Method for separating and determining key intermediate of non-steroidal anti-inflammatory drug and related impurities thereof 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 intermediate and related impurities of a non-steroidal anti-inflammatory drug by HPLC (High Performance Liquid Chromatography), which comprises the following steps: carrying out gradient elution by adopting a chromatographic column taking octadecylsilane chemically bonded silica as a filler, a mixed solvent of a phosphate buffer and acetonitrile as a mobile phase A and acetonitrile as a mobile phase B, and entering a detector for detection. The non-steroidal anti-inflammatory drug key intermediate 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] The present invention relates to the field of analytical chemistry, and relates to a detection method for an intermediate of a non-steroidal anti-inflammatory drug, in particular to an HPLC method for separating and determining a key intermediate of a non-steroidal anti-inflammatory drug and its related impurities. Background Art

[0002] Imrecoxib (CAS: 395683-14-4) is the first COX-2 selective inhibitor with independent intellectual property rights in China and belongs to non-steroidal anti-inflammatory drugs (NSAIDs). Traditional NSAIDs are prone to cause gastrointestinal adverse reactions due to excessive inhibition of COX-1, while highly selective COX-2 inhibitors (such as rofecoxib and valdecoxib) may increase the cardiovascular risk. Imrecoxib is developed based on the concept of "moderate inhibition". While exerting anti-inflammatory and analgesic effects, it can maintain the balance of prostaglandins in the body, thereby reducing the risks of gastrointestinal and cardiovascular adverse reactions. It is clinically used to relieve the pain symptoms of osteoarthritis.

[0003] At present, the quality standards of Imrecoxib are not included in the United States Pharmacopeia, the Japanese Pharmacopeia, the import drug registration standards of the National Medical Products Administration, etc., and there are relatively few reports on the related analysis methods of its key intermediates. Lu Yanfang et al., Determination of the content of Imrecoxib in Imrecoxib tablets by HPLC, reported using methanol-water (80:20) as the mobile phase and isocratic elution. However, this method is only applicable to measuring the content of Imrecoxib and does not involve the detection method of Imrecoxib intermediates.

[0004] Generally speaking, the total content of drug impurities should be less than 1.0%, and the content of a single impurity should be less than 0.1%. For the impurities generated or related substances introduced during the preparation of Imrecoxib, strict control is required both in the raw material drug and in the preparation. 2-Bromo-1-(4-(methylsulfonyl)phenyl)ethan-1-one is a key intermediate of Imrecoxib. The quality control of this compound plays an important role in the preparation of high-quality raw materials of a non-steroidal anti-inflammatory drug. Therefore, developing an efficient and accurate analytical method for quality analysis of the key intermediate of a non-steroidal anti-inflammatory drug is of great significance for the subsequent preparation of high-purity non-steroidal anti-inflammatory drug products. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a method for separating and determining a key intermediate of a non-steroidal anti-inflammatory drug and its related impurities by HPLC; the method of the present invention can effectively separate the key intermediate of Imrecoxib and its related impurities, and the method has high sensitivity and resolution, good repeatability and durability, simple operation, and stable and reliable results.

[0006] To achieve the above object, after a large number of experiments, the technical solution of the present invention determined by the applicant is as follows:

[0007] A method for separating and determining a key intermediate of a non-steroidal anti-inflammatory drug and its related impurities by HPLC. The chromatographic column used in the method is packed with octadecylsilane-bonded silica gel, gradient elution is carried out with mobile phase A and mobile phase B, and detection is carried out by entering a detector; the related impurities include one or more of impurity A, impurity B, impurity C, impurity D, impurity E, and impurity F, and the specific structural formulas are as follows:

[0008] Impurity Code Impurity Name Impurity A 2,2-Dibromo-1-(4-(methylsulfonyl)phenyl)ethyl-1-one Impurity B Ethyl 2-(4-(methylsulfonyl)phenyl)-2-oxoacetate Impurity C 2-Hydroxy-1-(4-(methylsulfonyl)phenyl)ethyl-1-one Impurity D 2-Bromo-4'-methylthioacetophenone Impurity E 2-Bromo-1-(4-(methylsulfinyl)phenyl)ethyl-1-one Impurity F 1-(4-(methylsulfonyl)phenyl)ethyl-1-one

[0009] The mobile phase A is a mixed solvent of phosphate buffer solution and organic solvent, and the mobile phase B is an organic solvent.

[0010] The key intermediate of ericoxib is 2-bromo-1-(4-(methylsulfonyl)phenyl)ethyl-1-one, which is a key intermediate for synthesizing ericoxib. Its chemical formula is C9H9BrO3S, and the structural formula is as shown in formula (I) below. In the present invention, it is also abbreviated as "compound of formula I" or "key intermediate of ericoxib":

[0011]

[0012] Further, the mobile phase A is a mixed solvent of phosphate buffer solution and acetonitrile, and the volume ratio of the phosphate buffer solution to acetonitrile is 90:10;

[0013] Further, the pH value of the phosphate buffer solution is adjusted to 6.8 - 7.2 with potassium hydroxide solution;

[0014] As a preference, the pH value of the phosphate buffer solution is adjusted to 7.0 with phosphoric acid;

[0015] Further, the phosphate buffer solution is an aqueous solution of potassium dihydrogen phosphate, and the phosphate concentration is 5 - 15 mmol / L;

[0016] As a preference, the phosphate concentration in the phosphate buffer solution is 10 mmol / L;

[0017] Further, the mobile phase B is one or more of acetonitrile, ethanol, and methanol;

[0018] Further, the mobile phase B is acetonitrile.

[0019] The flow rate of the mobile phase for elution is 0.8 - 1.2 ml / min, preferably 1.0 ml / min.

[0020] The gradient elution is set as follows:

[0021]

[0022] As an optimization, the gradient elution is set as follows:

[0023]

[0024] Furthermore, the particle size of the packing particles of octadecylsilyl silica gel is 2 - 5 μm; the column temperature of the chromatographic column is 30 - 40 °C.

[0025] As an optimization, the particle size of the packing particles of octadecylsilyl silica gel is 5 μm; the column temperature of the chromatographic column is 35 °C.

[0026] Furthermore, the detection wavelength of the detector is 244 ± 5 nm.

[0027] As an optimization, the detection wavelength of the detector is 244 nm.

[0028] Furthermore, for the method for separating and determining the key intermediate of etoricoxib and its related impurities by HPLC, the related impurities are reference substances of impurity A, impurity B, impurity C, impurity D, impurity E, and impurity F, and specifically include the following steps:

[0029] 1) Prepare the test solution: Dissolve the test sample in the diluent to prepare a solution containing approximately 0.5 mg per 1 ml to obtain the test solution;

[0030] 2) Prepare the control solution: Take an appropriate amount of the test solution and dilute it with the diluent to prepare a solution containing approximately 5 μg per 1 ml as the control solution.

[0031] 3) Prepare the system suitability solution: Take the key intermediate of etoricoxib and its impurities A, B, C, D, E, and F, and dissolve and dilute them with the diluent to prepare the system suitability solution;

[0032] 4) Inject the system suitability solution prepared in step 3) for high - performance liquid chromatography analysis, record the chromatogram, determine the retention times of the key intermediate of etoricoxib and its impurities, and then inject the test solution prepared in step 1) and the control solution prepared in step 2) respectively, and calculate the content of the impurities in the key intermediate of etoricoxib in the test solution by the self - control method of the main component.

[0033] 5) The linear correlation coefficients of the key intermediate of etoricoxib and its impurities A, B, C, D, and E are shown in the following table:

[0034]

[0035]

[0036] The diluent is a solution of acetonitrile-water (volume ratio is 50:50).

[0037] The second object of the present invention is to provide a reagent composition for solid-liquid separation and determination of key intermediates of erlotinib and its impurities, which is composed of the following reagents:

[0038] Reagent A: a mixed solvent of phosphate buffer and organic solvent;

[0039] Reagent B: organic solvent;

[0040] The relevant impurities include one or more of impurity A, impurity B, impurity C, impurity D, impurity E, and impurity F;

[0041] The phosphate concentration in the phosphate buffer is 5-15 mmol / L; the organic solvent is one or more of acetonitrile, ethanol and methanol.

[0042] Preferably, the phosphate concentration in the phosphate buffer is 10 mmol / L; and the organic solvent is acetonitrile.

[0043] The reagent composition for solid-liquid separation determination of key intermediates of erlotinib and their impurities provided by the present invention can effectively separate key intermediates of erlotinib and their impurities, and is of great significance for achieving quality control of key intermediates of erlotinib and erlotinib.

[0044] The beneficial effects of the present invention are:

[0045] 1) The present invention provides a method for separating and determining the key intermediates of erlotinib and its related impurities by HPLC. The method of the present invention can effectively separate the key intermediates of erlotinib and its related impurities, and the method has high sensitivity and separation degree, good repeatability and durability, simple operation, and stable and reliable results.

[0046] 2) It has been found through research that the impurity A of the key intermediate of formula I is very similar to the structure of the key intermediate. If it is not controlled at this step, it will be difficult to remove it if it is brought into the raw material drug during the continued reaction. Therefore, it is necessary to strictly monitor each impurity in this step of the reaction. That is, the analysis and research of the key intermediate of Errecoxib in the present invention plays a vital role in the control of the synthesis reaction and the improvement of the quality, and also directly affects the quality of the Errecoxib finished product. Therefore, this method is extremely important for achieving the quality control of the key intermediate of Errecoxib and Errecoxib. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1Under the conditions of the embodiments of the present invention, the chromatogram of the system suitability solution shows the elution order as follows: impurity C (retention time Rt 6.599 minutes), impurity F (retention time Rt 9.815 minutes), impurity B (retention time Rt 10.178 minutes), impurity E (retention time Rt 10.598 minutes), compound of formula I (retention time Rt 12.567 minutes), impurity A (retention time Rt 14.728 minutes), impurity D (retention time Rt 17.448 minutes);

[0048] Figure 2 Chromatogram of the system suitability solution under the conditions of Comparative Example 1;

[0049] Figure 3 Chromatogram of the system suitability solution under the conditions of Comparative Example 2;

[0050] Figure 4 Chromatogram of the system suitability solution under the conditions of Comparative Example 3. Detailed Description of the Invention

[0051] Hereinafter, with reference to the accompanying drawings, the preferred embodiments of the present invention will be described in detail. For the experimental methods without specific conditions indicated in the preferred embodiments, they are generally carried out under conventional conditions. The examples are given to better illustrate the content of the present invention, but the content of the present invention is not limited to the given examples. Therefore, those skilled in the art who make non-essential improvements and adjustments to the implementation schemes based on the above-mentioned inventive content still fall within the protection scope of the present invention. The key intermediate of ixekizumab in this example refers to the compound of formula I.

[0052] Example 1

[0053] 1 Chromatographic conditions:

[0054] Using octadecylsilane-bonded silica gel as the filler (Agilent ZORBAX Eclipse plus-C18, 4.6 mm × 250 mm, 5 μm or a chromatographic column with equivalent performance); using 10 mmol / L potassium dihydrogen phosphate solution (adjusting the pH value to 7.0 with potassium hydroxide solution)-acetonitrile (90:10) as mobile phase A, and acetonitrile as mobile phase B; performing gradient elution according to the following table; the column temperature is 35 °C; the flow rate is 1.0 ml per minute; the detection wavelength is 244 nm; the injection volume is 10 μl.

[0055]

[0056] 2 Methods and Results

[0057] 2.1 Solution preparation

[0058] Weigh an appropriate amount of the key intermediate of imrecoxib accurately, dissolve it in acetonitrile - water (volume ratio 50:50) and quantitatively dilute it to prepare a solution containing about 0.5 mg per 1 ml as the test solution; accurately measure an appropriate amount of the test solution and dilute it with the diluent to prepare a solution containing about 5 μg per 1 ml as the control solution.

[0059] 2.2 Specificity

[0060] Weigh appropriate amounts of the key intermediate of imrecoxib and reference substances of impurity A, impurity B, impurity C, impurity D, impurity E, and impurity F accurately, place them in the same volumetric flask, dissolve and dilute with the solvent to prepare a solution containing about 0.5 mg of the compound of formula I per 1 ml, 25 μg of impurity A, and about 10 μg of each of the other impurities as the system suitability solution. Accurately measure 10 μl and inject it into the liquid chromatograph, record the chromatogram, and the results of the system suitability solution are shown in the appendix Figure 1 In the system suitability solution, the resolution between the key intermediate of imrecoxib and the adjacent impurity peak is 2.0, and the retention time Rt is 12.567.

[0061] 2.3 Repeatability

[0062] Weigh appropriate amounts of the key intermediate of imrecoxib and its impurity reference substances, dissolve and dilute with the solvent to prepare a solution containing about 0.5 mg of the compound of formula I per 1 ml, 25 μg of impurity A, and about 10 μg of each of the other impurities as the spiked test solution; accurately measure 1 ml of the spiked test solution and place it in a 100 - ml volumetric flask, dilute with the solvent to prepare a solution containing about 5 μg of the compound of formula I per 1 ml as the control solution. Accurately measure the above - mentioned spiked test solution and control solution for injection, record the chromatogram. Calculate the RSD of the contents of each related substance in 6 portions of the spiked test solution by the self - control method of the main component with correction factors. The RSDs of the contents of each related impurity detected in the 6 portions of the spiked test solution are all less than 10.0%, meeting the requirements for the detection of related substances by high - performance liquid chromatography.

[0063] 2.4 Linearity

[0064] Appropriately weigh the key intermediate of imrecoxib and its impurity reference substances, dissolve and dilute with a solvent to prepare a mixed solution containing 0.5 mg of A and 0.2 mg of each of Formula I and other impurities per 1 ml as the linear stock solution (1). Accurately transfer 1 ml of the linear stock solution (1) into a 20-ml volumetric flask, and dilute to the mark with the diluent to obtain the linear stock solution (2). Respectively and accurately transfer 1 ml of the linear stock solution (1) into 50-ml, 25-ml, 20-ml, and 20-ml volumetric flasks containing 3 ml, and dilute to the mark with the diluent; respectively and accurately transfer 1 ml of the linear stock solution (2) into 10-ml and 10-ml volumetric flasks containing 2 ml, and dilute to the mark with the diluent. Additionally, take the quantitation limit solution as the linear solution. Perform linear regression with the peak area against the concentration to obtain the linear equation (Table 1). The key intermediate of imrecoxib and its impurities have good linear relationships within the linear range.

[0065] Table 1 Results of Linear Determination

[0066]

[0067] 2.5 Detection Limit and Quantitation Limit

[0068] Appropriately weigh the key intermediate of imrecoxib and its impurity reference substances, and prepare a series of solutions. When S / N ≥ 10, it is used as the quantitation limit solution, and when S / N ≥ 3, it is used as the detection limit solution. The results of the quantitation limit and detection limit of the key intermediate of imrecoxib and its impurities are shown in Table 2.

[0069] Table 2 Results of Quantitation Limit and Detection Limit

[0070]

[0071] 2.6 Accuracy

[0072] Accurately weigh appropriate amounts of Impurity A, Impurity B, Impurity C, Impurity D, Impurity E, and Impurity F, dissolve them by ultrasonic treatment with a solvent and dilute to prepare a mixed solution containing 0.5 mg of Impurity A and 0.2 mg of the other impurities per 1 ml as the mixed impurity reference substance solution. Prepare 3 parallel portions; weigh an appropriate amount of the key intermediate of imrecoxib, and accurately measure 0.5 ml of the mixed impurity reference substance stock solution into 200-ml, 50-ml, 20-ml, 20-ml, and 20-ml volumetric flasks containing 1 ml and 2 ml respectively, dilute to the mark with the solvent, and shake well to obtain the test solutions for recovery. Each concentration is prepared in 3 parallel portions. Calculate the recovery rate and RSD of each impurity according to the method of self-control of the main component with a correction factor. The results show that the recovery rates of each impurity at each concentration are between 80.0% and 120.0%, and the RSDs are all less than 10.0%, meeting the requirements for the determination of related substances by high performance liquid chromatography.

[0073] 3 Conclusion:

[0074] Under these chromatographic conditions, the key intermediate of ixekib and its impurities can be completely separated. This method has strong specificity, good accuracy, high sensitivity, good repeatability, and good system suitability, meeting the technical requirements of drug quality research standards, and the results obtained are stable and reliable.

[0075] Control Example 1:

[0076] 1 Chromatographic conditions:

[0077] Chromatographic column: Agilent ZORBAX Eclipse Plus C18 (4.6 mm × 250 mm, 5 μm), mobile phase A: water, mobile phase B: acetonitrile, perform linear gradient elution, and the gradient elution settings are as follows:

[0078]

[0079] Flow rate: 1.0 ml / min; column temperature: 35 °C; detection wavelength: 244 nm; injection volume: 10 μl; the solvent is 50% acetonitrile.

[0080] 2 Method: Take appropriate amounts of the key intermediate of ixekib and impurities A, B, C, D, E, and F, weigh accurately, place them in the same volumetric flask, dissolve and dilute with the solvent to prepare a solution containing about 0.5 mg of the compound of formula I and about 0.5 μg of each impurity per 1 ml as the system suitability solution. Accurately measure 10 μl and inject it into the liquid chromatograph, record the chromatogram, and the chromatogram of the system suitability solution is shown in the appendix Figure 2 .

[0081] 3 Conclusion: Under these chromatographic conditions, the peak shape of the main peak with a retention time of Rt 12.643 minutes is poor, and it is not baseline separated from the adjacent peak, affecting the quantitative results and qualitative judgment of the impurities.

[0082] Control Example 2:

[0083] 1 Chromatographic conditions:

[0084] Chromatographic column: Agilent ZORBAX Eclipse XDB C18 (4.6 mm × 250 mm, 5 μm), mobile phase A: 0.1% phosphoric acid aqueous solution, mobile phase B: acetonitrile, perform linear gradient elution, and the gradient elution settings are as follows:

[0085]

[0086] Flow rate: 1.0 ml / min, column temperature: 35 °C, detection wavelength: 244 nm, injection volume: 10 μl.

[0087] 2 Method: Weigh appropriate amounts of the key intermediate of imrecoxib and impurities A, B, C, D, E, and F accurately, place them in the same volumetric flask, dissolve with a solvent and dilute to prepare a solution containing approximately 0.5 mg of the compound of Formula I and approximately 4 μg of each impurity per 1 ml as the system suitability solution. Accurately measure 10 μl and inject it into the liquid chromatograph, record the chromatogram, and the chromatogram of the system suitability solution is shown in the appendix Figure 3 。

[0088] 3 Conclusion: Under these chromatographic conditions, the peak shape of the main peak with a retention time of Rt 12.173 minutes is poor, and the peak shape of impurity A with a retention time of Rt 14.527 minutes is poor, affecting the quantitative results and qualitative judgment of the impurities.

[0089] Control Example 3:

[0090] 1 Chromatographic conditions:

[0091] Chromatographic column: Agilent ZORBAX Eclipse XDB C18 - 16# (4.6 mm × 250 mm, 5 μm), mobile phase A: 10 mmol / L potassium dihydrogen phosphate solution (pH = 4.15), mobile phase B: acetonitrile, perform gradient elution, and the gradient elution settings are as follows:

[0092]

[0093]

[0094] Flow rate: 1.0 ml / min, column temperature: 35 °C, detection wavelength: 244 nm, injection volume: 10 μl.

[0095] 2 Method: Weigh appropriate amounts of the key intermediate of imrecoxib and impurities A, B, C, D, E, and F accurately, place them in the same volumetric flask, dissolve with a solvent and dilute to prepare a solution containing approximately 0.5 mg of the compound of Formula I and approximately 4 μg of each impurity per 1 ml as the system suitability solution. Accurately measure 10 μl and inject it into the liquid chromatograph, record the chromatogram, and the chromatogram of the system suitability solution is shown in the appendix Figure 4 。

[0096] 3 Conclusion: Under these chromatographic conditions, the peak shape of impurity A with a retention time of Rt 12.22 minutes is poor and it is not completely separated from the adjacent unknown impurity, affecting the quantitative results and qualitative judgment of the impurities.

[0097] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A detection method for a key intermediate of a non-steroidal anti-inflammatory drug. The method is a method for separating and determining a key intermediate of a non-steroidal anti-inflammatory drug and its related impurities by HPLC. The chromatographic column used is packed with octadecylsilane-bonded silica gel, and gradient elution is carried out using mobile phase A and mobile phase B, and then it enters the detector for detection; the related impurities include one or more of impurity A, impurity B, impurity C, impurity D, impurity E, and impurity F, and the specific structural formulas are shown as follows: The mobile phase A is a mixed solvent of phosphate buffer solution and organic solvent, and the mobile phase B is an organic solvent. The key intermediate of ixabepilone is 2-bromo-1-(4-(methylsulfonyl)phenyl)ethyl-1-one, which is a key intermediate for synthesizing ixabepilone. Its chemical formula is C9H9BrO3S, and the structural formula is as shown in formula (Ⅰ) below. In the present invention, it is also simply referred to as "compound of formula Ⅰ" or "key intermediate of ixabepilone": 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 according to claim 1, wherein The mobile phase A is a mixed solvent of phosphate buffer solution and acetonitrile, and the volume ratio of the phosphate buffer solution to acetonitrile is 90:

10. The mobile phase B is acetonitrile.

3. The method according to claim 2, wherein The pH value of the phosphate buffer solution is adjusted to 6.8 - 7.2 with potassium hydroxide solution.

4. The method according to claim 1 or 2, characterized in that, The phosphate buffer solution is an aqueous solution of potassium dihydrogen phosphate, and the concentration of phosphate in the phosphate buffer solution is 5 - 15 mmol / L.

5. The method according to claim 1, wherein The gradient elution is set as follows:

6. The method according to claim 1, wherein The particle size of the packing particles of the octadecylsilane-bonded silica gel is 2 - 5 μm; the column temperature of the chromatographic column is 30 - 40 °C.

7. The method according to claim 1, characterized in that, The detection wavelength of the detector is 244 ± 5 nm.

8. The method according to claim 1, characterized in that, The related impurities are reference substances of impurity A, impurity B, impurity C, impurity D, impurity E, and impurity F. The specific steps are as follows: 1) Prepare a test solution: Take the test sample and dissolve it in a diluent to make a solution containing about 0.5 mg per 1 ml to obtain the test solution; 2) Prepare a control solution: Take an appropriate amount of the test solution and dilute it with a diluent to make a solution containing about 5 μg per 1 ml as the control solution; 3) Prepare a system suitability solution: Take the key intermediate of ixabepilone and its impurities A, B, C, D, E, and F, and dissolve and dilute them with a diluent to make a system suitability solution; 4) Inject the system suitability solution prepared in step 3) for high-performance liquid chromatography analysis, record the chromatogram, determine the retention times of the key intermediate of ixabepilone and its impurities, and then inject the test solution prepared in step 1) and the control solution prepared in step 2) respectively, and calculate the content of impurities in the key intermediate of ixabepilone in the test solution according to the self-control method of the main component. The diluent is a solution of acetonitrile - water (volume ratio 50:50).

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