CN112526017A

CN112526017A - Method for detecting contents of various impurities in amimetu-yusu compound preparation by using HPLC (high performance liquid chromatography) method

Info

Publication number: CN112526017A
Application number: CN202011328664.2A
Authority: CN
Inventors: 何德中; 杜鸿儒; 曲韵智; 钟茂团; 钟小莉; 刘亚
Original assignee: SICHUAN FENGCHUN PHARMACEUTICAL CO LTD
Current assignee: SICHUAN FENGCHUN PHARMACEUTICAL CO LTD
Priority date: 2020-11-24
Filing date: 2020-11-24
Publication date: 2021-03-19

Abstract

The invention discloses a method for detecting contents of various impurities in a compound preparation of amimetu, which comprises the following steps of: performing gradient elution by using octadecylsilane chemically bonded silica as a filling agent, phosphate buffer solution-methanol-acetonitrile as a mobile phase A and phosphate buffer solution-methanol-acetonitrile as a mobile phase B at a flow rate of 0.8-1.5 ml per minute, a column temperature of 25-35 ℃ and a detection wavelength of 217-225 nm. The method can simultaneously detect the content of 13 impurities in the amimexican compound preparation, has the advantages of simple operation, quick analysis, good repeatability and good specificity, improves the efficiency and saves the cost. By adopting the mobile phase and the gradient elution condition, the peak shape is improved, the separation degree is increased, the tailing phenomenon of individual components is effectively improved, the mobile phase has good tolerance to different chromatographic columns, and all components can be completely separated with good peak shape.

Description

Method for detecting contents of various impurities in amimetu-yusu compound preparation by using HPLC (high performance liquid chromatography) method

Technical Field

The invention relates to the technical field of pharmaceutical analysis, in particular to a method for detecting the content of various impurities in a compound preparation of ametocin by using an HPLC method.

Background

The amanitol compound preparation is composed of active ingredients of acetaminophen, dextromethorphan hydrobromide, guaiacol glyceryl ether and phenylephrine hydrochloride, and a certain amount of inactive ingredients such as auxiliary materials of cosolvent, flavoring agent, PH regulator and the like, is a chemical medicine compound preparation, is an oral solution preparation which is more suitable for children to take, and has the functions of treating and relieving the symptoms of common cold and influenza.

Any substance that affects the purity of the drug is collectively referred to as an impurity. Chemical drugs inevitably bring more or less impurities to raw materials in the synthesis process, and some impurities are also generated in the preparation process. The existence of impurities affects the curative effect of the medicine on the one hand, and possibly increases the adverse reaction of the medicine on the other hand, and certain impurities can interact with the medicine to affect the safety of the medicine and even generate toxic effect. Therefore, research and development of a control and detection method of impurities have important significance for controlling the quality of the medicine and ensuring the clinical curative effect of the medicine. In particular, for children's medication, there should be a higher demand for controlling impurities, which is beneficial to improve the safety and reliability of children's medication.

Some researches have been made on impurity control of similar drugs, but general impurity detection is to measure the content of known impurities with relatively high content or to perform limited detection of unknown impurities by using a principal component self-control method. The former method needs to be carried out one by one, but for compound preparations with extremely many components, if the content of each component is detected by the method, the method is not feasible, and even if the method is barely feasible, the problems of manpower and material resources waste, resource waste, environmental pollution, long detection period and the like exist. In the latter case, although the total amount of impurities can be controlled by the principal component self-control method, since each impurity is not controlled individually, individual impurities having particularly high biological activity and not high content are not controlled effectively, which may have serious consequences. If a plurality of components can be detected at one time, the problems are effectively solved. Some experiments have also been performed in relation to methods for detecting multiple impurities at one time.

Methods for simultaneously detecting a plurality of impurities also exist in the prior art, but the number of detections is small and the accuracy is low, such as: the application number is CN202010248908.X, a method for measuring related substances of a pharmaceutical preparation containing acetaminophen, dextromethorphan hydrobromide and phenylephrine hydrochloride, and provides a method for detecting various impurities at one time. However, this method has some problems, such as it does not detect acetaminophen as an impurity, and dextromethorphan hydrobromide and phenylephrine hydrochloride as impurities, which are only easily controlled. Related substances in dextromethorphan hydrobromide guaiacol glyceryl ether granules with the application number of CN201610650073.4 and an analysis and detection method thereof also provide a method for detecting various impurities at one time. However, this method has some disadvantages, such as that triethylamine is added to the mobile phase in the high performance liquid detection method, and the specification states that "mobile phase: a is acetonitrile and B is an aqueous solution (pH adjusted to 7.8 with triethylamine) ", which has a certain problem. Triethylamine, N-diethylethylamine, has the chemical properties of tertiary amine, and the aqueous solution is alkaline and reacts with alkyl halide to produce quaternary ammonium salt, which is unstable to oxidant. The chromatographic column of high performance liquid is mostly the packed column of octadecylsilane chemically bonded silica, and triethylamine easily damages the post, leads to detecting impurity's the degree of accuracy lower.

Disclosure of Invention

The invention aims to provide a method for detecting contents of various impurities in a compound preparation of amikacin by an HPLC (high performance liquid chromatography) method, and solves the problems of small measurement quantity and low accuracy caused by the existing method for measuring the impurities.

The invention is realized by the following technical scheme:

a method for detecting the content of various impurities in the amimexican compound preparation by an HPLC method comprises the following chromatographic column conditions:

performing gradient elution by using octadecylsilane chemically bonded silica as a filling agent, phosphate buffer solution-methanol-acetonitrile as a mobile phase A and phosphate buffer solution-methanol-acetonitrile as a mobile phase B at a flow rate of 0.8-1.2 ml per minute, a column temperature of 25-35 ℃ and a detection wavelength of 217-225 nm.

Those skilled in the art are aware of:

the type of the chromatographic column, the composition of the mobile phase and the flow rate all can significantly influence the retention time, the separation degree, the tailing factors and the like related to the HPLC method, and influence the accuracy of the detection results of main components and impurities.

The applicant found through experiments that:

by adopting the specific HPLC conditions, the peak emergence time between each known impurity and the main drug in an HPLC map has no obvious interference, and particularly has better separation degree on each impurity peak to be detected, and can simultaneously detect 13 impurities: p-chlorophenylacetamide, p-aminophenol, guaiacol glyceryl ether impurity A, guaiacol glyceryl ether impurity B, guaiacol glyceryl ether impurity C, guaiacol glyceryl ether impurity D, phenylephrine hydrochloride impurity A, phenylephrine hydrochloride impurity C, phenylephrine hydrochloride impurity D, phenylephrine hydrochloride impurity E, dextromethorphan hydrobromide impurity A, dextromethorphan hydrobromide impurity B, and dextromethorphan hydrobromide impurity C.

Further, the volume ratio of the phosphate buffer solution to the methanol to the acetonitrile in the mobile phase A is 89-91: 2.8-3.2: 6.8-7.2.

Further, the volume ratio of the phosphate buffer solution, methanol and acetonitrile in the mobile phase a was 90:3: 7.

Furthermore, the volume ratio of the phosphate buffer solution to the methanol to the acetonitrile in the mobile phase B is 19-28: 26-28: 31-35.

Further, the volume ratio of the phosphate buffer solution, methanol and acetonitrile in the mobile phase B was 25:33.75: 41.25.

Further, the flow rate was 1.0ml per minute, the column temperature was 30 ℃ and the detection wavelength was 220 nm.

Further, the gradient elution conditions were:

0-18 min, 100-80% of mobile phase A and 0-20% of mobile phase B; 42-55 minutes, 80-45% of mobile phase A and 20-55% of mobile phase B; at 60-75 min, the mobile phase A is 35-32%; 65-68% of mobile phase B; 75-77 min, 32-20% of mobile phase A and 65-80% of mobile phase B.

Further, the phosphate buffer solution is 30mmol/L sodium dihydrogen phosphate and 2.50g/L sodium octane sulfonate, and the pH is adjusted to 3.0 buffer solution by phosphoric acid.

Further, the method comprises the following steps:

s1, preparation of control solutions: using mobile phase A as solvent to respectively prepare solutions containing 0.000325mg of p-chlorophenylacetamide impurity, 0.000325mg of p-aminophenol impurity, A0.008mg of guaiacol glyceryl ether impurity, B0.008mg of guaiacol glyceryl ether impurity, C0.008mg of guaiacol glyceryl ether impurity, D0.008mg of guaiacol glyceryl ether impurity, A0.0005mg of phenylephrine hydrochloride impurity, C0.0005mg of phenylephrine hydrochloride impurity, D0.0005mg of phenylephrine hydrochloride impurity, E0.0005mg of phenylephrine hydrochloride impurity, A0.0008mg of dextromethorphan hydrobromide impurity, B0.0008mg of dextromethorphan hydrobromide impurity and C0.0008mg of dextromethorphan hydrobromide impurity in each 1ml of the solution, filtering, and taking the filtrate as a reference solution;

s2, preparation of a test solution: the mobile phase A is taken as a solvent to prepare the amimex compound preparation into a test solution containing 2ml of the amimex compound preparation in each 10 ml;

s3, detection: precisely sucking 20 μ l of each of the reference solution and the sample solution, continuously injecting sample 5 needles, and injecting into a liquid chromatograph to obtain chromatogram.

Further, the amimex compound preparation is an oral solution.

The specific formulation of the oral solution is as follows:

active ingredients: 350g of acetaminophen 280-one, 250g of guaiacol glyceryl ether 150-one, 4-6g of phenylephrine hydrochloride and 8-12g of dextromethorphan hydrobromide;

other auxiliary components: 1.5-2.5g stabilizer, 400.0-600.0g cosolvent, 95.0-110.0g flavoring agent 0.8-1.0g, thickening agent 2.5-3.2g, pH regulator 0.4-0.6g, complexing agent 1.9-2.1g, pigment 30.0-35.0mg, aromatic 3.0-5.0ml and preservative;

the solvent was added to a total volume of 1000 ml.

The stabilizer is propyl gallate, the cosolvent is a mixture of glycerol and propylene glycol, the flavoring agent is a mixture of sorbitol and sucralose, the thickening agent is xanthan gum, the pH regulator is a mixture of anhydrous citric acid and sodium citrate, the preservative is sodium benzoate, the complexing agent is disodium edetate, the flavoring agent is a mixture of grape essence and blueberry essence, the pigment is a mixture of allura red pigment and brilliant blue pigment, and the solvent is purified water.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. the method can simultaneously detect the content of 13 impurities in the amimexican compound preparation, has the advantages of simple operation, quick analysis, good repeatability and good specificity, improves the efficiency and saves the cost.

2. The method of the invention improves the accuracy of measurement by reasonably setting the chromatographic column conditions.

3. The mobile phase and the gradient elution condition of the invention improve the peak shape, increase the separation degree, effectively improve the tailing phenomenon of individual components, have good tolerance to different chromatographic columns, can completely separate each component, have good peak shape, and the response value of each component measured by a test sample is in a proper range.

4. The method does not adopt harmful solvents such as triethylamine and the like, and avoids the damage to the chromatographic column.

Drawings

The accompanying drawings, which are included to provide a further understanding of the embodiments of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principles of the invention. In the drawings:

FIG. 1 is a chromatogram from an example, wherein 1 is: guaifenesin B; 2 is as follows: p-aminophenol; 3 is as follows: guaifenesin; 4 is as follows: guaiacol; 5 is as follows: phenylephrine hydrochloride a; 6 is as follows: phenylephrine hydrochloride; 7 is as follows: sodium benzoate; 8 is as follows: phenylephrine hydrochloride C; 9 is as follows: propyl gallate; 10 is as follows: p-chlorophenyl acetamide; 11 is as follows: phenylephrine D hydrochloride; 12 is as follows: dextromethorphan hydrobromide B; 13 is as follows: phenylephrine E hydrochloride; 14 is as follows: dextromethorphan hydrobromide C; 15 is as follows: guaifenesin C; 16 is as follows: dextromethorphan hydrobromide; 17 is as follows: dextromethorphan hydrobromide a; 18 is as follows: guaifenesin D.

FIG. 2 is a chromatogram of example 2, wherein 5 is the guaifenesin and feed impurity peak; 8 is phenylephrine hydrochloride; 9 is phenylephrine hydrochloride impurity C; and 11 is a propyl gallate and propyl gallate raw material impurity peak.

FIG. 3 is a chromatogram of example 3, wherein 5 is the guaifenesin and feed impurity peak; 8 is phenylephrine hydrochloride; 9 is phenylephrine hydrochloride impurity C; and 11 is a propyl gallate and propyl gallate raw material impurity peak.

FIG. 4 is a chromatogram of example 4, wherein 11 is acetaminophen; 32 is phenylephrine hydrochloride impurity E and dextromethorphan hydrobromide impurity B; guaifenesin impurity C and dextromethorphan hydrobromide impurity C.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.

Example 1:

octadecylsilane chemically bonded silica was used as a filler (ACE Excel C18, 4.6 mm. times.250 mm, 5 μm), phosphate buffer solution-methanol-acetonitrile was used as a mobile phase A, phosphate buffer solution-methanol-acetonitrile was used as a mobile phase B, gradient elution was performed at a flow rate of 1.0ml per minute, a column temperature of 30 ℃ and a detection wavelength of 220 nm.

In this example, the volume ratio of phosphate buffer solution, methanol and acetonitrile in mobile phase a was 90:3:7, the volume ratio of phosphate buffer solution, methanol and acetonitrile in mobile phase B was 20:27:33, and the gradient elution conditions were:

0-18 min, 100-80% of mobile phase A and 0-20% of mobile phase B; 42-55 minutes, 80-45% of mobile phase A and 20-55% of mobile phase B; at 60-75 min, the mobile phase A is 35-32%; 65-68% of mobile phase B; 75-77 minutes, 32-20% of mobile phase A and 68-80% of mobile phase B;

as shown in table 1:

TABLE 1

Time (unit: minute)	Mobile phase A (unit: V/V)	Mobile phase B (unit: V/V)
			0.0	100	0
18.0	80	20
			42.0	80	20
55.0	45	60
			60.0	35	65
75.0	32	68
			77.0	20	80

The phosphate buffer solution is a buffer solution containing 30mmol/L sodium dihydrogen phosphate and 2.50g/L sodium octane sulfonate, and the pH value is adjusted to 3.0 by using phosphoric acid.

The detection process is as follows:

preparation of a test solution:

precisely measuring 2ml of a compound preparation, placing into a 10ml measuring flask, adding mobile phase A to dilute to scale, shaking, filtering, and collecting the filtrate as sample solution;

the formula of the liquid preparation of the ammonia Meiyusu is as follows:

active ingredients: 28-35g of acetaminophen, 15-25g of guaiacol glyceryl ether, 0.4-0.60g of phenylephrine hydrochloride and 0.8-1.2g of dextromethorphan hydrobromide;

other auxiliary components: 15-25g of stabilizer, 4000-6000g of cosolvent, 950-1100g of flavoring agent, 25-32g of thickening agent, 4-6g of pH regulator, 19-21g of complexing agent, 350mg of pigment 300-50 ml of aromatic and preservative;

the solvent was added to a total volume of 1000 ml.

Preparation of control solutions:

using mobile phase A as solvent to prepare solution containing about 0.000325mg of p-chlorophenylacetamide impurity, 0.000325mg of p-aminophenol impurity, A0.008mg of guaiacol glyceryl ether impurity, B0.008mg of guaiacol glyceryl ether impurity, C0.008mg of guaiacol glyceryl ether impurity, D0.008mg of guaiacol glyceryl ether impurity, A0.0005mg of phenylephrine hydrochloride impurity, C0.0005mg of phenylephrine hydrochloride impurity, D0.0005mg of phenylephrine hydrochloride impurity, E0.0005mg of phenylephrine hydrochloride impurity, A0.0008mg of dextromethorphan hydrobromide impurity, B0.0008mg of dextromethorphan hydrobromide impurity and C0.0008mg of dextromethorphan hydrobromide impurity in each 1ml, filtering, and taking filtrate as control solution;

the detection method comprises the following steps:

precisely sucking the reference solution and the sample solution respectively, continuously feeding 20 μ l, introducing 5 needles, injecting into a liquid chromatograph, calculating peak area according to external standard method, and recording chromatogram (see figure 1).

As can be seen from fig. 1, by using the HPLC conditions described in this embodiment, there is no significant interference in the HPLC profile at the peak emergence time between each known impurity and the main drug, and particularly, the HPLC profile has a better resolution for each impurity peak to be detected.

The above implementation was verified using a partial methodology study experiment:

1. system suitability test

Sample preparation:

the mobile phase A is taken as a solvent to prepare the compound which contains 6.5mg, 200.0 mu g, 4.0mg, 100 mu g, 400 mu g and 400 mu g of acetaminophen, dextromethorphan hydrobromide, guaiacol glyceryl ether, phenylephrine hydrochloride, sodium benzoate and propyl gallate in each 1ml, known impurities such as 0.000325mg of p-chlorophenylacetamide, 0.000325mg of p-aminophenol, 0.008mg of guaiacol glyceryl ether impurity, C0.008mg of guaiacol glyceryl ether impurity, D0.008mg of guaiacol glyceryl ether impurity, A0.0005mg of phenylephrine impurity hydrochloride, C0.0005mg of phenylephrine impurity hydrochloride, D0.0005mg of phenylephrine impurity hydrochloride, E0.0005mg of phenylephrine impurity hydrochloride, A0.0008mg of dextromethorphan impurity hydrobromide, B0.0008mg of dextromethorphan impurity hydrobromide and C0.0008mg of dextromethorphan impurity hydrobromide are solution, filtered, and a filtrate is taken, a sample injection 5 needle is continuously fed by 20 μ l, and the detection results are shown in Table 2:

TABLE 2

And (4) conclusion: RSD is less than 2 percent, and the design requirement is met.

2. And (3) stability test:

taking the liquid preparation of the amitocin prepared by the preparation method, preparing the liquid preparation according to a related substance method, measuring the related substance after 0, 12, 24, 48, 72 and 96 hours, and inspecting the stability of the solution.

And (4) conclusion: the area of the sample solution is basically unchanged within 12 hours, which indicates that the solution of related substances of the product is good in stability and suitable for routine analysis.

3. And (3) repeatability test:

taking the liquid preparation of the amitocin prepared according to the preparation method, preparing the liquid preparation according to a related substance method, measuring the related substance, and inspecting the repeatability of the method for measuring the related substance, wherein the result is shown in a table 3:

TABLE 3

Name (R)	RSD％	Name (R)	RSD％
				Para-aminophenol	3.67％	Phenylephrine hydrochloride C	1.26％
P-chlorobenzene acetamide	1.53％	Phenylephrine hydrochloride D	2.44％
				Guaiacol	0.44％	Phenylephrine hydrochloride E	1.93％
Guaifenesin B	1.73％	Dextromethorphan hydrobromide A	0.29％
				Guaifenesin C	0.49％	Dextromethorphan hydrobromide B	1.35％
Guaifenesin D	0.66％	Dextromethorphan hydrobromide C	0.58％
				Phenylephrine hydrochloride A	0.38％

Under the conditions of the liquid chromatography, the minimum detection limit is measured according to the response value three times of the noise of the instrument, the minimum quantification limit is measured according to the response value ten times of the noise of the instrument, the mobile phase is made into a proper concentration, and the results of the quantification limit and the detection line are respectively shown in tables 4 and 5:

TABLE 4

TABLE 5

Name (R)	Concentration (μ g/ml)	s/n	Name (R)	Concentration (μ g/ml)	s/n
						Para-aminophenol	0.033	5.34	Phenylephrine hydrochloride C	0.016	2.14
P-chlorobenzene acetamide	0.016	4.35	Phenylephrine hydrochloride D	0.013	2.64
						Guaiacol	0.01	3.13	Phenylephrine hydrochloride E	0.0075	2.85
Guaifenesin B	0.024	3.75	Dextromethorphan hydrobromide A	0.050	4.73
						Guaifenesin C	0.074	3.45	Dextromethorphan hydrobromide B	0.025	3.44
Guaifenesin D	0.075	2.93	Dextromethorphan hydrobromide C	0.075	3.84
						Phenylephrine hydrochloride A	0.025	3.23

High temperature, strong light, strong acid, strong base and oxidation are used to accelerate the degradation of the product, so as to investigate the specificity of the chromatographic condition.

Taking a compound preparation, respectively carrying out high temperature, strong light, strong acid, strong base and oxidation destruction tests according to the following methods, and then investigating the specificity of the method for measuring related substances of the product under the HPLC conditions.

4. Detection wavelength:

scanning all impurities and bulk drugs at the wavelength of 190-400nm to determine that all components have better absorption at 220 nm. Durability, results are shown in table 6:

TABLE 6

And (3) detection results:

RSD value: 1.56 parts of p-chlorophenylacetamide, 2.03 parts of p-aminophenol, 0.96 part of guaiacol glyceryl ether impurity A, 1.22 parts of guaiacol glyceryl ether impurity B, 0.34 part of guaiacol glyceryl ether impurity C, 2.76 parts of guaiacol glyceryl ether impurity D, 1.96 parts of phenylephrine hydrochloride impurity A, 0.25 part of phenylephrine hydrochloride impurity C, 1.89 parts of phenylephrine hydrochloride impurity D, 0.95 part of phenylephrine hydrochloride impurity E, 0.47 part of dextromethorphan hydrobromide impurity A, 1.64 parts of dextromethorphan hydrobromide impurity B and 1.28 parts of dextromethorphan hydrobromide impurity C, wherein all the impurities are less than 2%, and the research requirements are met.

Example 2:

the ratio of the mobile phase was different, and other conditions and detection methods were the same as those used in example 1, and detection was performed by the HPLC method.

The detection method comprises the following steps: in this example, the volume ratio of phosphate buffer solution, methanol and acetonitrile in mobile phase A was 89:2.8:7.2, the volume ratio of phosphate buffer solution, methanol and acetonitrile in mobile phase B was 19:26:31,

the results show that: the separation degree of the No. 5 peak guaifenesin and the raw material impurity peak is unqualified; the separation degree of the phenylephrine hydrochloride with the No. 8 peak and the impurity C of the phenylephrine hydrochloride with the No. 9 peak is unqualified; the separation degree of the 11-peak propyl gallate and the impurity peak of the propyl gallate raw material is unqualified, and is shown in figure 2.

Example 3:

the column temperature and the detection wavelength were different, and other conditions and detection methods were the same as those used in example 1, and detection was performed by the HPLC method.

In this example, the column temperature was 32 ℃ and the detection wavelength was 215 nm.

The results show that: no. 44 guaiacol glyceryl ether impurity C and dextromethorphan hydrobromide impurity C are not separated and completely coincide. See figure 3.

Example 4:

the elution conditions were different from those of example 1, and other conditions were the same, and HPLC was used for detection.

The elution conditions are shown in table 7:

TABLE 7

The results show that: peak 11 acetaminophen is not isolated; no. 32 peak phenylephrine hydrochloride impurity E and dextromethorphan hydrobromide impurity B are not separated; guaifenesin impurity C and dextromethorphan hydrobromide impurity C are not separated, and are shown in figure 4.

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. A method for detecting the content of various impurities in a meceutralin compound preparation by using an HPLC method is characterized in that the conditions of a chromatographic column are as follows:

2. The method for detecting the content of various impurities in the amimexican compound preparation by the HPLC method according to claim 1, wherein the volume ratio of the phosphate buffer solution, the methanol and the acetonitrile in the mobile phase A is 89-91: 2.8-3.2: 6.8-7.2.

3. The method for detecting the content of various impurities in the amimexican compound preparation by the HPLC method as claimed in claim 2, wherein the volume ratio of the phosphate buffer solution, methanol and acetonitrile in the mobile phase A is 90:3: 7.

4. The method for detecting the content of various impurities in the amimexican compound preparation by the HPLC method according to claim 1, wherein the volume ratio of the phosphate buffer solution, the methanol and the acetonitrile in the mobile phase B is 19-28: 26-28: 31-35.

5. The method for detecting the content of various impurities in the amitocin compound preparation by the HPLC method as claimed in claim 4, wherein the volume ratio of phosphate buffer solution, methanol and acetonitrile in the mobile phase B is 25:33.75: 41.25.

6. The method for detecting the contents of various impurities in the amitocin compound preparation by the HPLC method as claimed in claim 1, wherein the flow rate is 1.0ml per minute, the column temperature is 30 ℃, and the detection wavelength is 220 nm.

7. The method for detecting the content of various impurities in the ammetin compound preparation by the HPLC method according to claim 1, wherein the gradient elution conditions are as follows:

8. The method for detecting the content of various impurities in the amimexican compound preparation by the HPLC method as claimed in claim 1, wherein the phosphate buffer solution is 30mmol/L sodium dihydrogen phosphate and 2.50g/L sodium octane sulfonate, and the pH is adjusted to 3.0 by phosphoric acid.

9. The method for detecting the content of various impurities in the amimexican compound preparation by the HPLC method according to any one of claims 1 to 8, comprising the following steps:

10. The method for detecting the content of various impurities in the ammetin compound preparation by using the HPLC method according to claim 9, wherein the ammetin compound preparation is an oral solution.