Method for determining related substances of compound preparation cefprozil and probenecid tablets

Abstract

The invention relates to a method for determining related substances of a compound preparation cefprozil and probenecid tablet, which is characterized in that the related substances are detected by high performance liquid chromatography, the related substances are impurities in the compound preparation cefprozil and probenecid, and the content of the impurity cefadroxil, other single impurities and total impurities is determined; wherein the mobile phase A is a 0.23% ammonium dihydrogen phosphate solution, and the mobile phase B is HPLC (High Performance Liquid Chromatography) grade methanol; a sample is diluted by a diluent and then is injected, the diluent is a mixed solution of a 0.23% ammonium dihydrogen phosphate solution and HPLC-grade methanol, the sample injection amount of the sample is 5 [mu] L, the concentration of cefprozil is 1.5 mg / mL, and the concentration of probenecid is 2.25 mg / ml; according to the present invention, the related substances in the cefprozil and the probenecid can be simultaneously detected, the detection cost is reduced, the method has advantages of strong specificity, high sensitivity, good reproducibility and good accuracy, has certain feasibility and practicality, is easy to popularize and apply, and can provide the research reference for the quality control and evaluation of the product.

Description

Technical Field

[0001] The invention relates to a method for determining related substances of a compound preparation of cefprozil-probenecid tablets, and provides a new, highly sensitive and highly specific analytical detection method. Background Art

[0002] Cefprozil-probenecid tablets are a compound preparation containing two ingredients, cefprozil and probenecid. Among them, cefprozil is the one that exerts pharmacological effects. The mechanism of action of cefprozil is similar to that of other cephalosporins, and it mainly exerts its antibacterial effect by hindering the biosynthesis of bacterial cell walls. The characteristics of cefprozil's action are that its anti-Gram-negative bacillus activity and stability against Gram-negative bacillus β-lactamase are stronger than those of the first-generation cephalosporins. The combined use of cefprozil and probenecid can prolong the excretion time of cefprozil and increase the blood drug concentration. Therefore, the development of a method that can detect cefprozil and probenecid is of great significance to improving drug safety and quality control of cefprozil.

[0003] The quality control of compound cefprozil-probenecid tablets is inseparable from the control of impurities, which are the key quality attributes of drugs and can affect the safety and effectiveness of products. Impurities in drug quality standards refer to impurities introduced by the production process or raw materials of drugs produced in accordance with the process and raw materials approved by the national drug supervision and administration department in accordance with the law, or impurities generated during storage.

[0004] At present, regarding the detection method of related substances in the compound preparation of cefprozil and probenecid, no detection method of related substances of this variety is found in the pharmacopoeias of various countries. The detection method of related substances in the quality standards of cefprozil and probenecid included in the pharmacopoeias of various countries is only applicable to single-preparation preparations, and cannot take into account the effective separation of the two components and related impurities at the same time, and cannot reflect the true quality of the product, thereby affecting the safety of the clinical use of the drug. In the prior art, the detection method of related substances in the quality standards of the compound preparation of cefprozil and probenecid tablets included in the pharmacopoeias of various countries is only applicable to single-preparation preparations, and cannot take into account the effective separation of the two components and related impurities at the same time; although the relevant literature has not reported the detection method of related substances in the compound preparation of cefprozil and probenecid tablets, it cannot reflect the true quality of the product.

[0005] In summary, there is still no effective detection method for cefprozil-probenecid-related substances, and the impurities in compound cefprozil-probenecid tablets cannot be effectively controlled. In order to ensure the product quality of compound cefprozil-probenecid tablets, it is urgent to develop an effective method suitable for the impurity detection of this product. Summary of the invention

[0006] The invention aims to provide a method for determining related substances of a compound preparation of cefprozil and probenecid tablets. The compound preparation of cefprozil and probenecid tablets is a compound preparation. Cefprozil raw materials and probenecid raw materials are all included in the United States Pharmacopoeia, the European Pharmacopoeia and the British Pharmacopoeia, but the compound preparation of cefprozil and probenecid tablets is a newly developed preparation of our company and is not included in the pharmacopoeias of various countries. In order to meet the quality research of this product, the detection method is screened and optimized with reference to the pharmacopoeia standards of various countries and the methods of raw material manufacturers and combined with the actual situation of this product, and a new method for detecting related substances is established, which can simultaneously meet the detection of two main component contents and impurities, has high sensitivity and strong specificity, and can be applicable to the quality control of this product.

[0007] To achieve the above object, the technical solution of the present invention is implemented as follows: a method for determining related substances in a compound preparation of cefprozil and probenecid tablets, characterized in that: the related substances are detected by high performance liquid chromatography, wherein the related substances are impurities in the compound preparation of cefprozil and probenecid, including the impurity cefadroxil, other single impurities and total impurities for content determination; the chromatographic conditions include the following steps: (1) Preparation of control solution Take appropriate amounts of cefprozil and probenecid reference substances respectively, weigh them accurately, and quantitatively dilute them with diluent 2 to prepare a solution containing about 7.5 μg of cefprozil and 11.25 μg of probenecid per 1 ml, as the reference substance solution; (2) Preparation of test solution Take 3 tablets of this product, put them in a 250ml volumetric flask, add 75ml mobile phase A for ultrasonic disintegration, add 0.2ml 20% phosphoric acid solution, add appropriate amount of diluent 1 for ultrasonic disintegration for about 5 minutes, take out and restore to room temperature, dilute to the scale with diluent 1, shake well, centrifuge at 16000 rpm for 2 minutes, take the supernatant, and obtain a solution containing about 1.5mg of cefprozil and 2.25mg of probenecid per 1ml, which is used as the test solution; (3) Sensitivity solution Accurately measure an appropriate amount of the reference solution and quantitatively dilute it with diluent 2 to produce a solution containing approximately 0.75 μg of cefprozil per 1 ml; (4) Chromatographic conditions Octadecylsilane bonded silica gel is used as filler; Mobile phase A is 0.23% diammonium phosphate solution (pH adjusted to 4.4 with phosphoric acid or ammonia); Mobile phase B was methanol; The flow rate is 1.0 ml per minute; The column temperature was 25 °C; The detection wavelength is 225nm; Injection volume 5 μl; Sample pan temperature: 5°C; Gradient elution Perform linear gradient elution as shown in the table below. Determination method: Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram; Limit: If there are impurity peaks in the chromatogram of the test solution, calculate them according to the main component external standard method based on the peak area of ​​cefuroxil in the reference solution. The cefadroxil shall not exceed 0.5% of the labeled amount, the largest unknown single impurity shall not exceed 0.5% of the labeled amount, the total impurities shall not exceed 3.0%, and the peaks smaller than the main peak area of ​​the sensitivity solution shall be ignored (reporting limit: 0.05%).

[0008] In the liquid chromatography analysis and detection process, the sample is diluted with a diluent and then injected, the diluent 1 is methanol-acetonitrile (1:1]; the diluent 2 is mobile phase A-methanol-acetonitrile (30:35:35); the injection volume of the sample is 5 μL, wherein the concentration of cefprozil is 1.5 mg / mL, and the concentration of probenecid is 2.25 mg / ml.

[0009] The total volume of the diluent is 100%, and the 0.23% diammonium phosphate solution and the methanol are HPLC grade methanol, with a volume ratio of 30:70.

[0010] In the liquid chromatography analysis and detection process, taking the total volume of the mobile phase as 100%, the content gradient of the mobile phase A and the mobile phase B is: At minute 0, the volume ratio of the mobile phase A to the mobile phase B was 85:15; At the 15th minute, the volume ratio of the mobile phase A to the mobile phase B was 85:15; At the 25th minute, the volume ratio of the mobile phase A to the mobile phase B was 60:40; At the 30th minute, the volume ratio of the mobile phase A to the mobile phase B was 40:60; At the 50th minute, the volume ratio of the mobile phase A to the mobile phase B was 15:85; At the 60th minute, the volume ratio of the mobile phase A to the mobile phase B was 15:85; At 60.1 minutes, the volume ratio of the mobile phase A to the mobile phase B was 85:15; At the 70th minute, the volume ratio of the mobile phase A to the mobile phase B was 85:15.

[0011] The flow rate of the mobile phase during the liquid chromatography analysis and detection process is 0.8 ml / min to 1.0 ml / min.

[0012] A C18 chromatographic column is used in the liquid chromatography analysis and detection process.

[0013] The specifications of the chromatographic column are: length 250 mm, inner diameter (ID) 4.6 mm, particle diameter S-5 μm, i.e. 5 microns.

[0014] Application of liquid chromatography in the detection or separation of related substances in the compound preparation of cefprozil and probenecid.

[0015] The positive effect of the present invention is that compared with the prior art, the present invention adopts a related substance analysis method to analyze various impurities in cefprozil and probenecid, the method is simple and easy to implement, and the detection and separation of known impurities in cefprozil and probenecid are realized at the same time, and compared with the conventional compound preparations using two or more related substance analysis methods, a large amount of analysis and detection time is saved. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the system suitability solution chromatogram of related substances of cefprozil and probenecid. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and Examples. The embodiments described below are only used to explain the present invention and should not be construed as limiting the present invention. If no specific technology or conditions are indicated in the embodiments, the technology or conditions described in the document in this area or the product specification are carried out. The reagents used or the instruments that do not indicate the manufacturer are all conventional products that can be obtained by commercial purchase.

[0018] The following describes in detail a method for determining the content of each impurity in compound cefprozil probenecid tablets according to an embodiment of the present invention. The specific steps are as follows: Example 1: Method establishment (1) Optimization and screening of mobile phase and gradient elution program ①Chromatographic condition screening 1: Method source: Ch.P Cefprozil Pharmacopoeia Standard Mobile phase A: 0.23% ammonium dihydrogen phosphate solution; Mobile phase B: methanol; Chromatographic column: octadecylsilane bonded silica gel as filler; Detection wavelength: 225nm Column temperature: 25℃ Flow rate: 1.0ml / min Injection volume: 20 μl Probenecid test solution: Accurately weigh 25.13 mg of probenecid API into a 50 ml volumetric flask, add probenecid diluent 2 to dissolve to the scale, shake well and mark as RS-BA2312001-2-API (0.5 mg / ml). Cefprozil sample solution (5 mg / ml): Accurately weigh 125.06 mg of cefprozil API into a 25 ml volumetric flask, add 1 ml of 103 g / L hydrochloric acid solution to dissolve, add cefprozil diluent to dissolve to the scale, shake well, and mark as RS-230402-1-API (5 mg / ml). The gradient schedule is as follows: Table 2 Gradient schedule for chromatographic condition screening 1 Detection conclusion: The impurities in the Chinese Pharmacopoeia related substance method for cefprozil are well separated, and no peak is detected for probenecid by this method.

[0019] ②Chromatographic condition screening 2: Method source: Ch.P Pharmacopoeia standard of probenecid Mobile phase: 0.05 mol / L sodium dihydrogen phosphate (added with 1% glacial acetic acid, pH adjusted to 3.0 with phosphoric acid)-acetonitrile (50:50); Chromatographic column: octadecylsilane bonded silica gel as filler; Detection wavelength: 245nm Column temperature: 30°C Flow rate: 1.0ml / min Injection volume: 20 μl Detection conclusion: The retention time of the main peak of probenecid in the Chinese Pharmacopoeia method is about 10 minutes; from the gradient elution table, it can be seen that a low initial organic phase ratio is conducive to the separation of impurities in cefprozil, and a high organic phase ratio is conducive to the elution of probenecid. Because this product is a compound preparation, the cost of using two detection methods is relatively high. Therefore, it is considered to optimize the detection method of cefprozil-related substances in the Chinese Pharmacopoeia and develop a detection method that can simultaneously detect cefprozil and probenecid-related substances.

[0020] ③Chromatographic condition screening 3: Mobile phase A: 0.23% ammonium dihydrogen phosphate solution; Mobile phase B: methanol; Chromatographic column: octadecylsilane bonded silica gel as filler; Detection wavelength: 225nm Column temperature: 25℃ Flow rate: 1.0ml / min Injection volume: 20 μl The gradient schedule is as follows: Table 3 Gradient schedule for chromatographic condition screening 3 Detection conclusion: By comparing the gradient 3 in the above figure with the method for related substances of cefuroxime in the Chinese Pharmacopoeia, it can be seen that the main peak time is slightly advanced, which is still not good, and the method optimization will continue.

[0021] ④Chromatographic condition screening 4: Mobile phase A: 0.23% ammonium dihydrogen phosphate solution; Mobile phase B: methanol; Chromatographic column: octadecylsilane bonded silica gel as filler; Detection wavelength: 225nm Column temperature: 25℃ Flow rate: 1.0ml / min Injection volume: 20 μl The gradient schedule is as follows: Table 4 Gradient schedule for chromatographic condition screening 4 Detection conclusion: In the HPLC, DB1 is 6% methanol; DB2 is 0.23% (NH4)H2PO4: methanol = 94:6, RS-230402-1-API (0.75mg / ml) and RS-230402-2-API (0.75mg / ml) are diluted with 6% methanol and 0.23% (NH4)H2PO4: methanol = 94:6 respectively; the difference between the two diluents used in this method is not large, and the main peak impurities of the sample are well separated. The retention time of probenecid detected by this method is about 39min, and it has no effect on the detection of cefprozil and its impurities. In order to facilitate the preparation of solvents, 50% methanol is considered as the diluent to detect the mixed sample of cefprozil and probenecid. However, when testing the 23010501 batch of samples, it was found that after using 50% methanol as a diluent to dissolve probenecid, it would precipitate after standing for a period of time, so the diluent was changed to 50% acetonitrile. Take an appropriate amount of probenecid API and 23010501 batches of homemade samples and dissolve them in 50% acetonitrile. After standing, there is no precipitation phenomenon. However, because the organic phase in the diluent is acetonitrile and the organic phase in the mobile phase is methanol, a solvent effect occurs when the sample is detected, so the diluent is changed to a mixed solvent of methanol and mobile phase A. Because the polarity of cefprozil and probenecid is quite different, cefprozil cannot be dissolved when the organic phase ratio is high. By adjusting the ratio of methanol to mobile phase A and observing the dissolution of the mixed API, it is finally found that when the ratio of mobile phase A to methanol is 30:70, cefprozil and probenecid can be dissolved. However, when using this gradient detection, impurity Z17 did not peak, but was eluted in the next injection of ImpA. This method cannot elute all probenecid-related impurities, and needs to be further optimized.

[0022] (2) Determination of HPLC conditions Octadecylsilane bonded silica gel is used as filler; Mobile phase A is 0.23% ammonium dihydrogen phosphate solution Mobile phase B was methanol; The flow rate is 1.0 ml per minute. Column temperature was 25°C The detection wavelength is 225nm, Injection volume 5μ1 Sample plate temperature: 5°C Gradient elution Perform linear gradient elution as shown in the table below. Determination method: Precisely measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram. If there are impurity peaks in the chromatogram of the limit test solution, calculate the peak area of ​​cefoperiprole in the reference solution according to the main component external standard method. The cefadroxil shall not exceed 0.5% of the labeled amount, the largest unknown single impurity shall not exceed 0.5% of the labeled amount, and the total impurities shall not exceed 3.0%. Peaks smaller than the main peak area of ​​the sensitivity solution shall be ignored (report limit: 0.05%) The application of the above method in the detection or separation of related substances in the compound preparation of cefprozil and probenecid is also within the protection scope of the present invention.

[0023] Example 2: Methodological Validation (1): Specificity and system applicability Sample preparation blank solution: Solvent 1 [mobile phase A - methanol - acetonitrile (30:35:35)]: measure 1200 ml of mobile phase A, 1400 ml of methanol and 1400 ml of acetonitrile, mix well, and degas by ultrasonication. Blank excipient: Accurately weigh 412.35 mg of blank excipient and place it in a 250 ml volumetric flask. First add 75 ml of mobile phase A, add an appropriate amount of solvent 1, ultrasonicate for 5 min, add solvent 1 to dilute to the scale, shake well, centrifuge at 16,000 rpm for 2 minutes, and take the supernatant. Test solution: Take 3 tablets of this product and place them in a 250ml volumetric flask. First add 75ml of mobile phase A. After ultrasonic disintegration of the sample, add 0.2ml of 20% phosphoric acid solution and then add appropriate amount of solvent 2. Ultrasonicate for about 5min, dilute to the scale with solvent 2, shake well, and centrifuge at 16,000 rpm for 2 minutes. Stock solutions of cefprozil and probenecid reference substances: accurately weigh 9.386 mg of cefprozil reference substance and 14.123 mg of probenecid reference substance, place in a 25 ml volumetric flask, add solvent 1 and dissolve by ultrasonication, then dilute to the scale and shake well. Prepare two portions in parallel. The second portion of cefprozil reference substance and probenecid reference substance weighed 9.407 mg and 14.086 mg, respectively, and named them Stock#1 and Stock#2, respectively. Cefprozil and probenecid reference solution: Accurately pipette 5.0 ml of the cefprozil and probenecid reference stock solution into a 250 ml volumetric flask, dilute to the mark with solvent 1, shake well, and mark them as RS-STD#1 and RS-STD#2 respectively. Sensitivity solution: Accurately pipette 2.0 ml of cefprozil / probenecid reference solution RS-STD#1 into a 20 ml volumetric flask, dilute to the mark with solvent 1, shake well, and mark as RS-SEN. Cefadroxil reference substance stock solution: accurately take 15.678 mg of cefadroxil reference substance, place it in a 25 ml volumetric flask, add an appropriate amount of mobile phase A and ultrasonically dissolve it, then dilute it to the scale with mobile phase A and shake well. Cefadroxil localization solution: Pipette 0.3 ml of cefadroxil reference substance stock solution into a 25 ml volumetric flask, dilute to the mark with solvent, and shake well. Conclusion: System suitability: The blank solution has no obvious interference at the retention time position of cefprozil, probenecid and cefadroxil peaks. The specific HPLC chromatogram is attached. Figure 1 ; The signal-to-noise ratio (S / N) of the cefprozil (Z) isomer in the sensitivity solution was 40.6; Specificity: In the chromatograms of the blank solution and the blank excipient solution, there was no interference at the retention time position of the cefprozil, probenecid and impurity cefadroxil peaks;

[0024] (2): Limit of quantification, limit of detection Accurately weigh the impurities cefadroxil and cefprozil, dissolve them in diluent and dilute them to make a mixed solution of appropriate concentration, dilute them step by step, accurately measure 5μl and inject them into the liquid chromatograph, record the chromatogram, when the signal-to-noise ratio (S / N) is about 3:1, it is the detection limit, when the signal-to-noise ratio (S / N) is about 10:1, it is the quantification limit. Take the quantification limit solution and inject it 6 times continuously, record the chromatogram Table 6: Detection limit and quantification limit results Table 7: Results of precision test of quantitative limit of each component (n=6)

[0025] Conclusion: The quantification limit of cefprozil is 0.7222μg / mL, which is 0.048% of the test sample concentration, the quantification limit signal-to-noise ratio S / N is 33.5, and the RSD value of the quantification limit peak area of ​​6 injections is 1.7%; Cefadroxil: The quantification limit is 0.7227μg / mL, which is 0.048% of the test sample concentration, the quantification limit signal-to-noise ratio S / N is 12.0, and the RSD value of the quantification limit peak area of ​​6 injections is 6.0%. Detection limit: The signal-to-noise ratios s / n of cefprozil and cefdroxil peaks are 9.3 and 18.7, respectively, which are 0.016% of the test sample concentration. Both meet the requirements.

[0026] (3) Linearity and range Cefprozil reference substance stock solution: Accurately weigh 9.493 mg of cefprozil reference substance, place in a 25 ml volumetric flask, add mobile phase A and ultrasonically dissolve, then dilute to the scale with mobile phase A and shake well. Cefadroxil reference substance stock solution: accurately take 15.883 mg of cefadroxil reference substance, place it in a 25 ml volumetric flask, add an appropriate amount of mobile phase A and ultrasonically dissolve it, then dilute it to the scale with mobile phase A and shake well. Linear stock solution: Accurately pipette 5.0 ml of cefprozil reference stock solution and 3.0 ml of cefadroxil reference stock solution into a 25 ml volumetric flask, dilute to the mark with solvent 1, and shake well. Linear solution: Accurately pipette an appropriate amount of linear stock solution, add solvent 1 to dilute to a certain volume, and prepare a series of linear solutions of different concentrations. See the table below for detailed preparation scheme. Table-14 Linear solution preparation table Table 3 Linear range study results name Regression equation Correlation coefficient r Correction Factor Linear range (μg / ml) Cefadroxil y=14918.5930x-864.4612 1.000 0.92 0.7227～10.840 Cefprozil y=13770.7592x-1773.6844 1.000 1.00 0.7222～10.830 Conclusion: In the range of 0.7222μg / ml~10.830μg / ml, the linear equation of cefuroxil is y=13770.7592x-1773.6844, and the linear correlation coefficient r value is 1.000; in the range of 0.7227μg / ml~10.84μg / ml, the linear equation of cefuroxil is y=14,918.5930x-864.4612, and the linear correlation coefficient r value is 1.000; 10% of the peak area of ​​the limit concentration of cefuroxil (10283.16)> the absolute value of intercept b (1733.68), and 10% of the peak area of ​​the limit concentration of cefadroxil (11267.02)> the absolute value of intercept b (864.46); the correction factor of cefadroxil is 0.92, which is 1.0 after rounding.

[0029] (4) Accuracy (repeatability) Solution preparation Test solution: Take 3 tablets of this product and place them in a 250ml volumetric flask. First add 75ml of mobile phase A, disintegrate the sample by ultrasonication, add 0.2ml of 20% phosphoric acid solution, then add 3.0ml of cefadroxil reference substance stock solution in "8.2.1 Sample preparation", add appropriate amount of solvent 2, ultrasonicate for about 5min to dissolve, take out, dilute to the scale with solvent 2, shake well, centrifuge at 16000 rpm for 2min, take the supernatant and prepare 6 copies in parallel. Test results and conclusions Table-19 Summary of Cefadroxil in Accuracy Solution Conclusion: (1) The average recovery of cefadroxil in 6 accuracy solutions was 109.8%, and the RSD% of the recovery was 0.6%; (2) The RSD% of the content of total impurities in 6 accuracy solutions was 6.8%.

[0030] (5) Solution stability test Freshly prepared control solution and test solution were injected and analyzed at time 0 under the condition that the system suitability requirements were met, and the stability of the samples at different time points under the condition of 2-8°C was examined. Reference solution: Compared with the determination result at the starting point, the recovery rate of the peak area of ​​the reference solution at each time point should be between 90.0% and 110.0%. Test solution: Compared with the determination result at the starting point, the recovery rate of cefadroxil content is between 90.0% and 110.0%; the recovery rate of total impurities is between 90.0% and 110.0%, and no new impurities greater than the reporting limit are generated. Test results and conclusions Table-20 Solution stability test results - test solution Table-21 Solution stability test results - reference solution Conclusion: Test solution: At 2-8℃, the test solution is stable within 1.2h and unstable within 3.5h. Reference solution: At 2-8℃, the reference solution is stable for at least 45.0h. The present invention provides a method and idea for analyzing related substances of compound cefprozil probenecid tablets. There are many methods and approaches to implement the technical solution. 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. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for determining related substances in a compound preparation of cefprozil and probenecid tablets, characterized in that: The relevant substances are detected by high performance liquid chromatography, wherein the relevant substances are impurities in the compound preparation of cefprozil and probenecid, including the impurity cefadroxil, other single impurities and total impurities for content determination; the chromatographic conditions include the following steps: (1) Preparation of control solution Take appropriate amounts of cefprozil and probenecid reference substances respectively, weigh them accurately, and quantitatively dilute them with diluent 2 to prepare a solution containing about 7.5 μg of cefprozil and 11.25 μg of probenecid per 1 ml, as the reference substance solution; (2) Preparation of test solution Take 3 tablets of this product, put them in a 250ml volumetric flask, add 75ml mobile phase A for ultrasonic disintegration, add 0.2ml 20% phosphoric acid solution, add appropriate amount of diluent 1 for ultrasonic disintegration for about 5 minutes, take out and restore to room temperature, dilute to scale with diluent 1, shake well, centrifuge at 16000 rpm for 2 minutes, take the supernatant, and obtain a solution containing about 1.5mg of cefprozil and 2.25mg of probenecid per 1ml, which is used as the test solution; (3) Sensitivity solution Accurately measure an appropriate amount of the reference solution and quantitatively dilute it with diluent 2 to produce a solution containing approximately 0.75 μg of cefprozil per 1 ml; (4) Chromatographic conditions Octadecylsilane bonded silica gel is used as filler; Mobile phase A is 0.23% ammonium dihydrogen phosphate solution, i.e., the pH value is adjusted to 4.4 with phosphoric acid or ammonia; Mobile phase B was methanol; The flow rate is 1.0 ml per minute; The column temperature was 25°C; The detection wavelength is 225nm; Injection volume 5 μl; Sample pan temperature: 5°C; Gradient elution Determination method: Accurately measure the test solution and reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram; Limits: If there are impurity peaks in the chromatogram of the test solution, calculate the cefoperazone peak area in the reference solution according to the main component external standard method. The cefadroxil shall not exceed 0.5% of the labeled amount, the largest unknown single impurity shall not exceed 0.5% of the labeled amount, and the total impurities shall not exceed 3.0%. Peaks smaller than the main peak area of ​​the sensitivity solution shall be ignored. Report limit: 0.05%.

2. The method for determining the related substances of the compound preparation cefprozil probenecid tablets according to claim 1, characterized in that During the liquid chromatography analysis and detection process, the sample was diluted with a diluent and then injected, wherein the diluent 1 was methanol-acetonitrile (1:1); the diluent 2 was mobile phase A-methanol-acetonitrile (30:35:35); the injection volume of the sample was 5 μL, wherein the concentration of cefprozil was 1.5 mg / mL, and the concentration of probenecid was 2.25 mg / ml.

3. The method for determining the related substances of the compound preparation cefprozil probenecid tablets according to claim 1, characterized in that The total volume of the diluent is 100%, and the 0.23% diammonium phosphate solution and the methanol are HPLC grade methanol, with a volume ratio of 30:

70.

4. The method for determining the related substances of the compound preparation cefprozil probenecid tablets according to claim 2, characterized in that In the liquid chromatography analysis and detection process, taking the total volume of the mobile phase as 100%, the content gradient of the mobile phase A and the mobile phase B is: At minute 0, the volume ratio of the mobile phase A to the mobile phase B was 85:15; At the 15th minute, the volume ratio of the mobile phase A to the mobile phase B was 85:15; At the 25th minute, the volume ratio of the mobile phase A to the mobile phase B was 60:40; At the 30th minute, the volume ratio of the mobile phase A to the mobile phase B was 40:60; At the 50th minute, the volume ratio of the mobile phase A to the mobile phase B was 15:85; At the 60th minute, the volume ratio of the mobile phase A to the mobile phase B was 15:85; At 60.1 minutes, the volume ratio of the mobile phase A to the mobile phase B was 85:15; At the 70th minute, the volume ratio of the mobile phase A to the mobile phase B was 85:

15.

5. The method for determining the related substances of the compound preparation cefprozil probenecid tablets according to claim 4, characterized in that The flow rate of the mobile phase during the liquid chromatography analysis and detection process is 0.8ml / min~1.0ml / min.

6. The method for determining the related substances of the compound preparation cefprozil probenecid tablets according to claim 1, characterized in that A C18 chromatographic column is used in the liquid chromatography analysis and detection process.

7. The method for determining the related substances in the compound preparation cefprozil probenecid tablets according to claim 6, characterized in that The specifications of the chromatographic column are: length 250 mm, inner diameter (ID) 4.6 mm, particle diameter S-5 μm, i.e. 5 microns.

8. The method for determining the related substances of the compound preparation cefprozil probenecid tablets according to claim 1, characterized in that The liquid chromatography analysis method is used in the detection or separation of related substances in the compound preparation cefprozil and probenecid.

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