CN113671048A

CN113671048A - Method for detecting methyl p-toluenesulfonate and ethyl p-toluenesulfonate in high piperazine residue

Info

Publication number: CN113671048A
Application number: CN202010402175.0A
Authority: CN
Inventors: 刘蓓蓓; 陈云建; 陈斌; 杨建玲; 涂继辉
Original assignee: KPC Pharmaceuticals Inc
Current assignee: KPC Pharmaceuticals Inc
Priority date: 2020-05-13
Filing date: 2020-05-13
Publication date: 2021-11-19

Abstract

The invention discloses a method for detecting methyl p-toluenesulfonate and ethyl p-toluenesulfonate in high piperazine residues, which comprises the following steps of: (1) weighing a high piperazine product, adding a solvent for dissolving, fixing the volume, and uniformly mixing to obtain a test solution; (2) preparing a reference substance solution and a sensitivity solution; (3) the determination is carried out by adopting a liquid chromatography-mass spectrometry combined method: the liquid chromatography determination conditions were: gradient elution is carried out on a mobile phase A which is a pure water solution of 0.1% formic acid and a mobile phase B which is a methanol solution of 0.1% formic acid, the column temperature is 40 ℃, the flow rate is 0.6ml per minute, the sample injection temperature is 5 ℃, and the sample injection volume is 5 mu l; the mass spectrum conditions are as follows: an ion source API-ES adopts a mass spectrum scanning mode of multi-reaction monitoring; the inlet voltage is 8V, the outlet voltage is 10V, the air curtain pressure is 25Psi, the collision gas CAD is 10Psi, the spray voltage is 5500V, and the ion source temperature is 430 ℃; the atomization gas pressure was 55Psi and the assist gas pressure was 55 Psi. The method is suitable for detecting the two impurities in the homopiperazine.

Description

Method for detecting methyl p-toluenesulfonate and ethyl p-toluenesulfonate in high piperazine residue

Technical Field

The invention belongs to the field of medicine quality control, particularly relates to a detection method of residues in homopiperazine, and more particularly relates to a detection method of residues in homopiperazine, namely methyl p-toluenesulfonate and ethyl p-toluenesulfonate.

Background

The homopiperazine is synthesized by using ethylenediamine as a starting material and performing three-step reactions of sulfonylation, cyclization and desulfonylation, wherein methyl p-toluenesulfonate (MTS), ethyl p-toluenesulfonate (ETS) and other unknown impurities may remain, and the homopiperazine is used as a starting material for medicine synthesis, so that the residues of methyl p-toluenesulfonate (MTS) and ethyl p-toluenesulfonate (ETS) need to be detected and controlled in quality control.

The application number 2015100770097 discloses a method for preparing homopiperazine, which comprises the steps of using ethylenediamine as a raw material, carrying out acylation reaction with p-toluenesulfonyl chloride in an N-butanol solvent to obtain N, N '-di-p-toluenesulfonyl ethylenediamine, directly carrying out cyclization reaction with bromochloropropane under the action of sodium hydroxide without separating a product from a reaction solution to obtain N, N' -di-p-toluenesulfonyl homopiperazine, then carrying out desulfonylation under the action of hydrobromic acid and phenol to obtain homopiperazine hydrobromide, and finally carrying out sodium hydroxide dissociation and toluene carrying water to obtain high-purity homopiperazine. The technical scheme does not disclose a method for detecting residues in homopiperazine.

The present invention has been made in view of this situation.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a method for detecting residues in homopiperazine, in particular a method for detecting residues in homopiperazine, namely methyl p-toluenesulfonate and ethyl p-toluenesulfonate.

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

the invention aims to provide a method for detecting methyl p-toluenesulfonate and ethyl p-toluenesulfonate in high piperazine residue, which comprises the following steps of:

(1) weighing a high piperazine product, adding a solvent for dissolving, fixing the volume, and uniformly mixing to obtain a test solution;

(2) respectively weighing reference substances of methyl p-toluenesulfonate and ethyl p-toluenesulfonate, respectively placing in a measuring flask, adding a solvent for dissolving, fixing the volume, uniformly mixing, and diluting to obtain reference substance stock solution; diluting the reference substance stock solution to a set concentration to serve as a reference substance solution, and diluting the reference substance solution to serve as a sensitivity solution;

(3) the determination is carried out by adopting a liquid chromatography-mass spectrometry combined method:

the liquid chromatography determination conditions were: gradient elution is carried out on a mobile phase A which is a pure water solution of 0.1% formic acid and a mobile phase B which is a methanol solution of 0.1% formic acid, the column temperature is 40 ℃, the flow rate is 0.6ml per minute, the sample injection temperature is 5 ℃, and the sample injection volume is 5 mu l;

the mass spectrum conditions are as follows: an ion source API-ES adopts a mass spectrum scanning mode of multi-reaction monitoring; the inlet voltage is 8V, the outlet voltage is 10V, the air curtain pressure is 25Psi, the collision gas CAD is 10Psi, the spray voltage is 5500V, and the ion source temperature is 430 ℃; the atomization gas pressure was 55Psi and the assist gas pressure was 55 Psi.

In the further scheme, in the step (1) and the step (2), the solvent is a mixed solution of acetonitrile and water with the volume percentage of 0.5% formic acid.

In a further embodiment, the volume ratio of 0.5% formic acid in acetonitrile to 0.5% formic acid in water in the mixed solution is 30-50: 50-70.

In a further embodiment, the volume ratio of 0.5% formic acid in acetonitrile to 0.5% formic acid in water in the mixed solution is 30: 70.

In a further scheme, in the step (3), the time and the mobile phase ratio of gradient elution are as follows: 0.01-6.00min, the volume percentage of the mobile phase A is reduced from 60% to 5%, the volume percentage of the mobile phase B is increased from 40% to 95% and 6.00-6.10min, the volume percentage of the mobile phase A is increased from 5% to 60%, the volume percentage of the mobile phase B is reduced from 95% to 40% and 6.10-10.00min, the volume percentage of the mobile phase A is maintained at 60%, and the volume percentage of the mobile phase B is maintained at 40%.

In a further embodiment, in the liquid chromatography determination conditions of step (3), the chromatographic column used is an Agilent Zorbax SB-C8 column.

In a further scheme, in the step (3), acetonitrile-pure water is adopted as the needle washing liquid, wherein the volume ratio of the acetonitrile to the pure water is 30: 70.

In a further embodiment, in the step (3), the running time under the liquid chromatography measurement condition is 10 min.

In a further scheme, the signal-to-noise ratio S/N of the methyl p-toluenesulfonate and the ethyl p-toluenesulfonate in the sensitivity solution is not less than 10.

In a further scheme, the peak area is calculated according to an external standard method, and the p-toluenesulfonic acid methyl ester and the p-toluenesulfonic acid ethyl ester are not more than 8 ppm.

The limit requirements for methyl p-toluenesulfonate (MTS) and ethyl p-toluenesulfonate (ETS) in high piperazine products are each not more than 8 ppm. The structural formula of methyl p-toluenesulfonate (MTS) is shown as the following formula I, and the structural formula of ethyl p-toluenesulfonate (ETS) is shown as the following formula II.

As a specific embodiment, the method for detecting the residues of methyl p-toluenesulfonate (MTS) and ethyl p-toluenesulfonate (ETS) in homopiperazine comprises the following steps:

(1) precisely weighing 50mg of the product, placing the product in a 10ml measuring flask, adding a solvent to dissolve and dilute the product to a scale, and shaking the product uniformly to obtain a test solution. Precisely weighing about 50mg of MTS and about 50mg of ETS respectively, placing the MTS and the ETS into a 50ml measuring flask, dissolving the MTS and the ETS by using a solvent and diluting the MTS and the ETS to scales, shaking up, precisely weighing 80 mu l of the MTS and the ETS, placing the MTS and the ETS into a 20ml measuring flask, diluting the MTS and the ETS to scales by using the solvent and shaking up to obtain a reference substance stock solution; another 0.1ml sample was removed precisely and placed in a 10ml measuring flask, diluted to the mark with solvent and shaken up to give a control solution. 2.0ml of the control solution was precisely measured, placed in a 10ml measuring flask, diluted to the mark with a solvent and shaken up to give a sensitive solution.

(2) The mobile phase A was a 0.1% formic acid solution in pure water (v/v) and the mobile phase B was a 0.1% formic acid solution in methanol (v/v) as determined by liquid chromatography-mass spectrometry (LC-MS), and the conditions of gradient elution and mass spectrometry are shown in Table 1. The column temperature was 40 ℃, the flow rate was 0.6ml per minute, the injection temperature was 5 ℃ and the injection volume was 5. mu.l. The signal-to-noise ratio (S/N) of MTS and ETS in the sensitive solution is not less than 10. The MTS and the ETS both have to exceed 8ppm calculated by peak area according to an external standard method.

TABLE 1

The solvent in step (1) was 0.5% formic acid in acetonitrile-water (30: 70, v/v).

The chromatographic column used in step (2) was an Agilent Zorbax SB-C8 column (4.6X 75mm, 3.5 μm).

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

1. in the detection method, the diluent adopts a mixed solution of 0.5% of formic acid acetonitrile and 0.5% of formic acid water (the volume ratio is 30-50:50-70) as the diluent, so that the hydrolysis of methyl p-toluenesulfonate (MTS) and ethyl p-toluenesulfonate (ETS) is inhibited, the methyl p-toluenesulfonate (MTS) and the ethyl p-toluenesulfonate (ETS) are directly detected, and the method is simple.

2. The detection method of the residues in the high piperazine, namely methyl p-toluenesulfonate (MTS) and ethyl p-toluenesulfonate (ETS), provided by the invention, proves that the method can be used for the detection method of the residues in the high piperazine, namely methyl p-toluenesulfonate (MTS) and ethyl p-toluenesulfonate (ETS), through the methodological research on the specificity, the system applicability, the linear range, the quantitative limit/detection limit, the recovery rate, the accuracy, the precision, the solution stability and the method durability of the method, the separation detection effect is good, and the guarantee can be provided for the quality control of the high piperazine.

The following describes 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, 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 without limiting the invention to the right. It is obvious that the drawings in the following description are only some embodiments, and that for a person skilled in the art, other drawings can be derived from them without inventive effort. In the drawings:

FIG. 1 is a chromatogram obtained by detection using mobile phase 1 in example 1; a is a chromatogram of a sample obtained by adopting mobile phase 1 detection; b is a chromatogram of a positive control, and C is a chromatogram of a negative control;

FIG. 2 is a chromatogram obtained by detection using mobile phase 2 in example 1; a is a chromatogram of a sample obtained by adopting mobile phase 2 detection; b is a chromatogram of a positive control, and C is a chromatogram of a negative control;

FIG. 3 is a chromatogram obtained by detection using mobile phase 3 in example 1; a is a chromatogram of a sample obtained by adopting mobile phase 3 detection; b is a chromatogram of a positive control, and C is a chromatogram of a negative control;

FIG. 4 shows diluent 1 (50% H) used in example 2₂O-50% ACN) for detecting chromatograms of 1000ng/mL MTS (A) and ETS (B) control solutions;

FIG. 5 shows diluent 1 (50% H) used in example 2₂O-50% ACN) to detect the chromatogram of the test solution added with 1000ng/mL MTS and ETS; wherein MTS is not detected (A), ETS is detected (B);

FIG. 6 shows diluent 2 (50% H) used in example 2₂O-50% ACN-0.1% FA) detecting the chromatogram of the control solution of MTS and ETS of 1000 ng/mL; wherein MTS is not detected (A), ETS is detected (B);

FIG. 7 shows diluent 2 (50% H) used in example 2₂O-50% ACN-0.1% FA) detection spiked 1Chromatograms of test solutions of 000ng/mL MTS and ETS; wherein MTS is not detected (A), ETS is detected (B);

FIG. 8 shows diluent 3 (50% H) used in example 2₂O-50% ACN-0.5% FA) detecting the chromatogram of the control solution of MTS and ETS of 1000 ng/mL; wherein A is an MTS contrast solution chromatogram, and B is an ETS contrast solution chromatogram;

FIG. 9 shows diluent 3 (50% H) used in example 2₂O-50% ACN-0.5% FA) to detect the chromatogram of the test solution of MTS and ETS added with standard 1000 ng/mL; wherein MTS (A) and ETS (B) are measured;

FIG. 10 is a chromatogram of a control obtained by the assay of example 3; wherein, STD is 40 ng/mL;

FIG. 11 is a chromatogram of a spiked sample detected in example 3; wherein, 100% Spiked is 5 mg/mL;

FIG. 12 is a chromatogram of blank, control solution and spiked sample solution in the specificity test in test example 2;

FIG. 13 is a graph showing the linearity (20% to 200%) of methyl p-toluenesulfonate in test example 5;

FIG. 14 is a graph showing the linearity (20% to 200%) of ethyl p-toluenesulfonate in test example 5.

It should be noted that the drawings and the description are not intended to limit the scope of the inventive concept in any way, but to illustrate it by a person skilled in the art with reference to specific embodiments.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and the following embodiments are used for illustrating the present invention and are not intended to limit the scope of the present invention.

Example 1

In this example, methyl p-toluenesulfonate (MTS) and ethyl p-toluenesulfonate (ETS) were hydrolyzed to p-toluenesulfonic acid under alkaline conditions, and MTS and ETS were indirectly quantified by detecting p-toluenesulfonic acid.

Precisely weighing 50mg of the product, placing the product in a 10ml measuring flask, adding a solvent to dissolve and dilute the product to a scale, and shaking the product uniformly to obtain a test solution. Precisely weighing about 50mg of MTS and about 50mg of ETS respectively, placing the MTS and the ETS into a 50ml measuring flask, dissolving the MTS and the ETS by using a solvent and diluting the MTS and the ETS to scales, shaking up, precisely weighing 80 mu l of the MTS and the ETS, placing the MTS and the ETS into a 20ml measuring flask, diluting the MTS and the ETS to scales by using the solvent and shaking up to obtain a reference substance stock solution; another 0.1ml sample was removed precisely and placed in a 10ml measuring flask, diluted to the mark with solvent and shaken up to give a control solution.

Several basic mobile phases were tried in this example, and the liquid phase conditions in table 2 below were specifically used for the detection.

TABLE 2

As a result:

the chromatogram, positive control (MS positive scan), and negative control (MS negative scan) of the sample obtained by mobile phase 1 assay are shown in A, B, C in FIG. 1, the chromatogram, positive control, and negative control chromatogram obtained by mobile phase 2 assay are shown in A, B, C in FIG. 2, and the chromatogram, positive control, and negative control chromatogram obtained by mobile phase 3 assay are shown in A, B, C in FIG. 3.

And (4) conclusion: in the three mobile phase systems, MTS and ETS are not easy to hydrolyze when the control is tested, and MTS and ETS cannot be detected by measuring p-toluenesulfonic acid.

TABLE 3

Example 2

MTS hydrolyzed in the spiked solution at a sample concentration of 10mg/mL, and ETS was not hydrolyzed. An attempt was made to adjust the pH of the sample solution by adding formic acid to the diluent in an amount such that M isTS and ETS can keep the prototype, inhibit its hydrolysis, and directly detect it. 3 different diluents (i.e. solvents to formulate the sample) were tried, respectively: diluent 1: 50% H₂O-50% ACN, diluent 2: 50% H₂O-50% ACN-0.1% FA, diluent 3: 50% H₂O-50% ACN-0.5% FA, and were tested using the test conditions as set forth in Table 4 below.

TABLE 4

As a result:

the chromatogram obtained by the detection with the diluent 1 is shown in fig. 4-5, MTS in the labeled sample is not detected, and ETS can be detected; the chromatogram obtained by the detection of the diluent 2 is shown in fig. 6-7, the MTS peak value in the labeled sample is very small, and ETS can be detected; the chromatograms obtained by using diluent 3 for detection are shown in FIGS. 8-9, and MTS and ETS in the labeled sample can be detected.

TABLE 5

Diluent (solvent)	Whether MTS in the spiked solution is hydrolyzed	Whether ETS in the target solution is hydrolyzed
			50％H₂O-50％ACN	Is that	Whether or not
50％H₂O-50％ACN-0.1％FA	Is that	Whether or not
			50％H₂O-50％ACN-0.5％FA	Whether or not	Whether or not

The results in three different diluents show that at 50% H₂Both MTS and ETS were detected in spiked samples in O-50% ACN-0.5% FA. This solvent was therefore selected for subsequent process studies.

The MTS in spiked solution was found to be more stable when the diluent water ratio was high, using 0.5% FA in H₂O: 0.5% FA in ACN 7:3 as solvent, while the temperature of the sample plate is controlled at 5 ℃ under which the MTS and ETS in the standard solution are stabilized for at least 12 h.

Example 3

The detection method for MTS and ETS residues in homopiperazine is pre-verified, and the detection conditions and results are shown in Table 6.

TABLE 6

The chromatograms obtained by detection are shown in FIG. 10(STD 40ng/mL) and FIG. 11 (100% Spiked 5 mg/mL).

The method has the following characterization results:

1. specificity: no interference was observed during the retention time of MTS, ETS.

2. Sensitivity solution: MTS 8ng/mL (1.6ppm), signal-to-noise ratio S/N54; ETS 8ng/mL (1.6ppm) and signal-to-noise ratio S/N20.

3. Linearity: MTS 8ng/mL to 80ng/mL (1.6to 16ppm, r 0.9989)

ETS:8ng/mL to 80ng/mL(1.6to 16ppm,r＝0.9983)

4. Precision:

MTS:

RSD% of 6 repeated injections_RT0.1% (n ═ 6);

RSD% of 6 repeated injections_AreaIs 5.6% (n ═ 6)

ETS:

RSD% of 6 repeated injections_RT0.2% (n ═ 6);

RSD% of 6 repeated injections_AreaIs 4.3% (n ═ 6)

5. Reproducibility of

MTS, standard addition 4 ppm: the recovery rate is 111%, (n is 2)

Adding standard 8 ppm: the recovery rate is 95%, (n is 2)

Adding standard 12 ppm: the recovery rate is 102%, (n is 2)

ETS, standard addition 4 ppm: the recovery rate is 87%, (n is 2)

Adding standard 8 ppm: the recovery rate is 86%, (n is 2)

Adding standard 12 ppm: the recovery rate is 89%, (n is 2)

6. Stability of

MTS is stable in the diluent for at least 12 hours, (initial content/12 h content)% > 110.8%

MTS was stable in the spiked solution for at least 12 hours, (initial content/12 h content)% > 108.0%

The ETS is stable in the diluent for at least 12 hours, (initial content/12 h content)% > 91.1%

The ETS was stable in the spiked solution for at least 12 hours, (initial content/12 h content)% > 103.4%

And (4) conclusion: the method pre-verification result shows that the result meets the acceptance standard and the method verification can be carried out.

Test example 1 System Adaptation/method parameters

(1) Experimental methods

Validation studies using chromatographic systems that meet system adaptability requirements in methods

(2) Acceptance criteria

The system is clean and stable, and has no interference at the peak position of the main peak. The peak area of the interference peak, if any, is no more than 30% of the peak area of the main peak in the sensitivity solution.

The signal-to-noise ratio (S/N) of MTS and ETS in the sensitive solution is not less than 10

The RSD% of the peak area in the initial 6-pin reference solution is not more than 15.0%

The retention time RSD% in the initial 6-pin control solution was not greater than 1.0%

The recovery rate of the inserted control solution is between 70.0 and 130.0 percent.

In the formula: h represents the peak height from the peak top to the baseline; h represents background noise in the chromatogram

As a result: all verification parameters in the system adaptability test result of the method of the invention meet the acceptance standard, and detailed data are shown in tables 7 and 8.

TABLE 7 results of system adaptability test

Remarking: ND is not detected

TABLE 8 results of system adaptability test

Test example 2 specificity

(1) Experimental methods

The following representative samples were prepared: blank (solvent), 100% limit control solution for each test article,

a spiked sample solution to the 100% limit;

and (5) feeding and analyzing the special detection solution.

(2) Acceptance criteria

In the blank chromatogram, there was no significant interference at the target peak. The peak area of the interference peak, if any, must not be greater than 30% of the average integrated area of the main peak of the LOQ.

A single test article control solution at the 100% limit shows the target peak.

The control solution at the 100% limit shows the target peak.

And no interference exists near a target peak in the chromatogram of the standard sample with the limit of 100 percent.

(3) Results

In the blank chromatogram, there was no interfering peak at the retention time of the target peak, and the specificity test sample chromatogram is shown in fig. 12.

The target peaks were well localized and the Retention Times (RT) are shown in Table 9.

TABLE 9 Retention time and degree of separation

Test example 3 detection Limit (LOD)

(1) Experimental methods

Prepare a 10% limit control solution as LOD solution

LOD solution sample injection analysis 3 times

Calculating the signal-to-noise ratio of the LOD

(2) Acceptance criteria

The signal-to-noise ratio (S/N) of LOD solution per needle is more than or equal to 3

(3) The resulting signal to noise ratio (S/N) calculations are shown in Table 10.

TABLE 10 detection Limit (LOD)

Test example 4 quantitative Limit (LOQ)

(1) Experimental methods

A 20% limit control solution was prepared as the LOQ solution,

the sample introduction of the LOQ solution is analyzed for 6 times,

the signal-to-noise ratio of the LOQ is calculated,

the% RSD of the target peak-to-peak area in the six needles LOQ was calculated.

(2) Acceptance criteria

The signal-to-noise ratio (S/N) of each LOQ solution is more than or equal to 10;

the% RSD of the peak-to-peak area of the target peak in the continuous 6-pin LOQ is less than or equal to 30%

(3) Results

The results of the signal to noise ratio (S/N) calculations are shown in tables 11 (limits for quantitation of methyl p-toluenesulfonate) and 12 (limits for quantitation of ethyl p-toluenesulfonate).

TABLE 11 limits of quantitation (methyl p-toluenesulfonate)

TABLE 12 limits of quantitation (ethyl p-toluenesulfonate)

Test example 5 linearity

(1) Experimental methods

A series of linear test solutions (20%, 50%, 100%, 150%, 200%) were prepared from LOQ to 200% concentration, each solution was analyzed by injection,

calculation equation and regression coefficient (r)

(2) Acceptance criteria

The regression coefficient (r) is more than or equal to 0.990,

reporting regression coefficients, slopes, y-intercept and residual sum of squares

(3) Results

The linearity test results meet the acceptance criteria, see table 13 and fig. 13 and 14.

TABLE 13 results of the Linear test

Test example 6 accuracy

(1) Experimental methods

Three spiked sample solutions were prepared with each of the three limiting levels of 50%, 100% and 150% standard solution

Three sample solutions were prepared according to the method

Each solution is injected and analyzed once

Calculate the recovery per solution and the average recovery per concentration level

(2) Acceptance criteria

The recovery of the individual spiked solutions was between 75-120%.

(3) Results

The results of the accuracy experiments are shown in tables 14 (methyl p-toluenesulfonate) and 15 (ethyl p-toluenesulfonate).

TABLE 14 accuracy (methyl p-toluenesulfonate)

TABLE 15 accuracy (ethyl p-toluenesulfonate)

Test example 7 reproducibility

(1) Experimental methods

6 parts of the spiked sample solution were prepared at the 100% limit level. The three solutions prepared in the accuracy test can also be used for calculating the repeatability, and each solution is injected and analyzed once

% RSD to calculate recovery of 6 spiked solutions

(2) Acceptance criteria

The percent RSD of the recovery rate of 6 parts of 100 percent standard-adding solution is less than or equal to 20 percent

(3) Results

The results are shown in Table 16 (methyl p-toluenesulfonate) and 17 (ethyl p-toluenesulfonate).

TABLE 16 repeatability (methyl p-toluenesulfonate)

TABLE 17 repeatability (ethyl p-toluenesulfonate)

Experimental example 8 intermediate precision

(1) Experimental methods

Additional investigators prepared 6 solutions of the spiked sample at the 100% limit level at different times.

Each solution is injected and analyzed once

Calculating% RSD for recovery of 12 aliquots of the spiked sample solution at the 100% limit level

(2) Acceptance criteria

The percent RSD of the recovery rate of the solution of the standard added sample is less than or equal to 20 percent

(3) Results

Intermediate precision results are shown in tables 18 (methyl p-toluenesulfonate) and 19 (ethyl p-toluenesulfonate).

TABLE 18 intermediate precision (methyl p-toluenesulfonate)

TABLE 19 intermediate precision (ethyl p-toluenesulfonate)

Test example 9 solution stability

(1) Experimental methods

A 100% limit level of each of the control solution and spiked sample solution is prepared and these solutions are analyzed at 24 hours or other time periods and compared to freshly prepared control solutions. If there is no stability problem, a 24 hour investigation is required.

The stability period is the time interval for which the acceptance criterion is met.

(2) Acceptance criteria

The content of the stabilizing solution is 70-130% of the initial content.

(3) Results the solution stability results are shown in tables 20 and 21.

Table 20100% solution stability of control solution

TABLE 21100% level spiked sample solution stability

Test example 10 Range

(1) Experimental methods

Data determination according to method linearity and accuracy (50% -150%)

(2) Acceptance criteria

Is free of

(3) Results

The method range is 50% to 150% as demonstrated by the method linearity and accuracy verification results. The results are shown in Table 22.

TABLE 22 method detection Range

Name (R)	Range
		P-toluenesulfonic acid methyl ester	19.33ng/mL～57.98ng/mL
P-toluenesulfonic acid ethyl ester	19.40ng/mL～58.19ng/mL

Test example 11 durability

(1) Experimental methods

According to the chromatographic conditions in the following table, the influence of small changes of the analytical method on the system is examined.

TABLE 23

The system adaptability of the method after change needs to be examined during testing.

One aliquot of the spiked sample solution was prepared at the 100% limit, and injected once per condition.

The recovery of the spiked sample solution under durability conditions was calculated.

(2) Acceptance criteria

If the recovery rate of the standard sample solution is between 75 and 120 percent under the durability condition, if the durability method does not meet the requirement, a description can be made.

(3) Results

The results of the system suitability are shown in tables 24-16, and the results of the durability are shown in Table 27.

TABLE 24 results of system adaptability test

TABLE 25 results of System Adaptation test

TABLE 26 results of System Adaptation testing

TABLE 27 durability test results

Conclusion

All validation parameters met the acceptance criteria, so the method is applicable to residue testing of methyl p-toluenesulfonate (MTS) and ethyl p-toluenesulfonate (ETS) in high piperazine residues.

Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. A method for detecting methyl p-toluenesulfonate and ethyl p-toluenesulfonate in high piperazine residues is characterized by comprising the following steps of:

2. The method for detecting methyl p-toluenesulfonate and ethyl p-toluenesulfonate in high piperazine residue according to claim 1, wherein in step (1) and step (2), the solvent is a mixed solution of acetonitrile and water containing 0.5% by volume of formic acid.

3. The method for detecting methyl p-toluenesulfonate and ethyl p-toluenesulfonate in high piperazine residue according to claim 2, wherein the volume ratio of 0.5% formic acid in acetonitrile to 0.5% formic acid in water in the mixed solution is 30-50: 50-70.

4. The method for detecting methyl p-toluenesulfonate and ethyl p-toluenesulfonate in high piperazine residue according to claim 3, wherein the volume ratio of 0.5% formic acid in acetonitrile to 0.5% formic acid in water in the mixed solution is 30: 70.

5. The method for detecting methyl p-toluenesulfonate and ethyl p-toluenesulfonate in high piperazine residue according to any one of claims 1 to 4, wherein in step (3), the gradient elution time and the mobile phase ratio are as follows: 0.01-6.00min, the volume percentage of the mobile phase A is reduced from 60% to 5%, the volume percentage of the mobile phase B is increased from 40% to 95% and 6.00-6.10min, the volume percentage of the mobile phase A is increased from 5% to 60%, the volume percentage of the mobile phase B is reduced from 95% to 40% and 6.10-10.00min, the volume percentage of the mobile phase A is maintained at 60%, and the volume percentage of the mobile phase B is maintained at 40%.

6. The method for detecting methyl p-toluenesulfonate and ethyl p-toluenesulfonate in high piperazine residue according to any one of claims 1 to 5, wherein the chromatographic column used in the liquid chromatography determination conditions of step (3) is an Agilent Zorbax SB-C8 column.

7. The method for detecting methyl p-toluenesulfonate and ethyl p-toluenesulfonate in high piperazine residue according to any one of claims 1 to 6, wherein acetonitrile-pure water is used as the needle washing liquid in step (3), wherein the volume ratio of acetonitrile to pure water is 30: 70.

8. The method for detecting methyl p-toluenesulfonate and ethyl p-toluenesulfonate in the high piperazine residue according to any one of claims 1 to 7, wherein in the step (3), the operation time under the liquid chromatography measurement condition is 10 min.

9. The method for detecting methyl p-toluenesulfonate and ethyl p-toluenesulfonate in the high piperazine residue according to any one of claims 1 to 8, wherein the S/N ratio of methyl p-toluenesulfonate to ethyl p-toluenesulfonate in the sensitive solution is not less than 10.

10. The method for detecting methyl p-toluenesulfonate and ethyl p-toluenesulfonate in the high piperazine residue according to any one of claims 1 to 9, wherein the amount of methyl p-toluenesulfonate and ethyl p-toluenesulfonate is not more than 8ppm in terms of peak area by an external standard method.