CN113552257B

CN113552257B - Method for determining isomer impurities in p-fluorobenzothiophenol by HPLC (high performance liquid chromatography) method

Info

Publication number: CN113552257B
Application number: CN202110820160.0A
Authority: CN
Inventors: 白玮丽; 杨玉娜; 刘洪明; 许多; 温乐
Original assignee: Jari Pharmaceutical Co ltd
Current assignee: Jari Pharmaceutical Co ltd
Priority date: 2021-07-20
Filing date: 2021-07-20
Publication date: 2023-03-14
Anticipated expiration: 2041-07-20
Also published as: CN113552257A

Abstract

The invention relates to the technical field of chemical pharmacy, in particular to a method for measuring isomer impurities in p-fluorothiophenol by an HPLC (high performance liquid chromatography) method, which is used for quantitatively detecting an isomer A, an isomer B and thiophenol in the p-fluorothiophenol, wherein the isomer A is 2-fluorothiophenol, and the isomer B is 3-fluorothiophenol; adding TCEP solution into p-fluorophenylthiol sample, diluting to obtain sample solution, and detecting the sample solution by high performance liquid chromatography to obtain the content of three impurities in the p-fluorophenylthiol sample. The method can effectively separate the p-fluorophenylthiophenol and related substances thereof, and the generation of disulfide is prevented by adding the TCEP solution before the test, so that the p-fluorophenylthiophenol and impurities can exist in a single molecular state, and the method is simple and convenient to operate, strong in specificity, high in sensitivity and accuracy, and controllable in quality of the p-fluorophenylthiophenol.

Description

Method for determining isomer impurities in p-fluorobenzothiophenol by HPLC (high performance liquid chromatography) method

Technical Field

The invention relates to the technical field of chemical pharmacy, in particular to a method for determining isomer impurities in p-fluorobenzenethiol by an HPLC method.

Background

The p-fluorobenzothiaol is an important intermediate of medicines, pesticides and polymers, and can also be used for detecting chemiluminescence agents and preparing nonlinear optical materials and the like. Isomer impurities are introduced in the production process of p-fluorophenylthiophenol, and p-fluorophenylthiophenol and the isomer impurities are unstable and easily generate disulfide compounds with each other.

At present, no report is available on the detection method of isomer impurities in p-fluorophenylthiol, so that the development of an HPLC method capable of effectively detecting the isomer impurities in p-fluorophenylthiol is necessary.

Disclosure of Invention

The invention aims to overcome the problems in the prior art and provide a method for determining isomer impurities in p-fluorobenzenethiol by an HPLC method.

In order to achieve the technical purpose and achieve the technical effect, the invention is realized by the following technical scheme:

a method for determining isomer impurities in p-fluorothiophenol by an HPLC method is used for quantitatively detecting an isomer A, an isomer B and thiophenol in p-fluorothiophenol, wherein the isomer A is 2-fluorothiophenol, and the isomer B is 3-fluorothiophenol;

adding TCEP solution into the p-fluorophenylthiophenol sample, diluting to obtain a test solution, detecting the test solution by adopting high performance liquid chromatography to obtain the contents of three impurities in the p-fluorophenylthiophenol sample, wherein the chromatographic conditions are as follows:

and (3) chromatographic column: C18-PHP column, 4.6mm × 250mm,5 μm;

mobile phase: methanol and 0.05% aqueous trifluoroacetic acid;

column temperature: 25-45 ℃;

detection wavelength: 200-300nm;

mobile phase elution mode: the elution is carried out at an equal rate,

further, the method for determining isomer impurities in p-fluorobenzenethiol by the HPLC method is as described above, and the mass concentration of the TCEP solution is 8-12mg/ml.

Further, the HPLC method is used for determining the isomer impurities in the p-fluorobenzothiophenol, and the column length of the chromatographic column is 100-250mm.

Further, the HPLC method as described above is used for measuring the isomer impurities in p-fluorophenylthiol, and the flow rate of the mobile phase is 0.5 to 1.5ml/min.

Further, the HPLC method as described above is used for the determination of the isomer impurities in p-fluorophenylthiol, and the flow rate of the mobile phase is 1ml/min.

Further, the HPLC method described above was used to determine the isomeric impurities in p-fluorophenylthiol, and the volume ratio of methanol to 0.05% aqueous trifluoroacetic acid in the mobile phase was 1.

Further, the HPLC method as described above is used for determining the isomer impurities in p-fluorophenylthiol, and the column temperature is 35 ℃.

Further, the method for measuring isomer impurities in p-fluorophenylthiol by the HPLC method as described above, wherein the detection wavelength is 238nm.

The beneficial effects of the invention are:

the invention can effectively separate p-fluorothiophenol and related substances thereof, and prevents the generation of disulfide compounds by adding TCEP solution before testing, so that p-fluorothiophenol and impurities can exist in a single molecular state, thereby providing a method for quantitatively detecting 3 impurity contents of thiophenol, 2-fluorothiophenol and 3-fluorothiophenol in p-fluorothiophenol.

Of course, it is not necessary for any one product that embodies the invention to achieve all of the above advantages simultaneously.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the description below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 shows the results of the system applicability test of the present invention;

FIG. 2 is a result of the specificity test of the present invention;

FIG. 3 is a quantification limit test result of the present invention;

FIG. 4 is a graph of the results of the linearity test of the present invention;

FIG. 5 shows the result of the accuracy test of the present invention;

FIG. 6 is a blank solution profile of the present invention;

FIG. 7 is a diagram of a solution for the applicability of the system of the present invention;

FIG. 8 is a reference solution profile of the present invention;

FIG. 9 is a sample solution profile of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention provides a method for detecting isomer impurities in p-fluorobenzothiacol by an HPLC method, which specifically comprises the following steps:

1) Selecting an instrument: a high performance liquid chromatograph; the chromatographic column is C18-PFP,4.6mm multiplied by 250mm,5 μm or the chromatographic column with equivalent performance; mobile phase: 0.05% aqueous trifluoroacetic acid-methanol (50; an ultraviolet detector is adopted, and the detection wavelength is 200-300nm; the flow rate of the mobile phase is 0.5-1.5ml/min; the column temperature is 25-45 ℃; the length of the chromatographic column is 100-250mm;

2) Preparing a solution, namely preparing the following solutions:

a. TCEP solution: taking a proper amount of tris (2-carboxyethyl) phosphine hydrochloride (TCEP), adding water to dissolve and dilute the solution to prepare a solution containing 10mg of the tris (2-carboxyethyl) phosphine hydrochloride per 1 ml; (TCEP is intended for the selective reduction of disulfide compounds);

b. blank solution: taking 2ml of TCEP solution, placing the TCEP solution in a 50ml measuring flask, and diluting the TCEP solution to the scale with methanol;

c. impurity stock solution: precisely weighing about 30mg of sodium thiophenol, about 25mg of 3-fluorophenylthiophenol and about 25mg of 2-fluorophenylthiophenol respectively, placing the weighed materials into a same 100ml measuring flask, adding methanol to dissolve and dilute the materials to a scale, and shaking up the materials to be used as impurity stock solutions;

d. system applicability solution: taking about 50mg of the product, placing into a 50ml measuring flask, adding a proper amount of methanol to dissolve, adding 1.0ml of impurity stock solution and 2ml of TCEP solution, shaking up, and diluting with methanol to scale;

e. control solution: precisely measuring 1.0ml of impurity stock solution, placing the impurity stock solution in a 50ml measuring flask, adding a proper amount of methanol to dissolve the impurity stock solution, adding 2ml of TCEP solution, shaking up, and diluting the solution to scale with methanol;

f. test solution: taking about 50mg of the product, placing the product in a 50ml measuring flask, adding a proper amount of methanol to dissolve the product, adding 2ml of TCEP solution, shaking up, and diluting the solution to the scale with methanol.

Injecting samples according to the following sequence table:

name (R)	Number of sample introduction needles
		Blank solution	Needle with a diameter of not less than 1
System applicability solution	1 needle
		Control solution
	6 needles
		Test solution
	1 needle

And calculating each impurity in the p-fluorophenylthiol test sample solution by peak area according to an external standard method.

(1) The chromatographic conditions of the high performance liquid chromatography comprise: an ultraviolet detector is adopted, and the detection wavelength is preferably 238nm; the optimal flow rate of the mobile phase is 1.0ml/min; the column temperature was 35 ℃; the chromatographic column is preferably ACEExclec18-PFP,4.6mm × 250mm,5 μm; the column length was 250mm.

(2) The TCEP solution is intended for the selective reduction of disulfide compounds.

The related substances comprise thiophenol, isomer impurities of 2-fluorobenzothiophenol and 3-fluorobenzothiophenol.

The following are related to specific embodiments of the present invention:

example 1

And (3) investigating system precision and specificity:

a method for detecting isomer impurities in p-fluorobenzenethiol by an HPLC method comprises the following steps: instrument and chromatographic conditions, instrument: a high performance liquid chromatograph; a chromatographic column: ACE Excel C18-PFP,4.6mm x 250mm,5 μm; mobile phase: 0.05% aqueous trifluoroacetic acid-methanol (50: 238nm, column temperature: 35 ℃, flow rate: 1.0ml/min, sample size: 20 μ L, isocratic elution, recording chromatogram to 2 times the retention time of the main peak.

Firstly, solution preparation is carried out, and each impurity positioning solution: precisely measuring 1.0ml of each impurity stock solution, respectively placing the impurity stock solutions into 50ml measuring flasks, respectively adding 2ml of TCEP solution, shaking up, and diluting with methanol to scale.

Precisely measuring blank solution, respectively measuring 20 μ L of system applicability solution, reference solution and impurity positioning solution, injecting into liquid chromatograph, and recording liquid chromatogram. The system precision results are shown in FIG. 1: in the table of FIG. 1, the RSD of the peak area of the needle control solution is 0.5% at most; the results of the specificity test are shown in FIG. 2: the experimental results of the positioning and separation degree of each impurity in the table of fig. 2 show that the blank solution has no interference to the sample detection, other impurities have no interference to the sample determination, and the minimum separation degree among the impurities is 1.8.

Example 2

Quantitative investigation:

precisely transferring an appropriate amount of impurity stock solution, adding 2ml of TCEP solution, adding a diluent to dissolve and dilute the TCEP solution to prepare solutions with the concentration of each impurity of about 0.05 mu g/ml, and using the solutions as quantitative limit solutions. And taking 20 mu L of the quantitative limit solution, carrying out continuous sample injection for 6 times, and recording a chromatogram, wherein the specific result is shown in figure 3, and the measurement result of the quantitative limit of each impurity shows that the ratio of the quantitative limit concentration of each impurity to the concentration of the test sample is 0.005%, and the RSD of the peak area of 6 parts of the quantitative limit solution is 8.1% at the maximum and less than 10%. The detection sensitivity of the impurities of the thiophenol, the 2-fluorobenzothiophenol and the 3-fluorobenzothiophenol is high, and the limit detection requirement of the impurities of various isomers in the fluorobenzothiophenol can be met.

Example 3

Linear investigation:

precisely transferring appropriate amount of impurity stock solution, dissolving with diluent, gradually diluting to obtain solutions with impurity concentrations of 0.05 μ g/ml, 1 μ g/ml, 2.5 μ g/ml, 5 μ g/ml, 7.5 μ g/ml and 10 μ g/ml, respectively, and preparing according to the method of "preparation of reference solution" to obtain linear solution. And (3) taking 20 mu L of each linear solution, injecting the linear solution into a liquid chromatograph, recording a chromatogram, and carrying out linear regression analysis by taking the concentration as a horizontal coordinate and the peak area as a vertical coordinate, wherein the linearity is good, the correlation coefficients are all 1.0000, and the specific result is shown in figure 4.

Example 4

And (3) accuracy examination:

precisely weighing 50mg of p-fluorophenylthiophenol sample, 9 parts in total, respectively placing the p-fluorophenylthiophenol sample into 50ml measuring bottles, respectively placing 3 parts of the p-fluorophenylthiophenol sample into 1 group, respectively adding 0.5ml, 1.0ml and 1.5ml of impurity stock solutions according to low, medium and high concentrations, preparing solutions according to a method of preparing a system applicability solution, respectively carrying out sample injection analysis, recording a chromatogram, calculating recovery rates, and enabling the average recovery rates of thiophenol, 2-fluorophenylthiophenol and 3-fluorophenylthiophenol to be within the range of 92% -108%, and obtaining a result shown in figure 5.

The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for determining isomer impurities in p-fluorophenylthiol by an HPLC method is characterized by being used for quantitatively detecting an isomer A, an isomer B and thiophenol in p-fluorophenylthiol, wherein the isomer A is 2-fluorophenylthiol, and the isomer B is 3-fluorophenylthiol;

adding TCEP solution into a p-fluorophenylthiol sample, diluting to obtain a test solution, detecting the test solution by adopting a high performance liquid chromatography to obtain the contents of three impurities in the p-fluorophenylthiol sample, wherein the chromatographic conditions are as follows:

a chromatographic column: C18-PHP column, 4.6mm × 250mm,5 μm;

mobile phase: methanol and 0.05% aqueous trifluoroacetic acid;

column temperature: 25-45 ℃;

detection wavelength: 200-300nm;

mobile phase elution mode: isocratic elution;

the volume ratio of methanol to 0.05% aqueous trifluoroacetic acid in the mobile phase was 1.

2. The method for measuring isomeric impurities in p-fluorophenylthiophenol by HPLC as claimed in claim 1, wherein: the mass concentration of the TCEP solution is 8-12mg/ml.

3. The method for measuring isomer impurities in p-fluorophenylthiol according to claim 1, which comprises: the length of the chromatographic column is 100-250mm.

4. The method for measuring isomer impurities in p-fluorophenylthiol according to claim 1, which comprises: the flow rate of the mobile phase is 0.5-1.5ml/min.

5. The method for measuring isomer impurities in p-fluorophenylthiol according to claim 4, which comprises: the flow rate of the mobile phase was 1ml/min.

6. The method for measuring isomer impurities in p-fluorophenylthiol according to claim 1, which comprises: the column temperature was 35 ℃.

7. The method for measuring isomer impurities in p-fluorophenylthiol according to claim 1, which comprises: the detection wavelength was 238nm.