Method for detecting genotoxic impurity o-tolualdehyde in indobufen

Abstract

The invention provides a method for detecting a genotoxic impurity o-tolualdehyde in indobufen, which comprises the following steps of: firstly dissolving raw material medicines and then carrying out low-temperature treatment to obtain a test solution with detectable concentration. According to the method, a common ultraviolet detector is used for detection, the method is simple, accurate, efficient and easy to operate, interference of various other organic impurities and genotoxic impurities in the indobufen is avoided, and the problems of high cost and operation complexity caused by pre-column derivatization ultra-high performance liquid chromatography in the prior art are solved.

Description

Technical Field

[0001] The invention relates to the field of drug analysis, and in particular to a method for evaluating drug quality by using high performance liquid chromatography. Background Art

[0002] Indobufen is an isoindoline phenylbutyric acid derivative, belonging to the class of nonsteroidal anti-inflammatory drugs, with a chemical name of 2-[4-(1-oxo-2-isoindoline)phenyl]butyric acid and a molecular formula of C18H17NO3. It is an antithrombotic drug developed by Farmfa lia Carlo Erba in Italy and first marketed in Italy in August 1984. It is currently the only reversible, selective, multi-target antithrombotic drug. Most of the reported synthetic processes for indobufen involve phthalic anhydride, and patent CN106631974A discloses the following synthetic process:

[0003]

[0004] As shown above, in the process route for synthesizing indobufen, phthalic anhydride is the raw material of the penultimate step to obtain intermediate 2. o-Tolualdehyde is an oxidation product produced during the synthesis of phthalic anhydride. o-Tolualdehyde is a genotoxic impurity with a warning structure. Genotoxic impurities, i.e., low levels, can directly cause DNA damage, thereby leading to DNA mutations, and are therefore DNA-reactive substances that may cause cancer. Since o-Tolualdehyde is similar in nature to phthalic anhydride and intermediate 2, there is a possibility that it will be introduced into the final product indobufen, so it is necessary to detect its content. However, the current domestic and international quality standards for indobufen and preparations do not measure and control o-Tolualdehyde.

[0005] According to ICHM7 guidelines, and in combination with the maximum daily dose of indobufen tablets (indobufen 400mg), the maximum daily intake of o-methylbenzaldehyde shall not be greater than 1.5 μg, so the control limit of o-methylbenzaldehyde in indobufen raw materials shall not exceed 3.75ppm, and this limit is relatively low, requiring detection methods to have higher sensitivity and specificity. CN112034067A discloses a method for determining the content of o-phthalaldehyde, a genotoxic impurity in indobufen, by LC-MS / MS method. However, the method needs to be derivatized, and a functional group that can produce a mass spectrometry signal is introduced, so that it can be further detected by mass spectrometry. The derivatization reaction solution preparation method is complicated to operate, and the introduction of reagents is numerous, and the LC-MS / MS method is adopted to measure, and the equipment is expensive, and the test cost is high, which is not suitable for daily detection in industrial production. Yin Yanmin et al. reported in "Determination of 13 aldehydes and ketones in ambient air by ultra-high performance liquid chromatography" (Chemical Analysis and Metering, Vol. 30, No. 2, February 2021, pp32-36) that the use of pre-column derivatization ultra-high performance liquid chromatography can accurately determine 0.08 mg / L, or 80 ng / ml, of m-methylbenzaldehyde. Shi Xiaona et al. reported in the literature "Simultaneous determination of four genotoxic impurities in indobufen by HPLC-MS / MS" (Journal of Pharmaceutical Analysis, 2022, 42(2), pp312-319) that the use of ultra-high performance liquid chromatography to determine four genotoxic impurities in indobufen: 2-(4-nitrophenyl)butyric acid, 2-(4-aminophenyl)butyric acid, 2-(3-aminophenyl)butyric acid, and 2-(2-aminophenyl)butyric acid. These methods are not suitable for the determination of genotoxic impurities in indobufen APIs. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a method for detecting o-methylbenzaldehyde, a genotoxic impurity in indobufen. The method uses a common ultraviolet detector for detection, and the method is simple, accurate, efficient, and easy to operate. It is not interfered by various other organic impurities and genotoxic impurities in indobufen, and overcomes the problems of high cost and operation complexity caused by the use of pre-column derivatization ultra-high performance liquid chromatography in the prior art.

[0007] According to the present invention, the method for detecting the genotoxic impurity o-methylbenzaldehyde in indobufen comprises the following test solution preparation steps:

[0008] 1) dissolving the indobufen bulk drug with a sodium hydroxide solution, and then diluting it with a diluent to a first concentration between 5 and 50 g / L to obtain a diluent;

[0009] 2) subjecting the dilution to an optional ultrasonic treatment, followed by a low temperature treatment at -2-0°C for 0.5-5 hours;

[0010] 3) Filter the diluted solution to obtain a filtrate having a second concentration as a test solution.

[0011] In a preferred embodiment, the chromatographic column is an octadecylsilane bonded silica gel chromatographic column, the detector is a diode array detector, and o-methylbenzaldehyde is detected by gradient elution. The gradient elution procedure is as follows:

[0012]

[0013]

[0014] In a preferred embodiment, the detection wavelength is 190-400 nm, preferably 250 nm.

[0015] In a preferred embodiment, the flow rate ranges from 1.0 to 1.2 ml / min, preferably 1.1 ml / min.

[0016] In a preferred embodiment, the test solution is prepared as follows: take about 1 g of the product, accurately weigh it, place it in a 100 ml volumetric flask, add 8 ml of 0.5 mol / L sodium hydroxide solution, dissolve it by ultrasound, then slowly add 0.1% phosphoric acid solution-acetonitrile (80:20) to dilute to the scale, shake well, cool it in an ice bath for more than 1 hour, filter it, and take the filtrate.

[0017] In a preferred embodiment, the reference solution is prepared by taking an appropriate amount of o-methylbenzaldehyde reference, accurately weighing it, and diluting it with 0.1% phosphoric acid solution-acetonitrile (80:20) to make a solution containing about 0.0375 μg per 1 ml.

[0018] In the present invention, the column temperature of the chromatographic column is 29-31°C, preferably 30°C; the injection volume is 80-130 μl, preferably 100 μl. Through the method of the present invention, the genotoxic impurity o-methylbenzaldehyde in indobufen at around 3.75 ng / ml can be detected by high performance liquid chromatography, without the need for structural derivatization or mass spectrometry detection, with low analysis cost and simple operation, meeting the needs of industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is an overlay of the method specificity test results under the conditions of Example 1;

[0020] Figure 2 is a linear relationship diagram of o-methylbenzaldehyde according to the method of the present invention;

[0021] Figure 3 It is an overlay diagram of the method specificity test results under 2 conditions of the chromatograms obtained in Examples 1 and 3;

[0022] Figure 4It is an overlay diagram of different flow rate screening under the conditions of the present invention;

[0023] Figure 5 This is an overlay of the test results of other potential impurities for method specificity under the conditions of Example 1. DETAILED DESCRIPTION

[0024] In order to make the purpose, features and advantages of the present invention more obvious and easy to understand, the technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments and drawings. Obviously, the described embodiments are only exemplary, not all implementation methods. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0025] The present invention proposes based on such technical difficulties, on the one hand, the detection object genotoxic substance o-methylbenzaldehyde of the detection method of the present invention, the allowed content in the medicine indobufen is very low, must not exceed 3.75ppm. Under normal conditions, even if o-methylbenzaldehyde reaches the highest allowed content, but because the bulk drug itself is diluted by a high multiple in the test solution, the content of o-methylbenzaldehyde in the test solution is further reduced, so that it is not displayed by conventional detector. On the other hand, the absorption value of o-methylbenzaldehyde itself in the spectral range of the commonly used ultraviolet detector of liquid chromatography is not ideal, therefore, if you want to use ultraviolet detector to detect, then require the absolute concentration of o-methylbenzaldehyde in the test solution can not be too low.

[0026] In order to solve the above outstanding contradiction, in the detection method of the present invention, the preparation method of the test solution is improved to obtain a test solution with a detectable concentration. Herein, the detectable concentration means that if the raw material drug to be tested contains an unexpected amount (e.g., about 3.75 ppm) of o-methylbenzaldehyde, its absolute concentration in the test solution is sufficient to be quantitatively detected under the conditions defined in the present invention.

[0027] The method for detecting the genotoxic impurity o-methylbenzaldehyde in indobufen of the present invention comprises the preparation of a test solution, wherein indobufen is prepared into an alkaline solution with a first concentration, then the alkaline solution is treated at a low temperature, and a filtrate is obtained as the test solution after filtration.

[0028] In the present invention, the first concentration is the sum of the concentrations of indobufen and its sodium salt, which can be between 5-50g / L, preferably between 10-30g / L. In a typical embodiment of the present invention, the first concentration is between 10-20g / L. This is different from the test solution concentration in the prior art. In the prior art, the sample is generally prepared into a test solution of 0.1-1g / L to avoid the instability, easy precipitation and filtration adsorption caused by a high concentration solution. In the present invention, in order to obtain a test solution suitable for testing, the drug is first dissolved with a sodium hydroxide solution to make indobufen sodium salt, then diluted to the first concentration with a diluent, and then the sodium salt solution is treated at low temperature for a predetermined time to precipitate crystals therein as much as possible, and finally filtered to obtain a subsequent filtrate with a second concentration, i.e., the test solution. Herein, the term second concentration is the sum of the concentrations of indobufen and its sodium salt in the solution.

[0029] In the present invention, sodium hydroxide is preferably added in an equimolar amount of indobufen, but is not limited thereto, and the amount of sodium hydroxide can be 0.8-2.0 times, preferably 0.9-1.2 times, of the molar amount of indobufen. The test solution is prepared into a sodium salt, which can improve the solubility of the test sample and is conducive to the complete release of the test substance in the test sample. The concentration of the sodium hydroxide solution for dissolving indobufen can be between 0.1-1mol / L, typically between 0.3-0.7mol / L. The volume of the sodium hydroxide used for fully dissolving the medicine per unit mass (g) is preferably between 4-20 times of volume (ml). The test result shows that there is no significant impact on the analysis result within this range.

[0030] The dissolved solution can be optionally subjected to ultrasonic treatment for 1-2 minutes or shaken for dissolution. Ultrasonic treatment can promote the rapid dissolution of the main components of the drug and various impurities without destroying the structure of the substance to be tested. When the sample is completely dissolved, manual shaking and ultrasonic treatment are used without affecting the analysis results.

[0031] In a typical embodiment of the present invention, the diluent used to prepare the test solution is a mixed solvent of 0.1% phosphoric acid solution and acetonitrile in a volume ratio of 8:2. However, the present invention is not limited thereto, and the ratio of 0.1% phosphoric acid solution to acetonitrile can be controlled between 9:1 and 6:4, which is similar to the initial ratio of the mobile phase gradient elution program, and can effectively avoid the solvent effect. Preferably, in Example 1, the peak shape of o-methylbenzaldehyde is good and there is no solvent effect. After the introduction of the sodium hydroxide solution and the diluent, the first concentration is controlled within the selected range.

[0032] In the present invention, as a further preference, the diluent is subjected to low temperature treatment for a predetermined time. The low temperature treatment is to keep the diluent obtained above at a temperature of -2-0°C for 0.5-5 hours, preferably between 1-4 hours, such as 1, 2 hours, 3 hours and 3.5 hours. The purpose of the low temperature treatment is to precipitate part of indobufen or its sodium salt as much as possible to reduce the damage that the precipitation of high concentration raw materials may cause to the system, such as blocking the six-way valve. In a typical embodiment, 1g of indobufen is dissolved with 10ml sodium hydroxide solution and 90ml diluent, and its preparation concentration is 0.0338mol / L (first concentration). After keeping in an ice water bath for 1 hour and filtering, the concentration of indobufen in the test solution is detected to be 0.0025mol / L (second concentration), which indicates that the low temperature treatment leads to the partial precipitation of indobufen.

[0033] The test solution prepared by the method of the present invention increases the collection amount of the raw material drug, so that the trace amount of o-methylbenzaldehyde genotoxic substances with a content limit of about can be detected sensitively enough, while avoiding interference caused by excessively high content of the raw material drug in the test solution. The detection can be completed only with the help of a high performance liquid chromatograph, without the need to use a chromatographic-mass spectrometry detection device, and without the need to perform structural derivatization on the test object.

[0034] When the applicant adopts the aforementioned synthetic route to prepare indobufen, the obtained indobufen API may also contain one or more of the following impurities: intermediate 2: 2-[4-(1,3-dioxo-2-isoindolyl)phenyl]butyric acid; impurity 11: 2-[4-(1-hydroxy-3-oxo-1H-isoindol-2-yl)phenyl]butyric acid; impurity 13: 2-[4-(isoindol-2-yl)phenyl]butyric acid; impurity 14: 2-[4-(1-oxoisoindolyl)phenyl]butyric acid; 2-(4-nitrophenyl)butyric acid; 2-(4-nitrophenyl)butyric acid; 2-(4-nitrophenyl)butyric acid; 2-(4-nitrophenyl)butyronitrile; 2-(2-nitrophenyl)butyric acid; 2-(4-acetylaminophenyl)butyric acid; 2-(4-aminophenyl)butyric acid and intermediate 2 are raw material impurities, and the others are by-product impurities.

[0035] In order to separate the above substances with high resolution, in a preferred embodiment of the present invention, the chromatographic conditions are optimized. The chromatographic column uses an octadecylsilane bonded silica gel chromatographic column, and examples that can be used include AgilentZorbax SB-C18 chromatographic column, Kromasil C18 chromatographic column, and Sunfire C18 chromatographic column. The mobile phase uses the gradient elution shown in the table below, wherein mobile phase A is 0.1% phosphoric acid aqueous solution, and mobile phase B is acetonitrile.

[0036] Table 1

[0037] Time / min Mobile phase A (%) Mobile phase B (%) 0 80 20 5 80 20 50 70 30 55 20 80 60 20 80 60.1 80 20 70 80 20

[0038] The gradient elution conditions in Table 1 are particularly suitable for the Agilen Zorbax SB-C18 column.

[0039] In the following examples, the mobile phase conditions in Table 1 were used, the high performance liquid chromatograph was a Waters from Shimadzu, Japan, and the electronic balance was a MS205DU electronic balance from Mettler. The reagents used are listed in Table 2 below.

[0040] Table 2

[0041]

[0042]

[0043] Example 1

[0044] Detector: Light Emitting Diode Array Detector

[0045] Chromatographic column: Agilen Zorbax SB-C18 (4.6mm×250mm, 5μm)

[0046] Flow rate: 1.1 mL / min

[0047] Detection wavelength: 250nm

[0048] Column temperature: 30°C

[0049] Injection volume: 100 μL

[0050] Preparation of test solution: Take about 1 g of the raw material drug, weigh it accurately, put it in a 100 ml volumetric flask, add 8 ml of 0.5 mol / L sodium hydroxide solution, dissolve it by ultrasonication, then slowly add 0.1% phosphoric acid solution-acetonitrile (80:20) to dilute to the scale, shake well, cool in an ice bath for more than 1 hour, filter, and take the filtrate as the test solution.

[0051] The following methodological validation results confirm the reliability of the analytical conditions of this example.

[0052] Specificity test

[0053] Prepare diluent: measure 1600 ml of 0.1% phosphoric acid solution and 400 ml of acetonitrile respectively and mix well.

[0054] Prepare blank solvent: measure 0.8 ml of 0.5 mol / L sodium hydroxide solution, place it in a 10 ml volumetric flask, add diluent to dilute to the scale, shake well, and filter.

[0055] Preparation of reference solution: Take an appropriate amount of o-methylbenzaldehyde reference substance, weigh accurately, and dilute with 0.1% phosphoric acid solution-acetonitrile (80:20) to make a solution containing approximately 0.0375 μg per 1 ml.

[0056] Prepare mixed solution: Take appropriate amount of intermediate 2, impurity 11, impurity 13, impurity 14, intermediate 1, 2-(2-nitrophenyl)butyric acid, impurity 1, 2-(4-nitrophenyl)butyronitrile, 2-(4-nitrophenyl)butyric acid, 2-[4-(hydroxylamine)phenyl]butyric acid, 2-(2-aminophenyl)butyric acid, 2-[(4-acetylamino)phenyl]butyric acid, and o-methylbenzaldehyde reference substance, add diluent to dilute to scale, shake well (concentration of intermediate 2, impurity 11, impurity 13, impurity 14 The concentration of each is about 15 μg / ml, the concentration of 2-(4-aminophenyl)butyric acid, 2-(2-nitrophenyl)butyric acid, 2-(4-nitrosophenyl)butyric acid, 2-(4-nitrophenyl)butyronitrile, 2-(4-nitrophenyl)butyric acid, 2-[4-(hydroxylamino)phenyl]butyric acid, and 2-(2-aminophenyl)butyric acid is about 0.05 μg / ml, the concentration of 2-[(4-acetylamino)phenyl]butyric acid is about 0.06 μg / ml, and the concentration of o-tolualdehyde is about 0.0375 μg / ml.

[0057] Test solution: Take about 1 g of the product, weigh accurately, place in a 100 ml volumetric flask, add 8 ml of 0.5 mol / L sodium hydroxide solution, dissolve by ultrasonication, then slowly add diluent to dilute to the scale, shake well, cool in an ice bath for more than 1 hour, filter, and take the filtrate.

[0058] According to the above conditions and operations, the chromatogram is as follows: Figure 1 and Figure 2 shown.

[0059] The results showed that the blank solvent and other possible impurities did not interfere with the determination. The separation of o-tolualdehyde and adjacent peaks in the mixed solution and the spiked test solution met the requirements. The method had good specificity, and the main component analysis solution preparation method was not easy to clog the six-way valve, allowing continuous sampling.

[0060] Limit of quantitation, limit of detection

[0061] Take an appropriate amount of o-methylbenzaldehyde reference substance to prepare the detection limit solution and quantification limit solution of the following concentrations. Inject them into the liquid chromatograph according to the above determination method and record the chromatogram. The results are shown in Tables 3 and 4.

[0062] Table 3. Detection limit and quantitative limit determination results of Example 1

[0063]

[0064] Table 4: Quantitation limit determination results

[0065]

[0066] The results showed that the detection limit of o-tolualdehyde was 0.19 ppm and the quantification limit was 0.38 ppm, which could meet the detection requirements of o-tolualdehyde in indobufen, indicating that the method had high sensitivity.

[0067] Linear range

[0068] Take an appropriate amount of o-methylbenzaldehyde reference substance and prepare a linear solution of the following series of concentrations. According to the determination method in Example 1, inject it into a liquid chromatograph, record the chromatogram, and perform linear regression. The results are shown in Table 5 and Figure 2 .

[0069] Table 5. Linearity results of o-methylbenzaldehyde

[0070]

[0071] The results showed that for o-methylbenzaldehyde in the concentration range of 3.7511ng / ml-75.0220ng / ml (equivalent to the limit concentration of 10.00%-200.06%), y=494.59x+33.53, the correlation coefficient r=0.9999, which is greater than 0.999, and the intercept deviation is 0.18%, which is less than 20%, indicating a good linear relationship.

[0072] Accuracy

[0073] Take about 1g of this product, accurately weigh it, put it in a 100ml volumetric flask, accurately add an appropriate amount of o-methylbenzaldehyde reference substance, add 8ml of 0.5mol / L sodium hydroxide solution, dissolve it by ultrasound, slowly add diluent to dilute to the scale, shake well, cool it in an ice bath for more than 1 hour, filter, take the filtrate, and prepare the spiked test solution with low, medium and high o-methylbenzaldehyde concentration. According to the determination method in Example 1, inject it into the liquid chromatograph, record the chromatogram, and perform linear regression. The results are shown in Table 6.

[0074] Table 6: o-Tolualdehyde accuracy test results

[0075]

[0076]

[0077] The results showed that the recoveries of o-tolualdehyde at three concentration levels (50%, 100%, 150%) were between 92.11% and 105.77%, with means between 94.3% and 99.4%, and between 80% and 115%. The RSD of the recoveries of nine spiked test solutions was 4.2%, which was less than 10%, indicating that the method had good accuracy.

[0078] Precision

[0079] Six injections of o-tolualdehyde reference solution were made continuously, and the injection precision of the instrument was investigated using the o-tolualdehyde peak area and retention time RSD as evaluation indicators. The results showed that after six continuous injections, the RSD of the main peak area of ​​the reference solution was 3.01%, less than 5.0%, and the RSD of the retention time was 0.12%, less than 1.0%, indicating that the injection precision was good (see Table 7).

[0080] Table 7: Results of injection precision investigation

[0081] Inspection Project 1 2 3 4 5 6 RSD Retention time 35.719 35.709 35.787 35.793 35.751 35.820 0.12％ Peak area 19587 18892 18591 19261 18306 18116 3.01％

[0082] Six spiked test solutions were prepared in parallel by different people using different instruments on different dates to investigate the repeatability and intermediate precision of the method. The results showed that the RSD of the impurity recoveries in the six test solutions was 4.8%, which was less than 10%. Compared with the repeatability results, the RSD of the impurity recoveries in the 12 test solutions was 4.6%, which was less than 10%, indicating that the repeatability and intermediate precision of the method were good (see Table 8).

[0083] Table 8: Intermediate precision inspection results

[0084]

[0085] Solution stability study

[0086] Take the same portion of o-methylbenzaldehyde reference solution, test solution and spiked test solution, place at room temperature for different time periods according to the determination method in Example 1, inject into the liquid chromatograph, record the chromatogram, and evaluate the stability of the solution by the change in the o-methylbenzaldehyde peak area. The results show that the RSD of the peak area in the reference solution and the spiked test solution is less than 10%, the test solution is not detected, and the test solution is stable within 35 hours.

[0087] Durability

[0088] The durability of the method was investigated by fine-tuning the flow rate, column temperature, etc., and the recovery rate of o-tolualdehyde was examined (see Tables 11-12). The results showed that fine-tuning the chromatographic conditions had no effect on the detection of o-tolualdehyde, indicating that the method has good durability and is suitable for the application and promotion of the determination of genotoxic impurities in indobufen.

[0089] Table 11: Method robustness

[0090]

[0091] Table 12: Results of the robustness of the assay method

[0092]

[0093] Example 2

[0094] The method of Example 1 was repeated, except that the detection wavelength was 210 nm. The results showed that o-tolualdehyde had maximum absorption at this wavelength, but the signal-to-noise ratio was slightly worse than that at 250 nm.

[0095] Example 3

[0096] The method of Example 1 was repeated, except that the chromatographic column was a Kromas il C18 chromatographic column (4.6×250 mm, 5 μm). Figure 3 ,like Figure 3 As shown, the absorption peak of o-methylbenzaldehyde can also be separated from the adjacent peaks, but the separation degree is not as good as the chromatographic column used in Example 1.

[0097] Example 4

[0098] Using the same conditions as in Example 1, three different flow rates, 0.9 ml / min, 1.0 ml / min, and 1.2 ml / min, were used to compare the results. The test results are shown in Figure 4 The results show that the present invention can be implemented in the flow rate range of 1.0-1.2 ml / min. At a flow rate of 1.1 ml / min, the separation degree between the o-methylbenzaldehyde peak and the adjacent peaks is better than the separation degrees measured under other flow rate conditions.

[0099] The method of the invention is simple to operate, accurate and reliable, highly sensitive, has a good linear relationship, and has good precision and durability, and meets the requirements for the detection of o-methylbenzaldehyde in indobufen, and can be used as a basis for its quality control.

[0100] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art who is familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for detecting the genotoxic impurity o-methylbenzaldehyde in indobufen, characterized in that: The test solution is prepared by the following steps: 1) dissolving the indobufen API with a sodium hydroxide solution, and then diluting it with a diluent to a first concentration between 5 and 50 g / L to obtain a diluent; 2) subjecting the dilution to an optional ultrasonic treatment, followed by a low temperature treatment at -2-0°C for 0.5-5 hours; 3) Filtering the diluted solution to obtain a subsequent filtrate having a second concentration as a test solution.

2. The method according to claim 1, wherein: The first concentration is between 10-30 g / L, preferably between 10-20 g / L.

3. The method according to claim 1, wherein: The amount of sodium hydroxide used is 0.8-2.0 times, preferably 0.9-1.2 times, of indobufen in terms of mole. The concentration of the sodium hydroxide solution used to dissolve the raw material drug can be between 0.1-1 mol / L, preferably between 0.3-0.7 mol / L.

4. The method according to claim 1, wherein: The low temperature treatment is to maintain the mixture in an ice water bath for a predetermined time.

5. The method according to claim 1, wherein: The diluent is a mixed solution of 0.1% phosphoric acid aqueous solution and acetonitrile in a volume ratio of 9:1 to 6:4, preferably 8:

2.

6. The method according to any one of claims 1 to 5, wherein: The chromatographic column is an octadecylsilane bonded silica gel chromatographic column, the detector is a diode array detector, and the o-methylbenzaldehyde is detected by gradient elution. The gradient elution procedure is as follows: Wherein, mobile phase A is 0.1% phosphoric acid aqueous solution, and mobile phase B is acetonitrile.

7. The method according to claim 6, wherein: The chromatographic column is Agilent Zorbax SB-C18 or Kromasil C18, both with dimensions of 4.6 mm × 250 mm, 5 μm.

8. The method according to claim 6, wherein: The flow rate range is 1.0-1.2 ml / min, preferably 1.1 ml / min.

9. The method according to claim 6, wherein: The column temperature of the chromatographic column is 29-31°C, preferably 30°C; the injection volume is 80-130 μl, preferably 100 μl.

10. The method according to claim 6, wherein: The following conditions were used: Detector: Light Emitting Diode Array Detector Chromatographic column: Agilent Zorbax SB-C18, size 4.6 mm × 250 mm, 5 μm; Flow rate: 1.1 mL / min; Detection wavelength: 250nm; Column temperature: 30°C; Injection volume: 100 μL; Preparation of reference solution: Take an appropriate amount of o-methylbenzaldehyde reference substance, weigh accurately, add a diluent of 0.1% phosphoric acid solution-acetonitrile mixed in a volume ratio of 80:20, and make a solution containing about 0.0375μg per 1ml. Preparation of test solution: Take about 1g of the product, weigh accurately, place in a 100ml volumetric flask, add 8ml of 0.5mol / L sodium hydroxide solution, dissolve by ultrasonication, then slowly add a diluent of 0.1% phosphoric acid solution-acetonitrile mixed in a volume ratio of 80:20 to the scale, shake well, cool in an ice bath for more than 1 hour, filter, and take the filtrate.

Images