CN116223688A - Analysis method of related substances in aminoglycoside antibiotics - Google Patents

Abstract

The invention discloses an analysis method of related substances in aminoglycoside antibiotics, which adopts sodium perchlorate buffer solution as a mobile phase A and acetonitrile as a mobile phase B, and carries out gradient elution by a mixture of the mobile phase A and the mobile phase B; quantitative analysis is carried out by adopting an external standard method of reversed phase liquid chromatography, a chromatographic column adopts a Waters Atlantis T column, the length of the chromatographic column is 150mm, the detection wavelength is 200nm to 230nm, the flow rate is 0.6ml/min to 1.0ml/min, the diameter is 4.6mm, the particle size of the filler is 3 mu m, the column temperature is 30 ℃ to 40 ℃, and the sample injection amount is 10 mu L to 20 mu L; the sample temperature was controlled to be 2 ℃ to 8 ℃. The method disclosed by the invention can simply, rapidly and accurately monitor the aminoglycoside antibiotics and related substances in the preparation thereof, and has the advantages of strong specificity, high sensitivity, high precision, good stability and good reproducibility; the method can be used for batch detection, improves the detection efficiency, and meets the requirements of research, development and production.

Description

Analysis method of related substances in aminoglycoside antibiotics

Technical Field

The invention belongs to the technical field of chemical drug analysis methods, and particularly relates to an analysis method of related substances in aminoglycoside antibiotics.

Background

Aminoglycoside antibiotics (amino acids) are glycoside antibiotics formed by connecting amino sugar and amino cyclic alcohol through oxygen bridge, and include natural antibiotics such as Streptomycin, gentamicin, tobramycin, etc.; also semi-synthetic antibiotics such as Netilmicin (Netilmicin), amikacin (Amikacin).

Although most antibiotics inhibiting microbial protein synthesis are antibacterial agents, aminoglycoside antibiotics can play a role in sterilization, and belong to stationary phase bactericides. Currently, aminoglycoside antibiotics are mainly used for treating systemic infections caused by sensitive aerobic gram-negative bacilli, including respiratory tract, gastrointestinal tract, urinary tract, meningitis, burns, wounds, bone and joint infections, etc.; it can also be made into eye ointment, topical ointment or rinse solution for treating topical bacterial infection. The post-antibiotic effect on common gram-negative bacilli such as pseudomonas aeruginosa, pneumobacillis, escherichia coli and the like is long.

In the existing detection process of the aminoglycoside antibiotics, the main peak (aminoglycoside antibiotics) and the impurity peak behind the main peak cannot be effectively separated. In order to ensure the research, development and production quality of the aminoglycoside antibiotics and the preparation thereof, a detection method for measuring the related substances of the aminoglycoside antibiotics needs to be researched and obtained.

Disclosure of Invention

In order to solve the problems, the invention provides a method for analyzing related substances in aminoglycoside antibiotics.

The invention discloses an analysis method of related substances in aminoglycoside antibiotics, which adopts sodium perchlorate buffer solution as a mobile phase A and acetonitrile as a mobile phase B, and carries out gradient elution by a mixture of the mobile phase A and the mobile phase B; quantitative analysis is carried out by adopting an external standard method of reversed phase liquid chromatography, wherein,

the chromatographic column adopts a Waters Atlantis T column, the length of the chromatographic column is 150mm, the detection wavelength is 200nm to 230nm, the flow rate is 0.6ml/min to 1.0ml/min, the diameter is 4.6mm, the particle size of the filler is 3 mu m, the column temperature is 30 ℃ to 40 ℃, and the sample injection amount is 10 mu L to 20 mu L;

the sample temperature was controlled to be 2 ℃ to 8 ℃.

In some embodiments, the mobile phase a is formulated by: every 1000mL of aqueous solution contains 14.046g of sodium perchlorate, and the pH value of the mobile phase A is regulated to 2.95-3.05 by perchloric acid.

In some embodiments, the foregoing method further comprises preparing a solution prior to liquid chromatography detection, the solution comprising:

blank solution, the blank solution is made up of thinner;

the sensitivity solution consists of an aminoglycoside antibiotic reference substance and a diluent;

the system applicability solution consists of an aminoglycoside antibiotic reference substance, an aminoglycoside antibiotic impurity reference substance and a diluent;

the sample solution consists of the aminoglycoside antibiotics and a diluent; about 8mg of aminoglycoside-containing antibiotic per 1ml of the test solution;

a reference substance solution, wherein the reference substance solution consists of an aminoglycoside antibiotic reference substance and a diluent;

the diluent is selected from methanol, acetonitrile, water or mixtures thereof;

and (5) injecting the mixture into a high performance liquid chromatograph according to the chromatographic conditions, and recording a chromatogram.

In some embodiments, the gradient elution is performed by:

at 0 minutes, the volume ratio of mobile phase A to mobile phase B is 100:0;

the volume ratio of the mobile phase A to the mobile phase B gradually changes from 100:0 to 80:20 at a constant speed within 0-60 minutes;

the volume ratio of the mobile phase A to the mobile phase B of the mobile phase A gradually changes from 80:20 to 40:60 at a constant speed within 60-65 minutes;

in 65-65.1 minutes, the volume ratio of the mobile phase A to the mobile phase B gradually changes from 40:60 to 100:0 at a constant speed;

the volume ratio of mobile phase A to mobile phase B is kept constant at 100:0 within 65.1-75 minutes.

Compared with the prior art, the invention has the beneficial effects that:

the analysis method of related substances in aminoglycoside antibiotics provided by the invention adopts reversed phase liquid chromatography for determination, adopts Waters Atlantis T3 chromatographic column as chromatographic column, adopts sodium perchlorate buffer solution as mobile phase A, acetonitrile as mobile phase B, and adopts a mixture of the mobile phase A and the mobile phase B for gradient elution. The method disclosed by the invention can simply, rapidly and accurately monitor the aminoglycoside antibiotics and related substances in the preparation thereof, and has the advantages of strong specificity, high sensitivity, high precision, good stability and good reproducibility; the method can be used for batch detection, improves the detection efficiency, and meets the requirements of research, development and production.

Drawings

FIG. 1 is a chromatogram of a hollow white solution according to an embodiment of the present invention;

FIG. 2 is a chromatogram of an aminoglycoside antibiotic sensitivity solution in an embodiment of the invention;

FIG. 3 is a chromatogram of an aminoglycoside antibiotic control solution according to an embodiment of the present invention;

FIG. 4 is a chromatogram of an aminoglycoside antibiotic system applicability solution in an embodiment of the invention;

FIG. 5 is a chromatogram of a test solution of an aminoglycoside antibiotic according to an embodiment of the present invention.

Detailed Description

The present invention will be described in detail with reference to the following embodiments, but it should be understood that these embodiments are not limiting, and functional, method, or structural equivalents and alternatives thereof by those skilled in the art are within the scope of the present invention. Unless specifically stated in the specification, the components and raw materials in the invention are all of chemical purity grade. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1

The present example discloses a method for measuring related substances of aminoglycoside antibiotics by high performance liquid chromatography.

Chromatographic conditions of the detection method described in this example:

the instruments used were: island jin 20-A high performance liquid chromatograph, PDA detector; chromatographic column: waters Atlantis T3, specification 150 mm. Times.3 μm. Times.4.6 mm.

Solvent: the mobile phase A is sodium perchlorate water solution, and the preparation method of the mobile phase A is as follows: every 1000mL of aqueous solution contains 14.046g of sodium perchlorate, and the pH value of the mobile phase A is regulated to 3.0 by perchloric acid; mobile phase B was acetonitrile.

The flow rate is 0.8ml/min; the column temperature is 35 ℃; the sample injection volume is 10 μl; the detection wavelength is 210nm; the sample temperature was controlled to 5 ℃.

The gradient elution process is as follows:

at 0 minutes, the volume ratio of mobile phase A to mobile phase B is 100:0;

the volume ratio of the mobile phase A to the mobile phase B gradually changes from 100:0 to 80:20 at a constant speed within 0-60 minutes;

the volume ratio of the mobile phase A to the mobile phase B of the mobile phase A gradually changes from 80:20 to 40:60 at a constant speed within 60-65 minutes;

in 65-65.1 minutes, the volume ratio of the mobile phase A to the mobile phase B gradually changes from 40:60 to 100:0 at a constant speed;

the volume ratio of mobile phase A to mobile phase B is kept constant at 100:0 within 65.1-75 minutes.

The experimental steps are as follows:

solution preparation

Blank solution (diluent): pure water.

Preparation of a sensitivity solution: taking a proper amount of aminoglycoside antibiotics reference substance, precisely weighing, adding water for dissolving and diluting to prepare a solution containing about 0.004mg of aminoglycoside antibiotics in each 1ml, and taking the solution as a sensitivity solution.

Preparation of control solution (prepared for clinical use): taking a proper amount of aminoglycoside antibiotics reference substance, precisely weighing, adding water for dissolving and diluting to prepare a solution containing about 0.08mg of aminoglycoside antibiotics in each 1ml serving as a reference substance solution.

Preparing a system applicability solution: taking an aminoglycoside antibiotic reference substance and a proper amount of an aminoglycoside antibiotic impurity reference substance, precisely weighing, adding water for dissolving and diluting to prepare a solution which contains 8mg of aminoglycoside antibiotic, 0.08mg of aminoglycoside antibiotic impurity A, 0.08mg of aminoglycoside antibiotic impurity B, 0.08mg of aminoglycoside antibiotic impurity C, 0.08mg of aminoglycoside antibiotic impurity D, 0.08mg of aminoglycoside antibiotic impurity E and 0.08mg of aminoglycoside antibiotic impurity F in each 1ml, and taking the solution as a system applicability solution.

Preparing a test solution: taking a proper amount of aminoglycoside antibiotics, precisely weighing, adding water for dissolving and diluting to prepare a solution containing 8mg of aminoglycoside antibiotics in 1ml as a test sample solution.

Experimental operation:

precisely sucking blank solution, sensitivity solution, reference solution, system applicability solution, and test solution (10 μl) respectively, injecting into high performance liquid chromatograph, and recording chromatograms as shown in fig. 1, 2, 3, 4, and 5.

According to the chromatogram, the content of each impurity in the aminoglycoside antibiotics is calculated according to the external standard method of the main component without adding correction factors.

The results show that: as shown in fig. 1, 2, 3 and 5, the blank solution does not interfere with the detection of impurities of aminoglycoside antibiotics; in the sensitivity solution, the S/N of the main peak of the aminoglycoside antibiotics is more than 10, which meets the requirements. As shown in fig. 4, in the system applicability solution, the order of the outlet peaks of the known impurity peaks and the main peak is impurity a (2.955 min), impurity B (17.977 min), main peak (21.661 min), impurity C (36.429 min), impurity D (37.105 min), impurity E (39.518 min), impurity F (52.558 min); the separation degree of the main peak and the known impurity peak of the aminoglycoside antibiotics and the adjacent peaks meets the requirements, and the peak shape is good and meets the standard.

While only certain embodiments of the present invention have been described, it will be apparent to those skilled in the art that other modifications and improvements can be made without departing from the inventive concept of the present invention.

Claims (4)

1. The method is characterized in that sodium perchlorate buffer solution is adopted as a mobile phase A, acetonitrile is adopted as a mobile phase B, and gradient elution is carried out by a mixture of the mobile phase A and the mobile phase B; quantitative analysis is carried out by adopting an external standard method of reversed phase liquid chromatography, wherein,

the chromatographic column adopts a Waters Atlantis T column, the length of the chromatographic column is 150mm, the detection wavelength is 200nm to 230nm, the flow rate is 0.6ml/min to 1.0ml/min, the diameter is 4.6mm, the particle size of the filler is 3 mu m, the column temperature is 30 ℃ to 40 ℃, and the sample injection amount is 10 mu L to 20 mu L;

the sample temperature was controlled to be 2 ℃ to 8 ℃.

2. The method for analyzing related substances in aminoglycoside antibiotics according to claim 1, wherein the preparation method of the mobile phase a is as follows: every 1000mL of aqueous solution contains 14.046g of sodium perchlorate, and the pH value of the mobile phase A is regulated to 2.95-3.05 by perchloric acid.

3. The method for analyzing a substance of interest in an aminoglycoside according to claim 2, further comprising preparing a solution before liquid chromatography detection, said solution comprising:

a blank solution, the blank solution comprising a diluent;

the sensitivity solution consists of an aminoglycoside antibiotic reference substance and a diluent;

the system applicability solution consists of an aminoglycoside antibiotic reference substance, an aminoglycoside antibiotic impurity reference substance and a diluent;

a test solution comprising 8mg of the aminoglycoside antibiotic per 1ml of the test solution;

the reference substance solution consists of an aminoglycoside antibiotic reference substance and a diluent;

the diluent is selected from methanol, acetonitrile, water or a mixture thereof;

and (5) injecting the mixture into a high performance liquid chromatograph according to the chromatographic conditions, and recording a chromatogram.

4. A method for analyzing substances of interest in an aminoglycoside according to claim 3, wherein said gradient elution is performed by:

at 0 minutes, the volume ratio of mobile phase A to mobile phase B is 100:0;

the volume ratio of the mobile phase A to the mobile phase B gradually changes from 100:0 to 80:20 at a constant speed within 0-60 minutes;

the volume ratio of the mobile phase A to the mobile phase B of the mobile phase A gradually changes from 80:20 to 40:60 at a constant speed within 60-65 minutes;

in 65-65.1 minutes, the volume ratio of the mobile phase A to the mobile phase B gradually changes from 40:60 to 100:0 at a constant speed;

the volume ratio of mobile phase A to mobile phase B is kept constant at 100:0 within 65.1-75 minutes.

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