CN115993415A - Novel method for detecting content of polymer in cefcapene pivoxil hydrochloride - Google Patents

Abstract

The invention belongs to the technical field of medical analysis, and particularly discloses a novel method for detecting the content of a polymer in cefcapene pivoxil hydrochloride. The method mainly adopts a high performance molecular exclusion chromatography (HPSEC) method to measure the size of the polymer in the cefcapene pivoxil hydrochloride by using an ultraviolet detector, and the chromatographic conditions are as follows: chromatographic column: a BioCoreEC-120,7.8X 300mm,5 μm or equivalent performance chromatographic column; mobile phase: a mixed solution of the mobile phase A and the mobile phase B according to the volume ratio of 95:5; wherein the mobile phase A is a mixed solution of 0.05mol/L disodium hydrogen phosphate aqueous solution and 0.05mol/L sodium dihydrogen phosphate aqueous solution according to a volume ratio of 1:1; the mobile phase B is acetonitrile; elution mode: isocratic elution; flow rate: 0.7-0.9 ml/min; column temperature: 25-35 ℃; detection wavelength: 254nm; sample injection amount: 10ul.

Description

Novel method for detecting content of polymer in cefcapene pivoxil hydrochloride

Technical Field

The invention relates to the technical field of medical analysis, in particular to a novel method for detecting the content of a polymer in cefcapene pivoxil hydrochloride.

Background

Cefcapene pivoxil hydrochloride is a fourth-generation orally-taken cephalosporin antibiotic, belongs to beta-lactam antibiotics, and is suitable for infection caused by streptococcus, staphylococcus, gonococcus and the like. Research shows that the immediate allergic reaction caused by the beta-lactam antibiotics is not caused by the medicine itself, and is related to the high molecular polymer existing in the medicine. The various polymeric impurities in the beta-lactam antibiotics have similar biological characteristics (such as hypersensitivity) although the structures are different, so that the polymers with different structures are not required to be controlled one by one in quality control, and only the total amount of the polymers is required to be controlled. The high molecular polymer in cephalosporin antibiotics refers to the total term of impurities with molecular weight larger than that of the drug, and the molecular weight is generally 1000-5000Da, and the molecular weight can reach about 15000Da respectively.

Because the high molecular polymer has high heterogeneity and uncertainty, a reference substance with fixed content cannot be prepared, no related cefcapene pivoxil hydrochloride polymer detection method exists in the prior patent and literature, and related determination methods are not recorded in foreign pharmacopoeias such as USP and EP. The method for measuring the high polymer in the beta-lactam antibacterial drug by using the self-contrast external standard method is carried out in the 2020 edition of Chinese pharmacopoeia, and adopts a gel chromatography method which takes glucose gel Sephadex G-10 as a chromatographic column. The high molecular polymer in the drug is detected by using the drug self-reference substance to replace the high molecular polymer reference substance through switching of two mobile phases, which is a common technology at present and is used for detecting the polymer in the cephalosporin antibiotics. However, the method has the disadvantages of low column efficiency (about 1000), tailing peak shape, long analysis time, poor separation effect of high polymer impurities (all high polymer is one peak, and the separation effect of the peak and a main peak baseline is poor), very low detection amount of the high polymer, and the like of the gel chromatographic column. Therefore, the production process of cefcapene pivoxil hydrochloride bulk drug and the quality control of finished products are very inconvenient, and the content of the polymer in the cefcapene pivoxil hydrochloride cannot be rapidly and accurately quantified.

Therefore, the detection method for rapidly and effectively determining the polymer content in the cefcapene pivoxil hydrochloride, which is simple to operate and high in sensitivity, is very important.

Disclosure of Invention

The invention aims at: the invention overcomes the defects in the prior gel chromatography technology, provides a detection method for rapidly and effectively determining the content of the polymer in cefcapene pivoxil hydrochloride, and is different from the traditional gel chromatography method applied to the detection of the polymer in antibiotics.

The invention adopts a High Performance Size Exclusion Chromatography (HPSEC) method to measure the polymer content in the cefuroxime axetil hydrochloride. By differentiating the sizes of the component molecules, the high-efficiency molecular exclusion chromatography can permeate into the macropores of the gel, while the small component molecules can permeate into the macropores and the micropores of the macropores, even enter deeply, and are not easy to elute. Therefore, the large component molecules have short residence time in the chromatographic column and are eluted quickly, and the large component molecules and the small component molecules are separated according to the molecular size in the size exclusion chromatographic system to complete the elution process.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

a novel method for detecting the content of a biliary polymer in cefcapene pivoxil hydrochloride adopts a high-performance molecular exclusion chromatography (HPSEC) method to detect the size of the polymer in the cefcapene pivoxil hydrochloride by using an ultraviolet detector, and the chromatographic conditions are as follows:

chromatographic column: a BioCoreEC-120,7.8X 300mm,5 μm or equivalent performance chromatographic column;

mobile phase: a mixed solution of the mobile phase A and the mobile phase B according to the volume ratio of 95:5;

wherein the mobile phase A is a mixed solution of 0.05mol/L disodium hydrogen phosphate aqueous solution and 0.05mol/L sodium dihydrogen phosphate aqueous solution according to a volume ratio of 1:1; the mobile phase B is acetonitrile;

elution mode: isocratic elution;

flow rate: 0.7-0.9 ml/min;

column temperature: 25-35 ℃;

detection wavelength: 254nm;

sample injection amount: 10ul.

In the invention, the ultraviolet detector is a differential refraction detector, and the temperature of the detector is 35 ℃.

In the invention, the column temperature of the chromatographic column is preferably 30 ℃; the flow rate is preferably 0.8ml/min.

In the invention, the novel method for detecting the content of the liner polymer in the cefcapene pivoxil hydrochloride comprises the following steps:

a. preparing a blank solution: using a diluent as a blank solution;

b. preparing a sample solution: taking a cefcapene pivoxil hydrochloride sample, and dissolving the cefcapene pivoxil hydrochloride sample into a sample solution (prepared by clinical use) with the concentration of 0.1mg/ml by using a diluent;

c. preparing self-control solution: b, precisely measuring 1ml of the sample solution in the step b, placing the sample solution in a 100ml measuring flask, adding a diluent to dilute to a scale, and shaking uniformly to serve as a self control solution;

d. and respectively taking 10ul of blank solution, self-control solution and sample solution, respectively injecting into a liquid chromatograph, respectively recording the peak area of cefcapene pivoxil hydrochloride in the chromatogram, and calculating the content of the polymer in the cefcapene pivoxil hydrochloride sample according to the peak area self-control external standard method.

The diluent is a mixed solution of a mobile phase and methanol according to the volume of 1:1.

In summary, the beneficial effects of the invention are as follows:

the method can rapidly and accurately determine the content of the polymer in the cefcapene pivoxil hydrochloride. Compared with the existing gel chromatography method using glucose gel Sephadex G-10 as chromatographic column. The method of the invention uses high-efficiency molecular exclusion chromatography and cooperates with SEC chromatographic column, and the method has strong specificity, high accuracy and high sensitivity. The ultraviolet detector (UV) with higher cost performance is selected, and the reversed-phase ultra-high performance liquid chromatography is applied, so that the method has the advantages of strong reliability and low detection cost, and is suitable for quality inspection of intermediate products and finished products in cefcapene pivoxil hydrochloride production.

Drawings

FIG. 1 is a chromatogram of a white solvent S1 in the example;

FIG. 2 is a chromatogram of cefcapene pivoxil hydrochloride sample solution S2 in the example;

FIG. 3 is a chromatogram of cefcapene pivoxil hydrochloride control solution S3 in the example;

FIG. 4 is a chromatogram of a cefcapene pivoxil hydrochloride control solution in comparative example 1;

FIG. 5 is a chromatogram of the cefcapene pivoxil hydrochloride control solution of comparative example 2.

Detailed Description

The invention is further illustrated, but not limited, by the following examples.

Examples

The test sample of cefcapene pivoxil hydrochloride selected in the embodiment is cefcapene pivoxil hydrochloride self-made by Zhongshan lark biotechnology Co., ltd.

The chromatographic conditions used in this example are shown in table 1:

TABLE 1 chromatographic System conditions

The method for measuring the content of the cefcapene pivoxil hydrochloride polymer comprises the following steps:

a. preparing a blank solution: as blank solution S1, a mobile phase, alcohol=1:1 diluent was used;

b. preparing a sample solution: taking a cefcapene pivoxil hydrochloride sample, and dissolving the cefcapene pivoxil hydrochloride sample into a sample solution S2 (prepared by clinical use) with the concentration of 0.1mg/ml by using a diluent;

c. preparing self-control solution: b, precisely measuring 1ml of the sample solution S2 in the step b, placing the sample solution into a 100ml measuring flask, adding a diluent to dilute to a scale, and shaking uniformly to serve as self-control solution S3;

the diluent is a mixed solution of a mobile phase and methanol according to the volume ratio of 1:1.

d. And respectively taking 10ul of blank solution S1, self control solution S3 and sample solution S2, respectively, injecting into a liquid chromatograph, recording the peak area of cefcapene pivoxil hydrochloride in a cefcapene pivoxil hydrochloride control solution chromatogram (shown in figure 3) and the peak area of a polymer in the cefcapene pivoxil hydrochloride sample solution chromatogram (shown in figure 2), and calculating the content C of the polymer according to a peak area self-control external standard method.

The calculation formula is as follows:

wherein: as is the peak area of the main component in the self-control solution;

ai is the peak area of the polymer in the sample solution

The total content of the polymer in the cefcapene pivoxil hydrochloride test sample is 1.0% according to the formula.

Methodology investigation

To further verify the feasibility of the method, the following methodological investigation was performed:

1. specificity test

The cefcapene pivoxil hydrochloride sample is precisely weighed, a proper amount of the cefcapene pivoxil hydrochloride sample is prepared into 0.05mg/ml of test solution by using a diluent, 10ul of sample is taken, and the separation degree and theoretical plate number of the chromatogram polymer are recorded, and the result is shown in Table 2.

TABLE 2 results of specificity experiments

The results show that: the number of theoretical pedals of each peak is not less than 2000, and the separation degree is not less than 1.5.

2. Limit of detection and limit of quantification test

Precisely weighing cefcapene pivoxil hydrochloride reference substance, preparing into a certain concentration with diluent, respectively sucking 10ul solution, injecting into a liquid chromatograph, and recording a chromatogram. The results are shown in Table 3, with the concentration at a signal to noise ratio of about 3:1 as the limit of detection and the concentration at a signal to noise ratio of about 10:1 as the limit of quantification.

TABLE 3 detection limit and quantitative limit test results

The results show that: the detection limit of the polymer in the cefcapene pivoxil hydrochloride is 0.000050mg/ml, and the quantitative limit is 0.00010mg/ml.

3. Linearity test

Precisely weighing cefcapene pivoxil hydrochloride reference substance, and respectively preparing into linear solutions of 0.00010mg/ml, 0.00030mg/ml, 0.00040mg/ml, 0.00050mg/ml, 0.00060mg/ml and 0.00070mg/ml by using a diluent. 10ul of the solution was taken and poured into a liquid chromatograph, and the chromatogram was recorded, and the peak areas of the concentrations are shown in Table 4.

TABLE 4 Linear experiment results

Linear regression was performed on peak area with concentration to obtain a linear equation correlation coefficient r=0.9997.

The results show that: cefcapene pivoxil hydrochloride has good linearity within the concentration range of 0.00010mg/ml to 0.00070 mg/ml.

4. Precision test

Precisely weighing a proper amount of cefcapene pivoxil hydrochloride reference substance, respectively dissolving and diluting to 0.0005mg/ml with a diluent, respectively taking 10ul solution, injecting into a liquid chromatograph, and continuously sampling for 6 times. The chromatogram was recorded and the results are shown in table 5.

TABLE 5 repeatability test results

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

TABLE 6 results of intermediate precision test

The results show that: the method has good precision.

5. Solution stability test

Accurately weighing cefcapene pivoxil hydrochloride reference substance, dissolving and diluting to 0.0005mg/ml with diluent, standing at normal temperature for 0h, 4h and 8h respectively, sampling, and recording peak area change of main component, and the result is shown in Table 7.

TABLE 7 results of solution stability test

The results show that: the cefcapene pivoxil hydrochloride solution is unstable at normal temperature and needs to be prepared newly.

6. Method durability test

Precisely weighing a proper amount of biliverdin reference substance, dissolving and diluting to 0.005mg/ml with diluent, performing small-scale modification on the flow rate in chromatographic conditions and chromatographic column temperature conditions, examining the influence on the retention time, the separation degree and the theoretical plate number of the main component, and examining the results shown in Table 8.

Table 8 method durability test results

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The theoretical plate numbers of the polymer peak and the main component peak under each condition are larger than 2000, and the separation degree between the adjacent two peaks is larger than 1.5, which shows that the measurement conditions meet the durability requirement when the measurement conditions have small variation.

Comparative example

In this comparative example, two comparative examples 1 and 2, which are different from the chromatographic conditions in the examples, were set.

The chromatographic conditions used in comparative example 1 are shown in table 9.

TABLE 9 chromatographic System conditions

The chromatographic conditions used in comparative example 2 are shown in table 10.

TABLE 10 chromatographic system conditions

The control solutions themselves were prepared as described in the examples, and the peak areas of cefcapene pivoxil hydrochloride in the chromatograms (shown in fig. 4 and 5) of the cefcapene pivoxil hydrochloride control solutions were recorded according to the chromatographic conditions in table 9 of comparative example 1 and table 10 of comparative example 2, respectively.

According to the figure 3 in the embodiment, the peak shape of each peak of the comparison is good, the separation degree meets the requirement, and the analysis time is proper; as can be seen from FIG. 4, in comparative example 1, although the analysis time was short, the separation of each peak was poor, and the degree of separation was unsatisfactory; it can be seen from fig. 5 that the analysis time was long and the peak shape was also poor in comparative example 2.

Claims (6)

1. A novel method for detecting the content of a biliary polymer in cefcapene pivoxil hydrochloride is characterized in that an ultraviolet detector is applied to the determination of the size of the polymer in the cefcapene pivoxil hydrochloride by adopting a high-efficiency molecular exclusion chromatography, and the chromatographic conditions are as follows:

chromatographic column: a BioCoreEC-120,7.8X 300mm,5 μm or equivalent performance chromatographic column;

mobile phase: a mixed solution of the mobile phase A and the mobile phase B according to the volume ratio of 95:5;

wherein the mobile phase A is a mixed solution of 0.05mol/L disodium hydrogen phosphate aqueous solution and 0.05mol/L sodium dihydrogen phosphate aqueous solution according to a volume ratio of 1:1; the mobile phase B is acetonitrile;

elution mode: isocratic elution;

flow rate: 0.7-0.9 ml/min;

column temperature: 25-35 ℃;

detection wavelength: 254nm;

sample injection amount: 10ul.

2. The method for detecting the content of the liner polymer in the cefcapene pivoxil hydrochloride according to claim 1, wherein the ultraviolet detector is a differential refraction detector, and the temperature of the detector is 35 ℃.

3. The novel method for detecting the content of the biliary polymer in cefcapene pivoxil hydrochloride according to claim 1, wherein the column temperature of the chromatographic column is 30 ℃.

4. The novel method for detecting the content of the biliary polymer in cefcapene pivoxil hydrochloride according to claim 1, wherein the flow rate is 0.8ml/min.

5. The novel method for detecting the content of the biliary polymer in the cefcapene pivoxil hydrochloride according to claim 1, which is characterized by comprising the following steps:

a. preparing a blank solution: using a diluent as a blank solution;

b. preparing a sample solution: taking a cefcapene pivoxil hydrochloride sample, and dissolving the cefcapene pivoxil hydrochloride sample into a sample solution with the concentration of 0.1mg/ml by using a diluent;

c. preparing self-control solution: precisely measuring 1ml of the sample solution in the step b, placing the sample solution in a 100ml measuring flask, adding a diluent to dilute to a scale, and shaking uniformly to serve as a self control solution;

d. and respectively taking 10ul of blank solution, self-control solution and sample solution, respectively injecting into a liquid chromatograph, respectively recording the peak area of cefcapene pivoxil hydrochloride in the chromatogram, and calculating the content of the polymer in the cefcapene pivoxil hydrochloride sample according to the peak area self-control external standard method.

6. The method for detecting the content of the biliary polymer in cefcapene pivoxil hydrochloride according to claim 5, wherein the diluent is a mixed solution of a mobile phase and methanol in a volume ratio of 1:1.

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