A new method for detecting polymer content in cefcapone hydrochloride

Abstract

The present invention belongs to the field of pharmaceutical analysis technology, specifically discloses a new method for detecting polymer content in cefcapene pivoxil hydrochloride. The method mainly uses high-efficiency molecular exclusion chromatography (HPSEC) method to apply ultraviolet detector to determine the size of polymer in cefcapene pivoxil hydrochloride, and its chromatographic conditions are: chromatographic column: BioCoreSEC‑120, 7.8×300mm, 5μm or chromatographic column with equivalent performance; Mobile phase: mobile phase A and mobile phase B are mixed by volume ratio 95:5 solution; Wherein, the mobile phase A is 0.05mol / L sodium dihydrogen phosphate aqueous solution and 0.05mol / L sodium dihydrogen phosphate aqueous solution by volume ratio 1:1 mixed solution; The mobile phase B is acetonitrile; Elution mode: isocratic elution; Flow rate: 0.7~0.9ml / min; Column temperature: 25~35℃; Detection wavelength: 254nm; Sample size: 10ul.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceutical analysis, and in particular to a novel method for detecting the polymer content in cefcapone hydrochloride. Background Art

[0002] Cefuroxime hydrochloride is a fourth-generation oral cephalosporin antibiotic, belonging to the β-lactam antibiotics, suitable for infections caused by Streptococcus, Staphylococcus, Neisseria gonorrhoeae, etc. Studies have shown that the immediate allergic reaction caused by β-lactam antibiotics is not caused by the drug itself, but is related to the high molecular polymers present in the drug. Although the various high molecular impurities in β-lactam antibiotics have different structures, they have similar biological properties (such as high allergenicity), so in quality control, it is generally not necessary to control polymers of different structures one by one, but only to control their total amount. The high molecular polymers in cephalosporin antibiotics refer to the general term for impurities with a molecular weight greater than the drug itself, and their molecular weight is generally between 1000-5000Da, and some can reach about 15000Da.

[0003] Due to the high heterogeneity and uncertainty of high molecular polymers, it is impossible to prepare a reference substance with a fixed content. There are no relevant cefuroxime hydrochloride polymer detection methods in existing patents and literature, and foreign pharmacopoeias such as USP and EP have not included relevant determination methods. The method for determining high molecular polymers in β-lactam antibiotics by self-control external standard method included in the 2020 edition of the "Chinese Pharmacopoeia" adopts gel chromatography with glucose gel Sephadex G-10 as the chromatographic column. By switching between two mobile phases, the high molecular polymer reference substance is replaced by the drug's own reference substance to detect the high molecular polymer therein. This is a commonly used technology for detecting polymers in cephalosporin antibiotics. However, this method has the disadvantages of low column efficiency of gel chromatography column (about 1000), peak tailing, long analysis time, poor separation of polymer impurities (all high molecular polymers are one peak, and the peak is poorly separated from the main peak baseline), and very low detection of high molecular polymers. Therefore, there are great inconveniences in the production process of cefcapone hydrochloride raw materials and the quality control of finished products, and it is impossible to quickly and accurately quantify the content of polymers in cefcapone hydrochloride.

[0004] Therefore, it is very important to provide a method for determining the polymer content in cefcapone hydrochloride with simple operation, high sensitivity, rapidity and effectiveness. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the existing gel chromatography technology and provide a method for quickly and effectively determining the polymer content in cefcapene pivoxil hydrochloride. Different from the traditional gel chromatography used for polymer detection in antibiotics, the present invention adopts high performance molecular exclusion chromatography HPSEC method, which has the advantages of simple operation, strong specificity, high sensitivity, etc.

[0006] The present invention adopts high performance molecular exclusion chromatography (HPSEC) method to measure the polymer content in cefuroxime hydrochloride. High performance molecular exclusion chromatography distinguishes the size of each component molecule, and the large component molecule can penetrate into the macropores of the gel, while the small component molecule can penetrate into the macropores and small pores, and even penetrates very deeply, and is not easy to elute out. Therefore, the large component molecule has a short residence time in the chromatographic column and is quickly eluted out. The large component molecule and the small component molecule complete the elution process separated by molecular size in the molecular exclusion chromatography system.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A new method for detecting the content of bile polymers in cefcapene pivoxil hydrochloride uses high performance size exclusion chromatography (HPSEC) with an ultraviolet detector to determine the size of polymers in cefcapene pivoxil hydrochloride. The chromatographic conditions are as follows:

[0009] Chromatographic column: BioCoreSEC-120, 7.8×300mm, 5μm or other columns with equivalent performance;

[0010] Mobile phase: a mixed solution of mobile phase A and mobile phase B in a volume ratio of 95:5;

[0011] Wherein, the mobile phase A is a mixed solution of 0.05 mol / L disodium hydrogen phosphate aqueous solution and 0.05 mol / L sodium dihydrogen phosphate aqueous solution in a volume ratio of 1:1; the mobile phase B is acetonitrile;

[0012] Elution mode: isocratic elution;

[0013] Flow rate: 0.7~0.9ml / min;

[0014] Column temperature: 25-35°C;

[0015] Detection wavelength: 254nm;

[0016] Injection volume: 10ul.

[0017] The ultraviolet detector in the present invention is a differential refractive index detector, and the detector temperature is 35°C.

[0018] In the present invention, the column temperature of the chromatographic column is preferably 30° C.; the flow rate is preferably 0.8 ml / min.

[0019] In the present invention, the novel method for detecting the content of bile polymer in cefcapene pivoxil hydrochloride comprises the following steps:

[0020] a. Prepare blank solution: Use diluent as blank solution;

[0021] b. Prepare the test solution: Take the cefoperazone hydrochloride test sample and dissolve it in a diluent to a concentration of 0.1 mg / ml test solution (prepared fresh for use);

[0022] c. Prepare the self-control solution: accurately measure 1 ml of the test solution in step b, place it in a 100 ml volumetric flask, add diluent to dilute to the mark and shake well as the self-control solution;

[0023] d. Take 10ul of each of the blank solution, self-control solution and test solution and inject them into the liquid chromatograph, record the peak area of ​​cefcapene hydrochloride in the chromatogram, and calculate the content of the polymer in the cefcapene hydrochloride sample according to the peak area self-control external standard method.

[0024] The diluent in the present invention is a mixture of mobile phase and methanol in a volume ratio of 1:1.

[0025] In summary, the beneficial effects of the present invention are as follows:

[0026] The method of the present invention can quickly and accurately determine the content of polymers in cefcapene pivoxil hydrochloride. Relative to the existing gel chromatography method using glucose gel Sephadex G-10 as a chromatographic column. By switching two mobile phases, the high molecular polymer reference substance is replaced by the drug's own reference substance to detect the high molecular polymer therein. The method of the present invention uses high-efficiency molecular exclusion chromatography, cooperates with SEC chromatographic columns, and the method has strong specificity, high accuracy and high sensitivity. Selecting a high-cost-effective ultraviolet detector (UV), using reversed-phase ultra-high performance liquid chromatography, the method has strong reliability and low detection cost, and is suitable for the quality inspection of intermediate products and finished products in the production of cefcapene pivoxil hydrochloride. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The chromatogram of blank solvent S1 in the embodiment;

[0028] Figure 2 It is the chromatogram of the cefcapone hydrochloride test solution S2 in the embodiment;

[0029] Figure 3 It is a chromatogram of cefcapone hydrochloride reference solution S3 in the embodiment;

[0030] Figure 4 It is the chromatogram of the cefcapone hydrochloride reference solution in Comparative Example 1;

[0031] Figure 5 It is the chromatogram of the cefcapone hydrochloride reference solution in Comparative Example 2. DETAILED DESCRIPTION

[0032] The present invention will be further described below with reference to specific embodiments, but is not limited thereto.

[0033] Example

[0034] The cefcapone hydrochloride test sample selected in this example is cefcapone hydrochloride produced by Zhongshan Bailing Biotechnology Co., Ltd.

[0035] The chromatographic conditions used in this example are shown in Table 1:

[0036] Table 1 Chromatographic system conditions

[0037]

[0038] The method for determining the content of cefcapene pivoxil hydrochloride polymer is as follows:

[0039] a. Prepare blank solution: Use a diluent of mobile phase: alcohol = 1:1 as blank solution S1;

[0040] b. Configure the test solution: Take the cefoperazone hydrochloride test sample and dissolve it in a diluent to a concentration of 0.1 mg / ml of the test solution S2 (newly prepared for clinical use);

[0041] c. Prepare the self-control solution: accurately measure 1 ml of the S2 test solution in step b, place it in a 100 ml volumetric flask, dilute to the mark with diluent and shake well, as the self-control solution S3;

[0042] The diluent is a mixture of mobile phase and methanol in a volume ratio of 1:1.

[0043] d. Take 10ul of each of blank solution S1, self-control solution S3 and test solution S2 and inject them into the liquid chromatograph, and record the peak area of ​​cefuroxime hydrochloride in the chromatogram of cefuroxime hydrochloride control solution (such as Figure 3 as shown) and the polymer peak area in the chromatogram of the cefcapone hydrochloride test solution (as shown Figure 2 The polymer content C was calculated by the peak area self-reference external standard method.

[0044] The calculation formula is as follows:

[0045]

[0046] Where: As is the peak area of ​​the main component in the self-reference solution;

[0047] Ai is the peak area of ​​the polymer in the test solution

[0048] The total polymer content in the cefoperaxifen hydrochloride test sample was calculated to be 1.0% by the formula.

[0049] Methodological investigation

[0050] In order to further verify the feasibility of this method, the following methodological investigations were conducted:

[0051] 1. Specificity test

[0052] Accurately weigh an appropriate amount of cefcapone hydrochloride sample and prepare a 0.05 mg / ml test solution with a diluent. Take 10 ul for injection and record the separation degree and theoretical plate number of the polymer in the chromatogram. The results are shown in Table 2.

[0053] Table 2 Specificity test results

[0054]

[0055] The results show that the theoretical pedal number of each peak is not less than 2000, and the separation degree is not less than 1.5.

[0056] 2. Limit of detection and limit of quantification test

[0057] Accurately weigh an appropriate amount of cefuroxime hydrochloride reference substance, use a diluent to make a certain concentration, draw 10ul of solution respectively, inject into the liquid chromatograph, and record the chromatogram. The concentration when the signal-to-noise ratio is about 3:1 is taken as the detection limit, and the concentration when the signal-to-noise ratio is about 10:1 is taken as the quantitative limit. The results are shown in Table 3.

[0058] Table 3 Detection limit and quantification limit test results

[0059]

[0060] The results showed that the detection limit of polymers in cefcapene pivoxil hydrochloride was 0.000050 mg / ml, and the quantification limit was 0.00010 mg / ml.

[0061] 3. Linearity test

[0062] Accurately weigh an appropriate amount of cefcapone hydrochloride reference substance and prepare linear solutions of 0.00010 mg / ml, 0.00030 mg / ml, 0.00040 mg / ml, 0.00050 mg / ml, 0.00060 mg / ml, and 0.00070 mg / ml with diluent. Take 10 ul of each solution and inject it into the liquid chromatograph to record the chromatogram. The peak area of ​​each concentration is shown in Table 4.

[0063] Table 4 Linearity test results

[0064]

[0065]

[0066] Linear regression was performed using concentration versus peak area, and the linear equation correlation coefficient was r=0.9997.

[0067] The results showed that cefcapone hydrochloride had good linearity in the concentration range of 0.00010 mg / ml to 0.00070 mg / ml.

[0068] 4. Precision test

[0069] Accurately weigh an appropriate amount of cefcapone hydrochloride reference substance, dissolve and dilute to 0.0005 mg / ml with diluent, take 10 ul of solution, inject into liquid chromatograph, and inject 6 times continuously. Record the chromatogram, and the results are shown in Table 5.

[0070] Table 5 Reproducibility test results

[0071]

[0072]

[0073] The above test was repeated by different personnel at different times and the results are shown in Table 6.

[0074] Table 6 Intermediate precision test results

[0075]

[0076] The results showed that the method had good precision.

[0077] 5. Solution stability test

[0078] Accurately weigh an appropriate amount of cefcapone hydrochloride reference substance, dissolve it in a diluent and dilute it to 0.0005 mg / ml, place it at room temperature for 0 h, 4 h, and 8 h, inject the sample, and record the change in the peak area of ​​the main component. The results are shown in Table 7.

[0079] Table 7 Solution stability test results

[0080]

[0081] The results showed that the cefoperazone hydrochloride solution was unstable at room temperature and had to be prepared fresh before use.

[0082] 6. Method Durability Test

[0083] Accurately weigh an appropriate amount of biliverdin reference substance, dissolve it in a diluent and dilute it to 0.005 mg / ml. Make small changes to the flow rate and column temperature conditions in the chromatographic conditions to examine the effects on the retention time of the main component, separation degree and theoretical plate number. The results are shown in Table 8.

[0084] Table 8 Method durability test results

[0085]

[0086]

[0087] The theoretical plate numbers of the polymer peak and the main component peak under each condition were all greater than 2000, and the separation between two adjacent peaks was greater than 1.5, indicating that the measurement conditions met the durability requirements when there were small changes in the measurement conditions.

[0088] Comparative Example

[0089] In this comparative example, two comparative examples 1 and 2 are provided, whose chromatographic conditions are different from those in the embodiment.

[0090] The chromatographic conditions adopted in Comparative Example 1 are shown in Table 9.

[0091] Table 9 Chromatographic system conditions

[0092]

[0093] The chromatographic conditions adopted in Comparative Example 2 are shown in Table 10.

[0094] Table 10 Chromatographic system conditions

[0095]

[0096] The self-reference solution was prepared according to the preparation method of the reference solution described in the embodiment, and the chromatogram of the cefcapene pivoxil hydrochloride reference solution was recorded according to the chromatographic conditions in Table 9 in Comparative Example 1 and Table 10 in Comparative Example 2 (such as Figure 4 and Figure 5 The peak area of ​​cefcapone hydrochloride in the above sample was shown.

[0097] According to the embodiment Figure 3 It can be seen that the peak shapes of the control peaks are good and the separation meets the requirements, and the analysis time is appropriate; Figure 4 It can be seen that although the analysis time in Comparative Example 1 is short, the peaks are poorly separated and the separation degree does not meet the requirements; Figure 5 It can be seen that the analysis time in Comparative Example 2 is long and the peak shape is poor.

Claims

1. A new method for detecting the content of bile polymer in cefcapene pivoxil hydrochloride, characterized in that: The size of the polymer in cefcapone hydrochloride was determined by high performance size exclusion chromatography using a UV detector, and the chromatographic conditions were: Column: BioCoreSEC-120, 7.8×300 mm, 5 μm; Mobile phase: a mixed solution of mobile phase A and mobile phase B in a volume ratio of 95:5; Wherein, the mobile phase A is a mixed solution of 0.05 mol / L disodium hydrogen phosphate aqueous solution and 0.05 mol / L sodium dihydrogen phosphate aqueous solution in a volume ratio of 1:1; the mobile phase B is acetonitrile; Elution mode: isocratic elution; Flow rate: 0.7~0.9ml / min; Column temperature: 25-35°C; Detection wavelength: 254nm; Injection volume: 10ul; The ultraviolet detector is a differential refractive index detector, and the detector temperature is 35°C.

2. The novel method for detecting the content of bile polymer in cefcapene pivoxil hydrochloride according to claim 1, characterized in that The column temperature of the chromatographic column is 30°C.

3. The novel method for detecting the content of bile polymer in cefcapene pivoxil hydrochloride according to claim 1, characterized in that The flow rate is 0.8 ml / min.

4. The novel method for detecting the content of bile polymer in cefcapene pivoxil hydrochloride according to claim 1, characterized in that The following steps are involved: a. Prepare blank solution: Use diluent as blank solution; b. Prepare the test solution: Take the cefoperazone hydrochloride test sample and dissolve it in a diluent to a concentration of 0.1 mg / ml test solution; c. Prepare the self-control solution: accurately measure 1 ml of the test solution in step b, place it in a 100 ml volumetric flask, add diluent to dilute to the mark and shake well as the self-control solution; d. Take 10ul of each of the blank solution, self-control solution and test solution and inject them into the liquid chromatograph, record the peak area of ​​cefcapene hydrochloride in the chromatogram, and calculate the content of the polymer in the cefcapene hydrochloride sample according to the peak area self-control external standard method.

5. The novel method for detecting the content of bile polymer in cefcapene pivoxil hydrochloride according to claim 4, characterized in that The diluent is a mixture of mobile phase and methanol in a volume ratio of 1:1.

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