CN117517545A

CN117517545A - Method for detecting trazodone hydrochloride related substances

Info

Publication number: CN117517545A
Application number: CN202311687434.9A
Authority: CN
Inventors: 曾令高; 韩娟; 余剑; 匡敏; 钟世利
Original assignee: Chongqing Ruien Pharmaceutical Co ltd
Current assignee: Chongqing Ruien Pharmaceutical Co ltd
Priority date: 2023-12-08
Filing date: 2023-12-08
Publication date: 2024-02-06

Abstract

The invention provides a method for detecting trazodone hydrochloride related substances, which adopts a high performance liquid chromatography to detect the trazodone hydrochloride related substances, wherein the chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 chromatographic column, the mobile phase A is a mixed solution of glacial acetic acid, triethylamine and water, and the mobile phase B is acetonitrile, and the gradient elution is carried out. The high performance liquid chromatography determination method provided by the invention can realize the baseline separation of E05-ZZD and the peak of the trazodone, and the column pressure of the chromatographic column is proper.

Description

Method for detecting trazodone hydrochloride related substances

Technical Field

The invention belongs to the technical field of analysis and test, and particularly relates to a method for detecting trazodone hydrochloride related substances.

Background

Trazodone hydrochloride (C) ₁₉ H ₂₂ ClN ₅ O.hcl, molecular weight 408.32;2- [3- [4- (3-chlorophenyl) -1-piperazinyl]Propyl-1, 2, 4-triazolo [4,3-a ]]The pyridin-3 (2H) -one hydrochloride is an antidepressant with sedative-hypnotic effect, and is widely used for patients with depression, insomnia and depression anxiety complicated with or accompanied with insomnia clinically. In addition, it has effects of resisting anxiety, improving nightmare disorder and sexual dysfunction. Trazodone hydrochloride has a structure shown in formula I:

the detection method of relevant substances of trazodone hydrochloride bulk drug has the harvest in USP43, BP2020 and EP11.0, and is specifically shown in table 1

Table 1 method for detecting related substances in legal standards

Note 1: the detection chromatographic column of the trazodone hydrochloride related substances is referred to in USP pharmacopoeia, and the adopted chromatographic column is an XBIdge C18 column.

TABLE 2 elution procedure

Time (min)	Mobile phase a (%)	Mobile phase B (%)
			0	80	20
12	32	68
			12.01	80	20
15	80	20

Impurity A, B, C, D, E, H, I, J, K, L, M was studied in trazodone hydrochloride EP11.0 mass standard; in order to accurately control the quality of the crude drug, the research is also carried out on known impurities listed in legal standards.

Wherein, specific information of the impurity A, B, C, D, E, H, I, J, K, L, M, S, P is shown in Table 3

TABLE 3 impurity Structure information Table

In the prior art, E05-ZZD in a system applicability solution of the trazodone hydrochloride related substance analysis method shows a peak at 6.035min, the separation degree between E05-ZZD and a main peak is 2.69, and the separation degree is more than 1.5; however, in the experimental investigation of the recovery rate, the recovery rate of E05-ZZD is more than 150%; the finding of the cause is that E05-ZZD is not separated from the base line of the trazodone peak, so that the integration is inaccurate. Meanwhile, the flow rate is 2.0ml/min, the inner diameter of the chromatographic column is 3.5 mu m, the column pressure of the chromatographic column is maintained at 210bar, and the pressure is high. Combining the two above points requires the redevelopment of the method for detecting trazodone hydrochloride related substances.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a method for detecting trazodone hydrochloride related substances, and the method for determining high performance liquid chromatography provided by the present invention can realize baseline separation of E05-ZZD and trazodone peaks, and the column pressure of the chromatographic column is suitable.

The invention provides a method for detecting trazodone hydrochloride related substances, which adopts a high performance liquid chromatography to detect the trazodone hydrochloride related substances, wherein the chromatographic conditions of the high performance liquid chromatography are as follows:

the chromatographic column is a C18 chromatographic column, the mobile phase A is a mixed solution of glacial acetic acid, triethylamine and water, and the mobile phase B is acetonitrile, and the gradient elution is carried out.

The preparation method preferably comprises the following steps:

diluting trazodone hydrochloride with a diluent to obtain a sample solution;

and detecting the sample solution by adopting a high performance liquid chromatography.

Preferably, the concentration of the sample solution is 0.8 to 1.5mg/ml.

Preferably, the preparation method further comprises the following steps:

preparing a reference substance solution and preparing a recovery rate solution:

diluting a mother solution of a related substance reference substance by using a diluent to obtain a reference substance solution, wherein the related substance reference substance comprises 2- [3- [4- (3-bromophenyl) piperazin-1-yl ] propyl ] -1,2, 4-triazole [4,3-a ] pyridin-3 (2H) -one hydrochloride;

mixing trazodone hydrochloride with a mother solution of a related substance reference substance, and diluting with a diluent to obtain a recovery rate solution;

and detecting the reference substance solution and the recovery rate solution by adopting a high performance liquid chromatography, and analyzing and detecting the trazodone hydrochloride related substances through a chromatography result.

Preferably, the diluent is selected from mixed solution of water and acetonitrile with the volume ratio of 85:15-75:25.

Preferably, the method comprises the steps of, the related substance reference substances also comprise 4- (3-chlorophenyl) -1- [3- (3-oxygen-2, 3-dihydro-1, 2, 4-triazolo [4,3-a ] pyridine-2-alkyl) propyl ] piperazine-N-oxide, 2- [3- (4-phenylpiperazin-1-yl) -propyl ] [1,2,4] triazolo [4, 3-alpha ] pyridin-3 (2H) -one hydrochloride, 2- [3- [4- (4-chlorophenyl) piperazin-1-yl ] propyl ] [1,2,4] triazolo [4, 3-alpha ] pyridin-3 (2H) -one, 2- [3- [4- (3-chloro-4-ethylphenyl) piperazin-1-yl ] propyl ] [1,2,4] triazolo [4,3-a ] pyridin-3 (2H) -one hydrochloride, 1' - (propane-1, 3-diyl) bis [4- (3-chlorophenyl) piperazine, 3- [4- (3-chlorophenyl) piperazin-1-yl ] propane, 2- [3- (3-chlorophenyl) piperazin-1-yl ] propyl ] [1,2, 4- [4- (3-chlorophenyl) piperazin-1, 3-alpha ] triazolo [4- (3-chlorophenyl) piperazin-1-yl ] hydrochloride, 2- [3- [4- (3-chloro-4-ethyl) piperazin-1-yl ] propyl ] [1, 3- [4, 3-alpha ] triazolo [1, 3-yl ] pyridin-1-yl ] hydrochloride 1- (3-chlorophenyl) piperazine hydrochloride, 1- [3- [4- (3-chlorophenyl) piperazin-1-yl ] propyl ] [1,2,4] triazolo [4,3-a ] pyridin-1-ium-3-alkoxide, 1- (3-chlorophenyl) -4- (3-isopropoxypropyl) piperazine, and 2- [3- [4- (2-chlorophenyl) piperazin-1-yl ] propyl ] [1,2,4] triazolo [4,3-a ] pyridin-3 (2H) -one.

Preferably, the C18 column is selected from ACE Excel 3C18-AR column, with a specification of 4.6X100 mm.

Preferably, the mobile phase a is formulated as follows:

5.0 to 7.0ml of glacial acetic acid, 13 to 15ml of triethylamine and 850ml of water are mixed, the pH value is regulated to 6.5 to 7.5 by using the triethylamine, and the water is added to 1L.

Preferably, the gradient elution is performed by the following steps:

0-20 min, mobile phase A:65%, mobile phase B:35%;

20-30 min, mobile phase A:65% -35%, mobile phase B:35% -65%;

30-40 min, mobile phase A:35%, mobile phase B:65%;

40-40.1 min, mobile phase A:35% -65%, mobile phase B:65% -35%;

40.1-45 min, mobile phase A:65%, mobile phase B:35%.

Preferably, the flow rate of the high performance liquid chromatography is 0.8-1.5 ml/min, the column temperature is 25-35 ℃, the detection wavelength is 254nm, the sample injection amount is 20 μl, and the column pressure of the chromatographic column is 120-165 bar.

Compared with the prior art, the invention provides a method for detecting trazodone hydrochloride related substances, which adopts a high performance liquid chromatography to detect the trazodone hydrochloride related substances, wherein the chromatographic conditions of the high performance liquid chromatography are as follows: the chromatographic column is a C18 chromatographic column, the mobile phase A is a mixed solution of glacial acetic acid, triethylamine and water, and the mobile phase B is acetonitrile, and the gradient elution is carried out. The high performance liquid chromatography determination method provided by the invention can realize the baseline separation of E05-ZZD and the peak of the trazodone, and the column pressure of the chromatographic column is proper.

Drawings

FIG. 1 is a liquid chromatogram of the mixed solution in example 1;

FIG. 2 is a liquid chromatogram of the sample solution in example 1;

FIG. 3 is a liquid chromatogram of the control solution of example 1;

FIG. 4 is a liquid chromatogram of the 25% recovery solution of example 1;

FIG. 5 is a liquid chromatogram of a 50% recovery solution in example 1;

FIG. 6 is a liquid chromatogram of a 100% recovery solution in example 1;

FIG. 7 is a liquid chromatogram of a 150% recovery solution in example 1;

FIG. 8 is a graph showing the pressure change of a 20% acetonitrile-water blank solution in example 2;

FIG. 9 is a liquid chromatogram of the 0.5mg/ml test solution of example 2;

FIG. 10 is a liquid chromatogram of the sample solution of example 2 at 0.8 mg/ml;

FIG. 11 is a liquid chromatogram of 1.0mg/ml of the test solution in example 2;

FIG. 12 is a liquid chromatogram of 1.2mg/ml of the test solution in example 2;

FIG. 13 is a liquid chromatogram of 1.5mg/ml of the test solution in example 2;

FIG. 14 is a liquid chromatogram of a 15% acetonitrile aqueous blank solution in example 2;

FIG. 15 is a liquid chromatogram of a 15% acetonitrile aqueous sample solution in example 2;

FIG. 16 is a liquid chromatogram of a 25% acetonitrile aqueous blank solution in example 2;

FIG. 17 is a liquid chromatogram of a 25% acetonitrile aqueous sample solution in example 2;

FIG. 18 is a liquid chromatogram of the mobile phase pH (6.5) space-time white solution of example 2

FIG. 19 is a liquid chromatogram of System suitability solution 1 at mobile phase pH (6.5) in example 2;

FIG. 20 is a liquid chromatogram of System suitability solution 2 at mobile phase pH (6.5) in example 2;

FIG. 21 is a liquid chromatogram of the background solution at mobile phase pH (6.5) in example 2;

FIG. 22 is a liquid chromatogram of the background control solution at mobile phase pH (6.5) of example 2;

FIG. 23 is a liquid chromatogram of the sample solution (1) for labeling at mobile phase pH (6.5) in example 2;

FIG. 24 is a liquid chromatogram of the control solution of example 2 with the addition of the labeled test solution (1) at pH (6.5) of the mobile phase;

FIG. 25 is a liquid chromatogram of the sample solution (2) for labeling at mobile phase pH (6.5) in example 2;

FIG. 26 is a liquid chromatogram of the control solution of example 2 with the addition of the labeled test solution (2) at the mobile phase pH (6.5);

FIG. 27 is a liquid chromatogram of the mobile phase pH (7.5) space-time white solution of example 2;

FIG. 28 is a liquid chromatogram of System suitability solution 1 at mobile phase pH (7.5) in example 2;

FIG. 29 is a liquid chromatogram of System suitability solution 2 at mobile phase pH (7.5) in example 2;

FIG. 30 is a liquid chromatogram of the background solution at mobile phase pH (7.5) in example 2;

FIG. 31 is a liquid chromatogram of the background control solution at mobile phase pH (7.5) of example 2;

FIG. 32 is a liquid chromatogram of the sample solution (1) for labeling at mobile phase pH (7.5) in example 2;

FIG. 33 is a liquid chromatogram of the control solution of example 2 with the addition of the labeled test solution (1) at pH (7.5) of the mobile phase;

FIG. 34 is a liquid chromatogram of the sample solution (2) for labeling at mobile phase pH (7.5) in example 2;

FIG. 35 is a liquid chromatogram of the control solution of example 2 with the addition of the labeled test solution (2) at pH (7.5) of the mobile phase;

FIG. 36 is a liquid chromatogram of the mobile phase medium glacial acetic acid to triethylamine volume ratio 5:13 space-time white solution of example 2;

FIG. 37 is a liquid chromatogram of System suitability solution 1 for a mobile phase medium glacial acetic acid to triethylamine volume ratio of 5:13 in example 2;

FIG. 38 is a liquid chromatogram of System suitability solution 2 for a mobile phase medium glacial acetic acid to triethylamine volume ratio of 5:13 in example 2;

FIG. 39 is a liquid chromatogram of the background solution at a mobile phase medium glacial acetic acid to triethylamine volume ratio of 5:13 in example 2;

FIG. 40 is a liquid chromatogram of a background control solution at a mobile phase medium glacial acetic acid to triethylamine volume ratio of 5:13 in example 2;

FIG. 41 is a liquid chromatogram of the sample solution (1) with a mobile phase medium glacial acetic acid to triethylamine volume ratio of 5:13 in example 2;

FIG. 42 is a liquid chromatogram of a control solution of the sample solution (1) with a mobile phase medium glacial acetic acid and triethylamine in a volume ratio of 5:13 in example 2;

FIG. 43 is a liquid chromatogram of sample solution (2) with a mobile phase medium glacial acetic acid to triethylamine volume ratio of 5:13 in example 2;

FIG. 44 is a liquid chromatogram of the control solution of example 2 with the addition of the standard sample solution (2) at a mobile phase medium glacial acetic acid to triethylamine volume ratio of 5:13;

FIG. 45 is a liquid chromatogram of a mobile phase medium glacial acetic acid to triethylamine volume ratio 7:15 space-time white solution of example 2;

FIG. 46 is a liquid chromatogram of System suitability solution 1 for a mobile phase medium glacial acetic acid to triethylamine volume ratio of 7:15 in example 2;

FIG. 47 is a liquid chromatogram of System suitability solution 2 for a mobile phase medium glacial acetic acid to triethylamine volume ratio of 7:15 in example 2;

FIG. 48 is a liquid chromatogram of the background solution at a mobile phase medium glacial acetic acid to triethylamine volume ratio of 7:15 in example 2;

FIG. 49 is a liquid chromatogram of a background control solution at a mobile phase medium glacial acetic acid to triethylamine volume ratio of 7:15 in example 2;

FIG. 50 is a liquid chromatogram of sample solution (1) with a mobile phase medium glacial acetic acid to triethylamine volume ratio of 7:15 in example 2;

FIG. 51 is a liquid chromatogram of a control solution of the sample solution (1) with a mobile phase medium glacial acetic acid and triethylamine in a volume ratio of 7:15 in example 2;

FIG. 52 is a liquid chromatogram of sample solution (2) with a mobile phase medium glacial acetic acid to triethylamine volume ratio of 7:15 in example 2;

FIG. 53 is a liquid chromatogram of the control solution of sample solution (2) with a mobile phase medium glacial acetic acid to triethylamine volume ratio of 7:15 in example 2;

FIG. 54 is a liquid chromatogram of the background solution at mobile phase pH (6.8) in example 3;

FIG. 55 is a liquid chromatogram of the mobile phase pH (6.8) of example 3 with the addition of a labeled test solution;

FIG. 56 is a liquid chromatogram of the mobile phase pH (6.8) space-time white solution of example 3;

FIG. 57 is a liquid chromatogram of the system applicability solution (1) at mobile phase pH (6.8) in example 3;

FIG. 58 is a liquid chromatogram of the background solution at mobile phase pH (7.2) in example 3;

FIG. 59 is a liquid chromatogram of the sample solution with labeling at mobile phase pH (7.2) in example 3;

FIG. 60 is a liquid chromatogram of the mobile phase pH (7.2) space-time white solution of example 3;

FIG. 61 is a liquid chromatogram of the system applicability solution (1) at mobile phase pH (7.2) in example 3;

FIG. 62 is a liquid chromatogram of the background solution at a column temperature of 25℃in example 3;

FIG. 63 is a liquid chromatogram of the sample solution of example 3 with a column temperature of 25 ℃;

FIG. 64 is a liquid chromatogram of the space-time white solution of example 3 at a column temperature of 25 ℃;

FIG. 65 is a liquid chromatogram of the system applicability solution (1) at a column temperature of 25℃in example 3;

FIG. 66 is a liquid chromatogram of the background solution at a column temperature of 35℃in example 3;

FIG. 67 is a liquid chromatogram of the sample solution of example 3 with a column temperature of 35 ℃;

FIG. 68 is a liquid chromatogram of the space-time white solution of example 3 at a column temperature of 35 ℃;

FIG. 69 is a liquid chromatogram of the system applicability solution (1) at a column temperature of 35℃in example 3;

FIG. 70 is a liquid chromatogram of the mixed solution of comparative example 1;

FIG. 71 is a liquid chromatogram of the mixed solution in comparative example 2;

FIG. 72 is a liquid chromatogram of a prior art blank solution;

FIG. 73 is a liquid chromatogram of a prior art test solution;

FIG. 74 is a liquid chromatogram of a prior art system applicability solution;

FIG. 75 is a liquid chromatogram of a background solution;

FIG. 76 is a liquid chromatogram of a quantitative limit recovery solution;

FIG. 77 is a liquid chromatogram of a 50% recovery solution;

FIG. 78 is a liquid chromatogram of a 100% recovery solution;

FIG. 79 is a liquid chromatogram of a 150% recovery solution.

Detailed Description

Specifically, the method for detecting trazodone hydrochloride related substances comprises the following steps:

diluting trazodone hydrochloride with a diluent to obtain a sample solution;

Wherein the diluent is selected from mixed solution of water and acetonitrile with the volume ratio of 85:15-75:25. Wherein the volume ratio of water to acetonitrile may be any value between 85:15, 80:20, 75:25, or 85:15 to 75:25. Therefore, the acetonitrile water solutions with volume concentrations of 15%, 20% and 25% can completely dissolve the test sample into 1mg/ml solution; and each diluent (blank solution) does not interfere with the test sample detection.

The concentration of the sample solution is 0.8-1.5 mg/ml, and can be any value between 0.8, 1, 1.2, 1.4, 1.5, or 0.8-1.5 mg/ml. When the concentration of the sample solution is 0.8mg/ml to 1.5mg/ml, the main peak shape is good.

In the invention, the preparation method and the trazodone hydrochloride related substance detection method also comprise the following steps:

in some embodiments of the present invention, the related substance reference substances also comprise 4- (3-chlorophenyl) -1- [3- (3-oxygen-2, 3-dihydro-1, 2, 4-triazolo [4,3-a ] pyridine-2-alkyl) propyl ] piperazine-N-oxide, 2- [3- (4-phenylpiperazin-1-yl) -propyl ] [1,2,4] triazolo [4, 3-alpha ] pyridin-3 (2H) -one hydrochloride, 2- [3- [4- (4-chlorophenyl) piperazin-1-yl ] propyl ] [1,2,4] triazolo [4, 3-alpha ] pyridin-3 (2H) -one, 2- [3- [4- (3-chloro-4-ethylphenyl) piperazin-1-yl ] propyl ] [1,2,4] triazolo [4,3-a ] pyridin-3 (2H) -one hydrochloride, 1' - (propane-1, 3-diyl) bis [4- (3-chlorophenyl) piperazine, 3- [4- (3-chlorophenyl) piperazin-1-yl ] propane, 2- [3- (3-chlorophenyl) piperazin-1-yl ] propyl ] [1,2, 4- [4- (3-chlorophenyl) piperazin-1, 3-alpha ] triazolo [4- (3-chlorophenyl) piperazin-1-yl ] hydrochloride, 2- [3- [4- (3-chloro-4-ethyl) piperazin-1-yl ] propyl ] [1, 3- [4, 3-alpha ] triazolo [1, 3-yl ] pyridin-1-yl ] hydrochloride 1- (3-chlorophenyl) piperazine hydrochloride, 1- [3- [4- (3-chlorophenyl) piperazin-1-yl ] propyl ] [1,2,4] triazolo [4,3-a ] pyridin-1-ium-3-alkoxide, 1- (3-chlorophenyl) -4- (3-isopropoxypropyl) piperazine, and 2- [3- [4- (2-chlorophenyl) piperazin-1-yl ] propyl ] [1,2,4] triazolo [4,3-a ] pyridin-3 (2H) -one.

The specific structural formulas and codes of the related substances are shown in Table 3.

The related substance reference substance mother liquor is prepared according to the following method:

wherein 2.5mg of each of E05-ZZZL, trazodone hydrochloride E05-ZZZB, E05-ZZD and E05-ZZJ is dissolved and diluted to 100ml by a diluent.

2.5mg of each of E05-ZZZA, E05-ZZI, E05-ZZM, E05-ZZS, E05-ZZZE, E05-ZZP and E05-ZZH is completely dissolved by adding 20ml of acetonitrile, and then diluted to 100ml by adding water, and the mixture is used as each mother liquor.

After mother liquor of the related substance reference substance is obtained, 1.0ml of each mother liquor is precisely measured, the mother liquor is placed in a 25ml measuring flask, diluted to a scale by a diluent, and the mother liquor is uniformly shaken to obtain reference substance solution.

Wherein the diluent is selected from mixed solution of water and acetonitrile with the volume ratio of 85:15-75:25. Wherein the volume ratio of water to acetonitrile may be any value between 85:15, 80:20, 75:25, or 85:15 to 75:25.

The 25% recovery solution, the 50% recovery solution, the 100% recovery solution and the 150% recovery solution were prepared, respectively.

Wherein, 25% recovery rate solution is precisely weighed 25mg of trazodone hydrochloride test sample, 0.25ml of each mother solution is added, and then diluent is added to 25ml.

The 50% recovery solution is precisely weighed 25mg of trazodone hydrochloride test sample, 0.5ml of each mother solution is added, and then the diluent is added to 25ml.

The 100% recovery solution is precisely weighed 25mg of trazodone hydrochloride test sample, 1.0ml of each mother solution is added, and then the diluent is added to 25ml.

The 150% recovery solution was precisely weighed 25mg of trazodone hydrochloride test sample, 1.5ml of each mother liquor was added, and then the diluent was added to 25ml.

And after the reference substance solution and the recovery rate solution are obtained, detecting the reference substance solution and the recovery rate solution by adopting a high performance liquid chromatography, and analyzing and detecting the trazodone hydrochloride related substances through a chromatographic result.

The chromatographic column of the high performance liquid chromatography is a C18 chromatographic column, wherein the C18 chromatographic column is selected from Inertsil ODS-3 chromatographic columns, and the specification is 4.6X250 mm and 5 mu m; or ACE Excel 3C18-AR chromatography column, specification 4.6X100 mm. Preferably, the C18 column is selected from ACE Excel 3C18-AR columns, with a specification of 4.6X100 mm. The ACE Excel 3C18-AR short column was selected, the detection time was short, the degree of separation between E05-ZZD and the trazodone peak was 3.20, and a baseline separation between E05-ZZD and the trazodone peak was achieved.

The mobile phase of the high performance liquid chromatography is as follows: mobile phase a is a mixed solution of glacial acetic acid, triethylamine and water, and mobile phase B is acetonitrile.

Wherein, the mobile phase A is prepared according to the following method:

after glacial acetic acid, triethylamine and water were mixed, the pH was adjusted with triethylamine and water was added to 1L.

Wherein, in the mobile phase A, the volume ratio of glacial acetic acid, triethylamine and the mobile phase A is 5.0-7.0: 13 to 15:1000. preferably 6.3:14: 1000. 5.0:13: 1000. 7.0:15:1000, or 5.0 to 7.0:13 to 15: any value between 1000.

In the present invention, the gradient elution procedure is:

0-20 min, mobile phase A:65%, mobile phase B:35%;

20-30 min, mobile phase A:65% -35%, mobile phase B:35% -65%;

30-40 min, mobile phase A:35%, mobile phase B:65%;

40-40.1 min, mobile phase A:35% -65%, mobile phase B:65% -35%;

40.1-45 min, mobile phase A:65%, mobile phase B:35%.

The flow rate of the high performance liquid chromatography is 0.8-1.5 ml/min, can be any value between 0.8, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 or 0.8-1.5 ml/min, the column temperature is 25-35 ℃, can be any value between 25, 30, 35 or 25-35 ℃, the detection wavelength is 254nm, and the sample injection amount is 20 mul.

The blank solution used in the invention does not interfere with the detection of the sample and each impurity; and the minimum value of the separation degree between the main peak and each impurity peak (except the coincident peak) in the system applicability solution is 1.5. In the sample labeling solution, the recovery rate of the impurity E05-ZZD is 90% -108%, the absolute difference between other single impurities and the measurement result under normal conditions is not more than 0.02%, and the total sum of the impurities is not more than 0.05%.

The detection method of the trazodone hydrochloride related substance can realize the baseline separation of E05-ZZD and the trazodone peak, and the chromatographic column pressure is 120-165 bar.

In order to further understand the present invention, the following examples are provided to illustrate the method for detecting trazodone hydrochloride related substances according to the present invention, and the scope of protection of the present invention is not limited by the following examples.

Instrument and equipment

TABLE 4 Table 4

Instrument name	Model number	Manufacturer' s
			High performance liquid chromatograph	Agilent1260	Agilent
One part per million electronic balance	MSA6.6-OCE-DM	Sidoris (Sidoris)
			Electronic balance of ten thousandth	XSE105DU	Metrehler tolidol
PH meter	S220-K	Metrehler tolidol

Reagent and reagent

TABLE 5

Control, sample information

TABLE 6

Example 1

(1) High performance liquid chromatography conditions

TABLE 7

TABLE 8

(2) Solution preparation

Blank solution (diluent): water-acetonitrile (80:20)

Accurately weighing 2.5mg of E05-A, E mg, E05-ZZZA, E05-ZZB, E05-ZZI, E05-ZZM, E05-ZZZS, E05-ZZZC, E05-ZZD, E05-ZZJ, E05-ZZE, E05-ZZP and E05-ZZH into each 100ml measuring flask, wherein the E05-A, E mg, E05-ZZB, E05-ZZD and E05-ZZJ diluents are dissolved and diluted to the scales. 20ml of acetonitrile is firstly added into E05-ZZZA, E05-ZZI, E05-ZZM, E05-ZZS, E05-ZZZC, E05-ZZZE, E05-ZZP and E05-ZZH to be completely dissolved, and then water is added to be diluted to the scale. As each mother liquor.

Each impurity stock solution: precisely weighing about 2.5mg of E05-ZZZA, E05-ZZZB, E05-ZZI, E05-ZZM, E05-ZZZS, E05-ZZZC, E05-ZZD, E05-ZZJ, E05-ZZE, E05-ZZP and E05-ZZH, respectively placing into 100ml measuring bottles, adding 20ml of acetonitrile for ultrasonic dissolution, diluting to a scale with water, and shaking uniformly.

System applicability solution (mixed solution in table 1): precisely weighing 25mg of trazodone hydrochloride reference substance, placing into a 25ml measuring flask, precisely adding 1ml of each impurity stock solution, adding diluent, dissolving, diluting to scale, and shaking.

Control solution: precisely measuring 1.0ml of each mother solution, placing into a 25ml measuring flask, diluting to scale with diluent, and shaking to obtain the final product. Referring to fig. 3, fig. 3 is a liquid chromatogram of the control solution in example 1.

Test solution: precisely weighing 25mg of the product, placing into a 25ml measuring flask, adding diluent to dissolve and dilute to scale, and shaking. Referring to fig. 2, fig. 2 is a liquid chromatogram of the sample solution in example 1.

Control solution: precisely measuring 1ml of the sample solution, placing in a 50ml measuring flask, diluting to a scale with a diluent, shaking uniformly, precisely measuring 1ml, placing in a 20ml measuring flask, diluting to the scale with the diluent, and shaking uniformly.

Sensitivity solution: 3ml of control solution is precisely measured, placed in a 10ml measuring flask, diluted to a scale with a diluent and shaken well.

Recovery rate solution: in each 25ml volumetric flask in the table below, about 25mg of the sample is precisely weighed first, each impurity mother liquor is added according to table 9, and then the diluent is added for dissolution and volume fixation to scale, and the solution is used as each recovery rate solution. Referring to fig. 4 to 7, fig. 4 is a liquid chromatogram of a 25% recovery solution in example 1, fig. 5 is a liquid chromatogram of a 50% recovery solution in example 1, fig. 6 is a liquid chromatogram of a 100% recovery solution in example 1, and fig. 7 is a liquid chromatogram of a 150% recovery solution in example 1.

TABLE 9

(3) Experimental results

Table 10

Example 2

1. Measurement method

Injecting a blank solution into a liquid chromatograph until a baseline is stable, sampling and detecting a sensitivity solution and a system applicability solution, wherein the signal to noise ratio of the peak height of the trazodone peak in the sensitivity solution is not lower than 10, and the separation degree of the trazodone peak and adjacent known impurities in the system applicability solution is not less than 1.5; the peak-out sequence of the system applicability solution is E05-ZZA, E05-ZZB, E05-ZZI, E05-ZZM, E05-ZZZS, E05-ZZZC, trazodone, E05-ZZD, E05-ZZJ, E05-ZZE, E05-ZZP and E05-ZZH in sequence, then the sample solution and the control solution are precisely measured and sampled, the chromatogram is recorded, and the content of each impurity is calculated by the control method of adding correction factors.

2. Calculation formula

Note that: E05-A and E05-ZZA are completely or partially overlapped, and the two impurities are combined and controlled;

single impurity% = impurity a F/a vs. self-control content 100%

Other impurities (total)% = (total peak-main peak a-1 a hetero 2- … … -a hetero n)/a content of 100% for self-control

Total impurity% = hetero 1% + hetero 2% + … … + hetero n% + other total impurities%

Wherein: a, impurity: peak area of single impurity

F: correction factor

And A pair: peak area of self-control

3. Standard requirements

The sum of E05-ZZA and E05-A is less than 0.1%; E05-ZZB is less than 0.1%; E05-ZZI is less than 0.10%; E05-ZZM is less than 0.10%; E05-ZZS is less than 0.1%; E05-ZZC is less than 0.1%; E05-ZZD is less than 0.1%; E05-ZZJ is less than 0.10% E05-ZZE is less than 0.1%; E05-ZZP is less than 0.1%; E05-ZZH is less than 0.1%; less than 0.10% of other single impurities; the total impurity content is not more than 0.3%.

4. Analysis method

4.1 sample concentration

On the basis of example 1, the test sample concentration was varied:

preparing a solution:

blank solution: water-acetonitrile (80:20), chromatogram see FIG. 8, FIG. 8 is a graph of pressure change of 20% acetonitrile water blank solution.

0.5mg/ml test solution: precisely weighing about 10mg of the sample E05-A, placing into a 20ml measuring flask, adding diluent to dissolve and dilute to scale, and shaking to obtain the final product.

0.8mg/ml test solution: precisely weighing about 16mg of the sample E05-A, placing into a 20ml measuring flask, adding diluent to dissolve and dilute to scale, and shaking to obtain the final product.

1.0mg/ml test solution: precisely weighing about 20mg of the sample E05-A, placing into a 20ml measuring flask, adding diluent to dissolve and dilute to scale, and shaking to obtain the final product.

1.2mg/ml test solution: precisely weighing about 24mg of the sample E05-A, placing into a 20ml measuring flask, adding diluent to dissolve and dilute to scale, and shaking to obtain the final product.

1.5mg/ml test solution: precisely weighing about 30mg of the sample E05-A, placing into a 20ml measuring flask, adding diluent to dissolve and dilute to scale, and shaking to obtain the final product.

Taking the solution sample injection detection and recording chromatograms, referring to fig. 9-13, fig. 9 is a liquid chromatogram of 0.5mg/ml sample solution in example 2; FIG. 10 is a liquid chromatogram of the sample solution of example 2 at 0.8 mg/ml; FIG. 11 is a liquid chromatogram of 1.0mg/ml of the test solution in example 2; FIG. 12 is a liquid chromatogram of 1.2mg/ml of the test solution in example 2; FIG. 13 is a liquid chromatogram of the sample solution of example 2 at 1.5mg/ml.

Test results:

see Table 11

TABLE 11 main peak statistics for each sample concentration solution

Concentration of test sample	Symmetry factor	Theoretical plate number	Whether or not to form a flat top
				0.5mg/ml	0.92	13741	Whether or not
0.8mg/ml	0.92	13645	Whether or not
				1.0mg/ml	0.92	13465	Whether or not
1.2mg/ml	0.94	13066	Whether or not
				1.5mg/ml	0.98	12037	Whether or not

Conclusion of the test:

the main peak shape of the sample under each concentration is good, and no flat top phenomenon exists; wherein, the quantitative limit concentration of partial impurities is about 0.20 mug/ml, calculated according to the limit of 0.1 percent, and the quantitative limit concentration is not more than 30 percent of the limit concentration, and the concentration of the solution of the test sample is more than 0.6mg/ml, so that the requirement of the quantitative limit concentration can be met. Therefore, the protection range of the patent concentration of the method is 0.8 mg/ml-1.5 mg/ml, the instrument operation pressure of the method is 120 bar-165 bar, and the pressure is in the acceptable range of the instrument and the chromatographic column.

4.2 Diluent ratio

Preparing a solution:

blank solution: water-acetonitrile (80:20), water-acetonitrile (85:15), water-acetonitrile (75:25)

Test solution: E05-A test pieces were dissolved in each of the above diluents to 1mg/ml solutions.

Taking the solution, sampling, detecting and recording a chromatogram. Referring to fig. 14 to 17, fig. 14 is a liquid chromatogram of a 15% acetonitrile water blank solution in example 2; FIG. 15 is a liquid chromatogram of a 15% acetonitrile aqueous sample solution in example 2; FIG. 16 is a liquid chromatogram of a 25% acetonitrile aqueous blank solution in example 2; FIG. 17 is a liquid chromatogram of a 25% acetonitrile aqueous sample solution in example 2.

Test results and conclusions:

the diluent (blank solution) can dissolve the test sample into a solution of 1mg/ml, and the blank solution does not interfere with the detection of the test sample, so the concentration range of the diluent is 15% -25% acetonitrile aqueous solution.

4.3 flow rate, column temperature, mobile phase pH, blank solution proportion, mobile phase medium proportion

Preparing a solution:

blank solution: water-acetonitrile (80:20)

Impurity stock solution: precisely weighing about 2.5mg of each of E05-ZZA, E05-ZZB, E05-ZZI, E05-ZZS, E05-ZZZC, E05-ZZD, E05-ZZJ, E05-ZZE, E05-ZP, E05-ZZH, E05-SM2 and E05-ZN, putting into a 100ml measuring flask, adding 20ml of acetonitrile, ultrasonically dissolving, diluting to a scale with water, and shaking uniformly.

E05-ZZM stock solution: accurately weighing E05-ZZM about 2.5mg, placing into a 100ml measuring flask, adding acetonitrile 20ml for ultrasonic dissolution, diluting with water to scale, and shaking.

E05-A, E05-ZZE mixing stock solution: precisely weighing about 2.5mg of each reference substance E05-A, E05-ZZE, placing into a 100ml measuring flask, adding diluent, dissolving, diluting to scale, and shaking.

System applicability solution (1): precisely measuring 1ml of impurity stock solution, placing 1.0ml of E05-ZZM stock solution into 25ml measuring flask containing 25mg of E05 reference substance, adding diluent, dissolving, diluting to scale, and shaking.

System applicability solution (2): precisely measuring 1.0ml of E05-A, E-ZZE mixed stock solution, placing into 25ml measuring flask containing about 25mg of E05 reference substance, adding diluent, dissolving, diluting to scale, and shaking to obtain

Background solution: precisely weighing 25mg of the sample, placing in a 25ml measuring flask, adding diluent, dissolving, diluting to scale, and shaking.

Adding a labeled test sample solution (1): precisely weighing 25mg of sample, placing in 25ml measuring flask, adding 1.0ml of impurity stock solution and E05-ZZM stock solution, respectively, adding diluent, dissolving, diluting to scale, and shaking.

Adding a labeled test sample solution (2): precisely weighing 25mg of the sample, placing in a 25ml measuring flask, adding 1.0ml of E05-A, E-ZZE mixed stock solution, adding diluent, dissolving, diluting to scale, and shaking.

Self-control solution: precisely measuring 1ml of sample solution (background solution and each labeled sample solution), placing into a 50ml measuring flask, diluting to scale with diluent, shaking, precisely measuring 1ml, placing into a 20ml measuring flask, diluting to scale with diluent, and shaking.

Taking blank solution, system applicability solution, background solution, adding a labeled sample solution and self-contrast solution, and carrying out sample injection detection and chromatogram recording. See fig. 18-53.

Investigation conditions:

table 12

Test results:

TABLE 13 durability results of substances on E05 Table-degree of separation

Table 14E05 durability results of substances Table-recovery/content (%)

Table 15E05 durability results of substances Table-recovery/content (%)

Conclusion of the test:

under each chromatographic condition, the separation degree of the separation degree between a main peak and adjacent known impurity peaks in the system applicability solution is not less than 1.5. In the sample adding standard solution, the recovery rate of each known impurity is within the range of 90% -108%, the absolute difference between the maximum single impurity and the measurement result under normal conditions is not more than 0.02%, and the total sum of all impurities is not more than 0.05%. The method is therefore applicable under chromatographic conditions from condition 1 to condition 11.

Example 3

The test process comprises the following steps:

blank solution (diluent): water-acetonitrile (80:20)

System applicability solution (1): is prepared into a mixed solution containing E05-ZZZA, E05-ZZZB, E05-ZZI, E05-ZZM, E05-ZZS, E05-ZZZC, E05-ZZD, E05-ZZJ, E05-ZZE, E05-ZZP and E05-ZH, which each contain about 1.0 mug/ml and about 1mg/ml of E05 reference substance.

System applicability solution (2): is formulated as a mixed solution containing E05-A, E-ZZE each containing about 1.0 μg/ml and E05 control about 1 mg/ml.

Reference mother liquor (1): the preparation method is the same as that of the impurity stock solution in example 2.

Control mother liquor (2): the preparation method is the same as E05-A and E05-ZZE stock solution in example 2.

Adding a labeled test sample solution (1): precisely weighing 25mg of the sample, placing in 25ml measuring flask, adding 1.0ml of reference mother liquor (1), adding diluent, dissolving, diluting to scale, and shaking.

Adding a labeled test sample solution (2): precisely weighing 25mg of sample, placing in 25ml measuring flask, adding reference mother liquor (2) (1.0 ml), adding diluent, dissolving, diluting to scale, and shaking.

Investigation of conditions

See Table 16

Table 16

Durability inspection condition	Conditions of the verification method	Verification ofDurability range
			Chromatographic column temperature	30℃	25℃、35℃
pH of mobile phase A	7.0	6.8、7.2

Experimental results

Table 17 e05 related substance durability results table-degree of separation

Varying conditions	Minimum degree of separation between main peak and known impurities
		Column temperature 35 DEG C	3.0
Column temperature 25 DEG C	3.1
		pH 6.8 of mobile phase A	3.0
pH 7.2 of mobile phase A	3.0

Table 18E 05-related substance durability results Table-recovery/content (%)

Conclusion of the test:

under the condition of each durability investigation chromatograph, the separation degree between a main peak and each known impurity peak in the system applicability solution is more than 1.5, and the separation degree between each known impurity peak is more than 1.5 (except the separation degree between the combined control impurities). The recovery rate of each known impurity in the test sample is within the range of 90% -108%, the absolute difference between the maximum single impurity and the measurement result under the proposed condition is not more than 0.02%, and the total sum of all impurities is not more than 0.05%. The method has good durability.

The chromatograms of the solutions under the respective conditions are shown in fig. 54 to 69.

Comparative example 1

(1) High performance liquid chromatography conditions

TABLE 19 high performance liquid chromatography conditions

TABLE 20 gradient elution procedure

Comparative example 2

(1) High performance liquid chromatography conditions

TABLE 21 high performance liquid chromatography conditions

Table 22 gradient elution procedure

From the experimental data and chromatograms of the embodiment 1, the comparative example 1 and the comparative example 2, the chromatographic column ACE Excel 3C18-AR is adopted, the separation degree between the trazodone peak and the adjacent impurity peak is good, the listed impurities are detected, the time required for gradient elution is shorter, and the rapid and accurate detection of the trazodone hydrochloride related substances is facilitated.

Comparative example 3-analysis method of trazodone hydrochloride related substance in the prior art

The analytical method of the trazodone hydrochloride related substances is specifically shown in Table 23

TABLE 23 analysis method of trazodone hydrochloride related substances

Table 24

The sample preparation method is as follows:

blank solution (diluent): mobile phase a-mobile phase B (80:20) (see fig. 72, fig. 72 is a liquid chromatogram of a prior art blank solution)

Test solution: accurately weighing a proper amount of trazodone hydrochloride serving as a test sample, and adding a diluent to dissolve the trazodone hydrochloride to prepare a solution of about 1 mg/ml. (see FIG. 73. FIG. 73 is a liquid chromatogram of a sample solution in the prior art)

E05-ZZZA mother liquor: precisely weighing about 2mg of E05-ZZZA, placing in a 100ml measuring flask, adding about 10ml of acetonitrile for complete dissolution, and adding a diluent for constant volume to obtain E05-ZZZA mother liquor;

preparing mother solutions of E05-ZZB, E05-ZZZC, E05-ZZD, E05-ZZE, E05-ZZH, E05-ZZI, E05-ZZJ, E05-ZZM and E05-ZZS by the same method.

System applicability solution: accurately weighing about 20mg of trazodone hydrochloride reference substance, placing in a 20ml measuring flask, transferring into 1ml of each impurity mother liquor of E05-ZZB, E05-ZZC, E05-ZZD, E05-ZZE, E05-ZZH, E05-ZZI, E05-ZZJ, E05-ZZM and E05-ZZS, adding diluent to dissolve and fix volume. Referring to fig. 74, fig. 74 is a liquid chromatogram of a prior art system applicability solution.

Recovery solutions were prepared as follows table 25:

table 25 recovery rate solution 1: (FIGS. 75-79)

In Table 5, A-LOQ is named 30% recovery solution; A-L is named 50% recovery solution; A-M is named 100% recovery solution; A-H was named 150% recovery solution.

Referring to fig. 75 to 79, fig. 75 is a liquid chromatogram of a background solution, fig. 76 is a liquid chromatogram of a quantitative limit recovery solution, fig. 77 is a liquid chromatogram of a 50% recovery solution, fig. 78 is a liquid chromatogram of a 100% recovery solution, and fig. 79 is a liquid chromatogram of a 150% recovery solution.

The recovery rate test results of E05-ZZD are shown in Table 26.

Table 26E 05-ZZD recovery test results Table

E05-ZZD in the system applicability solution shows a peak at 6.035min, the separation degree between E05-ZZD and the main peak is 2.69, and the separation degree is more than 1.5; however, in the experimental investigation of the recovery rate, the recovery rate of E05-ZZD is more than 150%; the finding of the cause is that E05-ZZD is not separated from the base line of the trazodone peak, so that the integration is inaccurate. Meanwhile, the flow rate is 2.0ml/min, the inner diameter of the chromatographic column is 3.5 mu m, the column pressure of the chromatographic column is maintained at 210bar, and the pressure is high.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. The method for detecting the trazodone hydrochloride related substances is characterized by adopting a high performance liquid chromatography to detect the trazodone hydrochloride related substances, wherein the chromatographic conditions of the high performance liquid chromatography are as follows:

2. The method of manufacturing according to claim 1, comprising the steps of:

diluting trazodone hydrochloride with a diluent to obtain a sample solution;

3. The method according to claim 2, wherein the concentration of the sample solution is 0.8 to 1.5mg/ml.

4. The method of manufacturing according to claim 1, further comprising:

5. The method according to claim 4, wherein the reference substance further comprises 4- (3-chlorophenyl) -1- [3- (3-oxo-2, 3-dihydro-1, 2, 4-triazolo [4,3-a ] pyridin-2-alkyl) propyl ] piperazine-N-oxide, 2- [3- (4-phenylpiperazin-1-yl) -propyl ] [1,2,4] triazolo [4,3- α ] pyridin-3 (2H) -one hydrochloride, 2- [3- [4- (4-chlorophenyl) piperazin-1-yl ] propyl ] [1,2,4] triazolo [4,3- α ] pyridin-3 (2H) -one, 2- [3- [4- (3-chloro-4-ethylphenyl) piperazin-1-yl ] propyl ] [1,2,4] triazolo [4,3-a ] pyridin-3 (2H) -one hydrochloride, 1' - (propane-1, 3-diyl) bis [4- (3-chlorophenyl) piperazin-1-yl ] propyl ] [1,2, 4- [3- α ] triazolo [4, 3-chlorophenyl ] piperazin-1-yl ] propyl ] [1,2, 4- [3- [4- (3-chloro-4-ethylphenyl) piperazin-1-yl ] propyl ] [1,2, 4- [3- [ 4-a ] triazolo [ 2H) -one hydrochloride, 1- [3 ] bis [4- (3-phenyl) phenyl ] piperazin ] propyl ] hydrochloride One or more of 1,2, 4-triazolo [4,3-a ] pyridin-3 (2H) -one, 1- (3-chlorophenyl) piperazine hydrochloride, 1- [3- [4- (3-chlorophenyl) piperazin-1-yl ] propyl ] [1,2,4] triazolo [4,3-a ] pyridin-1-ium-3-alkoxide, 1- (3-chlorophenyl) -4- (3-isopropoxypropyl) piperazine, and 2- [3- [4- (2-chlorophenyl) piperazin-1-yl ] propyl ] [1,2,4] triazolo [4,3-a ] pyridin-3 (2H) -one.

6. The method according to claim 2 or 4, wherein the diluent is selected from a mixed solution of water and acetonitrile in a volume ratio of 85:15 to 75:25.

7. The method according to any one of claims 1 to 6, wherein the C18 column is selected from ACE Excel 3C18-AR columns, with a specification of 4.6x100 mm.

8. The preparation method according to any one of claims 1 to 6, wherein the mobile phase a is formulated as follows:

9. The method according to any one of claims 1 to 6, wherein the gradient elution procedure is:

0-20 min, mobile phase A:65%, mobile phase B:35%;

20-30 min, mobile phase A:65% -35%, mobile phase B:35% -65%;

30-40 min, mobile phase A:35%, mobile phase B:65%;

40-40.1 min, mobile phase A:35% -65%, mobile phase B:65% -35%;

40.1-45 min, mobile phase A:65%, mobile phase B:35%.

10. The method according to any one of claims 1 to 6, wherein the high performance liquid chromatography has a flow rate of 0.8 to 1.5ml/min, a column temperature of 25 to 35 ℃, a detection wavelength of 254nm, a sample injection amount of 20 μl, and a column pressure of 120 to 165bar.