CN109307716B

CN109307716B - Detection method of brexpiprazole related substance

Info

Publication number: CN109307716B
Application number: CN201710622591.XA
Authority: CN
Inventors: 柯潇; 张伟
Original assignee: Sichuan Hongyuan Pharmaceutical Co ltd
Current assignee: Sichuan Hongyuan Pharmaceutical Co ltd
Priority date: 2017-07-27
Filing date: 2017-07-27
Publication date: 2022-03-25
Anticipated expiration: 2037-07-27
Also published as: CN109307716A

Abstract

The invention provides a detection method of an brexpiprazole related substance. The detection of related substances is carried out by high performance liquid chromatography, wherein the liquid chromatography determination conditions are as follows: the chromatographic column uses octadecylsilane chemically bonded silica as a filler, the mobile phase A is acetonitrile, the mobile phase B is phosphate buffer solution-acetonitrile, and gradient elution is adopted for detection. The method has the characteristics of specificity, durability, good reproducibility, high sensitivity and the like, and is suitable for qualitative and quantitative detection of relevant substances of the brexpiprazole.

Description

Detection method of brexpiprazole related substance

Technical Field

The invention relates to a high performance liquid chromatography analysis method, in particular to a method for separating and analyzing related substances of brexpiprazole by using high performance liquid chromatography.

Background

Epipezole, the first dopamine, partial 5-HT1A receptor agonist and 5-HT2A receptor antagonist compound developed first by Otsuka Denshi pharmaceutical Co., Ltd, and later co-developed with Denmark North pharmaceuticals, is considered to be a heavy breed after aripiprazole, a popular drug developed by this company. Compared with aripiprazole, the product shows higher affinity with dopamine D2 receptor, 5-HT2A receptor and 5-HT1A receptor, and relatively lower affinity with H1 and M1 receptor. The product submits a new drug application to the FDA in 7-month and 14-2014, is sold on the market in 10-7-month and 2015, and is used for the auxiliary treatment of major depression and the treatment of schizophrenia. The chemical name of the brexpiprazole is 7- (4- (4 benzo [ b ] thiophene-4-yl piperazine-1-yl) butoxy-1H-quinoline-2-ketone, and the chemical structural formula is as follows:

a method for synthesizing epipiprazole is disclosed in WO2006112464A for preparing an Otsuka drug, and the route is as follows:

however, the compound of formula (2) reacts with 1-bromo-4-chlorobutane with poor selectivity, easily forming two impurities of formula a and formula b, and the compound of formula (3) reacts with the compound of formula (4) in the presence of a base to undergo a side reaction with ipiprazole, forming impurity c.

Also, in the case of the compound of the formula (4a) and the compound of the formula (4b) in the above patent, it is disclosed in WO2013015456A that impurities of the formulae d and e are liable to occur as side reactions.

Byproducts of each step may remain in the product ipiprazole to affect the purity and yield of the product, and even through complicated post-treatment and purification processes, it may be difficult to completely remove impurities. In the research process of new drugs, accurate determination and analysis of the content of the drugs and impurities thereof are important conditions for ensuring the quality of the drugs. Therefore, the quality control of the crude drug of the brexpiprazole is necessary, and the qualitative and quantitative detection of related substances of the brexpiprazole is accurately carried out.

The chromatography method is simple, convenient and rapid, has high sensitivity, can reflect the characteristics of chemical composition, content and the like, and is one of the most important methods with the widest application range. The following are commonly used chromatography methods: high Performance Liquid Chromatography (HPLC), Thin Layer Chromatography (TLC), Gas Chromatography (GC), and High Performance Capillary Electrophoresis (HPCE). Among them, HPLC is most widely used because of its high separation efficiency, fast and sensitive analysis speed, and diverse detection means, and is not limited by the volatility and thermal stability of the sample.

Although HPLC is commonly used for quantitative detection of epiprazole, only a few reports are reported in the literature regarding methods for detection of epiprazole-related substances. For example, CN105399736A uses octadecylsilane chemically bonded silica as filler, and mobile phase A is 5mmol/L sodium octane sulfonate solution (pH 3.0 adjusted by phosphoric acid); the mobile phase B is acetonitrile, the detection wavelength is 290nm, the flow rate is 1.0mg/min, and the quinoline dimer can be successfully detected by performing linear gradient elution. However, the inventor repeats the patent condition to detect 7 related substances of the brexpiprazole, and only 5 components of the brexpiprazole can be detected. CN105175401A uses octadecylsilane chemically bonded silica as filler, acetonitrile: and (3) taking 50ml of water as an eluent, detecting the total peak area of the impurities of the brexpiprazole at the temperature of 30 ℃ at the detection wavelength of 290nm and the flow rate of 1.0 mg/min. The inventor repeats the condition to detect 7 related substances of the brexpiprazole, and as a result, no chromatographic peak of the brexpiprazole is detected after 60 min.

Since aripiprazole and ipiprazole have similar structures, the inventors also tried to test the related substances of the aripiprazole by referring to the quality standard of aripiprazole and the method in the import registration standard of aripiprazole bulk drug in the second part of the chinese pharmacopoeia 2015 edition, however, the results showed that the peak time of the aripiprazole is too fast, and the aripiprazole impurities, the aripiprazole impurities and the adjacent impurities cannot be separated. Indicating that the quality control method for aripiprazole is not applicable to ipipiprazole. Therefore, the invention provides a method for effectively analyzing the brexpiprazole and related substances thereof.

Disclosure of Invention

The invention aims to provide a high performance liquid chromatography analysis method of an brexpiprazole related substance, thereby realizing quality control of brexpiprazole.

The method adopts a chromatographic column with octadecylsilane chemically bonded silica as a filler, adopts acetonitrile as a mobile phase A, adopts phosphate buffer solution-acetonitrile as a mobile phase B, and adopts gradient elution for detection.

The inventors have tried to use phosphate-methanol as a mobile phase, but no matter how the ratio, pH, etc. of the mobile phase are adjusted, the relevant substances in ipiprazole can be detected completely.

The inventors have tried isocratic elution with phosphate-acetonitrile as mobile phase and replacing phosphate with ammonium formate, sodium sulfate, citric acid buffer, etc., which did not achieve the purpose of effective separation of related substances.

Through a large number of experimental designs and verifications, acetonitrile is finally determined to be used as a mobile phase A, phosphate buffer solution with a certain proportion of acetonitrile is added to be used as a mobile phase B, gradient elution is carried out, and various related substances in the brexpiprazole are successfully separated. The gradient elution conditions of the invention are as follows:

time min	Mobile phase A%	Mobile phase B%
			0	25	75
55	70	30
			85	70	30
85.01	25	75
			90	25	75

The volume ratio of the phosphate buffer solution to the acetonitrile in the mobile phase B is 100: 0-80: 20, preferably 90: 10-80: 20. When the ratio of acetonitrile in mobile phase B was further increased, the time for impurity I to peak was too fast (4.522min), and could not be separated from the solvent peak, and the organic phase in phase B was too high, resulting in inorganic salt precipitation from the end-of-gradient buffer, resulting in baseline drift, which was not suitable for detection.

The pH value of the phosphate buffer solution is 6.5-8.0, preferably 6.7-7.0. The pH of the phosphate buffer has an effect on the separation of the relevant substances, and when the pH is less than 6.5, for example, when the pH is 6.2, the brexpiprazole cannot be effectively separated from the adjacent impurities.

The phosphate buffer of the present invention is selected from sodium dihydrogen phosphate or potassium dihydrogen phosphate, preferably sodium dihydrogen phosphate.

The concentration of the phosphate buffer solution is 0.01-0.04 mol/L.

In the liquid chromatography condition, the column temperature is 30-40 ℃, and preferably 35 ℃; the detection wavelength is 254 nm; the flow rate is 0.8-1.2 ml/min, preferably 1.0 ml/min.

The invention further provides a detection method of brexpiprazole and related substances thereof, which comprises the following steps:

1) taking a proper amount of an brexpiprazole sample, adding methanol for dilution to prepare a solution containing about 0.2mg of brexpiprazole in each 1 ml;

2) precisely measuring 50 mu l of the sample solution in the step 1), and injecting the sample solution into a liquid chromatograph to complete the measurement of related substances;

wherein, the chromatographic conditions are as follows: the chromatographic column takes octadecylsilane chemically bonded silica as a filler, the mobile phase A is acetonitrile, the mobile phase B is phosphate buffer solution-acetonitrile, the volume ratio of the phosphate buffer solution to the acetonitrile is 90:10, the pH value of phosphate is 6.7, the column temperature is 35 ℃, the flow rate is 0.1ml/min, the detection wavelength is 254nm, and the gradient elution procedure is as follows:

The detection method of the related substances in the bulk drug of the brexpiprazole is simple, convenient and fast, has good specificity, high sensitivity and good repeatability, is suitable for qualitative and quantitative detection of the related substances in the brexpiprazole, can provide powerful guarantee for controlling the quality of the brexpiprazole, and can also provide a basis for the occurrence and control of side reactions in the synthesis process of the brexpiprazole.

Drawings

FIG. 1 is an HPLC chromatogram of a sample solution in comparative example 1;

FIG. 2 is an HPLC chromatogram of the sample solution in comparative example 2;

FIG. 3 is an HPLC chromatogram of the sample solution in comparative example 3;

FIG. 4 is an HPLC chromatogram of comparative example 4, in which 4a is a sample solution and 4b is a control solution; (ii) a

FIG. 5 is an HPLC chromatogram of the sample solution in comparative example 5;

FIG. 6 is an HPLC chromatogram of the sample solution in comparative example 6;

FIG. 7 is an HPLC chromatogram of the sample solution in comparative example 7;

FIG. 8 is an HPLC chromatogram of the sample solution in comparative example 8;

FIG. 9 is an HPLC chromatogram of the sample solution in comparative example 9;

FIG. 10 is an HPLC chromatogram of the sample solution in comparative example 10;

FIG. 11 is an HPLC chromatogram of the sample solution in comparative example 11;

FIG. 12 is an HPLC chromatogram of the sample solution in comparative example 12;

FIG. 13 is an HPLC chromatogram of the control solution of the sample added with impurities in example 1;

FIG. 14 is an HPLC chromatogram of the sample solution of test No. 1 in example 2;

FIG. 15 is an HPLC chromatogram of the sample solution of test number 2 in example 2;

FIG. 16 is an HPLC chromatogram of the sample solution of test No. 4 in example 2;

FIG. 17 is an HPLC chromatogram of the sample solution of test No. 1 in example 3;

FIG. 18 is an HPLC chromatogram of a sample solution of assay number 3 in example 3;

FIG. 19 is an HPLC chromatogram of a sample solution of test No. 4 in example 3;

FIG. 20 is an HPLC chromatogram of a sample solution of assay number 5 in example 3;

FIG. 21 is an HPLC chromatogram of the sample solution in example 4;

FIG. 22 is an HPLC chromatogram of the sample solution of test No. 1 in example 5;

FIG. 23 is an HPLC chromatogram of the sample solution of test No. 3 in example 5;

the number of each peak in the chromatogram is respectively as follows: peak 1-ipiprazole impurity I, peak 2-ipiprazole impurity II, peak 3-ipiprazole impurity III, peak 4-ipiprazole, peak 5-ipiprazole impurity V, peak 6-ipiprazole impurity IV, peak 7-ipiprazole impurity VI, and peak 8-ipiprazole impurity VII

Detailed Description

The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention.

Drugs, reagents and instruments

1. Drugs and reagents

TABLE 1 drugs and reagents

TABLE 2 impurity structure

2. Main instrument

TABLE 3 Main Instrument List

Comparative example 1

1. Sample preparation

Sample solution: accurately weighing an appropriate amount of an brexpiprazole sample, brexpiprazole-impurity I, brexpiprazole-impurity II, brexpiprazole-impurity III, brexpiprazole-impurity IV, brexpiprazole-impurity V, brexpiprazole-impurity VI and brexpiprazole-impurity VII, and quantitatively diluting with methanol to prepare a solution containing 0.2mg of brexpiprazole and 0.4 mu g of impurity in each 1 ml.

2. Detection of

The detection conditions of the liquid chromatography refer to the conditions in CN105399736A, specifically, octadecylsilane chemically bonded silica is used as a filler (5 μm,250 × 4.6mm), acetonitrile is used as a mobile phase A, 0.05mol/L sodium octane sulfonate solution (pH is adjusted to 3.0 by phosphoric acid) is used as a mobile phase B, the detection wavelength is 290nm, and the flow rate is 1.0ml per minute; the temperature is 35 ℃; a50. mu.l solution was measured and injected into a liquid chromatograph for elution as follows:

time min	A％	B％
				0→5	30	70
30	65	35
			40	65	35

3. Results

The results of the HPLC analysis under the above conditions are shown in FIG. 1. As can be seen from the figure, the chromatographic conditions were able to detect only 4 components out of 7 impurities of Epipprazole, in addition to Epipprazole.

Comparative example 2

1. Sample preparation

As in comparative example 1.

2. Detection of

The liquid chromatography detection was performed under the chromatography conditions of CN105175401A, specifically, octadecylsilane chemically bonded silica was used as a filler (5 μm,250 × 4.6mm), and acetonitrile: 50ml of water is used as eluent, and the detection wavelength is 290 nm; the flow rate was 1.0ml per minute; the temperature is 35 ℃; 50 mul of the solution was measured and injected into a liquid chromatograph for detection.

3. Results

The results of the HPLC analysis under the above conditions are shown in FIG. 2. From the figure, it can be seen that no eipiprazole chromatographic peak has been detected yet by running for 60 min.

Comparative example 3

1. Sample preparation

As in comparative example 1.

2. Detection of

According to the detection conditions of related substances in the second aripiprazole quality standard of Chinese pharmacopoeia 2015 edition, octadecylsilane chemically bonded silica is used as a filler (5 μm,250 × 4.6 mm); citrate buffer (pH adjusted to 4.7 with ammonia water) -acetonitrile (55: 45) is used as mobile phase; the detection wavelength is 254mn, and the flow rate is 1.0ml per minute; the column temperature was 35 ℃; a50. mu.l injection liquid chromatograph was measured.

3. Results

According to the high performance liquid chromatography detection under the conditions, as shown in FIG. 3, the peak time of the ipiprazole is too fast (less than 10 minutes), and the impurities of the ipiprazole, the impurities of the ipiprazole and the adjacent impurities can not be separated.

Comparative example 4

1. Sample preparation

Sample solution: accurately weighing an brexpiprazole sample and a proper amount of brexpiprazole-impurity V, and quantitatively diluting with methanol to prepare a mixed solution containing 0.2mg of brexpiprazole and 2 mu g of brexpiprazole-impurity V per 1 ml.

Impurity control solution: an appropriate amount of brexpiprazole-impurity V is precisely weighed, and diluted quantitatively with methanol to prepare a solution containing 0.2mg per 1ml as a reference solution.

2. Detection of

And (3) establishing chromatographic conditions by referring to a content detection method in an aripiprazole bulk drug import registration standard (JX 20040158). Octadecylsilane chemically bonded silica was used as filler (5 μm,250 × 4.6mm) for the experiment; using 20mmol/L sodium sulfate solution (pH 3.5) -acetonitrile (75:25) as a mobile phase; the detection wavelength is 254 mn; the column temperature was 35 ℃; the flow rate was 1.0ml per minute; a50. mu.l injection liquid chromatograph was measured.

3. Results

The HPLC analysis was performed under the above conditions, and the results of the analysis of the sample solution and the control solution are shown in FIGS. 4a and 4b, respectively. As can be seen from FIG. 4b, the retention times of the ipiprazole and the impurity V are consistent, and the ipiprazole and the impurity peaks overlap in the mixed solution and cannot be separated effectively.

Comparative examples 5 to 8

1. Sample preparation

As in comparative example 1.

2. Detection of

Octadecylsilane bonded silica gel as filler (5 μm,250 × 4.6 mm); with 0.02mol/L NaH₂PO4 (containing 0.2% triethylamine) buffer solution-methanol as mobile phase, and using phosphoric acid to condition pH; the flow rate was 1.0ml per minute; the detection wavelength is 254 nm; the column temperature was 35 ℃. 50 mul of the solution was measured and injected into a liquid chromatograph for detection. The mobile phase pH and proportions in comparative examples 5 to 8 are shown in the following table:

TABLE 4 partial HPLC chromatographic conditions

3. Results

The results of the HPLC analysis were shown in FIGS. 5 to 8, by varying the mobile phase ratio and pH under the above conditions. As can be seen from fig. 5 to 6 and 8, only 5 of the 8 components were detected in the sample solutions of comparative examples 5 to 6 and 8, and the separation effect was poor in fig. 6. In comparative example 7, only 7 peaks were separated, and the separation effect was not satisfactory.

Comparative examples 9 to 10

1. Sample preparation

Sample solution: accurately weighing an brexpiprazole sample and a proper amount of brexpiprazole-impurity V, and quantitatively diluting with methanol to prepare a solution containing 0.2mg of brexpiprazole and 0.4 mu g of impurity per 1 ml.

Control solution: an appropriate amount of the brexpiprazole-impurity V is precisely weighed, and is quantitatively diluted by methanol to prepare a solution containing 0.2mg of the impurity per 1ml as a reference solution.

2. Detection of

Bonding with octadecylsilanesSilica gel as filler (5 μm,250 × 4.6 mm); with acetonitrile-0.02 mol/L NaH₂PO4 is mobile phase, and NaH is regulated by phosphoric acid₂pH of PO4 solution; the flow rate was 1.0ml per minute; the detection wavelength is 254 nm; the column temperature was 35 ℃. 50 mul of the solution was measured and injected into a liquid chromatograph for detection. The mobile phase pH and the proportions in comparative examples 9 to 10 are shown in the following table:

TABLE 5 partial HPLC chromatographic conditions

3. Results

The results of the HPLC analysis by adjusting the pH of the buffer under the above conditions are shown in FIGS. 9 to 10. As can be seen from FIG. 9, when the phosphate pH was 3.9, epipiprazole and impurity V exhibited a peak-off phenomenon, and the degree of separation was 1.2. When the pH value of the phosphate is 6.7, the main component peak of the ipiprazole cannot be detected after running for 70 minutes.

Comparative example 11

1. Sample preparation

Sample solution: accurately weighing an brexpiprazole sample and a proper amount of brexpiprazole-impurity V, and quantitatively diluting with methanol to prepare solutions containing 0.2mg of brexpiprazole and 0.4 mu g of impurity in each 1 ml.

Control solution: an appropriate amount of the brexpiprazole-impurity V is precisely weighed and quantitatively diluted by methanol to prepare a solution containing 0.4 mu g of the impurity per 1ml as a reference solution.

2. Detection of

Octadecylsilane bonded silica gel as filler (5 μm,250 × 4.6 mm); acetonitrile-0.05 mol/L ammonium formate solution (30:70) is used as a mobile phase; the flow rate was 1.0ml per minute; the detection wavelength is 254 nm; the column temperature was 35 ℃. 50 mul of the solution was measured and injected into a liquid chromatograph for detection.

3. Results

The results of the HPLC analysis under the above conditions are shown in FIG. 11. As can be seen from fig. 11, when the mobile phase of comparative example 9 or 10 was changed to ammonium formate from phosphate, the separation of ipiprazole from impurity V was still only 1.17.

Comparative example 12

1. Sample preparation

As in comparative example 1.

2. Detection of

Octadecylsilane bonded silica gel as filler (5 μm,250 × 4.6 mm); acetonitrile-0.05% trifluoroacetic acid as mobile phase B; the flow rate was 1.0ml per minute; the detection wavelength is 254 nm; the column temperature was 35 ℃. Measuring 50 μ l of the solution, injecting into a liquid chromatograph, and eluting under the following conditions:

time min	A％	B％
				0	15	85
50	80	20
			70	80	20
70.01→80	15	85

3. Results

The results of the HPLC analysis under the above conditions are shown in FIG. 12. From the figure, it can be seen that a large unknown peak appears in the baseline shift before the main peak of the ipiprazole, and the ipiprazole cannot be separated from adjacent impurities.

Example 1

1. Sample preparation

Impurity localization solution: and (3) respectively placing proper amounts of BPZ-impurity reference substances into measuring bottles, dissolving with methanol and diluting to scale to obtain the BPZ-impurity reference substance.

TABLE 6 BPZ-impurity concentration

Sample solution: BPZ (151101)11.32mg, placed in a 50ml volumetric flask, dissolved and diluted to the mark with methanol.

Sample plus impurity control solution: a sample solution was taken and 50. mu.l each of the impurity-localizing solutions was added.

Blank: methanol

Note: preparing BPZ-impurity II: dissolve with dimethylsulfoxide and dilute to scale.

Preparation of BPZ-impurity VII: dissolve with N, N dimethylformamide and dilute to the mark.

2. Detection of

Octadecylsilane bonded silica gel as filler (5 μm,250 × 4.6 mm); using acetonitrile as mobile phase A, pH value of 6.7 and concentration of 0.02mol/L NaH₂PO₄Buffer-acetonitrile (90:10) as mobile phase B; the flow rate was 1.0ml per minute; the detection wavelength is 254 nm; the column temperature was 35 ℃. Measuring 50 μ l of the solution, injecting into a liquid chromatograph, and eluting under the following conditions:

3. results

The results are shown in fig. 13, where the order of peak appearance for each main peak is: brexpiprazole-impurity I, brexpiprazole-impurity II, brexpiprazole-impurity III, brexpiprazole-impurity V, brexpiprazole-impurity VI, brexpiprazole-impurity VII, and brexpiprazole-impurity VIII. The blank solvent is free from interference, and the separation degree between the brexpiprazole and adjacent impurities and known impurities is more than or equal to 1.5.

TABLE 7 Agripprazole specificity survey data

Example 2

1. Sample preparation

As in comparative example 1.

2. Detection of

Octadecylsilane bonded silica gel as filler (5 μm,250 × 4.6 mm); the flow rate was 1.0ml per minute; the column temperature was 35 ℃; the detection wavelength is 254 nm; NaH with 0.02mol/L phosphate₂PO4 buffer, other conditions are as follows:

TABLE 8 partial HPLC chromatographic conditions

Measuring 50 μ l of the solution, injecting into a liquid chromatograph, and eluting under the following conditions:

3. results

The results under the detection conditions of experiment numbers 1 to 4 are shown in FIGS. 14, 15, 13 and 16, respectively. The pH value of the buffer solution is 6.5-6.9, the separation degree between the brexpiprazole and adjacent impurities and among the impurities is more than 1.5; the separation degree of the ipiprazole from adjacent impurity peaks decreases with decreasing pH, and the ipiprazole cannot be separated from the impurity V at pH values lower than 6.5, for example, at pH 6.2. And the pH value is alkaline, which can cause the stationary phase of the chromatographic column to be lost and the column efficiency to be reduced, so the pH value range of the detection method is 6.5-8.0, and the preferable pH value is 6.7 or 6.9.

Example 3

1. Sample preparation

As in comparative example 1.

2. Detection of

Octadecylsilane bonded silica gel as filler (5 μm,250 × 4.6 mm); the mobile phase A is acetonitrile, the mobile phase B is 0.02mol/L NaH containing no acetonitrile or a certain proportion₂PO4 buffer; the flow rate was 1.0ml per minute; the column temperature was 35 ℃; the detection wavelength is 254 nm; the acetonitrile ratio was as follows:

TABLE 9 partial HPLC chromatographic conditions

3. Results

The results of the experiments carried out under the conditions of numbers 1 to 5 by HPLC as described above are shown in FIGS. 17, 13, 18, 19 and 20, respectively. As can be seen from fig. 17, when acetonitrile was not added to mobile phase B, the ipiprazole and each impurity peak could be completely detected and each peak could be effectively separated, but the retention time of the ipiprazole-impurity vii was 87.406min, which was close to the end of the gradient procedure. As can be seen from FIGS. 13, 18 and 19, when the ratio of the phosphate buffer solution to the acetonitrile is 90:10 to 80:20, the eipiprazole and each impurity peak can be effectively separated. Whereas when the ratio of phosphate buffer to acetonitrile was 70:30, the time for impurity I to peak was too fast (4.522min), it could not separate from the solvent peak, and the organic phase in phase B was too high resulting in inorganic salt precipitation from the end of the gradient buffer, resulting in baseline drift.

Example 4

1. Sample preparation

The same as in example 1.

2. Detection of

Except that the phosphate buffer in example 1 was replaced with NaH₂PO₄Can be changed into KH₂PO₄Otherwise, the conditions were the same as in example 1.

3. Results

The results are shown in FIG. 21. As can be seen from the figure, under the detection condition, the peak of the ipiprazole and each impurity can be completely detected, wherein the separation degree of the ipiprazole and the impurity V is 1.08.

Example 5

1. Sample preparation

As in comparative example 1.

2. Detection of

Octadecylsilane bonded silica gel as filler (5 μm,250 × 4.6 mm); acetonitrile as mobile phase A, NaH of certain concentration₂PO₄Buffer-acetonitrile (90:10) as mobile phase B; the flow rate was 1.0ml per minute; the column temperature was 35 ℃; the detection wavelength is 254 nm; the phosphate concentrations were as follows:

TABLE 10 partial HPLC chromatographic conditions

3. Results

The results when the concentration of the phosphate buffer was 0.01mol/L, 0.02mol/L, and 0.04mol/L are shown in FIGS. 22, 13, and 23, respectively. The results show that the salt concentration has less influence on the separation of impurities, and NaH in the mobile phase B₂The concentration of PO4 is in the range of 0.01-0.04mol/L, and the ipiprazole can be effectively separated from adjacent impurities and impurities.

Example 6

This example is to examine the reproducibility of the method.

1. Sample preparation

Impurity control solution: the impurity localization solution of example 1, 100. mu.l each of impurities I to VI and 200. mu.l of impurity VII were taken and placed in a 100ml measuring flask and diluted to the scale with methanol.

Sample solution: taking 11.32mg of BPZ (151101), putting the BPZ into a 50ml measuring flask, adding methanol to dissolve and diluting to the scale mark to obtain the product.

Self-control solution: precisely measuring 0.1ml of sample solution, placing the sample solution into a 100ml measuring flask, adding methanol for dissolving, and diluting to scale.

2. Detection of

The same as in example 1.

3. Results

Impurity control solution, sample solution and self control solution are continuously injected into a sample 6 needles, and the peak area RSD is calculated and shown in the table. The results show that the peak areas RSD of the BPZ-impurity, the self-control and the impurity detected in the sample all accord with the specification (less than or equal to 3%).

TABLE 11 impurity, self-control repeatability data

TABLE 12 sample repeatability

Example 7

1. Sample preparation

Impurity control and sample solutions were as in example 6.

2. Detection of

The same as in example 1.

3. Results

The results of the HPLC analysis were as shown in the table. As can be seen from the table, the content of the impurity VI in the brexpiprazole sample is 0.023%, the total impurity is 0.046%, and the sensitivity of the method is very high.

Table 13 BPZ three sample assay results.

Batch number	BPZ-impurity VI%	Unknown impurities%	Other impurities%	The total amount of impurities%
					151101	0.02	0.02	Not detected out	0.04
151201	0.03	0.02	Not detected out	0.05
					151202	0.02	0.03	Not detected out	0.05
Average	0.023	0.023	-	0.046

In conclusion, the analysis method provided by the invention has the characteristics of specificity, durability, good reproducibility, high sensitivity and the like in the aspect of measuring the relevant substances of the brexpiprazole.

Claims

1. The detection method of the brexpiprazole related substances is characterized by adopting high performance liquid chromatography, wherein the liquid chromatography determination conditions are as follows: the chromatographic column uses octadecylsilane chemically bonded silica as a filler, a mobile phase A is acetonitrile, a mobile phase B is phosphate buffer solution-acetonitrile, the volume ratio of the phosphate buffer solution to the acetonitrile in the mobile phase B is 90: 10-80: 20, the pH value of the phosphate buffer solution is 6.5-8.0, the phosphate buffer solution is sodium dihydrogen phosphate, and the detection is performed by adopting gradient elution, wherein the gradient elution conditions are as follows:

The related substances comprise:

2. the assay of claim 1 wherein said phosphate buffer has a pH of 6.7 to 6.9.

3. The detection method according to claim 1, wherein the concentration of the phosphate buffer is 0.01 to 0.04 mol/L.

4. The detection method according to claim 3, wherein the liquid chromatography is performed at a column temperature of 30 to 40 ℃.

5. The detection method according to claim 4, wherein the liquid chromatography is performed under conditions in which the column temperature is 35 ℃.

6. The detection method according to claim 1, wherein the detection wavelength in the liquid chromatography is 254 nm.

7. The detection method according to claim 1, wherein the flow rate is 0.8 to 1.2ml/min under the liquid chromatography condition.

8. The detection method according to claim 1, wherein the flow rate in the liquid chromatography is 1.0 ml/min.

9. The detection method according to any one of claims 1 to 8, characterized by comprising the steps of:

wherein, the chromatographic conditions are as follows: the chromatographic column takes octadecylsilane chemically bonded silica as a filler, the mobile phase A is acetonitrile, and a phosphate buffer solution-acetonitrile, wherein the volume ratio of the phosphate buffer solution to the acetonitrile is 90:10, the pH of phosphate is 6.9, the column temperature is 35 ℃, the flow rate is 1ml/min, the detection wavelength is 254nm, and the gradient elution procedure is as follows:

。