A method for detecting related substances in oseltamivir phosphate starting materials

Abstract

The present invention relates to a method for detecting related substances in an oseltamivir phosphate starting material. The oseltamivir phosphate starting material is ethyl 5-(pentan-3-yloxy)-7-oxo-bicyclo[4.1.0]hept-3-ene-3-carboxylate. The detection method comprises the following steps: using reverse-phase high performance liquid chromatography (RP-HPLC) and acetonitrile-water as a mobile phase to detect ethyl 5-(pentan-3-yloxy)-7-oxo-bicyclo[4.1.0]hept-3-ene-3-carboxylate and seven related substance impurities thereof; wherein C10-SM1-ZZ9 is the process source of impurity C10-ZZF (impurity F in EP9.0) in an oseltamivir phosphate finished product; the detection method can quantitatively detect a content of C10-SM1-ZZ9 equivalent to 0.0084% of a test sample concentration, and can effectively control the content of impurity F in the finished product; the method is highly practical, and the detection process is simple and quick.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical analytical chemistry and relates to a method for detecting oseltamivir phosphate starting material 5-(pentan-3-yloxy)7-oxo-bicyclo[4.1.0]hept-3-ene-3-carboxylic acid ethyl ester (C10-SM1) and its impurities. Background Art

[0002] Oseltamivir phosphate, chemical name: (3R,4R,5S)-4-acetamido-5-amino-3-(1-ethylpropoxy)-1-cyclohexene-1-carboxylic acid ethyl ester phosphate, its chemical structure is:

[0003]

[0004] Oseltamivir phosphate is a neuraminidase inhibitor that inhibits the activity of influenza virus neuraminidase, preventing the virus from being released from infected cells, thereby achieving control. It has therapeutic and preventive effects on influenza caused by influenza A, B, H5N1, H9N2, and other subtypes of influenza viruses. The drug was approved for marketing by the US FDA in 1999 under the trade name Tamiflu. Oseltamivir phosphate is currently the main drug used internationally for the prevention and treatment of influenza. Its main indications include the treatment of influenza A and B in adults and children aged 1 year and above, and the prevention of influenza A and B in adults and adolescents aged 13 years and above. Oseltamivir is also the only FDA-approved drug for the treatment of influenza in neonates for more than 14 days.

[0005] Roche-Basel's process route (Organic Process Research & Development 2004, 8, 86-91) uses 5-(pentan-3-yloxy)7-oxo-bicyclo[4.1.0]hept-3-ene-3-carboxylic acid ethyl ester (C10-SM1) as the starting material, magnesium chloride as a complexing agent, tert-butylamine for ring opening, methanesulfonyl chloride as an activator, and subsequent ring closure. Diallylamine is used for ring opening, acetyl group is added, tert-butyl group is removed, deallyl group is removed, and then the product is salted with phosphoric acid to obtain oseltamivir phosphate.

[0006]

[0007] From the above route, it can be seen that the epoxy compound 5-(pentan-3-yloxy)7-oxo-bicyclo[4.1.0]hept-3-ene-3-carboxylic acid ethyl ester is a key starting material for the synthesis of oseltamivir phosphate. Its impurity composition and content have a great influence on the impurities of oseltamivir phosphate drugs, and greatly affect the safety of medication. Therefore, by targeted preparation of the target impurity of 5-(pentan-3-yloxy)7-oxo-bicyclo[4.1.0]hept-3-ene-3-carboxylic acid ethyl ester, establishing a corresponding analytical method, and controlling the quality of the raw material starting material can ensure the quality of oseltamivir phosphate raw materials and preparations, thereby ensuring the safety of medication. Summary of the Invention

[0008] The current mature process route for ethyl 5-(pentan-3-yloxy)7-oxa-bicyclo[4.1.0]hept-3-ene-3-carboxylate (C10-SM1) is as follows:

[0009]

[0010] The impurities that may be introduced are mainly the starting material C10-SM1-ZZ4 and the intermediates C10-SM1-ZZ1 and C10-SM1-ZZ3; the C10-SM1-ZZ3 isomer impurity C10-SM1-ZZ2, the optical isomer C10-SM1-ZZ5 of C10-SM1, the impurity C10-SM1-ZZ9 derived from the possible presence of 2-butanone in 3-pentanone, and the hydrolysis impurity C10-A-ZZ1 of the activated sulfonate of C10-SM1-ZZ1.

[0011] The present invention provides a detection method for the epoxy compound 5-(pentan-3-yloxy)-7-oxo-bicyclo[4.1.0]hept-3-ene-3-carboxylic acid ethyl ester (i.e., C10-SM1), a starting material for oseltamivir phosphate, and its related impurities. In particular, an effective method is provided for the detection and quantitative determination of seven process impurities, including C10-SM1-ZZ9. The impurity C10-SM1-ZZ9 is derived from the impurity C10-ZZF (impurity F in EP9.0) in the finished product of the API oseltamivir phosphate, so controlling it can effectively control the quality of the finished product.

[0012] In the technical solution of the present invention, the present invention provides a method for detecting the epoxy compound 5-(pentan-3-yloxy)7-oxa-bicyclo[4.1.0]hept-3-ene-3-carboxylic acid ethyl ester (i.e., C10-SM1) and its related impurities (i.e., related substances) of the starting material of oseltamivir phosphate, the detection method comprises the following steps:

[0013] (1) Reverse-phase high performance liquid chromatography was used, and the chromatographic conditions were set as follows:

[0014] Chromatographic column: octylsilane bonded silica gel liquid chromatography column (C8);

[0015] Detector: UV detector VWD;

[0016] Detection wavelength: 195-215nm;

[0017] Flow rate: 0.5~1.5ml / min;

[0018] Column temperature: 20-40°C;

[0019] Injection volume: 10-50 μl;

[0020] Diluent: acetonitrile;

[0021] Mobile phase A: purified water;

[0022] Mobile phase B: acetonitrile;

[0023] The elution method is gradient elution, and the linear elution procedure is as follows:

[0024] Time (minutes) Mobile phase A (volume %) Mobile phase B (volume %) 0～10 70～55 30～45 10～35 55 45 35～36 55～20 45～80 36～41 20 80 41～42 20～70 80～30 42～48 70 30

[0025] (2) Preparation of system suitability solution: Take impurity C10-SM1-ZZ1, C10-SM1-ZZ2, C10-SM1-ZZ3, C10-SM1-ZZ4, C10-SM1-ZZ5, C10-SM1-ZZ9, and C10-A-ZZ1 reference substances and add diluent to prepare impurity stock solutions, then take the test sample and impurity stock solutions and add diluent to prepare system suitability solution;

[0026] (3) Preparation of test solution: Take an appropriate amount of the test sample (i.e., the starting material of oseltamivir phosphate, the epoxy compound 5-(pentan-3-yloxy)-7-oxa-bicyclo[4.1.0]hept-3-ene-3-carboxylic acid ethyl ester (i.e., C10-SM1)), add a diluent to dissolve and prepare a test solution;

[0027] (4) Prepare the self-control solution: Take an appropriate amount of the test solution and dilute it 100 to 1000 times with diluent to prepare the self-control solution;

[0028] (5) Determination method: Accurately measure the system suitability solution, test solution, and self-control solution, inject them into the liquid chromatograph, record the chromatogram, and read at least one of the following information about the impurities: the amount of impurities, the type of impurities, the separation between the chromatographic peaks of the impurities, and the peak area of each chromatographic peak;

[0029] (6) Calculating the content of each impurity according to the impurity information read in step (5) using the principal component self-reference method with a correction factor added;

[0030] (7) The calculation formula for the content of each impurity is: the peak area of the impurity in the test solution in step (3) / the peak area of the control solution in step (4) / the dilution factor of the test solution in step (4) × the correction factor of each impurity; the correction factors of each impurity are as follows: C10-SM1-ZZ1: 1.45, C10-SM1-ZZ2: 1.22, C10-SM1-ZZ3: 1.21, C10-SM1-ZZ4: 1.07, C10-SM1-ZZ5: 1.00, C10-SM1-ZZ9: 0.96, C10-A-ZZ1: 1.16;

[0031] The diluent is acetonitrile.

[0032] In the technical solution of the present invention, the C10-SM1 related substances or impurities are shown in the following table:

[0033]

[0034] In step (3) of the above technical solution, the concentration of the test solution is 0.1 mg / ml to 10 mg / ml, preferably 2 to 5 mg / ml, and more preferably 3 mg / ml.

[0035] In step (4) of the above technical solution, the concentration of the self-control solution can be prepared by diluting the test solution 100 times or 1000 times, preferably 100 times.

[0036] In step (2) of the above technical solution, the concentration of the system suitability solution C10-SM1 is 3 mg / ml, and the concentrations of impurities C10-SM1-ZZ1, C10-SM1-ZZ2, C10-SM1-ZZ3, C10-SM1-ZZ4, C10-SM1-ZZ5, C10-SM1-ZZ9 and C10-A-ZZ1 are all 3 μg / ml.

[0037] In step (1) of the above technical solution, the column temperature of the chromatographic column is 20-40°C, preferably 25-35°C, and more preferably 30°C.

[0038] In step (1) of the above technical solution, the flow rate of the mobile phase in the high performance liquid chromatography method is 0.5 ml / min to 1.5 ml / min, preferably 0.8 to 1.2 ml / min, and more preferably 1.0 ml / min.

[0039] In step (1) of the above technical solution, the detection wavelength of the detector in the high performance liquid chromatography method is 195 nm to 215 nm, and the preferred detection wavelength is 207 nm.

[0040] In step (1) of the above technical solution, the injection volume is 10 to 50 μl, preferably 10 μl.

[0041] In the above technical solution, the retention time of C10-SM1 is 20 to 28 minutes, and preferably the retention time of C10-SM1 is 23 to 25 minutes.

[0042] The beneficial effects of the present invention are mainly reflected in: the method of the present invention is simple and effective, has good reproducibility and high sensitivity, can simultaneously detect C10-SM1 and its seven related impurities, among which the detection of C10-SM1-ZZ9 can effectively control the content of impurity E in the finished product of oseltamivir phosphate, and indirectly has important significance for the quality control of oseltamivir phosphate raw materials and related preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Separation chromatogram of system suitability solution

[0044] Figure 2 Liquid chromatography of the test sample Figure 1

[0045] Figure 3 Self-reference chromatogram of the test product

[0046] Figure 4 LOD diagram of the test sample and each impurity

[0047] Figure 5 LOQ diagram of test sample and each impurity

[0048] Figure 6 Liquid chromatogram of the sample in the precision experiment

[0049] Figure 7 Self-reference chromatogram of the test sample in the precision experiment Specific embodiments

[0050] The present invention is further described by the following examples, but the protection scope of the present invention is not limited thereto.

[0051] The reagents used in the following examples are readily available on the market.

[0052] The detection method was determined according to the high performance liquid chromatography method (Chinese Pharmacopoeia 2015 edition, Part IV, General Chapter 0512).

[0053] Example 1 Separation and determination of the main component and its related impurities in the test sample

[0054] 1. Equipment and methods:

[0055] Instrument: Reversed-phase high performance liquid chromatography

[0056] Detector: UV detector, detection wavelength is 207nm;

[0057] Chromatographic column: Octylsilane bonded silica gel liquid chromatography column (Agilent ZORBAX SB-C8, 4.6×250 mm, 5 μm);

[0058] Flow rate: 1.0ml / min

[0059] Column temperature: 30°C

[0060] Injection volume: 10 μl

[0061] Diluent: acetonitrile

[0062] Mobile phase A: purified water; mobile phase B: acetonitrile

[0063] Perform gradient elution according to the table below. Elution conditions:

[0064] Time (minutes) Mobile phase A (volume %) Mobile phase B (volume %) 0 70 30 10 55 45 35 55 45 36 20 80 41 20 80 42 70 30 48 70 30

[0065] 2. Solution preparation:

[0066] Blank solution: acetonitrile, i.e. diluent.

[0067] Impurity stock solution: Weigh an appropriate amount of each relevant substance impurity reference substance, place it in an appropriate volumetric bottle, add diluent to dissolve and dilute to the scale, shake well, and obtain 0.3 mg / ml impurity stock solution.

[0068] Impurity mixed solution: Take 5 ml of each impurity stock solution and place it in the same 50 ml volumetric flask, dissolve it with diluent and dilute it to the scale, shake well to obtain a 30 μg / ml impurity mixed solution.

[0069] System suitability solution: Weigh about 30 mg of the test sample and place it in a 10 ml volumetric flask. Accurately add 1 ml of the impurity mixed solution, dissolve it in diluent and dilute to the scale. Shake well to obtain the solution.

[0070] Test solution: Weigh about 30 mg of the test sample (oseltamivir phosphate starting material epoxy compound 5-(pentan-3-yloxy)7-oxo-bicyclo[4.1.0]hept-3-ene-3-carboxylic acid ethyl ester (i.e., C10-SM1), the same below) and place it in a 10 ml volumetric flask. Add diluent to dissolve and dilute to the scale. Shake well to obtain a 3 mg / ml solution.

[0071] Self-control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the scale with diluent, shake well, and obtain.

[0072] 3. Separation and determination method

[0073] Accurately measure 10 μl of each of the blank solution, system suitability solution, test solution, and self-control solution, inject them into the liquid chromatograph, and record the chromatogram. The measurement results are shown in the table below. The chromatograms are as follows: Figures 1 to 3 shown.

[0074] Experimental results:

[0075] Impurity name Relative retention time Separation Theoretical plates C10-A-ZZ1 0.48 \ 21745 C10-SM1-ZZ4 0.55 5.82 26827 C10-SM1-ZZ9 0.73 9.77 16496 C10-SM1-ZZ2 0.82 3.97 21249 C10-SM1-ZZ1 0.91 3.52 20015 C10-SM1 1.00 3.31 16035 C10-SM1-ZZ5 1.13 3.86 17449 C10-SM1-ZZ3 1.20 2.15 17387

[0076] The above test results show that the separation between the main peak and adjacent impurity peaks, and between adjacent known impurities is greater than 1.5, and the blank solution, C10-SM1 and other impurities do not interfere with the detection, indicating that the method has good specificity.

[0077] Example 2

[0078] Equipment and methods:

[0079] Instrument: Reversed-phase high performance liquid chromatography

[0080] Detector: UV detector, detection wavelength is 207nm;

[0081] Chromatographic column: Octylsilane bonded silica gel liquid chromatography column (Agilent ZORBAX SB-C8, 4.6×250 mm, 5 μm);

[0082] Flow rate: 1.0ml / min

[0083] Column temperature: 30°C

[0084] Injection volume: 10 μl

[0085] Diluent: acetonitrile

[0086] Mobile phase A: purified water; mobile phase B: acetonitrile

[0087] Perform gradient elution according to the table below. Elution conditions:

[0088] Time (minutes) Mobile phase A (volume %) Mobile phase B (volume %) 0 70 30 10 55 45 35 55 45 36 20 80 41 20 80 42 70 30 48 70 30

[0089] 1. Limit of quantification and limit of detection

[0090] Impurity stock solution 2: Measure 1 ml of each impurity stock solution (0.3 mg / ml) under Example 1 and place it in a 100 ml volumetric flask, dissolve it with diluent and dilute it to the scale, shake well to obtain a 3 μg / ml solution.

[0091] C10-SM1 stock solution: Weigh an appropriate amount of C10-SM1 reference substance, place it in an appropriate volumetric flask, add diluent to dissolve and dilute to the scale, shake well to obtain 0.3 mg / ml impurity stock solution; accurately measure 1 ml of impurity stock solution 1 and place it in a 100 ml volumetric flask, dissolve and dilute to the scale with diluent, shake well to obtain 3 μg / ml C10-SM1 stock solution.

[0092] Quantitation limit solution: Accurately measure 6 ml of C10-SM1-ZZ1 stock solution, 10 ml of C10-SM1-ZZ2 stock solution, 6 ml of C10-SM1-ZZ3 stock solution, 3 ml of C10-SM1-ZZ4 stock solution, 6 ml of C10-SM1-ZZ5 stock solution, 4 ml of C10-SM1-ZZ9 stock solution, 2 ml of C10-A-ZZ1 stock solution, and 6 ml of C10-SM1 stock solution from the above impurity stock solution 2, place them in the same 50 ml volumetric flask, dissolve and dilute to the mark with diluent, shake well, and obtain. (Equivalent to 0.0045% to 0.0152% of the test sample concentration)

[0093] Detection limit solution: Accurately measure 8 ml of quantitative limit solution and place it in a 20 ml volumetric flask. Dissolve it with diluent and dilute to the mark. Shake well to obtain the solution. (Equivalent to 0.0018% to 0.0061% of the test sample concentration)

[0094] Accurately measure 10 μl of the detection limit and quantification limit solutions, inject them into the liquid chromatograph, and record the chromatograms. The measurement results are shown in the table below. The chromatograms are as follows: Figure 4 and Figure 5 shown.

[0095] Impurity name Detection limit signal-to-noise ratio (S / N) Detection amount (%) Quantitation limit signal-to-noise ratio (S / N) Quantitative amount (%) C10-A-ZZ1 8.1 0.0018 18.8 0.0045 C10-SM1-ZZ4 16.2 0.0023 27.7 0.0058 C10-SM1-ZZ9 10.6 0.0033 25.5 0.0084 C10-SM1-ZZ2 14.9 0.0061 35.8 0.0152 C10-SM1-ZZ1 7.7 0.0046 19.4 0.0116 C10-SM1 11.0 0.0050 26.0 0.0124 C10-SM1-ZZ5 9.2 0.0042 20.2 0.0105 C10-SM1-ZZ3 8.1 0.0054 18.5 0.0136

[0096] Note: Both the detected amount and the quantitative amount are calculated as a percentage of the sample concentration.

[0097] The above results show that the minimum detection amount of each impurity is 0.0061%, the minimum detection concentration of each impurity limit is 0.0152%, and the impurity limit is 0.1%; therefore, the detection limit and quantification limit can meet the requirements.

[0098] 2. Repeatability Experiment

[0099] Blank solution: acetonitrile, i.e. diluent.

[0100] Test solution: Weigh about 30 mg of the test sample into a 10 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and obtain a 3 mg / ml solution. Prepare 6 copies in parallel.

[0101] Self-control solution: Accurately measure 1 ml of each test solution and place it in different 100 ml volumetric flasks, dilute to the scale with diluent, shake well, and obtain.

[0102] Accurately measure 10 μl of the blank solution, each test solution, and each self-control solution, inject them into the liquid chromatograph, and record the chromatogram. The measurement results are shown in the table below. The chromatograms are as follows: Figures 2-3 and Figures 6-7 shown.

[0103]

[0104] Note: ND means not detected, NA means not applicable.

[0105] Example 3

[0106] 1. Equipment and methods:

[0107] Instrument: Reversed-phase high performance liquid chromatography

[0108] Detector: UV detector, detection wavelength is 207nm;

[0109] Chromatographic column: Octylsilane bonded silica gel liquid chromatography column (Agilent ZORBAX SB-C8, 4.6×250 mm, 5 μm);

[0110] Flow rate: 0.9ml / min

[0111] Column temperature: 30°C

[0112] Injection volume: 10 μl

[0113] Diluent: acetonitrile

[0114] Mobile phase A: purified water; mobile phase B: acetonitrile

[0115] Perform gradient elution according to the table below. Elution conditions:

[0116]

[0117]

[0118] 2. Solution preparation:

[0119] Blank solution: acetonitrile, i.e. diluent.

[0120] Impurity stock solution: Weigh an appropriate amount of each relevant substance impurity reference substance, place it in an appropriate volumetric bottle, add diluent to dissolve and dilute to the scale, shake well, and obtain 0.3 mg / ml impurity stock solution;

[0121] Impurity mixed solution: Take 5 ml of each impurity stock solution and place it in the same 50 ml volumetric flask, dissolve it with diluent and dilute it to the scale, shake well to obtain a 30 μg / ml impurity mixed solution.

[0122] System suitability solution: Weigh about 30 mg of the test sample and place it in a 10 ml volumetric flask. Accurately add 1 ml of the impurity mixed solution, dissolve it with diluent and dilute to the scale, shake well, and obtain the solution.

[0123] Test solution: Weigh about 30 mg of the test sample into a 10 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well to obtain a 3 mg / ml solution.

[0124] Self-control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the scale with diluent, shake well, and obtain.

[0125] 3. Separation and determination method

[0126] Accurately measure 10 μl each of the above blank solution, system suitability solution, test solution, and self-control solution, inject them into the liquid chromatograph, and record the chromatogram; experimental results: the blank solution, C10-SM1 and other impurities do not interfere with the detection, and the separation between the relevant substances is good.

[0127] Example 4

[0128] 1. Equipment and methods:

[0129] Instrument: Reversed-phase high performance liquid chromatography

[0130] Detector: UV detector, detection wavelength is 207nm;

[0131] Chromatographic column: Octylsilane bonded silica gel liquid chromatography column (Agilent ZORBAX SB-C8, 4.6×250 mm, 5 μm);

[0132] Flow rate: 1.1 ml / min

[0133] Column temperature: 30°C

[0134] Injection volume: 10 μl

[0135] Diluent: acetonitrile

[0136] Mobile phase A: purified water; mobile phase B: acetonitrile

[0137] Perform gradient elution according to the table below. Elution conditions:

[0138]

[0139]

[0140] 2. Solution preparation:

[0141] Blank solution: acetonitrile, i.e. diluent.

[0142] Impurity stock solution: Weigh an appropriate amount of each relevant substance impurity reference substance, place it in an appropriate volumetric bottle, add diluent to dissolve and dilute to the scale, shake well, and obtain 0.3 mg / ml impurity stock solution;

[0143] Impurity mixed solution: Take 5 ml of each impurity stock solution and place it in the same 50 ml volumetric flask, dissolve it with diluent and dilute it to the scale, shake well to obtain a 30 μg / ml impurity mixed solution.

[0144] System suitability solution: Weigh about 30 mg of the test sample and place it in a 10 ml volumetric flask. Accurately add 1 ml of the impurity mixed solution, dissolve it with diluent and dilute to the scale, shake well, and obtain the solution.

[0145] Test solution: Weigh about 30 mg of the test sample into a 10 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well to obtain a 3 mg / ml solution.

[0146] Self-control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the scale with diluent, shake well, and obtain.

[0147] 3. Separation and determination method

[0148] Accurately measure 10 μl each of the above blank solution, system suitability solution, test solution, and self-control solution, inject them into the liquid chromatograph, and record the chromatogram; experimental results: the blank solution, C10-SM1 and other impurities do not interfere with the detection, and the separation between the relevant substances is good.

[0149] Example 5

[0150] 1. Equipment and methods:

[0151] Instrument: Reversed-phase high performance liquid chromatography

[0152] Detector: UV detector, detection wavelength is 207nm;

[0153] Chromatographic column: Octylsilane bonded silica gel liquid chromatography column (Agilent ZORBAX SB-C8, 4.6×250 mm, 5 μm);

[0154] Flow rate: 1.0ml / min

[0155] Column temperature: 25°C

[0156] Injection volume: 10 μl

[0157] Diluent: acetonitrile

[0158] Mobile phase A: purified water; mobile phase B: acetonitrile

[0159] Perform gradient elution according to the table below. Elution conditions:

[0160] Time (minutes) Mobile phase A (volume %) Mobile phase B (volume %) 0 70 30 10 55 45 35 55 45 36 20 80 41 20 80 42 70 30 48 70 30

[0161] 2. Solution preparation:

[0162] Blank solution: acetonitrile, i.e. diluent.

[0163] Impurity stock solution: Weigh an appropriate amount of each relevant substance impurity reference substance, place it in an appropriate volumetric bottle, add diluent to dissolve and dilute to the scale, shake well, and obtain 0.3 mg / ml impurity stock solution;

[0164] Impurity mixed solution: Take 5 ml of each impurity stock solution and place it in the same 50 ml volumetric flask, dissolve it with diluent and dilute it to the scale, shake well to obtain a 30 μg / ml impurity mixed solution.

[0165] System suitability solution: Weigh about 30 mg of the test sample and place it in a 10 ml volumetric flask. Accurately add 1 ml of the impurity mixed solution, dissolve it with diluent and dilute to the scale, shake well, and obtain the solution.

[0166] Test solution: Weigh about 30 mg of the test sample into a 10 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well to obtain a 3 mg / ml solution.

[0167] Self-control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the scale with diluent, shake well, and obtain.

[0168] 3. Separation and determination method

[0169] Accurately measure 10 μl each of the above blank solution, system suitability solution, test solution, and self-control solution, inject them into the liquid chromatograph, and record the chromatogram; experimental results: the blank solution, C10-SM1 and other impurities do not interfere with the detection, and the separation between the relevant substances is good.

[0170] Example 6

[0171] 1. Equipment and methods:

[0172] Instrument: Reversed-phase high performance liquid chromatography

[0173] Detector: UV detector, detection wavelength is 207nm;

[0174] Chromatographic column: Octylsilane bonded silica gel liquid chromatography column (Agilent ZORBAX SB-C8, 4.6×250 mm, 5 μm);

[0175] Flow rate: 1.0ml / min

[0176] Column temperature: 35°C

[0177] Injection volume: 10 μl

[0178] Diluent: acetonitrile

[0179] Mobile phase A: purified water; mobile phase B: acetonitrile

[0180] Perform gradient elution according to the table below. Elution conditions:

[0181] Time (minutes) Mobile phase A (volume %) Mobile phase B (volume %) 0 70 30 10 55 45 35 55 45 36 20 80 41 20 80 42 70 30 48 70 30

[0182] 2. Solution preparation:

[0183] Blank solution: acetonitrile, i.e. diluent.

[0184] Impurity stock solution: Weigh an appropriate amount of each relevant substance impurity reference substance, place it in an appropriate volumetric bottle, add diluent to dissolve and dilute to the scale, shake well, and obtain 0.3 mg / ml impurity stock solution;

[0185] Impurity mixed solution: Take 5 ml of each impurity stock solution and place it in the same 50 ml volumetric flask, dissolve it with diluent and dilute it to the scale, shake well to obtain a 30 μg / ml impurity mixed solution.

[0186] System suitability solution: Weigh about 30 mg of the test sample and place it in a 10 ml volumetric flask. Accurately add 1 ml of the impurity mixed solution, dissolve it with diluent and dilute to the scale, shake well, and obtain the solution.

[0187] Test solution: Weigh about 30 mg of the test sample into a 10 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well to obtain a 3 mg / ml solution.

[0188] Self-control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the scale with diluent, shake well, and obtain.

[0189] 3. Separation and determination method

[0190] Accurately measure 10 μl each of the above blank solution, system suitability solution, test solution, and self-control solution, inject them into the liquid chromatograph, and record the chromatogram; experimental results: the blank solution, C10-SM1 and other impurities do not interfere with the detection, and the separation between the relevant substances is good.

Claims

1. A method for separating oseltamivir phosphate starting material 5-(pentan-3-yloxy)7-oxa-bicyclo[4.1.0]hept-3-ene-3-carboxylic acid ethyl ester (C10-SM1) and its related substances. The impurities of C10-SM1 related substances are shown in the following table: ， The separation method comprises the following steps: (1) Reverse-phase high performance liquid chromatography was used for detection, and the parameters were set as follows: Chromatographic column: octylsilane bonded silica gel liquid chromatography column (C8); Detector: UV detector; Detection wavelength: 195~215nm; Flow rate: 0.5~1.5ml / min; Column temperature: 20~40℃; Injection volume: 10~50μl; Diluent: acetonitrile; Mobile phase A: purified water; Mobile phase B: acetonitrile; The elution method is gradient elution, and the linear elution procedure is as follows: ； (2) Solution preparation method Diluent: acetonitrile; System suitability solution: Take impurities C10-SM1-ZZ1, C10-SM1-ZZ2, C10-SM1-ZZ3, C10-SM1-ZZ4, C10-SM1-ZZ5, C10-SM1-ZZ9 and C10-A-ZZ1 reference substances and add diluent to prepare impurity stock solutions. Then, take the oseltamivir phosphate starting material 5-(pentan-3-yloxy)-7-oxo-bicyclo[4.1.0]hept-3-ene-3-carboxylic acid ethyl ester and the impurity stock solution and use diluent to prepare a solution containing C10-SM1 2-5 mg / ml and each impurity 2-5 μg / ml. The oseltamivir phosphate starting material 5-(pentan-3-yloxy)-7-oxo-bicyclo[4.1.0]hept-3-ene-3-carboxylic acid ethyl ester is C10-SM1. (3) Measurement method Accurately measure the system suitability solution and inject it into the liquid chromatograph, and record the chromatogram.

2. The method for separating oseltamivir phosphate starting material 5-(pentan-3-yloxy)-7-oxo-bicyclo[4.1.0]hept-3-ene-3-carboxylic acid ethyl ester (C10-SM1) and its related substances according to claim 1, characterized in that: The chromatographic column stationary phase in step (1) is an octylsilane bonded silica gel liquid chromatography column with a particle size of 3 to 5 μm, a length of 150 to 250 mm, and an inner diameter of 2.1 to 4.6 mm.

3. The method for separating oseltamivir phosphate starting material 5-(pentan-3-yloxy)-7-oxo-bicyclo[4.1.0]hept-3-ene-3-carboxylic acid ethyl ester (C10-SM1) and its related substances according to claim 1, characterized in that: The liquid chromatography conditions described in step (1) are: Agilent ZORBAX SB-C8, 4.6 mm × 250 mm, 5 μm, test time: 48 min, column temperature of 25-35 °C, flow rate of mobile phase of 0.9 ml / min-1.1 ml / min, detection wavelength of detector of 207 nm, injection volume of 10 μl, mobile phase A of purified water, mobile phase B of acetonitrile, gradient separation.

4. The method for separating oseltamivir phosphate starting material 5-(pentan-3-yloxy)7-oxo-bicyclo[4.1.0]hept-3-ene-3-carboxylic acid ethyl ester (C10-SM1) and its related substances according to claim 3, characterized in that: The liquid chromatography conditions are as follows: the column temperature is 30° C., and the flow rate of the mobile phase is 1 ml / min.

5. The method for separating oseltamivir phosphate starting material 5-(pentan-3-yloxy)7-oxo-bicyclo[4.1.0]hept-3-ene-3-carboxylic acid ethyl ester (C10-SM1) and its related substances according to claim 1, characterized in that: The solution prepared in step (2) has a concentration of 2-5 mg / ml of C10-SM1 and 2-5 μg / ml of each impurity for system suitability.

6. A method for separating oseltamivir phosphate starting material 5-(pentan-3-yloxy)7-oxo-bicyclo[4.1.0]hept-3-ene-3-carboxylic acid ethyl ester (C10-SM1) and its related substances according to claim 1, characterized in that: The solution prepared in step (2) has a concentration of 3 mg / ml of C10-SM1 and 3 μg / ml of each impurity for system suitability.

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