CN114076804A

CN114076804A - Method for measuring content of impurity D in celecoxib by HPLC separation and application thereof

Info

Publication number: CN114076804A
Application number: CN202111052514.8A
Authority: CN
Inventors: 殷浩; 曾晓君; 黄丽华; 谭慧; 谭银合; 张龙辉
Original assignee: Guangzhou Huiyuan Pharmaceutical Technology Co ltd
Current assignee: Guangzhou Huiyuan Pharmaceutical Technology Co ltd
Priority date: 2021-09-08
Filing date: 2021-09-08
Publication date: 2022-02-22

Abstract

The invention discloses a method for separating and measuring the content of impurity D in celecoxib by adopting HPLC (high performance liquid chromatography) and application thereof, and relates to the technical field of drug detection, wherein the method comprises the following steps: adding celecoxib into the organic solvent to prepare a test solution; detecting the sample solution by adopting a high performance liquid chromatograph, and adopting an ultraviolet detector as a monitor, wherein the chromatographic conditions are as follows: octadecylsilane bonded silica gel column, mobile phase a: 0.05% -0.15% phosphoric acid solution, mobile phase B: methanol, acetonitrile and aqueous solution, and the mobile phase A and the mobile phase B are subjected to gradient elution according to a program. The method for detecting the genotoxic impurity D in the celecoxib by using the high performance liquid chromatography is high in separation efficiency, high in analysis speed and high in detection sensitivity, and can effectively control the content of the impurity D in the celecoxib raw material and the preparation thereof.

Description

Method for measuring content of impurity D in celecoxib by HPLC separation and application thereof

Technical Field

The invention relates to the technical field of drug detection, in particular to a method for separating and determining the content of impurity D in celecoxib by adopting HPLC (high performance liquid chromatography) and application thereof.

Background

Any substance that affects the purity of a drug is collectively referred to as impurities, and impurities in a drug broadly refer to process impurities or degradation products, etc. generated during the production, storage and transportation of the drug. The adverse reaction generated by the medicine in clinical use is not only related to the pharmacological activity of the main component, but also has great relation to impurities in the medicine, and the control of the impurities in the medicine is an important aspect of medicine research and development and is also the guarantee of the safety of clinical use. Therefore, in order to ensure the safety and effectiveness of the medicine and also consider the actual production situation, the impurity detection is taken as an important index for controlling the quality of the medicine in the research process of the medicine at home and abroad. The drug impurity detection research is one of the weak points in the current drug research and development in China. To comprehensively improve the level of drug development in China and practically ensure the safety of public drug administration, attention must be paid to and the research on related impurities in drugs must be strengthened.

Celecoxib, 4[5-4 (4-tolyl) -3- (trifluoromethyl) -1 h-1-pyrazol-1-yl ] benzenesulfonamide, is the first selective COX-2 inhibitor and is also the most widely prescribed non-steroidal anti-inflammatory analgesics (NSAIDs) worldwide at present, not only has a significant analgesic effect, but also can significantly reduce joint tenderness, pain and joint swelling, and is very safe to the gastrointestinal tract in the human body. The impurity D can be generated in the celecoxib raw material medicine production process, and the structural formulas of the celecoxib and the celecoxib impurity D are as follows:

the impurity compound has a genotoxic impurity warning structure phenylhydrazine structure, is defined as 3 genotoxic impurities by ICH M7, namely has a warning structure, is irrelevant to the structure of a raw material drug and has no mutagenicity data, the quantity of the genotoxic impurities taken into a human body per day according to the ICH specification cannot exceed 1.5 mu g, strict limit control needs to be carried out on the genotoxic impurities, the quality of a celecoxib product is better monitored, and effective separation of celecoxib and reaction byproducts is realized.

However, since the detection of such impurities has special requirements in terms of sensitivity, selectivity, stability of the analyte, and complexity of the gene, the development and selection of the analysis method brings about a greater challenge in detection of other drug impurities.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a method for measuring the content of impurity D in celecoxib by HPLC separation.

In order to achieve the purpose, the invention provides the following technical scheme:

a method for separating and determining the content of impurity D in celecoxib by HPLC comprises the following steps:

taking celecoxib, and adding an organic solvent to prepare a test solution;

detecting the test solution by adopting a high performance liquid chromatograph, and adopting an ultraviolet detector as a monitor, wherein the chromatographic conditions are as follows:

a chromatographic column: octadecylsilane chemically bonded silica gel column;

wavelength: 320 nm-340 nm;

mobile phase A: 0.05 to 0.15 percent of phosphoric acid solution;

mobile phase B: methanol-acetonitrile-water solution;

the mobile phase a and the mobile phase B were programmed for gradient elution:

time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	50	50
20	40	60
			25	30	70
30	5	95
			60	5	95
62	50	50
			70	50	50

。

Further, the organic solvent is one or more of methanol solution and acetonitrile solution.

Further, the detection wavelength of the high performance liquid chromatograph is 330 nm.

Further, the mobile phase a is a 0.1% phosphoric acid solution.

Further, the content ratio of methanol to acetonitrile to water in the mobile phase B is 50:20: 30.

Further, the column temperature of the high performance liquid chromatography is 25-40 ℃, and the flow rate is 0.8-1.2 mL/min.

Further, the column temperature was 30 ℃ and the flow rate was 1.0 ml/min.

The invention also aims to provide the application of the method in controlling the quality of celecoxib, and the method is used for separating and quantitatively determining the content of the impurity D in a celecoxib raw material or preparation so as to control the quality of a medicine.

Furthermore, the content of the impurity D in the celecoxib raw material or the preparation is less than 2.5ppm, so that the quality of the celecoxib raw material or the preparation can be better controlled.

Compared with the prior art, the invention has the beneficial effects that:

1. the method adopts the high performance liquid chromatograph to measure the content of the impurity D in the celecoxib, and has the advantages of high separation efficiency, high analysis speed and high sensitivity; the detector is used as a monitor to detect the content of the impurity D in the celecoxib, and is used for converting the change of the composition and the content of a sample in column effluent into a signal which can be detected, and the detector is characterized by high sensitivity, wide linear range, low noise and suitability for gradient elution, the detection limit of a strong absorption substance can reach 1ng, and the sample can not be damaged after the content of the impurity D in the celecoxib is detected, so that the quality of the celecoxib can be better controlled; by using the gradient elution program of the mobile phase A and the mobile phase B, the separation degree of the impurities D is improved, the separation time is shortened, the minimum detection amount is reduced, and the separation precision is improved.

2. According to the method, the content of the impurity D in the celecoxib capsules is detected, so that the impurity D in each celecoxib capsule is controlled not to exceed 2.5ppm, and the quality of the celecoxib capsules is favorably and better controlled.

Drawings

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

FIG. 1 is an HPLC chart of a white solution in example 1;

FIG. 2 is a HPLC chart of the control solution of example 1;

FIG. 3 is a HPLC chart of the test solution in example 1;

FIG. 4 is an HPLC chart of a white solution in example 2;

FIG. 5 is an HPLC chart of the sensitive solution in example 2;

FIG. 6 is a HPLC chart of the control solution in example 2;

FIG. 7 is a HPLC chart of the test solution in example 2;

FIG. 8 is a linear regression plot for example 6.

Detailed Description

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

The materials required in the embodiment of the invention are purchased from commercial channels; the chromatographic column is Purospher STAR RP-18e (4.0mm multiplied by 5.5cm, 3 mu m) produced by Merck company; the experimental methods not mentioned in the examples are conventional experimental methods, and are not described in detail herein.

The invention is further illustrated by the following examples:

determination of experimental conditions for chromatographic systems

Example 1:

the instrument comprises the following steps: high performance liquid chromatography, PDA detector;

a chromatographic column: phenyl bonded silica gel;

the content ratio of the components of the mobile phase is acetonitrile to water is 35: 65;

the flow rate settings are as in table 1 below.

Table 1:

time (min)	Flow rate (ml/min)
		0-30	0.8
30-50	1.5

Wavelength: 205nm

Column temperature: 30 deg.C

Sample introduction amount: 20 μ l

Solvent: acetonitrile-water (pH adjusted to 8.5 with 1mol/L sodium hydroxide) 80: 20;

test solution: taking about 0.56g of celecoxib, precisely weighing, placing in a 25ml measuring flask, adding 5ml of methanol for dissolving, adding a solvent for diluting to a scale, and shaking up to obtain a test solution.

Control solution: taking about 56mg of celecoxib impurity D reference substance, precisely weighing, placing in a 100ml measuring flask, adding a solvent to dilute to a scale, and shaking up to obtain a solution I; precisely measuring the solution I1ml, placing in a 100ml measuring flask, adding solvent to dilute to scale, shaking to obtain solution II, precisely measuring the solution II 1ml, placing in a 100ml measuring flask, adding solvent to dilute to scale, and shaking to obtain reference solution.

Sample introduction procedure: and (3) taking 20 mu l of each solution to be detected, injecting the solution to be detected into a liquid chromatograph, and respectively recording chromatograms.

The blank solution (solvent) has no interference to the detection of the impurity D; impurity D in the chromatogram of the control solution peaks at 23.2 min; no impurity D is detected in the test solution, and interference peaks are suspected to be detected at 22.2min and 24.0 min. The chromatograms are shown in detail in fig. 1, 2 and 3.

And (4) conclusion: the method is characterized in that phenyl bonded silica gel filler is used as a fixed phase, 35% acetonitrile is used as a mobile phase, the impurity D is detected by adopting the flow velocity gradient, under the condition of the method, the position of the impurity D in a sample solution, where the peak appears, is interfered by an adjacent impurity peak, and the elution of a strongly-retained component by increasing the flow velocity risks incomplete elution.

Example 2:

the instrument comprises the following steps: high performance liquid chromatograph, ultraviolet detector;

a chromatographic column: octadecylsilane chemically bonded silica;

flow rate: 1 ml/min;

mobile phase A: 0.10% phosphoric acid solution;

mobile phase B: methanol-acetonitrile-water (50:20:30)

Gradient elution procedure for mobile phase a and mobile phase B is shown in table 2:

table 2:

wavelength: 330nm

Column temperature: 30 deg.C

Sample introduction amount: 20 μ l

Diluting liquid: mixing methanol 800ml and acetonitrile 200ml, and mixing.

Control solution: taking about 10mg of celecoxib impurity D reference substance, precisely weighing, placing in a 100ml measuring flask, adding methanol for dissolving, diluting to a scale, and shaking uniformly to obtain a solution I; precisely measuring the solution I1ml, transferring to a 100ml measuring flask, diluting to scale with diluent, and shaking to obtain a solution II; and precisely measuring 3ml of the solution II, transferring the solution II to a 10ml measuring flask, diluting the solution II to a scale with a diluent, and shaking up the solution II to obtain the reagent.

Sensitivity solution: and (4) taking 4.0ml to 25ml of the impurity D reference substance solution, diluting the impurity D reference substance solution to a scale with a diluent, and shaking up to obtain the impurity D reference substance.

Test solution: precisely weighing about 2.2g (equivalent to 1.6g of celecoxib) of celecoxib capsule content, placing into a 20ml measuring flask, adding diluent for dissolving (ultrasonic treatment if necessary) and diluting to scale, shaking up, filtering, and taking the subsequent filtrate.

Sample introduction procedure: the above solutions to be measured were each taken in an amount of 20. mu.l and injected into a liquid chromatograph. The chromatograms were recorded separately.

As can be seen from FIGS. 4 and 6, the blank solution (diluent) did not interfere with the detection of impurities, and the impurity D peaked at 22.0min in the HPLC chart of the control solution; as can be seen from the baseline fluctuation height and the peak height of FIG. 5, the signal-to-noise ratio of the sensitive solution is 81.4, which is much greater than the requirement that the quantitative limit signal-to-noise ratio must be greater than 10, and meets the experimental requirements; as can be seen from FIG. 7, the sample solution was found to be free from interference at the peak of impurity D at 22.0 min.

Through tests, the column temperature in the embodiment 2 can adopt other temperatures in the range of 25-40 ℃, and the flow rate can adopt other flow rates in the range of 0.8-1.2 ml/min.

And (4) conclusion: comparing example 1 with example 2, it can be known that, the separation impurity D is detected by a flow gradient method using phenyl bonded silica gel filler as a stationary phase and 35% acetonitrile as a mobile phase, under the conditions of the method, the impurity D in the HPLC image of the sample solution has interference of adjacent impurity peaks, so that the impurity is eluted by increasing the flow rate in example 1, and there is a risk of incomplete elution, so the method of example 1 has obvious defects, and thus the method is not adopted; in the embodiment 2, octadecylsilane chemically bonded silica is used as a fixed phase, a phosphoric acid solution is used as a mobile phase A, and a methanol-acetonitrile-water solution is used as a mobile phase B, and impurities are detected and separated in a gradient elution mode, under the condition of the method, a blank solution has no interference on impurity detection, and an impurity D in a contrast solution chromatogram shows a peak at 22.0 min; according to the fluctuation height and peak height of the base line, the signal-to-noise ratio of the sensitivity solution is 81.4, which is far greater than the requirement that the quantitative limit signal-to-noise ratio is more than 10, and the requirement of the experiment is met; the impurity D is not detected in the test solution, and no interference of adjacent impurity peaks exists, the method can obtain a chromatogram of the test solution with a smooth base line, and the method has the characteristic of high sensitivity, so that the method in the embodiment 2 is suitable for separating and detecting the impurity D in the celecoxib.

System precision test

Example 3:

according to the liquid chromatography experimental method of the

embodiment

2, 20 μ l of each of 6 reference substance solutions is continuously injected for 6 times, the peak area of the impurity D in 6 spectrograms is recorded, and the average peak area and the relative standard deviation are calculated, wherein the relative standard deviation is not more than 5.0%. The test results are shown in table 3 below:

table 3: results of System suitability test

And (4) conclusion: the system applicability test continuously samples 6 times of reference substance solution, and the peak area relative standard deviation of the impurity D calculated according to the chromatographic result is 0.25% (required to be less than or equal to 5.0%), which indicates that the precision of the liquid chromatography experimental method of the embodiment 2 is good.

Systematic reproducibility test

Example 4:

according to the liquid chromatography experimental method of the

embodiment

2, 6 parts of celecoxib capsule samples are prepared, each sample is injected for 1 time, and the chromatogram is recorded. Calculating the content of the impurity D and obtaining the relative standard deviation, wherein the relative standard deviation is not more than 5%. The test results are shown in table 4:

table 4: results of precision test

And (4) conclusion: the result of the systematic reproducibility test shows that no impurity D is detected in the celecoxib capsules in 6 liquid phase chromatographic experiments, which indicates that the method has good reproducibility.

Accuracy test

Example 5:

weighing 9 parts of celecoxib capsules, each 2.2g (equivalent to 1.6g of celecoxib) and placing the celecoxib capsules into a 20ml measuring flask respectively, dividing 9 parts of celecoxib capsules into 3 groups, wherein each group comprises 3 parts, diluting each group to a scale by using a methanol solution containing 240ng/ml, 300ng/ml and 360ng/ml of impurities D to prepare a standard recovery solution which is equivalent to limit levels containing the impurities D80%, 100% and 120%, wherein the standard recovery rate refers to adding a quantitative standard substance into a blank sample matrix without a substance to be detected, analyzing according to the processing steps of the samples to obtain the ratio of the obtained result to a theoretical value, respectively measuring the prepared solutions according to the method of the example 2, respectively injecting each sample for 1 time, recording a chromatogram to obtain the content of the impurities D, calculating the recovery rate according to the following formula, wherein the recovery rate is 75-120%, and the test results are shown in a table 5.

Table 5: results of recovery test

And (4) conclusion: the accuracy test result shows that the single recovery rate of 9 parts of the standard-added recovery solution is between 92.18% and 99.31%, the recovery rate requirement is met, the relative standard deviation is 2.92% and is not more than 5%, and the method has good accuracy in conclusion.

Linearity and range

Example 6:

taking impurity D solutions with the concentrations of 30.75ng/ml, 153.75ng/ml, 246.00ng/ml, 307.50ng/ml, 369.00ng/ml and 461.25ng/ml as reference solutions with the limits of 10%, 50%, 80%, 100%, 120% and 150%, respectively determining according to the method of example 2, repeatedly feeding samples for 3 times for each concentration, recording a chromatogram, determining a peak area, taking the peak area as a vertical coordinate and taking the concentration as a horizontal coordinate to make a linear regression graph, and obtaining a linear regression equation, a correlation coefficient r and a linear graph, wherein the correlation coefficient r is required to be not less than 0.990. The test results are shown in Table 6, and the corresponding linear regression graph is shown in FIG. 8.

Table 6: results of impurity D Linear test

And (4) conclusion: the linear and range test results and the linear regression graph show that the regression equation of the impurity D is that y is 0.0448x-0.1103, r is 0.9998, and the correlation coefficient r meets the requirement of not less than 0.990; in conclusion, the impurity D has good linear relation between the sample injection concentration and the peak area within the concentration range of 30.75 ng/ml-461.25 ng/ml.

Detection limit and quantification limit

Example 7:

taking the reference solution with the limit concentration of 10% in example 6 as the limit solution, taking 3.0ml of the limit solution and adding distilled water to dilute to 10ml as the limit solution, respectively measuring according to the method of example 2, repeatedly injecting the limit solution for 6 times, injecting the limit solution for 1 time, recording the chromatogram, and the test results of the limit solution and the limit solution are shown in Table 7:

table 7: test results of detection limit and quantification limit

And (4) conclusion: the detection limit and quantification limit test results show that when the concentration of the quantification limit solution of the impurity D is 30.75ng/ml, the sample introduction is repeated for 6 times, the relative standard deviation of the peak area in the obtained chromatogram is 1.46 percent, the detection experiment requirement is met, the S/N ratio of the quantification limit solution is more than or equal to 10 according to the experiment requirement, the measured S/N ratio of the quantification limit solution is 50, and the detection experiment requirement is met; the detection limit solution requires that the signal-to-noise ratio S/N is more than or equal to 3, while the signal-to-noise ratio of the detection limit solution in the experiment is 14.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for separating and measuring the content of impurity D in celecoxib by adopting HPLC (high performance liquid chromatography), which is characterized by comprising the following steps:

taking celecoxib, and adding an organic solvent to prepare a test solution;

a chromatographic column: octadecylsilane chemically bonded silica gel column;

wavelength: 320 nm-340 nm;

mobile phase A: 0.05 to 0.15 percent of phosphoric acid solution;

mobile phase B: methanol-acetonitrile-water solution;

the mobile phase a and the mobile phase B were programmed for gradient elution:

。

2. The method for determining the content of impurity D in celecoxib by HPLC separation of claim 1, wherein the organic solvent is a mixture of one or more of methanol solution and acetonitrile solution.

3. The method for determining the content of impurity D in celecoxib by HPLC separation according to claim 1, wherein the detection wavelength of the HPLC is 330 nm.

4. The method for determining the content of impurity D in celecoxib by HPLC separation of claim 1, wherein the mobile phase A is 0.1% phosphoric acid solution.

5. The method for determining the content of impurity D in celecoxib by HPLC separation according to claim 1, wherein the content ratio of methanol to acetonitrile to water in the mobile phase B is 50:20: 30.

6. The method for separating and determining the content of the impurity D in the celecoxib by HPLC according to claim 1, wherein the column temperature of the high performance liquid chromatography is 25-40 ℃, and the flow rate is 0.8-1.2 mL/min.

7. The method for determining the content of impurity D in celecoxib by HPLC separation of claim 6, wherein the column temperature is 30 ℃.

8. The method for determining the content of impurity D in celecoxib by HPLC separation of claim 6, wherein the flow rate is 1.0 ml/min.

9. Use of the method for determining the content of impurity D in celecoxib by HPLC separation according to any one of claims 1-8 for controlling the quality of celecoxib, wherein the method is used for separating and quantifying the content of impurity D in celecoxib starting materials or preparations.

10. The use according to claim 9, wherein the celecoxib starting material or formulation has an impurity D content of less than 2.5 ppm.