An analytical method for detecting related substances of fluticasone propionate intermediate thiocarboxylic acid

Abstract

The invention discloses an analytical method for detecting thiocarboxylic acid-related substances of fluticasone propionate intermediates, using high performance liquid chromatography, a chromatographic column with octylsilane bonded silica gel as a filler, 0.1% trifluoroacetic acid as a mobile phase A, and acetonitrile as a mobile phase B. The invention uses high performance liquid chromatography to achieve complete separation between a main component and its nine impurities, and the solvent and the main component have no interference with the inspection of each impurity. The method has good specificity, high accuracy, precision and sensitivity, simple sample preparation and analysis operations, and high detection efficiency.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to an analysis method for detecting thiocarboxylic acid-related substances of a fluticasone propionate intermediate. Background Art

[0002] Thiocarboxylic acid is an intermediate for preparing fluticasone propionate. Its synthetic route is as follows, and thiocarboxylic acid is intermediate IV in the synthetic route:

[0003]

[0004] The compound is prone to introduce 9 impurities during the preparation process; the structures of the thiocarboxylic acid and the 9 impurities are as follows:

[0005]

[0006]

[0007] Impurity H and Impurity I: Intermolecular condensation reactions may occur during the preparation of thiocarboxylic acids, generating impurities H and I, which may remain in thiocarboxylic acids and need to be studied. The generation pathways of impurities H and I are as follows:

[0008]

[0009] Impurity IV-3: Thiocarboxylic acid may degrade under high temperature conditions to produce impurity IV-3, which may remain in the thiocarboxylic acid and needs to be studied. The generation pathway of impurity IV-3 is as follows:

[0010]

[0011] Impurity B: Thiocarboxylic acid may be oxidized under alkaline conditions to produce impurity B, which may remain in the thiocarboxylic acid and needs to be studied. The generation pathway of impurity B is as follows:

[0012]

[0013] Flumethasone acid, 17-propionate, thiopropionate, impurity IV-1, impurity IV-2, impurity IV-3, impurity B, impurity H and impurity I all contain unsaturated ketones and halogenated alkanes genetic toxicity warning structures, and there is no clear toxicological data. This product has many impurities, and the impurities and the main components are similar in structure, making method development difficult.

[0014] At present, no relevant reports on the detection methods of thiocarboxylic acid-related substances have been retrieved. Summary of the invention

[0015] In order to fill the gap in the prior art, the present invention provides an analytical method for detecting thiocarboxylic acid-related substances of fluticasone propionate intermediates, which achieves complete separation and accurate detection between the main component and its nine impurities. The technical solution of the present invention is as follows:

[0016] The invention discloses an analytical method for detecting thiocarboxylic acid-related substances of fluticasone propionate intermediates. The method adopts high performance liquid chromatography, wherein the chromatographic column uses octylsilane bonded silica gel as a filler, trifluoroacetic acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, and the initial volume ratio of mobile phase A to mobile phase B is 52:48 to 58:42, preferably 55:45.

[0017] In the present invention, the impurities that may be contained in the thiocarboxylic acid include flumethasone acid, 17-propionate, impurity IV-1, impurity B, thiopropionate, impurity IV-3, impurity IV-2, impurity H, and impurity I. The content of each impurity may be different in different batches of samples. The effect of the present invention is that when the impurity exists, it can be effectively and accurately detected.

[0018] As an embodiment of the present invention, the flow rate of the mobile phase is 1.1-1.2 ml / min, preferably 1.1 ml / min.

[0019] As a preferred embodiment, the column temperature of the chromatographic column is 35-40°C, preferably 35°C, and the injector temperature is 4-8°C, preferably 5°C.

[0020] Preferably, the trifluoroacetic acid is a reagent with a higher purity, preferably 99.5% or above; the concentration range of the trifluoroacetic acid aqueous solution is 0.09% to 0.11%, preferably 0.1%.

[0021] Preferably, the gradient program of mobile phase elution is:

[0022] Within 0 to 15 minutes, the volume percentage of mobile phase A linearly changes from 55% to 48%, within 15 to 25 minutes, the volume percentage of mobile phase A remains unchanged at 48%, within 25 to 35 minutes, the volume percentage of mobile phase A linearly changes from 48% to 25% to 30%, within 35 to 50 minutes, the volume percentage of mobile phase A remains unchanged at 25% to 30%, and then within 1 minute, the volume percentage of mobile phase A linearly changes to 55%, and then equilibrates for 10 minutes.

[0023] As an embodiment of the present invention, within 0 to 15 minutes, the volume percentage of mobile phase A linearly changes from 55% to 48%, within 15 to 25 minutes, the volume percentage of mobile phase A remains unchanged at 48%, within 25 to 35 minutes, the volume percentage of mobile phase A linearly changes from 48% to 30%, within 35 to 50 minutes, the volume percentage of mobile phase A remains unchanged at 30%, within 50 to 51 minutes, the volume percentage of mobile phase A linearly changes from 30% to 55%, within 51 to 60 minutes, the volume percentage of mobile phase A remains unchanged at 55%, that is, the chromatographic column equilibrium time is reached after 50 minutes.

[0024] Preferably, the analytical method comprises chromatographic conditions:

[0025] Use octylsilane bonded silica gel as filler (Kromasil Classic C8, 4.6mm×250mm, 3.5μm or chromatographic column with equivalent performance is recommended); use 0.1% trifluoroacetic acid as mobile phase A and acetonitrile as mobile phase B, and perform linear gradient elution according to the table below; the flow rate is 1.1ml per minute; the column temperature is 35℃; the injector temperature is 5℃; the detector is a UV detector with a detection wavelength of 239nm; the injection volume is 10μl.

[0026] Preferably, the analysis method further comprises:

[0027] Preparation of test solution: Take the product and add acetonitrile to dissolve it by ultrasonic and dilute it with solvent to prepare a solution with a concentration of 0.4 mg / ml. The solvent is a water-acetonitrile solution with a volume ratio of 50:50.

[0028] Control solution: Accurately measure an appropriate amount of the test sample solution and quantitatively dilute it with solvent to make a solution with a concentration of 0.8 μg / ml.

[0029] Determination method: Accurately measure the test solution and the control solution, inject them into the liquid chromatograph respectively, and record the chromatogram; if the test sample contains impurities, the impurities will appear in the order of flumethasone acid, 17-propionic acid ester, impurity IV-1, impurity B, thiopropionic acid ester, impurity IV-3, impurity IV-2, impurity H, and impurity I; except for impurity IV-3, the principal component self-control method with correction factor is used for calculation, and other known impurities and other single impurities are calculated by the principal component self-control method without correction factor.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1. The present invention uses high performance liquid chromatography to achieve complete separation between the main component and its nine impurities, and the solvent and the main component have no interference with the inspection of each impurity. The method has good specificity, high accuracy, precision and sensitivity, simple sample preparation and analysis operations, and high detection efficiency.

[0032] 2. The study found that impurity I and impurity H cannot coexist, and through verification, it was found that the two do not affect each other in the test solution. This analytical method can ensure the stability of each impurity and ensure accurate detection and quantification. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0034] Figure 1 It is a chromatogram of the specific test in Example 2; wherein, the spectrum is blank solvent, spiked test solution, impurity I reference solution and 8 impurity mixed solutions from bottom to top, wherein the peak order in the 8 impurity mixed solutions is 1 is flumethasone acid, 2 is 17-propionic acid ester, 3 is impurity IV-1, 4 is impurity B, 5 is thiopropionic acid ester, 6 is impurity IV-3, 7 is impurity IV-2, 8 is impurity H, and 9 is impurity I.

[0035] Figure 2 This is a chromatogram of the impurity localization solution of the specificity test in Example 2; wherein, the spectrum from bottom to top is flumethasone acid, impurity IV-3, 17-propionic acid ester, impurity IV-1, impurity B, thiopropionic acid ester, impurity IV-2, impurity H, and impurity I.

[0036] Figure 3 The chromatograms of two time points under different elution gradient conditions of Comparative Example 1 are shown, wherein the spectra are 5h and 0h from bottom to top.

[0037] Figure 4 This is the chromatogram of Comparative Example 2 in which low-purity trifluoroacetic acid was used as the mobile phase for injection. DETAILED DESCRIPTION

[0038] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0039] It should be noted that the terms used herein are for describing specific embodiments only and are not intended to be limiting of exemplary embodiments according to the present invention.

[0040] Example 1, an analytical method for detecting related substances of fluticasone propionate intermediate thiocarboxylic acid

[0041] Chromatographic conditions: UV detector; octylsilane bonded silica gel as filler (Kromasil Classic C8, 4.6 mm × 250 mm, 3.5 μm); 0.1% trifluoroacetic acid as mobile phase A, acetonitrile as mobile phase B, linear gradient elution according to the following table; flow rate 1.2 ml per minute; column temperature 35°C; injector temperature 5°C; detection wavelength 239 nm; injection volume 10 μl. Trifluoroacetic acid purity 99.5%.

[0042]

[0043] Sample preparation:

[0044] Solvent: water-acetonitrile (50:50).

[0045] Test solution: Take about 8 mg of the product, place it in a 20 ml volumetric flask, add 2 ml of acetonitrile and dissolve it by ultrasonication, dilute to the scale with solvent, and shake well.

[0046] Control solution: Accurately measure an appropriate amount of the test sample solution and quantitatively dilute it with solvent to make a solution containing approximately 0.8 μg per 1 ml.

[0047] Sensitivity solution: Accurately measure 5 ml of the control solution, place it in a 20 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0048] Determination method: Accurately measure the test solution and the control solution, inject them into the liquid chromatograph respectively, and record the chromatogram; if the test sample contains impurities, the impurities will appear in the order of flumethasone acid, 17-propionic acid ester, impurity IV-1, impurity B, thiopropionic acid ester, impurity IV-3, impurity IV-2, impurity H, and impurity I; except for impurity IV-3, the principal component self-control method with correction factor is used for calculation, and other known impurities and other single impurities are calculated by the principal component self-control method without correction factor.

[0049] Calculation formula for each impurity:

[0050]

[0051] Where: A 样 is the peak area of ​​the known impurity in the chromatogram of the test solution;

[0052] A 对 is the peak area of ​​the main peak in the chromatogram of the reference solution;

[0053] f: Correction factor for known impurities. The correction factor for impurity IV-3 is 0.64.

[0054]

[0055] Where: A 样is the peak area of ​​other individual impurities in the chromatogram of the test solution;

[0056] A 对 is the peak area of ​​the main peak in the chromatogram of the reference solution;

[0057] Total impurities (%) = ∑ known impurities (%) + ∑ other individual impurities (%)

[0058] The test results of three batches of samples are shown in Table 1.

[0059] Table 1 Sample test results

[0060]

[0061] Example 2: Methodology Validation Test

[0062] 1. System suitability and specificity test

[0063] Solvent: Water-Acetonitrile (50:50)

[0064] Stock solutions of each impurity reference substance: Take an appropriate amount of each impurity reference substance, dissolve it in solvent or acetonitrile according to its solubility and prepare a stock solution of the reference substance with the following concentrations:

[0065] Flumethasone acid reference stock solution: 80 μg / ml.

[0066] 17-Propionate reference stock solution: 200 μg / ml.

[0067] Impurity IV-1 reference substance stock solution: 80 μg / ml.

[0068] Impurity B reference stock solution: 200 μg / ml.

[0069] Thiopropionate reference stock solution: 80 μg / ml.

[0070] Impurity IV-3 reference substance stock solution: 400 μg / ml.

[0071] Impurity IV-2 reference substance stock solution: 80 μg / ml.

[0072] Impurity H reference stock solution: 120μg / ml.

[0073] Impurity I reference substance stock solution: 200 μg / ml.

[0074] (Note: The detection amounts of 17-propionate, impurity IV-1, impurity IV-3 and impurity I in the validation samples were all approximately 50% of the limit, and the detection amount of impurity H was approximately 30% of the limit. No more impurities were added to the spiked test solution.)

[0075] Test solution: Use the test results of the test solution at 0h (TS-0h) under "4 Solution stability test".

[0076] Spiked test solution: Use the test result of spiked test solution 0h (MIX-0h) under "4 Solution stability test".

[0077] Control solution: Use the test results of the control solution 0h (RS-0h) under "4 Solution stability test".

[0078] Impurity localization solutions: Measure 1 ml of the impurity reference stock solution and place it in 9 different 100 ml volumetric flasks, dilute to the scale with solvent, shake well, and use them as the localization solutions of flumethasone acid, 17-propionic ester, impurity IV-1, impurity B, thiopropionic ester, impurity IV-3, impurity IV-2, impurity H and impurity I respectively.

[0079] Sensitivity solution: Accurately measure 5 ml of the control solution, place it in a 20 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0080] Precisely measure the solvent, sensitivity solution and control solution, and inject the control solution 5 times continuously, and record the chromatogram. Precisely measure each impurity location solution, test solution and spiked test solution, and inject them into the liquid chromatograph respectively, and record the chromatogram. The system suitability test results are shown in Table 2, and the chromatograms are shown in Figure 1 The results of the impurity location specificity test are shown in Table 3, and the impurity location chromatogram is shown in Figure 2 .

[0081] Table 2 System suitability test results

[0082]

[0083] Verification results: The control solution was injected 5 times in a row, the RSD of the retention time of the main peak of thiocarboxylic acid was 0.008%, less than 1.0%, the RSD of the main peak area of ​​thiocarboxylic acid was 0.8%, less than 5.0%, the theoretical plate number calculated by the thiocarboxylic acid peak was above 70,000, greater than 5,000, the signal-to-noise ratio S / N of the peak height was above 500, greater than 100, and the tailing factor was 1.0, less than 1.5. In the sensitivity solution chromatogram, the signal-to-noise ratio S / N of the main component peak height was 167.4, greater than 10, indicating that the system has good applicability.

[0084] Table 3 Specificity test results

[0085]

[0086] Verification results: The blank solvent did not interfere with the detection of impurities, and the separation between the peaks of each component was greater than 1.5, indicating that the method had good specificity.

[0087] 2. Linearity and range test

[0088] Solvent: Water-Acetonitrile (50:50)

[0089] Stock solutions of each impurity reference substance: Prepare the stock solutions of each impurity reference substance according to the method under "1 System suitability and specificity test".

[0090] Take an appropriate amount of thiocarboxylic acid reference substance, add acetonitrile to dissolve by ultrasonication and dilute to prepare the reference substance stock solution of the following concentration:

[0091] Thiocarboxylic acid reference substance stock solution①: 400μg / ml.

[0092] Thiocarboxylic acid reference substance stock solution②: 200μg / ml.

[0093] Thiocarboxylic acid reference substance stock solution ③: 80μg / ml.

[0094] Linear mixed stock solution ①: Accurately measure 5 ml each of flumethasone acid reference stock solution, 17-propionate reference stock solution, impurity IV-1 reference stock solution, impurity B reference stock solution, thiopropionate reference stock solution, impurity IV-2 reference stock solution, impurity H reference stock solution and thiocarboxylic acid reference stock solution ③, place in the same 50 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0095] Linear stock solution ②-1: Accurately measure 5 ml of impurity I reference stock solution, place it in a 50 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0096] Linear stock solution ②-2: Accurately measure 5 ml of thiocarboxylic acid reference stock solution ②, place in a 50 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0097] Linear mixed stock solution ③: Accurately measure 5 ml of impurity IV-3 reference stock solution and 5 ml of thiocarboxylic acid reference stock solution ①, place in a 50 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0098] Linear solution ①: Accurately measure 1.0ml, 3.0ml, 5.0ml, 8.0ml and 12.0ml of the linear mixed stock solution ① respectively, place them in different 50ml volumetric flasks, dilute to the scale with solvent, shake well, and use them as 20%, 60%, 100%, 160%, 240% linear solutions respectively (as the 2nd to 6th points of the linear test respectively).

[0099] Linear solution ②: Accurately measure 1.0ml, 3.0ml, 5.0ml, 8.0ml and 12.0ml of linear stock solution ②-1 and linear stock solution ②-2 respectively, place them in different 50ml volumetric flasks, dilute to the scale with solvent, shake well, and use them as 20%, 60%, 100%, 160%, 240% linear solutions (as the 2nd to 6th points of the linear test respectively).

[0100] Linear solution ③: Accurately measure 1.0ml, 3.0ml, 5.0ml, 8.0ml and 12.0ml of linear mixed stock solution ③ respectively, place them in different 50ml volumetric flasks, dilute to the scale with solvent, shake well, and use them as 20%, 60%, 100%, 160%, 240% linear solutions respectively (as the 2nd to 6th points of the linear test respectively).

[0101] Accurately measure an appropriate amount of each impurity reference stock solution and thiocarboxylic acid reference stock solution, and quantitatively dilute them with a solvent to prepare a solution with a signal-to-noise ratio of about 10 for the peak height of each component, which is used as the quantitative limit solution. The quantitative limit solution is used as the first linear point.

[0102] Accurately measure the above linear solutions, inject them into the liquid chromatograph respectively, and record the chromatogram. Perform linear regression with the concentration C (μg / ml) of each component as the abscissa and the peak area A of each component peak as the ordinate. Calculate the correction factor of each impurity by the ratio of the slope of the linear relationship curve of thiocarboxylic acid to the slope of the linear relationship curve of each impurity.

[0103] Verification results: The linear equations of each component peak (concentration range, linear correlation coefficient r, the absolute value of the intercept is equivalent to the percentage (%) of the 100% response value) are:

[0104] Thiocarboxylic acid: A = 20802.0081C-494.6223 (0.031 μg / ml-1.910 μg / ml, 0.9999, 3.063), flumethasone acid: A = 22721.6269C-99.3965 (0.008 μg / ml-1.934 μg / ml, 1.0000, 0.550), 17-propionic acid: A = 20472.6671C-82.2134 (0.019 μg / ml-4.567 μg / ml, 1.0000, 0.201), impurity IV-1: A = 2371 4.4850C-244.5575 (0.016μg / ml~1.897μg / ml, 1.0000, 1.306), impurity B: A=20674.1709C-242.6741 (0.020μg / ml~4.773μg / ml, 1.0000, 0.590), thiopropionate: A=20424.0714C-13.3898 (0.016μg / ml~1.959μg / ml, 1.0000, 0.082), impurity H: A=22380.6481C+16.3503

[0105] (0.024μg / ml~2.849μg / ml, 1.0000, 0.061), impurity IV-2: A=18778.6678C-946.3693(0.078μg / ml~1.876μg / ml, 1.0000, 6.723), impurity I: A=23126.4199C-122.9490

[0106] (0.019 μg / ml~4.675 μg / ml, 1.0000, 0.267), impurity IV-3: A=33928.4535C+28.1419

[0107] (0.162μg / ml~8.937μg / ml, 1.0000, 0.021), the linear correlation coefficient r was greater than 0.99, and the absolute value of the intercept was equivalent to the percentage of 100% response value was no more than 25%, indicating that the method had a good linear relationship.

[0108] Except for the correction factor f=0.64 of impurity IV-3, the impurities are quantitatively calculated by the main component self-control method with correction factors. The correction factors of other known impurities (correction factor f=0.92 for flumethasone acid, correction factor f=0.88 for impurity IV-1, correction factor f=1.02 for thiopropionate, correction factor f=1.11 for impurity IV-2, correction factor f=0.94 for impurity H, correction factor f=1.02 for 17-propionate, correction factor f=1.01 for impurity B, correction factor f=0.91 for impurity I) are all within the range of 0.8 to 1.1, and the impurities can be quantitatively calculated by the main component self-control method without correction factors.

[0109] 3. Limit of Quantitation and Limit of Detection Test

[0110] The signal-to-noise ratio S / N≈10 of the peak height of each component was used as the limit of quantification, and the signal-to-noise ratio S / N≈3 was used as the limit of detection. The lowest quantitative concentration (μg / ml) and the lowest detection concentration (μg / ml) were reported as percentages relative to the test sample concentration and relative to the limit concentration. Six solutions of the limit of quantification concentration were prepared and injected in sequence, and the chromatograms were recorded to examine the repeatability of the limit of quantification test.

[0111] Verification results: Quantitative limit: The minimum quantitative concentration (quantitative limit) of thiocarboxylic acid, flumethasone acid, 17-propionic acid ester, impurity IV-1, impurity B, thiopropionic acid ester, impurity IV-3, impurity IV-2, impurity H and impurity I were 0.031μg / ml (0.31ng), 0.008μg / ml (0.08ng), 0.019μg / ml (0.19ng), 0.016μg / ml (0.16ng), 0.020μg / ml (0.20ng), 0.016μg / ml (0.16ng), 0.162μg / ml (1.62ng), 0.078μg / ml (0.78ng), 0.024μg / ml (0.24ng), 0.019μg / ml (0.19ng), which are equivalent to 3.88%, 1.00%, 0.95%, 2.00%, 1.00% of the limit concentration respectively. %, 2.00%, 4.05%, 9.75%, 2.00%, 0.95%; when the test sample concentration is 0.4 mg / ml, thiocarboxylic acid, flumethasone acid, 17-propionic ester, impurity IV-1, impurity B, thiopropionic ester, impurity IV-3, impurity IV-2, impurity H and impurity I are equivalent to 0.008%, 0.002%, 0.005%, 0.004%, 0.005%, 0.004% of the test sample concentration, respectively. , 0.041%, 0.020%, 0.006%, 0.005%; the signal-to-noise ratios of the quantification limits of thiocarboxylic acid, flumethasone acid, 17-propionic ester, impurity IV-1, impurity B, thiopropionic ester, impurity IV-3, impurity IV-2, impurity H and impurity I were 13.3, 10.7, 16.0, 16.4, 12.5, 9.8, 14.1, 22.4, 26.4, 17.9, respectively, all of which were around 10 or higher than 10.

[0112] Detection limit: The minimum detection concentration (detection limit) of thiocarboxylic acid, flumethasone acid, 17-propionate, impurity IV-1, impurity B, thiopropionate, impurity IV-3, impurity IV-2, impurity H and impurity I were 0.016μg / ml (0.16ng), 0.004μg / ml (0.04ng), 0.010μg / ml (0.10ng), 0.008μg / ml (0.08ng), 0. 0.010μg / ml (0.10ng), 0.008μg / ml (0.08ng), 0.081μg / ml (0.81ng), 0.039μg / ml (0.39ng), 0.012μg / ml (0.12ng), 0.010μg / ml (0.10ng), which are equivalent to 2.00%, 0.50%, 0.50%, 1.00%, 0. 50%, 1.00%, 2.03%, 4.88%, 1.00%, 0.50%; when the test sample concentration was 0.4 mg / ml, thiocarboxylic acid, flumethasone acid, 17-propionic ester, impurity IV-1, impurity B, thiopropionic ester, impurity IV-3, impurity IV-2, impurity H and impurity I were equivalent to 0.004%, 0.001%, 0.003%, 0.002%, 0.003%, 0.002%, 0.020%, 0.010%, 0.003%, 0.003%; the signal-to-noise ratios of the detection limits of thiocarboxylic acid, flumethasone acid, 17-propionic ester, impurity IV-1, impurity B, thiopropionic ester, impurity IV-3, impurity IV-2, impurity H and impurity I were 9.0, 2.8, 9.2, 6.2, 5.8, 7.7, 5.3, 15.8, 10.3, 3.3, respectively, all around 3 or higher than 3.

[0113] In the repeatability test of the limit of quantification, the RSDs of the peak areas of thiocarboxylic acid, flumethasone acid, 17-propionic ester, impurity IV-1, impurity B, thiopropionic ester, impurity IV-3, impurity IV-2, impurity H and impurity I were 7.9%, 8.6%, 9.9%, 4.9%, 6.0%, 9.7%, 7.2%, 8.9%, 9.4% and 3.1%, respectively, all less than 10%, indicating that the repeatability results of the limit of quantification were good.

[0114] 4. Solution stability test

[0115] 4.1 Stock solutions of each impurity reference substance: Prepare the stock solutions of each impurity reference substance according to the method under “1 System suitability and specificity test”.

[0116] Mixed reference solution: Measure 5 ml each of flumethasone acid reference stock solution, 17-propionate reference stock solution, impurity IV-1 reference stock solution, impurity B reference stock solution, thiopropionate reference stock solution, impurity IV-3 reference stock solution, impurity IV-2 reference stock solution, and impurity H reference stock solution, place in the same 50 ml volumetric flask, dilute to the scale with solvent, shake well, measure 5 ml, place in a 50 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0117] Impurity I reference solution: Measure 5 ml of the Impurity I reference stock solution, place it in a 50 ml volumetric flask, dilute to the mark with solvent, shake well, measure 5 ml, place it in a 50 ml volumetric flask, dilute to the mark with solvent, shake well.

[0118] Impurity mixed stock solution (for spiking): Measure 2 ml each of flumethasone acid reference stock solution, impurity B reference stock solution, thiopropionate reference stock solution and impurity IV-2 reference stock solution, place in a 20 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0119] (Note: The detection amounts of 17-propionate, impurity IV-1, impurity IV-3 and impurity I in the validation samples are all about 50% of the limit, and the detection amount of impurity H is about 30% of the limit. No more impurities are added to the spiked test solution.)

[0120] Test solution: weigh about 8 mg of the product, place it in a 20 ml volumetric flask, add 2 ml of acetonitrile and dissolve it by ultrasonication, dilute to the scale with solvent, and shake well.

[0121] Control solution: Weigh about 8 mg of this product, place it in a 20 ml volumetric flask, add 2 ml of acetonitrile and dissolve it by ultrasonication, dilute to the scale with solvent, shake well, accurately measure an appropriate amount, and dilute with solvent to make a solution containing about 0.8 μg per 1 ml.

[0122] Spiked test solution: weigh about 8 mg of the product, place it in a 20 ml volumetric flask, add 2 ml of acetonitrile and ultrasonically dissolve it, measure 2 ml of the impurity mixed stock solution, place it in the same 20 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0123] The spiked test solution, test solution and reference solution were placed at low temperature (5°C) and injected and analyzed at 0h, 3h and 8h after preparation. The mixed reference solution and impurity I reference solution were placed at low temperature (5°C) and injected and analyzed at 0h, 3h, 6h and 13h after preparation. The chromatograms were recorded and the ratio of the peak area of ​​each component in the chromatograms of the reference solution and the reference solution at each time point to the peak area at 0h was calculated; the absolute value of the difference between the content of each impurity in the test solution and the spiked test solution at each time point and the content at 0h was calculated by the peak area normalization method. The test results are shown in Tables 4 to 7.

[0124] Table 4 Stability test results of mixed reference solution and impurity I reference solution

[0125]

[0126]

[0127] Table 5 Stability test results of spiked test solution

[0128]

[0129] Table 6 Control solution stability test results

[0130] Time (h) Main component peak area A / A0(%) 0 15028 / 3 14830 98.7 8 15325 102.0

[0131] Table 7 Test solution stability test results

[0132]

[0133] Verification results: The reference solution (mixed reference solution and impurity I reference solution) was placed at a low temperature of 5°C for 13 h. In the chromatogram of the reference solution, the ratio of the peak area of ​​each component at each time point to the peak area at 0 h was between 90% and 108%, indicating that the reference solution had good stability for at least 13 h when placed at a low temperature of 5°C; the reference solution was placed at a low temperature of 5°C for 8 h. In the chromatogram of the reference solution, the ratio of the peak area of ​​the main component at each time point to the peak area at 0 h was between 90% and 108%, indicating that the reference solution had good stability for at least 8 h when placed at a low temperature of 5°C.

[0134] The test solution was placed at low temperature of 5°C for 8 hours. In the chromatogram of the test solution, the absolute value of the difference between the content of known impurities at each time point and the content at 0h was no more than 0.03%, the absolute value of the difference between the content of other individual impurities and the content at 0h was no more than 0.02%, the absolute value of the difference between the total amount of impurities and the content at 0h was no more than 0.1%, and no new impurities greater than the reporting limit were generated, indicating that the test solution was stable for at least 8 hours when placed at low temperature of 5°C. In view of the increasing trend of unknown impurities less than the reporting limit, it is recommended that the test solution be prepared fresh before use.

[0135] The spiked test solution was placed at a low temperature of 5°C for 8h. In the chromatogram of the spiked test solution, the absolute value of the difference between the content of known impurities at each time point and the content at 0h was not greater than 0.03%, the absolute value of the difference between the content of other individual impurities and the content at 0h was not greater than 0.02%, the absolute value of the difference between the total amount of impurities and 0h was not greater than 0.1%, and no new impurities greater than the reporting limit were produced, indicating that the spiked test solution was stable for at least 8h when placed at a low temperature of 5°C.

[0136] 4.2, using the chromatographic conditions of Example 1, using a Kromasil Classic C8 (4.6mm×250mm) 3.5μm chromatographic column, and using water-acetonitrile (50:50) as the solvent, respectively prepare reference solutions and conduct solution stability tests. The reference solution preparation method is as follows:

[0137] Reference substance stock solution: Take about 2 mg each of flumethasone acid, 17-propionate, thiopropionate, impurity IV-1, impurity IV-2, impurity IV-3 and impurity B, place them in the same 10 ml volumetric flask, add 2 ml of acetonitrile to dissolve, dilute to the scale with solvent, and shake well.

[0138] Impurity H stock solution: Take about 2 mg of impurity H reference substance, place it in a 10 ml volumetric flask, add 2 ml of acetonitrile to dissolve, dilute to the scale with solvent, and shake well.

[0139] Impurity I stock solution: Take about 2 mg of impurity I reference substance, place it in a 10 ml volumetric flask, add 3 ml of acetonitrile to dissolve, dilute to the scale with solvent, shake well, measure 500 μl and 500 μl of solvent into the same injection vial, and mix well.

[0140] Reference substance mixed solution 1: Measure 200 μl of reference substance stock solution, impurity H stock solution and impurity I stock solution, place them in the same 20 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0141] Impurity I solution: Measure 200 μl of the impurity I stock solution, place it in a 20 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0142] Impurity H solution: Measure 200 μl of the impurity H stock solution, place it in a 20 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0143] Reference substance mixed solution 2 (without impurity I): Measure 200 μl of reference substance stock solution and impurity H stock solution respectively, place them in the same 20 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0144] Determination method: Precisely measure the solvent, impurity I solution, impurity H solution, reference substance mixed solution 1 and reference substance mixed solution 2, inject them into the liquid chromatograph respectively, and record the chromatogram. The results are shown in Table 8.

[0145] Table 8 Reference solution stability test results (low temperature 5°C)

[0146]

[0147]

[0148] Conclusion: The peak areas of impurity H in reference substance mixed solution 1, reference substance mixed solution 2 (without impurity I) and impurity H solution are 38644, 38488 and 38371 respectively, which are basically consistent, indicating that impurity H is stable in reference substance mixed solutions (with impurity I and without impurity I). When the concentration of impurity I is the same, the peak area of ​​impurity I in reference substance mixed solution 1 (containing impurities H and I) and the peak area of ​​impurity I solution are 10247 and 41899 respectively, indicating that impurity I is unstable in reference substance mixed solution 1 (containing impurities H and I); thiocarboxylic acid peaks were detected in reference substance mixed solution 1. From the impurity I, impurity H and thiocarboxylic acid structural formula, it can be seen that impurity I may be partially converted into thiocarboxylic acid under the condition of the presence of impurity H, and the stability of impurity I reference substance needs to be investigated separately.

[0149] Combined with the stability test results in 4.1, the inventors unexpectedly found that when mixed with the test sample, impurity I and impurity H can coexist. According to the impurity structure, it is speculated that impurity H is a dimeric impurity and impurity I is a trimeric impurity. There is only one S difference between H and I. The polymer impurities themselves may not be stable in the solution and are easily converted. After adding the test sample, it may be because of the pH or structural properties of the test sample that the impurity I is prevented from encountering H and converting into thiocarboxylic acid H polymerization.

[0150] 5. Repeatability Test

[0151] Solvent: Water-Acetonitrile (50:50)

[0152] Prepare the impurity mixed stock solution (for spiking), mixed reference solution and impurity I reference solution according to the method under "4. Solution stability test".

[0153] Spiked test solution: Take about 8 mg of the product, weigh accurately, place in a 20 ml volumetric flask, add 2 ml of acetonitrile and dissolve by ultrasonic, accurately measure 2 ml of impurity mixed stock solution (for spike), place in the same 20 ml volumetric flask, dilute to the mark with solvent, shake well. Prepare 6 portions in parallel.

[0154] Control solution: Accurately measure an appropriate amount of the spiked test solution and dilute it with solvent to make a solution containing approximately 0.8 μg per 1 ml.

[0155] Analyst 1 accurately measured the solvent, mixed reference solution, impurity I reference solution, spiked test solution and reference solution, respectively, and injected them into the liquid chromatograph to record the chromatogram. The content of each known impurity was calculated by the impurity reference external standard method and the main component self-reference method with correction factors, and the content of other single impurities was calculated by the self-reference method.

[0156] Verification results: In the chromatograms of the 6 spiked test solutions, the RSD of each known impurity content was less than 3%, the RSD of other single impurity contents was less than 10%, and the RSD of the total impurity content was less than 2%. The results calculated by the main component self-control method with correction factor and the impurity reference external standard method were basically consistent (both were no more than ±10% of the results of the impurity reference external standard method), indicating that the method has good repeatability.

[0157] 6. Intermediate precision test

[0158] The same batch of samples was used by analysts from the same laboratory. At different times, six spiked test solutions and control solutions were prepared in the same way according to the repeatability test method. The content of each impurity was determined on different liquid chromatographs according to the repeatability test method. The content of known impurities was calculated by the principal component self-control method with correction factors, and the content of other single impurities was calculated by the self-control method. The results of the repeatability test were combined, a total of 12 results, and the RSD of the impurity detection results was calculated to investigate the intermediate precision of the method.

[0159] Verification results: Using the same batch of samples, analysts from the same laboratory prepared 6 spiked test solutions and control solutions at different times according to the repeatability test method. The content of each impurity was determined on different liquid chromatographs according to the repeatability test method. In the chromatograms of the 6 spiked test solutions, the RSD of each known impurity content was less than 3%, the RSD of other single impurity contents was less than 10%, and the RSD of the total impurity content was less than 2%. The 6 test results were compared with the 6 results of the repeatability test. In a total of 12 spiked test solutions, the RSD of each known impurity content was less than 6%, the RSD of other single impurity contents should be less than 10%, and the RSD of the total impurity content should be less than 4%, indicating that the method has good precision.

[0160] 7. Accuracy Test

[0161] Solvent: water-acetonitrile (50:50).

[0162] Stock solutions of each impurity reference substance: Take an appropriate amount of each impurity reference substance, dissolve it in solvent or acetonitrile according to its solubility and prepare a stock solution of the reference substance with the following concentrations:

[0163] Flumethasone acid reference stock solution: 80 μg / ml.

[0164] 17-Propionate reference stock solution: 200 μg / ml.

[0165] Impurity IV-1 reference substance stock solution: 80 μg / ml.

[0166] Impurity B reference stock solution: 200 μg / ml.

[0167] Thiopropionate reference stock solution: 80 μg / ml.

[0168] Impurity IV-3 reference substance stock solution①: 200μg / ml.

[0169] Impurity Ⅳ-3 reference substance stock solution ②: 400μg / ml.

[0170] Impurity IV-2 reference substance stock solution: 80 μg / ml.

[0171] Impurity H reference stock solution: 120μg / ml.

[0172] Impurity I reference substance stock solution: 200 μg / ml.

[0173] Mixed reference solution: Accurately measure 2 ml each of flumethasone acid reference stock solution, 17-propionate reference stock solution, impurity IV-1 reference stock solution, impurity B reference stock solution, thiopropionate reference stock solution, impurity IV-2 reference stock solution, and impurity H reference stock solution, and 4.0 ml of impurity IV-3 reference stock solution ①, place in the same 20 ml volumetric flask, dilute to the scale with solvent, shake well, accurately measure 5.0 ml, place in a 50 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0174] Impurity I reference solution: Accurately measure 5.0 ml of the impurity I reference stock solution, place it in a 50 ml volumetric flask, dilute it to the mark with solvent, shake well, accurately measure 5.0 ml, place it in a 50 ml volumetric flask, dilute it to the mark with solvent, shake well.

[0175] Impurity mixed stock solution (for spiking): Accurately measure 10.0 ml each of flumethasone acid reference stock solution, impurity B reference stock solution, thiopropionate reference stock solution, impurity IV-2 reference stock solution and impurity H reference stock solution, and 5.0 ml each of 17-propionate reference stock solution, impurity IV-1 reference stock solution and impurity IV-3 reference stock solution②, place in the same 100 ml volumetric flask, dilute to the scale with solvent and shake well.

[0176] Impurity I reference substance recovery rate stock solution ①: Accurately measure 5.0 ml of impurity I reference substance stock solution, place it in a 100 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0177] Impurity I reference substance recovery rate stock solution ②: Take about 5 mg of impurity I reference substance, accurately weigh, place in a 25 ml volumetric flask, add 5 ml of acetonitrile and ultrasonically dissolve, dilute to the scale with solvent, shake well, accurately measure 5.0 ml, place in a 100 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0178] Test solution: Take about 8 mg of the product, accurately weigh it, place it in a 20 ml volumetric flask, add 2 ml of acetonitrile and dissolve it by ultrasonication, dilute it to the scale with solvent, and shake well.

[0179] Low concentration recovery solution-1: Take about 8 mg of this product, weigh accurately, place in a 20 ml volumetric bottle, add 2 ml of acetonitrile and dissolve by ultrasonic, accurately measure 1.0 ml of impurity mixed stock solution, place in the same 20 ml volumetric bottle, dilute to the scale with solvent, shake well. Prepare 3 copies in parallel.

[0180] Low concentration recovery solution-2: Take about 8 mg of this product, weigh accurately, place in a 20 ml volumetric bottle, add 2 ml of acetonitrile and dissolve by ultrasonic, accurately measure 1.0 ml of impurity I reference substance recovery stock solution ①, place in the same 20 ml volumetric bottle, dilute to the mark with solvent, shake well. Prepare 3 copies in parallel.

[0181] Medium concentration recovery solution-1: Take about 8 mg of this product, weigh accurately, place in a 20 ml volumetric bottle, add 2 ml of acetonitrile and dissolve by ultrasonic, accurately measure 2.0 ml of impurity mixed stock solution, place in the same 20 ml volumetric bottle, dilute to the scale with solvent, shake well. Prepare 3 copies in parallel.

[0182] Medium concentration recovery solution-2: Take about 8 mg of this product, weigh accurately, place in a 20 ml volumetric bottle, add 2 ml of acetonitrile and dissolve by ultrasonic, accurately measure 2.0 ml of impurity I reference substance recovery stock solution ①, place in the same 20 ml volumetric bottle, dilute to the mark with solvent, shake well. Prepare 3 copies in parallel.

[0183] High concentration recovery solution-1: Take about 8 mg of this product, weigh accurately, place in a 20 ml volumetric bottle, add 2 ml of acetonitrile and dissolve by ultrasonic, accurately measure 4.0 ml of impurity mixed stock solution, place in the same 20 ml volumetric bottle, dilute to the scale with solvent, shake well. Prepare 3 copies in parallel.

[0184] High concentration recovery solution-2: Take about 8 mg of this product, weigh accurately, place in a 20 ml volumetric bottle, add 2 ml of acetonitrile and dissolve by ultrasonic, accurately measure 4.0 ml of impurity I reference substance recovery stock solution ②, place in the same 20 ml volumetric bottle, dilute to the mark with solvent, shake well. Prepare 3 copies in parallel.

[0185] Accurately measure the solvent, mixed reference solution, impurity I reference solution, test solution and three concentrations of recovery solutions, respectively inject them into the liquid chromatograph, record the chromatogram, and calculate the detection amount of each known impurity and the recovery rate by peak area according to the impurity reference external standard method.

[0186]

[0187] Verification results: In the low, medium and high concentration ranges, the average recoveries of flumethasone acid, 17-propionate, impurity IV-1, impurity B, thiopropionate, impurity IV-3, impurity IV-2, impurity H and impurity I were 103.8%, 102.1%, 102.0%, 99.9%, 98.5%, 98.4%, 101.6%, 99.2% and 102.9%, respectively.

[0188] The average recovery rates of various impurities were between 90% and 108%, all in compliance with regulations, indicating that the method has high accuracy.

[0189] 8. Durability test

[0190] Solvent: water-acetonitrile (50:50).

[0191] Impurity mixed stock solution (for spiking): Prepare impurity mixed stock solution (for spiking) according to the method under "4. Solution stability test".

[0192] Spiked test solution: Take about 8 mg of the product, accurately weigh it, place it in a 20 ml volumetric flask, add 2 ml of acetonitrile and ultrasonically dissolve it, accurately measure 2 ml of the impurity mixed stock solution (for spike addition), place it in the same 20 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0193] Control solution: Accurately measure an appropriate amount of the spiked test solution and dilute it with solvent to make a solution containing approximately 0.8 μg per 1 ml.

[0194] Sensitivity solution: Accurately measure 5 ml of the control solution, place it in a 20 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0195] By fine-tuning the chromatographic conditions, the effects of slight changes in the chromatographic conditions on the system suitability test results and the inspection results of known impurities, other single impurities and total impurities are examined according to Tables 46 to 47.

[0196] Precisely measure the solvent, sensitivity solution, spiked test solution and reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram. Calculate the content of each known impurity by the main component self-reference method with correction factor, calculate the content of other single impurities by the self-reference method, and calculate the total amount of impurities. The results are shown in Tables 9 to 15.

[0197] Table 9 Summary of durability inspection items

[0198]

[0199] Table 10 Durability Information of Chromatographic Columns Used

[0200]

[0201]

[0202] Table 11 Durability test results of the influence of different conditions on the system suitability test results

[0203]

[0204] Table 12 Durability test results of the influence of different conditions on the determination of impurity content in spiked test solution

[0205]

[0206] Table 13 Durability test results of the influence of different conditions on the absolute value of the difference between the impurity content in the spiked test solution and the impurity content under standard conditions

[0207]

[0208]

[0209] Table 14 Durability test results of the effects of different conditions on the relative retention time of each impurity

[0210]

[0211] Table 15 Durability test results of the effect of different conditions on the separation between the peaks of each component in the spiked test solution

[0212]

[0213] Verification results: Under each condition, in the chromatogram of the sensitive solution, the signal-to-noise ratio of the main component peak height was greater than 10; in the chromatogram of the control solution, the RSD of the retention time of the main peak of thiocarboxylic acid was less than 1.0%, the RSD of the peak area was less than 5.0%, the theoretical plate number calculated based on the thiocarboxylic acid peak was greater than 5000, and the tailing factor was less than 1.5; in the spiked test solution, the separation between the peaks of each component was greater than 1.5. Under each condition, in the chromatogram of the spiked test solution, the absolute value of the difference between the known impurity content and the standard condition content was less than ±20% of the known impurity limit concentration, the absolute value of the difference between the other single impurity content and the standard condition content was less than ±20% of the limit concentration, and the absolute value of the difference between the total amount of impurities and the standard condition was less than ±20% of the limit concentration. The method has good durability when the flow rate (1.0ml / min~1.2ml / min), column temperature (33℃~37℃) and trifluoroacetic acid ratio (0.09%~0.11%) are changed and when the chromatographic column of the same brand, model, specification but different batches is replaced.

[0214] Comparative Example 1, different elution gradients

[0215] Solvent: water-acetonitrile (50:50).

[0216] Test solution: Take about 10 mg of the product, place it in a 25 ml volumetric flask, add 2 ml of acetonitrile and dissolve it by ultrasonication, dilute to the scale with solvent, and shake well.

[0217] Chromatographic conditions: octylsilane bonded silica gel was used as filler (using Kromasil Classic C8 (4.6 mm×250 mm, 3.5 μm) chromatographic column); 0.1% trifluoroacetic acid was used as mobile phase A, acetonitrile was used as mobile phase B, and linear gradient elution was performed according to the table below; the flow rate was 1.2 ml per minute; the column temperature was 35° C.; the detection wavelength was 239 nm; and the injection volume was 10 μl.

[0218]

[0219] Determination method: The test solution was placed at room temperature and injected at 0h and 5h after preparation. The results are shown in Table 16 and the chromatogram is shown in Figure 3 .

[0220] Table 16 Test solution stability test results (room temperature)

[0221]

[0222] Results: Under this method, the separation degree between the main peak and the impurity immediately after the peak (impurity IV-3) was 2.7, which met the requirements; the separation degree between 17-propionate and the impurity after the peak was 1.3, and the separation degree needed to be improved, so the gradient needed to be adjusted; the test solution was placed at room temperature for 5 hours, and the absolute values ​​of the content differences of 17-propionate, impurity IV-1 and thiocarboxylic acid compared with 0 hours were calculated by the area normalization method, which showed that the test solution was unstable within 5 hours at room temperature.

[0223] Comparative Example 2, changing the purity of trifluoroacetic acid

[0224] The same chromatographic conditions as in Example 1 were used, with the only difference being that the purity of the trifluoroacetic acid used was 99%.

[0225] Sample preparation:

[0226] Test solution: Take about 8 mg of the product, place it in a 20 ml volumetric flask, add 2 ml of acetonitrile and dissolve it by ultrasonication, dilute to the scale with solvent, and shake well.

[0227] Solvent: water-acetonitrile (50:50).

[0228] Results: Figure 4As shown, the chromatographic conditions are the same as those in Example 1, except that the purity of the trifluoroacetic acid used is changed to 99%. As a result, the main peak is bifurcated. The reason may be that the purity of the trifluoroacetic acid is low, the ionic strength is reduced, and the diffusion of the sample in the chromatographic column is affected, resulting in the bifurcation of the main peak.

[0229] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An analytical method for detecting thiocarboxylic acid-related substances in fluticasone propionate intermediates, characterized in that: High performance liquid chromatography was used, with octylsilane bonded silica gel as the filler of the chromatographic column, trifluoroacetic acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, and the initial volume ratio of mobile phase A to mobile phase B being 52:48 to 58:

42.

2. The analysis method according to claim 1, characterized in that The impurities that may be contained in thiocarboxylic acid include flumethasone acid, 17-propionate, impurity IV-1, impurity B, thiopropionate, impurity IV-3, impurity IV-2, impurity H, and impurity I, and the structural formulas are: Flumethasone acid: 17-Propionate: Impurity IV-1: Impurity B: Thiopropionate: Impurity IV-3: Impurity IV-2: Impurity H: Impurity I:

3. The analysis method according to claim 1, characterized in that The flow rate of the mobile phase is 1.1-1.2 ml / min.

4. The analysis method according to claim 1, characterized in that The column temperature of the chromatographic column is 33-37°C, and the injector temperature is 4-8°C.

5. The analysis method according to claim 1, characterized in that The detection wavelength was 239 nm and the injection volume was 10 μl.

6. The analysis method according to claim 1, characterized in that The trifluoroacetic acid has a purity of 99.5% or above; the concentration range of the trifluoroacetic acid aqueous solution is 0.09% to 0.11%.

7. The analysis method according to claim 1, characterized in that The gradient program of mobile phase elution was: Within 0 to 15 minutes, the volume percentage of mobile phase A linearly changes from 55% to 48%, within 15 to 25 minutes, the volume percentage of mobile phase A remains unchanged at 48%, within 25 to 35 minutes, the volume percentage of mobile phase A linearly changes from 48% to 25% to 30%, within 35 to 50 minutes, the volume percentage of mobile phase A remains unchanged at 25% to 30%, and then within 1 minute, the volume percentage of mobile phase A linearly changes to 55%, and then equilibrates for 9 minutes.

8. The analysis method according to claim 1, characterized in that The gradient program of mobile phase elution is: within 0 to 15 minutes, the volume percentage of mobile phase A linearly changes from 55% to 48%, within 15 to 25 minutes, the volume percentage of mobile phase A remains unchanged at 48%, within 25 to 35 minutes, the volume percentage of mobile phase A linearly changes from 48% to 30%, within 35 to 50 minutes, the volume percentage of mobile phase A remains unchanged at 30%, within 50 to 51 minutes, the volume percentage of mobile phase A linearly changes from 30% to 55%, within 51 to 60 minutes, the volume percentage of mobile phase A remains unchanged at 55%, that is, the chromatographic column equilibrium time is after 50 minutes.

9. The analysis method according to claim 1, characterized in that The analytical method includes the following chromatographic conditions: Octylsilane bonded silica gel was used as filler; 0.1% trifluoroacetic acid was used as mobile phase A, acetonitrile was used as mobile phase B, and linear gradient elution was performed; the flow rate was 1.1 ml per minute; the column temperature was 35°C; the injector temperature was 5°C; the detector was an ultraviolet detector with a detection wavelength of 239 nm; and the injection volume was 10 μl.

10. The analysis method according to claim 1, characterized in that The analysis method further comprises: Preparation of test solution: dissolve the product in acetonitrile by ultrasonication and dilute with solvent to obtain a solution with a concentration of 0.4 mg / ml. The solvent is a water-acetonitrile solution with a volume ratio of 50:

50. Control solution: Accurately measure an appropriate amount of the test solution and quantitatively dilute it with a solvent to make a solution with a concentration of 0.8 μg / ml; Determination method: Accurately measure the test solution and the control solution, inject them into the liquid chromatograph respectively, and record the chromatogram; if the test sample contains impurities, the impurities will appear in the order of flumethasone acid, 17-propionic acid ester, impurity IV-1, impurity B, thiopropionic acid ester, impurity IV-3, impurity IV-2, impurity H, and impurity I; except for impurity IV-3, which is calculated by the principal component self-control method with a correction factor, other known impurities and other single impurities are calculated by the principal component self-control method without a correction factor.

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