Method for separating and determining key starting materials and related impurities of non-steroidal anti-inflammatory drugs by HPLC (High Performance Liquid Chromatography) method

Abstract

The invention belongs to the field of analytical chemistry, and particularly relates to a method for separating and determining a key starting material and related impurities of a non-steroidal anti-inflammatory drug by an HPLC (High Performance Liquid Chromatography) method, gradient elution is carried out by adopting a chromatographic column taking octadecylsilane chemically bonded silica as a filler, a 0.1% phosphoric acid solution as a mobile phase A and acetonitrile as a mobile phase B, and detection is carried out in a detector. The non-steroidal anti-inflammatory drug key starting material and related impurities can be effectively separated, and the method has the advantages of high sensitivity and separation degree, good repeatability and durability, simple operation and stable and reliable result, and is of great significance for realizing the quality control of the non-steroidal anti-inflammatory drug.

Description

Technical Field

[0001] This invention relates to the field of analytical chemistry, and specifically to a method for detecting starting materials of nonsteroidal anti-inflammatory drugs (NSAIDs), particularly an HPLC method for separating and determining key starting materials of NSAIDs and their related impurities. Background Technology

[0002] Any substance that affects the purity of a drug is collectively referred to as an impurity. Impurities in pharmaceuticals generally refer to process impurities or degradation products generated during the production and storage of drugs. Adverse reactions to drugs in clinical use are not only related to the pharmacological activity of the main component but also significantly influenced by impurities present in the drug. Controlling impurities in drugs is a crucial aspect of drug development and a guarantee of safety in clinical use. Therefore, to ensure the safety and efficacy of drugs while also considering practical production conditions, impurity detection is considered a vital indicator for controlling drug quality in both domestic and international drug research processes.

[0003] Imrecoxib is a nonsteroidal anti-inflammatory drug (NSAID) used to relieve pain symptoms of osteoarthritis. It is a first-line treatment for joint pain and osteoarthritis, and is an ideal selective COX-2 inhibitor. Compared to other drugs, imrecoxib has a higher safety profile and better patient compliance. p-Tolueneacetic acid is a key starting material in the synthesis of imrecoxib. In recent years, impurity research has been a focus of active pharmaceutical ingredient (API) research, and as the source of impurities in APIs, the quality control of starting materials is particularly important. Currently, the quality standard for imrecoxib is not included in the Chinese Pharmacopoeia, the United States Pharmacopeia, the European Pharmacopoeia, or the Japanese Pharmacopoeia, and there are relatively few reports on analytical methods related to its key starting materials.

[0004] Generally, the total impurity content of a drug should be less than 1.0%, and the content of a single impurity should be less than 0.1%. Impurities or related substances generated or introduced during the preparation of etoricoxib require strict control, whether in the active pharmaceutical ingredient (API) or the finished product. p-Tolueneacetic acid is a key starting material for nonsteroidal anti-inflammatory drugs (NSAIDs), and its quality control plays a crucial role in the preparation of high-quality NSAID APIs. Therefore, developing an efficient and accurate analytical method for the quality analysis of key starting materials for NSAIDs is of great significance for the subsequent preparation of high-purity NSAID products. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a method for separating and determining key starting materials and related impurities of nonsteroidal anti-inflammatory drugs by HPLC. The method of this invention can effectively separate key starting materials and related impurities of etoricoxib, and the method has high sensitivity and resolution, good repeatability and robustness, simple operation, and stable and reliable results.

[0006] To achieve the above objectives, the applicant, through extensive experimentation, has determined the technical solution of this invention as follows:

[0007] A method for separating and determining key starting materials and related impurities of nonsteroidal anti-inflammatory drugs (NSAIDs) using HPLC, wherein the chromatographic column is packed with octadecylsilane-bonded silica gel, and gradient elution is performed between mobile phase A and mobile phase B, followed by detection by a detector; the related impurities include one or more of impurity A, impurity B, impurity C, impurity D, and impurity E, with the specific structural formulas shown below:

[0008]

[0009] Impurity A is the raw material used in the synthesis of p-methylphenylacetic acid, a key starting material for the preparation of etoricoxib;

[0010] Impurity B is an intermediate in the synthesis of p-toluidine, a key starting material for arbutin.

[0011] Impurity C is an intermediate in the synthesis of p-methylphenylacetic acid, a key starting material for atorcob.

[0012] Impurity D is a hydrolyzed impurity of impurity A;

[0013] Impurity E is a degradation impurity of p-methylphenylacetic acid, the key starting material for etoricoxib.

[0014] Impurities A, B, C, D, and E may remain in the starting material and be transferred to the finished product, Eritrexate.

[0015] The mobile phase A is an aqueous solution of phosphoric acid, and the mobile phase B is an organic solvent.

[0016] The key starting material for the synthesis of atorcitabine is p-methylphenylacetic acid, which has the chemical formula C9H. 10 O2, with the structural formula as shown in Formula (I), is referred to in this invention as "Formula I compound" or "Irrexib key starting material":

[0017]

[0018] The synthesis process is as follows:

[0019]

[0020] Furthermore, the mobile phase A is an aqueous solution of phosphoric acid, and the concentration of the aqueous solution of phosphoric acid is 0.05% to 0.15%.

[0021] As a preferred embodiment, the concentration of the phosphoric acid aqueous solution is 0.1%;

[0022] Furthermore, the mobile phase B is one or more of acetonitrile, ethanol, and methanol;

[0023] Furthermore, the mobile phase B is acetonitrile.

[0024] The elution flow rate of the mobile phase is 0.8-1.2 ml / min, preferably 1.0 ml / min.

[0025] The gradient elution settings are as follows:

[0026]

[0027] As a preferred embodiment, the gradient elution is configured as follows:

[0028]

[0029] Furthermore, the particle size of the octadecylsilane-bonded silica gel packing material is 2-5 μm; the column temperature of the chromatographic column is 30-40℃.

[0030] As a preferred embodiment, the octadecylsilane-bonded silica gel packing material has a particle size of 5 μm; and the column temperature of the chromatographic column is 35 °C.

[0031] Furthermore, the detection wavelength of the detector is 215±5nm.

[0032] As a preferred embodiment, the detection wavelength of the detector is 215 nm.

[0033] Furthermore, the method for separating and determining the key starting materials and related impurities of etoricoxib using HPLC, wherein the related impurities are reference standards for impurity A, impurity B, impurity C, impurity D, and impurity E, specifically includes the following steps:

[0034] 1) Preparation of test solution: Take the test sample of p-methylphenylacetic acid, the starting material of etoricoxib, add acetonitrile-water (volume ratio 80:20) to dissolve and dilute to prepare a solution containing about 0.5 mg per 1 ml, thus obtaining the test solution;

[0035] 2) Preparation of control solution: Take an appropriate amount of the test solution from step 1), add acetonitrile-water (volume ratio 80:20) to dissolve and dilute to prepare a solution containing approximately 1 μg per 1 ml, which will serve as the control solution.

[0036] 3) Preparation of system suitability solution: Take the key starting material of etoricoxib and its impurity A, impurity B, impurity C, impurity D, and impurity E reference standards, and dissolve and dilute them with acetonitrile-water (volume ratio 80:20) to prepare system suitability solution;

[0037] 4) Inject the system suitability solution described in step 3) and perform high performance liquid chromatography analysis. Record the chromatogram and determine the retention time of the key starting materials and impurities of etoricoxib. Then, inject the test solution described in step 1) and the control solution described in step 2) respectively. Calculate the content of impurities in the key starting materials of etoricoxib in the test solution according to the principal component self-comparison method.

[0038] 5) The linear correlation coefficients of the key starting materials of Arixicob and their impurities A, B, C, D, and E are shown in the table below:

[0039] name Concentration range (μg / ml) Linear equations Correlation coefficient (r) Formula I compound 0.0807～5.0455 y = 27.2080x + 0.1237 1.0000 Impurity A 0.0787～4.9213 y = 28.0626x + 0.1782 1.0000 Impurity B 0.0816～5.1028 y = 31.2903x + 0.1444 1.0000 Impurity C 0.0804～5.0249 y = 27.3866x + 0.0258 1.0000 Impurity D 0.0811～5.0714 y = 37.5883x + 0.0300 1.0000 Impurity E 0.0841～5.2592 y = 39.6971x + 0.2253 1.0000

[0040] The second objective of this invention is to provide a reagent composition for the solid-liquid separation determination of key starting materials and impurities of etoricoxib, comprising the following reagents:

[0041] Reagent A: Phosphoric acid aqueous solution;

[0042] Reagent B: Organic solvent;

[0043] The relevant impurities include one or more of impurity A, impurity B, impurity C, impurity D, and impurity E;

[0044] The concentration of the phosphoric acid aqueous solution is 0.05% to 0.15%; the organic solvent is one or more of acetonitrile, ethanol and methanol.

[0045] As a preferred embodiment, the concentration of the phosphoric acid aqueous solution is 0.1%; and the organic solvent is acetonitrile.

[0046] The reagent composition provided by this invention for solid-liquid separation determination of key starting materials and impurities of etoricoxib can effectively separate key starting materials and impurities of etoricoxib, which is of great significance for achieving quality control of key starting materials and etoricoxib.

[0047] The beneficial effects of this invention are as follows:

[0048] 1) This invention provides an HPLC method for separating and determining key starting materials and related impurities of etoricoxib. The method of this invention can effectively separate key starting materials and related impurities of etoricoxib, and the method has high sensitivity and resolution, good repeatability and durability, simple operation, and stable and reliable results.

[0049] 2) Research has revealed that the structures of impurities A, B, C, D, and E in the key starting material of Formula I are very similar to those of the key starting material. If this step is not controlled, the impurities will be difficult to remove if they are introduced into the active pharmaceutical ingredient during the reaction. Therefore, it is necessary to strictly monitor each impurity in this step. In other words, the analysis and research of the key starting material of etoricoxib in this invention plays a crucial role in the control of the synthesis reaction and the improvement of quality, and also directly affects the quality of the finished etoricoxib product. Therefore, this method is of great significance for achieving the control of the key starting material and quality of etoricoxib. Attached Figure Description

[0050] Figure 1 The chromatogram of the system suitability solution under the conditions of this embodiment shows that the peak elution order is as follows: impurity C (retention time Rt 6.356 min), impurity D (retention time Rt 8.129 min), compound of formula I (retention time Rt 9.828 min), impurity B (retention time Rt 13.181 min), impurity A (retention time Rt 17.903 min), and impurity E (retention time Rt 18.972 min).

[0051] Figure 2 Chromatogram of the system suitability solution under the conditions of Example 1;

[0052] Figure 3 Chromatogram of the system suitability solution under the conditions of Example 2;

[0053] Figure 4 Chromatogram of the system suitability solution under the conditions of Example 3. Detailed Implementation

[0054] The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Experimental methods in the preferred embodiments that do not specify specific conditions are generally performed under conventional conditions. The examples provided are for better illustration of the present invention, but are not intended to limit the scope of the invention to the examples given. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention. In this embodiment, the key starting material for etoricoxib refers to compound of formula I.

[0055] Example 1

[0056] 1. Chromatographic conditions:

[0057] The column was packed with octadecylsilane-bonded silica gel (YMC Pack ODS AM, 150 mm × 4.6 mm, 5 μm or equivalent column); mobile phase A was 0.1% phosphoric acid solution and mobile phase B was acetonitrile; linear gradient elution was performed according to the table below; column temperature was 35 °C; flow rate was 1.0 mL per minute; detection wavelength was 215 nm; injection volume was 10 μl.

[0058]

[0059] 2. Methods and Results

[0060] 2.1 Solution Preparation

[0061] Take an appropriate amount of the key starting material of etoricoxib, accurately weigh it, dissolve it in acetonitrile-water (volume ratio 80:20) and quantitatively dilute it to prepare a solution containing about 0.5 mg per ml, as the test solution; accurately measure an appropriate amount of the test solution, dilute it with acetonitrile-water (volume ratio 80:20) to prepare a solution containing about 1 μg per ml, as the control solution.

[0062] 2.2 Specificity

[0063] Accurately weigh appropriate amounts of the key starting material of etoricoxib and reference standards of impurities A, B, C, D, and E, and place them in the same volumetric flask. Dissolve and dilute with acetonitrile-water (80:20 v / v) to prepare a solution containing approximately 0.5 mg of compound I and approximately 1 μg of each impurity per 1 ml. This solution is used as the system suitability solution. Accurately inject 10 μl into the liquid chromatograph and record the chromatogram. The results of the system suitability solution are shown in the appendix. Figure 1 In the system suitability solution, the separation degree between the key starting material of etoricoxib and the adjacent impurity peak was 10.5, and the retention time Rt was 9.828 minutes.

[0064] 2.3 Repeatability

[0065] Accurately weigh appropriate amounts of the key starting material of etoricoxib and its various impurity reference standards, place them in the same volumetric flask, dissolve and dilute with acetonitrile-water (80:20 v / v) to prepare a solution containing approximately 0.5 mg of compound I, approximately 1 μg of impurity A, approximately 1 μg of impurity B, approximately 1 μg of impurity C, approximately 1 μg of impurity D, and approximately 1 μg of impurity E per 1 ml, as the spiked test solution; accurately measure 1 ml of the spiked test solution and place it in a 500 ml volumetric flask, dilute with acetonitrile-water (80:20 v / v) to prepare a solution containing approximately 1 μg of compound I per 1 ml, as the control solution. Accurately inject the above spiked test solution and control solution, and record the chromatograms. Calculate the RSD of each related substance in the six spiked test solutions using the principal component self-comparison method with correction factor. The RSDs of the detected related impurities in the six spiked test solutions are all less than 10.0%, which meets the requirements of high performance liquid chromatography for the detection of related substances.

[0066] 2.4 Linear

[0067] Accurately weigh appropriate amounts of the key starting materials of etoricoxib and their respective impurity reference standards, place them in the same volumetric flask, dissolve and dilute with acetonitrile-water (80:20 v / v) to prepare a solution containing approximately 20 μg of compound I, impurity A, impurity B, impurity C, impurity D, and impurity E per 1 ml, as the linearity stock solution. Take 1 ml of the stock solution and place it in 100 ml, 1 ml in 25 ml, 1 ml in 20 ml, 1.5 ml in 20 ml, 3 ml in 20 ml, and 5 ml in 20 ml volumetric flasks respectively, dilute to the mark with acetonitrile-water (80:20 v / v), and shake well. Separately, take the limit of quantitation solution (prepared by dissolving and diluting compound I and each impurity reference standard with acetonitrile-water (80:20 v / v) to prepare a solution with the limit of quantitation concentration), as the linearity test solution. Linear regression of peak area against concentration yielded the linear equation (Table 1), showing that the key starting materials and their impurities for etoricoxib exhibit a good linear relationship within the linear range.

[0068] Table 1. Results of linearity determination

[0069]

[0070]

[0071] 2.5 Limit of Detection and Limit of Quantification

[0072] Take appropriate amounts of the key starting materials and impurity reference standards of etoricoxib and prepare a series of solutions. Solutions with a S / N ≥ 10 are used as the limit of quantitation (LOQ) solutions, and solutions with an S / N ≥ 3 are used as the limit of detection (LOD) solutions. The LQ and LOD results for the key starting materials and impurities of etoricoxib are shown in Table 2.

[0073] Table 2 Results of Limit of Quantitation and Limit of Detection

[0074]

[0075] 2.6 Accuracy

[0076] Take appropriate amounts of reference standards for impurities A, B, C, D, and E, dissolve and dilute them in acetonitrile-water (80:20 v / v) to prepare a solution containing 20 μg of each impurity per ml, as the stock solution of the mixed impurity reference standard. Take appropriate amounts of the key starting material of etoricoxib, and accurately measure 0.5 ml of the mixed impurity reference standard solution into 50 ml, 0.5 ml into 20 ml, 1 ml into 20 ml, 3 ml into 20 ml, and 4.5 ml into 20 ml volumetric flasks respectively. Dilute to the mark with solvent and shake well to prepare the test solution for recovery. Perform three replicates for each concentration. Calculate the recovery rate and RSD of each impurity using the principal component self-comparison method with correction factor. The results show that the recovery rates of each impurity at all concentrations are between 80.0% and 120.0%, and the RSDs are all less than 10.0%, meeting the requirements of high performance liquid chromatography for the detection of related substances.

[0077] 3. Conclusion:

[0078] Under these chromatographic conditions, the key starting materials and impurities of etoricoxib can be completely separated. This method is highly specific, accurate, sensitive, repeatable, and system-compatible, meeting the technical requirements of drug quality research standards, and the results obtained are stable and reliable.

[0079] Compare with Example 1:

[0080] 1. Chromatographic conditions:

[0081] Chromatographic column: Agilent Zorbax Eclipse XDB-C18 (250 mm × 4.6 mm, 5 μm); mobile phase A: 10 mmol / L potassium dihydrogen phosphate buffer (pH adjusted to 6.0 with potassium hydroxide solution); mobile phase B: acetonitrile; gradient elution was performed as follows:

[0082]

[0083] Flow rate: 1 ml / min, column temperature: 35℃, detection wavelength: 220 nm, injection volume: 10 μl.

[0084] Method 2: Accurately weigh appropriate amounts of the key starting material of etoricoxib and impurities A, B, C, and D, place them in the same volumetric flask, dissolve and dilute with 40% acetonitrile to prepare a solution containing approximately 0.5 mg of compound I and approximately 1 μg of each impurity per 1 ml, which serves as the system suitability solution. Accurately inject 10 μl into the liquid chromatograph and record the chromatogram. The chromatogram of the system suitability solution is shown in the appendix. Figure 2 .

[0085] 3. Conclusion: Under these chromatographic conditions, the peak shapes of each impurity were poor, and the tailing factor of the impurity at Rt 6.934 minutes was 2.5, which affected the quantification of the impurities.

[0086] Compare with Example 2:

[0087] 1. Chromatographic conditions:

[0088] Chromatographic column: Agilent Zorbax Eclipse XDB-C18 (250 mm × 4.6 mm, 5 μm); mobile phase A: 10 mmol / L potassium dihydrogen phosphate buffer (pH adjusted to 7.0 with potassium hydroxide solution); mobile phase B: acetonitrile; gradient elution was performed as follows:

[0089]

[0090] Flow rate: 1 ml / min, column temperature: 35 ℃, detection wavelength: 215 nm, injection volume: 10 μl.

[0091] Method 2: Accurately weigh appropriate amounts of the key starting material of etoricoxib and impurities A, B, C, and D, place them in the same volumetric flask, dissolve and dilute with 80% acetonitrile to prepare a solution containing approximately 0.5 mg of compound I and approximately 1 μg of each impurity per 1 ml, which serves as the system suitability solution. Accurately inject 10 μl into the liquid chromatograph and record the chromatogram. The chromatogram of the system suitability solution is shown in the appendix. Figure 3 .

[0092] 3. Conclusion: Under these chromatographic conditions, the main peak bifurcated and the baseline fluctuated significantly, affecting the quantitative results and qualitative judgment of impurities.

[0093] Compare with Example 3:

[0094] 1. Chromatographic conditions:

[0095] Chromatographic column: Agilent Zorbax Eclipse XDB-C18 (250 mm × 4.6 mm, 5 μm); mobile phase A: 10 mmol / L potassium dihydrogen phosphate buffer (pH adjusted to 5.0 with potassium hydroxide solution); mobile phase B: acetonitrile; gradient elution was performed as follows:

[0096]

[0097] Flow rate: 1 ml / min, column temperature: 35 ℃, detection wavelength: 215 nm, injection volume: 10 μl.

[0098] 2. The method is the same as in Control Example 2. The chromatogram of the system suitability solution is shown in Appendix 2. Figure 4 .

[0099] 3. Conclusion: Under these chromatographic conditions, the separation between the main peak (Rt 9.592 min) and the impurities (Rt 9.878 min) was poor, affecting the quantitative results and qualitative judgment of the impurities.

[0100] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for detecting a non-steroidal anti-inflammatory drug key starting material, the method being a method for determining a non-steroidal anti-inflammatory drug key starting material and related impurities thereof by HPLC separation, using a chromatographic column filled with octadecylsilane-bonded silica gel, gradient elution with mobile phase A and mobile phase B, and detection by a detector; the related impurities include one or more of impurity A, impurity B, impurity C, impurity D, and impurity E, and the specific names are as follows: The mobile phase A is an aqueous phosphoric acid solution, and the mobile phase B is an organic solvent. The key starting material of the eribulin is p-methyl phenylacetic acid, which is a key starting material for synthesizing eribulin, and its chemical formula is C9H 10 O2, and its structural formula is as shown in formula (I) below, which is also referred to as "formula I compound" or "key starting material of eribulin" in the present application: The gradient elution is set as follows: The flow rate of the mobile phase for elution is 0.8-1.2 ml / min.

2. The method of claim 1, wherein, The mobile phase A is an aqueous phosphoric acid solution, and the mobile phase B is acetonitrile.

3. The method of claim 2, wherein, The concentration of the aqueous phosphoric acid solution is 0.05%-0.15%.

4. The method of claim 1, wherein, The gradient elution is set as follows:

5. The method of claim 1, wherein, The particle size of the filler of the octadecylsilane-bonded silica gel is 2-5 μm; and the column temperature of the chromatographic column is 30-40℃.

6. The method of claim 1, wherein, The detection wavelength of the detector is 215±5 nm.

7. The method of claim 1, wherein, The related impurities are impurity A, impurity B, impurity C, impurity D, and impurity E reference substances, and the method comprises the following steps: 1) Preparation of a test solution: a test sample is dissolved in a diluent to prepare a solution containing about 0.5 mg per 1 ml, to obtain a test solution; 2) Preparation of a control solution: an appropriate amount of the test solution is diluted with a diluent to prepare a solution containing about 1 μg per 1 ml, as a control solution; 3) Preparation of a system suitability solution: the key starting material of Erixi Xibu and impurities A, B, C, D, and E thereof are dissolved and diluted with a diluent to prepare a system suitability solution; 4) The system suitability solution of step 3) is injected for high performance liquid chromatography analysis, the chromatogram is recorded, the retention time of the key starting material of Erixi Xibu and its impurities is determined, and then the test solution of step 1) and the control solution of step 2) are injected, and the content of impurities in the key starting material of Erixi Xibu in the test solution is calculated by the main component self-control method, The diluent is a solution of acetonitrile-water (volume ratio 80:20).

Images