CN113671047B

CN113671047B - Method for detecting residues in homopiperazine

Info

Publication number: CN113671047B
Application number: CN202010402174.6A
Authority: CN
Inventors: 尤梅; 陈云建; 彭劭; 杨建玲
Original assignee: KPC Pharmaceuticals Inc
Current assignee: KPC Pharmaceuticals Inc
Priority date: 2020-05-13
Filing date: 2020-05-13
Publication date: 2023-05-23
Anticipated expiration: 2040-05-13
Also published as: CN113671047A

Abstract

The invention discloses a method for detecting residues in homopiperazine, which comprises the following steps: (1) Weighing the piperazine product, adding a diluent for dissolution, fixing the volume, and uniformly mixing to obtain a sample solution; (2) Respectively weighing reference substances N, N '-di-p-toluenesulfonyl ethylenediamine, N' -1, 4-di-p-toluenesulfonyl homopiperazine and phenol, placing in a same container, adding diluent for dissolving, fixing volume, mixing, and diluting to obtain reference substance solution; (3) The measurement is carried out by adopting high performance liquid chromatography, and the measurement conditions comprise: the chromatographic column with octadecylsilane chemically bonded silica as filler uses ammonium acetate buffer solution as mobile phase A and acetonitrile as mobile phase B, and carries out gradient elution with detection wavelength of 225nm, and the control solution and the sample solution are injected into a liquid chromatograph, and the chromatogram is recorded. The detection method of phenol and two intermediates in the residues can avoid interference of solvent peaks, and has stable base line and good separation degree.

Description

Method for detecting residues in homopiperazine

Technical Field

The invention belongs to the field of medicine quality control, and particularly relates to a method for detecting residues in homopiperazine, in particular to a method for detecting residues of phenol, N '-di-p-toluenesulfonyl ethylenediamine, N' -1, 4-di-p-toluenesulfonyl homopiperazine and other unknown impurities in homopiperazine.

Background

The homopiperazine is synthesized from ethylenediamine as a starting material through three steps of sulfonylation, cyclization and desulfonylation, and the reagents phenol, N '-di-p-toluenesulfonyl ethylenediamine (intermediate 1), N' -1, 4-di-p-toluenesulfonyl homopiperazine (intermediate 2) and other unknown impurities may remain, and the homopiperazine is used as a starting material for drug synthesis, so that the residues of phenol, N '-di-p-toluenesulfonyl ethylenediamine (intermediate 1), N' -1, 4-di-p-toluenesulfonyl homopiperazine (intermediate 2) and other unknown impurities need to be detected and controlled in quality control.

The Chinese patent with application number 2015100770097 discloses a preparation method of homopiperazine, which takes ethylenediamine as a raw material, and carries out acylation reaction with p-toluenesulfonyl chloride in N-butanol solvent to obtain N, N '-di-p-toluenesulfonyl ethylenediamine, wherein a product is not required to be separated from a reaction solution, and is directly subjected to cyclization reaction with bromochloropropane under the action of sodium hydroxide to obtain N, N' -di-p-toluenesulfonyl homopiperazine, then the product is subjected to desulfonylation under the action of hydrobromic acid and phenol to obtain homopiperazine hydrobromide, and finally the product is subjected to sodium hydroxide dissociation and toluene hydration to obtain high-purity homopiperazine. There is no disclosure in this technical scheme of how to detect residues in homopiperazine.

The present invention has been made in view of this.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the defects of the prior art and providing a method for detecting residues in homopiperazine, in particular to a method for detecting residues of phenol, N '-di-p-toluenesulfonyl ethylenediamine, N' -1, 4-di-p-toluenesulfonyl homopiperazine and other unknown impurities in homopiperazine.

In order to solve the technical problems, the invention adopts the basic conception of the technical scheme that:

the application provides a detection method of residues in homopiperazine, which comprises the following steps:

(1) Weighing the piperazine product, adding a diluent for dissolution, fixing the volume, and uniformly mixing to obtain a sample solution;

(2) Respectively weighing reference substances N, N '-di-p-toluenesulfonyl ethylenediamine, N' -1, 4-di-p-toluenesulfonyl homopiperazine and phenol, placing in a same container, adding diluent for dissolving, fixing volume, mixing, and diluting to obtain reference substance solution;

(3) The measurement is carried out by adopting high performance liquid chromatography, and the measurement conditions comprise: the chromatographic column with octadecylsilane chemically bonded silica as filler uses ammonium acetate buffer solution as mobile phase A and acetonitrile as mobile phase B, and carries out gradient elution with detection wavelength of 225nm, and the control solution and the sample solution are injected into a liquid chromatograph, and the chromatogram is recorded.

In a further scheme, in the step (3), the time and mobile phase ratio of gradient elution are as follows: 0-8min, the volume percentage of the mobile phase A is reduced from 95% to 25%, the volume percentage of the mobile phase B is increased from 5% to 75%,8-13min, the volume percentage of the mobile phase A is maintained at 25%, the volume percentage of the mobile phase B is maintained at 75%,13-13.1min, the volume percentage of the mobile phase A is increased from 25% to 95%, the volume percentage of the mobile phase B is reduced from 75% to 5%,13.1-15min, the volume percentage of the mobile phase A is maintained at 95%, and the volume percentage of the mobile phase B is maintained at 5%.

In the step (3), the concentration of the ammonium acetate buffer solution is 5mmol/L, and acetic acid is adopted to adjust the pH value to 5.0.

In a further scheme, the concentrations of N, N '-di-p-toluenesulfonyl ethylenediamine, N, N' -1, 4-di-p-toluenesulfonyl homopiperazine and phenol in the reference solution are all 0.01mg/mL; the concentration of homopiperazine in the test solution is 10mg/mL.

In a further embodiment, the sample volumes of the control solution and the test solution are 5. Mu.l.

Further, in the steps (1) and (2), the diluent is acetonitrile.

Further, the chromatographic column in step (3) is a Waters XB ridge ^TM C18,4.6 mm. Times.150 mm,3.5 μm, or equivalent columns.

In a further embodiment, the column temperature in the measurement conditions of step (3) is 30℃and the flow rate is 1.0ml per minute.

In a further scheme, in the measurement condition of the step (3), the acquisition time is 15min, and the equilibrium time is 5min.

In a further scheme, when chromatographic peaks of phenol, N '-di-p-toluenesulfonyl ethylenediamine and N, N' -1, 4-di-p-toluenesulfonyl homopiperazine are arranged in a chromatogram of the sample solution, the contents of the phenol, N '-di-p-toluenesulfonyl ethylenediamine and N, N' -1, 4-di-p-toluenesulfonyl homopiperazine are not more than 0.10% according to an external standard method and calculated according to peak areas; the other single impurities are not more than 0.10%, and the total amount of impurities is not more than 1.0%.

As a specific scheme, a method for detecting residual phenol, N '-di-p-toluenesulfonyl ethylenediamine (intermediate 1), N' -1, 4-di-p-toluenesulfonyl homopiperazine (intermediate 2) and other unknown impurities in homopiperazine comprises the following steps:

(1) Taking 0.5g of homopiperazine product, precisely weighing, placing into a 50ml measuring flask, adding about 30ml of acetonitrile, performing ultrasonic treatment (if necessary) until the homopiperazine product is dissolved, using acetonitrile to fix the volume to scale, and uniformly mixing to obtain a sample solution.

(2) Precisely weighing 20mg of each of the intermediate 1, the intermediate 2 and the phenol reference substance, placing into a same 100ml measuring flask, adding about 60ml of acetonitrile, performing ultrasonic treatment (if necessary) until the acetonitrile is dissolved, and using acetonitrile to fix the volume to scale, and uniformly mixing to obtain reference substance stock solution; precisely measuring 5ml, placing into a 100ml measuring flask, fixing volume to scale with acetonitrile, and mixing to obtain reference solution.

(3) According to high performance liquid chromatography, adopting octadecylsilane chemically bonded silica as filler, using 5mmol/L ammonium acetate buffer (pH value adjusted to 5.0) as mobile phase A, acetonitrile as mobile phase B, and performing gradient elution according to the following table 1; the detection wavelength was 225nm, the column temperature was 30℃and the flow rate was 1.0ml per minute. Precisely measuring 5 μl of each of the control solution and the sample solution, respectively injecting into a liquid chromatograph, and recording the chromatograms. The chromatograms of the test sample solutions include phenol, intermediate 1 and intermediate 2 peaks, and the peak areas calculated by an external standard method are not more than 0.10%. The content of other single impurities is less than 0.10 percent, and the total content of impurities is less than 1.0 percent. Preferably, the chromatographic column is a Waters XBridge ^TM C18,4.6 mm. Times.150 mm,3.5 μm or equivalent columns.

TABLE 1

After the technical scheme is adopted, compared with the prior art, the invention has the following beneficial effects:

The detection method of the residues phenol, N '-di-p-toluenesulfonyl ethylenediamine (intermediate 1), N' -1, 4-di-p-toluenesulfonyl homopiperazine (intermediate 2) and other unknown impurities in the homopiperazine can avoid the interference of solvent peaks, has stable base line, good separation degree of the intermediate I, II and phenol, good peak type of chromatographic peaks, short time and high efficiency, and can provide guarantee for the quality control of the homopiperazine.

The following describes the embodiments of the present invention in further detail with reference to the accompanying drawings.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. It is evident that the drawings in the following description are only examples, from which other drawings can be obtained by a person skilled in the art without the inventive effort. In the drawings:

FIG. 1 is a chromatogram of the mixed control of example 2.1 of the present invention;

FIG. 2 is a chromatogram of a 2.1 intermediate I localization solution of example 2 of the present invention;

FIG. 3 is a chromatogram of a 2.1 intermediate II positioning solution of example 2 of the present invention;

FIG. 4 is a chromatogram of a 2.1 phenol positioning solution of example 2 of the present invention;

FIG. 5 is a chromatogram of the mixed control of example 2.2 of the present invention;

FIG. 6 is a chromatogram of the mixed control of example 2.3 of the present invention;

FIG. 7 is a chromatogram of the mixed control of 3.1 of example 3 of the present invention;

FIG. 8 is a chromatogram of the mixed control of 3.2 of example 3 of the present invention;

FIG. 9 is a chromatogram of the actual sample in 3.3 of example 3 of the present invention;

FIG. 10 is a chromatogram of the mixed control of 3.4 of example 3 of the present invention;

FIG. 11 is a chromatogram of a 0.002% horizontal recovery test solution in 3.5 of example 3 of the present invention;

FIG. 12 is a chromatogram of the mixed control of 4.1 of example 4 of the present invention;

FIG. 13 is a chromatogram of the mixed control of 4.2 of example 4 of the present invention;

FIG. 14 is a chromatogram of the mixed control of 4.3 of example 4 of the present invention;

FIG. 15 is a chromatogram of the mixed control of 4.4 of example 4 of the present invention;

FIG. 16 is a chromatogram of the mixed control of 4.5 of example 4 of the present invention;

FIG. 17 is a chromatogram of the mixed control of 4.6 of example 4 of the present invention;

FIG. 18 is a chromatogram of the mixed control of 4.7 of example 4 of the present invention;

FIG. 19 is a chromatogram of the mixed control of example 5.1 of the present invention;

FIG. 20 is a chromatogram of the mixed control of example 5.2 of the present invention;

FIG. 21 is a chromatogram of the mixed control of example 5.3 of the present invention;

FIG. 22 is a graph showing the recovery rate at 0.001%, 0.002%, 0.1%, and 0.2% in example 6 of the present invention;

FIG. 23 is a chromatogram of the mixed control of comparative example 1.1 of the present invention;

FIG. 24 is a chromatogram of the mixed control of comparative example 1.2 of the present invention;

FIG. 25 is a chromatogram of the mixed control of comparative example 1.3 of the present invention;

FIG. 26 is a chromatogram of a hollow white solution, a mixed control, and a quantitative limiting solution in test example 1 of the present invention;

FIG. 27 is a chromatogram of an intermediate I localization solution of test example 1 of the present invention;

FIG. 28 is a chromatogram of an intermediate II positioning solution of test example 1 of the present invention;

FIG. 29 is a chromatogram of a phenol positioning solution of test example 1 of the present invention;

FIG. 30 is a chromatogram of a 0.1% level addition labeled recovery solution in test example 1 of the present invention.

It should be noted that these drawings and the written description are not intended to limit the scope of the inventive concept in any way, but to illustrate the inventive concept to those skilled in the art by referring to the specific embodiments.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments will be clearly and completely described with reference to the accompanying drawings in the embodiments of the present invention, and the following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

Sample information

Intermediate I (N, N' -di-p-toluenesulfonyl ethylenediamine), lot 201710Z07; intermediate II (N, N' -1, 4-di-p-toluenesulfonyl homopiperazine) with batch number XS180329-15; phenol, batch number G171215-06; homopiperazine product, lot 160201.

Taking 0.5g of homopiperazine product, precisely weighing, placing into a 50ml measuring flask, adding about 30ml of acetonitrile, performing ultrasonic treatment (if necessary) until the homopiperazine product is dissolved, using acetonitrile to fix the volume to scale, and uniformly mixing to obtain a sample solution.

Precisely weighing 20mg of each of the intermediate I, the intermediate II and the phenol reference substance, placing the mixture into a same 100ml measuring flask, adding about 60ml of acetonitrile, carrying out ultrasonic treatment (if necessary) until the mixture is dissolved, and using acetonitrile to fix the volume to a scale, and uniformly mixing the mixture to obtain a reference substance stock solution; precisely measuring 5ml, placing into a 100ml measuring flask, fixing volume to scale with acetonitrile, and mixing to obtain reference solution.

Example 1 initial detection method

1.1 initial liquid phase parameters

TABLE 2

EXAMPLE 2 preliminary optimization of liquid phase parameters and positioning solution, mixed control solution chromatograms

2.1 mobile phase A "0.5% ammonium acetate buffer (ammonia adjusted pH to 5.0)" was changed to "5mM ammonium acetate buffer (acetic acid adjusted pH to 5.0)". The specific preparation process is as follows: accurately weighing 385.4mg of anhydrous ammonium acetate in 1000mL of pure water, and dissolving; the pH was adjusted to 5.0.+ -. 0.05 with acetic acid. Waters XBridge TM C18,150 x 4.6mm,3.5 μm chromatography column was chosen for detection. The optimized liquid phase parameters are shown in the following table:

TABLE 3 Table 3

Mobile phase	A5 mM ammonium acetate buffer (pH adjusted to 5.0 with acetic acid) B acetonitrile A B=40:60
		Chromatographic column	Waters XBridge ^TM C18,150×4.6mm,3.5μm
Column temperature	30℃
		Flow rate	1.0mL/min
Wavelength of	225nm
		Sample injection amount	10μL
Acquisition time	10min
		Diluent and needle washing solution	Acetonitrile

In the prepared mixed reference substance solution, the concentrations of the intermediate I, the intermediate II and the phenol are all 0.01mg/mL. By using the above detection conditions, the mixed reference solution was detected, and the obtained chromatogram was shown in FIG. 1. The retention time of each sample was: phenol was 2.151min, intermediate I was 2.991min, intermediate ii was 5.444min.

The chromatogram of the intermediate I positioning solution (concentration: 1.0 mg/mL) is shown in FIG. 2, the chromatogram of the intermediate II positioning solution (concentration: 1.0 mg/mL) is shown in FIG. 3, and the chromatogram of the phenol positioning solution (concentration: 1.0 mg/mL) is shown in FIG. 4.

2.2 detection Limited solution chromatograms and System adaptive results

The liquid phase parameters were determined as in table 3.

0.02% level mixed control solution chromatogram, signal-to-noise ratio, separation degree and tailing factor result

The chromatogram of the 0.02% level (0.002 mg/mL for intermediate I, intermediate II and phenol) mixed control solution is shown in FIG. 5.

The signal to noise ratio, degree of separation, tailing factor results analysis of the 0.02% level (intermediate I, intermediate II and phenol concentrations of 0.002 mg/mL) mixed control solution are shown in Table 4.

TABLE 4 Table 4

Sample name	Signal to noise ratio	Degree of separation	Tailing factor
				Phenol (P)	253	N/A	1.1
Intermediate I	374	8.3	1.1
				Intermediate II	183	17.4	1.0

Conclusion:

the liquid phase parameters listed in table 3 can meet the requirements of intermediate I, intermediate II and phenol detection on the degree of separation and tailing factors. The separation degree is more than 1.5, and the tailing factors are all between 0.8 and 2.0. However, the peak time of phenol is about 2.1min, and in order to thoroughly avoid the interference of the solvent peak on the detection of phenol, the liquid phase parameters need to be further optimized, so that the peak time of phenol is prolonged.

The method has stronger detection capability on three compounds, the signal to noise ratio of 0.02% level (the concentrations of the intermediate I, the intermediate II and the phenol are all 0.002 mg/mL) is more than 180, the concentration has lost the meaning of detection capability evaluation, the sample injection amount is required to be reduced or the sample concentration is required to be reduced, and the actual detection limit level of the analysis method on the three compounds is tested.

2.3 Level 0.05% to level 0.3% assay

The liquid phase parameters were determined as in table 3.

The chromatogram of the 0.05% level (intermediate I, intermediate II, and phenol concentrations of 0.005 mg/mL) to the 0.3% level (intermediate I, intermediate II, and phenol concentrations of 0.03 mg/mL) solution is shown in FIG. 6.

As can be seen from fig. 6, from 0.05%,0.2% to 0.3%, the peak patterns of the intermediates I and II gradually worsen with increasing sample concentration, and in particular, the intermediate II is significantly broadened. It is desirable to reduce the sample concentration or optimize the liquid phase parameters.

Example 3 optimization of liquid phase parameters and corresponding chromatograms

3.1 liquid phase parameters were as per table 5. In the method, the sample injection amount is reduced from 10 mu L to 5 mu L. The proportion of the mobile phase B is reduced from 60% to 55%, so that the peak-out time of phenol can be delayed, and the phenol and the solvent peak can be completely separated.

TABLE 5

Mobile phase	A5 mM ammonium acetate buffer (pH adjusted to 5.0 with acetic acid) B acetonitrile A B=45:55
		Chromatographic column	Waters XBridge ^TM C18,150×4.6mm,3.5μm
Column temperature	30℃
		Flow rate	1.0mL/min
Wavelength of	225nm
		Sample injection amount	5μL
Acquisition time	15min
		Diluent and needle washing solution	Acetonitrile

The chromatogram of the mixed control solution is shown in FIG. 7, as determined by the above method.

3.2 liquid phase parameters were as per table 6. In the present process, the proportion of mobile phase B is reduced from 55% to 50%.

TABLE 6

Mobile phase	A5 mM ammonium acetate buffer (pH adjusted to 5.0 with acetic acid) B acetonitrile A B=50:50
		Chromatographic column	Waters XBridge ^TM C18,150×4.6mm,3.5μm
Column temperature	30℃
		Flow rate	1.0mL/min
Wavelength of	225nm
		Sample injection amount	5μL
Acquisition time	15min
		Diluent and needle washing solution	Acetonitrile

The chromatogram of the mixed control solution is shown in FIG. 8, as determined by the above method.

Conclusion: when the proportion of the mobile phase B is reduced from 55% to 50%, the phenol spike time is increased from 2.1min to 2.5min, the interference of solvent peaks can be avoided, and the intermediate I, II and phenol can reach baseline separation. The liquid phase conditions in table 6 were tentatively set as optimal parameters.

3.3 actual sample testing

The actual samples were examined for liquid phase parameters according to the conditions shown in Table 6, and the resulting chromatograms are shown in FIG. 9. As can be seen from the chromatogram, in the actual sample solution (sample solution concentration was 10 mg/mL), neither intermediate I, II nor phenol was detected.

3.4 actual detection limit level test

The concentration of the mixed control solution was reduced to give the actual detection capacity of the assay for intermediate I, II and phenol, i.e., the limit of detection level of the assay. The 0.05% level mixed control solution was diluted 50-fold and tested for signal to noise ratio.

The detection limit solution concentration is as follows:

TABLE 7

The liquid phase parameters were measured according to the conditions shown in Table 6, and the chromatogram of the mixed control solution is shown in FIG. 10. Conclusion: both intermediate I and phenol have a signal to noise ratio greater than 10, whereas intermediate II fails to reach a signal to noise ratio of 10 due to broadening of the chromatographic peaks. Intermediate II is diluted to 0.002% level (concentration of 0.0002 mg/mL) and the signal to noise ratio can meet the requirement of more than 10.

3.5 recovery test

3.5.1 preparing recovery test solution, the main compound homopiperazine concentration is 10mg/mL. The specific preparation process is as follows:

TABLE 8

The liquid phase parameters were measured according to the conditions in Table 6, and the chromatogram of the 0.002% horizontal recovery test solution is shown in FIG. 11.

Conclusion: the recovery rate test solution has obvious solvent peak in 2.0min, and produces interference to phenol, so that phenol quantification is inaccurate, and particularly, the recovery rate solution with low concentration is low. The liquid phase parameters need to be further optimized.

Example 4 further optimization of liquid phase parameters and corresponding chromatograms

The following liquid phase conditions are adopted to screen a better scheme, and the sample configuration mode is the same as the above.

4.1 measurements were made using the liquid phase parameters shown in Table 9 below

TABLE 9

The chromatogram of the mixed control solution is shown in FIG. 12, as determined by the above method. From this it can be seen that: intermediate I, II and phenol have good separation and the liquid phase parameters continue to be optimized. The inventors found that the peak formation of intermediate I, II can be accelerated and the analysis efficiency can be improved by increasing the proportion of mobile phase B.

4.2 measurements were made using the liquid phase parameters shown in Table 10 below

Table 10

The chromatogram of the mixed control solution is shown in FIG. 13, as determined by the above method.

4.3 measurements were performed using the liquid phase parameters shown in Table 11 below

TABLE 11

The chromatogram of the mixed control solution is shown in FIG. 14, as determined by the above method. From this it can be seen that: intermediate I, II and phenol have good separation, but have obvious baseline fluctuation at 8min, and the liquid phase parameters need to be continuously optimized, so that the peak time of intermediate I, II is reduced, and the baseline is optimized.

4.4 measurements were performed using the liquid phase parameters shown in Table 12 below

Table 12

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The chromatogram of the mixed control solution is shown in FIG. 15, as determined in the above manner. From this it can be seen that: intermediate I, II and phenol separated well, but intermediate I had a broader chromatographic peak.

4.5 measurements were performed using the liquid phase parameters shown in Table 13 below

TABLE 13

The chromatogram of the mixed control solution is shown in FIG. 16, as determined by the above method. From this it can be seen that: intermediate I, II and phenol separated well, but intermediate I had a broader chromatographic peak.

4.6 measurements were performed using the liquid phase parameters shown in Table 14 below

TABLE 14

The chromatogram of the mixed control solution is shown in FIG. 17, as determined by the above method.

4.7 measurements were performed using the liquid phase parameters shown in Table 15 below

TABLE 15

The chromatogram of the mixed control solution is shown in FIG. 18, as determined by the above method.

In the above results, the chromatograms (fig. 17 and 18) under the liquid phase parameters (table 14 and table 15) of the 4.6 and 4.7 schemes were close, and both the baseline and analysis time were superior to those obtained under the 4.5 scheme. Comparing the liquid phase parameters under the above 4.1-4.8 schemes with the corresponding chromatograms, it can be seen that the gradient change significantly improves the chromatographic peak pattern of phenol, but causes baseline fluctuation, and requires continuous optimization to obtain the optimal chromatographic peak pattern and baseline.

Example 5 still further optimization of the liquid phase parameters and chromatograms

5.1 measurements were made using the liquid phase parameters shown in Table 16 below

Table 16

The chromatogram of the mixed control solution is shown in FIG. 19, as determined by the above method.

5.2 measurements were performed using the liquid phase parameters shown in Table 17 below

TABLE 17

The chromatogram of the mixed control solution is shown in FIG. 20, as determined by the above method.

5.3 measurements were performed using the liquid phase parameters shown in Table 18 below

TABLE 18

The chromatogram of the mixed control solution is shown in FIG. 21, as determined by the above method.

As can be seen from the above schemes 5.1-5.3, the chromatograms (FIGS. 20 and 21) at the liquid phase parameters (Table 17 and Table 18) in the schemes 5.2 and 5.3 are close. And 5.3, the overall analysis time of the liquid phase parameter in the scheme is faster than 5.2, and the separation degree requirement can be met. In order to reduce unnecessary chromatographic acquisition time and ensure a state of sufficient balance of the system, the acquisition time is set to 15min, and the balance time is set to 5min.

EXAMPLE 6 optimal liquid phase parameters

6.1 optimization by screening, the optimal liquid phase parameters are shown in table 19 below.

TABLE 19

6.2 recovery and Linear testing Using final liquid phase method

6.2.1 preparing recovery test solution, the main compound homopiperazine concentration is 10mg/mL. The specific preparation process is as follows:

table 20

6.2.2 control solutions

The control solutions (intermediate I, intermediate II and phenol concentrations were all 0.005 mg/mL) were mixed at a level of 0.05% as control solutions for external standard quantification. The specific preparation process is as follows:

table 21

6.2.3 the liquid phase parameters in Table 19 were used for the determination.

6.2.4 results

(1) The 0.001% level, 0.002% level, 0.1% level, 0.2% level recovery solution chromatograms are shown in fig. 22.

(2) The system adaptation results are shown in table 22 below.

Table 22

(3) The signal to noise ratio results are shown in table 23 below.

Table 23

(4) The recovery results are shown in Table 24 below.

Table 24

(5) The linear results are shown in table 25 below.

Table 25

6.3 conclusion

(1) No interference is generated at the target peak in the blank solution chromatogram; the separation degree of three compounds in the 0.05% level mixed reference substance solution (the concentrations of the intermediate I, the intermediate II and the phenol are all 0.005 mg/mL) meets the requirement of more than 1.5, and the tailing factors meet the requirement of 0.8-2.0.

(2) Determination of actual detection limit concentration: the 0.001% level mixed reference substance solution (the concentrations of the intermediate I, the intermediate II and the phenol are all 0.0001 mg/mL), the signal to noise ratio of the three compounds is more than 3, and the requirement that the signal to noise ratio of the detection limit solution is more than or equal to 3 is met. A0.001% level mixed control solution (intermediate I, intermediate II and phenol concentrations of 0.0001 mg/mL) was determined as the limit of detection solution (LOD).

(3) Determination of the actual quantitative limit concentration: the 0.002% level mixed reference substance solution (the concentrations of the intermediate I, the intermediate II and the phenol are all 0.0002 mg/mL), the signal to noise ratio of the three compounds is more than 10, and the requirement that the signal to noise ratio of the quantitative limiting solution is more than or equal to 10 is met. A0.002% level of the mixed control solution (intermediate I, intermediate II and phenol concentrations of 0.0002 mg/mL) was determined as a limiting solution on quantification (LOQ).

(4) Determination of linear acceptance criteria: in the pre-verification experiment, the linear correlation coefficients of the three compounds all meet the requirement of more than 0.999 in the concentration range from 0.001% level (the concentration of the intermediate I, the concentration of the intermediate II and the concentration of the phenol are all 0.0001 mg/mL) to 0.2% level (the concentration of the intermediate I, the concentration of the intermediate II and the concentration of the phenol are all 0.02 mg/mL). According to our SOP AD-VAL-001 requirement, the linear correlation coefficient of impurity level should be greater than 0.995, so it is recommended that the acceptance criteria in the formal verification scheme be: the linear correlation coefficient was greater than 0.995 at a quantitative limit ranging from 0.02% level (intermediate I, intermediate II and phenol concentrations of 0.0002 mg/mL) to 0.2% level (intermediate I, intermediate II and phenol concentrations of 0.02 mg/mL). Specific linear concentrations and corresponding acceptance criteria are as follows:

Table 26

(5) Determination of recovery acceptance criteria: the recovery rate results in the pre-verification experiments are 102% -118% at the quantitative concentration limiting level, namely the level of 0.02% (the concentrations of the intermediate I, the intermediate II and the phenol are all 0.0002 mg/mL), and the reason is that the peak areas of three compounds at the level of 0.02% are smaller, and the difference of integral parameters has a larger influence on the recovery rate results. According to the SOP AD-VAL-001 requirement, when the impurity content is less than 0.15%, the difference between the theoretical content and the measured content is within +/-0.04%, and when the impurity content is more than or equal to 0.15%, the ratio of the theoretical content to the measured content is within +/-30%. In combination with the pre-validated recovery results, it is recommended that the acceptance criteria in the formal validation scheme be: the quantitative limit is 0.02% (0.0002 mg/mL for each of intermediate I, intermediate II and phenol) to 0.1% (0.01 mg/mL for each of intermediate I, intermediate II and phenol) and the recovery rate should be between 60% and 140%. Recovery acceptance criteria from 0.1% level to 0.2% level is 80% -120%. Specific recovery concentrations and corresponding acceptance criteria are as follows:

table 27

Comparative example 1 different chromatographic columns test 0.1% horizontal recovery solution

1.1 testing 0.1% horizontal recovery solution using ACE 5C 18, 25cm column

The liquid phase parameters of table 3 in reference example 2 are different in that: the mixed control solution was tested using a chromatographic column ACE 5C 18, 250X 4.6mm,5 μm, sample loading of 5ul, and 15min acquisition time.

The resulting chromatogram is shown in FIG. 23. It can be seen that none of intermediate I, intermediate ii and phenol was well quantified and detected using an ACE 5c 18,250 x 4.6mm,5 μm column and that the overall baseline was poor, indicating that the column was unsuitable for determining the content of intermediate I, intermediate ii and phenol in homopiperazine.

1.2 testing of 0.1% horizontal recovery solutions using a Agilent ZORBAX Eclipse XDB-C8,25 cm chromatographic column

The liquid phase parameters of table 3 in reference example 2 are different in that: the mixed control solution was tested using a chromatographic column Agilent ZORBAX Eclipse XDB-C8, 250X 4.6mm,5 μm, sample loading of 5ul, and 15min acquisition time.

The resulting chromatogram is shown in FIG. 24. It can be seen that using a Agilent ZORBAX Eclipse XDB-C8, 250X 4.6mm,5 μm column, the main compound produced a chromatographic peak that interfered with phenol for around 2.5min and a poor overall baseline between 0min and 4.5 min.

1.3 testing 0.1% horizontal recovery solution using a Waters Symmetry C, 25cm column

The liquid phase parameters of table 3 in reference example 2 are different in that: the mixed control solution was tested using a chromatographic column Waters Symmetry C, 250X 4.6mm,5 μm, 5ul sample, 15min acquisition time.

The resulting chromatogram is shown in FIG. 25. It can be seen that using Waters Symmetry C, 250×4.6mm,5 μm columns, the main compound produced a chromatographic peak that interfered with phenol for about 2.5min, the baseline varied, and there was a risk for subsequent quantitative testing of phenol under conditions where the durability of the process did not meet the target requirements.

Conclusion, using chromatographic column Waters XBridge ^TM C18,150 ×4.6mm,3.5 μm is the most preferred.

Test example 1 specificity

Sample: blank solution (diluent acetonitrile), 0.002% limit-on-quantity solution (LOQ), control solution. A blank solution (diluent acetonitrile), 0.002% limiting solution (LOQ) and control solution were prepared in one portion each according to the analytical method.

Intermediate I positioning solution, intermediate II positioning solution, and phenol positioning solution.

Preparing 0.05mg/mL of intermediate I positioning solution, intermediate II positioning solution and phenol positioning solution in one part each, wherein the preparation process is as follows:

for example: accurately weighing 10mg of intermediate I reference substance into a 200mL volumetric flask, adding 150mL of diluent, carrying out ultrasonic treatment (if necessary) until the diluent is dissolved, and carrying out constant volume mixing by using the diluent. Named I-ID.

Accurately weighing 10mg of intermediate II reference substance into a 200mL volumetric flask, adding 150mL of diluent, carrying out ultrasonic treatment (if necessary) until the diluent is dissolved, and fixing the volume by using the diluent, and uniformly mixing. Named Pheno II-ID.

Accurately weighing 10mg of phenol reference substance into a 200mL volumetric flask, adding 150mL of diluent, performing ultrasonic treatment (if necessary) until the diluent is dissolved, and fixing the volume by using the diluent, and uniformly mixing. Named Phenol-ID.

Acceptance criteria

In the blank solution chromatogram, no obvious interference exists in the intermediate I, the intermediate II and the phenol of the target peak; if there is interference, the peak area of the interference peak must not be greater than the peak area of the LOQ.

The intermediate I, the intermediate II and the phenol in the chromatograms of the reference substance solution and the 0.1 percent horizontal standard recovery rate solution can be completely separated, and the separation degree is more than or equal to 1.5.

Results: in the blank solution chromatogram, no obvious interference exists in the target peak intermediate I, intermediate II and phenol. The separation degree of the intermediate I, the intermediate II and the phenol in the chromatogram of the reference solution is shown in Table 28,0.1% and the separation degree of the intermediate I, the intermediate II and the phenol in the chromatogram of the solution with the standard recovery rate is shown in Table 29. The results are detailed in FIG. 26, and the results of intermediate I, intermediate II and phenol positioning solutions are shown in FIGS. 27-29. The 0.1% horizontal addition recovery solution is detailed in FIG. 30. The results meet the acceptance criteria.

Table 28

Name of the name	Degree of separation
		Phenol-ID (Phenol)	28.3
I-ID (intermediate I)	28.3,15.7
		II-ID (intermediate II)	15.7

Table 29

Name of the name	Degree of separation
		Phenol-ID (Phenol)	27.9
I-ID (intermediate I)	27.9,15.6
		II-ID (intermediate II)	15.6

Test example 2 limit of quantitation (LOQ), limit of detection (LOD)

(1) Quantitative limiting solution (LOQ): a0.002% quantitative limiting solution (LOQ) was prepared, and 6-needle quantitative limiting solutions (LOQ) were introduced to calculate the signal to noise ratio, the Relative Standard Deviation (RSD) of peak areas, and the Relative Standard Deviation (RSD) of retention times of the intermediates I, II and phenol in the quantitative limiting solutions, respectively.

Detection limit solution (LOD): a portion of a 0.001% limiting solution (LOD) was prepared and a two-step dilution was performed from a 0.002% limiting solution (LOQ) to obtain a limiting solution (LOD). The specific process is as follows: the limiting solution (LOD) was obtained by diluting the limiting solution (LOQ) twice (5 mL. Fwdarw.10 mL). And (3) injecting a 3-needle detection limiting solution (LOD), and respectively calculating the average value of the signal to noise ratios of the intermediate I, the intermediate II and the phenol in the quantitative limiting solution.

(2) Acceptance criteria

And the average value of the signal to noise ratio of the intermediate I, the intermediate II and the phenol in the LOD LOD solution is not less than 3.

The average value of the signal to noise ratio of the intermediate I, the intermediate II and the phenol in the LOQ LOQ solution is not less than 10. The RSD of the peak areas of the intermediate I, the intermediate II and the phenol in the continuous 6-needle LOQ is less than or equal to 15 percent, and the RSD of the retention time is less than or equal to 2.0 percent.

(3) Results: the limit of detection (LOD) and limit of quantification (LOQ) results are shown in tables 30-31. The results all meet the acceptance criteria.

Watch 30 limit of detection (LOD)

Name of the name	Intermediate I	Intermediate II	Phenol (P)
				First needle signal to noise ratio	5	4	1
Second needle signal to noise ratio	13	10	6
				Third needle signal to noise ratio	3	2	1
Average value of signal to noise ratio (n=3)	7	5	3

Table 31 quantitative limiting solution (LOQ)

Name of the name	Intermediate I	Intermediate II	Phenol (P)
				First needle signal to noise ratio	37	27	15
Second needle signal to noise ratio	51	40	21
				Third needle signal to noise ratio	13	10	5
Fourth needle signal to noise ratio	5	4	2
				Fifth needle signal to noise ratio	11	7	4
Sixth needle signal to noise ratio	28	21	11
				Average value of signal to noise ratio (n=6)	24	18	10
Peak area RSD (n=6)	2％	9％	8％
				Retention time RSD (n=6)	0.1％	0.1％	0.0％

Test example 3 linearity

Preparing a series of linear solutions, namely an intermediate I, an intermediate II and phenol, wherein the concentration levels of the intermediate I, the intermediate II and the phenol are respectively 0.002%,0.01%,0.05%,0.1% and 0.2% of the target concentration, feeding 1 needle into each solution, and calculating the respective linear correlation coefficients (r), slopes, intercept and residual square sum of the intermediate I, the intermediate II and the phenol.

A part of mixed reference substance solution with the concentration of 0.05mg/mL is prepared as follows: for example: accurately weighing 10mg of the intermediate I, the intermediate II and the phenol reference substance into the same 200mL volumetric flask, adding about 150mL of diluent, carrying out ultrasonic treatment (if necessary) until the diluent is dissolved, and fixing the volume by using the diluent, mixing uniformly, and naming the mixture as follows: stock solution #1.

Preparation of a linear solution: 0.002%,0.01%,0.05%,0.1% and 0.2% horizontal linear solutions were prepared in one portion each. The preparation process is as follows:

table 32

Linear solution name	Stock solution name	Two-stage dilution	Theoretical concentration (mg/mL)
				0.002％	0.1% horizontal linear solution	2mL→100mL	0.0002
0.01％	Stock solution #1	2mL→100mL	0.001
				0.05％	Stock solution #1	5mL→50mL	0.005
0.1％	Stock solution #1	5mL→25mL	0.01
				0.2％	Stock solution #1	10mL→25mL	0.02

Acceptance criteria:

the correlation coefficient (r) of each of the intermediate I, the intermediate II and the phenol is not lower than 0.995.

Slope, intercept and residual sum of squares are reported.

Results the linear results are detailed in table 33. The results meet the acceptance criteria.

Table 33

Name of the name	Intermediate I	Intermediate II	Phenol (P)
				Linear coefficient (r)	1.000	1.000	1.000
Slope of	17596727.097	13872713.910	8798348.199
				Intercept of (intercept of)	67.311	220.604	-142.773
Sum of squares of residuals	195637.496	255476.251	24577.779

Test example 4 accuracy

Homopiperazine drug substance sample control solution: 3 sample control solutions were prepared in parallel.

Preparing a mixed reference substance solution: a0.05 mg/mL portion of the mixed control solution (stock solution # 2) was prepared as follows:

for example: accurately weighing 10mg of the intermediate I, the intermediate II and the phenol reference substance into the same 200mL volumetric flask, adding about 150mL of diluent, carrying out ultrasonic treatment (if necessary) until the diluent is dissolved, and fixing the volume by using the diluent, mixing uniformly, and naming the mixture as follows: stock solution #2.

A0.005 mg/mL portion of the mixed control solution (stock solution # 3) was prepared as follows: 10mL of stock solution #2 is taken, added into a volumetric flask of 100mL, added with a diluent, and mixed evenly and named: stock solution #3.

Preparing a solution with a standard recovery rate: preparing a series of standard adding recovery rate solutions, wherein the standard adding concentration is 0.002%,0.05%,0.1% and 0.2% of the target concentration, 3 parts of standard adding recovery rate solutions at 0.002%,0.05% and 0.2% are respectively prepared, and 6 parts of standard adding recovery rate solutions at 0.1% are prepared according to the specific preparation process as follows:

watch 34

Each solution was injected 1 needle and individual recovery results and averages were calculated for each level of intermediate I, intermediate II and phenol, respectively.

Acceptance criteria

For standard recovery levels of 0.002% and 0.05%, the average recovery of each of intermediate I, intermediate II and phenol should be between 60% and 140%. For 0.1%, 0.2% level of standard recovery, the average of the individual recovery of intermediate I, intermediate II and phenol should be between 80% and 120%.

As a result, the actual formulation concentration of the phenol-spiked solution:

table 35

Actual configuration concentration of intermediate I plus standard solution:

table 36

Actual configuration concentration of intermediate II addition-labeling solution:

table 37

The accuracy results are shown in table 38. The results meet the acceptance criteria. Annotation: in the verification scheme, the titers of the reference substance intermediate I, the intermediate II and the phenol are not mentioned in the actual measurement concentration formula, and the titers in the actual calculation process participate in the calculation of accuracy, so that the requirement of accuracy calculation results is met, and the verification results are not influenced.

Table 38: accuracy results

Name of the name	Intermediate I	Intermediate II	Phenol (P)
				Average value of 0.002% horizontal standard recovery rate	100％	102％	110％
Average value of 0.05% horizontal standard recovery rate	101％	99％	102％
				Average value of 0.1% horizontal standard recovery rate	101％	99％	102％
Average value of 0.2% horizontal standard recovery rate	101％	99％	101％

Test example 5 precision

Precision of instrument

A control solution (STD#1) was prepared, and 6 needle control solutions (STD#1) were introduced to calculate the Relative Standard Deviation (RSD) of the peak areas of intermediate I, intermediate II and phenol, respectively.

Precision of the method

6 parts of 0.1% horizontal addition recovery solution were prepared in parallel according to the validation protocol, each solution was taken 1 needle, and the Relative Standard Deviation (RSD) of the results for recovery of intermediate I, intermediate II and phenol was calculated.

Acceptance criteria: RSD 5.0% of the peak area of each of intermediate I, intermediate II and phenol in the control solution.

The RSD 15% of the recovery results for intermediate I, intermediate II and phenol in the 0.1% level was added to the standard recovery solution.

Results: the results of the instrument precision and the method precision are shown in tables 39 and 40. The results meet the acceptance criteria.

Table 39

Name of the name	Intermediate I	Intermediate II	Phenol (P)
				01	175819.596	138874.837	88990.123
02	175625.691	139229.992	89170.853
				03	175173.269	139281.870	88672.074
04	175612.341	139330.697	88984.977
				05	175704.569	139404.501	89018.064
06	175671.088	141203.432	88871.857
				Peak surface	0.1％	0.6％	0.2％

Table 40

Name of the name	Recovery of intermediate I (%)	Recovery of intermediate II (%)	Phenol recovery (%)
				01	101	99	101
02	102	100	102
				03	101	99	101
04	102	100	102
				05	101	100	102
06	101	99	101
				RSD with 0.1% level of standard recovery	0％	0％	0％

Test example 6 working range

The working range will be established with an appropriate level of accuracy, precision and linearity.

Report working scope of acceptance criteria

Results working range results are shown in table 41. The results meet the acceptance criteria.

Table 41

Test example 7 tolerance

Preparing a solution: the following representative solutions were formulated for tolerability evaluation: blank solution (diluent), 0.002% limiting solution (LOQ), control solution (std#1), 0.1% level addition of standard recovery solution.

The following parameters of the liquid phase were varied, only one parameter was varied per test, and tolerability results were assessed.

1. Initial proportion of mobile phase, B%:7% (5% +2%) mobile phase gradient procedure is shown in table 42, with other liquid phase parameters referring to table 19:

table 42

2. Initial proportion of mobile phase, B%:7% (5% -2%) mobile phase gradient procedure is shown in table 43, with additional liquid phase parameters referring to table 19:

table 43

3. Mobile phase pH 5.2 (5.0+0.2), acetic acid adjusted pH to 5.2±0.05 in liquid phase parameters, other liquid phase parameters are referred to table 19;

4. the pH of the mobile phase is 4.8 (5.0-0.2), the pH value of acetic acid in the liquid phase parameters is adjusted to 4.8+/-0.05, and other liquid phase parameters are referred to in a table 19;

5. column temperature 33 ℃ (30 ℃ +3 ℃), column temperature 33 ℃ among liquid phase parameters, and other liquid phase parameters refer to table 19;

6. Column temperature 27 ℃ (30 ℃ -3 ℃), column temperature 27 ℃ in liquid phase parameters, and other liquid phase parameters refer to table 19;

7. the flow rate was 1.2mL/min (1.0 mL/min+20%), the flow rate was 1.2mL/min among the liquid phase parameters, and the other liquid phase parameters were as shown in Table 19;

8. the flow rate was 0.8mL/min (1.0 mL/min-20%), the flow rate was 0.8mL/min among the liquid phase parameters, and the other liquid phase parameters were as shown in Table 19.

Acceptance criteria

No obvious interference exists at the target peak in the blank solution chromatogram; if there is interference, the peak area of the interference peak must not be greater than the peak area of the LOQ.

The intermediate I, the intermediate II and the phenol in the chromatograms of the first reference substance solution and the 0.1 percent horizontal standard recovery rate solution can be completely separated, and the separation degree is more than or equal to 1.5.

Remarks: if the two requirements can not be met under a certain condition of liquid phase parameters, the variable is controlled in actual test, and the variable is strictly consistent with the method.

Results

1. Mobile phase pH4.8 (5.0-0.2) test results:

no obvious interference exists in the blank solution chromatogram at the target peak intermediate I, the intermediate II and the phenol (no chromatogram is provided and is described). The intermediate I, the intermediate II and the phenol in the chromatograms of the first reference solution and the 0.1 percent horizontal standard recovery rate solution can be completely separated, and the separation degree is more than 1.5. A blank solution, a 0.002% quantitative limiting solution (LOQ), a control solution and a 0.1% level addition standard recovery solution were used for tolerating tests, and the test results at pH4.8 mobile phase all met the acceptance criteria.

2. Mobile phase pH 5.2 (5.0+0.2) test results:

in the blank solution chromatogram, no obvious interference exists in the target peak intermediate I, intermediate II and phenol. The intermediate I, the intermediate II and the phenol in the chromatograms of the first reference substance solution and the 0.1 percent horizontal standard recovery rate solution can be completely separated, and the separation degree is more than or equal to 1.5. A blank solution, a 0.002% quantitative limiting solution (LOQ), a control solution and a 0.1% level standard recovery solution were used for tolerating tests, and the test results of the remaining parameters all met the acceptance criteria at pH 5.2 mobile phase.

3. Initial proportion of mobile phase, B%:7% (5% + 2%), the test results were as follows:

in the blank solution chromatogram, no obvious interference exists in the target peak intermediate I, intermediate II and phenol. The intermediate I, the intermediate II and the phenol in the chromatograms of the first reference solution and the 0.1 percent horizontal standard recovery rate solution can be completely separated, and the separation degree is more than 1.5. A blank solution, a 0.002% quantitative limiting solution (LOQ), a control solution and a 0.1% level addition standard recovery solution were used for tolerating tests, and the test results met the acceptance criteria with a change in mobile phase B (B%: 7% (5% +2%) parameters.

4. Initial proportion of mobile phase, B%:3% (5% -2%), the test results were as follows:

In the blank solution chromatogram, no obvious interference exists in the target peak intermediate I, intermediate II and phenol. The intermediate I, the intermediate II and the phenol in the chromatograms of the first reference substance solution and the 0.1 percent horizontal standard recovery rate solution can be completely separated, and the separation degree is more than or equal to 1.5. A blank solution, a 0.002% quantitative limiting solution (LOQ), a control solution and a 0.1% horizontal addition standard recovery solution were used for tolerance testing, and the test results met the acceptance criteria with the mobile phase B (B%: 3% (5% -2%) parameters changed.

5. Column temperature 33 ℃ (30 ℃ +3℃)

In the blank solution chromatogram, no obvious interference exists in the target peak intermediate I, intermediate II and phenol. The intermediate I, the intermediate II and the phenol in the chromatograms of the first reference substance solution and the 0.1 percent horizontal standard recovery rate solution can be completely separated, and the separation degree is more than or equal to 1.5. A blank solution, a 0.002% quantitative limiting solution (LOQ), a control solution and a 0.1% level addition standard recovery solution were used for the tolerability test, and the test results at the column temperature of 33 ℃ (30 ℃ +3 ℃) parameters all met the acceptance criteria.

6. Column temperature 27 ℃ (30 ℃ -3 ℃)

In the blank solution chromatogram, no obvious interference exists in the target peak intermediate I, intermediate II and phenol. The intermediate I, the intermediate II and the phenol in the chromatograms of the first reference solution and the 0.1 percent horizontal standard recovery rate solution can be completely separated, and the separation degree is more than 1.5. A blank solution, a 0.002% quantitative limiting solution (LOQ), a control solution and a 0.1% horizontal addition standard recovery rate solution are used for tolerance test, and test results under the parameters of column temperature 27 ℃ (30 ℃ -3 ℃) all meet the acceptance standard.

7. The flow rate was 1.2mL/min (1.0 mL/min+20%)

In the blank solution chromatogram, no obvious interference exists in the target peak intermediate I, intermediate II and phenol. The intermediate I, the intermediate II and the phenol in the chromatograms of the first reference solution and the 0.1 percent horizontal standard recovery rate solution can be completely separated, and the separation degree is more than 1.5. A blank solution, a 0.002% quantitative limiting solution (LOQ), a control solution and a 0.1% level addition standard recovery solution were used for tolerance testing, and the test results at a flow rate of 1.2mL/min (1.0 mL/min+20%) parameters all met the acceptance criteria.

8. The flow rate is 0.8mL/min (1.0 mL/min-20%)

In the blank solution chromatogram, no obvious interference exists in the target peak intermediate I, intermediate II and phenol. The intermediate I, the intermediate II and the phenol in the chromatograms of the first reference solution and the 0.1 percent horizontal standard recovery rate solution can be completely separated, and the separation degree is more than 1.5. A blank solution, a 0.002% quantitative limiting solution (LOQ), a control solution and a 0.1% level standard recovery solution were used for tolerance testing, and the test results at a flow rate of 0.8mL/min (1.0 mL/min-20%) parameters all met the acceptance criteria.

Test example 8 solution stability

Stability of control solution

A portion of the control solution was prepared. Storage under suitable conditions (e.g., normal temperature conditions, 2-8deg.C). The control solution was freshly prepared and analyzed after a specified period of time (e.g., 24 hours, 48 hours and 72 hours) of standing. The recovery of the control solution after the specified time of placement relative to the freshly prepared control solution was calculated and compared to the recovery at the initial time.

Stability of the solution at the recovery rate by addition of the standard

1 part of recovery solution with the 0.1 percent standard adding level is prepared according to a verification scheme, and the recovery solution with the 0.1 percent level is placed under proper conditions for storage (such as normal temperature conditions and 2-8 ℃).

The control solution was freshly prepared and analyzed for recovery at a 0.1% level after a specified period of time (e.g., 24 hours, 48 hours and 72 hours) of recovery. The recovery results of the 0.1% level recovery solution after the specified time of standing were compared with the ratio of the recovery results at the initial time.

Acceptance criteria

The recovery rate of the peak areas of the intermediate I, the intermediate II and the phenol in the reference substance solution after the specified time is placed is between 95.0 and 105.0 percent compared with the initial value.

The recovery of intermediate I, intermediate II and phenol in the solution after the specified time period was between 95.0 and 105.0% compared to the initial value.

Results

The stability results of the control solutions are detailed in Table 44, and the stability results of the spiked recovery solutions are detailed in Table 45. The results all meet the acceptance criteria.

Table 44

Name of the name	Intermediate I	Intermediate II	Phenol (P)
				Stability of control solution at room temperature for 1 day (31 h)	100％	101％	99％
Stability of control solution at room temperature for 5 days (129.5 h)	99％	100％	99％

Table 45

Name of the name	Intermediate I	Intermediate II	Phenol (P)
				Stability of 0.1% level addition of the standard recovery solution at room temperature for 1 day (20.5 h)	100％	101％	99％
0.1% level addition of standard recovery solution stability at room temperature for 5 days (119 h)	99％	100％	99％

The foregoing description is only illustrative of the preferred embodiment of the present invention, and is not to be construed as limiting the invention, but is to be construed as limiting the invention to any and all simple modifications, equivalent variations and adaptations of the embodiments described above, which are within the scope of the invention, may be made by those skilled in the art without departing from the scope of the invention.

Claims

1. The method for detecting the residues in the homopiperazine is characterized by comprising the following steps of:

(3) The measurement is carried out by adopting high performance liquid chromatography, and the measurement conditions comprise: the method comprises the steps of performing gradient elution by taking octadecylsilane chemically bonded silica as a chromatographic column of a filler, taking ammonium acetate buffer solution as a mobile phase A and acetonitrile as a mobile phase B, taking a control solution and a sample solution, injecting the control solution and the sample solution into a liquid chromatograph, and recording a chromatogram;

in the step (3), the concentration of the ammonium acetate buffer solution is 5 mmol/L, and acetic acid is adopted to adjust the pH value to 5.0;

in the step (3), the time of gradient elution and the proportion of mobile phases are as follows: 0-8 min, the volume percentage of the mobile phase A is reduced from 95% to 25%, the volume percentage of the mobile phase B is increased from 5% to 75%,8-13 min, the volume percentage of the mobile phase A is maintained at 25%, the volume percentage of the mobile phase B is maintained at 75%, 13-13.1 min, the volume percentage of the mobile phase A is increased from 25% to 95%, the volume percentage of the mobile phase B is reduced from 75% to 5%, 13.1-15 min, the volume percentage of the mobile phase A is maintained at 95%, and the volume percentage of the mobile phase B is maintained at 5%.

2. The method for detecting residues in homopiperazine according to claim 1, wherein the concentrations of N, N '-di-p-toluenesulfonyl ethylenediamine, N' -1, 4-di-p-toluenesulfonyl homopiperazine and phenol in the reference solution are all 0.01 mg/mL; the concentration of homopiperazine in the test solution was 10 mg/mL.

3. The method for detecting residues in homopiperazine according to claim 1, wherein the sample volumes of the control solution and the sample solution are 5 μl.

4. A method for detecting residues in homopiperazine according to any one of claims 1 to 3, wherein the diluent in steps (1) and (2) is acetonitrile.

5. A method for detecting residues in homopiperazine according to any one of claims 1 to 3, wherein the chromatographic column in step (3) is a Waters XBridge ^TM C18，4.6mm×150mm，3.5μm。

6. A method for detecting residues in homopiperazine according to any one of claims 1 to 3, wherein in the measurement condition of step (3), the column temperature is 30 ℃, and the flow rate is 1.0 ml per minute.

7. A method for detecting residues in homopiperazine according to any one of claims 1 to 3, wherein in the measurement condition of step (3), the acquisition time is 15 min, and the equilibration time is 5 min.

8. The method for detecting residues in homopiperazine according to any one of claims 1 to 3, wherein when a chromatogram of the sample solution has chromatographic peaks of phenol, N '-di-p-toluenesulfonyl ethylenediamine, N' -1, 4-di-p-toluenesulfonyl homopiperazine, the content of phenol, N '-di-p-toluenesulfonyl ethylenediamine, N' -1, 4-di-p-toluenesulfonyl homopiperazine is not more than 0.10% in terms of peak area by an external standard method; the other single impurities are not more than 0.10%, and the total amount of impurities is not more than 1.0%.