chromatogram was also calculated. The areas of each polymer impurity peak were compared with the areas of the corresponding ceftriaxone peaks in the control solution chromatograms to calculate the polymer impurity content in each sample. The calculation results are shown in Table 2 below.

[0125] Table 2. Polymer impurity content (%) in the samples

[0126]

[0127]

[0128] Example 16

[0129] In this embodiment, the degradation solution was prepared and tested according to the following steps:

[0130] (1) Take an appropriate amount of ceftriaxone reference standard with batch number 130480-201504, add acetonitrile-water solution with a volume ratio of 27:73 to dissolve it, and prepare a solution containing 10 mg of ceftriaxone reference standard per 1 mL.

[0131] (2) After heating in a water bath at 60℃ for 8 hours, measure 1 mL and transfer it to a 10 mL volumetric flask. Add an acetonitrile-water solution with a volume ratio of 27:73 to the water and bring it to the mark. Shake well to obtain the degradation solution.

[0132] (3) Take 20 μL of the degradation solution and inject it into a liquid chromatograph for chromatographic analysis. The chromatographic conditions are the same as those in Examples 1-13 above. Record the obtained chromatogram.

[0133] The chromatogram obtained in this embodiment is as follows: Figure 3 As shown in the figure, the five peaks marked from left to right are the ceftriaxone peak and polymer peaks 1-4. The retention times and resolutions of the ceftriaxone peak and polymer peaks 1-4 are as follows:

[0134] Chromatographic peak Retention time (min) Separation Ceftriaxone peak 14.957 -- Polymer 1 peak 25.955 25.11 Polymer 2 peak 29.341 7.15 Polymer 3 peak 33.358 7.45 Polymer 4 peak 34.838 1.67

[0135] As can be seen, using the detection method of the present invention, the resolution of each polymer impurity peak in the chromatogram meets the requirements, indicating that the fine separation of polymer impurities of different components has been achieved.

[0136] Example 17

[0137] In this embodiment, the degradation solution was prepared and tested according to the following steps:

[0138] (1) Take an appropriate amount of ceftriaxone reference standard with batch number 130480-201504, add methanol-water solution with a volume ratio of methanol to water of 27:73 to dissolve it, and prepare a solution containing 10 mg of ceftriaxone reference standard per 1 mL.

[0139] (2) After heating in a water bath at 60℃ for 8 hours, measure 1 mL and transfer it to a 10 mL volumetric flask. Add a methanol-water solution with a volume ratio of 27:73 (methanol to water) to the mark and shake well to obtain the degradation solution.

[0140] (3) Take 20 μL of the degradation solution and inject it into a liquid chromatograph for chromatographic analysis and record the obtained chromatogram.

[0141] The chromatographic conditions for the chromatographic analysis in step (3) above are as follows:

[0142] (1) The chromatographic column was a Kromasil 100-5-C8 column with dimensions of 250×4.6mm and 5μm;

[0143] (2) Mobile phase A is a 0.02 mol / L dipotassium hydrogen phosphate solution-methanol mixture (volume ratio 73:27), with the pH adjusted to 6.5 using phosphoric acid; mobile phase B is methanol.

[0144] (3) The flow rate is 1.0 mL / min;

[0145] (4) The detection wavelength is 254nm;

[0146] (5) The column temperature of the chromatographic column is 30℃;

[0147] (6) Perform gradient elution according to the following procedure:

[0148]

[0149] The chromatogram obtained in this embodiment is as follows: Figure 21 As shown in the figure, the five peaks marked from left to right are the ceftriaxone peak and polymer peaks 1-4. The retention times and resolutions of the ceftriaxone peak and polymer peaks 1-4 are as follows:

[0150] Chromatographic peak Retention time (min) Separation Ceftriaxone peak 14.076 -- Polymer 1 peak 25.969 31.54 Polymer 2 peak 27.991 5.02 Polymer 3 peak 29.843 4.53 Polymer 4 peak 32.951 6.56

[0151] As can be seen, using the detection method of the present invention, the resolution of each polymer impurity peak in the chromatogram meets the requirements, indicating that the chromatographic conditions achieve fine separation of polymer impurities of different components.

[0152] Experimental Example 1

[0153] This experimental example analyzes the composition of polymer impurities 1-4 detected in the above examples.

[0154] An appropriate amount of ceftriaxone reference standard was dissolved in an acetonitrile-water solution with a volume ratio of 27:73 (acetonitrile:water) to prepare a solution containing 30 mg of ceftriaxone reference standard per mL. The solution was heated in a water bath at 60°C for 8 hours to obtain a high-concentration degradation solution. The high-concentration degradation solution was analyzed by LC-MS with an injection volume of 10 μL, yielding the following results: Figure 4 The chromatogram shown has peaks numbered 1-4 corresponding to polymer impurities 1-4 that are to be detected.

[0155] Specifically, in this experimental example, the following method was used: Figure 20 The column-switching LC-MS system shown desaltes the components corresponding to the polymer impurity peaks obtained by RPLC detection of the sample before online MS analysis. The specific steps include:

[0156] (1) The high-concentration degradation solution described above was separated using chromatographic system 1, wherein the chromatographic conditions of chromatographic system 1 were the same as those in Examples 1-14 above;

[0157] (2) By switching the valve, the t of the target impurity peak is... R The eluting components within the ±0.1 min interval are switched to a 500 μL switching loop;

[0158] (3) Gradient elution was performed using chromatographic system 2 to desalt the target impurities, and then the impurities were sent to a mass spectrometer for MS analysis.

[0159] The two-dimensional chromatographic column of the chromatographic system 2 is a Zorbax SB-C18 (150×4.6mm, 5μm, Agilent); the switching valve is a six-way valve; the mobile phase includes Watson's distilled water as mobile phase IIA and acetonitrile as mobile phase; the flow rate is 0.5mL / min. The gradient elution process of the chromatographic system 2 is as follows:

[0160]

[0161] In step (1) above, the high-concentration degradation solution was separated, and the resulting chromatogram is shown below. Figure 4 As shown, the elution components corresponding to peaks 1-4 were sent to a mass spectrometer for MS analysis after desalting.

[0162] The mass spectrometry conditions were as follows: scanning voltage (+) 3000.0V, capillary temperature (+) 350.0℃, sheath gas (+) 35L / h, auxiliary gas (+) 10.00L / h, maximum spray current (+) 100.00, probe heating temperature (+) 350.00℃, S-prism RF level 50.00, and HESI ion source.

[0163] Primary mass spectrometry method: positive ion mode, resolution 7000, scan range m / z 150~1500; Secondary mass spectrometry method: resolution 17500, separation window 3.0 m / z, (N)CE 15V.

[0164] Studies have found that in ceftriaxone sodium polymers, ceftriaxone dimers may be present through methods such as... Figure 5 The shown polymerization methods produce, among which, Figure 5 (c) illustrates the dominant polymerization pathway. After dimer formation, the ceftriaxone fragment within it can undergo further secondary degradation reactions, commonly as follows: Figure 6 The compound obtained by removing the side chain at position 3 is still a ceftriaxone sodium polymer. Based on this, the mass spectra corresponding to chromatographic peaks 1-4 in this experimental example were analyzed.

[0165] Mass spectrometry analysis was performed on the eluent near chromatographic peak 1 (retention time 22.26 min), and its primary and secondary mass spectra are shown below. Figure 7 As shown. Mass spectrometry analysis was performed on the eluent near chromatographic peak 2 (retention time 25.89 min), and its primary and secondary mass spectra are shown below. Figure 8 As shown above. Figure 7 and Figure 8 In the images above, all are first-order mass spectra, and all are second-order mass spectra.

[0166] It can be seen that the primary mass spectra of chromatographic peaks 1 and 2 contain quasi-molecular particle peaks at m / z 950 and m / z 972, respectively, which are [M + H] + and [M] + Na] + The peak was observed, suggesting a molecular weight of 949. The secondary mass spectrum showed ceftriaxone fragments at m / z 555, exhibiting characteristic ceftriaxone fragments, indicating that the substances corresponding to chromatographic peaks 1 and 2 are ceftriaxone sodium polymers.

[0167] The polymer impurity 1 corresponding to chromatographic peak 1 has a molecular weight of... Figure 5 (c) shows a polymerization pathway where the polymers listed differ by 159, representing a 3-position side chain of one molecule. This indicates that polymer impurity 1 is... Figure 5(c) shows the polymerization pathway, from which a 3-position side chain is removed to obtain the lactone polymer. In the above dimer structure, the 3-position side chain can be removed at both sites. However, since the abundance of ceftriaxone fragments at m / z 555 in the secondary fragments of chromatographic peak 1 is very low, it is suggested that there is no complete ceftriaxone fragment in its molecule. Its structural formula is speculated to be as shown in Formula I.

[0168] The secondary fragments of chromatographic peak 2 contain fragments with m / z 636, which is... Figure 5 (a) shows a representative fragment of the dimer produced by the cleavage of one of the β-lactam rings, and its structure is presumed to be that of Formula II.

[0169]

[0170] Mass spectrometry analysis was performed on the eluent near chromatographic peak 3 (retention time 28.17 min), and its primary and secondary mass spectra are shown below. Figure 9 As shown. Mass spectrometry analysis was performed on the eluent near chromatographic peak 4 (retention time 28.57 min), and its primary and secondary mass spectra are shown below. Figure 10 As shown above. Figure 9 and Figure 10 In the images above, all are first-order mass spectra, and all are second-order mass spectra.

[0171] It can be seen that the mass spectral fragments corresponding to chromatographic peaks 3 and 4 are consistent, indicating that polymer impurities 3 and 4 are isomers. Specifically, the primary mass spectrum contains quasi-molecular particle peaks at m / z 968 and m / z 990, respectively [M... + H] + and [M] + Na] + The peak was observed, suggesting a molecular weight of 967. The secondary mass spectrum showed ceftriaxone fragments at m / z 555, exhibiting characteristic ceftriaxone fragments, indicating that the substances corresponding to peaks 3 and 4 are ceftriaxone sodium polymers.

[0172] because Figure 5 (c) shows the preferred polymerization pathway, and according to Figure 1 and Figure 2 As shown, the chromatographic peaks of polymer impurities 3 and 4 are clearly visible in the RPLC chromatogram of the actual sample, suggesting that polymer impurities 3 and 4 should be produced according to... Figure 5 The product polymerized via the polymerization pathway shown in (c). The molecular weights of polymer impurities 3 and 4 are... Figure 5 The polymers listed in the polymerization pathway shown in (c) differ by 141, meaning they have one more water molecule than polymer impurity 1, indicating that polymer impurities 3 and 4 are... Figure 5(c) shows the polymerization pathway, from which a 3-position side chain is removed to obtain a lactone hydrolysate. In the above dimer structure, both sites can remove the 3-position side chain to form a lactone hydrolysate. However, the presence of a ceftriaxone fragment at m / z 555 in the secondary mass spectrum indicates that a ceftriaxone fragment is retained in the polymer structure. It is speculated that polymer impurities 3 and 4 are a pair of isomers with the structural formula shown in Formula III.

[0173]

[0174] The analysis in this experimental example confirms that the impurity peaks in the RPLC chromatogram obtained by the detection method of the present invention are generated by ceftriaxone sodium polymer. Combined with the experimental results of good peak shape and resolution of each impurity peak in Example 14, it shows that the detection method of the present invention can effectively analyze ceftriaxone sodium polymer and has good specificity.

[0175] Experimental Example 2

[0176] This test example examines the precision of the ceftriaxone sodium polymer detection method of the present invention. Specifically, the degradation solution prepared in Example 14 was injected into a liquid chromatograph six times consecutively, and chromatographic analysis was performed under the same chromatographic conditions as in Examples 1-13. The test results are shown in Table 3 below.

[0177] Table 3 Precision test results

[0178]

[0179] The precision of the detection method of the present invention is 1.1% based on the area of ​​the main peak and 0.7% based on the area of ​​the polymer impurity peak, indicating that the detection method of the present invention has good precision.

[0180] Experimental Example 3

[0181] This experimental example examines the linear correlation of the ceftriaxone sodium polymer detection method of the present invention, specifically using the following method.

[0182] Preparation of linear test samples:

[0183] (1) Accurately weigh 0.0192 g of ceftriaxone reference standard, place it in a 25 mL volumetric flask, dissolve it in an acetonitrile-water solution with a volume ratio of 27:73 and dilute to the mark to obtain linearity test solution a;

[0184] (2) Accurately weigh 0.0116 g of ceftriaxone reference standard, place it in a 50 mL volumetric flask, dissolve it in an acetonitrile-water solution with a volume ratio of 27:73 and dilute to the mark to obtain linearity test solution b;

[0185] (3) Accurately weigh 0.0138 g of ceftriaxone reference standard and place it in a 10 mL volumetric flask. Dissolve it in an acetonitrile-water solution with a volume ratio of 27:73 and dilute to the mark. Transfer 1 mL of the solution to a 10 mL volumetric flask and dilute to the mark with an acetonitrile-water solution with a volume ratio of 27:73. This solution is used as linear test solution c.

[0186] (4) Measure 1 mL of linearity test solution c and transfer it to a 10 mL volumetric flask. Dilute to the mark with an acetonitrile-water solution with a volume ratio of acetonitrile to water of 27:73. This solution is used as linearity test solution d.

[0187] (5) Measure 0.25 mL of linear test solution d, transfer it to a 25 mL volumetric flask, and dilute it to the mark with an acetonitrile-water solution with a volume ratio of acetonitrile to water of 27:73 to obtain linear test solution e;

[0188] (6) Measure 1 mL of linearity test solution e and transfer it to a 10 mL volumetric flask. Dilute to the mark with an acetonitrile-water solution with a volume ratio of acetonitrile to water of 27:73. This solution is used as linearity test solution f.

[0189] RPLC chromatographic analysis: The above-mentioned linear test solution af was injected into the liquid chromatograph and chromatographic analysis was performed under the same chromatographic conditions as in Examples 1-13. The test results are shown in Table 4 below.

[0190] Table 4 Results of the linear experiment

[0191]

[0192] A linear fit was performed on the concentration against the area of ​​the main peak in Table 4 above, yielding the linear equation y = 5 × 10⁻⁶. 7 x+394455, R 2 =0.9971, the fitted straight line is as follows Figure 11 As shown. It can be seen that the detection method of the present invention is effective against ceftriaxone sodium at a concentration of at least 1.158 × 10⁻⁶. -5 Within the concentration range of ~0.644 mg / mL, the concentration and the area of ​​the main peak showed a good linear relationship.

[0193] Test Example 4

[0194] This experimental example examines the detection limit and quantitation limit of the ceftriaxone sodium polymer detection method of the present invention, specifically using the following method.

[0195] Determination of the limit of detection: The linear test solution e prepared in Example 3 above was accurately measured and serially diluted with an acetonitrile-water solution at a volume ratio of 27:73. After each dilution, 20 μL was accurately injected into the liquid chromatograph (chromatographic conditions were the same as in Examples 1-13). Using three times the noise level as an indicator, the limit of detection for the detection method was found to be 2.3 × 10⁻⁶. -4 μg.

[0196] Determination of the lowest limit of quantitation: The linearity test solution e prepared in Example 3 above was accurately measured and serially diluted with an acetonitrile-water aqueous solution at a volume ratio of 27:73. After each dilution, 20 μL was accurately injected into the liquid chromatograph (chromatographic conditions were the same as in Examples 1-13). Using 10 times the noise level as an indicator, the lowest limit of quantitation for the detection method was found to be 7.0 × 10⁻⁶. -5 μg.

[0197] Based on the lowest detection limit of ceftriaxone, the detection method of this invention can detect polymer impurities with a content of 0.005%, which meets the test requirements.

[0198] Experimental Example 5

[0199] This experimental example investigates the effect of the pH value of mobile phase A on the separation effect of ceftriaxone sodium polymer in the detection method of the present invention.

[0200] Specifically, after mixing the n-octylamine solution with acetonitrile, phosphoric acid was added to adjust the pH of mobile phase A to 6.3, 6.5, and 6.7, respectively. Other chromatographic conditions were the same as in Examples 1-13. At different pH values, 20 μL of the degradation solution prepared in Example 14 was injected into the liquid chromatograph for chromatographic analysis. The resulting chromatograms are shown below. Figure 12 As shown.

[0201] It can be seen that, Figure 12 In the three curves shown, the well-separated polymer peaks 1-4 can be clearly observed, indicating that the detection method of the present invention can effectively separate ceftriaxone sodium polymers when the pH values ​​of mobile phase A are 6.3, 6.5 and 6.7 respectively.

[0202] Experimental Example 6

[0203] This experimental example investigates the effect of column temperature on the separation efficiency of ceftriaxone sodium polymer in the detection method of the present invention.

[0204] Specifically, the column temperatures were controlled at 32°C, 30°C, and 28°C, while other chromatographic conditions remained the same as in Examples 1-13. At each of the different column temperatures, 20 μL of the degradation solution prepared in Example 14 was injected into the liquid chromatograph for chromatographic analysis. The resulting chromatograms are shown below.Figure 13 As shown.

[0205] It can be seen that, Figure 13 In the three curves shown, the well-separated polymer peaks 1-4 can be clearly observed, indicating that the detection method of the present invention can effectively separate ceftriaxone sodium polymers at column temperatures of 32℃, 30℃ and 28℃.

[0206] Experimental Example 7

[0207] This experimental example investigates the effect of different chromatographic columns on the separation effect of ceftriaxone sodium polymer in the detection method of the present invention.

[0208] Specifically, other chromatographic conditions were controlled the same as in Examples 1-13, except that Kromasil 100-C18, Capcell Pak C18 MG II, or Innoval C18 columns (all 250 × 4.6 mm, 5 μm) were used. 20 μL of the degradation solution prepared in Example 14 was injected into the liquid chromatograph for chromatographic analysis, and the resulting chromatograms are shown below. Figure 14 As shown.

[0209] It can be seen that, Figure 14 The three curves clearly show well-separated polymer peaks 1-4, indicating that the detection method of the present invention can effectively separate ceftriaxone sodium polymers when using Kromasil 100-C18, Capcell Pak C18 MG II or Innoval C18 columns.

[0210] The test results of Examples 5-7 above demonstrate that when the pH of mobile phase A is 6.3, 6.5, or 6.7, the column temperature is 32°C, 30°C, or 28°C, and when using Kromasil 100-C18, Capcell Pak C18 MG II, or Innoval C18 columns, the detection method of the present invention can effectively separate ceftriaxone sodium polymers, indicating that the detection method has good robustness.

[0211] Experimental Example 8

[0212] This test compares the generation of polymer impurities when a high-concentration solution of ceftriaxone sodium is left to stand at room temperature versus under water bath heating conditions. Specifically, the test was conducted as follows.

[0213] Preparation of room temperature degradation solution: Take an appropriate amount of ceftriaxone reference standard, dissolve it in an acetonitrile-water solution with a volume ratio of acetonitrile to water of 27:73, and prepare a 10 mg / mL solution. After standing at room temperature for 72 h, measure 1 mL and transfer it to a 10 mL volumetric flask. Add the acetonitrile-water solution with a volume ratio of 27:73 and dilute to the mark. Shake well to obtain the room temperature degradation solution.

[0214] Preparation of degradation solution: The degradation solution was obtained according to steps (1) and (2) in Example 14 of the present invention.

[0215] Chromatographic analysis: Equal amounts of the room temperature degradation solution and the degradation solution were injected into a liquid chromatograph, and the chromatographic conditions were the same as those in Examples 1-13 of the present invention. The resulting chromatograms were recorded.

[0216] The chromatogram obtained in this experimental example is as follows: Figure 15 As shown, the two curves exhibit largely consistent trends, with peaks appearing at several similar retention times, and the four polymer impurity peaks marked with an asterisk (*) can be observed in both. This indicates that a high-concentration solution of ceftriaxone reference standard, heated in a water bath at 60°C for 8 hours, produces the same number of polymer impurities as a solution left to stand at room temperature for 72 hours. Therefore, water bath heating can be used to reduce the preparation time of the degradation solution.

[0217] Comparative Example 1

[0218] Referring to the description of ceftriaxone sodium in ChP2020, the legal method for ceftriaxone sodium polymer was used, and the degradation solution prepared in Example 14 was analyzed using the G-10 method. The specific chromatographic conditions used included: a Sephadex-G10 gel chromatography column (Dalian Elite, 10.0 × 300 mm); a mobile phase of phosphate buffer (pH 7.0) [0.1 mol / L disodium hydrogen phosphate solution - 0.1 mol / L sodium dihydrogen phosphate solution (61:39)]; a flow rate of 1.5 mL / min; a detection wavelength of 254 nm; and a column temperature of 30 °C.

[0219] The chromatogram obtained using the G-10 method is as follows: Figure 16 As shown, the degradation solution eluted using the G-10 method yielded two chromatographic peaks, peak 1 and peak 2. In the G-10 system, peak 1 is generally considered to be the polymer peak, and peak 2 is the main peak.

[0220] Further analysis of the degradation solution was performed using two-dimensional liquid chromatography (HPLC). The elutions from chromatographic peaks 1 and 2 at various time points in the G-10 system were then incorporated into the RPLC system. The resulting chromatograms are shown below. Figure 17 . Figure 17 In the middle, from bottom to top, are:Figure 16 Chromatograms of the elutions from the middle section of chromatographic peak 1 (around 5.4 min), the beginning section of chromatographic peak 2 (around 11 min), the beginning section of chromatographic peak 2 (around 12 min), and the middle section of chromatographic peak 2 (around 17 min) in the RPLC system.

[0221] The results showed that both chromatographic peaks could be detected in the RPLC system. Furthermore, peak 1 and peak 2 obtained in the G-10 system both eluted more than one peak in the RPLC system, indicating that the peaks obtained by the G-10 method were co-eluting peaks of different substances and could not achieve fine separation of different substances, especially polymer impurities. Therefore, this demonstrates that the RPLC method used in this invention has superior separation efficiency and detection specificity compared to the G-10 method for detecting ceftriaxone sodium polymers.

[0222] Comparative Example 2

[0223] The degradation solution prepared in Example 14 was analyzed using the TSK method. Specific chromatographic conditions included: a spherical hydrophilic silica gel column (TSK gel G2000SWxl, 7.8 × 300 mm or equivalent column) as the packing material; phosphate buffer (pH 7.0) [0.005 mol / L disodium hydrogen phosphate solution - 0.005 mol / L sodium dihydrogen phosphate solution (61:39)] - acetonitrile (95:5) as the mobile phase; a flow rate of 0.8 mL / min; a detection wavelength of 254 nm; and a column temperature of 25 °C.

[0224] The chromatogram obtained using the TSK method is shown below. Figure 18 As can be seen, the degradation solution eluted five chromatographic peaks using the TSK method, designated as peaks 1-5. According to the retention mechanism of the TSK system, the elution order of chromatographic peaks is based on molecular weight; generally, the peaks preceding the main peak are considered polymer impurities. Based on the relative intensities of the peaks, peak 4 is the main peak, therefore peaks 1-3 are polymer impurities.

[0225] Further analysis of the degradation solution was performed using two-dimensional liquid chromatography (HPLC). The main chromatographic peaks from the TSK system were cut into the RPLC system, and the resulting chromatogram is shown below. Figure 19 shown. Specifically, Figure 19 In the middle, from bottom to top, are: Figure 18 Chromatograms of the elutions from the middle section of chromatographic peak 5 (around 11.3 min), the middle section of chromatographic peak 1 (around 8.9 min), the middle section of chromatographic peak 2 (around 9.2 min), the middle section of chromatographic peak 3 (around 9.7 min), and the middle section of chromatographic peak 4 (around 10.5 min) in the RPLC system.

[0226] The results showed that all chromatographic peaks could be detected in the RPLC system. Among the chromatographic peaks obtained using the TSK method, except for the main peak (peak 4) and the impurity following the main peak (peak 5), which were single peaks in the RPLC system, the other three peaks eluted as multiple peaks. This means that polymers 1-3 obtained using the TSK method were co-eluting peaks of different polymer impurities, failing to achieve fine separation of the polymer impurities. Therefore, this indicates that the RPLC method used in this invention has a superior separation capability for ceftriaxone sodium polymers compared to the TSK method.

[0227] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A novel method for detecting ceftriaxone sodium polymers, comprising reversing-phase high-performance liquid chromatography (RP-HPLC) for detecting ceftriaxone sodium samples, characterized in that... The ceftriaxone sodium polymer is a polymer produced by the polymerization reaction of ceftriaxone sodium during the standing process; the chromatographic conditions include: The chromatographic column uses either octadecylsilane-bonded silica gel or octylsilane-bonded silica gel as the packing material; the column temperature is 28~32℃. Mobile phase A consists of a mixed solution of aqueous and organic phases in a volume ratio of 73:

27. Phosphoric acid is added after mixing the aqueous and organic phases to adjust the pH to 6.3–6.

7. The aqueous phase is dipotassium hydrogen phosphate or n-octylamine solution, and the organic phase is methanol or acetonitrile. Mobile phase B is methanol or acetonitrile, and gradient elution is performed. The organic phase composition in mobile phase A is the same as that in mobile phase B. The gradient elution includes: The elution time is 0–5 min, the volume percentage of mobile phase A is maintained at 100%, and the volume percentage of mobile phase B is maintained at 0%. The elution time was 5–45 min, during which the volume percentage of mobile phase A decreased from 100% to 70%, and the volume percentage of mobile phase B increased from 0% to 30%. The elution time was 45-55 min, during which the volume percentage of mobile phase A decreased from 70% to 60%, and the volume percentage of mobile phase B increased from 30% to 40%. The detection method employs a diode array detector; The manufacturer of the tested ceftriaxone sodium samples was Jinhong Pharmaceutical Co., Ltd., and the batch numbers were 200707, 200708, 200709, 200710, 200711, 200712, 200713, 200714, 200715, B0476B03, B1844B05, or 8288IJ81HC for injectable ceftriaxone sodium. The ceftriaxone sodium polymer is a compound with the structural formulas shown in Formula I, Formula II and Formula III: 。 2. The novel detection method for ceftriaxone sodium polymer according to claim 1, characterized in that, The pH of the mobile phase A is adjusted to 6.

5.

3. The novel detection method for ceftriaxone sodium polymer according to claim 1, characterized in that, The concentration of the aqueous phase is 0.018 mol / L to 0.022 mol / L.

4. The novel detection method for ceftriaxone sodium polymer according to claim 3, characterized in that, The concentration of the aqueous phase is 0.020 mol / L.

5. The novel detection method for ceftriaxone sodium polymer according to claim 1, characterized in that, The chromatographic conditions also include one or more of the following: a) The flow rate is 0.5–1.5 mL / min; b) The detection wavelength is 249–261 nm; d) The injection volume is 10–30 μL.

6. The novel detection method for ceftriaxone sodium polymer according to claim 5, characterized in that, The flow rate was 1.0 mL / min.

7. The novel detection method for ceftriaxone sodium polymer according to claim 5, characterized in that, The detection wavelength is 254nm.

8. The novel detection method for ceftriaxone sodium polymer according to claim 1, characterized in that, The column temperature is 30℃.

9. The novel detection method for ceftriaxone sodium polymer according to claim 5, characterized in that, The injection volume was 20 μL.

10. A novel detection method for ceftriaxone sodium polymers according to any one of claims 1-9, characterized in that, The new detection method includes the following steps: (1) Take ceftriaxone sodium sample, dissolve it in methanol-water solution or acetonitrile-water solution, and prepare test solution and control solution; (2) Measure equal amounts of the test solution and the control solution and inject them into the liquid chromatograph for determination.

11. The novel detection method for ceftriaxone sodium polymer according to claim 10, characterized in that, The preparation of the test solution includes: taking an appropriate amount of ceftriaxone sodium sample, accurately weighing it, adding a methanol-water solution or acetonitrile-water solution with a volume ratio of organic phase to aqueous phase of 22:78 to 32:68 to dissolve it, and preparing a test solution containing 0.10 to 0.50 mg of ceftriaxone sodium sample per 1 mL.

12. The novel detection method for ceftriaxone sodium polymer according to claim 11, characterized in that, Dissolve the contents in a solution with a volume ratio of organic phase to aqueous phase of 27:

73.

13. The novel detection method for ceftriaxone sodium polymer according to claim 11, characterized in that, Each 1 mL of the test solution contains 0.22 mg of ceftriaxone sodium sample.

14. The novel detection method for ceftriaxone sodium polymer according to claim 10, characterized in that, The preparation of the control solution includes: accurately measuring the test sample solution. x mL, transfer to 100 x Add a solution with a volume ratio of organic phase to aqueous phase of 22:78 to 32:68 to a volumetric flask and dilute to the mark to obtain the control solution.

15. The novel detection method for ceftriaxone sodium polymer according to claim 14, characterized in that, Add a solution with a volume ratio of organic phase to aqueous phase of 27:73 and bring the volume to the mark.

16. The novel detection method for ceftriaxone sodium polymer according to any one of claims 1-9, characterized in that, It also includes system suitability testing: Take ceftriaxone reference standard, dissolve it in methanol-water solution or acetonitrile-water solution, heat in a water bath and then dilute to obtain degradation solution; The degradation solution was injected into a liquid chromatograph, and the resulting chromatogram was recorded.

17. The novel detection method for ceftriaxone sodium polymer according to claim 16, characterized in that, The preparation of the degradation solution specifically includes: Take an appropriate amount of ceftriaxone reference standard and dissolve it in a solution with a volume ratio of organic phase to aqueous phase of 22:78 to 32:68 to prepare a solution containing 5 to 15 mg of ceftriaxone reference standard per 1 mL. After heating in a water bath at 40–60℃ for 4–8 hours, measure... y mL, transferred to 10 y Add a solution with a volume ratio of organic phase to aqueous phase of 22:78 to 32:68 to a volumetric flask and bring the volume to the mark to obtain the degradation solution.

18. The novel detection method for ceftriaxone sodium polymer according to claim 17, characterized in that, When dissolving and adjusting the volume, add a solution with a volume ratio of organic phase to aqueous phase of 27:

73.

19. The novel detection method for ceftriaxone sodium polymer according to claim 17, characterized in that, The prepared solution contains 10 mg of ceftriaxone reference standard per 1 mL.

20. The novel detection method for ceftriaxone sodium polymer according to claim 17, characterized in that, Heating was performed using a 60℃ water bath for 8 hours.

21. The novel detection method for ceftriaxone sodium polymer according to claim 16, characterized in that, In the chromatogram of the degradation solution, the retention time of the ceftriaxone peak is 12-15 min. After the ceftriaxone peak, polymer peak 1, polymer peak 2, polymer peak 3 and polymer peak 4 appear sequentially. Among them, polymer peak 1 corresponds to the compound shown in formula I, polymer peak 2 corresponds to the compound shown in formula II, and polymer peaks 3 and 4 correspond to isomers shown in formula III.

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