CN116754664A

CN116754664A - Method for detecting aromatic amine genotoxic impurity residues in phloroglucinol

Info

Publication number: CN116754664A
Application number: CN202310554616.2A
Authority: CN
Inventors: 朱克玉; 肖云华; 万澄玉; 孙井龙; 梁溪
Original assignee: Nanjing Comer Biomedical Co ltd
Current assignee: Nanjing Comer Biomedical Co ltd
Priority date: 2023-05-17
Filing date: 2023-05-17
Publication date: 2023-09-15

Abstract

The application discloses a high performance liquid chromatography analysis method for detecting aromatic amine genotoxic impurity residues in phloroglucinol, which adopts a reversed phase C18 column, takes 8-12 mmol/L dipotassium hydrogen phosphate aqueous solution as a mobile phase A and takes methanol as a mobile phase B to carry out gradient elution; wherein the dissolved phloroglucinol diluent is 0.05-0.5% (w/v) Vc solution, and the pH value of the mobile phase A is adjusted to 4.7-4.9 by phosphoric acid. The method can carry out quick, accurate and stable qualitative and quantitative analysis on aromatic amine genotoxic impurities in phloroglucinol.

Description

Method for detecting aromatic amine genotoxic impurity residues in phloroglucinol

Technical Field

The application belongs to the field of medicine analysis, and particularly relates to a high performance liquid chromatography analysis method for detecting aromatic amine genotoxic impurity residues in phloroglucinol.

Background

Phloroglucinol (Phloroglucinol) can directly act on smooth muscle of gastrointestinal tract and genitourinary tract, and is a myophilic, non-atropine, non-papaverine smooth muscle antispasmodics. Compared with other smooth muscle spasmolytics, the preparation has the characteristics of no anticholinergic effect, no anticholinergic side effect, no symptoms such as hypotension, heart rate acceleration, arrhythmia and the like, and no influence on cardiovascular functions.

Phloroglucinol injection was marketed in france as batch at 12 months 1 of 1993 under the trade name Spasfon, specification 4ml:40mg, the company Teva Sante, is suitable for symptomatic treatment of acute pain associated with functional disorders of the gastrointestinal and biliary tract, acute painful spastic events and urinary system disorders (renal colic), symptomatic treatment of gynecological acute pain. The structural formula is as follows:

。

quality control of crude drugs and preparations has been a major point and difficulty in drug development, and research on impurities has been a major issue in quality control. The starting materials, intermediates, byproducts, degradation products and the like in the phloroglucinol synthesis process can become impurities remained in the final product, thereby affecting the product quality.

Excessive residues of potentially genotoxic impurities in a drug may affect drug safety. The "genotoxic impurity control guiding principle" is newly added in the pharmacopoeia of the people's republic of China "2020 edition, and is enough to be increasingly important in medicine quality research and control. In order to provide a phloroglucinol injection crude drug with controllable safety quality, the synthesis process of the phloroglucinol crude drug is researched, and the fact that 5- (dimethylamino) -resorcinol (abbreviated as impurity J) and 2, 6-dimethylamino-4-hydroxyaniline (abbreviated as impurity P) which possibly exist in the synthesis process are aromatic amine potential genotoxic impurities is found, the phloroglucinol injection crude drug has a genotoxic warning structure, the residual amount of the impurity J and the impurity P in the crude drug is required to be detected and controlled, and the structures of the impurity J and the impurity P are as follows:

。

according to the international pharmaceutical registration association (ICH) guidelines, for drugs with a drug administration time of more than 10 years, the acceptable toxicology attention threshold is 1.5 per person, and when the maximum daily dose in the administration instruction of the phloroglucinol injection is 0.2g, the acceptable impurity limit of the impurities J and P is 7.5ppm (1.5 mug/0.2 g), so that a high-sensitivity analysis method needs to be established for trace determination of aromatic amine genotoxic impurities in the phloroglucinol bulk drug. At present, researches on aromatic amine genotoxic impurities in phloroglucinol bulk drugs and researches on quality control analysis methods are rarely reported in literature.

Disclosure of Invention

The application aims to solve the problems of the prior art that the research and quality control analysis method of the phloroglucinol aromatic amine genotoxic impurities are lack, and provides a high performance liquid chromatography analysis method for rapidly and accurately detecting the aromatic amine genotoxic impurity residues in phloroglucinol.

In order to achieve the above purpose, the application adopts the following technical scheme:

the inventor carries out deep analysis on impurities possibly existing IN the phloroglucinol synthesis process, and combines European pharmacopoeia EP10.0 quality standard to identify 22 impurities IN total of phloroglucinol impurities A-IN 1-G, wherein the impurities comprise aromatic amine genotoxic impurities J and P, and the structure is as follows:

。

the high performance liquid chromatography method adopted by the application comprises the following steps:

(1) Setting chromatographic conditions: adopting a reversed-phase C18 column, taking dipotassium hydrogen phosphate aqueous solution as a mobile phase A, and methanol as a mobile phase B to perform gradient elution; the method comprises the steps of carrying out a first treatment on the surface of the

(2) Sample solution preparation: adopting a diluent to dissolve phloroglucinol raw materials to prepare a solution with the concentration of 1-10 mg/mL;

(3) And (3) detection: and (5) injecting the prepared phloroglucinol solution into a high performance liquid chromatograph, and recording a chromatogram.

Wherein, the gradient elution in the step (1) is preferably carried out by the following modes:

the sample injection amount in the step (3) is selected from 10-100 mu L;

according to an embodiment of the present application, the reversed phase C18 column in the liquid chromatography disclosed in the present application is preferably: horizon AQUA, 250X 4.6mm,5 μm.

According to the embodiment of the application, the inventor researches and discovers that the pH of the mobile phase A can influence the peak of the impurities J and P, and the inventor researches the influence of the pH of different mobile phases A on the detection of the impurities J and P in the sample solution: when the pH of the mobile phase A is 3.5, the main peak and the impurity P sequentially form peaks, but the impurity P is not separated from the base line of the main peak; when the pH of the mobile phase A is about 4.5, unknown small impurities contained in the sample solution coincide with the impurities J, and the pH of the mobile phase A can influence the peak time of the impurities J and P; when the pH of the mobile phase is 4.8, the specificity of the impurity J and the impurity P is good, and the impurity J and the impurity P are not overlapped with the 22 specific impurities and the unknown impurities in the sample. Comprehensive examination the pH of mobile phase a in the high performance liquid chromatography employed in the present application is preferably 4.8.

According to an embodiment of the present application, the inventors studied the effect of a diluent on the detection of impurity J and impurity P in a test sample solution: when the diluent in the step (2) is 0.1% Vc of 5% acetonitrile solution, the impurity P peak is split, and the solvent effect is achieved; when a proper amount of Vc aqueous solution is added into the diluent, not only the peak types of the impurity J and the impurity P are good, but also the degradation of the impurity P and the impurity J can be effectively inhibited by a proper amount of Vc, the peak areas of the impurity P and the impurity J are basically consistent within 24h, the diluent is preferably 0.05-0.5% (w/v) Vc aqueous solution, and further, the diluent is preferably 0.1% Vc aqueous solution.

According to the embodiment of the application, when the mobile phase A in the step (1) is selected from 8-12 mmol/L dipotassium hydrogen phosphate aqueous solution, the pH of the mobile phase A is regulated to 4.7-4.9 by phosphoric acid, and the temperature of a column Wen Xuanzi-35 ℃; the detection wavelength is selected from 210-240 nm; when the flow phase speed is selected from 0.9-1.1 mL/min, the peak shapes of the impurity J and the impurity P are good, and the separation degree is high. Further, mobile phase A is preferably 10mmol/L; mobile phase a is pH adjusted with phosphoric acid, preferably 4.8; the detection wavelength is preferably 210nm, and the mobile phase speed is preferably 1.0mL/min.

The application has the beneficial effects that:

(1) The method can effectively ensure that the peak types of the genotoxic impurity J and the impurity P are good, and has good solution stability;

(2) The detection technology disclosed by the application can realize the separation and measurement of the impurity J and the impurity P in phloroglucinol, the 22 impurities and other unknown impurities in the bulk drug;

(3) The method can rapidly and accurately perform qualitative and quantitative analysis on the potential genotoxic impurity J and the impurity P in the raw material phloroglucinol, and ensures the quality controllability of the raw material phloroglucinol.

Drawings

FIG. 1 is a chromatogram of a hollow white diluent of example 2;

FIG. 2 is a chromatogram of the system applicability solution in example 2;

FIG. 3 is a chromatogram of impurity J and impurity P solutions in example 2;

FIG. 4 is a graph showing the comparison of the phloroglucinol sample solution and the system-applicable solution (sample solution, system-applicable solution in order from top to bottom) of the crude drug in example 2;

FIG. 5 is a chromatogram of the mixed solution in example 3;

FIG. 6 is a chart showing the stacking of the phloroglucinol batches 1-3 and the mixed solution (mixed solution, batch 1 sample solution, batch 2 sample solution, batch 3 sample solution in order from top to bottom) in example 3;

FIG. 7 is a chromatogram of the system applicability solution of example 4 (5% acetonitrile solution with 0.1% Vc solvent);

FIG. 8 is a chromatogram of the system applicability solution (solution with 0.1% Vc solvent) in example 4;

FIG. 9 is a chromatogram of the system applicability solution in example 5;

FIG. 10 is a chromatogram of a test solution at a wavelength of 240nm in example 6;

FIG. 11 is a chromatogram of a system applicability solution at a wavelength of 240nm in example 6;

FIG. 12 is a chromatogram of sample solution of example 7 with an input of 10 μl;

FIG. 13 is a chromatogram of a 10 μl sample injection volume of the system applicability solution in example 7;

FIG. 14 is a chromatogram of sample solution at a flow rate of 1.1ml/min in example 8;

FIG. 15 is a chromatogram of a system applicability solution with a flow rate of 1.1ml/min in example 8;

FIG. 16 is a chromatogram of the sample solution of example 9 at a flow rate of 0.9 ml/min;

FIG. 17 is a chromatogram of a system applicability solution with a flow rate of 0.9ml/min in example 9;

FIG. 18 is a chromatogram of a sample solution of 0.05% Vc in example 10;

FIG. 19 is a chromatogram of a 0.05% Vc system applicability solution for the diluent in example 10;

FIG. 20 is a chromatogram of a sample solution of 0.5% Vc in example 11;

FIG. 21 is a chromatogram of a 0.5% Vc system applicability solution for the diluent of example 11;

FIG. 22 is a chromatogram of a sample solution at a column temperature of 35℃in example 12;

FIG. 23 is a chromatogram of a 35℃column temperature system applicability solution in example 12;

FIG. 24 is a chromatogram of a mobile phase A salt concentration 12mmol/L sample solution in example 13;

FIG. 25 is a chromatogram of a mobile phase A salt concentration 12mmol/L system-applicable solution in example 13;

FIG. 26 is a chromatogram of a mobile phase A salt concentration 8mmol/L sample solution in example 14;

FIG. 27 is a chromatogram of a mobile phase A salt concentration 8mmol/L system-applicable solution in example 14;

FIG. 28 is a chromatogram of mobile phase A pH4.9 sample solution in example 15;

FIG. 29 is a chromatogram of mobile phase A pH4.9 system applicability solution in example 15;

FIG. 30 is a chromatogram of mobile phase A pH4.7 sample solution in example 16;

FIG. 31 is a chromatogram of the mobile phase A pH4.7 system applicability solution of example 16.

Detailed Description

The following examples are merely illustrative of the present application and are not intended to limit the application. The method of the present application is also not limited to the impurities J and P, any combination of any aromatic amine genotoxic impurity in the isolated measurement of phloroglucinol by the method of the present application and 1 to 22 impurities selected from the above impurities falls within the scope of the present application, and any combination of any aromatic amine genotoxic impurity in the isolated measurement of phloroglucinol by the method of the present application and 1 to 22 impurities including the above impurities falls within the scope of the present application.

The preparation of impurity J and impurity P was carried out by the method of example 1.

EXAMPLE 1 preparation of impurity control

Preparation of impurity J

2g of the reactant is weighed and added into a reaction bottle, 20mL of dichloromethane is added, the temperature is reduced to 0 ℃, 2mol/L of boron tribromide dichloromethane solution (3 eq) is added dropwise, after the addition is finished, the reaction is completed at room temperature, the temperature is reduced to 0 ℃, the reactant is slowly added dropwise into the dichloromethane solution of methanol reduced to 0 ℃, the filtration is carried out, a filter cake is pulped with a small amount of water, the filtration is carried out, and 1.2g of solid is obtained, and the purity is 98.7%. ¹ H-NMR（DMSO-d6）：δ8.80（2H，s），δ5.59（3H，s），δ2.77（6H，s）。

Preparation of impurity P

2g of the reactant is weighed and added into a reaction bottle, 20mL of dichloromethane is added, the temperature is reduced to 0 ℃, 2mol/L of boron tribromide dichloromethane solution (2 eq) is added dropwise, after the addition is finished, the reaction is completed at room temperature, the temperature is reduced to 0 ℃, the reactant is slowly added dropwise into the dichloromethane solution of methanol reduced to 0 ℃, the filtration is carried out, a filter cake is pulped with a small amount of water, the filtration is carried out, and 1.5g of solid is obtained, and the purity is 97.9%.1H-NMR (DMSO-d 6): δ8.60 (1H, s), δ6.03 (2H, s), δ3.69 (6H, s), δ3.54 (2H, s).

Example 2

Instrument: thermo Scientific U3000 high performance liquid chromatograph

Chromatographic column: horizon AQUA (250X 4.6mm,5 μm)

Mobile phase: 10mmol/L dipotassium hydrogen phosphate buffer (pH value is adjusted to 4.8 by phosphoric acid) is taken as a mobile phase A; methanol as mobile phase B, linear gradient elution was performed as follows:

detection wavelength: 210nm of

Flow rate: 1mL/min

Column temperature: 25 DEG C

Sample injection amount: 100 mu L

A diluent: 0.1% Vc solution

Impurity J and impurity P stock solutions: taking proper amounts of impurity J and impurity P, precisely weighing, adding a diluent for dissolving and diluting to prepare a solution containing about 7.5 mug per 1 mL;

system applicability solution: weighing a proper amount of phloroglucinol, adding a proper amount of an impurity J and an impurity P stock solution, and preparing a solution containing about 10mg of phloroglucinol and about 0.075 mug of impurity J and impurity P in each 1mL of the solution as a system applicability solution (the solution is a mixed solution of a sample solution and the impurity J and P with the limit concentration of 7.5 ppm);

test solution: precisely weighing a proper amount of phloroglucinol, placing the phloroglucinol into a measuring flask, and dissolving and diluting the phloroglucinol with a diluent to prepare a solution of 10 mg/mL;

and (3) measuring: and (3) respectively injecting 100 mu L of each of the diluent, the system applicability solution, the impurity J and impurity P stock solution and the sample solution into a high performance liquid chromatograph, and recording a chromatogram.

Results: the blank diluent has no interference to the measurement; the chromatogram is shown in figure 1, the system applicability solution and the impurity J and P stock solution are shown in figures 2-3, the main peak of API (phloroglucinol), impurity P and impurity J in the system applicability solution are sequentially subjected to peak emission, and the retention time is 11.992min, 23.093min and 26.760min respectively; the comparison of the sample solution and the system applicability solution is shown in figure 4, and as the system applicability solution is a mixed solution of the sample solution and the impurity J/P with the limit concentration of 7.5ppn, the comparison shows that the impurity J and the impurity P are not detected in the phloroglucinol, and other unknown impurities in the sample solution do not interfere the detection of the impurity J and the impurity P;

。

example 3

Instrument: thermo Scientific U3000 high performance liquid chromatograph

Chromatographic column: horizon AQUA (250X 4.6mm,5 μm)

detection wavelength: 210nm of

Flow rate: 1mL/min

Column temperature: 25 DEG C

Sample injection amount: 100 mu L

A diluent: 0.1% Vc solution

Specific impurity monomer positioning solution: taking proper amounts of the 22 impurities, precisely weighing, respectively adding acetonitrile water mixed solution for dissolving and diluting to prepare a solution containing 5-15 mug per 1mL, and taking the solution as a monomer positioning solution of each specific impurity;

mixing solution: taking proper amounts of the impurity J and the impurity P, precisely weighing, adding a diluent for dissolving and diluting to prepare a solution containing about 0.075 mug per 1mL (the concentration of the impurity J/P in the mixed solution is 7.5ppm in the limit concentration);

test solution: accurately weighing a proper amount of phloroglucinol (multiple batches), placing the phloroglucinol into a measuring flask, and dissolving and diluting the phloroglucinol into 10mg/mL solution by using a diluent;

taking 100 mu L of each specific impurity monomer positioning solution and 100 mu L of each specific impurity monomer mixed solution, respectively injecting into a high performance liquid chromatograph, and recording a chromatogram.

The retention time results of the 22 specific impurities and the mixed solution are shown in the following table, and each specific impurity does not interfere with the detection of the impurities J and P, so that the specificity is good; according to the calculation of the external standard method, the mixed solution (the limit concentration of the impurity J and the impurity P is 7.5ppm, see figure 5) and the multi-batch sample map (see figure 6) are compared, and the impurity J and the impurity P are not detected in the multi-batch sample solution.

。

Example 4

Instrument: thermo Scientific U3000 high performance liquid chromatograph

Chromatographic column: horizon AQUA (250X 4.6mm,5 μm)

Mobile phase: 10mmol/L potassium dihydrogen phosphate buffer (pH value is adjusted to 3.5 by phosphoric acid) is taken as a mobile phase A; methanol as mobile phase B, linear gradient elution was performed as follows:

detection wavelength: 210nm of

Flow rate: 1mL/min

Column temperature: 25 DEG C

Sample injection amount: 100 mu L

A diluent: 0.1% Vc in 5% acetonitrile

system applicability solution: weighing a proper amount of phloroglucinol, adding a proper amount of impurity J and a proper amount of impurity P stock solution, and preparing a solution which contains about 10mg of phloroglucinol and about 0.75 mug of impurity J and impurity P in each 1mL of solution as a mixed solution;

and (3) measuring: 100 mu L of the system applicability solution is respectively injected into a high performance liquid chromatograph, and a chromatogram is recorded.

Results: when the pH value of the mobile phase A is 3.5 and the diluent is 5% acetonitrile solution with 0.1% Vc, the system applicability solution chromatogram is shown in FIG. 7, at the moment, the impurity P is not separated from the main peak baseline and the impurity P is split, the organic phase proportion in the diluent is considered to influence the impurity P, the solvent effect is realized, the pH value of the mobile phase A can influence the separation degree of the impurity P from the main peak, and when the diluent is not added with any organic solvent, the impurity P is good in peak type; when the diluent is pure water and a proper amount of Vc is added (the map is shown in fig. 8), not only are the impurity J and the P peaks good, but also the degradation of the impurity P and the impurity J can be effectively inhibited by the proper amount of Vc, the peak areas of the impurity P and the impurity J are basically consistent within 24h, but at the moment, the pH value of the mobile phase A is 3.5, and the impurity P and the main peak baseline are not separated.

Example 5

Instrument: thermo Scientific U3000 high performance liquid chromatograph

Chromatographic column: horizon AQUA (250X 4.6mm,5 μm)

Mobile phase: 10mmol/L potassium dihydrogen phosphate buffer solution is a mobile phase A; methanol as mobile phase B, linear gradient elution was performed as follows:

detection wavelength: 210nm of

Flow rate: 1mL/min

Column temperature: 25 DEG C

Sample injection amount: 100 mu L

A diluent: 0.1% Vc aqueous solution

and (3) measuring: 100 mu L of the sample solution and 100 mu L of the system applicability solution are respectively injected into a high performance liquid chromatograph, and chromatograms are recorded.

Results: the chromatogram of the solution with system applicability is shown in FIG. 9, when the mobile phase is 10mmol/L potassium dihydrogen phosphate buffer (pH value is about 4.5), unknown impurities in the test sample coincide with the impurity J, the detection of the impurity J is interfered, and the chromatographic condition is not feasible. Indicating that the pH of mobile phase A affects the peak appearance of impurities J and P.

Example 6

Instrument: thermo Scientific U3000 high performance liquid chromatograph

Chromatographic column: horizon AQUA (250X 4.6mm,5 μm)

detection wavelength: 240nm

Flow rate: 1mL/min

Column temperature: 25 DEG C

Sample injection amount: 100 mu L

A diluent: 0.1% Vc aqueous solution

control solution: precisely measuring a proper amount of stock solution of the impurity J and the impurity P, and diluting the stock solution with a diluent to prepare a solution with about 0.75 mug of each impurity J and each impurity P in 1mL serving as a reference substance solution;

sample solution: weighing appropriate amount of phloroglucinol, adding diluent to dissolve and dilute to obtain solution containing about 10mg per 1 mL;

Results: the chromatogram of the sample solution is shown in fig. 10, the chromatogram of the system applicability solution is shown in fig. 11, unknown impurities in the sample solution do not interfere with detection of the impurity J, P when the wavelength is 240nm, the impurities P and J in the system applicability solution sequentially come out of peaks, the specificity is good, and the impurity J, P is not detected in the sample solution.

Example 7

Instrument: thermo Scientific U3000 high performance liquid chromatograph

Chromatographic column: horizon AQUA (250X 4.6mm,5 μm)

detection wavelength: 210nm of

Flow rate: 1mL/min

Column temperature: 25 DEG C

Sample injection amount: 10 mu L

A diluent: 0.1% Vc aqueous solution

and (3) measuring: 10 mu L of the sample solution and 10 mu L of the system applicability solution are respectively injected into a high performance liquid chromatograph, and chromatograms are recorded.

Results: the chromatogram of the sample solution is shown in fig. 12, the chromatogram of the system applicability solution is shown in fig. 13, when the sample injection amount is 10 mu L, unknown impurities in the sample do not interfere with detection of the impurity J, P, the impurities P and J in the system applicability solution sequentially come out of peaks, the specificity is good, and the impurity J, P is not detected in the sample solution.

Example 8

Instrument: thermo Scientific U3000 high performance liquid chromatograph

Chromatographic column: horizon AQUA (250X 4.6mm,5 μm)

detection wavelength: 210nm of

Flow rate: 1.1mL/min

Column temperature: 25 DEG C

Sample injection amount: 100 mu L

A diluent: 0.1% Vc aqueous solution

Results: the chromatogram of the sample solution is shown in fig. 14, the chromatogram of the system applicability solution is shown in fig. 15, when the flow rate is 1.1mL/min, unknown impurities in the sample do not interfere with detection of the impurity J, P, the impurities P and J in the system applicability solution sequentially come out of peaks, the specificity is good, and the impurity J, P is not detected in the sample solution.

Example 9

Instrument: thermo Scientific U3000 high performance liquid chromatograph

Chromatographic column: horizon AQUA (250X 4.6mm,5 μm)

detection wavelength: 210nm of

Flow rate: 0.9mL/min

Column temperature: 25 DEG C

Sample injection amount: 100 mu L

A diluent: 0.1% Vc aqueous solution

Results: the chromatogram of the sample solution is shown in fig. 16, the chromatogram of the system applicability solution is shown in fig. 17, when the flow rate is 0.9mL/min, unknown impurities in the sample do not interfere with detection of the impurity J, P, the impurities P and J in the system applicability solution sequentially show peaks, the specificity is good, and the impurity J, P is not detected in the sample solution.

Example 10

Instrument: thermo Scientific U3000 high performance liquid chromatograph

Chromatographic column: horizon AQUA (250X 4.6mm,5 μm)

detection wavelength: 210nm of

Flow rate: 1.0mL/min

Column temperature: 25 DEG C

Sample injection amount: 100 mu L

A diluent: 0.05% Vc aqueous solution

Results: the chromatogram of the sample solution is shown in fig. 18, the chromatogram of the system applicability solution is shown in fig. 19, when the diluent is 0.05% Vc aqueous solution, unknown impurities in the sample do not interfere with detection of the impurity J, P, the impurities P and J in the system applicability solution sequentially come out of peaks, the specificity is good, and the impurity J, P is not detected in the sample solution.

Example 11

Instrument: thermo Scientific U3000 high performance liquid chromatograph

Chromatographic column: horizon AQUA (250X 4.6mm,5 μm)

detection wavelength: 210nm of

Flow rate: 1.0mL/min

Column temperature: 25 DEG C

Sample injection amount: 100 mu L

A diluent: 0.5% Vc aqueous solution

Results: the chromatogram of the sample solution is shown in fig. 20, the chromatogram of the system applicability solution is shown in fig. 21, when the diluent is 0.5% Vc aqueous solution, unknown impurities in the sample do not interfere with detection of the impurity J, P, the impurities P and J in the system applicability solution sequentially come out of peaks, the specificity is good, and the impurity J, P is not detected in the sample solution.

Example 12

Instrument: thermo Scientific U3000 high performance liquid chromatograph

Chromatographic column: horizon AQUA (250X 4.6mm,5 μm)

detection wavelength: 210nm of

Flow rate: 1.0mL/min

Column temperature: 35 DEG C

Sample injection amount: 100 mu L

A diluent: 0.1% Vc aqueous solution

Results: the chromatogram of the sample solution is shown in fig. 22, the chromatogram of the system applicability solution is shown in fig. 23, when the column temperature is 35 ℃, unknown impurities in the sample do not interfere with detection of the impurity J, P, the impurities P and J in the system applicability solution sequentially come out of peaks, the specificity is good, and the impurity J, P is not detected in the sample solution.

Example 13

Instrument: thermo Scientific U3000 high performance liquid chromatograph

Chromatographic column: horizon AQUA (250X 4.6mm,5 μm)

Mobile phase: 12mmol/L dipotassium hydrogen phosphate buffer (pH value is adjusted to 4.8 by phosphoric acid) is taken as a mobile phase A; methanol as mobile phase B, linear gradient elution was performed as follows:

detection wavelength: 210nm of

Flow rate: 1.0mL/min

Column temperature: 25 DEG C

Sample injection amount: 100 mu L

A diluent: 0.1% Vc aqueous solution

Results: the chromatogram of the sample solution is shown in fig. 24, the chromatogram of the system applicability solution is shown in fig. 25, when the salt concentration of the mobile phase A is 12mmol/L, unknown impurities in the sample do not interfere with detection of the impurity J, P, the impurities P and J in the system applicability solution sequentially come out of peaks, the specificity is good, and the impurity J, P is not detected in the sample solution.

Example 14

Instrument: thermo Scientific U3000 high performance liquid chromatograph

Chromatographic column: horizon AQUA (250X 4.6mm,5 μm)

Mobile phase: 8mmol/L dipotassium hydrogen phosphate buffer (pH value is adjusted to 4.8 by phosphoric acid) is taken as a mobile phase A; methanol as mobile phase B, linear gradient elution was performed as follows:

detection wavelength: 210nm of

Flow rate: 1.0mL/min

Column temperature: 25 DEG C

Sample injection amount: 100 mu L

A diluent: 0.1% Vc aqueous solution

Results: the chromatogram of the sample solution is shown in fig. 26, the chromatogram of the system applicability solution is shown in fig. 27, when the salt concentration of the mobile phase A is 8mmol/L, unknown impurities in the sample do not interfere with detection of the impurity J, P, the impurities P and J in the system applicability solution sequentially come out of peaks, the specificity is good, and the impurity J, P is not detected in the sample solution.

Example 15

Instrument: thermo Scientific U3000 high performance liquid chromatograph

Chromatographic column: horizon AQUA (250X 4.6mm,5 μm)

Mobile phase: 10mmol/L dipotassium hydrogen phosphate buffer (pH value is adjusted to 4.9 by phosphoric acid) is taken as a mobile phase A; methanol as mobile phase B, linear gradient elution was performed as follows:

detection wavelength: 210nm of

Flow rate: 1.0mL/min

Column temperature: 25 DEG C

Sample injection amount: 100 mu L

A diluent: 0.1% Vc aqueous solution

Results: the chromatogram of the sample solution is shown in fig. 28, the chromatogram of the system applicability solution is shown in fig. 29, when the pH of the mobile phase A is 4.9, unknown impurities in the sample do not interfere with detection of the impurity J, P, the impurities P and J in the system applicability solution sequentially show peaks, the specificity is good, and the impurity J, P is not detected in the sample solution.

Example 16

Instrument: thermo Scientific U3000 high performance liquid chromatograph

Chromatographic column: horizon AQUA (250X 4.6mm,5 μm)

Mobile phase: 10mmol/L dipotassium hydrogen phosphate buffer (pH value is adjusted to 4.7 by phosphoric acid) is taken as a mobile phase A; methanol as mobile phase B, linear gradient elution was performed as follows:

detection wavelength: 210nm of

Flow rate: 1.0mL/min

Column temperature: 25 DEG C

Sample injection amount: 100 mu L

A diluent: 0.1% Vc aqueous solution

Results: the chromatogram of the sample solution is shown in fig. 30, the chromatogram of the system applicability solution is shown in fig. 31, when the pH of the mobile phase A is 4.7, unknown impurities in the sample do not interfere with detection of the impurity J, P, the impurities P and J in the system applicability solution sequentially come out of peaks, the specificity is good, and the impurity J, P is not detected in the sample solution.

Claims

1. The high performance liquid chromatography analysis method for detecting the residue of the aromatic amine impurity J and the impurity P in the phloroglucinol is characterized by comprising the following steps of:

(1) Setting chromatographic conditions: adopting a reversed-phase C18 column, taking dipotassium hydrogen phosphate aqueous solution as a mobile phase A, taking methanol as a mobile phase B, setting column temperature, detection wavelength and mobile phase speed, and performing gradient elution;

(3) And (3) detection: taking the prepared phloroglucinol solution, injecting the phloroglucinol solution into a high performance liquid chromatograph, and recording a chromatogram;

wherein, the gradient elution adopts the following modes:

，

the structures of the impurity J and the impurity P are as follows:

。

2. the method of claim 1, wherein the reversed-phase C18 column in step (1) is selected from the group consisting of horizons AQUA, 250 x 4.6mm,5 μm.

3. The method for high performance liquid chromatography for detecting residues of aromatic amine impurities J and P in phloroglucinol according to claim 1, wherein the mobile phase A in the step (1) is 8-12 mmol/L dipotassium hydrogen phosphate aqueous solution, preferably 10mmol/L, and the pH is adjusted to 4.7-4.9 by phosphoric acid.

4. The method for high performance liquid chromatography for detecting residue of aromatic amine impurity J and impurity P in phloroglucinol according to claim 1, wherein the mobile phase A in the step (1) is 10mmol/L dipotassium hydrogen phosphate aqueous solution, preferably 10mmol/L, and the pH is adjusted to 4.8 with phosphoric acid.

5. The method for high performance liquid chromatography for detecting residue of aromatic amine impurity J and impurity P in phloroglucinol according to claim 1, wherein the column Wen Xuanzi to 35 ℃ in the step (1).

6. The method for high performance liquid chromatography for detecting residues of aromatic amine impurities J and P in phloroglucinol according to claim 1, wherein the detection wavelength in the step (1) is selected from 210-240 nm, preferably 210nm.

7. The method for high performance liquid chromatography for detecting residue of aromatic amine impurity J and impurity P in phloroglucinol according to claim 1, wherein the mobile phase velocity in step (1) is selected from 0.9-1.1 mL/min.

8. The method for high performance liquid chromatography for detecting residue of aromatic amine impurity J and impurity P in phloroglucinol according to claim 1, wherein the mobile phase velocity is selected from 1.0mL/min.

9. The method for high performance liquid chromatography for detecting residue of aromatic amine impurity J and impurity P in phloroglucinol according to claim 1, wherein the diluent in the step (2) is 0.05-0.5% (w/v) Vc solution, preferably 0.1% Vc aqueous solution.