CN113376294A

CN113376294A - Analysis method of ethyl cyanoacetate in tofacitinib citrate

Info

Publication number: CN113376294A
Application number: CN202110938566.9A
Authority: CN
Inventors: 谭稳; 毛倩; 马晓宁; 陈卉; 卢郭燕
Original assignee: HUNAN PUDAO MEDICAL TECHNOLOGY CO LTD; Hunan Jiudian Pharmaceutical Co Ltd
Current assignee: HUNAN PUDAO MEDICAL TECHNOLOGY CO LTD; Hunan Jiudian Pharmaceutical Co Ltd
Priority date: 2021-08-16
Filing date: 2021-08-16
Publication date: 2021-09-10

Abstract

The invention provides an analysis method of ethyl cyanoacetate in tofacitinib citrate, which adopts a gas chromatography, wherein a chromatographic column is selected from a capillary column taking 6% cyanopropylphenyl-94% dimethylpolysiloxane as a stationary liquid, a diluent is selected from acetonitrile-dilute acid solution, and the mass concentration of the dilute acid solution is 0.1-0.2%. The method for analyzing ethyl cyanoacetate in tofacitinib citrate has the advantages that the sample preparation is simple, the solvent is not required to be dissolved firstly and then diluted, the diluent can be directly used for dissolving and diluting to a constant volume, the experiment efficiency is improved in one step, the sample recovery rate is stable, and the accuracy is high.

Description

Analysis method of ethyl cyanoacetate in tofacitinib citrate

Technical Field

The invention belongs to the technical field of medicine detection and analysis, and particularly relates to an analysis method of ethyl cyanoacetate in tofacitinib citrate.

Background

Tofacitinib citrate, chemical name (3R,4R) -4-methyl-3- (methyl-7H-pyrrolo [2,3-d ] pyrimidin-4-ylamino) -beta-oxo-1-piperidinepropanil citrate, chemical structural formula as shown in the following figure:

tofacitinib citrate is the first small molecule non-receptor tyrosine protein kinase inhibitor approved for human autoimmune diseases, is mainly used for moderate to severe active Rheumatoid Arthritis (RA) adult patients with insufficient methotrexate curative effect or intolerance to the methotrexate, and can be used together with methotrexate or other non-biological disease-improving antirheumatic drugs (DMARDs).

Impurity research is an important part in the drug development process and is directly related to the safety and effectiveness of drugs. The main sources of impurities in the synthesis of tofacitinib citrate are impurities and process impurities introduced from starting raw materials, ethyl cyanoacetate is one of the starting materials of tofacitinib citrate, the substance has no chromogenic group, weak ultraviolet absorption, high boiling point, difficult gasification and high detection difficulty, and the content of the impurities needs to be strictly controlled in order to ensure the quality of the raw material medicines. Therefore, it is necessary to develop a method for detecting ethyl cyanoacetate in tofacitinib, which has simple operation, high accuracy and stable recovery rate.

Disclosure of Invention

The invention aims to provide an analysis method of ethyl cyanoacetate in tofacitinib citrate, which is simple to operate, stable in recovery rate and high in accuracy.

According to one aspect of the invention, the analysis method of ethyl cyanoacetate in tofacitinib citrate is realized by adopting a gas chromatography, a chromatographic column is a capillary column taking 6% cyanopropylphenyl-94% dimethylpolysiloxane as a stationary liquid, a diluent is an acetonitrile-dilute acid solution, and the mass concentration of the dilute acid solution is 0.1-0.2%.

Further, the diluted acid solution is selected from a phosphoric acid solution, a formic acid solution, an acetic acid solution or a trifluoroacetic acid solution, and the volume ratio of the acetonitrile to the diluted acid solution is 40-70: 60 to 30.

Further, the dilute acid solution is selected from a 0.1% phosphoric acid solution.

Further, the method for analyzing ethyl cyanoacetate in tofacitinib citrate comprises the following steps:

1) preparing a sample solution of tofacitinib citrate to be detected by using a diluent,

2) preparing a reference substance solution from ethyl cyanoacetate by using a diluent,

3) and (4) injecting the test solution and the reference solution into a gas chromatograph, and recording the chromatogram.

Further, in the step 3), the gas chromatography detection conditions are as follows: detector temperature: 250 ℃; sample inlet temperature: 250 ℃; column flow rate: 1.3-1.8 ml/min; the split ratio is as follows: 1-5: 1; the sample injection amount is 2-5 mu l; column temperature: the initial temperature is 100 +/-5 ℃, the temperature is maintained for 5-10 minutes, the temperature is increased to 200-220 ℃ at the rate of 10-20 ℃ per minute, and the temperature is maintained for 5-10 minutes.

Furthermore, the concentration of the ethyl cyanoacetate in the control solution is 1-20 mug/ml, preferably 4-20 mug/ml.

Further, the concentration of the test solution is 3-10 mg/ml.

Further, the detection limit concentration of the ethyl cyanoacetate is 0.2732 mu g/ml; the quantitative limit concentration was 1.0927. mu.g/ml.

Further, the concentration of the ethyl cyanoacetate solution was linear with peak area in the range of 0.8802. mu.g/ml to 23.4720. mu.g/ml.

The invention has the beneficial effects that:

the analysis method of ethyl cyanoacetate in tofacitinib citrate has the advantages that the sample preparation is simple, the sample does not need to be dissolved first and then diluted by a solvent, the sample can be directly dissolved and diluted by a diluent to a constant volume, the experiment efficiency is improved in one step, the sample recovery rate is stable and is in a concentration range of 50-150%, the recovery rate of ethyl cyanoacetate is 94.39-102.38%, the average recovery rate is 97.97%, the RSD is 2.78%, and the accuracy is high.

Drawings

FIG. 1-1 is a chromatogram of a control solution of example 1; FIGS. 1-2 are chromatograms of the test solution of example 1;

FIG. 2-1 is a chromatogram of the control solution of example 2; FIG. 2-2 is a chromatogram of the test solution of example 2;

FIG. 3-1 is a chromatogram of the control solution of example 3; FIG. 3-2 is a chromatogram of the test solution of example 3;

FIG. 4-1 is a chromatogram of the control solution of example 4; FIG. 4-2 is a chromatogram of the test solution of example 4.

Detailed Description

The present application will be described in further detail with reference to specific examples. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application.

The embodiment provides an analysis method of ethyl cyanoacetate in tofacitinib citrate, which comprises the steps of directly injecting samples by adopting a gas chromatography, and quantifying by adopting an external standard method, wherein a chromatographic column is selected from a capillary column taking 6% cyanopropyl phenyl-94% dimethyl polysiloxane as a stationary liquid, a diluent is selected from an acetonitrile-dilute acid solution, and the mass concentration of the dilute acid solution is 0.1-0.2%. A certain amount of dilute acid solution is added into acetonitrile, so that the solubility of the tofacitinib citrate is increased, the tofacitinib citrate can be directly dissolved by a diluent and diluted to a constant volume to prepare a sample solution, the prior art does not need to firstly dissolve by a solvent and then fix the volume by the diluent, the operation of experimenters is facilitated, and the experimental efficiency is improved. The volume ratio of the acetonitrile to the dilute acid solution is preferably 40-70: 60 to 30. The diluted acid solution is preferably a phosphoric acid solution, a formic acid solution, an acetic acid solution or a trifluoroacetic acid solution, and more preferably a phosphoric acid solution. The diluent adopts acetonitrile-dilute acid solution, so that the solubility of tofacitinib citrate can be increased, the dissolving dilution constant volume is in one step in sample preparation, the sample recovery rate of the analysis method is stable and is within the concentration range of 50-150%, the recovery rate of ethyl cyanoacetate is 94.39-102.38%, the average recovery rate is 97.97%, the RSD is 2.78%, the accuracy is high, the specificity is strong, the blank solution has no interference to the detection of ethyl cyanoacetate, and the separation degree between the ethyl cyanoacetate and the adjacent peak in the mixed solution is more than 1.5.

The method for analyzing ethyl cyanoacetate in tofacitinib citrate specifically comprises the following steps:

1) preparing a sample solution from a sample to be tested of tofacitinib citrate by using a diluent, wherein the concentration of the sample solution is 3-10 mg/ml.

2) Preparing a reference substance solution from the ethyl cyanoacetate reference substance by using a diluent, wherein the concentration of the ethyl cyanoacetate in the reference substance solution is 1-20 mu g/ml, and preferably 4-20 mu g/ml.

The gas chromatography detection conditions are as follows: detector temperature: 250 ℃; sample inlet temperature: 250 ℃; column flow rate: 1.3-1.8 ml/min; the split ratio is as follows: 1-5: 1; the sample injection amount is 2-5 mu l; column temperature: the initial temperature is 100 +/-5 ℃, the temperature is maintained for 5-10 minutes, the temperature is increased to 200-220 ℃ at the rate of 10-20 ℃ per minute, and the temperature is maintained for 5-10 minutes.

The analytical instrument used in this example was an Agilent 7890B gas chromatograph, phosphoric acid and trifluoroacetic acid were AR grade, acetonitrile was HPLC grade, a reference product ethyl cyanoacetate was AR grade, and tofacitinib citrate was prepared by oneself.

Example 1

Diluent solution: acetonitrile-0.1% aqueous phosphoric acid (50: 50).

Preparing a test solution: taking 0.1g of tofacitinib citrate, precisely weighing, placing in a 10ml measuring flask, adding a diluent solution, diluting to a scale, and shaking up to obtain the product.

Control solution: a suitable amount of ethyl cyanoacetate control was added with a diluent to prepare a solution containing about 10. mu.g of ethyl cyanoacetate per 1 ml.

Injecting 5 μ l of each of the sample solution and the reference solution into chromatograph, and recording chromatogram. Referring to FIGS. 1-1 and 1-2, FIG. 1-1 is a chromatogram of a control solution; FIG. 1-2 is a chromatogram of a test solution. As can be seen from the figure, in the analysis method of this example, ethyl cyanoacetate as a reference sample was effectively detected, and the peak in the sample did not interfere with the reference sample.

The chromatographic conditions were as follows:

a chromatographic column: 6% cyanopropylphenyl-94% dimethylpolysiloxane as a capillary column in a stationary liquid, in this example the column specification/model is Agilent DB-624 UI (30 m.times.0.32 mm.times.1.8 μm), FID detector;

detector temperature: 250 ℃; sample inlet temperature: 250 ℃; sample introduction amount: 5 mu l of the solution;

column flow rate: 1.3 ml/min; the split ratio is as follows: 3: 1; solvent: acetonitrile-0.1% aqueous phosphoric acid (50: 50);

column temperature: the temperature was started at 100 ℃ for 6 minutes, ramped up to 220 ℃ at a rate of 15 ℃ per minute, and held for 8 minutes.

Example 2

Diluent solution: acetonitrile-0.1% aqueous trifluoroacetic acid (50: 50).

Injecting 5 μ l of each of the sample solution and the reference solution into chromatograph, and recording chromatogram. Referring to FIGS. 2-1 and 2-2, FIG. 2-1 is a chromatogram of a control solution; FIG. 2-2 is a chromatogram of a test solution. As can be seen from the figure, in the analysis method of this example, ethyl cyanoacetate as a reference sample was effectively detected, and the peak in the sample did not interfere with the reference sample.

The chromatographic conditions were as follows:

column flow rate: 1.8 ml/min; the split ratio is as follows: 3: 1; solvent: acetonitrile-0.1% aqueous trifluoroacetic acid (50: 50);

column temperature: the temperature was started at 100 ℃ for 5 minutes, and increased to 220 ℃ at a rate of 15 ℃ per minute for 8 minutes.

Example 3

Diluent solution: acetonitrile-0.1% aqueous phosphoric acid (40: 60).

Control solution: a suitable amount of ethyl cyanoacetate control was added with a diluent to prepare a solution containing about 20. mu.g of ethyl cyanoacetate per 1 ml.

Injecting 5 μ l of each of the sample solution and the reference solution into chromatograph, and recording chromatogram. Referring to FIGS. 3-1 and 3-2, FIG. 3-1 is a chromatogram of a control solution; FIG. 3-2 is a chromatogram of a test solution. As can be seen from the figure, in the analysis method of this example, ethyl cyanoacetate as a reference sample was effectively detected, and the peak in the sample did not interfere with the reference sample.

The chromatographic conditions were as follows:

column flow rate: 1.5 ml/min; the split ratio is as follows: 3: 1; solvent: acetonitrile-0.1% aqueous phosphoric acid (50: 50);

column temperature: the temperature was started at 100 ℃ for 5 minutes, ramped up to 220 ℃ at a rate of 10 ℃ per minute for 8 minutes.

Example 4

Diluent solution: acetonitrile-0.2% aqueous phosphoric acid (60: 40).

Control solution: a suitable amount of ethyl cyanoacetate control was added with a diluent to prepare a solution containing about 4. mu.g of ethyl cyanoacetate per 1 ml.

Injecting 5 μ l of each of the sample solution and the reference solution into chromatograph, and recording chromatogram. Referring to FIGS. 4-1 and 4-2, FIG. 4-1 is a chromatogram of a control solution; FIG. 4-2 is a chromatogram of a test solution. As can be seen from the figure, in the analysis method of this example, ethyl cyanoacetate as a reference sample was effectively detected, and the peak in the sample did not interfere with the reference sample.

The chromatographic conditions were as follows:

column flow rate: 1.5 ml/min; the split ratio is as follows: 3: 1; solvent: acetonitrile-0.2% aqueous phosphoric acid (50: 50);

Methodology validation

(1) Specificity

Blank solution (diluent): acetonitrile-0.1% phosphoric acid aqueous solution (50: 50)

Positioning solution for each solution: taking 300mg of methanol, 500mg of ethanol, 500mg of acetone, 500mg of tert-butyl methyl ether, 500mg of dimethyl sulfoxide, 60mg of dichloromethane, 500mg of ethyl acetate, 72mg of tetrahydrofuran, 100mg of cyanoacetic acid, 89mg of toluene and 500mg of n-butanol, precisely weighing, respectively placing into 100ml measuring bottles, adding a diluent to dissolve and dilute to a scale, and shaking up to obtain the product.

Benzene localization solution: precisely weighing about 20mg of benzene, placing the benzene into a 100ml measuring flask, adding a diluent to dissolve and dilute the benzene to a scale, and shaking up; and (3) putting 1ml of the solution into a 100ml measuring flask, diluting the solution to a scale with a diluent, and shaking up to obtain the product.

Reference stock solution: accurately weighing about 20mg of ethyl cyanoacetate, placing in a 20ml measuring flask, dissolving and diluting to scale with diluent, and shaking up to obtain the final product. (1 mg/ml)

Control solution: taking 1ml of the reference stock solution, placing in a 100ml measuring flask, diluting with diluent to scale, and shaking up to obtain the final product. (10. mu.g/ml)

Test solution: weighing about 0.1g of the product, accurately weighing, placing in a 10ml measuring flask, adding a diluent to dissolve and dilute to scale, and shaking up to obtain the product. (10 mg/ml)

Mixing the solution: weighing about 0.2g of the product, precisely weighing, placing in a 20ml measuring flask, taking 1ml of each positioning solution of each solution, placing in the same 20ml measuring flask, adding diluent to dilute to scale, and shaking up to obtain the final product.

And (3) sampling and analyzing the blank solution, the reference solution, the test solution, the mixed solution and each solvent positioning solution, recording a chromatogram, and obtaining the result shown in the following table.

TABLE 1 results sheet for specialization

The result shows that the blank solvent has no interference on the detection of the ethyl cyanoacetate, each solution has no interference on the detection of the ethyl cyanoacetate, and the separation degree between the ethyl cyanoacetate and the adjacent solvent in the mixed solution meets the requirement.

(2) Detection limit and quantification limit

Precisely measuring a proper amount of reference solution, and gradually diluting to obtain a detection limit when the S/N is about 3; when the S/N is about 10, the quantitative limit is defined. Each solution was taken separately for analysis and the results are shown in the table below.

TABLE 2 detection limit and quantitation limit results

The results show that: in the method, the detection limit concentration of the ethyl cyanoacetate is 0.2732 mu g/ml; the quantitative limit concentration was 1.0927. mu.g/ml.

(3) Linearity

Quantitative limiting solutions, 40%, 60%, 80%, 100%, 120%, 160%, 200% linear solutions were sampled and analyzed (3 pins each), and chromatograms were recorded. Linear regression was performed with the concentration as abscissa (C) and the average peak area as ordinate (a), and a linear equation was obtained. The results of the experiments are shown in the following table.

TABLE 3 Linear results Table

From the table, the concentration of the ethyl cyanoacetate solution is in the range of 0.8802 μ g/ml to 23.4720 μ g/ml, the linear equation of the concentration and the peak area is y =32798.7461x-10920.8369, and the correlation coefficient r =0.9998, which shows that the linear relationship is good.

(4) Stability of solution

The comparison product and the test solution are taken for analysis at 0h, 2h, 4h, 6h, 8h and 12h, and the results are shown in the following table.

Table 4 solution stability results

The result shows that the solution of the reference substance and the test solution is stable within 12 hours at room temperature.

(5) Precision degree

Precision of sample introduction

And (4) sampling and analyzing the reference substance solution, recording a chromatogram, and calculating each peak area RSD, wherein the result is shown in the following table.

TABLE 5 sample introduction precision results table

From the above table, the retention time RSD obtained by repeating the sample injection 6 times for the control solution is 0.01%, and the RSD of the peak area is 0.34%, indicating that the precision of the instrument is good.

② repeatability

Taking the reference solution and the test solution (prepared in 6 parts in parallel), injecting and analyzing, recording the chromatogram, calculating the result according to an external standard method, and obtaining the result shown in the table.

TABLE 6 repeatability test results table

The results show that ethyl cyanoacetate is not detected in 6 samples, which indicates that the method has good repeatability.

Third intermediate precision

Different experimenters respectively prepare a reference solution and 6 test solution at different time, sample introduction analysis is carried out, a chromatogram is recorded, and the result is calculated according to an external standard method and is shown in the following table.

TABLE 7 intermediate precision test results table

As a result, ethyl cyanoacetate was not detected in 12 parts of the test sample.

(6) Accuracy of

Sample introduction analysis is carried out on the test solution, the reference solution and the solutions with various concentration levels (50%, 100% and 150%), a chromatogram is recorded, and the result is shown in the following table.

TABLE 8 recovery results table

From the table, in the concentration range of 50% -150%, the recovery rate of ethyl cyanoacetate is 94.39% -102.38%, the average recovery rate is 97.97%, and the RSD is 2.78%, which shows that the method has better accuracy.

(7) Durability

And evaluating the bearing degree of the measurement result which is not influenced when the measurement condition parameters have slight changes by changing the flow rate and the initial column temperature. The blank solution, the reference solution and the test solution were taken for analysis, and the results are shown in the following table.

TABLE 9 durability color spectrum condition table

TABLE 10 durability test results for different chromatographic conditions

And (4) conclusion: the flow rate of the column (1.3 ml/min-1.8 ml/min) and the initial column temperature (+ -5 ℃) are respectively adjusted to detect, and no ethyl cyanoacetate is detected in the test sample. When the measurement condition parameters were slightly changed, the measurement results were not substantially affected, i.e., the method was more durable.

The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. For those skilled in the art to which the present application pertains, several simple deductions or substitutions may be made without departing from the concept of the present application, and all should be considered as belonging to the protection scope of the present application.

Claims

1. An analysis method of ethyl cyanoacetate in tofacitinib citrate, which adopts gas chromatography and is characterized in that,

the chromatographic column is a capillary column taking 6% of cyanopropylphenyl-94% of dimethyl polysiloxane as a stationary liquid, the diluent is acetonitrile-dilute acid solution, and the mass concentration of the dilute acid solution is 0.1-0.2%.

2. The method of claim 1, wherein the method comprises the steps of,

the diluted acid solution is selected from a phosphoric acid solution, a formic acid solution, an acetic acid solution or a trifluoroacetic acid solution, and the volume ratio of the acetonitrile to the diluted acid solution is 40-70: 60 to 30.

3. The method for analyzing ethyl cyanoacetate in tofacitinib citrate according to claim 2, wherein the sample is a sample obtained by the method,

the dilute acid solution is selected from 0.1% phosphoric acid solution.

4. The method for analyzing ethyl cyanoacetate in tofacitinib citrate according to any one of claims 1 to 3, comprising the following steps:

5. The method of claim 4, wherein the method comprises the step of analyzing ethyl cyanoacetate in tofacitinib citrate,

in the step 3), the gas chromatography detection conditions are as follows: detector temperature: 250 ℃; sample inlet temperature: 250 ℃; column flow rate: 1.3-1.8 ml/min; the split ratio is as follows: 1-5: 1; the sample injection amount is 2-5 mu l; column temperature: the initial temperature is 100 +/-5 ℃, the temperature is maintained for 5-10 minutes, the temperature is increased to 200-220 ℃ at the rate of 10-20 ℃ per minute, and the temperature is maintained for 5-10 minutes.

6. The method of claim 4, wherein the method comprises the step of analyzing ethyl cyanoacetate in tofacitinib citrate,

the concentration of the ethyl cyanoacetate in the reference solution is 1-20 mu g/ml.

7. The method of claim 4, wherein the method comprises the step of analyzing ethyl cyanoacetate in tofacitinib citrate,

the concentration of the test solution is 3-10 mg/ml.

8. The method of claim 4, wherein the method comprises the step of analyzing ethyl cyanoacetate in tofacitinib citrate,

the detection limit concentration of the ethyl cyanoacetate is 0.2732 mu g/ml; the quantitative limit concentration was 1.0927. mu.g/ml.

9. The method of claim 4, wherein the method comprises the step of analyzing ethyl cyanoacetate in tofacitinib citrate,

the concentration of the ethyl cyanoacetate solution is in the range of 0.8802-23.4720 mu g/ml, and has linearity with peak area.