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 of a non-steroidal anti-inflammatory drug and related impurities thereof by an HPLC (High Performance Liquid Chromatography) method, gradient elution is performed by adopting a chromatographic column taking octadecylsilane chemically bonded silica as a filler, a phosphoric acid aqueous solution as a mobile phase A and acetonitrile as a mobile phase B, and detection is performed in a detector. The non-steroidal anti-inflammatory drug key intermediate and related impurities can be effectively separated, and the method has the advantages of very high sensitivity and separation degree, good repeatability and durability, simple operation and stable and reliable result, and has very important significance for realizing the quality control of the non-steroidal anti-inflammatory drug.

Description

Technical Field

[0001] The invention relates to the field of analytical chemistry, and relates to a method for detecting a non-steroidal anti-inflammatory drug intermediate, in particular to an HPLC method for separating and determining a key non-steroidal anti-inflammatory drug intermediate and related impurities. Background Art

[0002] Nabumetone, also known as naproxen, has a chemical name of 4-(6-methoxy-2-naphthyl)-2-butanone. It is a new generation of non-steroidal anti-inflammatory analgesics developed by the British company Beecham and was first launched in Ireland in 1985. Nabumetone is a keto-type prodrug, mainly used for the treatment of rheumatism. Its main active metabolite in the body can inhibit the biosynthesis of prostaglandins, and has strong antipyretic, analgesic and anti-inflammatory effects. The drug is much less irritating to the stomach than indomethacin and aspirin, has good tolerance for long-term use, and has a long half-life. It is a first-line therapeutic drug with good efficacy for various types of inflammation and pain.

[0003] At present, the quality standards of nabumetone are included in the United States Pharmacopoeia, the European Pharmacopoeia, and the Chinese Pharmacopoeia, but there are relatively few reports on the analysis methods of its key intermediates. Wu Wei et al., reported that 1,2-dichloroethane-n-hexane (3:2) was used as the mobile phase for the inspection of related substances of nabumetone, and isocratic elution was used. However, under this method, only nabumetone and one impurity were detected. Cai Lei et al., reported that the content of nabumetone and its related substances were determined by gradient elution high performance liquid chromatography. The mobile phase A = tetrahydrofuran: acetonitrile: 0.1% glacial acetic acid deionized water solution = 12:28:60, mobile phase B = tetrahydrofuran: acetonitrile: 0.1% glacial acetic acid deionized water solution = 24:56:20, gradient elution, but under this liquid chromatography condition, only nabumetone and two impurities can be detected. At present, the existing technologies do not take into account the impurities brought in by the raw materials and the impurities generated in the process, which is far from meeting the quality control requirements in the actual production process.

[0004] Generally speaking, 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 generated during the preparation of nabumetone or related substances introduced need to be strictly controlled in both raw materials and preparations. 6-Methoxy-2-acetylnaphthalene is a key intermediate of non-steroidal anti-inflammatory drugs. The quality control of this compound plays an important role in the preparation of high-quality non-steroidal anti-inflammatory drug raw materials. Therefore, developing an efficient and accurate analytical method to analyze the quality of key intermediates of non-steroidal anti-inflammatory drugs 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 object of the present invention is to provide a method for separating and determining key intermediates of non-steroidal anti-inflammatory drugs and related impurities thereof by HPLC; the method of the present invention can effectively separate the key intermediates of nabumetone and related impurities thereof, and the method has high sensitivity and separation degree, good repeatability and durability, simple operation, and stable and reliable results.

[0006] To achieve the above purpose, the applicant has conducted a large number of experiments and determined that the technical solution of the present invention is:

[0007] A method for separating and determining key intermediates of non-steroidal anti-inflammatory drugs and related impurities thereof by HPLC, wherein the chromatographic column used in the method is filled with octadecylsilane bonded silica gel, and mobile phase A and mobile phase B are used for gradient elution, and the mixture enters a detector for detection; the related impurities include one or more of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, and impurity I, and the specific structural formula is as follows:

[0008] Impurity code Impurity Name Impurity A 2-Methoxynaphthalene Impurity B 1-Methoxynaphthalene Impurity C 2-Methoxy-1-acetonapthone Impurity D 2-Naphthyl acetate Impurity E 6-Acetyl-2-naphthol Impurity F 1,1'-(2-methoxynaphthalene-1,6-diyl)bis(ethane-1-one) Impurity G 1-Naphthyl acetate Impurity H 1-Acetyl-2-naphthol Impurity I 1,1'-(2-Hydroxynaphthalene-1,6-diyl)bis(ethane-1-one)

[0009] The mobile phase A is a phosphoric acid aqueous solution, and the mobile phase B is an organic solvent.

[0010] The key intermediate of nabumetone is 6-methoxy-2-acetylnaphthalene, which is the key intermediate for synthesizing nabumetone. Its chemical formula is C 13 H 12 O2, the structural formula is as follows: Formula (I), which is also referred to as "the compound of Formula I" or "the key intermediate of nabumetone" in the present invention:

[0011]

[0012] Further, the phosphoric acid aqueous solution is a 0.1% phosphoric acid aqueous solution;

[0013] Further, the mobile phase B is one or more of acetonitrile, ethanol and methanol;

[0014] Furthermore, the mobile phase B is acetonitrile.

[0015] The flow rate of the mobile phase for elution is 0.8-1.2 ml / min, preferably 1.0 ml / min.

[0016] The gradient elution was set up as follows:

[0017]

[0018]

[0019] As a preference, the gradient elution is set as follows:

[0020] Furthermore, the filler particles of the octadecylsilane bonded silica gel have a particle size of 2-5 μm; and the column temperature of the chromatographic column is 25-35°C.

[0021] As a preference, the filler particles of the octadecylsilane bonded silica gel have a particle size of 5 μm; and the column temperature of the chromatographic column is 30°C.

[0022] Furthermore, the detection wavelength of the detector is 220±5 nm.

[0023] Preferably, the detection wavelength of the detector is 220 nm.

[0024] Further, the method for separating and determining the key intermediate of nabumetone and its related impurities by HPLC, wherein the related impurities are impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, and impurity I reference substances, specifically comprises the following steps:

[0025] 1) Prepare the test solution: dissolve the test sample in the diluent to make a solution containing about 0.3 mg per 1 ml to obtain the test solution;

[0026] 2) preparing a system suitability solution: taking the key intermediate of nabumetone and its impurities A, B, C, D, E, F, G, H and I, dissolving and diluting them with a diluent to prepare a system suitability solution;

[0027] 3) sampling the system suitability solution described in step 2), performing high performance liquid chromatography analysis, recording the chromatogram, determining the retention time of the key intermediate of nabumetone and its impurities, and then sampling the test solution described in step 1) respectively, and calculating the content of the impurities in the key intermediate of nabumetone in the test solution by the area normalization method.

[0028] 4) The linear correlation coefficients of the key intermediate of nabumetone and its impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H and impurity I are shown in the following table:

[0029]

[0030] The diluent is a solution of acetonitrile-water (volume ratio of 50:50).

[0031] The second object of the present invention is to provide a reagent composition for solid-liquid separation and determination of key intermediates of nabumetone and its impurities, which is composed of the following reagents:

[0032] Reagent A: phosphoric acid aqueous solution;

[0033] Reagent B: organic solvent;

[0034] The relevant impurities include one or more of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, and impurity I;

[0035] The phosphoric acid aqueous solution is a 0.1% phosphoric acid aqueous solution; the organic solvent is one or more of acetonitrile, ethanol and methanol.

[0036] As a preference, the organic solvent is acetonitrile.

[0037] The reagent composition for solid-liquid separation determination of nabumetone key intermediates and impurities thereof provided by the present invention can effectively separate nabumetone key intermediates and impurities thereof, and is of great significance for achieving quality control of nabumetone key intermediates and nabumetone.

[0038] The beneficial effects of the present invention are:

[0039] 1) The present invention provides a method for separating and determining the key intermediate of nabumetone and its related impurities by HPLC. The method of the present invention can effectively separate the key intermediate of nabumetone and its related impurities, and the method has high sensitivity and separation degree, good repeatability and durability, simple operation, and stable and reliable results.

[0040] 2) It has been found that impurities A, B, C, D, E, F, G, H and I of the key intermediate of formula I affect its purity, so 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 nabumetone 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 nabumetone finished product, so this method is of great significance for achieving the key intermediate of nabumetone and the quality control of nabumetone. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The system suitability solution chromatogram under the conditions of the embodiment of the present invention shows that the peak order is impurity A (retention time Rt 21.188 minutes), impurity B (retention time Rt 22.282 minutes), compound of formula I (retention time Rt 15.117 minutes), impurity C (retention time Rt 16.204 minutes), impurity D (retention time Rt 16.825 minutes), impurity E (retention time Rt 7.052 minutes), impurity F (retention time Rt 11.730 minutes), impurity G (retention time Rt 15.792 minutes), impurity H (retention time Rt 12.300 minutes), and impurity I (retention time Rt 8.536 minutes);

[0042] Figure 2 System suitability solution chromatogram under the conditions of Control Example 1;

[0043] Figure 3 System suitability solution chromatogram under the conditions of Control Example 2;

[0044] Figure 4 System suitability solution chromatogram under the conditions of Control Example 3. DETAILED DESCRIPTION

[0045] Below with reference to the accompanying drawings, preferred embodiments of the present invention are described in detail. The experimental methods of the unrecorded specific conditions in the preferred embodiments are usually according to normal conditions, and the examples are for better illustrating the content of the present invention, but are not limited to the examples. Therefore, those skilled in the art carry out non-essential improvements and adjustments to the embodiments according to the above-mentioned invention content, and still belong to the protection scope of the present invention. The nabumetone key intermediate in the present embodiment refers to a compound of formula I.

[0046] Embodiment 1

[0047] 1Chromatographic conditions:

[0048] Chromatographic column: ACE Excel 5C18-AR, 250mm×4.6mm, 5μm; 0.1% phosphoric acid aqueous solution as mobile phase A, acetonitrile as mobile phase B; linear gradient elution according to the table below; column temperature, 30℃; flow rate, 1.0ml per minute; detection wavelength, 220nm; injection volume, 10μl.

[0049]

[0050]

[0051] 2 Methods and Results

[0052] 2.1 Solution preparation

[0053] Take an appropriate amount of the key intermediate of nabumetone for the test solution, weigh it accurately, add the solvent acetonitrile-water (volume ratio 50:50), dissolve it by ultrasonication, and dilute it to make a solution containing about 0.3 mg per 1 ml.

[0054] 2.2 Exclusivity

[0055] Take appropriate amounts of nabumetone key intermediate and impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, and impurity I reference substances, accurately weigh them, place them in the same volumetric bottle, add solvent to dissolve and dilute to make a mixed solution containing 0.3 mg of compound I, 6 μg of impurity A, 12 μg of impurity F, 1.5 μg of impurity D and impurity E, and 0.3 μg of other impurities per 1 ml, as the system suitability solution. Accurately measure 10 μl and inject it into the liquid chromatograph, record the chromatogram, and the system suitability solution results are attached. Figure 1 In the system suitability solution, the separation degree of the key intermediate of nabumetone and the adjacent impurity peaks was 2.1, and the retention time Rt was 15.117 minutes.

[0056] 2.3 Repeatability

[0057] Take an appropriate amount of nabumetone key intermediate and its impurity reference substance, dissolve and dilute with solvent to make a mixed solution containing about 0.3 mg of compound I, 6 μg of impurity A, 1.5 μg of impurity D and impurity E, and 0.3 μg of other impurities per 1 ml, as a spiked test solution; accurately measure the above-mentioned spiked test solution for injection, and record the chromatogram. Calculate the RSD of the content of each related substance in 6 spiked test solutions by area normalization method. The RSD of the content of each related impurity detected in the 6 spiked test solutions is less than 10.0%, which meets the requirements of high performance liquid chromatography for related substance detection.

[0058] 2.4 Linearity

[0059] Take an appropriate amount of nabumetone key intermediate and its impurity reference substance, add diluent to dissolve and dilute to make a mixed solution containing about 120μg of impurity A, 30μg of impurity D and impurity E, and 6μg of other impurities per 1ml as a linear stock solution. Accurately pipette 1ml of linear stock solution into 50ml, 1ml into 25ml, 1ml into 20ml, 3ml into 50ml, 3ml into 20ml, and 5ml into 20ml volumetric bottles, dilute to the scale with diluent, and take the quantitative limit solution as the linear solution. Linear regression is performed on the concentration using the peak area to obtain a linear equation (Table 1). The key intermediates of nabumetone and its impurities have a good linear relationship within the linear range.

[0060] Table 1 Linearity determination results

[0061]

[0062]

[0063] 2.5 Limit of detection and limit of quantification

[0064] Take appropriate amount of nabumetone key intermediate and its impurity reference substance to prepare a series of solutions. When S / N≥10, it is used as the quantitative limit solution, and when S / N≥3, it is used as the detection limit solution. The quantitative limit and detection limit results of nabumetone key intermediate and its impurities are shown in Table 2.

[0065] Table 2 Results of limit of quantification and limit of detection

[0066]

[0067] 2.6 Accuracy

[0068] Take appropriate amount of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I reference substances, dissolve and dilute with solvent to make a mixed solution containing about 120μg of impurity A, 30μg of impurity D and impurity E, and 6μg of other impurities per 1ml, as impurity mixed stock solution; take appropriate amount of key intermediate of nabumetone, accurately measure 1ml of impurity mixed reference stock solution into 100ml, 1ml into 20ml, 1.5ml into 20ml, 5ml into 20ml volumetric bottle, dilute to scale with solvent, shake well, as recovery test solution, each concentration is parallel 3 times. The recovery rate and RSD of each impurity are calculated by area normalization method with correction factor. The results show that the recovery rate of each impurity at each concentration is between 80.0% and 120.0%, and the RSD is less than 10.0%, which meets the requirements of high performance liquid chromatography for the detection of related substances.

[0069] 3 Conclusion:

[0070] Under the chromatographic conditions, the key intermediates of nabumetone and their impurities can be completely separated. This method has strong specificity, good accuracy, high sensitivity, good repeatability, and good system applicability. It meets the technical requirements of drug quality research standards, and the results obtained are stable and reliable.

[0071] Comparative Example 1:

[0072] 1Chromatographic conditions:

[0073] Chromatographic column: Agilent Eclipse XDB-C18 (250 mm × 4.6 mm, 5 μm), mobile phase A: 0.1% phosphoric acid aqueous solution, mobile phase B: acetonitrile, gradient elution, gradient elution settings are as follows:

[0074]

[0075] Flow rate: 1 ml / min, column temperature: 30°C, injection volume: 10 μl, solvent: acetonitrile-water (volume ratio 50:50).

[0076] 2 Method: Take appropriate amounts of nabumetone key intermediate and impurities A, B, C, D, E, F, G, H and I, weigh them accurately, place them in the same volumetric bottle, add solvent to dissolve and dilute to make a solution containing about 0.2 mg of compound I and about 15 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 system suitability solution chromatogram is attached. Figure 2 .

[0077] 3 Conclusion: Under the chromatographic conditions, the retention time Rt 12.912 is the overlap of two impurity peaks, which affects the quantitative results and qualitative judgment of impurities C and G.

[0078] Comparative Example 2:

[0079] 1Chromatographic conditions:

[0080] Chromatographic column: Kromasil C18 (250 mm × 4.6 mm, 5 μm), mobile phase A: 0.1% phosphoric acid aqueous solution, mobile phase B: acetonitrile, gradient elution, gradient elution settings are as follows:

[0081]

[0082]

[0083] Flow rate: 1 ml / min, column temperature: 30°C, injection volume: 10 μl, solvent: acetonitrile-water (volume ratio 50:50).

[0084] 2 Method: Take appropriate amounts of nabumetone key intermediate and impurities A, B, C, D, E, F, G, H and I, weigh them accurately, place them in the same volumetric bottle, add solvent to dissolve and dilute to make a solution containing about 0.5 mg of compound 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 system suitability solution chromatogram is attached. Figure 3 .

[0085] 3 Conclusion: Under the chromatographic conditions, the retention time Rt 13.583 minutes is the overlap of two impurity peaks, which affects the quantitative results and qualitative judgment of impurities C and G.

[0086] Comparative Example 3:

[0087] 1Chromatographic conditions:

[0088] Chromatographic column: ACE Excel5 C18-AR (250mm×4.6mm, 5μm), mobile phase A: 0.1% phosphoric acid aqueous solution, mobile phase B: acetonitrile-methanol (4:1), gradient elution, gradient elution settings are as follows:

[0089]

[0090] Flow rate: 1 ml / min, column temperature: 30°C, injection volume: 10 μl, solvent: acetonitrile-water (volume ratio 50:50).

[0091] 2 Method: Take appropriate amounts of nabumetone key intermediate and impurities A, B, C, D, E, F, G, H and I, weigh them accurately, place them in the same volumetric bottle, add solvent to dissolve and dilute to make a solution containing about 0.15 mg of compound I and about 0.3 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 system suitability solution chromatogram is attached. Figure 4 .

[0092] 3 Conclusion: Under the chromatographic conditions, the retention time Rt 18.426 minutes is the overlap of two impurity peaks, which affects the quantitative results and qualitative judgment of impurities C and G.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A method for detecting 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 filled with octadecylsilane bonded silica gel, mobile phase A and mobile phase B are used for gradient elution, and the mixture enters a detector for detection; the related impurities include one or more of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, and impurity I, and the specific structural formula is as follows: The mobile phase A is a phosphoric acid aqueous solution, and the mobile phase B is an organic solvent. The key intermediate of nabumetone, 6-methoxy-2-acetylnaphthalene, is a key intermediate for synthesizing nabumetone, and its chemical formula is C 13 H 12 O2, the structural formula is as follows: Formula (I), which is also referred to as "the compound of Formula I" or "the key intermediate of nabumetone" in the present invention: The gradient elution was set up 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, characterized in that: The mobile phase A is 0.1% phosphoric acid aqueous solution, and the mobile phase B is acetonitrile.

3. The method according to claim 1, characterized in that: The gradient elution was set up as follows:

4. The method according to claim 1, characterized in that: The filler particles of the octadecylsilane bonded silica gel have a particle size of 2-5 μm; and the column temperature of the chromatographic column is 25-35°C.

5. The method according to claim 1, characterized in that The detection wavelength of the detector is 220±5 nm.

6. The method according to claim 1, characterized in that The relevant impurities are impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, and impurity I reference substances, and specifically include the following steps: 1) Prepare the test solution: dissolve the test sample in the diluent to make a solution containing about 0.3 mg per 1 ml to obtain the test solution; 2) preparing a system suitability solution: taking the key intermediate of nabumetone and its impurities A, B, C, D, E, F, G, H and I, dissolving and diluting them with a diluent to prepare a system suitability solution; 3) sampling the system suitability solution described in step 2), performing high performance liquid chromatography analysis, recording the chromatogram, determining the retention time of the key intermediate of nabumetone and its impurities, and then sampling the test solution described in step 1), and calculating the content of the impurities in the key intermediate of nabumetone in the test solution by the area normalization method, The diluent is a solution of acetonitrile-water (volume ratio is 50:50).

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