CN111487354A

CN111487354A - Method for detecting cefixime related impurities

Info

Publication number: CN111487354A
Application number: CN202010344563.8A
Authority: CN
Inventors: 苏燕琼; 胡海容; 蔡国伟; 王健松; 高琳; 叶伟文; 李卓
Original assignee: Guangzhou Baiyunshan Pharmaceutical Holdings Co ltd Baiyunshan Pharmaceutical General Factory
Current assignee: Guangzhou Baiyunshan Pharmaceutical Holdings Co ltd Baiyunshan Pharmaceutical General Factory
Priority date: 2020-04-27
Filing date: 2020-04-27
Publication date: 2020-08-04
Anticipated expiration: 2040-04-27
Also published as: CN111487354B

Abstract

The invention discloses a method for detecting cefixime related impurities, which adopts liquid chromatography to detect through reverse phase chromatography, and the detection conditions comprise that: the mobile phase comprises a mixed solution obtained by mixing a phase A and a phase B, wherein the phase A is an ammonium acetate aqueous solution with or without an added organic solvent, and the phase B is an organic solvent with or without an added ammonium acetate solution; wherein, the organic solvent in the phase A and the phase B is independently selected from at least one of acetonitrile or lower alcohol. The method can effectively detect cefixime related impurities, avoids using ion pair mobile phase, solves the problems of large damage to the chromatographic column, low detection sensitivity and the like of the original detection method, prolongs the service life of the chromatographic column, saves the detection cost, has low noise of a detection baseline by adopting an ammonium acetate solution, and has stronger detection capability on low-content impurities and better sensitivity.

Description

Method for detecting cefixime related impurities

Technical Field

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

Background

Cefixime is a third-generation oral cephalosporin, and the structures of related impurities (including impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G and the like) in cefixime are recorded in both British pharmacopoeia and European pharmacopoeia, but are not controlled independently. Regarding the detection of related impurities in the bulk drugs and preparations thereof, all national pharmacopoeias adopt ion pair reagent tetrabutyl ammonium hydroxide as a mobile phase, and the method can realize detection to a certain extent, but still has inherent defects, for example, chinese patent application CN109490440A discloses a method for detecting cefixime related impurities, and specifically discloses that a tetrabutyl ammonium hydroxide solution is used as an aqueous phase, acetonitrile or/and methanol are used as organic phases (not independently methanol), and the method can realize separation of a main peak from cefixime impurity a, cefixime impurity B, cefixime impurity C, cefixime impurity D and cefixime impurity E, but from the example data thereof, the method has many defects, such as: (1) the mobile phase is tetrabutyl ammonium hydroxide solution, the base line noise is high, and the detection capability of detecting impurities with low content is weak; (2) impurity B detected by the method appears as a shoulder peak and cannot achieve baseline separation; meanwhile, the method has the defects that the reagent is greatly damaged by ions, and the service life of the chromatographic column is short.

In addition, the pharmacopoeia of various countries only controls single impurities and total impurities related to cefixime and preparations thereof, and limits of various known impurities are not clear. Therefore, it is necessary to develop a new detection method capable of effectively separating various impurities to achieve effective detection.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides a method which can accurately determine the content of cefixime impurities A, B, C, D, E, F and G, can successfully separate a main peak from an impurity A, B, C, D, E, F, G, and ensures that the separation degree meets the specification.

According to the method provided by the embodiment of the invention, liquid chromatography is adopted for detection through reverse phase chromatography, and the detection conditions comprise:

the mobile phase comprises a mixed solution obtained by mixing a phase A and a phase B, wherein the phase A is an ammonium acetate aqueous solution with or without an added organic solvent, and the phase B is an organic solvent with or without an added ammonium acetate solution; wherein, the organic solvents in the phase A and the phase B are independently selected from at least one of acetonitrile or lower alcohol;

the elution procedure adopted in the detection process is gradient elution, the total gradient duration is not less than 65min, the volume percentage of the phase A in 0-50 min is 60-100%, and the volume percentage of the phase A in 50-65 min is 0-60%.

According to some embodiments of the invention, the mobile phase consists of a phase a and a phase B, and the elution gradient is set as follows:

according to some embodiments of the present invention, the organic solvent in phase a is present in an amount of no more than 10% (more preferably no more than 8%; more preferably no more than 6%; more preferably between 5 and 6%) by volume, and the water in phase B is present in an amount of no more than 60% (more preferably no more than 55%; more preferably no more than 53%; more preferably between 52 and 53%) by volume; preferably, the lower alcohol is selected from alcohols with 1-4 carbon atoms; preferably, the lower alcohol is methanol; more preferably, the volume ratio of methanol to water in the phase a is 52.5:1000, the volume ratio of water to methanol in the phase B is 1: 1; the mobile phase is composed of a phase A and a phase B, and the elution gradient is set as follows:

according to some embodiments of the invention, the lower alcohol is selected from 1 to 4 carbon alcohols; preferably, the lower alcohol is methanol.

According to some embodiments of the invention, the mobile phase has a pH in the range of 2.0 to 8.0; preferably, the pH range of the mobile phase is 2.5-8.0; more preferably, the pH of the mobile phase is in a range of 3.0-5.0; more preferably, the pH range of the mobile phase is 3.5-4.5; more preferably, the pH range of the mobile phase is 3.9-4.5; most preferably, the pH of the mobile phase is in the range of 3.9 to 4.2.

According to some embodiments of the invention, the mass concentration of ammonium acetate in the phase A is 0.01-1%; preferably, the mass concentration of ammonium acetate in the phase A is 0.1-1%; more preferably, the mass concentration of the ammonium acetate in the phase A is 0.1-0.5%.

According to some embodiments of the invention, the detection condition further comprises: the chromatographic column is an octadecylsilane chemically bonded silica gel column; preferably, the column size is 4.6 x 250mm, 5 μm; more preferably, the chromatographic column is Inertsil ODS-3V, and can be other chromatographic columns satisfying the conditions.

According to some embodiments of the invention, the detection condition further comprises: the column temperature is 30-50 ℃; preferably, the column temperature is 35-45 ℃; preferably, the column temperature is between 35 and 40 ℃.

According to some embodiments of the invention, the detection condition further comprises: the flow rate is 1.0-2.0 ml/min; preferably, the flow rate is 1.2-1.8 ml/min.

According to some embodiments of the invention, the detection condition further comprises: and detecting by using an ultraviolet detector, wherein the detection wavelength is 254 nm.

According to some embodiments of the invention, the method comprises the steps of:

s1, preparing a test solution and a standard solution;

s2, detecting the test solution and the standard solution through liquid chromatography;

and S3, analyzing the detection result.

According to some embodiments of the invention, the method and conditions for detection by liquid chromatography are as described above.

According to some embodiments of the invention, further comprising the steps of preparing a standard solution and detecting it by liquid chromatography; preferably, the solvent used for preparing the test solution and/or the standard solution is selected from at least one of ammonium acetate solution, phosphate buffer solution, methanol or acetonitrile.

According to some embodiments of the invention, the relevant impurity is selected from at least one of impurity a, impurity B, impurity C, impurity D, impurity E, impurity F or impurity G. The related impurities comprise impurities introduced in the process of synthesis, related impurities generated in the process of storage, transportation and use, and the like.

According to some embodiments of the invention, the test article is selected from at least one of a cefixime bulk drug, a cefixime capsule, a cefixime granule, a cefixime tablet, a cefixime dispersible tablet or a cefixime suspension. The method can be widely applied to detection of related impurities in cefixime bulk drugs and various dosage form drugs of cefixime, including cefixime bulk drugs, cefixime capsules, cefixime granules, cefixime tablets, cefixime dispersible tablets or cefixime suspension and the like, and has wide application range.

According to some embodiments of the invention, the analyzing in step S3 comprises qualitative and/or quantitative analysis; preferably, the analysis is performed by a method selected from at least one of area normalization, self-control, internal standard, or external standard. And (3) carrying out qualitative or quantitative analysis on the detection result by using analysis methods such as an area normalization method, a self-comparison method, an internal standard method or an external standard method and the like.

The method provided by the embodiment of the invention has at least the following beneficial effects: the method can effectively detect cefixime related impurities, simultaneously avoids using ion pair mobile phase, solves the problems of large damage to a chromatographic column, low detection sensitivity and the like of the original detection method, prolongs the service life of the chromatographic column, saves the detection cost, simultaneously has small noise of a detection base line by adopting an ammonium acetate solution, has stronger detection capability on low-content impurities and has better sensitivity; by adopting gradient elution, the impurity B can be effectively separated, and the detection method can better control single impurity and total impurity of cefixime and related impurities of the preparation of cefixime, thereby being beneficial to further controlling the purity of cefixime; the method can realize effective detection of the impurities A to G in one detection.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

FIG. 1 is a chromatogram for detection in example 1 of the present invention;

fig. 2 is a chromatogram of mobile phase a pH 4.5 and mobile phase B pH 4.2 in example 1 of the present invention;

fig. 3 is a chromatogram of mobile phase a pH 3.9 and mobile phase B pH 4.2 in example 1 of the present invention;

fig. 4 is a chromatogram of mobile phase a pH 4.2 and mobile phase B pH 4.2 in example 1 of the present invention;

fig. 5 is a chromatogram of mobile phase a pH 4.2 and mobile phase B pH 4.5 in example 1 of the present invention;

fig. 6 is a chromatogram of mobile phase a pH 4.2 and mobile phase B pH 3.9 in example 1 of the present invention;

fig. 7 is a chromatogram of mobile phase a pH 2.0 and mobile phase B pH 2.0 in example 1 of the present invention;

fig. 8 is a chromatogram of mobile phase a pH 8.0 and mobile phase B pH 8.0 in example 1 of the present invention;

FIG. 9 is a chromatogram at a flow rate of 1.2ml/min for an example of the present invention;

FIG. 10 is a chromatogram at a flow rate of 1.5ml/min for an example of the present invention;

FIG. 11 is a chromatogram at a flow rate of 1.8ml/min for an example of the present invention;

FIG. 12 is a chromatogram at a column temperature of 35 ℃ in an example of the present invention;

FIG. 13 is a chromatogram at a column temperature of 40 ℃ in an example of the present invention;

FIG. 14 is a chromatogram at a column temperature of 45 ℃ in an example of the present invention;

FIG. 15 is a chromatogram for detection in comparative example 1 of the present invention;

FIG. 16 is a chromatogram for detection in comparative example 2 of the present invention.

Detailed Description

In order to explain technical contents, achieved objects, and effects of the present invention in detail, the following description is made with reference to the accompanying drawings in combination with the embodiments.

The embodiment of the invention is as follows: the method for detecting cefixime related impurities comprises the following steps of (1):

TABLE 1

The reagent information used is shown in table 2 below:

TABLE 2

Name of reagent	Manufacturer of the product	Batch number	Specification of
				Ammonium acetate	Guangzhou chemical test	20150602 etc	Analytical purity
Phosphoric acid	Guangzhou chemical test	20160602-1 etc	Analytical purity
				Acetonitrile	Merck	I0990930, etc	Pure chromatography
Methanol	Merck	I0997507 and the like	Pure chromatography
				Anhydrous disodium hydrogen phosphate	Guangzhou chemical test	20180501-1, etc	Analytical purity
Anhydrous potassium dihydrogen phosphate	Guangzhou chemical test	20171001-1, etc	Analytical purity

The standard and control information used is shown in table 3 below:

TABLE 3

The detection process specifically comprises the following steps:

1. preparation of diluents

Phosphate buffer (9.08 g/L potassium dihydrogen phosphate solution: 23.8 g/L anhydrous disodium hydrogen phosphate solution: 39: 61).

2. System applicability solution preparation

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 of cefixime in each ml, heating the solution on a water bath at the temperature of 95 ℃ for 45 minutes, and cooling the solution to room temperature to obtain the system applicability solution.

3. Preparation of test solution

Taking cefixime granules (G1802001, after standing at 30 ℃ for about 18 months, the cefixime content is about 96% according to the marked amount), adding a proper amount of diluent, dissolving by ultrasonic treatment, diluting with the diluent to prepare a solution containing 1mg of cefixime in each 1ml, filtering, discarding 2.0ml of primary filtrate, and taking the subsequent filtrate as a test solution.

4. Preparation of a mobile phase:

mobile phase a was 3.85 g/L ammonium acetate-methanol solution (3.85 g of ammonium acetate was taken and 1000ml of water was added to dissolve it, 52.5ml of methanol was added thereto and the pH was adjusted with phosphoric acid), and mobile phase B was 3.85 g/L ammonium acetate-methanol solution (3.85 g of ammonium acetate was taken and 1000ml of water was added to dissolve it, 1000ml of methanol was added thereto and the pH was adjusted with phosphoric acid).

5. Detection was performed by liquid chromatography under the following conditions:

chromatographic column Inertsil ODS-3V 4.6mm × 250mm, 5 μm

Flow rate: 1.5ml/min

Detection wavelength: 254nm

Column temperature: the column temperature was 40 deg.C

The gradient settings are shown in table 4 below:

TABLE 4

Time (minutes)	Mobile phase A (%)	Mobile phase B (%)
			0	100	0
5	95	5
			10	95	5
20	92	8
			50	80	20
60	0	100
			65	0	100
70	100	0
			80	100	0

The detection result is shown in fig. 1, and as can be seen from fig. 1, the method can realize the detection of the impurities A, B, C, D, E, F and G, can completely separate the impurities from the main peak, has a separation degree meeting the quantitative detection requirement, and well meets the impurity monitoring requirement in the process of the bulk drug and the preparation and the impurity control requirement in the finished product of the bulk drug and the preparation. A1, A2, A3 and A4 marked in the figure represent different isomers of impurity A prescribed by pharmacopoeia, respectively; b1, B2 represent different isomers of impurity B.

To examine the effect of the detection conditions on the detection effect, the following experiment was performed (the test sample was cefixime particles G1802001, and the preparation process of the test solution was as follows: cefixime particles were taken and added with an appropriate amount of diluent, dissolved by ultrasound, diluted with diluent to make a solution containing 1mg of cefixime per 1ml, filtered, and 2.0ml of the primary filtrate was discarded, and the subsequent filtrate was taken as the test solution):

1) examination of pH conditions, experiments were performed with different pH conditions (other conditions were set with reference to the above examples), as follows:

i) the pH of mobile phase a is 4.5, the pH of mobile phase B is 4.2, and the detection results are shown in fig. 2;

ii) mobile phase a pH 3.9 and mobile phase B pH 4.2, the results are shown in fig. 3;

iii) mobile phase a pH 4.2 and mobile phase B pH 4.2, the results are shown in fig. 4;

iv) mobile phase a pH 4.2 and mobile phase B pH 4.5, the results are shown in fig. 5;

v) mobile phase a pH 4.2 and mobile phase B pH 3.9, the results are shown in fig. 6;

vi) mobile phase a pH 2.0 and mobile phase B pH 2.0, the results are shown in fig. 7; vii) mobile phase a pH 8.0, mobile phase B pH 8.0, and the results are shown in fig. 8.

The separation and relative retention times in FIGS. 2-6 were calculated as shown in Table 5 below:

TABLE 5

As can be seen from the above table, the change of pH value in this range has no significant influence on the determination of impurity content and the separation of impurities. In addition, as can be seen from fig. 2 to 8, when the pH of the mobile phase is 2.0 to 8.0, separation of cefixime-related impurities can be achieved, and when the pH is 2.0, a baseline is shifted to a certain extent, but separation of each impurity can also be substantially achieved.

2) Examination of flow rate conditions, experiments were carried out with different flow rate conditions (both phase a and phase B had a pH of 4.2, other conditions were set with reference to the above examples) as follows:

i) the flow rate is 1.2ml/min, and the detection result is shown in fig. 9;

ii) the flow rate is 1.5ml/min, and the detection result is shown in fig. 10;

iii) the flow rate was 1.8ml/min, and the results are shown in FIG. 11.

The separation and relative retention times in FIGS. 9-11 were calculated as shown in Table 6 below:

TABLE 6

Impurities	Flow rate 1.2ml/min	Flow rate 1.5ml/min	Flow rate 1.8ml/min
				Degree of separation	20.76	19.16	15.87
Control peak area RSD%	0.33	0.35	0.51
				A1，％	0.33	0.27	0.34
A2，％	0.11	0.10	0.12
				A3，％	0.06	/	0.08
A4，％	/	0.13	0.25
				B1，％	0.11	0.15	0.12
B2，％	0.12	0.17	0.12
				C，％	/	/	0.15
D，％	/	/	0.06
				E，％	0.10	0.10	0.12
F，％	/	/	/
				G，％	/	/	/
Maximum other single impurity (unknown)%	0.10	0.08	0.31
				Total impurities%	1.41	1.19	1.90

As can be seen from the above table, the change in flow rate in this range has no significant effect on the determination of the impurity content and the separation of impurities.

3) Examination of column temperature conditions, different column temperature conditions were set for the experiment (both the pH values of phase a and phase B were 4.2, and other conditions were set with reference to the above example), specifically as follows:

i) the column temperature was 35 ℃ and the results are shown in fig. 12;

ii) column temperature 40 ℃, the results are shown in fig. 13;

iii) column temperature 45 ℃, the results are shown in fig. 14.

The following table 7 is calculated for the degrees of separation and relative retention times in FIGS. 12-14:

TABLE 7

As can be seen from the above table, the temperature change in this range has no significant influence on the measurement of the content of impurities and the separation of impurities.

The second embodiment of the present invention is a method for detecting cefixime-related impurities, and the other conditions are the same as those in fig. 1 of embodiment 1, except that: the gradient settings are shown in table 8 below:

TABLE 8

Time (minutes)	Mobile phase A (%)	Mobile phase B (%)
			0	80	20
5	75	25
			10	75	25
20	72	28
			50	60	40
60	20	80
			65	20	80
70	80	20
			80	80	20

The detection result is similar to that of fig. 1, and therefore, the A, B, C, D, E, F and G can be effectively separated.

In addition, similar detection is carried out on cefixime granules and cefixime capsules (02180401, 02180501 and 02180502) of other batches (G1803001 and G1803002) produced by Baiyunshan pharmaceutical factories, and the effect is similar to that of the above (for avoiding redundancy, the specific map is not repeated here), thereby showing that the scheme of the invention can be better suitable for various cefixime medicines.

The first comparative example of the present invention is: a method for detecting cefixime related impurities adopts a traditional ion pair reagent, and other conditions are shown in the following table 9:

TABLE 9

The test results are shown in fig. 15, and it can be seen from the figure that the baseline noise detected by the method is large, the detection limit and the quantification limit of each impurity are high, and accurate detection is difficult when the impurity content is low.

The second comparative example of the present invention is: a method for detecting impurities related to cefixime adopts an ammonium acetate system, and other conditions are shown in the following table 10:

watch 10

The test results are shown in fig. 16, and it can be seen that the chromatogram obtained by the method does not achieve baseline separation between the main component and the adjacent impurities and between the impurities.

The related impurities referred to in the invention refer to impurities introduced in the process of synthesis technology and related impurities generated in the processes of storage, transportation and use.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent changes made by using the contents of the present specification and the drawings, or applied directly or indirectly to the related technical fields, are included in the scope of the present invention.

Claims

1. A method for detecting cefixime related impurities adopts liquid chromatography to detect through reverse phase chromatography, and is characterized in that: the detection conditions include:

2. The method for detecting cefixime-related impurities according to claim 1, wherein: the mobile phase in the detection process consists of a phase A and a phase B, and the elution gradient is set as follows:

3. the method for detecting cefixime-related impurities according to claim 1, wherein: the volume content of the organic solvent in the phase A is not higher than 10 percent, and the volume content of the water in the phase B is not higher than 60 percent; preferably, the lower alcohol is selected from alcohols with 1-4 carbon atoms; preferably, the lower alcohol is methanol; more preferably, the volume ratio of methanol to water in the phase a is 52.5:1000, the volume ratio of water to methanol in the phase B is 1: 1; the mobile phase is composed of a phase A and a phase B, and the elution gradient is set as follows:

4. the method for detecting cefixime-related impurities according to claim 1, wherein: the pH range of the mobile phase is 2.0-8.0; preferably, the pH range of the mobile phase is 2.5-8.0; more preferably, the pH of the mobile phase is in a range of 3.0-5.0; more preferably, the pH range of the mobile phase is 3.5-4.5; more preferably, the pH range of the mobile phase is 3.9-4.5; most preferably, the pH of the mobile phase is in the range of 3.9 to 4.2.

5. The method for detecting cefixime-related impurities according to claim 1, wherein: the mass concentration of ammonium acetate in the phase A is 0.01-1%; preferably, the mass concentration of ammonium acetate in the phase A is 0.1-1%; more preferably, the mass concentration of the ammonium acetate in the phase A is 0.1-0.5%.

6. The method for detecting cefixime-related impurities according to claim 1, wherein: the detection conditions further include at least one of the following i-v:

i. the chromatographic column is an octadecylsilane chemically bonded silica gel column;

column size 4.6 x 250mm, 5 μm;

iii, column temperature is 30-50 ℃;

iv, the flow rate is 1.0-2.0 ml/min;

v, detecting by using an ultraviolet detector, wherein the detection wavelength is 254 nm.

7. The method for detecting cefixime-related impurities according to any one of claims 1 to 6, wherein: the method comprises the following steps:

s1, preparing a test solution and a standard solution;

and S3, analyzing the detection result.

8. The method for detecting cefixime-related impurities according to claim 7, wherein: the related impurity is at least one selected from the group consisting of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F and impurity G.

9. The method for detecting cefixime-related impurities according to claim 7, wherein: the test sample is at least one selected from cefixime bulk drug, cefixime capsule, cefixime granules, cefixime tablets, cefixime dispersible tablets or cefixime suspension.

10. The method for detecting cefixime-related impurities according to claim 8, wherein: the analysis in step S3 includes qualitative and/or quantitative analysis; preferably, the analysis is performed by a method selected from at least one of area normalization, self-control, internal standard, or external standard.