CN114441666B

CN114441666B - Method for detecting impurities in 4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride

Info

Publication number: CN114441666B
Application number: CN202011221768.3A
Authority: CN
Inventors: 陈正龙; 蒲雯; 刘霞
Original assignee: Chengdu Baiyu Pharmaceutical Co Ltd
Current assignee: Chengdu Baiyu Pharmaceutical Co Ltd
Priority date: 2020-11-05
Filing date: 2020-11-05
Publication date: 2024-02-27
Anticipated expiration: 2040-11-05
Also published as: CN114441666A

Abstract

The invention discloses a method for detecting impurities in 4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride, which comprises the following steps: a. preparing a system applicability solution; b. preparing a sample solution; c. preparing a control solution; d. detecting the control solution and the sample solution by adopting a high performance liquid chromatography method; e. and adding correction factors according to self contrast and calculating according to peak areas to obtain the impurity content in the 4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride sample. The invention provides a novel detection method for impurities in 4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride, which has high separation degree of various chromatographic peaks, has no interference with each other, can realize accurate detection of impurities 1, 6 and 2-6, and provides an effective detection method for monitoring the impurity content in 4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride, thereby further ensuring the product quality of parecoxib sodium and the medication safety of patients.

Description

Method for detecting impurities in 4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride

Technical Field

The invention belongs to the field of chemical analysis and detection, and particularly relates to a method for detecting impurities in 4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride.

Background

4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride is a parecoxib sodium intermediate, and the chemical name of parecoxib sodium is N- [ [4- (5-methyl-3-phenyl-4-isoxazolyl) phenyl ]]Sulfonyl group]Propionamide sodium salt (formula: C) ₁₉ H ₁₇ N ₂ O ₄ SNa), a non-steroidal anti-inflammatory drug is developed by combining pyroxene and French, is a global first selective cyclooxygenase-2 inhibitor capable of being used for intravenous injection and intramuscular injection at the same time, has the characteristics of good analgesic effect, quick response, lasting effect, effective inhibition of hyperalgesia, high gastrointestinal safety, no influence on platelet function, no extra increase of cardiovascular risk and the like, and can obviously reduce the dosage of opioid drugs and related adverse reactions when being combined with opioid drugs. Parecoxib sodium was marketed in the european collection in 2002. In year 2008, parecoxib sodium for injection is marketed in China in batches, and is widely used for short-term treatment of postoperative pain in a plurality of departments.

In order to ensure the quality of parecoxib sodium products and further ensure the medication safety of patients, an accurate and effective liquid chromatography analysis method of intermediate impurities is needed, and the invention provides a detection method of the impurities in the intermediate 4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride.

Disclosure of Invention

The invention aims to provide a method for detecting impurities in 4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride.

The invention provides a method for detecting impurities in 4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride, which comprises the following steps:

a. taking a reference substance of 4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride and impurities thereof, and preparing a system applicability solution;

b. taking a 4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride sample to be detected, and preparing a sample solution;

c. diluting a sample solution to prepare a control solution;

d. the method comprises the steps of respectively detecting a sample solution and a control solution by adopting a high performance liquid chromatography method, wherein the detection conditions of the high performance liquid chromatography method are as follows:

chromatographic column: the stationary phase takes phenyl bonded silica gel as a filler;

mobile phase: 0.05% trifluoroacetic acid-acetonitrile (40-50:50-60);

detection wavelength: 200 nm-230 nm;

column temperature is 20-40 ℃;

e. and adding correction factors according to self contrast and calculating according to peak areas to obtain the impurity content in the 4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride.

Further, the impurities include at least 4- (5-methyl-3-phenyl-4-isoxazolyl) benzenesulfonic acid.

Further, the impurities include one or more of 4- (5-methyl-3-phenyl-4-isoxazolyl) benzenesulfonic acid, 5-methyl-3, 4-diphenylisoxazole, and 4- [4- [4- (5-methyl-3-phenylisoxazol-4-yl) phenylsulfonyl ] phenyl ] -5-methyl-3-phenylisoxazole.

Further, in the step a, the solvent for preparing the system applicability solution is acetonitrile; in the step b, the solvent for preparing the test sample solution is acetonitrile, and in the step c, the solvent for preparing the control solution is acetonitrile.

Further, in the step d, the column is YMC Pack Ph, the length is 250mm, the inner diameter is 4.6mm, and the particle size of the packing is 5 μm

Further, in the step d, the detection wavelength is 215nm.

Further, in the step d, the volume ratio of 0.05% trifluoroacetic acid to acetonitrile is 45:55;

further, in the step d, the flow rate of the mobile phase is 0.8 ml/min-1.2 ml/min; preferably, the flow rate of the mobile phase is 1.0ml/min.

Further, in the step d, the sample injection volume is 5-100 μl; preferably, the sample volume is 10. Mu.l.

The invention provides a novel detection method for the impurity content in 4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride, which has the advantages of high separation degree of each chromatographic peak, no interference between each chromatographic peak, capability of simultaneously realizing accurate detection of impurity 1, impurity 6 and impurity 2-6, simple operation, easy control, low detection cost, good linear relation, specificity, precision, stability, sensitivity and repeatability, high sample recovery rate, accurate and reliable detection result, and provides an effective detection method for monitoring 4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride impurities, and further ensures the product quality of parecoxib sodium and the medication safety of patients.

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.

Drawings

FIG. 1 is an HPLC chart of a reference solution of impurity 1 under the detection condition of example 1 of the present invention.

FIG. 2 is an HPLC chart of the impurity 6 control solution under the detection condition of example 1 of the present invention.

FIG. 3 is an HPLC chart of a reference solution of impurities 2-6 under the detection conditions of example 1 of the present invention.

FIG. 4 is an HPLC chart of the impurity mixed solution under the detection condition of example 1 of the present invention.

FIG. 5 is an HPLC chart of a sample solution under the test condition of example 1 of the present invention.

FIG. 6 is a graph showing the purity of the main peaks of the sample in the test solution under the test condition of example 1 according to the present invention.

FIG. 7 is an HPLC chart of a system applicability solution under the detection condition of example 1 of the present invention.

FIG. 8 is an HPLC plot of a control solution of impurity 1 under comparative run 1 chromatographic conditions.

FIG. 9 is an HPLC plot of a control solution of impurity 6 under comparative run 1 chromatographic conditions.

FIG. 10 is an HPLC chart of a comparative impurity 2-6 control solution under the chromatographic conditions of comparative run 1.

FIG. 11 is an HPLC chart of a system applicability solution under the chromatographic condition of comparative experiment 1.

FIG. 12 is an HPLC plot of a system applicability solution under comparative run 2 chromatographic conditions.

FIG. 13 is an HPLC plot of a system applicability solution under comparative run 3 chromatographic conditions.

FIG. 14 is an HPLC plot of a system applicability solution under comparative run 4 chromatographic conditions.

FIG. 15 is a graph of the purity of the main peaks of the samples in the system applicability solution under the chromatographic conditions of comparative experiment 4.

FIG. 16 is an HPLC chart of a system applicability solution under comparative test 5 chromatography conditions

FIG. 17 is an HPLC plot of a control solution of impurity 1 under comparative run 6 chromatographic conditions.

FIG. 18 is an HPLC plot of a control solution of impurity 6 under comparative run 6 chromatographic conditions.

FIG. 19 is an HPLC plot of a control solution of impurity 2-6 under comparative run 6 chromatographic conditions.

FIG. 20 is an HPLC chart comparing system applicability solutions under test 6 chromatographic conditions.

FIG. 21 is an HPLC plot of a control solution of impurity 1 under the chromatographic conditions of comparative run 7.

FIG. 22 is an HPLC plot of a control solution of impurity 6 under comparative run 7 chromatographic conditions.

FIG. 23 is an HPLC chart comparing impurity 2-6 control solutions under the chromatographic conditions of run 7.

FIG. 24 is an HPLC plot of a control solution of impurity 1 under the chromatographic conditions of comparative run 8.

FIG. 25 is an HPLC plot of a control solution of impurity 6 under comparative run 8 chromatographic conditions.

FIG. 26 is an HPLC plot of a comparative impurity 2-6 control solution under the chromatographic conditions of comparative run 8.

FIG. 27 is a standard curve of impurity 1 in test example 1 item 2.

Fig. 28 is a standard curve of impurity 6 in test example 1 item 2.

FIG. 29 is a standard curve of impurities 2-6 in test example 1 item 2.

Detailed Description

The raw materials and equipment used in the specific embodiment of the invention are all known products and are obtained by purchasing commercial products or self-making.

For example, 4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride has lot number 20190401; impurity 1 lot No. 2018121101, content: 92.7%; all from the adult pharmaceutical stock company. Impurity 6 lot No. 201804014, content: 99.6%; from Shanghai Biotechnology Inc. Impurity 2-6 lot No. 39224, content: 96.4%; from Beijing Kang Pasen pharmaceutical technologies Co.

Impurity 1 is named 4- (5-methyl-3-phenyl-4-isoxazolyl) benzenesulfonic acid.

Impurity 6 is named 5-methyl-3, 4-diphenylisoxazole.

Impurity 2-6 is named 4- [4- [4- (5-methyl-3-phenylisoxazol-4-yl) phenylsulfonyl ] phenyl ] -5-methyl-3-phenylisoxazole.

AUW220D precision electronic balance is available from Shimadzu corporation; LC-2010CHT type high performance liquid chromatography pump is available from Shimadzu corporation, LC-2030C type high performance liquid chromatography pump is available from Shimadzu corporation, empower3 workstation is available from Wo-specialty corporation; YMC Pack Ph (250 mm. Times.4.6 mm,5 μm) column was commercially available from YMC company;

EXAMPLE 1 high performance liquid chromatography for detecting 4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride related substances according to the invention

Chromatographic column: YMC Pack Ph,4.6 mm. Times.250 mm,5 μm;

mobile phase: 0.05% trifluoroacetic acid-acetonitrile (45:55)

Solvent: acetonitrile

Column temperature: 30 ℃; flow rate: 1.0ml/min; UV detector (detection wavelength 215 nm).

Sample injection volume: 10 mu L.

The detection step comprises:

and dissolving a proper amount of the impurity 1 reference substance with a solvent to prepare an impurity 1 reference substance solution containing about 130 mug per 1 mL.

And dissolving a proper amount of the impurity 6 reference substance with a solvent to prepare an impurity 6 reference substance solution containing about 130 mug per 1 mL.

And dissolving a proper amount of the impurity 2-6 reference substance with a solvent to prepare an impurity 2-6 reference substance solution containing about 130 mug of the impurity 2-6 reference substance per 1 mL.

The impurities 1, 6, 2-6 and 4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride were dissolved in a suitable amount of a solvent to prepare a solution containing 1.0mg of 4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride and 10.0. Mu.g of each impurity per 1mL of the solution as a system-applicable solution.

Taking a proper amount of 4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride sample to be detected, dissolving the 4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride sample with acetonitrile, and then adding a solvent for dilution to prepare a sample solution containing about 1.0mg per 1 mL.

And diluting the impurities 1, 6 and 2-3 with a proper amount of reference substances by using a solvent to prepare an impurity mixed solution.

Assay: 10. Mu.L of each of the above solutions was poured into a liquid chromatograph, and the chromatograms were recorded, and the results were shown in FIGS. 1 to 7.

FIG. 1 is an HPLC chart of a control solution of impurity 1, with impurity 1 retention time of 3.227min.

FIG. 2 is an HPLC chart of a control solution of impurity 6, with impurity 6 retention time of 10.313min.

FIG. 3 is an HPLC chart of a control solution of impurities 2-6, with retention time of impurities 2-6 being 22.663min.

FIG. 4 is an HPLC chart of an impurity mixed solution of impurity 1, impurity 6, and impurities 2 to 6, wherein the retention time of impurity 1 is 3.379min, the retention time of impurity 6 is 10.233min, and the retention time of impurities 2 to 6 is 22.204min.

FIG. 5 is an HPLC plot of the sample solution with a retention time of 12.887min for the main peak of the sample.

FIG. 6 is a graph showing the purity of the main peaks of the samples in the test sample solution.

FIG. 7 is an HPLC diagram of a system applicability solution, the retention time of the sample is 12.854min, the retention time of the impurity 1 is 3.385min, the retention time of the impurity 6 is 10.248min, the retention time of the impurity 2-6 is 22.243min, and the degree of separation between the impurity 1, the impurity 6, the sample and the impurity 2-6 is 29.6,8.1, 20.0, respectively.

The result shows that under the chromatographic condition of the invention, the separation degree between the 4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride and the impurity is high, and the detection of the 4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride related substances can be realized.

Comparative test 1:

chromatographic column: waters SunFire C18.6mm×250mm,3.5 μm;

mobile phase a:0.05% trifluoroacetic acid

Mobile phase B: acetonitrile

Solvent: acetonitrile

Gradient elution conditions were as follows:

each impurity control solution and system applicability solution were prepared as described in example 1.

Assay: 10. Mu.L of the above solution was injected into a liquid chromatograph, and the chromatogram was recorded, and the results are shown in FIGS. 8 to 11.

FIG. 8 compares the HPLC profile of the impurity 1 control solution under the chromatographic conditions of run 1, with a retention time of 12.197 min for impurity 1.

FIG. 9 compares the HPLC profile of the impurity 6 control solution under the chromatographic conditions of run 1, with a retention time of 17.364min for impurity 6.

FIG. 10 compares the HPLC profile of a control solution of impurities 2-6 under the chromatographic conditions of run 1, with impurities 2-6 not eluted.

FIG. 11 compares the HPLC plot of the system suitability solution under the chromatographic conditions of run 1, impurity 1 retention time of 12.121min, impurity 6 retention time of 17.382min, sample (i.e., 4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride) retention time of 17.721min, sample peak late, and this method failed to detect impurities 2-6.

Comparative test 2:

chromatographic column: waters SunFire C18.6mm×250mm,3.5 μm;

mobile phase a: acetonitrile

Mobile phase B:0.05% trifluoroacetic acid

Solvent: acetonitrile

Gradient elution conditions were as follows:

the system applicability solution was prepared as described in example 1.

Assay: 10. Mu.L of the above solution was poured into a liquid chromatograph, and the chromatogram was recorded, and the result is shown in FIG. 12.

FIG. 12 compares the HPLC plot of the system applicability solution under the chromatographic conditions of run 2, with a sample retention time of 1.691min, with the sample coming out of the peak before being subject to interference from the solvent peak.

Comparative test 3:

chromatographic column: waters SunFire C18.6mm×250mm,3.5 μm;

mobile phase: 0.05% trifluoroacetic acid-acetonitrile (45:55)

Solvent: acetonitrile

The system applicability solution was prepared as described in example 1.

Assay: 10. Mu.L of the above solution was poured into a liquid chromatograph, and the chromatogram was recorded, and the result is shown in FIG. 13.

FIG. 13 is an HPLC plot of a system-adapted solution under comparative run 3 chromatographic conditions with impurity 1 retention time of 9.292min, impurity 6 retention time 27.153min, sample retention time 35.463 min, impurity 2-6 not eluted within 60 min. Therefore, the method fails to detect the impurity 2-6.

Comparative test 4

Chromatographic column: YMC Pack Ph,4.6 mm. Times.250 mm,5 μm;

mobile phase: 0.05% trifluoroacetic acid-acetonitrile (35:65)

Solvent: acetonitrile

The system applicability solution was prepared as described in example 1.

Assay: 10. Mu.L of the above solution was injected into a liquid chromatograph, and the chromatogram was recorded, and the results are shown in FIGS. 14 to 15.

FIG. 14 compares the HPLC profile of a system-adapted solution under the chromatographic conditions of run 4 with impurity 1 retention time 3.486min, impurity 6 retention time 7.984min, sample retention time 9.084min, impurity 2-6 retention time 12.785min.

FIG. 15 is a graph comparing the purity of the main peaks of samples in the system applicability solution under the chromatographic condition of test 4, and it can be seen that the main peaks are not pure.

Comparative test 5

Chromatographic column: YMC Pack Ph,4.6 mm. Times.250 mm,5 μm;

mobile phase a:0.05% trifluoroacetic acid

Mobile phase B: acetonitrile

Solvent: acetonitrile

Gradient elution conditions were as follows:

the system applicability solution was prepared as described in example 1.

Assay: 10. Mu.L of the above solution was poured into a liquid chromatograph, and the chromatogram was recorded, and the result is shown in FIG. 16.

FIG. 16 is a comparison of HPLC graphs of system applicability solutions under test 5 chromatographic conditions, impurity 1 retention time 7.689min, impurity 6 retention time 14.968min, sample retention time 15.647min, impurity 2-6 retention time 16.827, late sample peak and interference from unknown impurities; impurity 1 peak front.

Comparative test 6

Chromatographic column: YMC Pack Ph,4.6 mm. Times.250 mm,5 μm;

mobile phase: 0.05% trifluoroacetic acid-methanol (45:55)

Solvent: acetonitrile

Assay: 10. Mu.L of the above solution was injected into a liquid chromatograph, and the chromatogram was recorded, and the results are shown in FIGS. 17 to 20.

FIG. 17 is a HPLC chart comparing impurity 1 control solutions under run 6 chromatography conditions, with impurity 1 retention time of 6.298 min.

FIG. 18 compares the HPLC profile of the impurity 6 control solution under the chromatographic conditions of run 6, with a retention time of 40.127min for impurity 6.

FIG. 19 compares the HPLC profile of a control solution of impurity 2-6 under run 6 chromatographic conditions, with impurity 2-6 not eluted.

FIG. 20 compares the HPLC profile of a system-adapted solution under test 6 chromatographic conditions with impurity 1 retention time of 6.310min and impurity 6 retention time of 40.011min, samples and impurities 2-6 not eluted within 60 min.

Comparative test 7

Chromatographic column: agilent Pursuilt 5PFP,4.6mm×150mm;

mobile phase a:0.01mol/1KH ₂ PO ₄ (pH was adjusted to 3.0 with phosphoric acid)

Mobile phase B: methanol

Solvent: acetonitrile

Gradient elution conditions were as follows:

each impurity control solution was prepared as described in example 1. Assay: 10. Mu.L of the above solution was poured into a liquid chromatograph, and the chromatogram was recorded, and the results are shown in FIGS. 21 to 23.

FIG. 21 is a HPLC chart comparing the impurity 1 control solution under the chromatographic condition of test 7, wherein the retention time of the impurity 1 is 8.743min, and the peak shape of the impurity 1 is abnormal.

FIG. 22 compares the HPLC profile of the impurity 6 control solution under the chromatographic conditions of run 7, with a retention time of 17.442min for impurity 6.

FIG. 23 compares the HPLC profile of a control solution of impurity 2-6 under chromatographic conditions of run 7, with impurity 2-6 retention time of 22.944min.

Comparative experiment 8

Chromatographic column: agilent Pursuilt 5PFP,4.6mm×150mm;

mobile phase a: water-methanol-trifluoroacetic acid (90:10:0.05)

Mobile phase B: water-methanol-trifluoroacetic acid (10:90:0.05)

Solvent: acetonitrile

Gradient elution conditions were as follows:

each impurity control solution was prepared as described in example 1. .

Assay: injecting 10 μl of the above solution into liquid chromatograph, and recording chromatogram, with the results shown in FIGS. 24-26

FIG. 24 is a HPLC chart comparing the impurity 1 control solution under the chromatographic condition of test 8, wherein the retention time of impurity 1 is 4.745min, and the peak shape of impurity 1 is abnormal.

FIG. 25 compares the HPLC profile of the impurity 6 control solution under the chromatographic conditions of run 8, with a retention time of impurity 6 of 16.756min.

FIG. 26 compares the HPLC profile of a control solution of impurities 2-6 under the chromatographic conditions of run 8, with impurities 2-6 not eluted.

Thus, the comparative test 8 method cannot detect the impurity 2-6.

Experimental example 1 methodological study of the detection method of the present invention

The following conditions were used for each test in this test example:

chromatographic column: YM 2-6P 12-6k Ph, 4.6mm.times.250 mm,5 μm;

mobile phase: 0.05% trifluoroacetic acid-acetonitrile (45:55)

Solvent: acetonitrile

Sample injection volume: 10 mu L.

1. Specificity test

Preparing each of the impurity reference substance solution, the system applicability solution, the test substance solution and the impurity mixed solution according to the method described in example 1, precisely taking 10 mu L of each of the above solutions, respectively, injecting into a liquid chromatograph, and recording a chromatogram. The results are shown in FIGS. 1 to 7.

The results show that under the condition of the detection method, the sample and other impurities in the sample have no interference on the determination of the impurity 1, the impurity 6 and the impurity 2-6, and the specificity of the detection method is proved to be strong.

2. Standard curve and linear range

Precisely measuring the right amount of the reference substance solutions of the impurities 1, 6 and 2-6, and diluting the reference substance solutions with mobile phases to prepare a series of reference substance solutions with the concentrations. Respectively precisely taking 10 μl of reference solutions with different concentrations, injecting into a liquid chromatograph, and recording chromatogram. Peak areas were measured separately and the results are shown in table 1.

TABLE 1 Linear relationship

And drawing a standard curve by taking the concentration of the impurity reference substance solution as an abscissa X and the peak area as an ordinate Y, and calculating a linear regression equation and a correlation coefficient r of the impurity, wherein the standard curve is shown in figure 25.

The result shows that the concentration of the impurity 1 in the detection method of the invention is in good linear relation with the peak area within the range of 1.8661 mu g/mL-55.9815 mu g/mL, and the linear equation is as follows: y=37, 715.9429x+4, 486.2919, r=1.0000; the concentration of the impurity 6 is in good linear relation with the peak area in the range of 0.1013 mu g/mL-3.0400 mu g/mL, and the linear equation is as follows: y=47, 678.0818x+85.6352, r=0.9998; the concentration of 4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride is 0.9963 mug/mL-29.8901 mug/mL, and has good linear relation with peak area, and the linear equation is that: y= 35451.5242X-446.8719, r=0.9999; the concentration of impurity 2-6, in the range of 0.4875 μg/mL-14.6248 μg/mL, has a good linear relationship with peak area, and the linear equation: y=49, 325.2930X-1, 881.7969, r=0.9999, and the method provided by the invention has the advantages of wide linear range and high accuracy.

3. Precision test

The system applicability solution in test example 1 item 1 was taken, 10. Mu.L was precisely taken, and injected into a high performance liquid chromatograph, and the peak areas were measured respectively by the detection method of the present invention 6 times continuously, and the results are shown in Table 2.

TABLE 2 precision trial results

The RSD of the impurity 1 peak area was calculated as: 1.18%, RSD of impurity 6 peak area is: 0.51%, RSD of impurity 2-6 peak area: 0.15% of the sample, and the detection method of the invention has excellent precision.

4. Quantitative limit

The mixed solution of the impurities in test example 1 was measured in an appropriate amount, diluted with a solvent, measured in an accurate amount of 10. Mu.l, injected into a liquid chromatograph, and the peak area and the baseline noise were measured according to the detection method of the present invention, and the results are shown in Table 3.

TABLE 3 quantitative limit test results

Impurity name	Concentration (μg/mL)	Quantitative limit (ng)
			Impurity 1	0.013	0.13
Impurity 6	0.027	0.27
			Impurity 2-6	0.072	0.72

The peak heights of the impurity 1, the impurity 6 and the impurity 2-6 are about 10 times of the baseline noise, the quantitative limit of the impurity 1 is 0.13ng, the quantitative limit of the impurity 6 is 0.27ng and the quantitative limit of the impurity 2-6 is 0.72ng according to the signal to noise ratio S/N=10, and the detection sensitivity of the method is high, so that the requirement of quantitative determination of the impurity can be fully met.

5. Repeatability test

6 parts of 4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride of a sample is precisely weighed, about 10mg of each part is placed in a 10mL measuring flask, and the mixture is dissolved by adding a solvent and diluted to a scale to obtain a sample solution. The above 6 parts of the sample solutions were measured precisely at 10. Mu.L each, and were examined by the method of the present invention, and the contents of impurity 1, impurity 6 and impurities 2 to 6 were calculated as peak areas by the external standard method, and the results are shown in Table 4.

TABLE 4 repeatability test results

Sample numbering

1

2

3

4

5

6

Average value of

Impurity 1

0.268％

0.265％

0.266％

0.261％

0.258％

0.262％

0.263％

Impurity 6

0.003％

0.002％

0.003％

Impurity 2-6

0.135％

0.132％

0.130％

0.131％

0.129％

0.130％

0.131％

From the above results, the reproducibility of the detection method of the present invention was good.

6. Solution stability test

10mg of 4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride sample is precisely weighed, placed in a 10mL measuring flask, dissolved by adding a solvent and diluted to a scale, and then a sample solution is obtained. Sample injection of 10 mu L is carried out at 0h, 1h, 2h, 3h, 4h and 5h after preparation, a chromatogram is recorded, and stability conditions of impurities 1, 6 and 2-6 in sample solutions are examined, and the results are shown in Table 5.

TABLE 5 stability test results Table of test sample solutions

Time of placement

0h

1h

2h

3h

4h

5h

Impurity 1

0.241％

0.246％

0.251％

0.244％

0.254％

0.250％

Impurity 6

0.003％

Impurity 2-6

0.859％

0.857％

0.852％

0.842％

0.855％

From the results, the impurities 1 and 6 in the sample solution are not detected within 5 hours after preparation, and the detection results of the impurities 2-6 are not obviously changed, so that the sample solution is stable.

7. Recovery test

9 parts of 4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride as a sampling product are precisely weighed, about 10mg of each is placed into a 10mL measuring flask, 3 parts of each of 0.5mL, 1.0mL and 2.0mL of the reference substance solution of each impurity under the test example 1 are added, and the mixture is dissolved and diluted to a scale by adding a solvent, and the mixture is uniformly shaken to serve as the sample solutions to be recovered. And respectively precisely taking 9 parts of the sample solution with the recovery rate and the impurity mixed solution under the test example 1, respectively carrying out 10 mu L sample injection measurement, recording a chromatogram, calculating the measured amounts of the impurity 1, the impurity 6 and the impurity 2-6, and the addition amount and the recovery rate of the reference substances, wherein the results are shown in tables 6-8.

The calculation formula is as follows:

wherein: a is the amount (mg) of impurities contained in the sample;

b is the addition amount (mg) of the isomer impurity reference substance;

c is the measured amount (mg) of the isomer impurity.

TABLE 6 results of impurity 1 recovery test

The result shows that the detection method of the invention detects the impurity 1 in 4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride, the recovery rate is 96.91% -98.48%, the relative standard deviation is 0.56%, and the detection method of the invention has good recovery rate and high accuracy.

TABLE 7 results of impurity 6 recovery test

The result shows that the detection method of the invention detects the impurity 6 in 4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride, the recovery rate is 97.00% -98.00%, the relative standard deviation is 0.66%, and the detection method of the invention has good recovery rate and high accuracy.

TABLE 8 results of impurity 2-6 recovery test

The result shows that the detection method of the invention detects 2-6 impurity in 4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride, the recovery rate is 97.64% -98.82%, and the relative standard deviation is 1.74%, which proves that the detection method of the invention has good recovery rate and high accuracy.

In summary, the invention provides a novel detection method for related substances in 4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride, which has high separation degree of each chromatographic peak, no interference between each chromatographic peak, can simultaneously realize accurate detection of impurity 6, impurity 1 and impurity 2-6, is simple and convenient to operate, easy to control, low in detection cost, has good linear relation, specificity, precision, stability, sensitivity and repeatability, high in sample recovery rate, accurate and reliable in detection result, provides an effective detection method for monitoring 4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride, and further ensures the product quality of parecoxib sodium and the medication safety of patients.

Claims

1. A method for detecting impurities in 4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride is characterized by comprising the following steps: it comprises the following steps:

c. diluting a sample solution to prepare a control solution;

chromatographic column: YMC Pack Ph has a length of 250mm, an inner diameter of 4.6mm and a filler particle size of 5 mu m;

mobile phase: 0.05% trifluoroacetic acid-acetonitrile, the volume ratio of the two is 45:55;

detection wavelength: 200 nm-230 nm;

column temperature is 20-40 ℃;

e. adding correction factors according to self contrast and calculating according to peak areas to obtain the impurity content in the 4- (5-methyl-3-phenyl-4-isoxazole) benzenesulfonyl chloride;

wherein,

the impurities include 4- (5-methyl-3-phenyl-4-isoxazolyl) benzenesulfonic acid, 5-methyl-3, 4-diphenylisoxazole and 4- [4- [4- (5-methyl-3-phenylisoxazol-4-yl) phenylsulfonyl ] phenyl ] -5-methyl-3-phenylisoxazole.

2. The method of claim 1, wherein: in the step a, acetonitrile is used as a solvent for preparing a system applicability solution; in the step b, acetonitrile is used as a solvent for preparing the sample solution; in the step c, the solvent for preparing the control solution is acetonitrile.

3. The method of claim 1, wherein: in the step d, the detection wavelength is 215nm.

4. The method of claim 1, wherein: in the step d, the flow rate of the mobile phase is 0.8 ml/min-1.2 ml/min.

5. The method of claim 4, wherein: the flow rate of the mobile phase was 1.0ml/min.

6. The method of claim 1, wherein: in the step d, the sample injection volume is 5-100 μl.

7. The method of detecting according to claim 6, wherein: the sample volume was 10. Mu.l.