Detection method for related substances of fluralana and application of detection method

Abstract

The invention provides a method for detecting related substances of fluralana, and relates to the technical field of pharmaceutical analysis. According to the method, a high performance liquid chromatograph is adopted to detect the fluralana related substances, and the detection conditions are as follows: octadecylsilane chemically bonded silica is adopted as a filling agent of a chromatographic column; the wavelength of the UV detector is 250 nm to 280 nm; the column temperature is 30-45 DEG C; the flow velocity of the mobile phase is 0.5-1.5 mL / min; the sample injection volume is 5-20 [mu] L; the mobile phase A is a formic acid aqueous solution; the mobile phase B is acetonitrile; the mobile phase C is methanol; the elution mode is gradient elution. According to the method provided by the invention, effective separation between main components and impurities can be realized, the specificity is high, the sensitivity is high, the accuracy and the reproducibility are good, the detection result is stable and reliable, and a plurality of impurities in the flurala raw material can be qualitatively and quantitatively, so that the quality of the flurala can be better controlled.

Description

Technical Field

[0001] The invention belongs to the field of drug analysis, and specifically relates to a method for detecting flurellan-related substances and an application thereof. Background Art

[0002] Fluralaner, English name: Fluralaner, chemical name is 4-(5(3,5-dichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)-2-methyl-N-(2-oxo-2-((2,2,2-trifluoroethyl)amino)ethyl)benzamide. The specific structural formula is shown below.

[0003]

[0004] Flurella is a new type of insecticidal active substance obtained in the process of studying phthalic acid formamide and o-formylaminobenzamide. As a new type of broad-spectrum veterinary drug, its mechanism of action is to exert its effect by intervening in the γ-aminobutyric acid (GABA) gated chloride ion channel. Compared with animal insecticides such as cyclopentadiene, phenylpyrazole and macrolide, flurella has significant differences in molecular structure, site of action, selectivity and cross-resistance. It has low toxicity and safety to non-target organisms such as mammals, zebrafish and poultry, and has excellent insecticidal activity against pests such as cicadas, Siphonaptera, Pediculus, Hemiptera and Diptera. In recent years, this drug has been used in the clinical treatment of external parasitic diseases of dogs and cats.

[0005] Fluorella has been commercialized as a veterinary drug and is used for the prevention and treatment of parasites. In 2014, Merck first commercialized fluorella as a veterinary drug Bravecto to kill dog parasites such as lice and fleas. A series of studies have shown that the drug can be quickly absorbed by the dog body, has the characteristics of rapid effect, long half-life, long duration and low metabolic rate, and is a very safe anti-ectoparasite drug.

[0006] As the domestic research on this product deepens, impurities generated in its production process are gradually discovered. During the synthesis of flurellana, the starting materials, process impurities generated in the reaction, and degradation impurities will affect the quality of flurellana. In order to strengthen the quality control of the raw materials and their intermediates and impurities, it is necessary to provide a sensitive, stable and reliable detection method. Summary of the invention

[0007] The invention provides a drug analysis method for efficiently determining related substances in flurellana, wherein the method adopts a chromatographic column using octadecylsilane bonded as a filler.

[0008] The flurellana-related substance is one or more of impurity A, impurity B, impurity C, impurity D, impurity E, and impurity F. The impurity structure is shown in Table 1:

[0009] Table 1 Structure of each impurity

[0010]

[0011]

[0012] The specific steps of the detection of related substances of Flurellana are as follows:

[0013] (1) Preparation of test solution: Take an appropriate amount of flurellanil, weigh accurately, and dissolve in acetonitrile to prepare the test solution;

[0014] (2) Preparation of system suitability solution: Take appropriate amount of crude flurellanine and various impurity reference substances, add acetonitrile to dissolve, and obtain system suitability solution;

[0015] (3) Use high performance liquid chromatography to detect the test solution and system suitability solution.

[0016] The chromatographic conditions for the high performance liquid chromatography detection in step (3) are as follows:

[0017] Chromatographic column: octadecylsilane bonded silica gel as filler;

[0018] Mobile phase: It consists of mobile phase A, mobile phase B and mobile phase C; mobile phase A is formic acid aqueous solution; mobile phase B is acetonitrile; mobile phase C is methanol;

[0019] UV detector wavelength: 250nm~280nm;

[0020] Column temperature: 30℃~45℃;

[0021] Mobile phase flow rate: 0.5~1.5mL / min;

[0022] Injection volume: 5~20μL.

[0023] The initial volume ratio of acetonitrile to methanol in the mobile phase was 3:1 to 5:1.

[0024] The elution method of the high performance liquid chromatography detection in step (3) is gradient elution, and the elution program is as shown in Table 2:

[0025] Table 2 Gradient elution program

[0026]

[0027]

[0028] Preferably, the elution method of the high performance liquid chromatography detection in step (3) is gradient elution, and the elution program is as shown in Table 3:

[0029] Table 3 Gradient elution program

[0030] Time (minutes) Mobile phase A (%) Mobile phase B (%) Mobile phase C (%) 0 40 48 12 14 35 52 13 30 25 60 15 34 0 100 0 38 0 100 0 38.1 40 48 12 48 40 48 12

[0031] In the system suitability solution chromatogram obtained by the chromatographic conditions described in step (3), the order of peaks is impurity A, flurella, impurity E, impurity C, impurity B, impurity D, impurity F, and the separation between the chromatographic peaks of each component should be greater than 2.0.

[0032] Preferably, the concentration of formic acid in the formic acid aqueous solution is 0.05% to 0.15%, more preferably 0.1%.

[0033] Preferably, the chromatographic column is a reverse phase chromatographic column selected from SHIMADZU Shim-packGIST, Agilent, and Alphasil.

[0034] Preferably, the specification of the chromatographic column is 4.6 mm×250 mm, 5 μm, or other chromatographic columns with equivalent performance.

[0035] Preferably, the column temperature of the method is 35°C and the detection wavelength is 265nm;

[0036] Preferably, the flow rate of the mobile phase is 1.0 ml / min.

[0037] Preferably, the injection volume is 10 μL.

[0038] Preferably, the initial volume ratio of acetonitrile to methanol is 4:1.

[0039] The system suitability solution in step (2) contains 0.5-2.0 mg of fluorouracil per 1 ml, and 1-5 μg of each of impurities A, B, C, D and F.

[0040] Preferably, the system suitability solution in step (2) contains 1.0 mg of fluorouracil and 2 μg of each impurity per 1 ml.

[0041] Another aspect of the present invention provides an application of a method for detecting flurellan-related substances in quality control of flurellan raw materials or preparations.

[0042] The beneficial effects of the present invention are as follows:

[0043] 1. The mobile phase uses formic acid aqueous solution. Formic acid has a modifier effect. When analyzing acidic and alkaline substances by HPLC, it can reduce secondary retention, reduce peak tailing, improve the separation between adjacent chromatographic peaks, and improve integration accuracy.

[0044] 2. Impurity E and flurana have similar structures and small polarity differences, making them difficult to separate. The mobile phase gradient and formic acid aqueous solution of the present invention can improve the peak shape of the main peak, effectively separate impurity E and flurana from each other, and improve the accuracy of quantitative detection.

[0045] 3. Impurity C, impurity B and impurity D have similar structures, small polarity differences, and close elution times on the chromatographic column, making them difficult to separate. The mobile phase gradient, flow rate and column temperature of the present invention can increase the differences in the retention capacity of each impurity on the chromatographic column, thereby effectively separating impurities C, impurity B and impurity D from each other and improving the accuracy of quantitative detection.

[0046] 4. Select the SHIMADZU Shim-packGIST C18 (4.6mm×250mm, 5μm) chromatographic column. This particle size chromatographic column has a strong separation effect, good separation of the sample components, and good chromatographic column stability.

[0047] 5. Selecting 265nm as the detection wavelength can take into account both fluorouracil and impurity C, which have a large difference in absorption wavelengths, and the detection limit and quantification limit of each component meet the requirements. At this wavelength, each impurity component has high sensitivity and can be accurately quantified.

[0048] 6. In the system suitability solution chromatogram obtained according to the chromatographic conditions, the order of peaks is impurity A, flurellane, impurity E, impurity C, impurity B, impurity D, impurity F, and the separation between the chromatographic peaks of each component is greater than 2.0.

[0049] 7. Through methodological verification, the method of the present invention can effectively separate flurana from its impurities and between impurities, accurately determine the content of flurana and each impurity, and the detection method is simple and reliable, with high sensitivity, strong specificity, and good precision and repeatability. It provides a reliable and effective detection method for quality monitoring of related impurities in the industrial production process of flurana. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 HPLC spectrum of the blank solution in Example 1;

[0051] Figure 2 The HPLC spectrum of the test solution in Example 1;

[0052] Figure 3 The HPLC spectrum of the system suitability solution in Example 1;

[0053] Figure 4 The HPLC spectrum of the acid destruction solution in Example 1;

[0054] Figure 5The HPLC spectrum of the alkali destruction solution in Example 1;

[0055] Figure 6 The HPLC spectrum of the oxidation destruction solution in Example 1;

[0056] Figure 7 This is the HPLC spectrum of the high temperature solid destruction solution in Example 1;

[0057] Figure 8 HPLC spectrum of the high temperature liquid destruction solution in Example 1;

[0058] Fig. 9 HPLC spectrum of the light-irradiated solid destruction solution in Example 1;

[0059] Fig.10 HPLC spectrum of the light-irradiated liquid destruction solution in Example 1;

[0060] Fig.11 The HPLC spectrum of the system suitability solution in Example 1 with the flow rate changed to 0.8 ml / min;

[0061] Fig.12 The flow rate was changed to 1.2 ml / min for Example 1. HPLC spectrum of system suitability solution;

[0062] Fig.13 The column temperature was changed to 30°C for Example 1. System suitability solution HPLC spectrum;

[0063] Fig.14 The column temperature was changed to 40°C for Example 1. System suitability solution HPLC spectrum;

[0064] Fig.15 The HPLC spectrum of the system suitability solution was modified from Example 1 to have a formic acid content of 0.05%;

[0065] Fig.16 The HPLC spectrum of the system suitability solution was modified from Example 1 to have a formic acid content of 0.15%;

[0066] Fig.17 The chromatographic conditions of Comparative Example 1 and the HPLC profile of the system suitability solution;

[0067] Fig.18 The chromatographic conditions of Comparative Example 2, the system suitability solution HPLC spectrum;

[0068] Fig.19 The chromatographic conditions of Comparative Example 3, the system suitability solution HPLC spectrum;

[0069] Fig. 20 The chromatographic conditions of Comparative Example 4, the system suitability solution HPLC spectrum;

[0070] Fig.21 The chromatographic conditions of Comparative Example 5, the system suitability solution HPLC spectrum;

[0071] Fig. 22 The chromatographic conditions of Comparative Example 6, the system suitability solution HPLC spectrum;

[0072] Fig.23 The chromatographic conditions of Comparative Example 7, the system suitability solution HPLC spectrum;

[0073] Fig.24 The chromatographic conditions of Comparative Example 8 and the system suitability solution HPLC spectrum. DETAILED DESCRIPTION

[0074] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention is described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. 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.

[0075] Example 1 Analytical Methodology Verification

[0076] 1. Method specificity investigation:

[0077] Blank solution: Use the diluent acetonitrile as the blank solution.

[0078] System suitability solution: Weigh appropriate amounts of crude fluororana (containing impurity E), impurity A, impurity B, impurity C, impurity D and impurity F reference substances, dissolve in acetonitrile and quantitatively dilute to prepare a mixed solution containing approximately 1.0 mg of fluororana and approximately 2 ug each of impurities A, B, C, D and F per ml, as the system suitability solution.

[0079] Test solution: Take about 0.1 g of fluorouracil raw material, weigh accurately, place in a 100 ml volumetric flask, add appropriate amount of acetonitrile to dissolve and dilute to the scale, and shake well.

[0080] Accurately measure 10 μL of blank solution, test solution, and system suitability solution, and inject them into the reversed-phase high-performance liquid chromatography instrument respectively, and record the chromatogram ( Figure 1-3 ). Impurity A, flurana, impurity E, impurity C, impurity B, impurity D, and impurity F are eluted in sequence, and the separation degree between impurities and flurana should meet the requirements.

[0081] The determination was carried out according to the specifications of high performance liquid chromatography recorded in Appendix 0512 of the Chinese Pharmacopoeia of Veterinary Medicine.

[0082] Reversed-phase HPLC conditions:

[0083] Chromatographic column: Octadecylsilane bonded silica gel as filler, model SHIMADZU Shim-packGISTC18 (4.6×250mm, 5μm), Shimadzu;

[0084] Testing conditions:

[0085] The UV detector wavelength is 265nm;

[0086] The column temperature was 35°C;

[0087] The mobile phase flow rate was 1.0 mL / min;

[0088] The injection volume was 10 μL.

[0089] Mobile phase: Mobile phase A is 0.1% formic acid in water; Mobile phase B is acetonitrile; Mobile phase C is methanol;

[0090] The gradient elution program is shown in Table 4:

[0091] Table 4 Gradient elution program

[0092] Time (minutes) Mobile phase A (%) Mobile phase B (%) Mobile phase C (%) 0 40 48 12 14 35 52 13 30 25 60 15 34 0 100 0 38 0 100 0 38.1 40 48 12 48 40 48 12

[0093] Conclusion: Figure 1-3 It can be seen that under the chromatographic conditions of this detection method, each impurity can be well separated from the main peak, and the blank solvent has no interference.

[0094] 2. Specificity inspection-destructive test

[0095] Strong acid destruction solution: Take about 0.1g of the test sample, weigh it accurately, put it in a 100ml volumetric flask, add 5ml of acetonitrile to dissolve it, then add 1.0ml of 5.0mol / L hydrochloric acid solution, shake it, and leave it at room temperature for 4 days. Adjust the pH value to neutral with 1.0mol / L sodium hydroxide solution, dilute it to the scale with acetonitrile, shake well, and use it as the acid destruction test solution.

[0096] Take the above acid-destroyed test solution, inject it into the liquid chromatograph, and record the chromatogram. The results are detailed in the instructions. Figure 4 .Depend on Figure 4 It can be seen that in the acid-destroyed solution, each impurity peak can be effectively separated from the main peak, and the main degraded impurity peaks are impurity A and impurity B.

[0097] Strong alkali destruction solution: Take about 0.1g of the test sample, weigh it accurately, put it in a 100ml volumetric flask, add 5ml of acetonitrile to dissolve it, then add 3.0ml of 1.0mol / L sodium hydroxide solution, shake it, and leave it at room temperature for 4 days. Adjust the pH value to neutral with 1.0mol / L hydrochloric acid solution, dilute it to the scale with acetonitrile, shake well, and use it as the alkali destruction test solution.

[0098] Take the above alkali-destroyed test solution, inject it into the liquid chromatograph, and record the chromatogram. The results are detailed in the instructions. Figure 5 .Depend on Figure 5 It can be seen that in the alkaline destruction solution, each impurity peak can be effectively separated from the main peak, and the main degraded impurity peak is impurity A.

[0099] Oxidative destruction solution: Take about 0.1g of the test sample, weigh it accurately, put it in a 100ml volumetric flask, add 5ml of acetonitrile to dissolve it, add 3.0ml of 3% hydrogen peroxide solution, shake it, leave it at room temperature for 4 days, dilute it to the scale with acetonitrile, shake well, and use it as the oxidative destruction test sample solution.

[0100] Take the above oxidatively damaged test solution, inject it into the liquid chromatograph, and record the chromatogram. The results are detailed in the instructions. Figure 6 .Depend on Figure 6 It can be seen that in the oxidative destruction solution, fluororana is relatively stable, no degradation impurities are produced, and each impurity peak can be effectively separated from the main peak.

[0101] High temperature (solid) destruction solution: Take an appropriate amount of the test sample, place it in a weighing bottle, open it, and place it in a 60°C oven for 10 days. Take about 0.1g of the test sample, accurately weigh it, place it in a 100ml volumetric flask, dissolve it in acetonitrile and dilute it to the scale, mix it well, and use it as the high temperature (solid) destruction test sample solution.

[0102] Take the above high temperature (solid) destroyed test sample solution, inject it into the liquid chromatograph, record the chromatogram, and the results are detailed in the instructions. Figure 7 .Depend on Figure 7 It can be seen that in the high temperature (solid) destruction solution, fluororana is relatively stable, no degradation impurities are produced, and each impurity peak can be effectively separated from the main peak.

[0103] High temperature (liquid) destruction solution: Take about 0.1g of the test sample, weigh it accurately, put it in a 100ml volumetric flask, add 5ml of acetonitrile to dissolve it, seal it, put it in a 60℃ oven for 10 days, take it out, dilute it to the scale with acetonitrile, shake it well, and use it as the high temperature (liquid) destruction test sample solution.

[0104] Take the above high temperature (liquid) destroyed test solution, inject it into the liquid chromatograph, record the chromatogram, and the results are detailed in the instructions. Figure 8 .Depend on Figure 8 It can be seen that in the high temperature (liquid) destruction solution, fluororana is relatively stable, no degradation impurities are produced, and each impurity peak can be effectively separated from the main peak.

[0105] Light (solid) destruction solution: Take an appropriate amount of the test sample, place it in a weighing bottle, open it, and place it in a light box at 5000lx, with near-ultraviolet energy not less than 83μw / cm 2After 10 days of irradiation, the light intensity should be 1.2×10 6 lx·h, near-ultraviolet lamp energy is not less than 200W·h / m 2 Take about 0.1g of the sample, weigh accurately, put it in a 100ml volumetric flask, dissolve it in acetonitrile and dilute it to the mark, shake well, and use it as the light (solid) destruction sample solution.

[0106] Take the above light-damaged (solid) test solution, inject it into the liquid chromatograph, and record the chromatogram. The results are detailed in the instructions. Fig. 9 .Depend on Fig. 9 It can be seen that in the light-irradiated (solid) destruction solution, fluororana is relatively stable, no degradation impurities are produced, and each impurity peak can be effectively separated from the main peak.

[0107] Light (liquid) destruction solution: Take about 0.1g of the test sample, weigh it accurately, put it in a 100ml volumetric flask, add 5ml of acetonitrile to dissolve it, shake it well, seal it, and place it in a light box at 5000lx, with near-ultraviolet energy not less than 83μw / cm 2 Light intensity illumination for 10 days (10 days of illumination meets the total illumination of 1.2×10 6 lx·h, near-ultraviolet lamp energy is not less than 200W·h / m 2 Dilute to scale with acetonitrile, shake well, and use it as the light (liquid) destruction test solution.

[0108] Take the above light-damaged (liquid) test solution, inject it into the liquid chromatograph, and record the chromatogram. The results are detailed in the instructions. Fig.10 .Depend on Fig.10 It can be seen that in the light-irradiated (liquid) destruction solution, flurellanine is relatively stable, no degradation impurities are produced, and each impurity peak can be effectively separated from the main peak.

[0109] 3. Durability inspection

[0110] The durability test was conducted using a system suitability solution. The system suitability solution was prepared according to the method of Example 1 and injected into the liquid chromatograph. According to the chromatographic conditions of Example 1, the parameters in the liquid chromatography system were appropriately adjusted to examine the separation of the components after the system conditions were changed.

[0111] (1) Chromatographic conditions of Example 1, the spectrum of the system suitability solution is as follows Figure 3 shown.

[0112] It can be seen from the figure that impurity peaks and impurity peaks can be effectively separated from the main peak, and the separation degree is greater than 2.0. The separation degree between impurity E and flurellane is 2.48, and the separation degrees between impurity B and impurity C and impurity D are 2.51 and 2.63 respectively.

[0113] (2) Change the flow rate to 0.8 ml / min. The spectrum of the system suitability solution is as follows: Fig.11 shown.

[0114] It can be seen from the figure that impurity peaks and impurity peaks, as well as impurity peaks and main peaks can be effectively separated, and the separation degrees are all greater than 2.0. The separation degree between impurity E and flurellane is 2.57, and the separation degrees between impurity B and impurity C and impurity D are 2.20 and 2.70 respectively.

[0115] (3) Change the flow rate to 1.2 ml / min. The spectrum of the system suitability solution is as follows: Fig.12 shown.

[0116] It can be seen from the figure that impurity peaks and impurity peaks, impurity peaks and main peaks can be effectively separated, and the separation degree is greater than 2.0. The separation degree between impurity E and flurellane is 2.38, and the separation degrees between impurity B and impurity C and impurity D are 3.24 and 2.24 respectively.

[0117] (4) Change the column temperature to 30°C. The spectrum of the system suitability solution is as follows: Fig.13 shown.

[0118] It can be seen from the figure that impurity peaks and impurity peaks, impurity peaks and main peaks can be effectively separated, and the separation degree is greater than 2.0. The separation degree between impurity E and flurellane is 2.29, and the separation degrees between impurity B and impurity C and impurity D are 2.58 and 2.71 respectively.

[0119] (5) Change the column temperature to 40°C. The spectrum of the system suitability solution is as follows: Fig.14 shown.

[0120] It can be seen from the figure that impurity peaks and impurity peaks can be effectively separated from the main peak, and the separation degree is greater than 2.0. The separation degree between impurity E and flurellane is 2.11, and the separation degrees between impurity B and impurity C and impurity D are 2.02 and 3.09 respectively.

[0121] (6) Change the formic acid content to 0.05%. The spectrum of the system suitability solution is as follows: Fig.15 shown.

[0122] It can be seen from the figure that impurity peaks and impurity peaks, impurity peaks and main peaks can be effectively separated, and the separation degree is greater than 2.0. The separation degree between impurity E and flurellane is 2.46, and the separation degrees between impurity B and impurity C and impurity D are 2.53 and 2.59 respectively.

[0123] (7) Change the formic acid content to 0.15%. The spectrum of the system suitability solution is as follows: Fig.16 shown.

[0124] It can be seen from the figure that impurity peaks and impurity peaks, as well as impurity peaks and main peaks can be effectively separated, and the separation degrees are all greater than 2.0. The separation degree between impurity E and flurellane is 2.53, and the separation degrees between impurity B and impurity C and impurity D are 2.58 and 2.53 respectively.

[0125] Conclusion: The above experiments show that this method has good durability.

[0126] Example 2 Determination of detection limit and quantification limit

[0127] Accurately measure an appropriate amount of each impurity reference stock solution and flurana reference solution, place them in a volumetric flask, quantitatively dilute them step by step, and measure them under the established chromatographic conditions. The concentration of each tested solution when S / N≈3 is taken as the detection limit, and the concentration of each tested solution when S / N≈10 is taken as the quantitative limit.

[0128] Table 5 Detection limit and quantification limit

[0129]

[0130]

[0131] Example 3 Stability Study

[0132] The test solution was sampled at 0, 8, 12, 24 and 48 hours. The individual impurity content and the total impurity content of the test solution were consistent, indicating that the solution was stable within 48 hours. The results are shown in the table below.

[0133] Table 6 Solution stability test results

[0134] Setting time (h) Impurity E content % Other single maximum impurity % Total impurities% 0 0.06 ND 0.06 8 0.06 ND 0.06 12 0.06 ND 0.06 24 0.06 ND 0.06 48 0.06 ND 0.06

[0135] Example 4 Specificity comparison test

[0136] Comparative Example 1

[0137] Chromatographic conditions:

[0138] Chromatographic column: SHIMADZU Shim-packGIST C18 (4.6×250mm, 5μm), Shimadzu;

[0139] Mobile phase: Mobile phase A is aqueous phosphoric acid; mobile phase B is acetonitrile:methanol (50:50);

[0140] The gradient elution program was:

[0141] Table 7 Gradient elution program

[0142]

[0143]

[0144] Detection wavelength: 265nm;

[0145] Flow rate: 1.0ml / min;

[0146] Column temperature: 35°C;

[0147] Injection volume: 10 μL.

[0148] In the system suitability solution, impurities B and C cannot be separated, and impurity E is completely contained in the main peak. Fig.17 shown.

[0149] Comparative Example 2

[0150] Chromatographic conditions:

[0151] Chromatographic column: SHIMADZU Shim-packGIST C18 (4.6×250mm, 5μm), Shimadzu;

[0152] Mobile phase: Mobile phase A is trifluoroacetic acid aqueous solution; Mobile phase B is acetonitrile;

[0153] The gradient elution program was:

[0154] Table 8 Gradient elution program

[0155] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 85 15 5 85 15 35 65 35 40 20 80 40.1 85 15 45 85 15

[0156] Detection wavelength: 265nm;

[0157] Flow rate: 1.0ml / min;

[0158] Column temperature: 35°C;

[0159] Injection volume: 10 μL.

[0160] Impurities B and D in the system suitability solution cannot be separated, and impurity E is completely contained in the main peak. Fig.18 shown.

[0161] Comparative Example 3

[0162] Chromatographic conditions:

[0163] Chromatographic column: SHIMADZU Shim-packGIST C18 (4.6×250mm, 5μm), Shimadzu;

[0164] Mobile phase: Mobile phase A is water; Mobile phase B is acetonitrile; Mobile phase C is methanol;

[0165] The gradient elution program was:

[0166] Table 9 Gradient elution program

[0167] Time (minutes) Mobile phase A (%) Mobile phase B (%) Mobile phase C (%) 0 40 48 12 14 35 52 13 30 25 60 15 34 0 100 0 38 0 100 0 38.1 40 48 12 48 40 48 12

[0168] Detection wavelength: 265nm;

[0169] Flow rate: 1.0ml / min;

[0170] Column temperature: 35°C;

[0171] Injection volume: 10 μL.

[0172] The separation between impurities B and D in the system suitability solution is only 1.48, and the peak shape of impurity D is abnormal. Fig.19 shown.

[0173] Comparative Example 4

[0174] Chromatographic conditions:

[0175] Chromatographic column: SHIMADZU Shim-packGIST C18 (4.6×250mm, 5μm), Shimadzu;

[0176] Mobile phase: Mobile phase A is 0.1% formic acid in water; Mobile phase B is acetonitrile; Mobile phase C is methanol;

[0177] The gradient elution program was:

[0178] Table 10 Gradient elution program

[0179] Time (minutes) Mobile phase A (%) Mobile phase B (%) Mobile phase C (%) 0 40 48 12 14 35 52 13 30 25 60 15 34 0 100 0 38 0 100 0 38.1 40 48 12 48 40 48 12

[0180] Detection wavelength: 265nm;

[0181] Flow rate: 1.8ml / min;

[0182] Column temperature: 35°C;

[0183] Injection volume: 10 μL.

[0184] Impurity E in the system suitability solution is not separated from the main peak. Fig. 20 shown.

[0185] Comparative Example 5

[0186] Chromatographic conditions:

[0187] Chromatographic column: SHIMADZU Shim-packGIST C18 (4.6×250mm, 5μm), Shimadzu;

[0188] Mobile phase: Mobile phase A is 0.1% formic acid in water; Mobile phase B is acetonitrile;

[0189] The gradient elution program was:

[0190] Table 11 Gradient elution program

[0191] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 40 60 14 35 65 30 25 75 34 0 100 38 0 100 38.1 40 60 48 40 60

[0192] Detection wavelength: 265nm;

[0193] Flow rate: 1.0ml / min;

[0194] Column temperature: 35°C;

[0195] Injection volume: 10 μL.

[0196] The separation degree of impurity B and impurity D in the system suitability solution is only 1.23, and the main peak and each impurity peak are tailing. Fig.21 shown.

[0197] Comparative Example 6

[0198] Chromatographic conditions:

[0199] Chromatographic column: SHIMADZU Shim-packGIST C18 (4.6×250mm, 5μm), Shimadzu;

[0200] Mobile phase: Mobile phase A is 0.1% formic acid in water; Mobile phase B is acetonitrile; Mobile phase C is methanol;

[0201] The gradient elution program was:

[0202] Table 12 Gradient elution program

[0203]

[0204]

[0205] Detection wavelength: 265nm;

[0206] Flow rate: 1.0ml / min;

[0207] Column temperature: 35°C;

[0208] Injection volume: 10 μL.

[0209] The separation between impurities B and D in the system suitability solution is only 1.48, and the peak shape of impurity D is abnormal. Fig. 22 shown.

[0210] Comparative Example 7

[0211] Chromatographic conditions:

[0212] Chromatographic column: SHIMADZU Shim-packGIST C18 (4.6×250mm, 5μm), Shimadzu;

[0213] Mobile phase: Mobile phase A is 0.1% formic acid in water; Mobile phase B is acetonitrile; Mobile phase C is methanol;

[0214] The gradient elution program was:

[0215] Table 13 Gradient elution program

[0216] Time (minutes) Mobile phase A (%) Mobile phase B (%) Mobile phase C (%) 0 30 56 14 30 25 60 15 34 0 100 0 38 0 100 0 38.1 30 56 14 48 30 56 14

[0217] Detection wavelength: 265nm;

[0218] Flow rate: 1.0ml / min;

[0219] Column temperature: 35°C;

[0220] Injection volume: 10 μL.

[0221] The separation between impurities B and C in the system suitability solution is only 1.33, and the separation between impurities B and D is only 0.98. Fig.23 shown.

[0222] Comparative Example 8

[0223] Chromatographic conditions:

[0224] Chromatographic column: SHIMADZU Shim-packGIST C18 (4.6×250mm, 5μm), Shimadzu;

[0225] Mobile phase: Mobile phase A is 0.1% formic acid in water; Mobile phase B is acetonitrile; Mobile phase C is methanol;

[0226] The gradient elution program was:

[0227] Table 14 Gradient elution program

[0228] Time (minutes) Mobile phase A (%) Mobile phase B (%) Mobile phase C (%) 0 60 30 10 30 20 60 20 34 0 100 0 38 0 100 0 38.1 60 30 10 48 60 30 10

[0229] Detection wavelength: 265nm;

[0230] Flow rate: 1.0ml / min;

[0231] Column temperature: 35°C;

[0232] Injection volume: 10 μL.

[0233] Impurities B and D in the system suitability solution are not separated, and the resolution is only 0.59. Fig.24 shown.

[0234] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting related substances of flurara sodium, characterized in that: High performance liquid chromatography was used for detection, and the specific chromatographic conditions were as follows: Chromatographic column: octadecylsilane bonded silica gel as filler; Mobile phase: Mobile phase A is formic acid aqueous solution; Mobile phase B is acetonitrile; Mobile phase C is methanol; The wavelength of UV detector is 250nm~280nm; Column temperature is 30℃~45℃; The flow rate of the mobile phase is 0.5-1.5 mL / min; The injection volume is 5 to 20 μL; Gradient elution was used.

2. The detection method according to claim 1, characterized in that: The gradient elution procedure is:

3. The detection method according to claim 2, characterized in that: The gradient elution procedure is:

4. The detection method according to claim 1, characterized in that: The sodium flurarafil related substance is one or more of impurity A, impurity B, impurity C, impurity D, impurity E, and impurity F, and its structure is as follows:

5. The detection method according to claim 1, characterized in that: The concentration of formic acid in the mobile phase A formic acid aqueous solution is 0.05-0.15%.

6. The detection method according to claim 5, characterized in that: The concentration of formic acid in the mobile phase A, the formic acid aqueous solution, is 0.1%.

7. The detection method according to claim 1, characterized in that: The chromatographic column model is one of SHIMADZU Shim-packGIST, Agilent, and Alphasil; the chromatographic column specifications are: inner diameter 4.6×250 mm, 5 μm.

8. The detection method according to claim 1, characterized in that: The wavelength of the UV detector is 265 nm; the column temperature is 35° C.; the flow rate of the mobile phase is 1.0 mL / min; and the injection volume is 10 μL.

9. The detection method according to claim 4, characterized in that: In the system suitability solution chromatogram obtained by testing under the chromatographic conditions, the order of peaks is impurity A, flurellane, impurity E, impurity C, impurity B, impurity D, impurity F, and the separation between the chromatographic peaks of each component should be greater than 2.

0.

10. Use of the method for detecting related substances of flurella sodium according to any one of claims 1 to 9 in the quality control of flurella sodium raw materials or preparations.

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