CN115856135A

CN115856135A - Method for separating and measuring related substances in aximinide hydrochloride by using HPLC (high performance liquid chromatography)

Info

Publication number: CN115856135A
Application number: CN202211596297.3A
Authority: CN
Inventors: 陈海兵; 葛德培; 吴其华; 李强
Original assignee: Anhui Lianchuang Biological Medicine Co ltd
Current assignee: Anhui Lianchuang Biological Medicine Co ltd
Priority date: 2022-12-13
Filing date: 2022-12-13
Publication date: 2023-03-28

Abstract

The invention relates to a method for separating and measuring related substances in aximinide hydrochloride by using HPLC (high performance liquid chromatography), which comprises the following steps of: octadecylsilane chemically bonded silica is used as a filling agent; performing gradient elution by using 0.01-0.12% (V/V) phosphoric acid aqueous solution as a mobile phase A and acetonitrile as a mobile phase B; the flow rate is 0.8ml/min to 1.2ml/min; the column temperature is 30-40 ℃; the invention optimizes the detection result by considering the analysis column, the mobile phase, the gradient elution program, the flow rate and the comprehensive influence of the column temperature on the separation detection, can quickly and efficiently separate the impurity A, the impurity B, the impurity D, the impurity E, the impurity F, the impurity G, the impurity H and the impurity I in the aximinide hydrochloride under the same chromatographic condition, has high sensitivity, strong specificity, is quick, simple and convenient, is convenient to operate, can effectively control the quality of the aximinide hydrochloride, and is suitable for separating and determining related substances of the aximinide hydrochloride.

Description

Method for separating and measuring related substances in aximinide hydrochloride by using HPLC (high performance liquid chromatography)

Technical Field

The invention relates to the technical field of analytical chemistry, in particular to a method for separating and determining related substances in aximinide hydrochloride by using HPLC (high performance liquid chromatography).

Background

Aximinib (Asciminib, trade name SCEMBLIX), a STAMP inhibitor developed by nova pharmaceutical company, is a CML therapeutic agent that acts by specifically targeting to overcome mutations at the ATP-binding site of BCR-ABL 1. On 29 months 10 in 2021, aximinine received U.S. FDA approval for 2 different indications for the treatment of Chronic Myelogenous Leukemia (CML): (1) treatment of philadelphia chromosome positive chronic myelogenous leukemia (Ph + CML) adult patients in the Chronic Phase (CP) who previously received 2 or more Tyrosine Kinase Inhibitors (TKI); (2) the patient described above (Ph + CML-CP) carrying the T315I mutation. Currently, amirninib tablets are also marketed in japan with PMDA approval.

The aximinide hydrochloride is a raw material drug for producing the aximinide tablet, and the molecular formula of the aximinide hydrochloride is as follows: c ₂₀ H ₁₈ ClF ₂ N ₅ O ₃ HCl, molecular weight: 486.30.

the chemical name is as follows: n- [4- (chlorodifluoromethoxy) phenyl ] -6- [ (3R) -3-hydroxypyrolidin-1-yl ] -5- (1H-pyrazol-3-yl) pyridine-3-carboxamide hydrochloride. The structural formula is shown as the following formula (1):

the asimiib hydrochloride is prepared by taking 5-bromo-6-chloronicotinic acid (SM 1), 4- (chloro-difluoro-methoxy) -aniline (SM 2), (R) -3-hydroxypyrrolidine (SM 3) and 1- (2-tetrahydropyranyl) -1H-pyrazole-5-boronic acid pinacol ester (SM 4) as starting materials and synthesizing through multi-step reaction. The synthesis process of the aximinide hydrochloride comprises the following steps:

during the preparation process of aximicib hydrochloride, a plurality of impurities are generated by the residues of starting materials and synthetic intermediates, by-products of the synthetic process and other factors. The residual impurities of the starting material and the synthesis intermediate are A, B, C, D and E, F, G; IM2 reacts with SM4 to generate IM3 and pinacol (impurity J), in the reaction, IM2 is subjected to dechlorination reaction to generate a dechlorinated compound, and the dechlorinated compound participates in subsequent reaction to finally generate impurity H; carrying out hydrolysis reaction on the IM3 to generate aximinide hydrochloride and tetrahydropyran-2-ol (impurity K); the impurity K is combined with aximinide under the reaction condition to generate the impurity I. The SM3 structure contains 1 chiral carbon atom and is in an R configuration, and an enantiomer (S configuration) of the R configuration participates in subsequent reaction to finally generate an impurity L. The source and nomenclature of each impurity is as follows:

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impurities generated in the process of preparing the aximicine hydrochloride or related substances introduced in the process need to be strictly controlled in both raw material medicaments and preparations. Therefore, the analysis and detection of the impurities such as the starting materials, the intermediates and the byproducts in the table play an important role in the quality control of the final product, namely the acipimenib hydrochloride.

At present, the quality standards of aciminib (Asciminib) have not been recorded in the latest edition of the united states pharmacopeia USP and japanese pharmacopeia JP since they are soon on the market. In published documents or patents, no method for analyzing and detecting impurities in aximicib (Asciminib) has been found. Therefore, no analysis method which can be referred to exists in the product at the present stage, and the application and popularization of the product are hindered. Therefore, it is necessary to establish a stable and effective analysis and detection method with simple operation for the analysis and detection of impurities in aximicide hydrochloride.

Through research, in an ultraviolet scanning spectrum, acipimox hydrochloride has strong absorption in the wavelength range of 200nm-230nm and maximum absorption in the wavelength range of 285nm, and impurities C, J and K almost have no ultraviolet absorption in the wavelength range of 200nm-400nm, and the detection sensitivity of the impurities can not reach the requirement of quality control limit (the limit of single impurity in the related substances is not more than 0.10%), so that the related substance method of acipimox hydrochloride does not perform the quality control of the impurities C, J and K, and the impurities C, J and K are alternatively selected and controlled by a GC method, which is not described herein. The impurity L is an enantiomer of the aximinide hydrochloride, and the peak of the impurity L is completely coincided with the peak of the aximinide hydrochloride in the related substance chromatogram, so the quality control of the impurity L is not carried out in the related substance method of the aximinide hydrochloride in the invention, and the impurity L can be alternatively controlled by a normal phase HPLC method, which is not described again. The impurity structure and analysis of related substance control of the aximinide hydrochloride are as follows:

disclosure of Invention

In order to overcome the technical problems in the prior art, the inventors of the present invention have conducted extensive and intensive studies and, as a result, have provided a method for separating and measuring related substances in aximicine hydrochloride by HPLC, which has the advantages of rapidness, simplicity, high sensitivity and good resolution.

The technical scheme adopted by the invention is as follows:

a method for separating and measuring related substances in aximinide hydrochloride by using HPLC comprises the following steps:

octadecylsilane chemically bonded silica is used as a filling agent; performing gradient elution by using 0.01-0.12% (V/V) phosphoric acid aqueous solution as a mobile phase A and acetonitrile as a mobile phase B; the flow rate is 0.8ml/min to 1.2ml/min; the column temperature is 30-40 ℃; and detecting by adopting an ultraviolet detector, wherein the detection wavelength of the ultraviolet detector is 200nm-230 nm.

The filler is octadecylsilane bonded silica, preferably Agilent 5TC-C18 (2) 250 × 4.6mm chromatographic column and nanop Chromocore 120C185 μm 250 × 4.6mm chromatographic column.

The mobile phase A is 0.01-0.12% (V/V) phosphoric acid aqueous solution. Changes in the concentration of phosphoric acid mainly affect the degree of separation of impurity B from adjacent components; preferably 0.05% (V/V) phosphoric acid aqueous solution.

The mobile phase B is acetonitrile;

the conditions of the gradient elution are as follows:

time, minutes	Mobile phase A,% by volume	Mobile phase B, volume%
			0	90～70	10～30
25	55	45
			35	15	85
50	15	85

Preferably:

time in minutes	Mobile phase A,% by volume	Mobile phase B, volume%
			0	80	20
25	55	45
			35	15	85
50	15	85

The separation obtained is the best and the peak shape is the best.

The flow rate is 0.8ml/min to 1.2ml/min, preferably 1.0ml/min.

The column temperature is 30 ℃ to 40 ℃, preferably 35 ℃.

The detection wavelength of the ultraviolet detector is 200nm-230nm, and the product has strong absorption at 200nm-230nm and maximum absorption at 285nm after ultraviolet scanning; the impurity B and the impurity D have weak or no absorption at 285nm and are difficult to detect; the absorption intensity of each component is stronger closer to the ultraviolet end, and 210nm is preferable.

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

the method for separating and determining the aximinide hydrochloride related substances by using HPLC optimizes the detection result by considering the comprehensive influence of an analytical column, a mobile phase, a gradient elution program, flow velocity and column temperature on separation and detection, can quickly and efficiently separate the impurity A, the impurity B, the impurity D, the impurity E, the impurity F, the impurity G, the impurity H and the impurity I in the aximinide hydrochloride under the same chromatographic condition, has high sensitivity and specificity, is quick, simple and convenient, is convenient to operate, can effectively control the quality of the detection method, and is suitable for separating and determining the aximinide hydrochloride related substances.

Drawings

FIG. 1 is a chromatogram of a blank solution tested under the conditions of example 1 in accordance with the present invention;

FIG. 2 is a chromatogram of a system suitability solution tested under the conditions of example 1 in the present invention;

FIG. 3 is a chromatogram of a test solution tested under the conditions of example 1 in accordance with the present invention;

FIG. 4 is a chromatogram of a limiting quantitation solution detected under the conditions of example 1 in the present invention;

FIG. 5 is a chromatogram of a detection limiting solution detected under the conditions of example 1 in the present invention;

FIG. 6 is a chromatogram of a system suitability solution (mobile phase A:0.01% phosphoric acid aqueous solution) measured under the conditions of example 2 in the present invention;

FIG. 7 is a chromatogram of a system suitability solution tested under the conditions of example 2 in the present invention (mobile phase A:0.12% phosphoric acid aqueous solution)

FIG. 8 is a chromatogram of a system suitability solution (column temperature-30 ℃ C.) measured under the conditions of example 3 in the present invention;

FIG. 9 is a chromatogram of a system suitability solution (column temperature-40 ℃ C.) measured under the conditions of example 3 in the present invention;

FIG. 10 is a chromatogram of a system suitability solution (column flow rate-0.8 ml/min) tested under the conditions of example 4 in the present invention;

FIG. 11 is a chromatogram of a system suitability solution (column flow rate-1.2 ml/min) measured under the conditions of example 4 in the present invention

FIG. 12 is a chromatogram of a system suitability solution tested under the conditions of example 5 in the present invention (gradient initiation ratio: mobile phase A-mobile phase B (90;

FIG. 13 is a chromatogram of a system suitability solution tested under the conditions of example 5 in the present invention (initial ratio of gradient: mobile phase A-mobile phase B (70)

FIG. 14 is a chromatogram of a test solution (column: nanop Chromocore 120C185 μm 250 mm. Times.4.6 mm) tested under the conditions of example 6 in the present invention

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will aid those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any manner. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention. The reagents and instruments used are not indicated by manufacturers, and conventional products can be obtained commercially.

The aximicine hydrochloride and the impurity reference substance used in the embodiment of the invention are prepared by the inventor.

Example 1

The instrument and chromatographic conditions were as follows:

the instrument comprises the following steps: agilent 1260 high performance liquid chromatograph

A chromatographic column: agilent 5TC-C18 (2) 250 x 4.6mm

Mobile phase A:0.05% phosphoric acid aqueous solution

Mobile phase B: acetonitrile

Column temperature: 35 deg.C

Flow rate: 1.0ml/min

Detection wavelength: 210nm

Sample introduction amount: 10 μ l

The gradient elution procedure was:

TABLE 1 gradient elution procedure

Time in minutes	Mobile phase A, volume%	Mobile phase B, volume%
			0	80	20
25	55	45
			35	15	85
50	15	85

Solution preparation:

impurity reference stock solution: precisely weighing about 12.5mg of each of an impurity A reference substance, an impurity B reference substance, an impurity D reference substance, an impurity E reference substance, an impurity F reference substance, an impurity G reference substance, an impurity H reference substance and an impurity I reference substance, placing the reference substances into a same 25ml measuring flask, adding a diluent to dissolve and dilute the reference substances to a scale, and shaking up to obtain the reagent; the diluent is as follows: acetonitrile-water (50.

System applicability solution: precisely weighing about 50mg of aximinine hydrochloride working reference substance, placing into a 100ml measuring flask, precisely adding 1ml of impurity reference substance stock solution into the 100ml measuring flask, adding diluent to dilute to scale, and shaking up to obtain the final product.

Test solution: taking about 50mg of aximicin hydrochloride, precisely weighing, placing in a 100ml measuring flask, adding a diluent to dissolve and dilute to a scale, and shaking up.

Acerninib hydrochloride control stock solution: precisely weighing about 50mg of the acernib hydrochloride working reference substance, placing the acernib hydrochloride working reference substance into a 100ml measuring flask, adding a diluent to dissolve and dilute the acernib hydrochloride working reference substance to a scale, and shaking up the solution to obtain the acernib hydrochloride working reference substance.

Quantitative limiting solution: precisely measuring the impurity reference substance storage solution and the aximinine hydrochloride reference substance storage solution in measuring bottles of 1ml to 100ml respectively, adding a diluent to dilute to scales, shaking up, precisely measuring 1ml to 25ml, adding the diluent to dilute to scales, and shaking up to obtain the product.

Detection limiting solution: precisely measuring 5ml of the quantitative limiting solution, putting the quantitative limiting solution into a 10ml measuring flask, adding a diluent to dilute to a scale, and shaking up to obtain the product.

And (3) determination: respectively injecting blank solution (namely diluent), system applicability solution, sample solution, quantitative limiting solution and detection limiting solution into a high performance liquid chromatograph for detection, using octadecylsilane chemically bonded silica as a filler (the particle size is 5 mu m, the inner diameter of a column is 4.6mm, the length of a chromatographic column is 250 mm), detecting according to a gradient elution program in Table 1, and recording a chromatogram.

Chromatograms of the blank solution (i.e., diluent), the system applicability solution, the sample solution, the quantification limit solution, and the detection limit solution are shown in fig. 1, 2, 3, 4, and 5, respectively, and it can be seen that fig. 1 shows that the blank does not interfere with the inspection of impurities; FIG. 2 shows that the separation degree between each impurity and the aximinide hydrochloride is good, and the applicability map data of the aximinide hydrochloride system are shown in Table 2; FIG. 3 shows that the self-made asimilini hydrochloride sample has no impurity A, B, D, E, F and G, and the detected impurities H and I are below 0.10wt%; the other single impurities detected are all below 0.10wt%; FIG. 4 shows that the quantitative limits of acipimenib hydrochloride and impurities A, B, D, E, F, G, H and I are 0.04wt%, 0.04wt% and 0.04wt%, respectively; FIG. 5 shows that the detection limits of aximicide hydrochloride and impurity A, impurity B, impurity D, impurity E, impurity F, impurity G, impurity H and impurity I are 0.02wt%, 0.02wt% and 0.02wt%, respectively, below the limit of 0.10wt% for each impurity; the method has high detection sensitivity.

Table 2 applicability profiles of aximinide hydrochloride system in example 1

Sample(s)	Retention time (min)	Peak area	Degree of separation	Number of theoretical plates
					Impurity D	7.113	99.011	--	19879
Impurity H	14.476	160.211	36.931	86929
					Impurity B	17.238	197.287	11.301	55864
Impurity A	20.669	256.717	11.971	86927
					Acimetinib hydrochloride	21.718	11788.052	3.630	85565
Impurity I	28.740	200.674	23.479	146631
					Impurity G	31.608	128.915	12.325	592324
Impurity F	34.966	146.556	21.139	836165
					Impurity E	38.218	233.743	22.150	1188900

Example 2

The instrument and chromatographic conditions were as follows:

A chromatographic column: agilent 5TC-C18 (2) 250 x 4.6mm

Mobile phase A:0.01% phosphoric acid aqueous solution or 0.12% phosphoric acid aqueous solution

Mobile phase B: acetonitrile (ACN)

Column temperature: 35 deg.C

Flow rate: 1.0ml/min

Detection wavelength: 210nm

Sample introduction amount: 10 μ l

The gradient elution procedure was:

TABLE 3 gradient elution procedure

Solution preparation:

impurity control stock solution: accurately weighing about 12.5mg of each of an impurity A reference substance, an impurity B reference substance, an impurity D reference substance, an impurity E reference substance, an impurity F reference substance, an impurity G reference substance, an impurity H reference substance and an impurity I reference substance, placing the reference substances into a same 25ml measuring flask, adding a diluent to dissolve and dilute the reference substances to a scale, and shaking up to obtain the reagent; the diluent is as follows: acetonitrile-water (50.

And (3) determination: and (4) injecting the system applicability solution into a high performance liquid chromatograph, and recording the chromatogram. See fig. 6, 7.

The results show that under the chromatographic conditions described above, the baseline is stable and 9 components reach baseline separation.

When the concentration of phosphoric acid in the mobile phase A is reduced, the retention time of the impurity B is increased, when the concentration of phosphoric acid is increased, the retention time of the impurity B is reduced, and the retention time of other components is unchanged. When the content of phosphoric acid is less than 0.01%, the separation degree of the impurity B peak and the impurity A peak is less than 1.5, the peak type change of the impurity A peak is large, and the lower the content of phosphoric acid is, the worse the impurity A peak type is (the smaller the number value of theoretical plates is); when the phosphoric acid is 0%, the peak emergence sequence of the impurity B is changed, the peak emergence of the impurity B is behind the aximinini peak and the separation degree of the impurity B from the aximinini is less than 1.5, the impurity A is kept in a chromatographic column and is difficult to elute, and the base line is poor. When the content of phosphoric acid is 0.01%, the retention time of the impurity B is 19.945min, the separation degree of the impurity B from the impurity A is smaller than 1.810, and the separation degree of the impurity B from the impurity H is 22.985 (note that when the mobile phase A is 0.05% phosphoric acid aqueous solution, the separation degree of the impurity B from the impurity A is 11.971, and the separation degree of the impurity B from the impurity H is 11.301), the retention time of the impurity A is stable, the peak shape is good, and the retention time and the separation degree of other components have no obvious difference. With the increase of the concentration of phosphoric acid, the retention time of the impurity B is reduced and tends to be stable, when the content of phosphoric acid is 0.12%, the retention time of the impurity B is 16.536min, the separation degree of the impurity B and the impurity A is 14.596, the separation degree of the impurity B and the impurity H is 8.973, the retention time of the impurity A is stable, the peak pattern is better, and the retention time and the separation degree of other components have no obvious difference (in consideration of the damage influence of strong acidity on a chromatographic column, a higher phosphoric acid concentration experiment is not carried out); based on the above experiments, it can be concluded that: the mobile phase A can realize baseline separation of 9 substances in 0.01-0.12% phosphoric acid aqueous solution. The most preferable mobile phase A is 0.05 percent phosphoric acid aqueous solution for chromatographic separation by combining the factors of separation degree, chromatographic column, phosphoric acid concentration and the like.

Table 4 data table of applicability profile (different mobile phase a) of aximicin hydrochloride system in example 2

Example 3

The instrument and chromatographic conditions were as follows:

A chromatographic column: agilent 5TC-C18 (2) 250 x 4.6mm

Mobile phase A:0.05% phosphoric acid aqueous solution

Mobile phase B: acetonitrile

Column temperature: 30 ℃ or 40 DEG C

Flow rate: 1.0ml/min

Detection wavelength: 210nm

Sample introduction amount: 10 μ l

The gradient elution procedure was:

TABLE 5 gradient elution procedure

Time, minutes	Mobile phase A,% by volume	Mobile phase B,% by volume
			0	80	20
25	55	45
			35	15	85
50	15	85

Solution preparation:

And (3) determination: and (4) injecting the system applicability solution into a high performance liquid chromatograph, and recording the chromatogram. See fig. 8, 9.

The results show that under the chromatographic conditions, the base line is stable, the column temperature is increased, and the separation degree of each component is not obviously changed; the column temperature is reduced, the separation degree of the impurity A and the axirnib is obviously reduced, the separation degrees of other components are not obviously different, and the base line separation of 9 substances can be realized at the column temperature of 30-40 ℃. By combining the factors of the separation degree, the column temperature and the like, the chromatographic separation is carried out at the most preferable column temperature of 35 ℃.

Table 6 applicability profiles (different column temperatures) of aximinini hydrochloride system in example 3

Example 4

The instrument and chromatographic conditions were as follows:

A chromatographic column: agilent 5TC-C18 (2) 250X 4.6mm

Mobile phase A:0.05% phosphoric acid aqueous solution

Mobile phase B: acetonitrile

Column temperature: 35 deg.C

Flow rate: 0.8ml/min or 1.2ml/min

Detection wavelength: 210nm

Sample introduction amount: 10 μ l

The gradient elution procedure was:

TABLE 7 gradient elution procedure

Solution preparation:

And (3) determination: and (4) injecting the system applicability solution into a high performance liquid chromatograph, and recording the chromatogram. See fig. 10, 11.

The result shows that under the chromatographic conditions, the base line is stable, the retention time of each peak is slightly increased along with the reduction of the flow rate, the separation degree of the impurity A and the axirninib is obviously reduced, the separation degrees of other components are not obviously different, and the base line separation of 9 substances can be realized within 0.8ml/min to 1.2ml/min of the flow rate of the column. In the invention, the chromatographic separation is carried out at the most preferable column flow rate of 1.0ml/min by combining the factors of the separation degree, the column flow rate and the like.

Table 8 data table for applicability profile (different flow rates) of aximinini hydrochloride system in example 4

Example 5

The instrument and chromatographic conditions were as follows:

the instrument comprises the following steps: agilent 1260 high performance liquid chromatograph column: agilent 5TC-C18 (2) 250 x 4.6mm

Mobile phase A:0.05% phosphoric acid aqueous solution

Mobile phase B: acetonitrile (ACN)

Column temperature: 35 deg.C

Flow rate: 1.0ml/min

Detection wavelength: 210nm

Sample introduction amount: 20 μ l

The gradient elution procedure was:

TABLE 9 gradient elution procedure

Or

TABLE 10 gradient elution procedure

Time in minutes	Mobile phase A, volume%	Mobile phase B, volume%
			0	70	30
25	55	45
			35	15	85
50	15	85

Solution preparation:

impurity reference stock solution: accurately weighing about 12.5mg of each of an impurity A reference substance, an impurity B reference substance, an impurity D reference substance, an impurity E reference substance, an impurity F reference substance, an impurity G reference substance, an impurity H reference substance and an impurity I reference substance, placing the reference substances into a same 25ml measuring flask, adding a diluent to dissolve and dilute the reference substances to a scale, and shaking up to obtain the reagent; the diluent is as follows: acetonitrile-water (50.

And (3) determination: and (4) injecting the system applicability solution into a high performance liquid chromatograph, and recording the chromatogram. See fig. 12, 13.

The result shows that under the chromatographic conditions, the base line is stable, the proportion of the initial organic phase (mobile phase B) is increased, the retention time of each component before the peak appears in the chromatogram is obviously reduced, the separation degree of the impurity A and the aximinide hydrochloride is obviously reduced, and the separation degrees of other components have no obvious difference; the proportion of an initial organic phase (mobile phase B) is reduced, the retention time of each component before the peak appears in the chromatogram is obviously increased, the separation degree of the impurity A and the aximicanide hydrochloride is obviously reduced, and the separation degrees of other components are not obviously different; the invention can realize baseline separation of 9 substances in the initial ratio of mobile phase A to mobile phase B (90-70. Combining the factors of separation and flow equality, the most preferred initial ratio of the gradient elution procedure of the present invention is mobile phase a-mobile phase B (80.

Table 11 data table of applicability profile (different gradients) of aximinini hydrochloride system in example 5

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Example 6

The instrument and chromatographic conditions were as follows:

the instrument comprises the following steps: agilent 1260 high performance liquid chromatograph column: nanop chromacore 120C185 μm 250mm x 4.6mm mobile phase a:0.05% phosphoric acid aqueous solution

And (3) mobile phase B: acetonitrile

Column temperature: 35 deg.C

Flow rate: 1.0ml/min

Detection wavelength: 210nm

Sample introduction amount: 10 μ l

The gradient elution procedure was:

TABLE 12 gradient elution procedure

Solution preparation:

impurity control stock solution: precisely weighing about 12.5mg of each of an impurity A reference substance, an impurity B reference substance, an impurity D reference substance, an impurity E reference substance, an impurity F reference substance, an impurity G reference substance, an impurity H reference substance and an impurity I reference substance, placing the reference substances into a same 25ml measuring flask, adding a diluent to dissolve and dilute the reference substances to a scale, and shaking up to obtain the reagent; the diluent is as follows: acetonitrile-water (50.

System applicability solution: precisely weighing about 50mg of aximicine hydrochloride working reference substance, placing into a 100ml measuring flask, precisely adding 1ml of the impurity reference substance stock solution into the 100ml measuring flask, adding diluent to dilute to scale, and shaking up to obtain the product.

And (3) determination: and (4) injecting the system applicability solution into a high performance liquid chromatograph, and recording the chromatogram. See fig. 14.

The results show that under the chromatographic conditions, the baseline is stable, the peak order of each component is not changed, the retention time is slightly different, the separation degree of the impurity A and the axirninib is obviously improved, the separation degree of other components is not obviously different, and the invention can realize the baseline separation of 9 substances on a chromatographic column (nano chroma core 120C185 mu m 250mm multiplied by 4.6 mm).

Table 13 data table of applicability profile (different chromatographic columns) of aximinini hydrochloride system in example 6

The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention by those skilled in the art should fall within the protection scope defined by the claims of the present invention without departing from the spirit of the present invention.

Claims

1. A method for separating and measuring related substances in aximinide hydrochloride by using HPLC is characterized in that: the method comprises the following steps: octadecylsilane chemically bonded silica is used as a filling agent, 0.01-0.12% (V/V) phosphoric acid aqueous solution is used as a mobile phase A, acetonitrile is used as a mobile phase B for gradient elution, the flow rate is 0.8-1.2 ml/min, the column temperature is 30-40 ℃, an ultraviolet detector is used for detection, and the detection wavelength of the ultraviolet detector is 200-230 nm.

2. The method of claim 1, wherein the packing agent is octadecylsilane bonded silica, preferably Agilent 5TC-C18 (2) 250 x 4.6mm chromatography column and nano chromocore 120C185 μm 250 x 4.6mm chromatography column.

3. The method according to claim 1, wherein the mobile phase A is 0.01-0.12% (V/V) phosphoric acid aqueous solution; preferably 0.05% (V/V) phosphoric acid aqueous solution.

4. The method of claim 1, wherein the conditions of the gradient elution are:

time, minutes Mobile phase A,% by volume Mobile phase B,% by volume 0 90～70 10～30 25 55 45 35 15 85 50 15 85

Preferably:

5. The method according to claim 1, wherein the flow rate is between 0.8ml/min and 1.2ml/min, preferably 1.0ml/min.

6. The process according to claim 1, wherein the column temperature is from 30 ℃ to 40 ℃, preferably 35 ℃.

7. The method according to claim 1, wherein the detection wavelength of the ultraviolet detector is 200nm to 230nm, preferably 210nm.

8. The process according to claim 1, wherein the degree of separation between 9 components, e.g. acipimox hydrochloride, impurity a, impurity B, impurity D, impurity E, impurity F, impurity G, impurity H and impurity I, is > 1.5.

9. The method of claim 1, wherein the impurities of aximinide hydrochloride comprise impurity A, impurity B, impurity D, impurity E, impurity F, impurity G, impurity H and impurity I, and the specific structural formula is as follows:

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