Detection method of cefoxitin related substances

Abstract

The invention provides a cefoxitin related substance detection method, which comprises: dissolving a cefoxitin bulk drug with a buffer solution, and carrying out high performance liquid chromatography detection by using an ammonium formate solution as a mobile phase A, acetonitrile as a mobile phase B and phenylsilane bonded silica gel as a stationary phase to obtain the chromatogram of the bulk drug. Compared with the prior art, the detection method provided by the invention has the advantages that the impurity and the cefoxitin have better separation degree, especially the separation degree of the impurity I and the cefoxitin is better, and the impurity F and the impurity L can achieve baseline separation.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection, and in particular relates to a method for detecting cefoxitin-related substances. Background Art

[0002] Cefoxitin Sodium, chemically named (6R,7S)-3-(carbamoyloxymethyl)7-methoxy-8-oxo-7-[2-(2-thienyl)acetylamino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid sodium salt, is a cephalosporin antibiotic. It differs from cephalosporins in that it contains a methoxy group at position 7 of the β-lactam ring. This structural difference makes cefoxitin different from existing cephalosporin derivatives in terms of resistance to degradation by β-lactamase produced by Gram-negative bacteria.

[0003] Currently, there is a serious abuse of antibiotics, which has led to a gradual increase in antibiotic resistance. The drug resistance produced by bacteria has posed a serious threat to beta-lactam antibiotics. Hydrolysis and ring opening under the action of beta-lactamase is the main reason for the inactivation of this type of antibiotics. As a second-generation cephalosporin, cefoxitin will play a greater role in the field of anti-infection because of its extremely high stability against β-lactamase. Cefoxitin has been widely used in a variety of infectious diseases due to its balanced antibacterial spectrum, wider coverage, no toxic reactions, and high safety.

[0004] The quality of APIs is the key and source of drug quality control. At the same time, the research and control of API impurities is one of the key elements of API quality assurance and an important evaluation indicator for ensuring drug safety and controllability.

[0005] Based on the analysis of process impurities and main component degradation impurities that may be produced in the synthesis process of cefoxitin raw materials, and combined with the control of related substances of this variety in the pharmacopoeias of various countries, the impurity spectrum that needs to be studied is determined and shown in Table 1.

[0006] Table 1 Impurities contained in cefoxitin API

[0007]

[0008]

[0009]

[0010] Many impurities in the detection method of related substances of cefoxitin raw materials in the Chinese Pharmacopoeia (ChP) cannot be completely separated, such as impurity I, impurity L and impurity F overlap, impurity N and impurity D overlap in most cases, and only specific impurities can be detected, and impurity I cannot be detected. Summary of the invention

[0011] In view of this, the technical problem to be solved by the present invention is to provide a method for detecting cefoxitin-related substances, which can separate multiple impurities.

[0012] The present invention provides a method for detecting related substances of cefoxitin, comprising the following steps: dissolving the cefoxitin raw material drug in a buffer solution, using ammonium formate solution as mobile phase A, acetonitrile as mobile phase B, and phenylsilane bonded silica gel as stationary phase, performing high performance liquid chromatography detection, and obtaining a chromatogram of the raw material drug.

[0013] The detection method of cefoxitin-related substances provided by the present invention is carried out by high performance liquid chromatography. Quantitative detection of substances by high performance liquid chromatography (HPLC) is a commonly used detection method at present, but the impurities in cefoxitin cannot be completely separated in the prior art. The detection method provided by the present invention enables the impurities and cefoxitin to have a better separation degree, especially the separation degree of impurity I and cefoxitin is better, and impurity F and impurity L can achieve baseline separation.

[0014] Specifically, in the system suitability solution chromatogram, the retention time of the cefoxitin peak is about 35 minutes, the separation between the impurity H and impurity E peaks should be no less than 2.0; the peak-to-valley ratio between the impurity I and the cefoxitin peak should be no less than 2.0; in the sensitivity solution chromatogram, the signal-to-noise ratio of the cefoxitin peak should be no less than 10.

[0015] In the present invention, the buffer is preferably a phosphate buffer; the pH value of the buffer is preferably 6.5 to 7, more preferably 6.7 to 6.9, and even more preferably 6.8.

[0016] In a specific embodiment provided by the present invention, the phosphate buffer is preferably prepared according to the following method: 34.836 g of dipotassium hydrogen phosphate is dissolved in 1000 mL of water, the pH value is adjusted to 6.5-7 with phosphoric acid, 20 mL is taken, and diluted to 1000 mL with water.

[0017] In the present invention, the cefoxitin raw material is dissolved in a buffer solution to obtain a test solution; the concentration of the cefoxitin raw material in the test solution is preferably 2-3 mg / mL, more preferably 2.2-2.8 mg / mL, further preferably 2.4-2.6 mg / mL, and most preferably 2.5 mg / mL.

[0018] In a specific embodiment provided by the present invention, the concentration of ammonium formate in the ammonium formate solution is preferably 0.5-1.5 g / L, more preferably 0.8-1.2 g / L, and more preferably 1.0 g / L; the pH value of the ammonium formate solution is preferably 2.5-3, more preferably 2.6-2.8, and more preferably 2.7; the pH value of the ammonium formate solution is preferably adjusted with formic acid.

[0019] In a specific embodiment provided by the present invention, the stationary phase is preferably an Inertsil ph-3 phenyl chromatographic column, more preferably GL Sciences Inertsil ph-3.

[0020] In a specific embodiment provided by the present invention, the specifications of the stationary phase are preferably 250×4.6 mm, 3 μm.

[0021] The invention uses ammonium formate solution as mobile phase A, acetonitrile as mobile phase B and phenylsilane bonded silica gel as stationary phase to perform high performance liquid chromatography detection on the test solution.

[0022] In a specific embodiment provided by the present invention, the injection volume of the HPLC is preferably 10 to 20 μL.

[0023] In a specific embodiment provided by the present invention, the elution program of the high performance liquid chromatography is: 0-12 min, the volume fraction of mobile phase A is 90%±2%; 12-37 min, the volume fraction of mobile phase A is from 90%±2% to 80%±2%; 37-50 min, the volume fraction of mobile phase A is from 80%±2% to 60%±2%; 50-55 min, the volume fraction of mobile phase A is from 60%±2% to 20%±2%; 55-60 min, the volume fraction of mobile phase A is 20%±2%; 60-62 min, the volume fraction of mobile phase A is from 20%±2% to 90%±2%; 62-72 min, the volume fraction of mobile phase A is 90%±2%.

[0024] Further specifically, the elution procedure of the high performance liquid chromatography is: 0-12min, the volume fraction of mobile phase A is 90%; 12-37min, the volume fraction of mobile phase A is from 90% to 80%; 37-50min, the volume fraction of mobile phase A is from 80% to 60%; 50-55min, the volume fraction of mobile phase A is from 60% to 20%; 55-60min, the volume fraction of mobile phase A is 20%; 60-62min, the volume fraction of mobile phase A is from 20% to 90%; 62-72min, the volume fraction of mobile phase A is 90%.

[0025] Mobile phase A and mobile phase B together constitute the mobile phase; in a specific embodiment provided by the present invention, the flow rate of the mobile phase is preferably 0.9-1.1 mL / min, more preferably 1.0 mL / min.

[0026] In a specific embodiment provided by the present invention, the column temperature of the HPLC detection is preferably 20°C to 30°C, more preferably 25°C.

[0027] In a specific embodiment provided by the present invention, the detection wavelength of the HPLC detection is preferably 250-260 nm, more preferably 254 nm.

[0028] In the present invention, the content of the cefoxitin-related substances is obtained by an external standard method.

[0029] In a specific embodiment provided by the present invention, the content of the cefoxitin-related substances is obtained by an external standard method, specifically: the cefoxitin reference substance is dissolved in a buffer solution, ammonium formate solution is used as mobile phase A, acetonitrile is used as mobile phase B, and phenylsilane bonded silica gel is used as the stationary phase, and high performance liquid chromatography is performed to obtain a chromatogram of the reference substance; according to the chromatogram of the reference substance and the chromatogram of the raw material drug, the content of the cefoxitin-related substances is obtained.

[0030] In a specific embodiment provided by the present invention, the cefoxitin reference substance is dissolved in a buffer solution to obtain a reference substance solution; the buffer solution is the same as described above and will not be described in detail herein; the concentration of the cefoxitin reference substance in the reference substance solution is preferably 2 to 3 μg / mL, more preferably 2.2 to 2.8 μg / mL, further preferably 2.4 to 2.6 μg / mL, and most preferably 2.5 μg / mL.

[0031] The reference substance solution is subjected to high performance liquid chromatography with ammonium formate solution as mobile phase A, acetonitrile as mobile phase B, and phenylsilane bonded silica gel as stationary phase to obtain a chromatogram of the reference substance; the conditions for the high performance liquid chromatography detection are the same as those for the high performance liquid chromatography detection of the test solution.

[0032] According to the chromatogram of the reference substance and the chromatogram of the raw material drug, the content of cefoxitin-related substances was obtained.

[0033] In a specific embodiment provided by the present invention, the content of cefoxitin-related substances is obtained according to the chromatogram of the reference substance and the chromatogram of the raw material drug according to the formulas shown in formula (I) and formula (II).

[0034]

[0035] m 对is the sample amount of cefoxitin reference substance in the reference solution, mg; α is the cefoxitin (C 16 H 17 N3O7S2) content, %; A 对 V is the peak area of ​​cefoxitin in the chromatogram of the reference solution; 对 A is the dilution volume multiple of the reference solution, mL; 杂 V is the peak area of ​​each impurity peak in the chromatogram of the test solution; 供 is the dilution volume multiple of the test solution, mL; m 供 is the weighed amount of cefoxitin sodium in the test solution, mg; 449.43 is the molecular weight of cefoxitin sodium; 427.43 is the molecular weight of cefoxitin. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is the positioning test chromatogram of the detection method of related substances of ChP raw materials;

[0037] Figure 2 It is the chromatogram of the localization test for the detection method of related substances of BP API;

[0038] Figure 3 The chromatogram is of the impurity mixed solution at pH 2.9 of phase A (other conditions are ChP standard conditions);

[0039] Figure 4 The chromatogram is of a 5 mg / mL test solution at pH 2.9 in phase A (other conditions are ChP standard conditions);

[0040] Figure 5 is the chromatogram of the mixed solution;

[0041] Figure 6 Chromatogram of 20 μL injection of 5 mg / mL test solution;

[0042] Figure 7 This is the chromatogram of a 10 μl injection of a 5 mg / mL test solution;

[0043] Figure 8 Typical chromatogram of mixed solution - 235nm;

[0044] Fig. 9 Chromatogram of 20 μL injection of 2.5 mg / mL test solution - 235 nm;

[0045] Fig.10 is the full wavelength scanning UV absorption spectrum of impurity A;

[0046] Fig.11 is the full wavelength scanning UV absorption spectrum of impurity B;

[0047] Fig.12 is the full wavelength scanning UV absorption spectrum of impurity C;

[0048] Fig.13 is the full wavelength scanning UV absorption spectrum of impurity D;

[0049] Fig.14 is the full wavelength scanning UV absorption spectrum of impurity E;

[0050] Fig.15 is the full wavelength scanning ultraviolet absorption spectrum of impurity F;

[0051] Fig.16 is the full wavelength scanning UV absorption spectrum of impurity H;

[0052] Fig.17 is the full wavelength scanning ultraviolet absorption spectrum of impurity I;

[0053] Fig.18 is the full wavelength scanning UV absorption spectrum of impurity K;

[0054] Fig.19 is the full wavelength scanning ultraviolet absorption spectrum of impurity L;

[0055] Fig. 20 is the full wavelength scanning ultraviolet absorption spectrum of impurity M;

[0056] Fig.21 is the full wavelength scanning ultraviolet absorption spectrum of impurity N;

[0057] Fig. 22 This is the full wavelength scanning UV absorption spectrum of cefoxitin;

[0058] Fig.23 It is the chromatogram of the mixed solution detected by dual wavelength;

[0059] Fig.24 This is a typical chromatogram of the system suitability solution;

[0060] Fig.25 This is a typical chromatogram of the system suitability solution;

[0061] Fig.26 This is the RRT1.67 mass spectrum obtained by the BP API method;

[0062] Fig. 27 This is the mass spectrum of RRT1.43 in the proposed method;

[0063] Fig.28 The chromatograms are related substances, of which the upper figure is the chromatogram of the proposed related substance method, and the lower figure is the chromatogram of the BP API related substance method;

[0064] Fig.29This is a typical spectrum of EP Cefoxitin for peak identification on CRS COA. DETAILED DESCRIPTION

[0065] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0066] In order to further illustrate the present invention, a method for detecting cefoxitin-related substances provided by the present invention is described in detail below in conjunction with examples.

[0067] The reagents used in the following examples are all commercially available.

[0068] Comparison of Pharmacopoeia Methods

[0069] A comparison of the detection methods for relevant substances in the pharmacopoeias of various countries is shown in Table 2.

[0070] Table 2 Detection methods of relevant substances in pharmacopoeias of various countries

[0071]

[0072]

[0073] Comparing the chromatographic conditions of various pharmacopoeias, USP does not control related substances; the detection methods for related substances of ChP APIs, ChP preparations and BP preparations are consistent, with slightly different limits; the detection method for BP APIs is different from other standard detection methods. Therefore, the subsequent research will focus on the detection methods for related substances in the standards of ChP APIs and BP APIs.

[0074] In the following embodiments of the present invention, unless otherwise specified, the ChP bulk drug corresponds to the following in Table 2: ChP2020 cefoxitin sodium / cefoxitin sodium for injection; the BP bulk drug corresponds to the following in Table 2: BP2022 / EP11.2 cefoxitin sodium.

[0075] 2) Changes in calculation method

[0076] Since the detection method for related substances in ChP raw materials takes 72 minutes per injection and the detection method for related substances in BP raw materials takes 95 minutes per injection, and both methods use the self-control method to calculate the content of each impurity, which is time-consuming, in order to save the detection cost, the study of the main component external standard method for calculating the content of each impurity was added. The results are shown in Table 3.

[0077] Reference solution for the main component external standard method: Accurately weigh an appropriate amount of cefoxitin reference substance, dissolve it in phosphate buffer and dilute it to make a solution containing approximately 2.5 μg of cefoxitin per 1 mL.

[0078] Table 3 Comparison of principal component calculations by external standard method and self-reference method

[0079]

[0080]

[0081]

[0082] From the above results, it can be seen that there is no significant difference in the test results using the main component external standard method and the self-control method. The calculation method of the related substances of this product can be changed to the main component external standard method. Since the main component reference substance provided by the China National Institute for Inspection and Quarantine is cefoxitin, it is necessary to convert the molecular weight with cefoxitin sodium, so the calculation formula after the modified calculation method is as follows:

[0083]

[0084] In the formula, m 对 is the sample amount of cefoxitin reference substance in the reference solution, mg; α is the cefoxitin (C 16 H 17 N3O7S2) content, %; A 对 V is the peak area of ​​cefoxitin in the chromatogram of the reference solution; 对 A is the dilution volume multiple of the reference solution, ml; 杂 V is the peak area of ​​each impurity peak in the chromatogram of the test solution; 供 is the dilution volume multiple of the test solution, ml; m 供 is the weighed amount of cefoxitin sodium in the test solution, mg; 449.43 is the molecular weight of cefoxitin sodium; 427.43 is the molecular weight of cefoxitin.

[0085] Note: The reference solution is prepared according to the limit concentration of other impurities, that is, 0.1% of the test solution concentration.

[0086] 3) Reproduction of pharmacopoeia methods

[0087] The localization results of the detection method of related substances of ChP and BP raw materials are shown in Table 4. The localization test chromatogram of the detection method of related substances of ChP raw materials is obtained as follows: Figure 1 As shown; the localization test chromatogram of the BP raw material drug related substance detection method is obtained as shown Figure 2 shown.

[0088] Table 4 Positioning test for detection methods of related substances of ChP and BP APIs

[0089]

[0090]

[0091] From the above results, it can be seen that the test solution concentration of the ChP API related substance detection method is 5 mg / mL, which contains impurity I, and impurity L and impurity F overlap, and impurity N and impurity D overlap in most cases. From previous studies, it can be seen that the test solution concentration of the BP API related substance detection method is 1 mg / mL, and the valley-to-height ratio of impurity I to cefoxitin is greater than 2.0, but impurity L and impurity F overlap.

[0092] Multiple batches of APIs were tested for impurities using the relevant substance methods in the ChP API and BP API standards. The results are shown in Table 5.

[0093] Table 5 Results of related substances testing of multiple batches of APIs by the ChP and BP API methods

[0094]

[0095]

[0096] For the detection of specific impurities, the ChP raw material related substance detection method cannot detect impurity I, and there is no significant difference in the detection results of other specific impurities; for the total impurities after removing impurity I, the total impurity content detected by the ChP raw material related substance detection method is slightly higher.

[0097] Considering that the maximum proportion of organic phase in the detection method of related substances of ChP raw materials (80%) is much larger than the maximum proportion of organic phase in the detection method of related substances of BP raw materials (26%), impurities with small polarity are more easily eluted, and considering that single-needle collection is time-consuming, it is considered to optimize the detection method of related substances of ChP raw materials.

[0098] 4) Preliminary test on the durability of the test methods for ChP API

[0099] Mixed solution: Take appropriate amounts of impurity K, impurity A, impurity I, impurity H, impurity E, impurity F, impurity L, impurity B, impurity M, impurity N, impurity C and cefoxitin reference substance respectively to prepare a solution containing 12.5 μg of each impurity and 2.5 mg of cefoxitin per 1 ml.

[0100] Table 6 Exploration of relevant detection methods

[0101]

[0102] According to Table 6, the mobile phase pH, method gradient, etc. were optimized to investigate the impurities in the mixed solution. The results are shown in Table 7. The chromatogram of the impurity mixed solution of phase A pH 2.9 (other conditions are ChP standard conditions) is as follows: Figure 3 As shown; the chromatogram of the 5 mg / mL test solution of phase A pH 2.9 (others are ChP standard conditions) is as follows Figure 4 As shown.

[0103] Table 7 ChP API method robustness mixed solution results

[0104]

[0105]

[0106] From Table 7, Figure 3 and Figure 4 It can be seen that under the ChP standard conditions, impurity L and impurity F were not separated, and the separation degrees of impurity L and impurity F were less than 1 or not separated when the mobile phase pH was changed to 2.5, the gradient was changed, and the ChP gradient was used; when the mobile phase and pH were changed to 2.9, the separation degree of impurity L and impurity F was greater than 1.5, but the peak shape of cefoxitin in the chromatogram of the 5 mg / mL test solution was poor; in addition, impurity I and cefoxitin were not separated under all chromatographic conditions.

[0107] 5) Replacement of chromatographic columns for ChP API related testing methods

[0108] Considering that the separation of impurity I and cefoxitin peak under the BP raw material drug related substance detection method is better than that under the chromatographic conditions of the ChP raw material drug related substance detection method, after comprehensive evaluation, the chromatographic column recommended for use under the BP raw material drug related substance detection method is GL Sciences Inertsil ph-3 250×4.6mm, 3μm, which has a smaller particle size than the chromatographic column recommended under the ChP raw material drug related substance detection method (Agilent ZORBAX SB-Phenyl 4.6×250mm, 5μm), and is more conducive to the separation of impurities. Therefore, the chromatographic column was changed to GL Sciences Inertsil ph-3 250×4.6mm, 3μm chromatographic column, and other chromatographic conditions remained unchanged (i.e., the ChP raw material drug related substance detection method). The mixed solution (i.e., a solution containing 12.5μg of each impurity and 1mg of cefoxitin per 1ml) was investigated. The results are shown in Table 8; the chromatogram of the mixed solution is shown in Table 8. Figure 5 As shown; the chromatogram of 20 μL of 5 mg / mL test solution injection is as follows Figure 6 As shown; the chromatogram of 10 μl of 5 mg / mL test solution injection is as follows Figure 7 shown.

[0109] Table 8 Determination results of mixed solution after replacing the chromatographic column

[0110] name RRT Tailing Factor Separation Impurity A 0.81 1.00 12.62 Impurity I 0.98 na 1.07 Cefoxitin 1.00 2.17 3.31 Impurity H 1.06 1.02 3.48 Impurity E 1.10 1.01 2.47 Impurity F 1.14 1.02 1.81 Impurity L 1.17 na 3.57 Impurity B 1.22 0.99 20.13 Impurity C 1.44 0.82 1.52 Impurity N 1.45 na na

[0111] From Table 8 and Figures 5 to 7 It can be seen that under this chromatographic column, in the chromatogram of the mixed solution, impurity F and impurity L can be separated, and the symmetry factor of cefoxitin is 2.17. However, when the cefoxitin concentration in the mixed solution injected in the above results is about 1 mg / mL and the sample concentration is 5 mg / mL (i.e., the concentration of the test solution under the ChP method), impurity I is wrapped by the cefoxitin peak.

[0112] 6) Change of mobile phase composition in the detection method of ChP API

[0113] The aqueous mobile phase of the ChP API related substance detection method is water with formic acid adjusted to pH 2.7, which has weak buffering capacity and poor reproducibility. The aqueous mobile phase under the reference BP API related substance detection method (i.e., 1 g / L ammonium formate solution, pH adjusted to 2.7 with formic acid) is still the GL Sciences Inertsil ph-3250×4.6mm, 3μm column, and other chromatographic conditions remain unchanged (i.e., ChP API related substance detection method). The results of the mixed solution (cefoxitin concentration is about 1 mg / mL, and each impurity is 12.5μg / L) are shown in Table 9; the typical chromatogram of the mixed solution is obtained as shown in -235nm. Figure 8 As shown; the chromatogram of 20 μL of 2.5 mg / mL test solution was obtained -235 nm as Fig. 9 shown.

[0114] Table 9 Measurement results of mixed solution after changing the mobile phase components

[0115]

[0116] From Table 9 and Figures 8-9 It can be seen that under this condition, impurity I and cefoxitin can meet the requirements, and impurity F and impurity L can achieve baseline separation. This chromatographic condition is tentatively determined as the chromatographic condition for the detection of related substances of this product.

[0117] 7) Detection wavelength

[0118] The full wavelength scanning results of each impurity and cefoxitin are shown in Table 10 and Figure 10 to Figure 22 As shown, Fig.10 is the full wavelength scanning UV absorption spectrum of impurity A; Fig.11 is the full wavelength scanning UV absorption spectrum of impurity B; Fig.12 is the full wavelength scanning UV absorption spectrum of impurity C; Fig.13 is the full wavelength scanning UV absorption spectrum of impurity D; Fig.14 is the full wavelength scanning UV absorption spectrum of impurity E; Fig.15 is the full wavelength scanning ultraviolet absorption spectrum of impurity F; Fig.16 is the full wavelength scanning UV absorption spectrum of impurity H; Fig.17 is the full wavelength scanning ultraviolet absorption spectrum of impurity I; Fig.18 is the full wavelength scanning UV absorption spectrum of impurity K; Fig.19 is the full wavelength scanning ultraviolet absorption spectrum of impurity L; Fig. 20 is the full wavelength scanning ultraviolet absorption spectrum of impurity M; Fig.21 is the full wavelength scanning ultraviolet absorption spectrum of impurity N; Fig. 22 This is the full wavelength scanning UV absorption spectrum of cefoxitin.

[0119] Table 10 Full wavelength scanning results of each impurity

[0120]

[0121]

[0122] Referring to the wavelengths of determination of related substances of cefoxitin API in ChP2020 and BP2022, each specific impurity has maximum absorption at wavelengths of 235nm and 254nm. From the ultraviolet absorption results of the above specific impurities and cefoxitin, it can be seen that at a wavelength of 254nm, the absorption of each specific impurity and cefoxitin at a wavelength of about 254nm is relatively gentle, indicating that the detection stability of this method at a wavelength of 254nm is better; comparing the dual wavelength results in the mixed solution (see Table 11 and Fig.23 , Fig.23 The figure is a chromatogram of the mixed solution detected by dual wavelengths. Since the baseline noise is smaller and the baseline is flatter at 254nm, the signal-to-noise ratio of each specific impurity at 254nm is larger than that at 235nm, indicating that the detection capability of related substances of cefoxitin sodium is stronger at 254nm, so the detection wavelength of related substances is selected as 254nm.

[0123] Chromatographic conditions in Table 11: Chromatographic column GL Sciences Inertsil ph-3 250×4.6mm, 3μm); mobile phase A is 1.0g / L ammonium formate solution (adjusted to pH 2.7 with formic acid), mobile phase B is acetonitrile, flow rate is 1.0mL per minute, column temperature is 25°C, injection volume is 20μL. Elution program is: 0-12min, volume fraction of mobile phase A is 90%; 12-37min, volume fraction of mobile phase A is from 90% to 80%; 37-50min, volume fraction of mobile phase A is from 80% to 60%; 50-55min, volume fraction of mobile phase A is from 60% to 20%; 55-60min, volume fraction of mobile phase A is 20%; 60-62min, volume fraction of mobile phase A is from 20% to 90%; 62-72min, volume fraction of mobile phase A is 90%.

[0124] Table 11 Proposed related substance detection method - mixed solution measurement results at different wavelengths

[0125]

[0126]

[0127] 8) Test solution

[0128] The concentration of the test solution under the ChP raw material related substance detection method is 5 mg / ml (detection wavelength is 235nm, injection volume is 20μL); the concentration of the test solution under the BP raw material related substance detection method is 1 mg / ml (detection wavelength is 254nm, injection volume is 20μL). Taking into account the detection sensitivity and impurity separation of the final proposed related substance detection method, the final proposed test solution concentration for related material detection is 2.5 mg / mL, and the injection volume is 20μL (detection wavelength 254nm).

[0129] The diluent used was phosphate buffer under the ChP API related substance detection method, i.e., 34.836 g of dipotassium hydrogen phosphate was weighed, dissolved in 1000 mL of water, the pH value was adjusted to 6.8 with phosphoric acid, 20 mL was taken, and diluted to 1000 mL with water.

[0130] 9) Sensitivity solution

[0131] In order to ensure the detection sensitivity of the final proposed related substance detection method, the sensitivity solution is added. According to the relevant requirements of the EP monograph: the concentration of the preparation of the sensitivity solution of the related substances of this product should not be higher than 1 / 20 of the impurity limit of concern, and not lower than the detection limit (the minimum impurity limit of the raw material drug is 0.10%), so the proposed sensitivity solution is 0.02% of the concentration of the test solution, that is, 0.5μg / mL. According to experimental research, at this concentration, the signal-to-noise ratio of the cefoxitin peak is about 125, which meets the detection requirements.

[0132] 10) System suitability solution

[0133] ①. The relevant contents of system suitability under the detection method of related substances of BP API are as follows:

[0134] System suitability solution: Dissolve 5 mg of cefoxitin identification peak reference (containing impurities A, B, E, H, I and J) in diluent and dilute to 5 mL with diluent.

[0135] System suitability requirements: The minimum resolution between the impurity H and impurity E peaks is 2.0; the minimum peak-to-valley ratio between the impurity I and cefoxitin peaks is 2.0.

[0136] ②. The relevant contents of system suitability under the detection method of related substances of ChP raw materials / preparation are as follows:

[0137] System suitability solution: Take an appropriate amount of cefoxitin reference substance, dissolve and dilute with phosphate buffer to make a solution containing approximately 0.25 mg per 1 mL, place in a 70°C water bath for 1 hour, and cool.

[0138] System suitability requirements: In the system suitability solution chromatogram, the retention time of the cefoxitin peak is about 35 minutes, and the separation between the cefoxitin peak and the impurity peak with a relative retention time of about 0.8 should be greater than 5.0.

[0139] ③. The relevant contents of system suitability under the detection method of related substances of BP preparations are as follows:

[0140] System suitability solution: Add 7 mL of water and 2 mL of methanol to 1 mL of the test solution, add 25 mg of sodium carbonate, stir at room temperature for 10 minutes, heat to 70°C in a water bath for 30 minutes, and then cool. Add 3 drops of glacial acetic acid and 0.4 mL of the above test solution to mix (to generate cefoxitin lactone, i.e., impurity C).

[0141] System suitability requirement: In the system suitability solution chromatogram, the resolution between the cefoxitin peak and the peak with a cefoxitin relative retention time of approximately 1.31 (cefoxitin lactone) is at least 5.0.

[0142] ④. Test the mixed solution using the final proposed method for testing related substances.

[0143] Table 12 Results of mixed solutions of various impurities under the final proposed related substance detection method

[0144]

[0145]

[0146] According to the test results of various mixed impurities under the final proposed relevant substance detection method and the system suitability requirements of various pharmacopoeias, the separation between the peaks of impurity H and impurity E, the separation between the peak of impurity I and cefoxitin, and the separation between the peak of impurity A and cefoxitin need to be focused on. Therefore, the system suitability related contents in the proposed standard are as follows:

[0147] System suitability solution: Weigh 2.5 mg of cefoxitin identification peak reference (containing impurities A, B, E, H, I and J), dissolve in 1 mL of phosphate buffer, and mix well.

[0148] System suitability requirements: In the system suitability solution chromatogram, the retention time of the cefoxitin peak is about 35 minutes, the separation between the impurity H and impurity E peaks should be no less than 2.0; the peak-to-valley ratio between the impurity I and the cefoxitin peak should be no less than 2.0. A typical spectrum of the system suitability solution chromatogram is as follows: Fig.24 and Fig.25 As shown, Fig.24 Only A / B / E / H / I / J are marked. Fig.25 Each impurity is assigned to a specific impurity according to its location.

[0149] 11) Results comparison

[0150] The final proposed related substance detection method was used to determine the related substances of the three batches of reference preparations. The results of the proposed detection method, the ChP raw material related substance determination method, and the BP raw material related substance determination method were compared. The results are shown in Table 13.

[0151] Proposed detection method: chromatographic column GL Sciences Inertsil ph-3 250×4.6mm, 3μm); mobile phase A is 1.0g / L ammonium formate solution (adjust pH to 2.7 with formic acid), mobile phase B is acetonitrile, flow rate is 1.0mL per minute, column temperature is 25℃, detection wavelength is 254nm; injection volume is 20μL. Elution program is: 0-12min, volume fraction of mobile phase A is 90%; 12-37min, volume fraction of mobile phase A is from 90% to 80%; 37-50min, volume fraction of mobile phase A is from 80% to 60%; 50-55min, volume fraction of mobile phase A is from 60% to 20%; 55-60min, volume fraction of mobile phase A is 20%; 60-62min, volume fraction of mobile phase A is from 20% to 90%; 62-72min, volume fraction of mobile phase A is 90%.

[0152] Table 13 Comparison of test results of different determination methods

[0153]

[0154]

[0155]

[0156] The results in Table 13 show that there is no significant difference between the test results of the proposed related substance detection method and the test results of the related substance detection method in the ChP raw material drug standard (total impurities excluding impurity I) and the related substance detection method in the BP raw material drug standard. The proposed related substance method can be used as the final method for the detection of related substances of this product.

[0157] (III) Prediction of retention time of impurity J

[0158] In each standard, the relative retention time of impurity J is specified only under the related substance detection method of BP API, which is RRT1.66, but the structure is unknown. According to the related substance detection method of cefoxitin API, EP Cefoxitin for peak identification CRS (EDQM batch number 1, specification 15mg / vial, containing impurities A / B / E / H / I / J) is used to locate impurity J. The detection spectrum of EP Cefoxitin for peak identification CRS reproduces well with the typical spectrum on its COA, and the number and size of peaks on the chromatogram can correspond one to one. The peak at the RRT1.67 position should be impurity J, and the m / z of the corresponding compound is 870; the related substance detection EP Cefoxitin for peak identification CRS is proposed, and the number and size of peaks on the detection chromatogram can correspond one to one with the spectrum reproduced under the related substance method of BP API, and the m / z of the compound with RRT1.43 in the proposed related substance method is 870, so it is inferred that the position is impurity J. The results are shown in Table 14. Figure 26 to Figure 29 ,in Fig.26 This is the RRT1.67 mass spectrum obtained by the BP API method; Fig. 27 This is the mass spectrum of RRT1.43 in the proposed method; Fig.28 The chromatograms are related substances, of which the upper figure is the chromatogram of the proposed related substance method, and the lower figure is the chromatogram of the BP API related substance method; Fig.29 This is a typical spectrum of EP Cefoxitin for peak identification on CRS COA.

[0159] Table 14 Research results of impurity J

[0160]

[0161] (IV) RRT of each impurity under the proposed method

[0162] The relative retention time of the peaks of each impurity under the proposed method for detecting related substances is formulated based on the actual measured results. The summary results of RRT when different people conducted the test at different times are shown in 15. The results show that the RRT is within 5% of the mean, so the mean is temporarily taken as the RRT of each impurity in the proposed method standard.

[0163] Table 15 Summary of RRT of each impurity peak when the proposed method was tested by the same person at different times

[0164]

[0165]

[0166] (V) Limit setting

[0167] Based on the control limits of various impurities in the pharmacopoeias of various countries (see Table 16), the most stringent limits are formulated to ensure the quality of this product.

[0168] Table 16 Limits of related substances in the pharmacopoeias of various countries for cefoxitin sodium injection

[0169]

[0170] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for detecting cefoxitin-related substances, characterized in that: The following steps are involved: The cefoxitin raw material drug was dissolved in a buffer solution, and high performance liquid chromatography was performed using ammonium formate solution as mobile phase A, acetonitrile as mobile phase B, and phenylsilane bonded silica gel as stationary phase to obtain a chromatogram of the raw material drug.

2. The detection method according to claim 1, characterized in that: The elution procedure of the high performance liquid chromatography is as follows: from 0 to 12 minutes, the volume fraction of the mobile phase A is 90%±2%; from 12 to 37 minutes, the volume fraction of the mobile phase A is from 90%±2% to 80%±2%; from 37 to 50 minutes, the volume fraction of the mobile phase A is from 80%±2% to 60%±2%; from 50 to 55 minutes, the volume fraction of the mobile phase A is from 60%±2% to 20%±2%; from 55 to 60 minutes, the volume fraction of the mobile phase A is 20%±2%; from 60 to 62 minutes, the volume fraction of the mobile phase A is from 20%±2% to 90%±2%; and from 62 to 72 minutes, the volume fraction of the mobile phase A is 90%±2%.

3. The detection method according to claim 1, characterized in that: The elution procedure of the high performance liquid chromatography is as follows: from 0 to 12 minutes, the volume fraction of the mobile phase A is 90%; from 12 to 37 minutes, the volume fraction of the mobile phase A is from 90% to 80%; from 37 to 50 minutes, the volume fraction of the mobile phase A is from 80% to 60%; from 50 to 55 minutes, the volume fraction of the mobile phase A is from 60% to 20%; from 55 to 60 minutes, the volume fraction of the mobile phase A is 20%; from 60 to 62 minutes, the volume fraction of the mobile phase A is from 20% to 90%; and from 62 to 72 minutes, the volume fraction of the mobile phase A is 90%.

4. The detection method according to claim 1, characterized in that: The buffer is a phosphate buffer; the pH value of the buffer is 6.5-7.

5. The detection method according to claim 1, characterized in that: The concentration of ammonium formate in the ammonium formate solution is 0.5-1.5 g / L; the pH value of the ammonium formate solution is 2.5-3.

6. The detection method according to claim 1, characterized in that: The flow rate of the mobile phase is 0.9-1.1 mL / min; The column temperature of the high performance liquid chromatography detection is 20°C to 30°C; The detection wavelength of the high performance liquid chromatography detection is 250 to 260 nm.

7. The detection method according to claim 1, characterized in that: The flow rate of the mobile phase is 1 mL / min; The column temperature of the HPLC detection is 25°C; The detection wavelength of the high performance liquid chromatography detection is 254 nm.

8. The detection method according to claim 1, characterized in that: The stationary phase is an Inertsilph-3 phenyl chromatographic column.

9. The detection method according to claim 1, characterized in that: The contents of the cefoxitin-related substances are obtained by using an external standard method.

10. The detection method according to claim 9, characterized in that: The cefoxitin reference substance was dissolved in a buffer solution, and high performance liquid chromatography was performed using ammonium formate solution as mobile phase A, acetonitrile as mobile phase B, and phenylsilane bonded silica gel as stationary phase to obtain a chromatogram of the reference substance; According to the chromatogram of the reference substance and the chromatogram of the raw material drug, the content of cefoxitin-related substances was obtained.

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