GCMS (Gas Chromatography Mass Spectrometry) detection method for ethyl cyanoacetate in tofacitinib citrate

Abstract

The invention belongs to the technical field of medicine detection, and particularly relates to a GCMS (Gas Chromatography Mass Spectrometry) detection method for ethyl cyanoacetate in tofacitinib citrate. Respectively injecting the blank solution, the reference solution and the test solution into a GCMS (Gas Chromatography Mass Spectrometer), recording a spectrogram, and calculating the content of ethyl cyanoacetate in tofacitinib citrate by adopting an external standard method; wherein the GCMS conditions comprise a chromatographic column, the column temperature of the chromatographic column is kept at 40 DEG C for 1 min, the temperature is increased to 240 DEG C at the heating rate of 15 DEG C / min, and the temperature is kept for 1 min. The method can accurately and quantitatively detect the content of ethyl cyanoacetate in tofacitinib citrate, and is simple to operate, high in precision, good in stability and good in reproducibility.

Description

Technical Field

[0001] This invention belongs to the field of drug detection technology, specifically relating to a GCMS method for the detection of ethyl cyanoacetate in tofacitinib citrate. Background Technology

[0002] Tofacitinib citrate is a novel oral small molecule targeted drug that is indicated for adult patients with moderate to severe active rheumatoid arthritis who have not responded adequately to or cannot tolerate methotrexate.

[0003] The production of tofacitinib citrate uses ethyl cyanoacetate. Ethyl cyanoacetate is highly toxic; at low concentrations, it may cause symptoms such as shortness of breath, tearing, drowsiness, lethargy, and slowed reflexes; at slightly higher concentrations, it may cause difficulty breathing, lateral decubitus position, and protruding eyeballs; at extremely high concentrations, it may lead to severe respiratory distress, convulsions, and even death. To ensure patient safety, the content of ethyl cyanoacetate in tofacitinib citrate must be strictly controlled.

[0004] Chinese patent CN 118777503A discloses a method for detecting cyanoacetic acid in tofacitinib citrate tablets, including reversing-phase chromatography to separate cyanoacetic acid, qualitative analysis using mass spectrometry, the reversing-phase chromatography using an octadecyl-bonded silica column as the separation column, formic acid aqueous solution as mobile phase A, and methanol as mobile phase B; the mass spectrometry detector uses an electrospray ionization (ESI) source or an atmospheric pressure chemical ionization (APCI) source; the flow rate is 0.1~2.0 ml / min, the column temperature is 20~60℃, and the injection volume is 0.2~100 μl. This patent only determines the content of cyanoacetic acid in tofacitinib citrate tablets and does not detect ethyl cyanoacetate in tofacitinib citrate. Summary of the Invention

[0005] The purpose of this invention is to provide a GCMS detection method for ethyl cyanoacetate in tofacitinib citrate, which can accurately and quantitatively detect the content of ethyl cyanoacetate in tofacitinib citrate. The method is simple to operate, has high precision, good stability, and good reproducibility.

[0006] The GCMS detection method for ethyl cyanoacetate in tofacitinib citrate of the present invention involves injecting blank solution, reference solution, and test solution into a GCMS, recording the chromatograms, and calculating the content of ethyl cyanoacetate in tofacitinib citrate using the external standard method. The GCMS conditions include a chromatographic column, a column temperature of 40°C for 1 min, a heating rate of 15°C / min to 240°C, and a holding time of 1 min.

[0007] The blank solution is prepared by measuring 3 ml of purified water and 5 ml of ethyl acetate, vortexing for 15 seconds to mix, letting stand for more than 15 minutes, vortexing again for 15 seconds to mix, and taking the upper layer solution after separation to obtain the blank solution.

[0008] The preparation method of the reference solution is as follows: accurately weigh ethyl cyanoacetate, dissolve and dilute it with purified water to prepare a solution containing 20-22 μg of ethyl cyanoacetate per 1 ml as the reference stock solution; measure 1.0 ml of the reference stock solution, add purified water to dilute to 10 ml, shake well to obtain the reference stock diluted solution; measure 3 ml of the reference stock diluted solution and 5 ml of ethyl acetate, vortex for 15 seconds to mix well, let stand for more than 15 minutes, vortex again for 15 seconds to mix well, and after separation, take the upper layer solution to obtain the reference solution.

[0009] The test solution is prepared by weighing 20-22 mg of tofacitinib citrate, adding purified water, sonicating to dissolve and diluting to 10 ml to obtain a diluted test solution; measuring 3 ml of the diluted test solution and 5 ml of ethyl acetate, vortexing for 15 seconds to mix, letting stand for more than 15 minutes, vortexing again for 15 seconds to mix, and taking the upper layer solution after separation to obtain the test solution.

[0010] The chromatographic column is a capillary column with 6% cyanopropylphenyl-94% dimethylpolysiloxane as the stationary phase; the column size is 30m × 0.32mm and the film thickness is 1.8μm.

[0011] The GCMS conditions also include the detector, which is MS, the detector scanning mode is SIM, and the quantitative ion is m / z 68.

[0012] The conditions for GCMS also include a carrier gas, which is helium.

[0013] GCMS conditions also include flow rate, which is 1.5 mL / min.

[0014] The GCMS conditions also include injection volume, injection port temperature, and split ratio. The injection volume is 1 μL, the injection port temperature is 220 °C, and the split ratio is 20:1.

[0015] The conditions for GCMS also include an ion source, with the ion source type being EI and the ion source temperature being 230℃.

[0016] The beneficial effects of this invention are as follows: This invention achieves dissolution of both the test sample and the ethyl cyanoacetate reference standard by adjusting the solvent ratio; it employs extraction to extract ethyl cyanoacetate from the test sample; it optimizes extraction conditions to ensure complete extraction and meet recovery requirements; it selects a chromatographic column and adjusts the flow rate and column temperature gradient program to ensure good retention of ethyl cyanoacetate in the column and effective separation from other impurity peaks in the test sample; and it adjusts the detection mode and selects quantitative ions to ensure good response of ethyl cyanoacetate. This invention exhibits high precision, good stability, and good reproducibility. Attached Figure Description

[0017] Figure 1 The spectrum is shown for the blank solution in Example 1.

[0018] Figure 2 This is the chromatogram of the reference solution in Example 1.

[0019] Figure 3 The spectrum is shown for the test solution in Example 1.

[0020] Figure 4 The spectrum is shown for the mixed solution in Example 1. Detailed Implementation

[0021] The present invention will be further described below with reference to embodiments.

[0022] Example 1 Blank solution: Measure 3 ml of purified water and 5 ml of ethyl acetate, vortex for 15 seconds to mix, let stand for more than 15 minutes, vortex again for 15 seconds to mix, and take the upper layer solution after separation to obtain the blank solution.

[0023] Reference solution: Accurately weigh 55.13 mg of ethyl cyanoacetate, place it in a 50 ml volumetric flask, add purified water, sonicate to dissolve and dilute to the mark, and shake well. Measure 1.0 ml of this solution, place it in a 50 ml volumetric flask, add purified water to the mark, and shake well to obtain the reference stock solution. Measure 1.0 ml of the reference stock solution, place it in a 10 ml volumetric flask, add purified water to the mark, and shake well to obtain the reference stock diluted solution. Measure 3 ml of the reference stock diluted solution, place it in a 15 ml centrifuge tube, add 5 ml of ethyl acetate, vortex for 15 seconds to mix, let stand for at least 15 minutes, vortex again for 15 seconds to mix, and after separation, take the upper layer solution to obtain the reference solution.

[0024] Test solution: Weigh 20.43 mg of tofacitinib citrate test sample, place it in a 10 ml volumetric flask, add purified water and sonicate to dissolve, then dilute to the mark to obtain the test sample dilution; measure 3 ml of the test sample dilution, place it in a 15 ml centrifuge tube, add 5 ml of ethyl acetate, vortex for 15 seconds to mix, let stand for more than 15 minutes, vortex again for 15 seconds to mix, and after separation, take the upper layer solution to obtain the test solution.

[0025] Mixed solution: Weigh 20.45 mg of tofacitinib citrate test sample and place it in a 10 ml volumetric flask. Add 1.0 ml of the reference stock solution, add purified water and sonicate to dissolve, then dilute to the mark. Take 3 ml of the solution and place it in a 15 ml centrifuge tube. Add 5 ml of ethyl acetate and vortex for 15 seconds to mix. Let stand for at least 15 minutes, then vortex again for 15 seconds to mix. After separation, take the upper layer solution to obtain the mixed solution.

[0026] Inject 1 μL each of blank solution, reference solution, test solution, and mixed solution into a GCMS, record the spectra, and see the results below. Figure 1-4 The content of ethyl cyanoacetate in tofacitinib citrate was calculated using the external standard method.

[0027] The conditions for GCMS are as follows: Column: Capillary column with stationary phase of 6% cyanopropylphenyl-94% dimethylpolysiloxane; Detector: MS; Carrier gas: Helium; Column dimensions: 30m × 0.32mm, membrane thickness 1.8μm; Flow rate: 1.5 mL / min; Column temperature: 40℃ for 1 min, then increase to 240℃ at a rate of 15℃ / min and hold for 1 min. Injection volume: 1 μL; Inlet temperature: 220℃; Flow split ratio: 20:1; Ion source type: EI; Ion source temperature: 230℃; Detector scan mode: SIM; Quantitative ion: m / z 68.

[0028] Depend on Figure 1-4 It can be seen that there is no significant interference near the ethyl cyanoacetate peak in the blank solution spectrum; the retention time of ethyl cyanoacetate is determined to be 9.403 minutes based on the reference solution spectrum; and there are no significant interfering peaks near the retention time of the ethyl cyanoacetate peak in the mixed solution spectrum, indicating that the present invention has good specificity.

[0029] The methodology was validated as follows: 1. Limit of detection and limit of quantitation Needle washing solution and blank solution: Measure 3 ml of purified water and 5 ml of ethyl acetate, vortex for 15 seconds to mix, let stand for more than 15 minutes, vortex again for 15 seconds to mix, and take the upper layer solution after separation; prepare two upper layer solutions in the same way, one as needle washing solution and the other as blank solution.

[0030] Limit of Quantitation (LOQ) Stock Solution: Weigh approximately 50 mg of ethyl cyanoacetate and place it in a 50 ml volumetric flask. Add purified water, sonicate to dissolve and dilute to the mark, and shake well. Take 1.0 ml of this solution and place it in a 50 ml volumetric flask. Add purified water to the mark and shake well to obtain the LOQ stock solution.

[0031] Limit of Quantitation (LOQ) Test Solution: Measure 1.0 ml of the LOQ stock solution and place it in a 10 ml volumetric flask. Dilute to the mark with purified water and shake well. Measure 1.0 ml of this solution and place it in a 10 ml volumetric flask. Dilute to the mark with purified water and shake well. Measure 3.0 ml of this solution and place it in a 15 ml centrifuge tube. Add 5 ml of ethyl acetate and vortex for 15 seconds to mix. Let stand for at least 15 minutes, then vortex again for 15 seconds to mix. After separation, take the upper layer solution to obtain the LOQ test solution. Prepare 6 parallel solutions using the same method.

[0032] Limit of Detection (LOD) test solution: Accurately measure 3.0 ml of the LOD test solution and place it in a 10 ml volumetric flask. Add 6.0 ml of purified water and mix well. Take 3 ml of this solution and place it in a 15 ml centrifuge tube. Add 5 ml of ethyl acetate and vortex for 15 seconds to mix well. Let it stand for at least 15 minutes, then vortex again for 15 seconds to mix well. After separation, take the upper layer solution to obtain the LOD test solution. Prepare 6 parallel solutions using the same method.

[0033] 1 μL of each of the above-mentioned needle washing solution, blank solution, limit of quantitation test solution and limit of detection test solution were precisely measured and injected into GCMS for detection. The results are shown in Table 1.

[0034] Table 1. Limits of detection and limits of quantitation for ethyl cyanoacetate in tofacitinib citrate.

[0035] 2. Linear range Needle washing solution and blank solution: Measure 3 ml of purified water and 5 ml of ethyl acetate, vortex for 15 seconds to mix, let stand for more than 15 minutes, vortex again for 15 seconds to mix, and take the upper layer solution after separation; prepare two upper layer solutions in the same way, one as needle washing solution and the other as blank solution.

[0036] Linear stock solution: Weigh about 50 mg of ethyl cyanoacetate, place it in a 50 ml volumetric flask, add purified water to dissolve by sonication and dilute to the mark, shake well, measure 1.0 ml of it, place it in a 50 ml volumetric flask, add purified water to dilute to the mark, shake well, and obtain the linear stock solution; prepare 3 parallel solutions in the same way.

[0037] Linearity test solution: Linearity test solution ①: Measure 1.0 ml of linearity stock solution into a 100 ml volumetric flask, dilute with purified water to the mark, shake well, measure 3 ml from it, put it into a 15 ml centrifuge tube, add 5 ml of ethyl acetate, vortex for 15 seconds to mix, let stand for more than 15 minutes, vortex again for 15 seconds to mix, after separation, take the upper layer solution to obtain linearity test solution ①; prepare 3 parallel solutions in the same way.

[0038] Linearity test solution ②: Measure 0.5 ml of the linearity stock solution into a 10 ml volumetric flask, dilute with purified water to the mark, shake well, measure 3 ml from it, put it into a 15 ml centrifuge tube, add 5 ml of ethyl acetate, vortex for 15 seconds to mix, let stand for more than 15 minutes, vortex again for 15 seconds to mix, after separation, take the upper layer solution to obtain linearity test solution ②; prepare 3 parallel solutions in the same way.

[0039] Linearity test solution ③: Measure 0.8 ml of the linearity stock solution into a 10 ml volumetric flask, dilute with purified water to the mark, shake well, measure 3 ml from it, put it into a 15 ml centrifuge tube, add 5 ml of ethyl acetate, vortex for 15 seconds to mix, let stand for more than 15 minutes, vortex again for 15 seconds to mix, after separation, take the upper layer solution to obtain linearity test solution ③; prepare 3 parallel solutions in the same way.

[0040] Linearity test solution ④: Measure 1.0 ml of the linearity stock solution into a 10 ml volumetric flask, dilute with purified water to the mark, shake well, measure 3 ml from it, put it into a 15 ml centrifuge tube, add 5 ml of ethyl acetate, vortex for 15 seconds to mix, let stand for more than 15 minutes, vortex again for 15 seconds to mix, after separation, take the upper layer solution to obtain linearity test solution ④; prepare 3 parallel solutions in the same way.

[0041] Linearity test solution ⑤: Measure 1.2 ml of the linearity stock solution into a 10 ml volumetric flask, dilute with purified water to the mark, shake well, measure 3 ml from it, put it into a 15 ml centrifuge tube, add 5 ml of ethyl acetate, vortex for 15 seconds to mix, let stand for more than 15 minutes, vortex again for 15 seconds to mix, after separation, take the upper layer solution to obtain linearity test solution ⑤; prepare 3 parallel solutions in the same way.

[0042] Precisely measure 1 μL of the needle washing solution, blank solution, and each linear test solution for injection, record the spectra, and calculate the linear regression equation with peak area as the abscissa and concentration (μg / mL) as the ordinate. The results are shown in Table 2. The ethyl cyanoacetate concentration showed good linearity in the range of 0.1234-1.480 ug / ml (equivalent to 0.01-0.12% of the test sample).

[0043] Table 2. Linearity test results of ethyl cyanoacetate in tofacitinib citrate

[0044] 3. Accuracy Needle washing solution and blank solution: Measure 3 ml of purified water and 5 ml of ethyl acetate, vortex for 15 seconds to mix, let stand for more than 15 minutes, vortex again for 15 seconds to mix, and take the upper layer solution after separation; prepare two upper layer solutions in the same way, one as needle washing solution and the other as blank solution.

[0045] Sample blank solution: Weigh 20.36 mg of tofacitinib citrate test sample, place it in a 10 ml volumetric flask, add purified water to dissolve by sonication and dilute to the mark, measure 3 ml of the solution, place it in a 15 ml centrifuge tube, add 5 ml of ethyl acetate, vortex for 15 seconds to mix, let stand for more than 15 minutes, vortex again for 15 seconds to mix, after separation, take the upper layer solution to obtain the sample blank solution.

[0046] Reference stock solution: Weigh about 50 mg of ethyl cyanoacetate and place it in a 50 ml volumetric flask. Add purified water, sonicate to dissolve and dilute to the mark, and shake well. Take 1.0 ml of the solution and place it in a 50 ml volumetric flask. Add purified water and dilute to the mark, and shake well to obtain the reference stock solution. Prepare 10 parallel solutions using the same method. Use 1 solution to prepare the reference solution and 9 solutions to prepare the accuracy test solution.

[0047] Reference solution: Measure 1.0 ml of the reference stock solution and place it in a 10 ml volumetric flask. Add purified water to dilute to the mark and shake well to obtain the reference stock diluent. Measure 3 ml of the reference stock diluent and place it in a 15 ml centrifuge tube. Add 5 ml of ethyl acetate and vortex for 15 seconds to mix. Let stand for at least 15 minutes, then vortex again for 15 seconds to mix. After separation, take the upper layer solution to obtain the reference solution.

[0048] Accuracy test solution 1: Measure 1.0 ml of the reference stock solution and place it in a 100 ml volumetric flask. Add purified water to dilute to the mark and shake well to obtain a solution. Weigh about 20 mg of the test sample and place it in a 10 ml volumetric flask. Add the above solution to dissolve and dilute to the mark, shake well, measure 3 ml of the solution, place it in a 15 ml centrifuge tube, add 5 ml of ethyl acetate, vortex for 15 seconds to mix, let stand for at least 15 minutes, vortex again for 15 seconds to mix, and after separation, take the upper layer solution for injection to obtain accuracy test solution 1. Prepare 3 parallel solutions using the same method.

[0049] Accuracy test solution 2: Weigh about 20 mg of the test sample and place it in a 10 ml volumetric flask. Add 1.0 ml of the reference stock solution, dissolve and dilute to the mark with purified water, shake well, measure 3 ml of the solution and place it in a 15 ml centrifuge tube. Add 5 ml of ethyl acetate, vortex for 15 seconds to mix, let stand for more than 15 minutes, vortex again for 15 seconds to mix, and after separation, take the upper layer solution for injection to obtain accuracy test solution 2; prepare 3 parallel solutions in the same way.

[0050] Accuracy test solution 3: Weigh about 20 mg of the test sample and place it in a 10 ml volumetric flask. Add 1.2 ml of the reference stock solution, dissolve and dilute with purified water to the mark, shake well, measure 3 ml of the solution and place it in a 15 ml centrifuge tube. Add 5 ml of ethyl acetate, vortex for 15 seconds to mix, let stand for more than 15 minutes, vortex again for 15 seconds to mix, and after separation, take the upper layer solution for injection to obtain accuracy test solution 3; prepare 3 parallel solutions in the same way.

[0051] Precisely measure 1 μL of the needle washing solution, blank solution, and each accuracy test solution and inject them into the solution. Record the spectra. The results are shown in Table 3. Based on the theoretical concentration and the actual concentration, the recovery rate of accuracy test solution 1 is in the range of 80-120%; the recovery rates of accuracy test solutions 2 and 3 are in the range of 90-108%.

[0052] Table 3. Accuracy test results of ethyl cyanoacetate in tofacitinib citrate

[0053] 4. Repeatability Needle washing solution and blank solution: Measure 3 ml of purified water and 5 ml of ethyl acetate, vortex for 15 seconds to mix, let stand for more than 15 minutes, vortex again for 15 seconds to mix, and take the upper layer solution after separation; prepare two upper layer solutions in the same way, one as needle washing solution and the other as blank solution.

[0054] Reference solution: Weigh 54.83 mg of ethyl cyanoacetate and place it in a 50 ml volumetric flask. Add purified water, sonicate to dissolve and dilute to the mark, and shake well. Take 1.0 ml of the solution and place it in a 50 ml volumetric flask. Add purified water to the mark and shake well to obtain the reference stock solution. Take 1.0 ml of the reference stock solution and place it in a 10 ml volumetric flask. Add purified water to the mark and shake well. Take 3 ml of the solution and place it in a 15 ml centrifuge tube. Add 5 ml of ethyl acetate, vortex for 15 seconds to mix, let stand for at least 15 minutes, vortex again for 15 seconds to mix, and take the upper layer solution after separation to obtain the reference solution.

[0055] Repeatability test solution: Weigh about 20 mg of tofacitinib citrate test sample and place it in a 10 ml volumetric flask. Add 1.0 ml of the reference stock solution, add purified water and sonicate to dissolve, then dilute to the mark. Take 3 ml of the solution and place it in a 15 ml centrifuge tube. Add 5 ml of ethyl acetate and vortex for 15 seconds to mix. Let stand for at least 15 minutes, then vortex again for 15 seconds to mix. After separation, take the upper layer solution to obtain the repeatability test solution. Prepare 6 parallel solutions using the same method.

[0056] Precisely measure 1 μL each of the above-mentioned injection washing solution, blank solution, reference solution, and repeatability test solution, and inject them into the solution. Record the spectra. The results are shown in Table 4. The RSD of the determination of ethyl cyanoacetate in the six repeatability test solutions was 5.2%, which is less than 10% and meets the requirements. The repeatability test results of ethyl cyanoacetate in tofacitinib citrate were good.

[0057] Table 4. Repeatability test results of ethyl cyanoacetate in tofacitinib citrate

[0058] 5. Intermediate precision Needle washing solution and blank solution: Measure 3 ml of purified water and 5 ml of ethyl acetate, vortex for 15 seconds to mix, let stand for more than 15 minutes, vortex again for 15 seconds to mix, and take the upper layer solution after separation; prepare two upper layer solutions in the same way, one as needle washing solution and the other as blank solution.

[0059] Reference solution: Weigh 51.51 mg of ethyl cyanoacetate and place it in a 50 ml volumetric flask. Add purified water, sonicate to dissolve and dilute to the mark, and shake well. Take 1.0 ml of the solution and place it in a 50 ml volumetric flask. Add purified water to the mark and shake well to obtain the reference stock solution. Take 1.0 ml of the reference stock solution and place it in a 10 ml volumetric flask. Add purified water to the mark and shake well. Take 3 ml of the solution and place it in a 15 ml centrifuge tube. Add 5 ml of ethyl acetate, vortex for 15 seconds to mix, let stand for at least 15 minutes, vortex again for 15 seconds to mix, and take the upper layer solution after separation to obtain the reference solution.

[0060] Intermediate precision test solution: Weigh approximately 20 mg of tofacitinib citrate test sample and place it in a 10 ml volumetric flask. Add 1.0 ml of the reference stock solution, add purified water, sonicate to dissolve, and dilute to the mark. Take 3 ml of this solution and place it in a 15 ml centrifuge tube. Add 5 ml of ethyl acetate, vortex for 15 seconds to mix, let stand for at least 15 minutes, vortex again for 15 seconds to mix, and after separation, take the upper layer solution to obtain the intermediate precision test solution. Prepare 6 parallel solutions using the same method.

[0061] Precisely measure 1 μL each of the above-mentioned needle washing solution, blank solution, reference solution, and intermediate precision test solution, and inject them into the solution, recording the chromatograms. The results are shown in Table 5. The RSD of the ethyl cyanoacetate determination results in the six intermediate precision test solutions was 5.0%, and the RSD of the 12 determinations (including the repeatability test solution) was 4.9%, less than 10%, meeting the requirements. The intermediate precision test results for ethyl cyanoacetate in tofacitinib citrate were good.

[0062] Table 5. Intermediate precision test results of ethyl cyanoacetate in tofacitinib citrate

[0063]

[0064] 6. Durability Needle washing solution and blank solution: Measure 3 ml of purified water and 5 ml of ethyl acetate, vortex for 15 seconds to mix, let stand for more than 15 minutes, vortex again for 15 seconds to mix, and take the upper layer solution after separation; prepare two upper layer solutions in the same way, one as needle washing solution and the other as blank solution.

[0065] Reference solution: Weigh approximately 50 mg of ethyl cyanoacetate and place it in a 50 ml volumetric flask. Add purified water, sonicate to dissolve, and dilute to the mark. Shake well. Take 1.0 ml of the solution and place it in a 50 ml volumetric flask. Add purified water to the mark and shake well to obtain the reference stock solution. Take 1.0 ml of the reference stock solution and place it in a 10 ml volumetric flask. Add purified water to the mark and shake well. Take 3 ml of the solution and place it in a 15 ml centrifuge tube. Add 5 ml of ethyl acetate, vortex for 15 seconds, and mix well. Let stand for at least 15 minutes, then vortex again for 15 seconds to mix well. After separation, take the upper layer solution to obtain the reference solution.

[0066] Durability test solution: Weigh about 20 mg of tofacitinib citrate test sample and place it in a 10 ml volumetric flask. Add 1.0 ml of the reference stock solution, add purified water and sonicate to dissolve, then dilute to the mark. Take 3 ml of the solution and place it in a 15 ml centrifuge tube. Add 5 ml of ethyl acetate and vortex for 15 seconds to mix. Let stand for at least 15 minutes, then vortex again for 15 seconds to mix. After separation, take the upper layer solution to obtain the durability test solution.

[0067] Based on the original GCMS conditions (the GCMS conditions of Example 1), the initial column temperature, flow rate, injection port temperature, and ion source temperature were changed for detection. 1 μL each of the above-mentioned needle washing solution, blank solution, reference solution, and robustness test solution was precisely measured and injected, and the spectra were recorded. The results are shown in Table 6. Under each condition, the RSD of the ethyl cyanoacetate content was 6.9%, less than 10%, which meets the requirements. The robustness of this invention meets the requirements.

[0068] Table 6. Durability test results of ethyl cyanoacetate in tofacitinib citrate

[0069] 7. Solution stability To investigate the stability of the solutions, the reference solution and the test solution were prepared and added to colorless vials at room temperature, placed in the instrument's injection tray, and left for 120 hours. Samples were injected at appropriate time intervals from 0 to 120 hours, and the spectra were recorded. The results are shown in Table 7. The preparation of the reference solution and the test solution was the same as in Example 1. Three parallel portions of the test solution were prepared, namely test solution 1, test solution 2, and test solution 3.

[0070] As shown in Table 7, the test solution is stable within 120 hours.

[0071] Table 7 Solution stability test data

Claims

1. A GCMS method for the detection of ethyl cyanoacetate in tofacitinib citrate, characterized in that... Blank solution, reference solution and test solution were injected into GCMS, and the spectra were recorded. The content of ethyl cyanoacetate in tofacitinib citrate was calculated using the external standard method. The GCMS conditions included the column temperature of 40℃ for 1 min, the temperature increase rate of 15℃ / min to 240℃, and the holding time of 1 min.

2. The GCMS detection method for ethyl cyanoacetate in tofacitinib citrate according to claim 1, characterized in that... The blank solution is prepared by measuring 3 ml of purified water and 5 ml of ethyl acetate, vortexing for 15 seconds to mix, letting stand for more than 15 minutes, vortexing again for 15 seconds to mix, and taking the upper layer solution after separation to obtain the blank solution.

3. The GCMS detection method for ethyl cyanoacetate in tofacitinib citrate according to claim 1, characterized in that... The preparation method of the reference solution is as follows: accurately weigh ethyl cyanoacetate, dissolve and dilute it with purified water to prepare a solution containing 20-22 μg of ethyl cyanoacetate per 1 ml as the reference stock solution; measure 1.0 ml of the reference stock solution, add purified water to dilute to 10 ml, shake well to obtain the reference stock diluted solution; measure 3 ml of the reference stock diluted solution and 5 ml of ethyl acetate, vortex for 15 seconds to mix well, let stand for more than 15 minutes, vortex again for 15 seconds to mix well, and after separation, take the upper layer solution to obtain the reference solution.

4. The GCMS detection method for ethyl cyanoacetate in tofacitinib citrate according to claim 1, characterized in that... The test solution is prepared by weighing 20-22 mg of tofacitinib citrate, adding purified water, sonicating to dissolve and diluting to 10 ml to obtain a diluted test solution; measuring 3 ml of the diluted test solution and 5 ml of ethyl acetate, vortexing for 15 seconds to mix, letting stand for more than 15 minutes, vortexing again for 15 seconds to mix, and taking the upper layer solution after separation to obtain the test solution.

5. The GCMS detection method for ethyl cyanoacetate in tofacitinib citrate according to claim 1, characterized in that... The chromatographic column is a capillary column with 6% cyanopropylphenyl-94% dimethylpolysiloxane as the stationary phase; the column size is 30m × 0.32mm and the film thickness is 1.8μm.

6. The GCMS detection method for ethyl cyanoacetate in tofacitinib citrate according to claim 1, characterized in that... The GCMS conditions also include the detector, which is MS, the detector scanning mode is SIM, and the quantitative ion is m / z 68.

7. The GCMS detection method for ethyl cyanoacetate in tofacitinib citrate according to claim 1, characterized in that... The conditions for GCMS also include a carrier gas, which is helium.

8. The GCMS detection method for ethyl cyanoacetate in tofacitinib citrate according to claim 1, characterized in that... GCMS conditions also include flow rate, which is 1.5 mL / min.

9. The GCMS detection method for ethyl cyanoacetate in tofacitinib citrate according to claim 1, characterized in that... The GCMS conditions also include injection volume, injection port temperature, and split ratio. The injection volume is 1 μL, the injection port temperature is 220 °C, and the split ratio is 20:

1.

10. The GCMS detection method for ethyl cyanoacetate in tofacitinib citrate according to claim 1, characterized in that... The conditions for GCMS also include an ion source, with the ion source type being EI and the ion source temperature being 230℃.

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