CN115684397A

CN115684397A - Method for determining content of genotoxic impurity hydroxylamine hydrochloride in parecoxib

Info

Publication number: CN115684397A
Application number: CN202211298820.4A
Authority: CN
Inventors: 徐国芹; 陈晓荔; 李月月; 王丽美
Original assignee: Shandong Langnuo Pharmaceutical Co ltd; Shandong Bestcomm Pharmaceutical Co ltd
Current assignee: Shandong Langnuo Pharmaceutical Co ltd; Shandong Bestcomm Pharmaceutical Co ltd
Priority date: 2022-10-24
Filing date: 2022-10-24
Publication date: 2023-02-03

Abstract

The invention provides a method for measuring the content of genetic toxic impurity hydroxylamine hydrochloride in parecoxib, which controls the residual quantity of hydroxylamine hydrochloride not to exceed twenty parts per million (20 ppm), meets the control requirement of parecoxib sodium, and ensures the quality safety of parecoxib sodium for injection of preparation products.

Description

Method for determining content of genotoxic impurity hydroxylamine hydrochloride in parecoxib

1. Field of the invention

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a content determination method of a genotoxic impurity hydroxylamine hydrochloride in parecoxib.

2. Background of the invention

Parecoxib sodium for injection is the first specific cyclooxygenase-2 (COX-2) inhibitor which can be intravenously and intramuscularly developed by the company of Famaxi (Pharmacia) and is used for treating mild and moderate postoperative pain with less side effects. The product was approved for marketing in Europe in the beginning of 2002 and imported into China in 2008.

The bulk drug parecoxib sodium of parecoxib sodium for injection, CAS number: 197502-82-2, chemical name is N- [ [4- (5-methyl-3-phenyl-4-isoxazolyl) phenyl ] sulfonyl ] propanamide sodium salt, structural formula is as follows:

parecoxib, CAS number: 198470-84-7, (N- [ [4- (5-methyl-3-phenyl-4-isoxazolyl) phenyl ] sulfonyl ] propionamide), which is the acid radical of parecoxib sodium, has the following structural formula:

hydroxylamine hydrochloride, CAS number: 5470-11-1, is an important reagent in the synthesis process of parecoxib, and has the following structural formula:

hydroxylamine hydrochloride has mutagenicity and carcinogenicity. According to the ICHM7 guiding principle, the medicine needs to be controlled according to genotoxic impurities, the acceptable intake amount of the medicine is controlled to be 1.7 mu g/day according to the toxicity data of hydroxylamine hydrochloride, and the control limit is as low as 20ppm according to the maximum daily dose (85 mg) of parecoxib sodium.

Currently, the detection methods searched for are ion chromatography, gas-phase derivatization chromatography and liquid-phase derivatization chromatography. The ion chromatograph is an extremely-used instrument, is high in price, cannot be configured in most pharmaceutical factories, is limited by the instrument, and cannot be popularized. The gas-phase derivatization method needs cyclohexanone derivatization reaction, and needs drying and extraction, the operation is complex, and the reproducibility of the method is difficult to guarantee. Patent CN202010834914.3 discloses a method for detecting hydroxylamine hydrochloride in azilsartan, which uses benzaldehyde as a derivatization reagent, the limit of quantitation is 18.9ppm, the control requirement of parecoxib cannot be met, and the benzaldehyde is used as the derivatization reagent, so that the derivatized solution needs to be stored at 8 ℃, the control requirement on test conditions is high, and the accuracy of the determination result has a risk. Patent CN201410646803.4 discloses an HPLC detection method for related substances of parecoxib, mainly for detecting related substances, and the sensitivity of the method is not enough to be used as an analysis control means for genotoxic impurities.

Therefore, a trace detection method for hydroxylamine hydrochloride, which is suitable for parecoxib, is effective, sensitive and accurate, and has more easily controlled conditions, needs to be established so as to realize effective control of genotoxic impurities in parecoxib sodium products.

3. Summary of the invention

The invention provides a method for measuring the content of genetic toxic impurity hydroxylamine hydrochloride in parecoxib, which controls the residual quantity of hydroxylamine hydrochloride not to exceed twenty parts per million (20 ppm) and meets the quality control requirement of parecoxib sodium.

The invention provides a method for determining the content of genotoxic impurity hydroxylamine hydrochloride in parecoxib, which is characterized by comprising the following steps:

(1) Preparing a test solution: taking a proper amount of parecoxib, adding a solvent for dissolving and diluting to prepare a test solution;

(2) Preparing a reference substance solution: taking a proper amount of hydroxylamine hydrochloride, adding a solvent to dissolve and dilute the hydroxylamine hydrochloride to prepare a reference substance solution;

(3) Preparation of derivatization reagent solution: taking a proper amount of 9-fluorenylmethoxycarbonyl acyl chloride, adding a solvent to dissolve and diluting to prepare a derivative reagent solution;

(4) Derivatization: taking a proper amount of a reference substance solution and a proper amount of a test solution, respectively mixing the reference substance solution and the test solution with a proper amount of a derivative reagent solution, and diluting the solutions into a reference substance solution and a test solution to be tested after reaction;

(5) Chromatographic experiments: and (3) taking appropriate amount of the reference solution and the to-be-detected solution of the test solution after derivatization, injecting the appropriate amount of the to-be-detected solution into a liquid chromatograph, recording a chromatogram, and calculating by peak area according to an external standard method.

The solvent in the steps (1) - (3) is selected from one or more of acetonitrile water and dimethyl sulfoxide, preferably acetonitrile water, and the volume ratio of acetonitrile to water is 50-100: 50 to 0.

In the step (4), the derivatization conditions are that the reaction temperature is 20-40 ℃ and the reaction time is more than 10 minutes, preferably 30 minutes.

In the step (5), the chromatographic conditions are analyzed by a reversed-phase high performance liquid chromatograph.

The specific chromatographic conditions are as follows:

a detector: an ultraviolet detector;

and (3) chromatographic column: a chromatographic column using octadecylsilane chemically bonded silica as a filler, agela Venusil MP C18 mm multiplied by 4.6mm,5 mu m;

detection wavelength: 265nm;

and (3) chromatographic column: chromatographic column with octadecylsilane chemically bonded silica as filler;

mobile phase: mobile phase A: a phosphate buffer solution, preferably a potassium dihydrogen phosphate solution, with a concentration of 5 to 20mmol/L, preferably 10mmol/L, and adjusting the pH value to 2.5 to 3.5, preferably 3.0 with phosphoric acid; mobile phase B: acetonitrile;

flow rate: 1.0ml/min;

column temperature: 35 ℃;

sample injection amount: the sample amount of the reference substance is not less than 0.3ng, and the sample amount of the test substance is not less than 15 mug;

sample introduction volume: 10 mu l of the mixture;

gradient elution: when the mobile phase program is 0-20 minutes, the volume ratio of the phosphate buffer solution to the acetonitrile water is 60-70: and after 20 minutes, increasing the proportion of acetonitrile to elute other substances in the chromatographic column. The specific elution procedure was performed as follows:

the invention further provides a method for measuring the content of the genetic toxicity impurity hydroxylamine hydrochloride in parecoxib, which is characterized by comprising the following steps:

(1) Preparing a test solution: taking about 0.75g of parecoxib, precisely weighing, placing in a 25ml measuring flask, adding 2ml of water, adding acetonitrile to dissolve and dilute to a scale, and shaking up;

(2) Preparation of a reference solution: taking about 15mg of hydroxylamine hydrochloride reference substance, accurately weighing, placing in a 100ml measuring flask, adding 2ml of water to dissolve, diluting to scale with acetonitrile, shaking up, accurately weighing a proper amount, and quantitatively diluting with acetonitrile to obtain a solution containing about 0.6 mu g of hydroxylamine hydrochloride in each 1 ml;

(3) Preparation of derivatization reagent solution: weighing a proper amount of 9-fluorenylmethoxycarbonyl chloride, adding acetonitrile to dissolve and dilute the mixture to prepare a solution containing about 5mg of 9-fluorenylmethoxycarbonyl chloride in each 1ml of the solution;

(4) Derivatization: taking 5ml of each of the reference substance solution and the sample solution, respectively mixing with 1ml of the derivative reagent solution, and diluting the mixture into the reference substance solution and the sample solution to-be-detected solution by using acetonitrile after reaction;

(5) Chromatographic experiments: and (3) taking 10 mu l of the reference substance solution and the to-be-detected solution of the test solution after the derivatization, injecting the solutions into a liquid chromatograph, recording a chromatogram, and calculating by peak area according to an external standard method.

In the step (4), the derivatization condition is that the reaction temperature is 20-40 ℃, and the reaction time is more than 10 minutes, preferably 30 minutes.

In the step (5), the chromatographic conditions are analyzed by a reversed-phase high performance liquid chromatograph. The specific chromatographic conditions are as follows:

a detector: an ultraviolet detector;

a chromatographic column: a chromatographic column using octadecylsilane chemically bonded silica as a filler, agela Venusil MP C18 mm multiplied by 4.6mm,5 mu m;

detection wavelength: 265nm;

mobile phase: mobile phase A: a phosphate buffer, preferably a potassium dihydrogen phosphate solution, having a concentration of 5 to 20mmol/L, preferably 10mmol/L, and adjusting the pH to 2.5 to 3.5, preferably 3.0 with phosphoric acid; and (3) mobile phase B: acetonitrile;

flow rate: 1.0ml/min;

column temperature: 35 ℃;

gradient elution was performed as follows:

the invention has the technical effects that:

the method takes the 9-fluorenylmethoxycarbonyl chloride as a derivative solvent, does not need to carry out special treatment on a sample to be detected, is simple to operate, and ensures complete reaction by controlling the reaction time.

According to the invention, by screening and optimizing chromatographic conditions, the separation of the hydroxylamine hydrochloride derivative product peak and other chromatographic peaks is realized, and the interference of other impurities in parecoxib on the hydroxylamine hydrochloride derivative peak is avoided.

The method adopts an external standard method to carry out quantitative determination on the hydroxylamine hydrochloride in the parecoxib, has good linearity, detection sensitivity, precision, accuracy and the like, realizes detection of the hydroxylamine hydrochloride with trace concentration, has the quantitative limit of two millionths, and can better meet the control requirement of the parecoxib sodium on the genetic toxicity impurity hydroxylamine hydrochloride.

The invention solves the defects in the prior art, provides an HPLC method for measuring trace genotoxic impurity hydroxylamine hydrochloride in parecoxib, can be used for quality control of parecoxib, and ensures the quality safety of parecoxib sodium for injection of preparation products.

4. Description of the drawings

Fig. 1 is a resolution solution chromatogram in example 1.

FIG. 2 is a chromatogram of the control solution in example 3.

FIG. 3 is a 25 hour chromatogram of the control solution of example 3.

FIG. 4 is a graph of the stability test of the control solution in example 3.

FIG. 5 is a chromatogram of the test solution in example 3.

FIG. 6 is a 24-hour chromatogram of the test solution in example 3.

FIG. 7 is a stability test pattern of the test article in example 3.

Figure 8 hydroxylamine hydrochloride in example 4 checks the line graph.

FIG. 9 is a chromatogram of the limiting quantitation solution in example 5.

FIG. 10 is a chromatogram of a detection limit solution in example 5.

FIG. 11 is a chromatogram of a test sample solution in the 2ppm concentration recovery test in example 6.

FIG. 12 is a chromatogram of a control solution for the 2ppm concentration recovery test in example 6.

FIG. 13 is a chromatogram of a test sample solution in the 10ppm concentration recovery test in example 6.

FIG. 14 is a chromatogram of a sample solution in the 20ppm concentration recovery test in example 6.

FIG. 15 is a chromatogram of a sample solution in the 30ppm concentration recovery test in example 6.

FIG. 16 is a chromatogram of a control solution for the concentration recovery test of 10 to 30ppm in example 6.

5. Detailed description of the preferred embodiments

The following detailed description of specific embodiments of the present invention is provided for illustrative purposes only and is not intended to limit the scope of the present invention.

Instrument and chromatographic conditions:

the instrument comprises the following steps: HITACHI CM5110 liquid chromatograph, ultraviolet detector;

mobile phase:

mobile phase A:10mmol/L potassium dihydrogen phosphate solution (pH value is adjusted to 3.0 by phosphoric acid),

mobile phase B: acetonitrile;

flow rate: 1.0ml/min;

column temperature: 35 ℃;

sample introduction volume: 10 mu l of the mixture;

gradient elution was performed as follows:

preparing a solution:

preparing a test solution: taking about 0.75g of parecoxib, accurately weighing, placing in a 25ml measuring flask, adding 2ml of water, adding acetonitrile to dissolve and dilute to a scale, and shaking up;

preparing a reference substance solution: taking about 15mg of hydroxylamine hydrochloride reference substance, precisely weighing, placing in a 100ml measuring flask, adding 2ml of water to dissolve, diluting with acetonitrile to scale, shaking up, precisely weighing an appropriate amount, and quantitatively diluting with acetonitrile to obtain a solution containing about 0.6 mu g of hydroxylamine hydrochloride in each 1 ml;

preparing a derivatization reagent solution: an appropriate amount of 9-fluorenylmethoxycarbonylcarbonyl chloride was weighed, and dissolved in acetonitrile and diluted to give a solution containing about 5mg of 9-fluorenylmethoxycarbonylcarbonyl chloride per 1 ml.

Example 1 degree of separation test

Resolution solution: adding the sample solution into 1.5ml of the reference solution, diluting to scale with acetonitrile, measuring 5ml, putting into a 10ml measuring flask, adding 1ml of the derivative reagent solution, shaking, standing for 30 minutes, diluting to scale with acetonitrile, and shaking.

And (4) injecting the separation degree solution into a liquid chromatograph, and recording the chromatogram. The results are shown in Table 1 and FIG. 1.

Table 1 example 1 results of the separation test

Name of impurity	Retention time (minutes)	Separation condition of hydroxylamine hydrochloride and adjacent impuritiesIn a state of being immersed in
			Hydroxylamine hydrochloride	15.796	Baseline separation

As a result: the hydroxylamine hydrochloride peak is well separated from other impurity peaks in the sample.

Example 2 derivation time investigation test

The instrument and chromatographic conditions were as above.

Precisely measuring 5ml of reference substance solution, weighing 6 parts in total, placing in 6 measuring bottles of 10ml, respectively adding 1ml of derivatization reagent solution, shaking up, sequentially placing for 0, 5, 10, 15, 20 and 30 minutes, and then respectively diluting with acetonitrile to scale. Respectively and precisely measuring 10 mu l of the 6 parts of reference substance solution, injecting the solution into a liquid chromatograph, recording a chromatogram, analyzing and comparing peak areas of derivative peaks, wherein the results are shown in table 2, when the reaction solution is placed at room temperature, the peak areas of the derivative products tend to increase along with the time extension, and the derivative products reach equilibrium after 15 minutes and do not increase any more, which indicates that the hydroxylamine hydrochloride can completely react after 15 minutes; to reduce the risk of the reaction being affected by fluctuations in room temperature, the derivatization time was determined to be 30 minutes.

TABLE 2 investigation results of different derivation times

Time	0 minute	5 minutes	10 minutes	15 minutes	20 minutes	30 minutes
							Peak area	18322	23087	35525	38561	38223	38656

EXAMPLE 3 solution stability test

1. Stability of control solutions

Precisely measuring 5ml of reference substance solution, placing the reference substance solution in a 10ml measuring flask, adding 1ml of derivatization reagent solution, shaking up, placing the solution at room temperature for 30 minutes, adding acetonitrile to dissolve and dilute the solution to a scale, and shaking up; and continuously placing at room temperature for 0, 2, 4, 6, 8 and 25 hours respectively, precisely measuring 10 mu l, injecting into a liquid chromatograph, recording a chromatogram, and comparing with 0 hour to calculate the change value of the peak area of the hydroxylamine hydrochloride. The results show that: the control solutions were stable over 25 hours, and the results are shown in Table 3 and FIGS. 2-4.

TABLE 3 stability test results of hydroxylamine hydrochloride inspection control solutions

Time (hours)	0	2	4	6	8	25	Rate of change (%)
								Hydroxylamine hydrochloride	38210	39852	39884	39655	39841	39958	4.57

2. Stability of test solution

Precisely measuring 5ml of a sample solution, placing the sample solution in a 10ml measuring flask, adding 1ml of a derivatization reagent solution, shaking up, placing the sample solution at room temperature for 30 minutes, adding acetonitrile to dissolve and dilute the sample solution to a scale, and shaking up; continuously standing at room temperature for 0, 2, 4, 6, 8, and 24 hr, precisely measuring 10 μ l of the sample solution, injecting into liquid chromatograph, recording chromatogram, and comparing with 0 hr. The results show that: when the test solution is placed at room temperature for 24 hours, no hydroxylamine hydrochloride is detected, the area of the impurity peak is enlarged about 21min, the solution is unstable, the detection of hydroxylamine hydrochloride is not influenced, and the solution does not need to be prepared newly for use. The results are shown in Table 4 and FIGS. 5 to 7.

TABLE 4 stability test results of hydroxylamine hydrochloride test sample solutions

Time (hours)

0

2.5

4

6.5

8

24

Rate of change (%)

Hydroxylamine hydrochloride (ppm)

Undetected

Not detected out

Undetected

－

Presence or absence of new impurities

－

Is free of

Is composed of

－

Example 4 Linear test

Control stock solution: taking about 15mg of hydroxylamine hydrochloride reference substance, placing into a 100ml measuring flask, adding 2ml of water to dissolve, diluting to scale with acetonitrile, shaking up, precisely measuring 2ml, placing into a 50ml measuring flask, diluting to scale with acetonitrile, and shaking up to obtain the final product.

Precisely measuring the reference substance storage solutions respectively 0.5ml, 1.5ml, 2.5ml, 5.0ml, 7.5ml and 10.0ml, respectively placing the reference substance storage solutions in 50ml measuring flasks, diluting the solutions to a scale by using acetonitrile, shaking up to be respectively used as linear 1-linear 6 storage solutions, precisely measuring the 5ml respectively, placing the solutions in different 10ml measuring flasks, respectively adding 1ml of derivative reagent solutions, shaking up, placing the flasks for 30 minutes, diluting the solutions to a scale by using the acetonitrile, shaking up to be respectively used as linear 1-linear 6 solutions. Precisely measuring 10 μ l of each of the linear solutions, respectively injecting into a liquid chromatograph, recording chromatogram, making a curve of peak area to concentration, and calculating regression equation and correlation coefficient according to least square method.

The results show that: hydroxylamine hydrochloride has good linearity in the range of concentration of 0.030. Mu.g/ml to 0.608. Mu.g/ml. The results are shown in Table 5 and FIG. 8.

TABLE 5 hydroxylamine hydrochloride Linearity test results

Sample name	Concentration Range (μ g/ml)	Linear equation of equations	Coefficient of correlation
				Hydroxylamine hydrochloride	0.030～0.608	y＝137239x-485.69	0.9991

Example 5 limit of quantitation and Limit of detection test

Preparation of a quantitative limiting solution: precisely measuring 1ml of the reference solution, placing the reference solution into a 10ml measuring flask, diluting the reference solution to the scale with acetonitrile, and shaking up.

Preparation of detection limiting solution: precisely measuring 0.3ml of the reference solution, placing the reference solution into a 10ml measuring flask, diluting the reference solution to a scale with acetonitrile, and shaking up.

Precisely measuring 5ml of each of the quantitative limit solution and the detection limit solution, respectively placing the quantitative limit solution and the detection limit solution into 10ml measuring flasks, adding 1ml of each of the derivatization reagent solutions, shaking up, standing at room temperature for 30 minutes, adding acetonitrile to dissolve and dilute the solutions to a scale, and shaking up; respectively and precisely measuring 10 mu l, injecting into a liquid chromatograph, recording a chromatogram, and measuring the response value of each sample to be about 10 times and 3 times of the noise signal, namely the quantitative limit and the detection limit, wherein the results are shown in a table 6 and figures 9-10.

TABLE 6 hydroxylamine hydrochloride examination quantitation limit of detection results

Name(s)	Concentration (μ g/ml)	Detection quantity (ng)	Signal-to-noise ratio (s/n)	Ratio to concentration of sample solution (ppm)
					Limit of quantification	0.030	0.30	19	2.0
Limit of detection	0.010	0.10	4	0.6

Example 6 accuracy test

Preparation of derivatization reagent solution: weighing a proper amount of 9-fluorenylmethoxycarbonyl chloride, adding acetonitrile to dissolve and dilute to prepare a solution containing about 5mg of 9-fluorenylmethoxycarbonyl chloride in every 1 ml;

blank test solution: taking about 0.75g of parecoxib, placing the parecoxib into a 25ml measuring flask, adding 2ml of water, adding acetonitrile to dissolve and dilute the parecoxib to a scale, precisely measuring 5ml of parecoxib, placing the parecoxib into a 10ml measuring flask, adding 1ml of derivative reagent solution, shaking up, placing the parecoxib for 30 minutes, diluting the parecoxib to the scale with the acetonitrile, and shaking up to obtain the parecoxib-containing liquid.

Reference stock solution: taking about 15mg of hydroxylamine hydrochloride reference substance, placing the hydroxylamine hydrochloride reference substance in a 100ml measuring flask, adding 2ml of water to dissolve the hydroxylamine hydrochloride reference substance, diluting the hydroxylamine hydrochloride reference substance to a scale by using acetonitrile, shaking up, precisely measuring 5ml of hydroxylamine hydrochloride reference substance, placing the hydroxylamine hydrochloride reference substance in a 50ml measuring flask, diluting the hydroxylamine hydrochloride reference substance to a scale by using acetonitrile, and shaking up to obtain the hydroxylamine hydrochloride reference substance.

Control solution: precisely measuring 1ml of the reference substance stock solution, placing the reference substance stock solution in a 25ml measuring flask, diluting the reference substance stock solution to a scale with acetonitrile, precisely measuring 5ml of the reference substance stock solution, placing the reference substance stock solution in a 10ml measuring flask, adding 1ml of the derivatization reagent solution, shaking up, placing the solution for 30 minutes, diluting the solution to a scale with acetonitrile, and shaking up to obtain the reagent.

Quantitative limiting solution: taking about 0.75g of parecoxib, placing the parecoxib into a 25ml measuring flask, adding 2ml of water, adding acetonitrile to dissolve the parecoxib, adding 0.3ml of a reference substance stock solution, diluting the parecoxib to a scale with the acetonitrile, precisely measuring 5ml of the parecoxib, placing the parecoxib into a 10ml measuring flask, adding 1ml of a derivative reagent solution, shaking up, placing the parecoxib for 30 minutes, diluting the parecoxib to the scale with the acetonitrile, shaking up, and taking the parecoxib as a recovery rate quantification limit solution. 3 parts are prepared in parallel.

Recovery 50% solution: taking about 0.75g of parecoxib, placing the parecoxib in a 25ml measuring flask, adding 2ml of water, adding acetonitrile to dissolve the parecoxib, adding 0.5ml of reference substance stock solution, diluting the parecoxib to a scale mark by using acetonitrile, precisely measuring 5ml of the parecoxib, placing the parecoxib in a 10ml measuring flask, adding 1ml of derivative reagent solution, shaking up, placing the parecoxib for 30 minutes, diluting the parecoxib to a scale mark by using acetonitrile, shaking up to obtain a solution with the recovery rate of 50%. 3 parts are prepared in parallel.

Recovery 100% solution: taking about 0.75g of parecoxib, placing the parecoxib into a 25ml measuring flask, adding 2ml of water, adding acetonitrile to dissolve the parecoxib, adding 1ml of a reference substance stock solution, diluting the parecoxib to a scale with the acetonitrile, precisely measuring 5ml of the parecoxib, placing the parecoxib into a 10ml measuring flask, adding 1ml of a derivative reagent solution, shaking up, placing the parecoxib for 30 minutes, diluting the parecoxib to the scale with the acetonitrile, shaking up to obtain a solution with the recovery rate of 100%. 3 parts are prepared in parallel.

Recovery 150% solution: taking about 0.75g of parecoxib, placing the parecoxib into a 25ml measuring flask, adding 2ml of water, adding acetonitrile to dissolve the parecoxib, adding 1.5ml of a reference substance stock solution, diluting the parecoxib to a scale with the acetonitrile, precisely measuring 5ml of the parecoxib, placing the parecoxib into a 10ml measuring flask, adding 1ml of a derivative reagent solution, shaking up, placing the parecoxib for 30 minutes, diluting the parecoxib to the scale with the acetonitrile, shaking up to obtain a solution with the recovery rate of 150%. 3 parts are prepared in parallel.

Precisely measuring 10 μ l of each of the reference solution, the blank sample solution and the recovery rate measuring solution, respectively injecting into a liquid chromatograph, and recording chromatogram. And calculating the detected amount of hydroxylamine hydrochloride according to an external standard method, correcting the detected amount by using a blank test sample solution, calculating the recovery rate, and calculating the relative standard deviation of the result. The results show that: the test result of the hydroxylamine hydrochloride in parecoxib on sample recovery rate is good, and the result is shown in table 7 and attached figures 11-16.

TABLE 7 hydroxylamine hydrochloride examination recovery test results

Claims

1. A method for measuring the content of genetic toxicity impurity hydroxylamine hydrochloride in parecoxib is characterized by comprising the following steps:

(1) Preparing a test solution: taking a proper amount of parecoxib, adding a solvent to dissolve and dilute the parecoxib to prepare a test solution;

(3) Preparing a derivatization reagent solution: taking a proper amount of 9-fluorenylmethoxycarbonyl acyl chloride, adding a solvent to dissolve and diluting to prepare a derivative reagent solution;

(4) Derivatization: taking a proper amount of each of a reference substance solution and a test solution, respectively mixing the reference substance solution and the test solution with a proper amount of a derivative reagent solution, and diluting the solutions into a reference substance solution and a test solution to be tested by using a solvent after reaction;

(5) Chromatographic experiments: taking appropriate amount of reference solution and sample solution to-be-detected solution after derivatization, injecting into a liquid chromatograph, recording chromatogram, and calculating according to peak area by an external standard method;

wherein, the chromatographic condition in the step (5) is to adopt a reversed phase high performance liquid chromatograph for analysis.

2. The method for determining the content of the genetic toxic impurity hydroxylamine hydrochloride in parecoxib according to claim 1, wherein in the step (4), the derivatization reaction is carried out under the conditions of a reaction temperature of 20-40 ℃ and a reaction time of 10 minutes or more.

3. The method for determining the content of the genotoxic impurity hydroxylamine hydrochloride in parecoxib according to claim 2, wherein the reaction time is 30 minutes.

4. The method for determining the content of the genotoxic impurity, namely hydroxylamine hydrochloride, in parecoxib according to claim 1, wherein the chromatographic conditions are as follows:

a detector: an ultraviolet detector;

detection wavelength: 265nm;

a chromatographic column: chromatographic column with octadecylsilane chemically bonded silica as filler;

mobile phase: phosphate buffer, acetonitrile;

flow rate: 1.0ml/min;

column temperature: 35 ℃;

gradient elution: when the mobile phase program is 0-20 minutes, the volume ratio of the phosphate buffer solution to the acetonitrile water is 60-70: and (3) 40-30, and after 20 minutes, the proportion of acetonitrile is increased.

5. The method for determining the content of the genotoxic impurity hydroxylamine hydrochloride in parecoxib according to claim 4, wherein the gradient elution is performed according to the following table.

6. The method for determining the content of the genetic toxic impurity, namely the hydroxylamine hydrochloride in the parecoxib according to claim 4, wherein the salt concentration of the mobile phase phosphate buffer solution is 5-20 mmol/L.

7. The method for determining the content of the genotoxic impurity, namely the hydroxylamine hydrochloride in the parecoxib according to claim 6, wherein the salt concentration of the mobile phase phosphate buffer is 10mmol/L.

8. The method for determining the content of the genetic toxicity impurity hydroxylamine hydrochloride in parecoxib according to claim 4, wherein the pH value of the mobile phase phosphate buffer solution is 2.5-3.5.

9. The method for determining the content of the genetic toxicity impurity of the hydroxylamine hydrochloride in the parecoxib according to claim 8, wherein the pH value of the mobile phase phosphate buffer is 3.0.

10. The method for determining the content of the genetic toxic impurity hydroxylamine hydrochloride in parecoxib according to claim 4, wherein the chromatographic column is Agela Venusil MP 150mm x 4.6mm,5 μm.