Quantitative detection method for degraded impurities in flurbiprofen gel plaster based on HPLC (High Performance Liquid Chromatography)

Abstract

The invention belongs to the field of medical analysis, and discloses a quantitative detection method for degradation impurities in flurbiprofen gel plaster. The HPLC correction factor method is adopted, and the detection method can effectively separate and quantitatively detect degraded impurities in the flurbiprofen gel plaster. According to the detection method, high performance liquid chromatography is adopted, a stationary phase is octadecyl bonded silica gel filler, a mobile phase is an acetonitrile-acetic acid aqueous solution, and gradient elution is carried out. The peak shape is good, the separation degree with an adjacent chromatographic peak is high, the collection time is reasonable, and the experiment efficiency can be effectively improved.

Description

Technical Field

[0001] The invention belongs to the field of medical analysis and discloses a quantitative detection method for degradation impurities in a flurbiprofen gel patch. Background Art

[0002] Flurbiprofen, chemical name is 2-fluoro-α-methyl (1,1'-diphenyl)-4-acetic acid, CAS number: 5104-49-4, molecular formula C15H13FO2 , molecular weight 244.27, structural formula is as follows: Flurbiprofen is an aromatic propionic acid non-steroidal analgesic and anti-inflammatory drug with prostaglandin synthesis inhibition. It has obvious analgesic and anti-inflammatory effects on local inflammations such as periarthritis of shoulder, tenosynovitis, peritendinitis, humeral granulation (tennis elbow, etc.), muscle pain, swelling and pain after trauma, etc. Its oral preparations are widely used in various painful and inflammatory diseases, especially in the field of plastic surgery. However, oral non-steroidal analgesic and anti-inflammatory drugs may cause systemic adverse reactions. In order to overcome the possible adverse reactions, Mikasa Pharmaceutical Co., Ltd. of Japan has developed a topical flurbiprofen gel patch. Compared with traditional patches, this type of patch has more excipients or other additives, some of which are less inert and more likely to produce degradation impurities during preparation and storage, which puts higher requirements on its quality control.

[0003] At present, flurbiprofen bulk drug is included in EP11.0, ChP2020, JP18, and USP-NF, and HPLC method is used for detection, but flurbiprofen gel patch preparation is not included for the time being. It is found in the actual detection process that the detection method of bulk drug cannot effectively control the degradation impurities generated in the gel patch preparation. Retrieving the prior art and related literature, CN 115932135A discloses a method for analyzing related substances in flurbiprofen sodium gel, but the method is directed to the related substances in flurbiprofen sodium ((±)-2-(2-fluoro-4-biphenyl)-propionic acid sodium salt dihydrate) gel, and the detection conditions are quite different from the present invention, the detection time is long, and the degradation impurities targeted by the present invention cannot be quickly and effectively separated and quantified.

[0004] Therefore, a detection method can be developed that can effectively detect degradation impurities in flurbiprofen gel patch, which is simple to operate, has readily available reagents, good reproducibility, and can be quickly and accurately quantified without the need for impurity reference substances. This is of great significance for the quality control of flurbiprofen gel patch. Summary of the invention

[0005] In view of the above shortcomings, the present invention proposes a method for quantitatively determining degradation impurities in flurbiprofen gel patch based on HPLC correction factor method. The impurities detected by the technical solution of the present invention are degradation impurities generated during the production and storage of flurbiprofen gel patch, which are commonly present in flurbiprofen gel patch preparations, and aim to provide a quantitative detection method for flurbiprofen gel patch to fill the gap in the prior art.

[0006] In order to solve the technical problem existing in the detection of degradation impurities in the above-mentioned flurbiprofen gel patch preparation, the present invention adopts the following technical solution: A quantitative detection method for degradation impurities in flurbiprofen gel patch based on HPLC, comprising the following steps: The chromatographic conditions were as follows: Chromatographic column: reverse phase chromatographic column, the filler is octadecyl bonded silica gel; Mobile phase A: acetic acid aqueous solution; Mobile phase B: acetonitrile; Column temperature: 28-32°C; Detection wavelength: 252-256nm; Flow rate: 1.0 ml / min; Gradient elution was performed, and the elution program is shown in Table 1 below: Mobile phase A: acetic acid-water (5:60 v / v) Mobile phase B: acetonitrile Table 1 Elution procedure Time (min) Mobile phase A (%) Mobile phase B (%) 0 55 45 10 55 45 18 0 100 28 0 100 31 55 45 35 55 45 Further preferably, the chromatographic column is Agilent ZORBAX XDB-C18 (4.6 mm×150 mm, 5 μm); Further preferably, the column temperature is 30°C; Further preferably, the detection wavelength is 254 nm; Further preferably, the injection volume is 20L; Further preferably, the method for detecting degradation impurities in the flurbiprofen gel patch is applicable to the compounds in the following Table 2: Table 2 Impurities

[0007] More preferably, the correction factor of impurity A is 1.3, and the correction factor of impurity B is 1.4.

[0008] Due to the adoption of the above technical solution, the beneficial effects of the present invention are: The present invention discloses a quantitative detection method of degradation impurities in flurbiprofen gel patch based on HPLC: 1) Provides an effective analytical method for quantitative detection of degradation impurities in flurbiprofen gel patch. A principal component self-control method with correction factor was established to quantitatively analyze degradation impurities in flurbiprofen gel patch.

[0009] 2) This analytical method has strong specificity and can effectively separate and quantify the two degradation impurities in flurbiprofen gel patch with a separation degree greater than 1.5, high sensitivity, good reproducibility, and a reasonable collection time, which can effectively improve the experimental efficiency and effectively control the quality of flurbiprofen gel patch. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 is the liquid chromatogram of blank solvent; Figure 2 is the system suitability solution chromatogram; Figure 3 It is the linear relationship diagram of the concentration of flurbiprofen impurity A and the average peak area; Figure 4 It is the linear relationship diagram of the concentration of flurbiprofen impurity B and the average peak area; Figure 5 It is the liquid chromatogram of the test solution. DETAILED DESCRIPTION

[0011] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0012] Example 1 1. Instruments and test drugs 1.1 Instruments Agilent 1260 liquid chromatograph (Agilent); chromatographic column Agilent ZORBAX XDB-C18 (4.6 mm × 150 mm, 5 μm); MS205DU electronic balance (Mettler Toledo); acidity meter (Seven Compact model) 1.2 Drug testing The experimental water was purified water, methanol and acetonitrile were chromatographically pure, glacial acetic acid was chromatographically pure (Aladdin Chemical Reagent Co., Ltd.), and other reagents were analytically pure. Flurbiprofen reference substance (China Food and Drug Inspection Institute, content 99.9%); Flurbiprofen impurity A (PharmaBlock Sciences, Inc, content 94.0%); Flurbiprofen impurity B (PharmaBlock Sciences, Inc, content 97.6%) can also be purchased from Shenzhen Hengfeng Wanda Pharmaceutical Technology Co., Ltd. https: / / www.hdimpurity.com / pro / 15668.html. Flurbiprofen gel patches were purchased from Sanrai Pharmaceutical Co., Ltd., a total of 3 batches, and the blank preparation was homemade.

[0013] Example 2 2. Solution Preparation 2.1 Diluent methanol-water (80:20) 2.2 Reference solution Flurbiprofen reference solution: Take 15 mg of Flurbiprofen reference, weigh accurately, place in a 25 mL volumetric flask, add 80% methanol to dissolve and dilute to scale, shake well. Take another 3 mL, place in a 100 mL volumetric flask, add 80% methanol to dilute to scale, shake well, and use as Flurbiprofen reference solution.

[0014] Flurbiprofen impurity A and B reference solution: Take 22.5 mg of Flurbiprofen impurity A and B reference respectively, weigh accurately, place in a 25 mL volumetric flask, add 80% methanol to dissolve and dilute to scale, shake well. Take another 2.5 mL, place in a 25 mL volumetric flask, add 80% methanol to dissolve and dilute to scale, shake well, and use as Flurbiprofen impurity A and B reference solution.

[0015] 2.3 System Suitability Solution Take an appropriate amount of the above reference substances, dissolve in diluent and dilute to a mixed solution containing approximately 0.3 mg / ml of Flurbiprofen Impurity A, 0.4 mg / ml of Flurbiprofen Impurity B and 0.2 mg / ml of Flurbiprofen per 1 mL.

[0016] 2.4 Test solution Take 1 flurbiprofen gel patch, cut it into pieces and accurately weigh 3.0 g, put it in a conical flask, add 50 mL of diluent, sonicate for 1 h, filter and take the filtrate as the test solution.

[0017] 2.5 Control solution Accurately measure 0.1 mL of the test solution and place it in a 50 mL volumetric flask. Add diluent to the mark and mix well.

[0018] 2.6 Blank preparation solution: Extract the blank patch according to the test solution extraction method in 2.4.

[0019] Example 3 3. Methodological Validation 3.1 System suitability Take 20 μL of blank solvent and system suitability solution and inject them into the liquid chromatograph. Figure 1 , Attachment Figure 2 The retention time of flurbiprofen (2) was 9.3, the retention time of impurity A (1) was 5.3min, and the retention time of impurity B (3) was 22.6 min; the relative retention times were 0.57 and 2.4, respectively. The results showed that other components in the sample had no interference with the determination of impurities A and B.

[0020] 3.2 Exclusivity Investigation Investigate the separation of the main peak of the preparation and the peak of degradation impurities to verify the specificity of the method. Take 1 tablet of this product, remove the covering release film, cut into pieces and accurately weigh 3.0g (equivalent to 9mg of flurbiprofen), place in a conical flask, and prepare 5 portions in parallel. Acid destruction (1 mol·L-1 HCL, 2 h): first add 3 mL 80% methanol and then add 1 mL 1 mol·L-1 HCl solution, leave it at room temperature for 2 hours, then add 1 mL 1 mol·L-1 NaOH solution to neutralize it, add 45 mL 80% methanol for ultrasonic extraction for 1 hour, filter and obtain; alkali destruction (1 mol·L-1 NaOH, 2 h): first add 3 mL 80% methanol and then add 1 mL 1 mol·L-1 NaOH solution, leave it at room temperature for 2 hours, then add 1 mL 1 mol·L-1 HCl solution to neutralize it, add 45 mL 80% methanol for ultrasonic extraction for 1 hour, filter and obtain; oxidative destruction (30% H2O2, 4 h): first add 3 mL 80% methanol and then add 2 mL 10% H2O2 Solution, after being placed at room temperature for 4 hours, add 45 mL of 80% methanol and ultrasonically extract for 1 hour, filter, and obtain; high temperature destruction (80℃, 5 h): add 50 mL of 80% methanol and ultrasonically extract for 1 hour, take out after 5 hours in 80℃ water bath, put to room temperature, filter, and obtain; light destruction (strong light + ultraviolet, 7 d): add 50 mL of 80% methanol and ultrasonically extract for 1 hour, place in a light (5000 1x±500 lx) stabilization box for 7 days, take out, filter, and obtain. Measure the above solutions respectively and inject and test according to the chromatographic conditions under "2.1". The results are shown below Figure 4Under the above-mentioned destruction conditions, the mass balance was between 90% and 110%, and the main peak and impurity peaks were well separated. The purity of the main peak met the requirements, and the impurity peaks produced by the destruction did not interfere with the experiment, indicating that this method has good specificity under all conditions.

[0021] 3.3 Linearity and correction factor determination Flurbiprofen linear stock solution: Accurately measure an appropriate amount of "Flurbiprofen reference solution under Item 2.2" and place it in a 10 mL volumetric flask. Dissolve and dilute with a diluent to prepare flurbiprofen linear solutions with flurbiprofen concentrations of approximately 0.2, 2.7, 3.6, 0.45, and 5.4 μg / mL. Accurately measure an appropriate amount of "Flurbiprofen impurity A and B reference solution under Item 2.2" and place them in a 25 mL volumetric flask. The linear concentrations of the impurity A series are 0.01, 1.8, 3.6, 4.32, and 5.4 μg / mL; the linear concentrations of the impurity B series are 0.1, 5.4, 10.8, 12.96, and 16.20 μg / mL. Take the above-mentioned flurbiprofen and flurbiprofen impurity A and impurity B linear solutions, inject them according to the chromatographic conditions under Item "2.1", record the chromatogram, and calculate the regression equation using concentration-peak area. See the attached document for details. Figure 3 , Attachment Figure 4 The correction factor was determined by the standard curve method, and the correction factor f was calculated using the slope of the flurbiprofen linear regression equation and the regression equations of flurbiprofen impurity A and impurity B, respectively. The results are shown in Table 3 below.

[0022] Table 3 Linearity and range test results of flurbiprofen and impurities name Regression equation R Correction Factor Linear range Flurbiprofen y=72.75x+2.737 0.9957 / 0.2-5.4 μg / mL Flurbiprofen Impurity A y=56.095x+0.3954 0.9999 1.3 0.01-5.4 μg / mL Flurbiprofen Impurity B y=52.84x+0.1115 1 1.4 0.1-16.2 μg / mL 3.4 Limit of detection and limit of quantification Accurately measure the linear solutions of Flurbiprofen impurity A and impurity B, dissolve them in diluent and gradually dilute them to the limit of quantification (S / N=10) and the limit of detection (S / N=3). Calculate the signal-to-noise ratio (S / N) of each chromatographic peak. The results show that the limit of detection and the limit of quantification of Flurbiprofen impurity A are 0.003 μg / mL and 0.01 μg / mL, respectively; the limit of detection and the limit of quantification of Flurbiprofen impurity B are 0.01 μg / mL and 0.09 μg / mL, respectively.

[0023] 3.5 Accuracy Prepare recovery test solutions at three concentration levels, accurately weigh appropriate amounts of flurbiprofen gel patches, place in 50 mL volumetric flasks, add impurity reference stock solutions and dilute to scale, shake well, and use as recovery test solutions. Prepare three portions at each concentration level in the same way, calculate the recovery, and the results show that the average recovery of impurity A is 98.9%, and the average recovery of impurity B is 97.9%. The above results show that the accuracy of the method is good.

[0024] 3.6 Precision The injection precision was determined by injection of the test solution under item "2.4", and 6 injections were made continuously to calculate the RSD of the peak areas of flurbiprofen impurity A and impurity B. The results showed that the RSD of the peak area of ​​flurbiprofen impurity A was 1.6%, and the RSD of the peak area of ​​flurbiprofen impurity B was 2.1%, indicating that the injection precision of the method was good.

[0025] Intermediate precision Different experimenters performed experimental operations at different times according to the precision experimental method and conducted analysis. The results showed that the RSD of the peak area of ​​flurbiprofen impurity A was 2.8%, and the RSD of the peak area of ​​flurbiprofen impurity B was 3.2%, indicating that the intermediate precision of the method was good.

[0026] 3.7 Stability The test solution was placed at room temperature for 0, 2, 4, 6, 10, 12, and 24 hours before sampling and testing, and the chromatogram was recorded. The results showed that the impurity peak area at each time point was within ±2.0% compared with the initial result, the RSD of the peak area of ​​flurbiprofen impurity A was 1.3%, and the RSD of the peak area of ​​flurbiprofen impurity B was 0.73%, indicating that the sample had good stability.

[0027] 3.8 Durability The chromatographic conditions were changed respectively, and the durability of the method was investigated by observing the separation between the main peak and each impurity peak and between each impurity peak in the chromatograms of the system suitability solution and the test solution. The results showed that the separation between the main peak and each impurity peak met the requirements by changing the column temperature (±2°C) and replacing the chromatographic column of the same model but different batches, and the change rate of the impurity content under each condition compared with the unchanged condition was ±0.1%, which had no significant effect on the results, indicating that the method has good durability.

[0028] Example 4 4. Sample testing The above analytical method was verified to be suitable for the quantitative detection of degradation impurities in flurbiprofen gel patch. The results are shown in Table 4 below. Figure 5 This is a typical liquid phase spectrum of the test solution.

[0029] Table 4 Detection results of sample degradation impurities batch Impurity A (%) Impurity B (%) 1 0.66 1.72 2 0.55 1.63 3 0.51 1.59 The specific contents of the above embodiments 1-4 are only for a clearer description of the present invention. The above embodiments are all analyzed under the conditions of Shimadzu, Agilent, and Thermo Fisher high performance liquid chromatographs. The quantitative detection method of other degradation impurities in flurbiprofen gel patch is similar to the above embodiments, which will not be repeated here. The above-described embodiments are only for better description of the use of the present invention, and are not intended to limit the scope of the patent of this application. Appropriate modifications or changes made by professionals and technicians in this field based on this application should be deemed to belong to the protection scope of this application.

Claims

1. A quantitative detection method for degradation impurities in flurbiprofen gel patch based on HPLC, characterized in that: The stationary phase of the chromatographic column in the HPLC method is octadecyl bonded silica gel filler, the mobile phase A is acetic acid-water solution 5:60 v / v, and the mobile phase B is acetonitrile, and gradient elution is performed; the gradient elution program used in the HPLC method is as follows: 。 2. The detection method according to claim 1, characterized in that: The chromatographic column is a C18 chromatographic column with a specification of 4.6×150 mm and 5 um.

3. The detection method according to claim 1, characterized in that: The elution flow rate was 1.0 ml / min.

4. The detection method according to claim 1, characterized in that: The injection volume is 5-20mL.

5. The detection method according to claim 1, characterized in that: The detection wavelength is 252-256nm.

6. The detection method according to claim 1, characterized in that: The column temperature was tested at 28-32°C.

7. The detection method according to claim 1, characterized in that: The degradation impurities are: 2,3-dihydroxypropane-2-yl-2-(2-fluoro-[1,1'diphenyl]-4-yl) propionate, i.e., impurity A and (1R, 2S, 5R)-2-isopropyl-5-methylcyclohexyl-2-(2-fluoro-[1,1'diphenyl]-4-yl) propionate, i.e., impurity B.

8. The detection method according to claim 7, characterized in that: The correction factors of impurity A and impurity B are 1.3 and 1.4 respectively.

9. The detection method according to any one of claims 1 to 8, characterized in that: The analysis steps include: 1) Preparation of test solution: Take an appropriate amount of flurbiprofen gel patch as the test sample, place the test sample in a diluent, dissolve the test component in the diluent by ultrasonication, shake well, and filter to obtain the test solution; 2) Determination: Use acetic acid aqueous solution as mobile phase A and acetonitrile as mobile phase B, accurately measure 20 mL of each of the above solutions, inject into high performance liquid chromatography, perform gradient elution, and record the chromatogram; 3) Draw a peak area-concentration standard curve based on the measured chromatogram; and quantify the degradation impurities using the HPLC correction factor method.

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