A method for detecting related substances of cefcapene pivoxil hydrochloride granules

Abstract

This invention provides a method for detecting related substances in cefoperazone hydrochloride particles. The method employs high-performance liquid chromatography (HPLC), wherein mobile phase A is phosphate buffer and mobile phase B is a methanol-water mixture; the chromatographic column is a column packed with octadecylsilane-bonded silica gel; and the sample is diluted with a methanol-water (1:1) mixture at pH 3-4. This method achieves separation of the four related substances in cefoperazone particles using a single analytical method (resolution > 1.5), and the use of a specific diluent ensures the stability of impurity A during detection, thereby guaranteeing the accuracy of the results.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation analysis, specifically relating to a method for detecting related substances in cefoperazone hydrochloride particles. Background Technology

[0002] Cefcaptin hydrochloride granules are a third-generation oral cephalosporin antibiotic with a broad antibacterial spectrum, showing significant efficacy against infections caused by a variety of susceptible bacteria. During the production and storage of cefcaptin hydrochloride granules, various impurities and degradation products may be generated, and the presence of these substances directly affects the quality and efficacy of the drug.

[0003] The 2020 edition of the Chinese Pharmacopoeia includes methods for detecting related substances in cefoperazone. However, cefoperazone contains numerous impurities that readily precipitate, and the methods in the pharmacopoeia suffer from poor separation of components, low detection efficiency, and low sensitivity, failing to meet the requirements for formulation detection. The 2018 edition of the Japanese Pharmacopoeia employs specific chromatographic conditions for quality control of cefoperazone hydrochloride; however, this method only detects relatively obvious main drug peaks.

[0004] The 2021 "Imported Drug Registration Standard" for cefoperazone granules stipulates △ 2 - The content of cefoperazone (relative retention time 1.3, correction factor 1.6) shall not exceed 0.6%, the content of cefoperazone trans isomer (relative retention time 1.5, correction factor 1.3) shall not exceed 1.4%, the content of cefoperazone (relative retention time 0.24) shall not exceed 0.3%, the content of cefoperazone ester dimer (relative retention time 1.7) shall not exceed 0.3%, the content of other individual impurities shall not exceed 0.2%, and the content of total impurities shall not exceed 3.2%. However, when using the imported registration standard, the detection time is 96 minutes, which is a long detection time and the separation is poor.

[0005] CN118146239A discloses a method for detecting cefoperazone and its related substances using high-performance liquid chromatography (HPLC); however, it can only simultaneously detect one related substance and cefoperazone. CN115791996A discloses a method for detecting the intermediate cefoperazone precursor acid and its related substances using HPLC; however, the resolution between the cefoperazone precursor acid and the related substances in this method is only about 1.5, and this chromatographic method cannot detect stable impurities Δ. 2 -Cefoperazone. Therefore, the above detection conditions may interfere with the detection of impurities.

[0006] Therefore, it is still necessary to develop an efficient and accurate detection method that can detect all related substances of cefcapine hydrochloride and has good separation effect, so as to ensure the quality and safety of the product is controllable. Summary of the Invention

[0007] Based on the defects and deficiencies in the existing technology, the main objective of this invention is to provide a method for detecting related substances in cefcapine hydrochloride particles. This method can detect impurities and main components with high accuracy.

[0008] The specific technical method of this invention is as follows:

[0009] A method for detecting cefoperazone hydrochloride particles-related substances, wherein the detection method employs high-performance liquid chromatography (HPLC), wherein mobile phase A is phosphate buffer and mobile phase B is a methanol-water mixture; the chromatographic column is an octadecylsilane-bonded silica gel column, and the sample to be tested is diluted with a methanol-water (1:1) mixture at pH 3-4.

[0010] Preferably, the mixing ratio of methanol and water in mobile phase B is 5:1-10:1, and more preferably 7-8:1.

[0011] Preferably, the diluent has a pH value of 3.5 ± 0.1.

[0012] Preferably, the resolution between the components of the cefoperazone-related substances in the detection method is >1.5, more preferably >2.

[0013] In some embodiments of the present invention, the flow rate of the mobile phase in the detection method is 0.5-2.0 ml / min, preferably 0.8 ± 0.2 ml / min;

[0014] Preferably, the elution method of the mobile phase is gradient elution, and the elution gradient is as follows:

[0015]

[0016] In some embodiments of the present invention, the chromatographic column temperature of the detection method is 10-40℃, preferably 20±5℃.

[0017] In some embodiments of the present invention, the detector wavelength of the detection method is 250-275nm, preferably 265±2nm.

[0018] In some embodiments of the present invention, the chiral chromatographic column of the detection method has a stationary phase selected from octadecylsilane, including but not limited to Waters XBridge C18, Waters XTERRA-MS C18, and Waters... Commercially available models include C18.

[0019] In some embodiments of the present invention, the pH value of the dilution reagent in the detection method is 3.5 ± 0.1.

[0020] In some embodiments of the present invention, the concentration of cefoperazone hydrochloride particle-related substances in the detection method is 0.2–4 μg / mL, preferably 0.6–2.8 μg / mL.

[0021] In some embodiments of the present invention, the injection volume of the detection method is 1-40 μL, preferably 30 μL.

[0022] This invention also provides a method for detecting cefoperazone impurities, which employs HPLC. The mobile phase A is phosphate buffer, and the mobile phase B is methanol-water (22:3). The flow rate of the mobile phase is 0.8 ml / min. The elution mode of the mobile phase is gradient elution. The column temperature is 20±5℃, and the detector wavelength is 265±2 nm. The sample to be tested is diluted with a methanol-water (1:1) mixed solvent with pH 3-4.

[0023] Preferably, the diluent has a pH value of 3.5 ± 0.1.

[0024] In this invention, the pH of the diluent is adjusted by hydrochloric acid.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] (1) By optimizing the mobile phase system, elution gradient and other mobile phase conditions and chromatographic column conditions such as detection wavelength, this invention has achieved the separation of four related substances of cefoperazone particles in the same analytical method (resolution > 1.5).

[0027] (2) The inventors were surprised to find that the sample to be tested was unstable in solution. By using a specific diluent, impurity A could be made to remain stable during the detection process, thereby ensuring the accuracy of the measurement results.

[0028] (3) This detection method does not rely on a single chromatographic column. It ensures that the blank excipient does not interfere with the detection of the above components, while improving the robustness, sensitivity and accuracy of the method. It also simplifies the analytical conditions, is easy to operate, and is very suitable for industrial operation. Detailed Implementation

[0029] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0030] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0031] Unless otherwise stated, all raw materials and equipment used in the specific embodiments of this invention are commercially available.

[0032] The structural formulas of related substances of cefoperazone in this invention are as follows:

[0033]

[0034] C 17 H 19 O6N5S2 453.49

[0035] Impurity C (cefotaxime), chemical name: (6R,7R)7-[(Z)2-(2-aminothiazo-4-yl)pent-2-enoic acid amino]-3-carbamoyloxymethyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-2-carboxylic acid

[0036]

[0037] C 23 H 29 O8 N5S2 567.63

[0038] Impurity B (trans isomer of cefoperazone), chemical name: (6R,7R)7-[(E)-2-(2-aminothiazo-4-yl)pent-2-enoic acid amino]-3-carbamoyloxymethyl-8-oxo-5-thia-1-aza-bicyclo[4.2.0]oct-2-en-2-carboxylic acid 2,2-dimethylpropionyloxymethyl ester

[0039]

[0040] C 23 H 29 O8 N5S2 567.63

[0041] Impurity A (△) 2 -Cefcapine ester), chemical name: (6R,7R)7-[(Z)-2-(2-aminothiazo-4-yl)pent-2-enoic acid amino]-3-carbamoyloxymethyl-8-oxo-5-thia-1-aza-bicyclo[4.2.0]oct-2-en-2-carboxylic acid 2,2-dimethylpropionyloxymethyl ester

[0042]

[0043] C 39 H 45 N9O 12 S4 960.08

[0044] Dimer (cefotaxime ester type dimer), chemical name: (6R,7R)7-[(E)-2-(2-aminothiazol-4-yl)-pent-2-enoic acid amino]-8-oxo-2[[(neopentyloxy)methoxy]carbonyl]-5-thia-1-aza-bicyclo[4.2.0]oct-2-en-3-yl]methyl(6R,7R)7-[(Z)-2-(2-aminothiazol-4-yl)-pent-2-enoic acid amino]-3-carbamoyloxymethyl8-oxo-5-thia-1-aza-bicyclo[4.2.0]oct-2-en-2-carboxylic acid ester

[0045] Example 1 Methodological Validation

[0046] 1.1 Exclusivity

[0047] 1.1.1 Chromatographic conditions

[0048] The column was packed with octadecylsilane-bonded silica gel (Waters XBridge C18, 4.6 mm × 150 mm, 5 μm or equivalent column); mobile phase A was phosphate buffer (approximately 5.99 g of potassium dihydrogen phosphate dissolved in 1100 ml of water; approximately 1.89 g of tetrapentylammonium bromide dissolved in 1000 ml of methanol; the tetrapentylammonium bromide methanol solution was added to the potassium dihydrogen phosphate aqueous solution and mixed well); mobile phase B was methanol-water (22:3), and linear gradient elution was performed according to Table 1; the flow rate was 0.8 ml / min; the column temperature was 20 °C; the detection wavelength was 265 nm; the injection volume was 30 μL, and the injection plate was set at 5 °C. The elution gradient was as follows:

[0049] Table 1 Elution gradient data

[0050]

[0051] 1.1.2 Solution Preparation

[0052] Diluent: Methanol-water solution with pH 3.6

[0053] Impurity localization solutions: Take appropriate amounts of impurity A, impurity B, impurity C, and dimer, accurately weigh them, dissolve them in an appropriate amount of diluent, and then dilute them with diluent to prepare impurity localization solutions with concentrations of 1.2 μg / mL, 2.8 μg / mL, 0.6 μg / mL, and 0.6 μg / mL, respectively.

[0054] System suitability solutions: Take appropriate amounts of impurity A, impurity B, impurity C, dimer and main component, accurately weigh them, dissolve them in an appropriate amount of diluent, and then dilute them with diluent to prepare impurity localization solutions with concentrations of 1.2 μg / mL, 2.8 μg / mL, 0.6 μg / mL, 0.6 μg / mL and 0.2 mg / mL respectively.

[0055] 1.1.3 Detection

[0056] Under the chromatographic conditions described above, accurately measure the target solutions for each impurity and the system suitability solution, inject them into the liquid chromatograph, record the chromatograms, and examine the method specificity.

[0057] Table 2 Specificity Data

[0058]

[0059] As can be seen from the table above, the elution order of this method is impurity C, cefoperazone hydrochloride, impurity A, impurity B and dimer. The resolution between each peak is greater than 1.5 and the tailing factor is less than 1.5, which meets the requirements and shows good specificity.

[0060] 1.2 Limit of Quantification

[0061] 1.2.1 Chromatographic conditions

[0062] Chromatographic conditions are the same as above.

[0063] 1.2.2 Solution Preparation

[0064] Limit of Quantitation (LOQ) Solutions: Accurately weigh appropriate amounts of impurity A, impurity B, impurity C, dimer, and main component. Dissolve them in an appropriate amount of diluent, and then dilute with the diluent to prepare LQ solutions with concentrations of 0.1 μg / mL, 0.1 μg / mL, 0.02 μg / mL, 0.05 μg / mL, and 0.05 μg / mL, respectively.

[0065] 1.2.3 Detection

[0066] Under the chromatographic conditions described above, accurately measure each limit of quantitation solution (samples should be prepared fresh), inject into the liquid chromatograph, record the chromatogram, prepare 6 parallel aliquots, and statistically analyze the signal-to-noise ratio and peak area of ​​each known impurity and cefoperazone hydrochloride in the 6 limit of quantitation solutions. Calculate the RSD of each known impurity and the main peak area in the 6 limit of quantitation solutions.

[0067] Table 3 Sensitivity data for limit of quantitation solutions

[0068] name Limit of Quantification (%) S / N RSD (%) Impurity A 0.06 16 2.1 Impurity B 0.06 18 1.5 Impurity C 0.01 19 1.4 Dimer 0.03 17 1.2 principal component 0.03 16 2.0

[0069] As can be seen from the table above, this method has high detection sensitivity. Impurities A, B, C, dimer, and cefotaxime can be quantified at the above concentrations, with a limit of quantitation ≤0.06%, RSD ≤2.1%, and S / N >15.

[0070] 1.3 Detection Limit

[0071] 1.3.1 Chromatographic conditions

[0072] Chromatographic conditions are the same as above.

[0073] 1.3.2 Solution Preparation

[0074] Take an appropriate amount of the limit of quantitation solution under “1.2 Limit of Quantitation” and dilute it with diluent to prepare a detection limit concentration where the signal-to-noise ratio of impurity A, impurity B, impurity C, dimer and main component should be greater than 3:1.

[0075] 1.3.3 Detection

[0076] Inject the sample into the liquid chromatograph under the chromatographic conditions described above and record the chromatogram.

[0077] Table 4. Detection data for limit of quantitation solutions

[0078] name Detection limit (%) S / N Impurity A 0.018 5 Impurity B 0.020 6 Impurity C 0.002 9 Dimer 0.009 8 principal component 0.008 6

[0079] As can be seen from the table above, this method has high detection sensitivity. Impurities A, B, C, dimer, and cefotaxime can be detected at the above concentrations, with a detection limit ≤0.02% and a signal-to-noise ratio (S / N) >3.

[0080] 1.4 Linear Relationship

[0081] 1.4.1 Chromatographic conditions

[0082] Chromatographic conditions are the same as above.

[0083] 1.4.2 Solution Preparation

[0084] Mixed stock solution: Take appropriate amounts of impurity A, impurity B, impurity C, dimer and main component, accurately weigh them, dissolve them with an appropriate amount of diluent, and dilute with diluent to prepare mixed stock solutions with concentrations of 2.4 μg / mL, 5.6 μg / mL, 1.2 μg / mL, 1.2 μg / mL and 4 μg / mL respectively.

[0085] Linear solutions: Accurately measure the mixed impurity stock solution into the corresponding volumetric flasks, dilute to the mark with the dissolution medium, and shake well. See the table below for specific preparation methods.

[0086] Table 5. Data on the preparation of linear solutions

[0087]

[0088] 1.4.3 Detection

[0089] Accurately measure the above linear solution, inject it into the liquid chromatograph, record the chromatogram, plot the linear regression equation with concentration as the x-axis and peak area as the y-axis, and calculate the correction factor for impurities.

[0090] Table 6 Impurity Correction Factor Data

[0091]

[0092] As can be seen from the table above, impurities A, B, C, dimer, and cefoperazone showed good linearity under the chromatographic conditions, all >0.99.

[0093] 1.5 Repeatability

[0094] 1.5.1 Chromatographic conditions

[0095] Chromatographic conditions are the same as above.

[0096] 1.5.2 Solution Preparation

[0097] Mixed stock solution: Take appropriate amounts of impurity A, impurity B, impurity C and dimer, accurately weigh them, dissolve them in an appropriate amount of diluent, and then dilute them with diluent to prepare mixed stock solutions with concentrations of 2.4 μg / mL, 5.6 μg / mL, 1.2 μg / mL and 1.2 μg / mL respectively.

[0098] Test solution: Accurately weigh an appropriate amount of the ground fine powder, place it in a suitable volumetric flask, add an appropriate amount of methanol and shake, then add an appropriate amount of diluent, vortex, measure an appropriate amount of the mixed impurity stock solution, dilute to the mark with diluent, shake well, filter, and the test solution is obtained. Prepare 6 parallel portions.

[0099] 1.5.3 Detection

[0100] Accurately measure the above-mentioned test solution, inject it into the liquid chromatograph, record the chromatogram, and calculate the content of known impurities and total impurities and their relative standard deviations (RSD) in the 6 test solutions using the principal component external standard method with a correction factor (if the correction factor is not in the range of 0.9 to 1.1).

[0101] Table 7. Data on the content of common impurities and relative standard deviations of the test samples.

[0102]

[0103] As can be seen from the table above, the content of impurities A, B, C, dimers, and total impurities in the six test solutions, as well as the relative standard deviation (RSD%), all meet the requirements, indicating that the repeatability of this method is good.

[0104] Comparative Example 1

[0105] 1.1.1 Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase; phosphate buffer was used as mobile phase A; methanol was used as mobile phase B, and linear gradient elution was performed according to the table below; the flow rate was 1.0 mL / min; the column temperature was 30 °C; the detection wavelength was 265 nm; and the injection volume was 30 μL. The elution gradient is as follows:

[0106] Table 8 Elution gradient data for Comparative Example 1

[0107]

[0108] 1.1.2 Solution Preparation

[0109] System suitability solutions: Take appropriate amounts of impurity A, impurity B, impurity C, dimer and main component, accurately weigh them, dissolve them in an appropriate amount of diluent, and then dilute them with diluent to prepare impurity localization solutions with concentrations of 1.2 μg / mL, 2.8 μg / mL, 0.6 μg / mL, 0.6 μg / mL and 0.2 mg / mL respectively.

[0110] 1.1.3 Detection

[0111] Under the chromatographic conditions described above, accurately measure the above system suitability solution, inject it into the liquid chromatograph, record the chromatogram, and examine the method specificity.

[0112] Table 9 shows the separation data for Comparative Example 1.

[0113]

[0114] As can be seen from Table 9, the separation degree is poor when using the above chromatographic conditions in routine testing, which may interfere with the detection of impurities.

[0115] The separation effect data from Example 1 and Comparative Example 1 show that the method in Example 1 results in a higher theoretical plate number for each component, a smaller tailing factor, and a separation degree greater than 1.5 for each peak of impurity C, cefoperazone hydrochloride, impurity A, impurity B, and dimer, as well as a shorter separation time.

[0116] Comparative Example 2

[0117] Chromatographic conditions are the same as in "Example 1"

[0118] 1.1.2 Solution Preparation

[0119] Diluent: Methanol-water (1:1).

[0120] Diluent ②: Methanol-pH3.6 aqueous solution (take 1L of purified water and adjust the pH to 3.6 with hydrochloric acid) (1:1).

[0121] Impurity A reference solution: Weigh approximately 3.080 mg of impurity A reference standard accurately, place it in a 25 ml volumetric flask, add diluent ② to dissolve and dilute to the mark, shake well, accurately measure 1 ml, place it in a 100 ml volumetric flask, dilute to the mark with diluent, shake well, and the solution is ready.

[0122] 1.1.3 Detection

[0123] Under the chromatographic conditions described above, accurately measure the above solution, inject it into the liquid chromatograph, record the chromatogram, and examine the stability of the solution.

[0124] Table 10 shows the stability data for Comparative Example 2.

[0125]

[0126] As can be seen from Table 10, the above-mentioned diluent used in this method has poor stability during routine testing and may interfere with the detection of impurities.

[0127] The stability data from Comparative Example 2 show that the stability of impurity A increases under the conditions of diluent ②.

[0128] Comparative Example 3

[0129] The chromatographic column was different (Waters XTERRA-MS C18, 4.6 mm × 150 mm, 5 μm), but the other chromatographic conditions were the same as in "Example 1".

[0130] 1.1.2 Solution Preparation

[0131] Mixed stock solution: Take appropriate amounts of impurity A, impurity B, impurity C and dimer, accurately weigh them, dissolve them in an appropriate amount of diluent, and then dilute them with diluent to prepare mixed stock solutions with concentrations of 2.4 μg / mL, 5.6 μg / mL, 1.2 μg / mL and 1.2 μg / mL respectively.

[0132] Reference solution: Measure an appropriate amount of the mixed impurity stock solution, dilute to the mark with diluent, and shake well to obtain the solution.

[0133] System suitability solutions: Take appropriate amounts of impurity A, impurity B, impurity C, dimer and main component, accurately weigh them, dissolve them in an appropriate amount of diluent, and then dilute them with diluent to prepare impurity localization solutions with concentrations of 1.2 μg / mL, 2.8 μg / mL, 0.6 μg / mL, 0.6 μg / mL and 0.2 mg / mL respectively.

[0134] 1.1.3 Detection

[0135] Under the chromatographic conditions described above, accurately measure the above solution, inject it into the liquid chromatograph, record the chromatogram, and examine the robustness of different chromatographic columns. Table 11 shows the stability data for Comparative Example 3.

[0136]

[0137] As can be seen from Table 11, the above-mentioned chromatographic column used in this method does not interfere with the detection of impurities during routine testing.

[0138] Based on the stability data of Comparative Example 3, it can be seen that under different chromatographic column conditions (Waters XTERRA-MS C18, 4.6mm×150mm, 5μm), the elution order in the system suitability solution is as follows: the resolution between each peak should be greater than 1.5, and the system suitability meets the requirements.

[0139] Comparative Example 4

[0140] The wavelengths are different (±2nm), but the other chromatographic conditions are the same as in "Example 1".

[0141] 1.1.2 Solution Preparation

[0142] Mixed stock solution: Take appropriate amounts of impurity A, impurity B, impurity C and dimer, accurately weigh them, dissolve them in an appropriate amount of diluent, and then dilute them with diluent to prepare mixed stock solutions with concentrations of 2.4 μg / mL, 5.6 μg / mL, 1.2 μg / mL and 1.2 μg / mL respectively.

[0143] Reference solution: Measure an appropriate amount of the mixed impurity stock solution, dilute to the mark with diluent, and shake well to obtain the solution.

[0144] System suitability solutions: Take appropriate amounts of impurity A, impurity B, impurity C, dimer and main component, accurately weigh them, dissolve them in an appropriate amount of diluent, and then dilute them with diluent to prepare impurity localization solutions with concentrations of 1.2 μg / mL, 2.8 μg / mL, 0.6 μg / mL, 0.6 μg / mL and 0.2 mg / mL respectively.

[0145] 1.1.3 Detection

[0146] Under the chromatographic conditions described above, accurately measure the above solution, inject it into the liquid chromatograph, record the chromatogram, and examine the durability of different chromatographic columns.

[0147] Table 12 shows the stability data for Comparative Example 4.

[0148]

[0149]

[0150] As can be seen from Table 12, the above-mentioned chromatographic column used in this method does not interfere with the detection of impurities during routine testing.

[0151] Based on the stability data of Comparative Example 4, it can be seen that under different wavelengths (±2nm), the peak order in the system suitability solution is as follows: the resolution between each peak should be greater than 1.5, and the system suitability meets the requirements.

Claims

1. A method for detecting a related substance of cefcapene pivoxil hydrochloride particles, wherein the detection is performed by high performance liquid chromatography, wherein, Mobile phase A is phosphate buffer and mobile phase B is a methanol-water mixture; the chromatographic column is an octadecylsilane-bonded silica gel column and the sample to be tested is diluted with a methanol-water (1:1) mixture with pH 3-4 as the diluent.

2. The detection method of claim 1, characterized in that, The mixing ratio of methanol and water in mobile phase B is 5:1-10:1, preferably 7-8:

1.

3. The detection method of claim 2, wherein, The diluent has a pH value of 3.5 ± 0.

1.

4. The detection method of claim 3, wherein, The flow rate of the mobile phase in the detection method is 0.5-2.0 ml / min, preferably 0.8±0.2 ml / min; preferably, the elution mode of the mobile phase is gradient elution.

5. The detection method of claim 4, wherein, The column temperature of the detection method is 10-40℃, preferably 20±5℃.

6. A method for detecting cefoperazone impurities, comprising HPLC, wherein mobile phase A is phosphate buffer and mobile phase B is methanol-water (22:3); the flow rate of the mobile phase is 0.8 ml / min; the elution mode of the mobile phase is gradient elution; the column temperature is 20±5℃; the detector wavelength is 265±2 nm; and the sample to be tested is diluted with methanol-water (1:1) at pH 3-4.

7. The detection method of claim 6, wherein, The diluent has a pH value of 3.5 ± 0.1.

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