CN116087361A

CN116087361A - Method for detecting cefixime related substances

Info

Publication number: CN116087361A
Application number: CN202310036017.1A
Authority: CN
Inventors: 陈燕苹; 姜安民; 黄梓洋; 黄论达
Original assignee: Baiyunshan Chemical Pharmaceutical Factory Of Guangzhou Baiyunshan Pharmaceutical Holding Co ltd; Baiyunshan Chemical Pharmaceutical Zhuhai Co ltd
Current assignee: Baiyunshan Chemical Pharmaceutical Factory Of Guangzhou Baiyunshan Pharmaceutical Holding Co ltd; Baiyunshan Chemical Pharmaceutical Zhuhai Co ltd
Priority date: 2023-01-06
Filing date: 2023-01-06
Publication date: 2023-05-09

Abstract

The invention relates to a method for detecting cefixime related substances, and relates to the technical field of medicine quality detection. The method comprises the steps of detecting by using high performance liquid chromatography, wherein the detection conditions of the high performance liquid chromatography comprise: mobile phase: consists of a mobile phase A and a mobile phase B, wherein the mobile phase A is ammonium acetate aqueous solution-methanol (95:5), and the mobile phase B is ammonium acetate aqueous solution-methanol (50:50). The method for detecting cefixime polymer impurities can realize simultaneous detection of related substances of cefixime, including impurities A1, A2, A3, A4, B1, B2, C, D, E, F, B1, B2, t-butyl cefixime A, B, D, 7-methyl cefixime, cefixime imp, oxidation impurities of cefixime and AVNA impurity 1, and has the advantages of strong detection capability, good stability and strong specificity.

Description

Method for detecting cefixime related substances

Technical Field

The invention relates to the field of detection methods, in particular to a method for detecting cefixime related substances.

Background

Cefixime is a third-generation cephalosporin antibiotic for oral administration, and is suitable for treating respiratory, urinary and biliary tract infections caused by sensitive bacteria. Related substances in cefixime can influence the safety and effectiveness of cefixime products, and detection is needed to control the quality of the products.

The existing detection method of cefixime related substances can only realize the independent detection of degraded impurities or polymer impurities, has poor separation capability, and can not realize the positioning of each known impurity. The related substances of cefixime are detected by adopting the current detection method, on one hand, the detection is needed for a plurality of times, so that the workload of a drug analysis staff is extremely high; on the other hand, the detection capability is limited, the quality can not be controlled well, and the safety of the product has hidden trouble.

Disclosure of Invention

Based on the above, the invention aims to provide a method for detecting cefixime related substances, which can realize the simultaneous detection of the cefixime related substances, including impurity A1, impurity A2, impurity A3, impurity A4, impurity B1, impurity B2, impurity C, impurity D, impurity E, impurity F, trans-impurity B1, trans-impurity B2, cefixime tert-butyl ester A, cefixime polymer B, cefixime dimer D, cefixime 7-bit methyl ester, cefixime imp impurity, cefixime oxidation impurity and AVNA impurity 1, can realize the complete separation of the cefixime related substances from the cefixime main peak, and the separation degree of each impurity can meet the quantitative detection requirement of pharmacopoeia, can well meet the impurity monitoring requirements in the process of bulk drugs and preparations and the impurity control requirements in the finished products of the bulk drugs and preparations, and has the advantages of strong detection capability, good stability and strong specificity.

A method for detecting cefixime related substances. Detecting by liquid chromatography, wherein the related substances are impurities A, B, C, D, E, F, trans-impurity B, t-butyl cefixime A, cefixime polymer B, cefixime dimer D, 7-methyl cefixime, cefixime imp impurity, cefixime oxidation impurity and AVNA impurity 1; wherein the impurity a includes impurity A1, impurity A2, impurity A3, and impurity A4; the impurity B comprises an impurity B1 and an impurity B2; the trans impurity B comprises a trans impurity B1 and a trans impurity B2; the detection conditions of the liquid chromatography method include:

chromatographic column: ODS column;

mobile phase a: the mobile phase A comprises an ammonium acetate aqueous solution and methanol, wherein the volume ratio of the ammonium acetate aqueous solution to the methanol in the mobile phase A is 95:5, the pH of the mobile phase A is 4.1;

mobile phase B: ammonium acetate aqueous solution and methanol, wherein the volume ratio of the ammonium acetate aqueous solution to the methanol in the mobile phase B is 50:50, the pH of the mobile phase B is 3.8-3.9;

the detection process is carried out by adopting a gradient elution program, wherein a mobile phase of the gradient elution program consists of a mobile phase A and a mobile phase B, and the gradient elution program is set as follows:

the volume percentage of the mobile phase A is 90-100% within 0-5 min;

the volume percentage of the mobile phase A is 85-95% within 5-10 min;

the volume percentage of the mobile phase A is 80-90% within 10-20 min;

the volume percentage of the mobile phase A is 70-80% within 20-30 min;

the volume percentage of the mobile phase A is 60-70% within 30-60 min;

the volume percentage of the mobile phase A is 30-40% within 60-75 min;

the volume percentage of the mobile phase A is 0-10% within 75-85 min;

the volume percentage of the mobile phase A is 90-100% within 85-95 min.

The method for detecting the cefixime related substances can realize simultaneous detection of the cefixime related substances, comprises an impurity A1, an impurity A2, an impurity A3, an impurity A4, an impurity B1, an impurity B2, an impurity C, an impurity D, an impurity E, an impurity F, a trans-impurity B1, a trans-impurity B2, cefixime tert-butyl ester A, a cefixime polymer B, a cefixime dimer D, cefixime 7-bit methyl ester, cefixime imp impurities, cefixime oxidation impurities and AVNA impurities 1, and has the advantages of strong detection capability, good stability and strong specificity.

Further, the gradient elution procedure was set as follows:

the volume percentage of the mobile phase A is 100 percent within 0 to 5 minutes;

the volume percentage of the mobile phase A is 95 percent within 5 to 10 minutes;

the volume percentage of the mobile phase A is 90 percent within 10 to 20 minutes;

the volume percentage of the mobile phase A is 80 percent within 20 to 30 minutes;

the volume percentage of the mobile phase A is 70 percent within 30 to 60 minutes;

the volume percentage of the mobile phase A is 40% within 60-75 min;

at 75-85 min, the volume percentage of the mobile phase A is 0%;

the volume percentage of the mobile phase A is 100 percent within 85 to 95 minutes.

Further, the detection conditions of the liquid chromatography further include:

specification of the chromatographic column: 4.6 x 250mm,5 μm;

column temperature: 40-45 ℃;

flow rate: 1.5ml/min;

injector temperature: 4-15 ℃;

sample injection amount: 20 μl;

detection wavelength: 254nm.

Further, the concentration of the ammonium acetate aqueous solution was 3.85g/L.

Further, the front end of the chromatographic column is also connected with a ghost peak collecting column. The front end of the chromatographic column is connected with the ghost peak trapping column, so that the interference of baseline noise on impurity detection can be reduced, and the workload of a practitioner is reduced.

Further, the method comprises the steps of:

preparing a blank solution, a sensitivity solution, a system applicability solution, a reference substance solution and a test sample solution;

detecting by liquid chromatography to obtain detection result, and performing qualitative and quantitative analysis.

Further, the content of the related substances is calculated by adopting a principal component self-comparison method added with correction factors. Compared with the area normalization method and the self-comparison method without correction factors adopted by the current pharmacopoeia, the main component self-comparison method with correction factors is adopted, so that all known impurities in cefixime can be accurately quantified, the actual content of the impurities can be more accurately known, and the actual quality of products can be more accurately reflected.

Further, the correction factor is determined using a standard curve method.

Further, the analysis includes qualitative and/or quantitative analysis.

For a better understanding and implementation, the present invention is described in detail below with reference to the drawings.

Drawings

FIG. 1 is a detection chromatogram of a method for detecting cefixime related substances described in example 1;

FIG. 2 is a graph showing the comparison of detection chromatograms of example 1 with and without the ghost peaks trapping column;

FIG. 3 is a detection chromatogram of one method for detecting cefixime related impurities described in comparative example 1.

Detailed Description

The instrument and equipment information used in the detection process of the invention is shown in the following table 1:

table 1 instrument information

The information on the reagents used in the detection process of the present invention is shown in table 2 below:

TABLE 2 reagent information

The information of the standard and the reference used in the detection process of the invention is shown in the following table 3:

TABLE 3 Standard and control information

Name of the name	Source	Lot number	Content (%)
				Cefixime reference substance	Middle examining yard	130503-202007	88.9
Impurity A	Biological cards	PITBKW-A-EP-20190426-01	92.28
				Impurity B	Biological cards	PITBKW-B-EP-20190420-01	89.76
Impurity C	Biological cards	PITBKW-C-EP-20200304-06	96.14
				Impurity D	Biological cards	PITBKW-D-EP-20180622-05	98.32
Impurity E	Biological cards	PITBKW-E-EP-20180509-01	80.51
				Impurity F	Biological cards	PITBKW-F-EP-20180715-01	96.63
Trans impurity B	Biological cards	PITBKW-B(7E)-20200323-01	95.80
				Cefixime tert-butyl ester A	Biological cards	PITBKW-TBE-A-20180529-01	86.05
Cefixime polymer B	Biological cards	PITBKW-IMPB-YN1110-01	81.80
				Cefixime dimer D	Biological cards	PITBKW-DimerD-YN0820-03	87.50
Cefixime 7-methyl ester	Biological cards	PITBKW-7-ME-20190525-01	88.77
				Cefixime imp impurity	Biological cards	PITBKW-Imp-20190113-01	82.67
Cefixime oxidation impurity	Biological cards	PITBKW-O-20200717-01	84.86
				AVNA impurity 1	Biological cards	PI7-AVCA ImpB-20190513-01	96.70

The mechanism of each impurity according to the present invention is shown in Table 4 below

TABLE 4 impurity Structure type

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Sample information used in the detection process of the present invention is shown in table 5 below:

TABLE 5 sample information

Blank solution (diluent) preparation: preparing a potassium dihydrogen phosphate solution with the concentration of 9.08g/L and a disodium hydrogen phosphate solution with the concentration of 23.8g/L, and adopting the potassium dihydrogen phosphate solution: disodium hydrogen phosphate solution=39: 61, a blank solution was prepared, which also served as a diluent.

Preparing a system applicability solution: taking a proper amount of cefixime reference substance, adding water to dissolve and dilute the cefixime reference substance to prepare a solution containing about 0.5mg in each 1ml, then placing a measuring flask on a boiling water bath to heat for 45min, and cooling to obtain the system applicability solution.

Preparing a test solution: precisely weighing 25mg of cefixime test (KWK 2111102), placing into a 25ml measuring flask, adding a diluent into the 25ml measuring flask, diluting to a scale, and shaking uniformly to obtain a test solution.

Preparing a reference substance solution: precisely weighing 10mg of cefixime reference substance, placing in a 10ml measuring flask, adding a diluent into the 10ml measuring flask, diluting to a scale, and shaking uniformly; precisely measuring 100 mu l of the solution in the 10ml measuring flask, placing the solution in a 20ml measuring flask, adding a diluent to dilute to a scale, and shaking uniformly to obtain a reference solution;

preparing a sensitivity solution: precisely measuring 1ml of reference substance solution, placing into a 10ml measuring flask, adding diluent, diluting to scale, and shaking to obtain the final product;

preparing an impurity stock solution: and precisely weighing the standard substances of the impurity A, the impurity B, the impurity C, the impurity D, the impurity E, the impurity F, the trans-impurity B, the cefixime tert-butyl ester A, the cefixime polymer B, the cefixime dimer D, the cefixime 7-bit methyl ester, the cefixime imp impurity, the cefixime oxidized impurity and the AVNA impurity 1 according to the dosage shown in the table 6, respectively placing the standard substances into measuring bottles with the specifications defined in the table 6, adding a diluent to dilute to a scale, and shaking uniformly to obtain the impurity stock solution of each impurity. The invention develops a preparation method of the impurity stock solution, so that the impurity locating solution can meet the impurity limit standard and the quantitative limit.

TABLE 6 dosage of impurity standard in impurity stock solution

Name of the name	Dosage/mg	Gauge bottle/ml
			Impurity A	6.8	10
Impurity B	5.6	20
			Impurity C	3.3	25
Impurity D	3.3	25
			Impurity E	3.1	10
Impurity F	5.2	20
			Trans impurity B	3.3	25
Cefixime tert-butyl ester A	5.8	20
			Cefixime polymer impurity impB	3.1	10
Cefixime dimer D	4.3	20
			Cefixime 7-methyl ester	2.8	20
Cefixime imp impurity	4.5	20
			Cefixime oxidation impurity	2.9	20
AVNA impurity 1	2.6	20

Preparing an impurity positioning solution: 200 mu l of impurity stock solution of each impurity is precisely measured, and is respectively placed in a 25ml measuring flask, diluted to a scale by a diluent and uniformly shaken, so that the impurity positioning solution of each impurity is obtained.

Preparing a sample adding and marking solution: precisely weighing cefixime sample 25mg, placing into a 25ml measuring flask, adding a proper amount of diluent for dissolution, respectively adding 200 μl of impurity stock solution of each impurity, adding the diluent for dilution to scale, and shaking uniformly to obtain the sample addition standard solution.

Mobile phase A is prepared: preparing an ammonium acetate aqueous solution with the concentration of 3.85g/L, wherein the ammonium acetate aqueous solution is as follows: methanol=95: mobile phase a was formulated at a volume ratio of 5 and pH was adjusted to ph=4.1 with phosphoric acid.

Preparing a mobile phase B: preparing an ammonium acetate aqueous solution with the concentration of 3.85g/L, wherein the ammonium acetate aqueous solution is as follows: methanol=50: 50 volume ratio mobile phase B was formulated and pH adjusted with phosphoric acid to ph=3.8.

Example 1

The embodiment provides a method for detecting cefixime related substances, which comprises the following steps:

detecting by liquid chromatography, taking blank solution, impurity positioning solution of each impurity, test sample solution, and test sample labeling solution, and detecting by 1 needle, and recording analysis chromatogram under the following conditions:

chromatographic column: island Inertsil ODS-3V 4.6 x 250mm,5 μm; front end of the Ghost peak trapping Column (Ghost-Buster Column, 4.6X10 mm)

Mobile phase: mobile phase a or mobile phase B or a mixture of mobile phase a and mobile phase B;

mobile phase a:3.85g/L aqueous ammonium acetate-methanol (95:5), pH 4.1 with phosphoric acid;

mobile phase B:3.85g/L aqueous ammonium acetate-methanol (50:50), pH 3.8 with phosphoric acid;

detection wavelength: 254nm;

flow rate: 1.5ml/min;

column temperature: 40 ℃;

sample injection amount: 20 μl;

injector temperature: 4 ℃;

elution gradient settings are shown in table 7 below:

TABLE 7 elution gradient

Time (min)	Mobile phase a (%)	Mobile phase B (%)
			0	100	0
5	95	5
			10	90	10
20	80	20
			30	70	30
60	40	60
			75	0	100
80	0	100
			85	100	0
95	100	0

As the detection result is shown in fig. 1, it can be seen that by adopting the method for detecting cefixime related substances described in this embodiment, it is possible to detect impurity a, impurity B, impurity C, impurity D, impurity E, impurity F, trans-impurity B, cefixime tert-butyl ester a, cefixime polymer B, cefixime dimer D, cefixime 7-bit methyl ester, cefixime imp impurity, cefixime oxidation impurity and AVNA impurity 1 simultaneously, wherein impurity a includes impurity A1, impurity A2, impurity A3 and impurity A4; the impurity B comprises an impurity B1 and an impurity B2; the trans impurity B comprises a trans impurity B1 and a trans impurity B2; and each impurity can be completely separated from the main peak, the separation degree of each impurity can meet the quantitative detection requirement of pharmacopoeia, and the impurity monitoring in the technological process of the raw material medicine and the preparation and the impurity control requirement in the finished products of the raw material medicine and the preparation can be well met. Wherein the impurity A1, the impurity A2, the impurity A3 and the impurity A4 are respectively different isomers of the impurity A specified in pharmacopoeia; the impurity B1, the impurity B2, the impurity trans-B1 and the impurity trans-B2 are respectively different isomers of the impurity B specified in pharmacopoeia.

The blank solution of the method for detecting cefixime related substances in the embodiment does not interfere with the determination of the main peak and each impurity, the separation degree of the main peak and the adjacent peak is 3.05, the minimum separation degree of each known impurity and the adjacent peak is 1.51, and the minimum separation degree is more than 1.5, thereby meeting the requirements of Chinese pharmacopoeia.

Because the detection method provided by the embodiment adopts gradient elution, and the impurity types are more, in order to make the impurity detection effect better, the front end of the chromatographic column is connected with the ghost peak trapping column, so that the interference of the baseline noise on the impurity detection is reduced, the influence of the connection of the ghost peak trapping column and the non-connection of the ghost peak trapping column blank solution is shown in the figure 2, and the interference exists at the positions of 24min, 55min, 67min and 80min in the spectrogram of the blank solution when the trapping column is not connected, so that the judgment of the impurity is not facilitated.

In other embodiments, the temperature of the injector can be directly adjusted at 4-15 ℃, if the temperature is lower than 4 ℃, the requirement on experimental instruments is too high, and the detection cost is too high; if the temperature is higher than 15 ℃, the sample solution and the sample labeling solution are unstable, so that new impurities are generated and the detection result is influenced.

In other embodiments, the gradient elution program settings may also be adjusted within the following ranges:

the volume percentage of the mobile phase A is 90-100% within 0-5 min;

the volume percentage of the mobile phase A is 85-95% within 5-10 min;

the volume percentage of the mobile phase A is 80-90% within 10-20 min;

the volume percentage of the mobile phase A is 70-80% within 20-30 min;

the volume percentage of the mobile phase A is 60-70% within 30-60 min;

the volume percentage of the mobile phase A is 30-40% within 60-75 min;

the volume percentage of the mobile phase A is 0-10% within 75-85 min;

the volume percentage of the mobile phase A is 90-100% within 85-95 min.

Example 2

The present example is a method for determining an impurity correction factor for each impurity detected by the method of example 1, specifically, a standard curve method, and the detection in the present example is carried out along with the chromatographic conditions of example 1.

Cefixime linear stock solution preparation: precisely weighing 14mg of cefixime reference substance, placing in a 20ml measuring flask, adding a diluent to dilute to a scale, and shaking uniformly to obtain linear stock solution of cefixime.

Preparing a linear solution: precisely measuring cefixime linear stock solution, impurity A, impurity B, trans-impurity B, impurity C, impurity D, impurity E, impurity F, cefixime tert-butyl A, imp impurity, polymer B and dimer D according to the stock solution dosage shown in table 8 to prepare linear 1 solutions with the linear level of 25%, 50%, 100%, 200% and 300%, wherein the cefixime stock solution is also required to prepare linear 1 solution with the linear level of 10%; next, precisely measuring cefixime linear stock solution and impurity A, impurity B, trans-impurity B, impurity C, impurity D, impurity E, impurity F, cefixime tert-butyl A, imp impurity, polymer B, dimer D, oxidized impurity, 7-methyl ester and AVNA impurity 1 impurity stock solution respectively according to the stock solution dosage shown in table 8 to prepare linear 2 solution with the linear level of 25%, 50%, 100%, 150%, 200% and 300%, wherein the cefixime stock solution also needs to prepare linear 2 solution with the linear level of 10%; thus obtaining a series of linear 1 solution and linear 2 solution.

TABLE 8 Linear solution formulation Table

Linear level	Remove impurity stock solution/cefixime linear stock solution volume (ul)	Constant volume (ml)
			10％	20	25
25％	50	25
			50％	100	25
100％	200	25
			150％	300	25
200％	400	25
			300％	500	25

Detection was performed by liquid chromatography: taking blank solution and each linear 1 solution sample injection 1 needle, taking the concentration C (mug/ml) as an abscissa and the peak area (A) as an ordinate, and carrying out linear regression by using a least square method to obtain a linear 1 solution regression equation and related coefficients, intercept and slope thereof; controlling other chromatographic conditions to be the same on the other level instrument and the other chromatographic column, taking a blank solution and each linear 2 solution sample injection 1 needle, taking the concentration C (mug/ml) as an abscissa and the peak area (A) as an ordinate, and carrying out linear regression on the detection result of the same impurity by using a least square method to obtain a linear 2 solution regression equation and related coefficients, intercept and slope thereof; the correction factors of the linear 1 solution and the correction factors of the linear 2 solution were calculated, respectively, and the average value of the correction factors of the linear 1 solution and the correction factors of the linear 2 solution of the same impurity was taken as the impurity correction factor of each impurity detected by the method of example 1. Some impurity correction factor of linear 1 = linear 1 solution regression equation slope of cefixime stock/linear 1 solution regression equation slope of some impurity stock, some impurity correction factor of linear 2 = linear 2 solution regression equation slope of cefixime stock/linear 2 solution regression equation slope of some impurity stock. The linear regression results are shown in table 9:

TABLE 9 Linear regression results

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When the content of cefixime related substances is calculated in the current pharmacopoeia of various countries, as the impurity A, the impurity B and the trans-impurity B are all mixtures and the correction factors are not in the range of 0.9-1.1, the main component self-comparison method added with the correction factors is not adopted for calculation, so that the accuracy of the calculation result of the impurity is limited, and the true quality of the product cannot be reflected; in the embodiment, the main component self-comparison method for adding the correction factors to all known impurities in cefixime is implemented, so that compared with the area normalization method adopted in the current pharmacopoeia and the self-comparison method without adding the correction factors, all the known impurities in cefixime can be accurately quantified, the actual content of the impurities can be more accurately known, and the actual quality of a product can be more accurately reflected.

Example 3

In this example, the influence of changes in chromatographic conditions such as pH, flow rate, column temperature, and column chromatography of the mobile phase on the detection effect of the method for detecting cefixime-related substance described in example 1 was examined, and in this example, the chromatographic conditions were carried out along the chromatographic conditions of example 1 except for the single chromatographic conditions which were varied.

Preparing a reference substance solution: precisely weighing 10mg of cefixime reference substance, placing in a 10ml measuring flask, adding diluent to dilute to scale, and shaking; precisely measuring 100 μl of the above solution, placing in 20ml measuring flask, diluting with diluent to scale, shaking, and concocting in parallel to 2 parts to obtain reference solution 1 and reference solution 2.

Liquid chromatography detection was performed by varying the chromatographic conditions according to the defined single factor of table 10: taking blank solution, sensitivity solution, system applicability solution 1 needle, control solution 1 needle 6 needle, control solution 2 needle 1 needle, and sample adding solution 1 needle, and recording chromatogram.

The single factor variation refers to that the chromatographic condition of the example 1 is used as a standard condition, only one chromatographic condition is changed at each variation, and only one chromatographic condition is different from the standard condition at each variation.

TABLE 10 chromatographic condition variation information

Durability system applicability results are shown in table 11, and durability test sample labeling solution results are shown in table 12:

table 11 durability system applicability results

Table 12 results of test sample labeling solutions for durability

When the pH change of the mobile phases A and B is +/-0.1, the flow rate change is +/-0.1 ml/min, the column temperature change is +/-5 ℃ and different chromatographic columns are replaced, the minimum signal to noise ratio of cefixime peaks in the sensitivity solution is 10.5, and the minimum signal to noise ratio is more than 10; in a system applicability solution, the minimum value of the separation degree between a cefixime (E) isomer peak and a cefixime peak is 10.88, both are larger than 8.0, the maximum value of the tailing factor of the cefixime peak is 1.15, both are smaller than 1.5; continuously injecting the control solution into 6 needles, wherein the maximum value of the peak area RSD is 0.40% and is less than 5.0%; the system applicability meets the requirements under all conditions.

When the pH of mobile phase A is 4.0, the pH of mobile phase B is 3.7, the column temperature is 35 ℃ and the flow rate is changed by +0.1ml/min, the imp impurity and the impurity F are not separated, and cannot be accurately and quantitatively calculated; when the pH of mobile phase a was 4.2, the impB and unknown mono-impurities (rrt=2.51) were not separated; when the pH of mobile phase B was 3.9, the column temperature was 45℃and the column was changed, the maximum value of the total impurity content and the total impurity content was 0.05, and the durability was good.

Example 4

In this example, based on the impurity correction factors of the respective impurities determined in example 3, the known impurities, the individual other impurities and the total impurity content in the cefixime test samples KWK2111102, KWK2111105 and KWK2111116 were calculated after the chromatographic condition test according to example 3.

The test method of this example was the same as that of example 1, and the test results are shown in table 13 below.

TABLE 13 test results of cefixime test specimens

Comparative example 1

The comparative example is a method for detecting cefixime related impurities, comprising the following steps:

Preparing a system applicability solution: taking a proper amount of cefixime reference substance, adding water to dissolve and dilute the cefixime reference substance to prepare a solution containing about 1mg per 1ml, then placing a measuring flask on a boiling water bath to heat for 45min, and cooling to room temperature to obtain the system applicability solution.

Preparing a test solution: precisely weighing a proper amount of cefixime sample, placing the sample into a 25ml measuring flask, adding a diluent to dilute the sample to a solution containing 1mg of cefixime sample per 1ml, filtering, discarding 2.0ml of primary filtrate, and taking the subsequent filtrate as a sample solution to obtain the sample solution.

Mobile phase A is prepared: 3.85g of ammonium acetate was taken, dissolved by adding 1000ml of water, mixed well with 52.5ml of methanol, and the pH was adjusted to pH=4.5 with phosphoric acid.

Preparing a mobile phase B:3.85g of ammonium acetate was taken, dissolved by adding 1000ml of water, mixed well by adding 1000ml of methanol, and the pH was adjusted to pH=4.2 with phosphoric acid.

Detection was performed by liquid chromatography under the following conditions:

chromatographic column: island Inertsil ODS-3V 4.6 x 250mm,5 μm;

mobile phase a:3.85g/L ammonium acetate in water-methanol, pH 4.5 with phosphoric acid;

mobile phase B:3.85g/L ammonium acetate in water-methanol, pH 4.2 with phosphoric acid;

detection wavelength: 254nm;

flow rate: 1.5ml/min;

column temperature: 40 ℃;

elution gradient settings are shown in table 14 below:

TABLE 14 elution gradient

As shown in fig. 3, it can be seen from the spectrum that the method for detecting cefixime related impurities described in this comparative example can only detect impurity A1, impurity A2, impurity A3, impurity A4, impurity B1, impurity B2, impurity C, impurity D, impurity E, impurity F and tert-butyl ester a simultaneously, and has obvious defects: the impurity F and the polymer B have low separation degree, the blank solvent has interference on the tert-butyl ester A, the polymer B and the dimer D are not confirmed, only the polymer impurity can be used as unknown impurity to control, and the like, and the polymer B and the dimer D are process impurities generated with starting materials in the synthesis process, have great influence on the quality of raw materials and can influence the safety and the effectiveness of products. Therefore, the method of comparative example 1 cannot identify a large impurity peak, and cannot control the quality of the raw material well.

After the detection method of comparative example 1 is adopted, other methods are additionally adopted for working in order to detect the polymer impurities, and compared with the method of example 1, the method of example 1 greatly increases the workload of drug analysis practitioners, and compared with the method of example 1, the method of example 1 has stronger detection capability on the known impurities of cefixime, more comprehensive quality control capability on the cefixime and better separation effect, can enable the detection of the cefixime impurities to be simpler and more convenient, and improves the working efficiency of the drug analysis practitioners.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that modifications and improvements can be made by those skilled in the art without departing from the spirit of the invention, and the invention is intended to encompass such modifications and improvements.

Claims

1. A method for detecting cefixime related substances is characterized in that liquid chromatography is adopted for detection, wherein the related substances are impurities A, B, C, D, E, F, trans-impurity B, cefixime tert-butyl ester A, cefixime polymer B, cefixime dimer D, 7-bit methyl ester of cefixime, cefixime imp impurity, cefixime oxidation impurity and AVNA impurity 1; wherein the impurity a includes impurity A1, impurity A2, impurity A3, and impurity A4; the impurity B comprises an impurity B1 and an impurity B2; the trans impurity B comprises a trans impurity B1 and a trans impurity B2; the detection conditions of the liquid chromatography method include:

chromatographic column: ODS column;

the volume percentage of the mobile phase A is 90-100% within 0-5 min;

the volume percentage of the mobile phase A is 85-95% within 5-10 min;

the volume percentage of the mobile phase A is 80-90% within 10-20 min;

the volume percentage of the mobile phase A is 70-80% within 20-30 min;

the volume percentage of the mobile phase A is 60-70% within 30-60 min;

the volume percentage of the mobile phase A is 30-40% within 60-75 min;

the volume percentage of the mobile phase A is 0-10% within 75-85 min;

the volume percentage of the mobile phase A is 90-100% within 85-95 min.

2. A method for detecting cefixime related substances according to claim 1, wherein the gradient elution procedure is set as follows:

the volume percentage of the mobile phase A is 40% within 60-75 min;

at 75-85 min, the volume percentage of the mobile phase A is 0%;

3. The method for detecting cefixime related substance according to claim 1, wherein the detection conditions of the liquid chromatography further comprise:

specification of the chromatographic column: 4.6 x 250mm,5 μm;

column temperature: 40-45 ℃;

flow rate: 1.5ml/min;

injector temperature: 4-15 ℃;

sample injection amount: 20 μl;

detection wavelength: 254nm.

4. The method for detecting cefixime related substances according to claim 1, wherein the concentration of the aqueous solution of ammonium acetate is 3.85g/L.

5. The method for detecting cefixime related substances according to claim 1, wherein the front end of the chromatographic column is further connected with a ghost peak trapping column.

6. A method for detecting cefixime related substances according to claim 1, characterized in that the method comprises the steps of:

preparing a blank solution, an impurity positioning solution of each impurity, a sample solution and a sample labeling solution;

detecting by liquid chromatography, sequentially injecting blank solution, impurity positioning solution of each impurity, sample solution, and sample adding solution to obtain detection result, and analyzing.

7. The method for detecting cefixime related substances according to claim 1, wherein the content of the related substances is calculated by a principal component self-contrast method with correction factors added.

8. The method for detecting cefixime related substances according to claim 7, wherein the correction factor is determined by standard curve method.

9. The method for detecting cefixime related substances according to claim 8, wherein the standard curve method comprises the steps of:

preparing a blank solution, an impurity stock solution of each related substance and a cefixime linear stock solution;

preparing a plurality of groups of linear solutions with linear level from impurity stock solution of each related substance and linear stock solution of cefixime;

detecting by liquid chromatography, and sequentially injecting the blank solution and the groups of linear solutions with linear level;

performing linear regression on the same group of linear solutions with the concentration as an abscissa and the peak area as an ordinate by using a least square method to obtain regression equations of a plurality of linear solutions and correlation coefficients, intercept and slope of the regression equations;

calculating the correction factor of the related substance, wherein the correction factor of the related substance is equal to the linear solution regression equation slope of the cefixime linear stock solution/the linear solution regression equation slope of the impurity stock solution of the related substance.

10. A method for detecting cefixime related substances according to claim 6, wherein the analysis comprises qualitative and/or quantitative analysis.