CN111595985B

CN111595985B - Analytical method for measuring related substances of rebamipide by using HPLC (high performance liquid chromatography)

Info

Publication number: CN111595985B
Application number: CN202010655949.0A
Authority: CN
Inventors: 侯叶婷; 董梦飞
Original assignee: Suzhou Zhengji Pharmaceutical Co ltd
Current assignee: Suzhou Zhengji Pharmaceutical Co ltd
Priority date: 2020-07-09
Filing date: 2020-07-09
Publication date: 2022-03-29
Anticipated expiration: 2040-07-09
Also published as: CN111595985A

Abstract

The invention relates to the technical field of analytical chemistry, and discloses an analytical method for determining related substances of rebamipide by using HPLC. The analysis method provided by the invention is characterized in that a reversed phase chromatographic column with octadecylsilane chemically bonded silica as a filler, a buffer salt solution as a mobile phase A and a buffer salt solution, methanol and tetrahydrofuran as a mobile phase B are used for carrying out gradient elution on a sample solution of rebamipide and carrying out HPLC analysis. The analysis method can effectively separate rebamipide and related substances thereof, so that impurity peaks and rebamipide peaks are not overlapped, the peak shape is good, the separation requirement is met, and the method is suitable for controlling related substances and researching impurities.

Description

Analytical method for measuring related substances of rebamipide by using HPLC (high performance liquid chromatography)

Technical Field

The invention relates to the technical field of analytical chemistry, in particular to an analytical method for determining related substances of rebamipide by using HPLC.

Background

Rebamipide (±)2- (4-chlorobenzamide) -3- [2(1H) -quinolinone-4]Propionic acid is a prostaglandin derivative, is a novel gastric mucosa protective agent, and has the CAS number: 90098-04-7 with molecular formula C₁₉H₁₅ClN₂O₄The molecular weight is: 370.79, the chemical structure is:

rebamipide is an anti-ulcer drug developed by tsukamur pharmaceutical company of japan, since 1990

The trade name is first marketed in japan as an oral protective antiulcer drug. Preventing and treating peptic ulcer by strengthening the defense function of gastric mucosa; can increase prostaglandin content in gastric mucosa, increase blood flow of gastric mucosa, and increase gastric mucus; can inhibit gastric reverse diffusion and promote gastric alkali secretion; eliminating and inhibiting active oxygen, and maintaining gastric mucosa superoxide dismutase (SOD) activity. The composition can be used for preventing and treating acute and chronic gastric ulcer, and also can be used for preventing damage of various injury factors (such as sodium hydroxide, hydrochloric acid, acetic acid, and non-steroidal anti-inflammatory drug) to gastric mucosa. Meanwhile, the medicine has regenerative and uniform repair effect on gastric mucosa tissues, can reduce the cure recurrence rate of gastric ulcer, which is not possessed by various anti-ulcer medicines used at present.

At present, the quality control of related substances of rebamipide is recorded in pharmacopoeia, but impurities generated in the following 4 technological synthesis processes cannot be considered in one analysis method:

impurity A: 2-amino-3- (2-oxo-1, 2-dihydroquinolin-4-yl) propionic acid;

impurity B: 2- (2-chlorobenzoylamino) -3- (1, 2-dihydro-2-oxo-4-quinolinyl) propionic acid;

impurity C: 2- (3-chlorobenzoylamino) -3- (1, 2-dihydro-2-oxo-4-quinolinyl) propionic acid;

impurity D: 2- (3, 4-dichlorobenzamido) -3- (1, 2-dihydro-2-oxo-4-quinolinyl) propionic acid;

therefore, it is required to provide an efficient HPLC method for analyzing and detecting related substances of rebamipide.

Disclosure of Invention

In view of the above, it is an object of the present invention to provide an analytical method for determining rebamipide-related substances by HPLC, which is capable of efficiently separating 2-amino-3- (2-oxo-1, 2-dihydroquinolin-4-yl) propionic acid, 2- (2-chlorobenzoylamino) -3- (1, 2-dihydro-2-oxo-4-quinolyl) propionic acid, 2- (3-chlorobenzoylamino) -3- (1, 2-dihydro-2-oxo-4-quinolyl) propionic acid and 2- (3, 4-dichlorobenzoylamino) -3- (1, 2-dihydro-2-oxo-4-quinolyl) propionic acid, so that impurity peaks and rebamipide peaks are not overlapped, the peak shape is good and the separation requirement is met.

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

an analytical method for determining related substances of rebamipide by HPLC (high performance liquid chromatography), which is characterized in that a reverse phase chromatographic column with octadecylsilane chemically bonded silica as a filler, a buffer salt solution as a mobile phase A and a buffer salt solution, methanol and tetrahydrofuran as a mobile phase B are used, and a sample solution of rebamipide is subjected to gradient elution and HPLC analysis.

Preferably, the volume ratio of the buffer salt solution, the methanol and the tetrahydrofuran in the mobile phase B is (0-40): (54-90): (6-10). When the buffered salt solution is other than 0, it is more advantageous for the column. In a specific embodiment of the invention, the mobile phase B is a buffered salt solution, methanol and tetrahydrofuran in a volume ratio of 40:54: 6; or methanol and tetrahydrofuran in a volume ratio of 90: 10.

Preferably, the buffered salt solution is a phosphate buffer solution, the concentration of the phosphate buffer solution is between 0.005M and 0.05M, and the pH value is between 6.2 and 6.8. More preferably, the buffered salt solution is one of a potassium dihydrogen phosphate solution and a sodium dihydrogen phosphate solution, and the preparation method refers to the method in pharmacopoeia. In a specific embodiment of the invention, the buffered salt solution has a concentration of 0.05M potassium dihydrogen phosphate at a pH of 6.5.

Preferably, the gradient elution procedure is as follows in table 1:

TABLE 1

Elution time (minutes)	Mobile phase A (%)	Mobile phase B (%)
			0	90～10	10～90
A	90～10	10～90
			B	50～0	50～100
C	50～0	50～100
			D	90～10	10～90
E	90～10	10～90

Wherein A is more than 0 and less than or equal to 30 minutes, B is more than 30 and less than or equal to 45 minutes, C is more than 45 and less than or equal to 55 minutes, D is more than 55 and less than or equal to 58 minutes, and E is more than 58 and less than or equal to 70 minutes.

In a specific embodiment of the invention, the gradient elution procedure shown in table 1 can be referred to table 3:

TABLE 3

Elution time	Mobile phase A (%)	Mobile phase B (%)
			0min	60	40
25min	60	40
			40min	40	60
54min	40	60
			56min	60	40
60min	60	40

Preferably, the gradient elution procedure is as follows in table 2:

TABLE 2

Elution time (minutes)	Mobile phase A (%)	Mobile phase B (%)
			0	70～20	30～80
A	70～20	30～80
			B	30～0	0～100
C	30～0	0～100
			D	70～20	30～80
E	70～20	30～80

Wherein A is more than 0 and less than or equal to 25 minutes, B is more than 25 and less than or equal to 40 minutes, C is more than 40 and less than or equal to 55 minutes, D is more than 55 and less than or equal to 58 minutes, and E is more than 58 and less than or equal to 60 minutes.

In a specific embodiment of the present invention, the gradient elution procedure shown in table 2 can be referred to table 4:

TABLE 4

Elution time	Mobile phase A (%)	Mobile phase B (%)
			0min	33	67
25min	33	67
			40min	0	100
54min	0	100
			56min	33	67
60min	33	67

Preferably, the reversed phase chromatographic column is a chromatographic column with the filler particle diameter of between 3.0 and 5.0 microns, the chromatographic column length of between 150 and 250mm and the chromatographic column diameter of between 2.0 and 4.6 mm. In the specific embodiment of the invention, the reversed phase chromatographic column is a chromatographic column which takes octadecylsilane chemically bonded silica as a filler, the particle size is 5.0 μm, the length of the chromatographic column is 250mm, and the diameter of the chromatographic column is 4.6 nm; for example Kromasil100-5c 184.6 mm x 250mm, 5 μm.

Preferably, the HPLC detection wavelength is 235nm, the column temperature is 40 ℃, and the flow rate is 0.8 ml/min.

The invention uses different analysis methods to carry out HPLC analysis on the same sample solution preparation formulation, and the result shows that the impurities A-D and rebamipide can be effectively separated under the analysis method of the invention, and the peak shapes are good and have no overlapping; and the HPLC results of other mobile phases show that part of impurity peaks and rebamipide peaks do not meet the separation requirement, and part of impurity peaks are split into two peaks.

According to the technical scheme, the analysis method provided by the invention adopts a reversed phase chromatographic column with octadecylsilane chemically bonded silica as a filler, adopts a buffer salt solution as a mobile phase A, adopts a buffer salt solution, methanol and tetrahydrofuran as a mobile phase B, and performs gradient elution on a sample solution of rebamipide and performs HPLC analysis. The analysis method can effectively separate rebamipide and related substances thereof, so that impurity peaks and rebamipide peaks are not overlapped, the peak shape is good, the separation requirement is met, and the method is suitable for control of related substances and research on impurities.

Drawings

Fig. 1 shows the HPLC detection result of example 1, where RT-8.733 min is the peak of impurity a, RT-6.796 min is the peak of impurity B, RT-24.9 min is the peak of impurity D, RT-12.159 min is the peak of rebamipide, the peak of impurity C is not detected, and the impurity C overlaps with the rebamipide peak;

fig. 2 shows the HPLC detection result of example 2, where RT-7.097 min is the peak of impurity B, RT-19.668 min is the peak of impurity C, RT-20.897 min is the peak of rebamipide, RT-36.779 min is the peak of impurity D, and impurity a splits into two peaks;

fig. 3 is a result of HPLC detection in example 3, where RT ═ 3.302min is a peak of impurity a, RT ═ 6.096min is a peak of impurity B, RT ═ 11.496min is a peak of impurity C, RT ═ 11.917min is a peak of rebamipide, RT ═ 24.623min is a peak of impurity D, and the peaks of impurity C and rebamipide do not meet the separation requirement;

fig. 4 shows the HPLC detection result of example 4, where RT-3.153 min is the peak of impurity a, RT-7.848 min is the peak of impurity B, RT-19.756 min is the peak of impurity C, RT-21.014 min is the peak of rebamipide, and RT-39.146 min is the peak of impurity D;

fig. 5 shows the HPLC detection result of example 5, where RT ═ 3.168min is the peak of impurity a, RT ═ 7.892min is the peak of impurity B, RT ═ 19.788min is the peak of impurity C, RT ═ 20.607min is the peak of rebamipide, and RT ═ 39.177min is the peak of impurity D.

Detailed Description

The embodiment of the invention discloses an analysis method for measuring related substances of rebamipide by using HPLC (high performance liquid chromatography), and a person skilled in the art can realize the analysis by properly improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to those skilled in the art are deemed to be included within the invention. While the assay methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the assay methods described herein, as well as suitable variations and combinations of methods, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.

The chemical names and structural formulas of the impurity A, the impurity B, the impurity C and the impurity D are as follows:

impurity A: 2-amino-3- (2-oxo-1, 2-dihydroquinolin-4-yl) propionic acid;

in a specific embodiment of the present invention, the present invention provides the following two examples to describe the analytical method of the present invention:

(1) preparing a sample into a solution containing rebamipide and a proper amount of impurities, detecting on a reversed-phase high performance liquid chromatograph by referring to a high performance liquid chromatography, analyzing by using a chromatographic column with octadecylsilane chemically bonded silica as a filler, the particle size of the chromatographic column being 5.0 mu M, the length of the chromatographic column being 250mm and the diameter of the chromatographic column being 4.6mm, and performing gradient elution under a mobile phase system with 0.05M phosphate buffer solution, pH 6.5 as a mobile phase A and methanol-tetrahydrofuran (90:10) as a mobile phase B, wherein the elution gradient is shown in Table 3; detecting at 235nm, 40 deg.C, and 0.8ml/min flow rate, injecting 20ul of sample solution into liquid chromatograph, and recording chromatogram.

(2) Preparing a sample into a solution containing rebamipide and a proper amount of impurities, detecting on a reversed-phase high performance liquid chromatograph by referring to a high performance liquid chromatography, using octadecylsilane chemically bonded silica as a filler, and performing analysis on a chromatographic column with the particle size of 5.0 mu M, the length of the chromatographic column of 250mm and the diameter of the chromatographic column of 4.6mm, and performing gradient elution under a mobile phase system which uses 0.05M phosphate buffer solution, the pH of which is 5.5 as a mobile phase A, and uses phosphate buffer salt-methanol-tetrahydrofuran (40:54:6) to adjust the pH value to 6.5 by using phosphoric acid as a mobile phase B, wherein the elution gradient is shown in Table 4; the detection wavelength is 235nm, the column temperature is 40 ℃, and the flow rate is 0.8 ml/min; and (4) injecting 20ul of the test solution into a liquid chromatograph, and recording the chromatogram.

In the comparative tests, unless otherwise specified, the various test conditions were consistent with the examples, except for the differences noted.

The analytical method for determining rebamipide related substances by HPLC provided by the present invention is further described below.

Example 1: control HPLC analytical method

High performance liquid chromatograph: shimadzu;

a chromatographic column: senecio CAPCELL PAK UG 120250 4.6mm 5 μm;

mobile phase: 2.44g of 1-sodium decane sulfonate is dissolved by 1000ml of water, and then 1000ml of methanol and 10ml of phosphoric acid are added;

detection wavelength: 232 nm;

flow rate: adjusting the flow rate so that the retention time of rebamipide is about 12 minutes;

column temperature: 25 ℃;

sample introduction amount: 10 mu l of the mixture;

isocratic elution;

preparing a mixed solution: taking appropriate amount of rebamipide, impurity A, impurity B, impurity C and impurity D, dissolving and diluting with diluent [ 0.05mol/L phosphate buffer solution-methanol (7:7:6) with water-pH6.0 ] to obtain solution containing rebamipide 0.4mg, impurity A, impurity B, impurity C and impurity D0.04 mg respectively per 1ml, and shaking up.

And (3) detection results: as a result, referring to fig. 1, it can be seen that in this example, RT-8.733 min is the peak of impurity a, RT-6.796 min is the peak of impurity B, RT-24.9 min is the peak of impurity D, RT-12.159 min is the peak of rebamipide, and the peak of impurity C is not detected and overlaps with the peak of rebamipide.

Example 2: control HPLC analytical method

High performance liquid chromatograph: shimadzu;

a chromatographic column: wakopak Wakosil-II 3 C184.6mm 150 mm;

mobile phase A: phosphate buffer solution with pH of 6.2 (9.08 g of monopotassium phosphate is weighed and dissolved by 1000ml of water, 9.46g of anhydrous disodium hydrogen phosphate is weighed and dissolved by 1000ml of water, 800ml of potassium dihydrogen phosphate solution is taken and 200ml of disodium hydrogen phosphate solution is taken to be uniformly mixed, if necessary, the pH is adjusted to 6.2 by the potassium dihydrogen phosphate solution or the disodium hydrogen phosphate solution), 300ml of water is added and uniformly mixed;

mobile phase B: acetonitrile;

detection wavelength: 222 nm;

column temperature: 25 ℃;

sample introduction amount: 10 mu l of the mixture;

gradient elution: the flow rate was adjusted so that the rebamipide retention time was about 20 minutes, see table 5.

TABLE 5

Elution time	Mobile phase A (%)	Mobile phase B (%)
			0min	83	17
30min	83	17
			33min	60	40
38min	60	40
			40min	83	17
45min	83	17

And (3) detection results: referring to fig. 2, it can be seen that in this example, RT-7.097 min is the peak of impurity B, RT-19.668 min is the peak of impurity C, RT-20.897 min is the peak of rebamipide, RT-36.779 min is the peak of impurity D, and impurity a splits into two peaks.

Example 3: control HPLC analytical method

High performance liquid chromatograph: agilent 1260;

a chromatographic column: kromasil100-5 C184.6mm 250mm, 5 μm;

mobile phase: methanol: phosphate buffer (potassium dihydrogen phosphate 6.8g, 152ml with 0.1mol/L sodium hydroxide solution, 20ml with tetrabutylammonium hydroxide 10% aqueous solution, diluted to 1000ml with water, pH adjusted to 6.5 with phosphoric acid) 52: 48;

detection wavelength: 235 nm;

flow rate: 0.8 ml/min;

column temperature: 40 ℃;

sample introduction amount: 20 mu l of the mixture;

isocratic elution;

preparing a mixed solution: taking appropriate amount of rebamipide, impurity A, impurity B, impurity C and impurity D, dissolving and diluting with mobile phase to obtain solution containing rebamipide 0.2mg, impurity A, impurity B, impurity C and impurity D0.02 mg respectively per 1ml, and shaking up.

And (3) detection results: as a result, referring to fig. 3, it can be seen that in this example, RT ═ 3.302min is the peak of impurity a, RT ═ 6.096min is the peak of impurity B, RT ═ 11.496min is the peak of impurity C, RT ═ 11.917min is the peak of rebamipide, RT ═ 24.623min is the peak of impurity D, and the peaks of impurity C and rebamipide do not meet the separation requirement.

Example 4: HPLC analysis method of the present invention

High performance liquid chromatograph: agilent 1260;

a chromatographic column: kromasil100-5 C184.6mm 250mm, 5 μm;

mobile phase A: phosphate buffer solution [ potassium dihydrogen phosphate 6.8g, adding 0.1mol/L sodium hydroxide solution 152ml, adding 20ml tetrabutylammonium hydroxide 10% water solution, diluting with water to 1000ml, adjusting pH to 6.5 with phosphoric acid ];

mobile phase B: methanol-tetrahydrofuran (90: 10);

detection wavelength: 235 nm;

flow rate: 0.8 ml/min;

column temperature: 40 ℃;

sample introduction amount: 20 mu l of the mixture;

gradient elution: table 3;

preparing a mixed solution: dissolving appropriate amount of rebamipide, impurity A, impurity B, impurity C, and impurity D with diluent [ mobile phase A-mobile phase B (60:40) ] and diluting to obtain solution containing rebamipide 0.2mg, impurity A, impurity B, impurity C, and impurity D0.02 mg respectively per 1ml, and shaking

And (3) detection results: referring to fig. 4, it can be seen that in this example, RT-3.153 min is the peak of impurity a, RT-7.848 min is the peak of impurity B, RT-19.756 min is the peak of impurity C, RT-21.014 min is the peak of rebamipide, and RT-39.146 min is the peak of impurity D. The mobile phase A, B (40:60) mixture was at a pH of about 7.6, which was detrimental to the chromatography column.

Example 5: HPLC analysis method of the present invention

High performance liquid chromatograph: agilent 1260;

a chromatographic column: kromasil100-5 C184.6mm 250mm, 5 μm;

mobile phase B: phosphate buffer-methanol-tetrahydrofuran (40:54:6) was adjusted to pH 6.5 detection wavelength with phosphoric acid: 235 nm;

flow rate: 0.8 ml/min;

column temperature: 40 ℃;

sample introduction amount: 20 mu l of the mixture;

gradient elution: table 4;

preparing a mixed solution: taking appropriate amount of rebamipide, impurity A, impurity B, impurity C and impurity D, dissolving and diluting with diluent (mobile phase A-methanol-tetrahydrofuran (60: 36: 4)) to obtain solution containing 1.0mg of rebamipide, 1.5ug/ml of impurity A, 1.5ug/ml of impurity B and 1.0ug/ml of impurity D per 1ml, and shaking up.

And (3) detection results: referring to fig. 5, it can be seen that in this example, RT ═ 3.168min is the peak of impurity a, RT ═ 7.892min is the peak of impurity B, RT ═ 19.788min is the peak of impurity C, RT ═ 20.607min is the peak of rebamipide, and RT ═ 39.177min is the peak of impurity D.

The foregoing is only for the purpose of understanding the method of the present invention and the core concept thereof, and it will be understood by those skilled in the art that various changes and modifications may be made without departing from the principle of the invention, and the invention also falls within the scope of the appended claims.

Claims

1. An analytical method for determining related substances of rebamipide by using HPLC is characterized in that a reverse phase chromatographic column using octadecylsilane chemically bonded silica as a filler and a buffer salt solution as a mobile phase A are used for carrying out gradient elution on a sample solution of rebamipide and carrying out HPLC analysis; the rebamipide related substances are 2-amino-3- (2-oxo-1, 2-dihydroquinolin-4-yl) propionic acid, 2- (2-chlorobenzoylamino) -3- (1, 2-dihydro-2-oxo-4-quinolyl) propionic acid, 2- (3-chlorobenzoylamino) -3- (1, 2-dihydro-2-oxo-4-quinolyl) propionic acid and 2- (3, 4-dichlorobenzoylamino) -3- (1, 2-dihydro-2-oxo-4-quinolyl) propionic acid;

the buffer salt solution is phosphate buffer solution, the concentration of the phosphate buffer solution is between 0.005M and 0.05M, and the pH =6.2 to 6.8;

the gradient elution procedure is as follows in table 3:

when table 3 is used as the gradient elution procedure, the mobile phase B is methanol and tetrahydrofuran in a volume ratio of 90: 10;

or table 4 below:

when table 4 is used as the gradient elution procedure, mobile phase B is a buffered salt solution, methanol and tetrahydrofuran in a volume ratio of 40:54: 6.

2. The analytical method of claim 1, wherein the reverse phase chromatography column is a column having a packing particle size of 3.0 μm to 5.0 μm, a column length of 150mm to 250mm, and a column diameter of 2.0mm to 4.6 mm.

3. The analytical method according to claim 1, wherein the HPLC detection wavelength is 235nm, the column temperature is 40 ℃, and the flow rate is 0.8 ml/min.