Detection method for separating Efiniprofen and Efiniprofen enantiomer

Abstract

The chemical name of the enantiomer is R-2-fluoro-alpha-methyl (1, 1 '-diphenyl)-4-acetic acid, a phosphoric acid aqueous solution and acetonitrile are adopted as mobile phases, and amylose-tri-(3, 3'-difluorophenyl)-3, 3 '-difluorophenyl)-3, 3'-difluorophenyl)-3, 3 '-difluorophenyl)-3, 3'-difluorophenyl-3, 3 '-difluorophenyl-3, 3'-difluorophenyl-3, 3 '-difluorophenyl is adopted as a solvent. According to the present invention, the detection is performed by using the reversed-phase high performance liquid chromatography using the 2, 5-dimethyl phenyl carbamate (2, 5-dimethyl phenyl carbamate) as the stationary phase, and the advantages of high sensitivity, accurate quantification and simple operation are provided.

Description

Technical Field

[0001] The invention relates to the field of drug analysis, and in particular to a method for separating iflorfen and detecting iflorfen enantiomers by reversed-phase high performance liquid chromatography. Background Art

[0002] Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used in conservative treatments aimed at relieving symptoms of knee OA. The SFPP is an innovative NSAID patch developed by Taisho Pharmaceutical Co., Ltd. of Japan and launched in 2015 for the local treatment of OA. Its active ingredient is S-flurbiprofen (SFP), which is an enantiomer of racemic flurbiprofen (FP) and a strong COX inhibitor, of which R-flurbiprofen (RFP) is an inactive ingredient with a chemical name of R-2-fluoro-α-methyl (1,1'-diphenyl)-4-acetic acid. The chemical structures of FP, SFP and RFP are as follows:

[0003]

[0004] Compared with the existing flurbiprofen (FP) patch, SFPP allows the active ingredient to penetrate better into the synovial tissue. The FP patch has been widely used to treat musculoskeletal pain in patients with OA and rheumatoid arthritis. Therefore, SFPP is considered to be a drug that shows strong efficacy while avoiding direct gastrointestinal diseases. At present, this product has not been launched in China, and there are few reports on its related research and development.

[0005] Chinese patent application CN118515549A discloses a preparation method and detection method of flurbiprofen and S-flurbiprofen, wherein dependent claim 11 discloses the use of a chiral Chiralpak IA chromatographic column to detect R-flurbiprofen in S-flurbiprofen using a normal phase system. Figure 2 The HPLC test chart of Example 11, the test sample concentration is 1 mg / ml, attached Figure 2 No specific test results were given. Figure 2 It can be seen that the peak width of the main component is about 2 minutes, and the peak width of R-flurbiprofen is about 1 minute. As common knowledge, since the normal phase system uses a pure organic phase, it is easy to produce a solvation effect, resulting in a wide peak. The wider the peak, the lower the signal-to-noise ratio, that is, the lower the sensitivity. At the same time, normal phase chromatography does not dominate in high performance liquid chromatography (HPLC), and is only used in less than 20% of HPLC applications. Compared with reversed-phase high performance liquid chromatography, it is more expensive, has higher solvent toxicity, requires a larger sample size, etc., which is not conducive to daily maintenance.

[0006] At present, there is no literature reporting the reversed-phase high-performance liquid chromatography analysis method of ifluprofen SFP and its enantiomer RFP. Therefore, it is of positive practical significance to establish a new detection method that can quantitatively and efficiently detect the enantiomers in ifluprofen. Summary of the invention

[0007] The invention provides an analytical method for detecting enantiomer RFP in ifluprofen SFP by reverse phase high performance liquid chromatography, which can quantitatively detect the enantiomer in ifluprofen, thereby effectively controlling the quality of ifluprofen.

[0008] The reversed-phase high performance liquid chromatography method of the present invention has the following technical scheme:

[0009] Chromatographic column: Filler: amylose-tris-(3,5-dimethylphenylcarbamate), Pheromone Chiral ND(2)-RH, 250 mm × 4.6 mm, 5 μm;

[0010] Detector: PDA detector or UV detector

[0011] Mobile phase A: 0.05-0.5% phosphoric acid aqueous solution, mobile phase B: acetonitrile, the concentration of phosphoric acid aqueous solution is preferably 0.1%, the ratio of A to B is 30-50:70-50, preferably 40:60;

[0012] Flow rate: 0.7-1.2 ml / min, preferably 0.8 ml / min;

[0013] Injection volume: 2-20 μl, preferably 5 μl;

[0014] Detection wavelength: 240-260 nm, preferably 254 nm;

[0015] Column temperature: 20-30°C, preferably 25°C;

[0016] Solvent: selected from methanol, water or acetonitrile, used to dissolve the sample, preferably methanol;

[0017] System suitability solution: Accurately weigh an appropriate amount of iflorfen and its enantiomers, dissolve and dilute with solvent to prepare a mixed solution of 0.01 mg / ml to 1 mg / ml, preferably 0.05 mg / ml.

[0018] Test solution: Take an appropriate amount of the product, dissolve it in solvent and dilute it to make a solution containing about 0.5-5 mg per 1 ml, preferably 1 mg / ml.

[0019] Control solution: Take an appropriate amount of the test solution and dilute it quantitatively to prepare a control solution with a concentration of 0.05% to 0.5% relative to the test solution, preferably 0.5%.

[0020] Spiked solution: Take appropriate amount of ifloroprofen and its enantiomers to prepare an ifloroprofen solution containing 0.05-1.0% of the enantiomers, wherein the content of the isomers relative to ifloroprofen is 0.05-1.0%, preferably 0.5%.

[0021] Determination method: accurately measure the blank solution, system suitability solution, control solution, test solution and spiked solution respectively, inject them into the liquid chromatograph, record the chromatogram, and calculate the content of the isomer RFP of iflorfen in the test solution by the external standard method.

[0022] The method for detecting enantiomers in ifluprofen by reversed-phase high performance liquid chromatography provided by the present invention has the beneficial effects of high sensitivity of ifluprofen and its isomers, a separation degree greater than 1.5, good repeatability and durability of the method, and can realize quantitative inspection of enantiomers RFP in ifluprofen. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Solvent spectrum of Example 1

[0024] Figure 2 System suitability profile of Example 1

[0025] Figure 3 Sample solution spectrum of Example 1

[0026] Figure 4 Spectrum of 0.5% control solution of Example 1

[0027] Figure 5 Spectrum of the spiked 0.5% test solution of Example 1 DETAILED DESCRIPTION

[0028] In order to further illustrate the present invention, the method for determining the enantiomers in ifluprofen provided by the present invention is described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0029] The ifluprofen, ifluprofen enantiomers, reagents, high performance liquid chromatograph, electronic analytical balance, etc. used in the specific embodiments of the present invention are all known products.

[0030] Example 1

[0031] (1) Chromatographic conditions:

[0032] High performance liquid chromatograph Agilent 1260

[0033] Chromatographic column: Phenomenon Chiral ND(2)-RH, 250 mm × 4.6 mm, 5 μm;

[0034] Detector: UV detector

[0035] Mobile phase A: 0.1 phosphoric acid aqueous solution, mobile phase B: acetonitrile, the ratio of A to B is 40:60, isocratic elution.

[0036] Flow rate: 0.8ml / min;

[0037] Injection volume: 5 μl;

[0038] Detection wavelength: 254nm;

[0039] Column temperature: 25°C;

[0040] (2) Solution preparation:

[0041] Blank solution: methanol

[0042] System suitability solution: Accurately weigh an appropriate amount of ifluprofen and its enantiomers, dissolve in methanol and dilute to make a 0.05 mg / ml mixed solution.

[0043] Test solution: Take an appropriate amount of this product, dissolve it in methanol and dilute it to make a solution containing about 1 mg per 1 ml.

[0044] 0.5% control solution: Take an appropriate amount of the test solution and dilute it quantitatively to prepare a 0.005 mg / ml control solution.

[0045] 0.5% spiked test solution: Take appropriate amount of iflorfen and its enantiomer to prepare a 1 mg / ml iflorfen solution containing 0.5% of the enantiomer.

[0046] (3) Determination method:

[0047] Precisely measure the blank solution, system suitability solution, control solution, test solution and spiked test sample respectively, inject them into the liquid chromatograph, record the chromatogram, and calculate the content of the isomers of iflorfen in the test solution by the external standard method.

[0048] (4) Conclusion:

[0049] Figure 1 The blank solution spectrum in the figure shows that the blank solution does not interfere with the detection of ifluprofen and its enantiomers.

[0050] Figure 2 In the system suitability spectrum, the retention times of ioprofen and its enantiomer were 6.253min and 7.611min, respectively, with a separation degree of 6.05. The peak shape was good and met the quantitative requirements.

[0051] Figure 3 In the spectrum of the test solution, when the concentration of the test solution is 1 mg / ml, the peak shape is good and the enantiomer is detected at 0.03%.

[0052] Figure 4 The 0.5% control solution spectrum shows that when the control solution concentration is 0.005 mg / ml, the signal-to-noise ratio is 274.7, which is much larger than the quantitative limit S / N 10. The sensitivity is good and meets the detection requirements of 0.1% residual isomers.

[0053] Figure 5 The chromatogram of 0.5% spiked solution was obtained and the recovery rate was 102.5%. The method had good accuracy.

[0054] Example 2 Repeatability Test

[0055] Chromatographic conditions: Chromatographic conditions are the same as in Example 1

[0056] Solution preparation:

[0057] 0.5% control solution: Take an appropriate amount of the test sample and dissolve it in methanol to prepare a 0.005 mg / ml control solution.

[0058] Test solution: Take appropriate amount of iflorfen and its enantiomer to prepare a 1 mg / ml iflorfen solution containing 0.5% of the enantiomer, and prepare 6 portions in parallel.

[0059] Accurately measure 10 μl of the test solution respectively, inject it into the liquid chromatograph, and calculate by the external standard method. The results of the repeatability study are 0.488%, 0.502%, 0.495%, 0.497%, 0.511%, 0.502%, and the RSD is 1.4%. According to the guidelines for methodological validation of the 2020 version of the Chinese Pharmacopoeia, for 0.1% impurities, the RSD should not be greater than 3%. The research results show that this method has good repeatability.

[0060] Example 3 Durability Test

[0061] Solution preparation:

[0062] Blank solution: methanol

[0063] System suitability solution: Accurately weigh an appropriate amount of ifluprofen and its enantiomers, dissolve in methanol and dilute to 0.05 mg / ml.

[0064] Test solution: Take an appropriate amount of this product, dissolve it in methanol and dilute it to make a solution containing about 1 mg per 1 ml.

[0065] 0.5% control solution: Take an appropriate amount of the test solution and dilute it quantitatively to prepare a 0.005 mg / ml control solution.

[0066] 0.5% spiked test sample: Take appropriate amount of iflorfen and its enantiomer to prepare a 1 mg / ml iflorfen solution containing 0.5% of the enantiomer.

[0067] Based on the chromatographic conditions of Example 1, the flow rate, wavelength, mobile phase ratio, etc. were adjusted to investigate the separation and recovery of fluprofen and its enantiomers under different experimental conditions.

[0068] Accurately measure 10 μl of each of the blank solution, system suitability solution, control solution, and 0.1% spiked test solution and inject them into the liquid chromatograph. Results of the robustness separation and recovery rate inspection:

[0069] Chromatographic conditions Separation degree of ioprofen and its enantiomers Recovery rate (%) Standard conditions (Example 1) 6.05 97.5 Starting ratio A:B = 30:70 3.57 98.5 Starting ratio A:B = 50:50 7.56 97.1 Flow rate 0.7ml / min 4.86 96.6 Flow rate 1.0ml / min 6.52 98.3 Wavelength 250mm 6.04 95.8 Wavelength 260mm 6.01 96.9

[0070] Conclusion: The separation degree of ifluprofen and its enantiomers was greater than 1.5, and the separation degree was good; the recovery rate under different conditions was greater than 90.0%, and there was no difference in the detection results. The method had good durability.

[0071] The above description is only a preferred embodiment of the present invention. It should be pointed out that a person skilled in the art can make several improvements without departing from the principle of the present invention, and these improvements should also be regarded as within the protection scope of the present invention.

Claims

1. A method for separating ifluprofen and its enantiomer R-2-fluoro-α-methyl (1,1'-diphenyl)-4-acetic acid, the structural formula of which is shown below: RFP, the method using reverse phase high performance liquid chromatography, characterized in that: The mobile phase was aqueous phosphoric acid / acetonitrile; The column stationary phase was amylose-tris-(3,5-dimethylphenylcarbamate), and the filler particle size was 5 μm; The concentration of the phosphoric acid aqueous solution is 0.05-0.5%; The volume ratio of the phosphoric acid aqueous solution to acetonitrile is 30-50:70-50, and the elution is isocratic; The detection wavelength is 240-260 nm.

2. The method for separating iflorfen and its enantiomers according to claim 1, characterized in that: The steps include: a. Prepare test solution and control solution; b. The sample solution was detected by reversed-phase high performance liquid chromatography using 0.05-0.5% phosphoric acid aqueous solution / acetonitrile as the fluidity. The chromatographic conditions of the reversed-phase high performance liquid chromatography are as follows: the column temperature is 20-30°C; the detection wavelength is 240-260nm; the volume ratio of 0.05-0.5% phosphoric acid aqueous solution to acetonitrile is 40:60; the flow rate is 0.7-1.2ml / min; and the injection volume is 2-20μl.

3. The method for separating iflorfen and detecting its enantiomers according to claim 2, characterized in that: The solvent for preparing the test solution and the control solution is selected from methanol, water or acetonitrile; the concentration of the test solution of ifluprofen is 0.5-5 mg / ml, and the concentration of the control solution relative to the test solution is 0.05-1.0%.

4. The method for separating and detecting iflorfen and its enantiomers according to claim 3, characterized in that: The solvent for preparing the test solution and the control solution was methanol; the concentration of the test solution of ifluprofen was 1.0 mg / ml, and the concentration of the control solution relative to the test solution was 0.5%.

5. The method for separating and detecting iflorfen and its enantiomers according to claim 2, characterized in that: The concentration of the phosphoric acid aqueous solution was 0.1%, the column temperature was 25°C, the detection wavelength was 255 nm, the flow rate was 0.8 ml / min, and the injection volume was 10 μl.

6. The method for separating and detecting iflorfen and its enantiomers according to claim 2, characterized in that: The chromatographic column was selected from Phenom Chiral ND(2)-RH, 250 mm×4.6 mm, and the filler particle size was 5 μm.

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