Method for determining impurity content in hydrocortisone acetate intermediate 2 by utilizing HPLC (High Performance Liquid Chromatography)

Abstract

The invention discloses a method for determining impurity content in a hydrocortisone acetate intermediate 2 by utilizing HPLC (High Performance Liquid Chromatography), and belongs to the technical field of pharmaceutical analysis. According to the method, a chromatographic column taking octadecyl bonded silica gel as a filler is adopted, water is taken as a mobile phase A, acetonitrile is taken as a mobile phase B, gradient elution is carried out, and the content of impurities in hydrocortisone acetate bromide is measured. According to the method disclosed by the invention, the hydrocortisone acetate intermediate 2 can be well separated from impurities, the spectral purity of a destroyed sample is greater than 990 through peak purity detection under various conditions, and the detection specificity of related substances is good; meanwhile, when the chromatographic condition parameters are slightly changed, the separation and inspection results of main components and impurities are not influenced, and the durability is good; according to the method, the related substances in the hydrocortisone acetate intermediate 2 can be rapidly, effectively, accurately and reliably separated and detected, the product quality of the hydrocortisone acetate intermediate 2 is improved, and the medication safety of a patient is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for determining the impurity content in intermediate 2 of hydrocortisone acetate by HPLC. Background Art

[0002] Hydrocortisone acetate tablets are adrenal cortical hormone drugs. Clinically, they are mainly used for the replacement therapy of adrenocortical insufficiency and the treatment of congenital adrenal hyperplasia. They have anti-inflammatory, immunosuppressive, antitoxin and other effects.

[0003] The process of hydrocortisone acetate uses anecortave acetate as the starting material to obtain intermediate 1 through a bromination reaction, intermediate 1 is obtained through a debromination reaction to get intermediate 2, intermediate 2 is purified to obtain the refined product of intermediate 2 (crude hydrocortisone acetate), and the debrominated refined product is further refined to obtain the finished product of hydrocortisone acetate.

[0004] The starting material of hydrocortisone acetate will produce process by-product impurities during the production process, and impurities may be generated during the production and storage of intermediate 2 of hydrocortisone acetate. Therefore, providing a method for determining the impurity content in intermediate 2 of hydrocortisone acetate is of great significance for the production and storage of hydrocortisone acetate. Summary of the Invention

[0005] The technical problem solved by the present invention is to provide a high-performance liquid chromatography analysis method with high feasibility, simple and convenient operation process, good applicability, which can be widely used to determine the impurity content in hydrocortisone acetate bromide, fully meet the qualitative and quantitative analysis of various impurities in the product, and effectively control the product quality.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for determining the impurity content in hydrocortisone acetate bromide by HPLC, comprising the following steps:

[0008] 1) Prepare a test solution and a control solution;

[0009] The preparation process of the test solution is as follows: Accurately measure the test sample of intermediate 2 of hydrocortisone acetate, and dilute it with a diluent to a solution containing 1 mg of intermediate 2 of hydrocortisone acetate per 1 mL as the test solution;

[0010] The preparation process of the control solution is as follows: Accurately measure the test solution, and dilute it with a diluent to a solution containing 10 μg of intermediate 2 of hydrocortisone acetate per mL as the control solution;

[0011] 2) Set the high performance liquid chromatography (HPLC) detection conditions: Use a chromatographic column filled with octadecylsilyl silica gel. The mobile phase consists of phase A and phase B. Phase A is water, and phase B is acetonitrile. Perform gradient elution.

[0012] 3) Accurately measure the test solution and the reference solution respectively, inject them into the liquid chromatograph, record the chromatogram, plot the standard curve, obtain the standard curve equation, and calculate the content of impurities in Hydrocortisone Acetate Intermediate 2.

[0013] The impurities in Hydrocortisone Acetate Intermediate 2 include impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity INT1, impurity SM1-2, cortisone, impurity SM-N, and impurity SM-N1-1. Impurity A is C 21 H 30 O5, impurity B is C 21 H 30 O4, impurity C is C 23 H 30 O6, impurity D is C 23 H 30 O6, impurity E is C 23 H 30 O5, impurity F is C 23 H 32 O6, impurity G is C 25 H 34 O7, impurity INT1 is C 23 H 31 B r O6, impurity SM1-2 is C 19 H 24 O2, impurity SM-N is C 21 H 27 ClO3, impurity SM-N1-1 is C 21 H 28 O3, cortisone is C 21 H 28 O5.

[0014] Preferably, in step 1), the diluent is a methanol-water-glacial acetic acid solution, where the volume ratio of methanol, water, and glacial acetic acid is 90:10:1.

[0015] Preferably, in the step 2), the gradient elution conditions are as follows: from 0 to 30 min, the volume percentage of mobile phase A is 75% and the volume percentage of mobile phase B is 25%; at 45 min, the volume percentage of mobile phase A is 60% and the volume percentage of mobile phase B is 40%; from 45 to 60 min, the volume percentage of mobile phase A is 60% and the volume percentage of mobile phase B is 40%; at 65 min, the volume percentage of mobile phase A is 50% and the volume percentage of mobile phase B is 50%; from 65 to 80 min, the volume percentage of mobile phase A is 50% and the volume percentage of mobile phase B is 50%.

[0016] Preferably, in the step 2), the chromatographic column is Agilent ZORBAX Eclipse Plus C18, with a specification of 250 nm × 4.6 mm, a packing particle size of 5 μm, and a column temperature of 30 °C.

[0017] Preferably, in the step 2), the gradient elution flow rate is 1.0 mL / min.

[0018] Preferably, in the step 3), the injection volume is 20 μL.

[0019] Preferably, in the step 3), the detection wavelength of the liquid chromatograph is 254 nm.

[0020] Preferably, in the step 3), according to the chromatogram, a linear regression equation is made with the peak area A as the ordinate and the concentration C as the abscissa. When the detection wavelength is 254 nm, the linear equation of impurity A is A = 0.7191C - 0.0122, the linear equation of impurity B is A = 0.7670C - 0.0096, the linear equation of impurity C is A = 0.7054C - 0.0125, the linear equation of impurity D is A = 0.5416C - 0.0081, the linear equation of impurity E is A = 0.6272C - 0.0660, the linear equation of impurity F is A = 0.6156C + 0.0019, the linear equation of impurity G is A = 0.5161C - 0.0098, the linear equation of impurity INT1 is A = 0.5559C - 0.0121, the linear equation of hydrocortisone acetate intermediate 2 is A = 0.6529C - 0.0188, the linear equation of impurity SM1-2 is A = 0.8779C - 0.0058, the linear equation of impurity SM-N is A = 0.7391C - 0.0071, the linear equation of impurity SM-N1-1 is A = 0.7750C - 0.0012, and the linear equation of cortisone is A = 0.6869C + 0.0084.

[0021] Preferably, the linear concentration range of impurity A is 0.1040 - 10.3987 μg / mL, the quantitation limit is 0.063 μg / mL, and the detection limit is 0.016 μg / mL; the linear concentration range of impurity B is 0.2562 - 10.2474 μg / mL, the quantitation limit is 0.126 μg / mL, and the detection limit is 0.031 μg / mL; the linear concentration range of impurity C is 0.1266 - 12.6587 μg / mL, the quantitation limit is 0.072 μg / mL, and the detection limit is 0.018 μg / mL; the linear concentration range of impurity D is 0.1160 - 11.6046 μg / mL, the quantitation limit is 0.062 μg / mL, and the detection limit is 0.015 μg / mL; the linear concentration range of impurity E is 0.6415 - 64.1528 μg / mL, the quantitation limit is 0.094 μg / mL, and the detection limit is 0.047 μg / mL; the linear concentration range of impurity F is 0.2579 - 10.3163 μg / mL, the quantitation limit is 0.125 μg / mL, and the detection limit is 0.031 μg / mL; the linear concentration range of impurity G is 0.2723 - 10.8911 μg / mL, the quantitation limit is 0.126 μg / mL, and the detection limit is 0.016 μg / mL; the linear concentration range of impurity INT1 is 0.1767 - 17.6697 μg / mL, the quantitation limit is 0.058 μg / mL, and the detection limit is 0.029 μg / mL; the linear concentration range of hydrocortisone acetate intermediate 2 is 0.1964 - 19.6413 μg / mL, the quantitation limit is 0.069 μg / mL, and the detection limit is 0.017 μg / mL; the linear concentration range of impurity SM1-2 is 0.1010 - 4.0398 μg / mL, the quantitation limit is 0.052 μg / mL, and the detection limit is 0.026 μg / mL; the linear concentration range of impurity SM-N is 0.0985 - 1.9709 μg / mL, the quantitation limit is 0.051 μg / mL, and the detection limit is 0.026 μg / mL; the linear concentration range of impurity SM-N1-1 is 0.0952 - 1.9050 μg / mL, the quantitation limit is 0.049 μg / mL, and the detection limit is 0.025 μg / mL; the linear concentration range of cortisone is 0.0989 - 1.9790 μg / mL, the quantitation limit is 0.099 μg / mL, and the detection limit is 0.025 μg / mL.

[0022] Advantages: Compared with the prior art, the present invention has the following advantages:

[0023] 1) In the method of the present invention, at a detection wavelength of 254 nm, hydrocortisone acetate intermediate 2 can be well separated from impurities. The excipient peaks and solvent peaks do not interfere with the detection of the main component and impurities. Moreover, after the stressed samples under various conditions are detected for peak purity, the spectral purity is greater than 990. The impurities generated under various conditions do not interfere with the detection of impurities and the main component, and the specificity of the related substances detection is good.

[0024] 2) Compared with the prior art, when there are minor changes in the chromatographic condition parameters of the present invention, it has no impact on the separation and inspection results of the related substance impurities of hydrocortisone acetate intermediate 2, and the durability is good.

[0025] 3) The method of the present invention uses high performance liquid chromatography to be able to quickly, effectively, accurately and reliably separate and detect the related substances in hydrocortisone acetate intermediate 2, which is beneficial to improving the product quality of hydrocortisone acetate intermediate 2 and enhancing the drug safety of patients. Description of the Drawings

[0026] Figure 1 Chromatograms for detecting the related substances of hydrocortisone acetate intermediate 2 using different methods in Example 2: Among them, 1a is the system suitability chromatogram of the original method, and 1b is the system suitability chromatogram of the method of the present invention.

[0027] Figure 2 System suitability chromatogram 1 of Example 3

[0028] Figure 3 Test chromatograms in the stress test of Example 3: Among them, 3a is the acid-damaged chromatogram, 3b is the base-damaged chromatogram, 3c is the light-damaged chromatogram, 3d is the pyrolysis-damaged chromatogram, 3e is the high-temperature-damaged chromatogram, and 3f is the oxidation-damaged chromatogram.

[0029] Figure 4 Stability chromatograms of the control solution in Example 3: Among them, 4a is the stability chromatogram of the impurity reference solution at 0 h, and 4b is the stability chromatogram of the impurity reference solution at 30 h; 4c is the stability chromatogram of the cortisone reference solution at 0 h, and 4d is the stability chromatogram of the cortisone reference solution at 21 h.

[0030] Figure 5 Stability chromatograms of the test solution in Example 3: Among them, 5a is the stability chromatogram of the test solution at 0 h, and 5b is the stability chromatogram of the test solution at 110 h.

[0031] Figure 6Durability chromatogram of Example 3; wherein, 6a is the chromatogram under normal conditions, 6b is the chromatogram at a flow rate of 0.95 mL / min, 6c is the chromatogram at a flow rate of 1.05 mL / min, 6d is the chromatogram at a column temperature of 28 °C, 6e is the chromatogram at a column temperature of 32 °C, 6f is the chromatogram with an initial ratio of water:acetonitrile = 74.5:25.5, and 6g is the chromatogram with an initial ratio of water:acetonitrile = 73.5:26.5;

[0032] Figure 7 Linear standard curve graph of the present invention. Detailed implementation manners

[0033] The following further clarifies the present invention in conjunction with specific embodiments. The embodiments are implemented on the premise of the technical solution of the present invention. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For those reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase.

[0034] The process of hydrocortisone acetate uses anecortave acetate as the starting material to obtain Intermediate 1 through a bromination reaction. Intermediate 1 undergoes a debromination reaction to obtain Intermediate 2. Intermediate 2 is purified to obtain a refined product of Intermediate 2 (crude hydrocortisone acetate). The refined product of debromination is further refined to obtain the finished product of hydrocortisone acetate. Table 1 below shows the information of Intermediate 1, Intermediate 2, and the refined product of Intermediate 2.

[0035] Table 1: Information on related substances

[0036]

[0037] In the following examples, the model of the high performance liquid chromatograph used is the Agilent 1260 series (VWD); the information on the relevant impurities in Intermediate 2 of hydrocortisone acetate in the following examples is shown in Table 2 below.

[0038] Table 2: Impurity information

[0039]

[0040]

[0041]

[0042] Example 1

[0043] A method for determining the impurity content in Intermediate 2 of hydrocortisone acetate by HPLC, comprising the following steps:

[0044] 1) Prepare a test solution and a reference solution:

[0045] The preparation process of the test solution is as follows: Take an appropriate amount of the hydrocortisone acetate intermediate 2 test sample, dissolve it with a diluent and quantitatively dilute it to the mark to prepare a solution containing 1.0 mg of hydrocortisone acetate intermediate 2 per 1 mL as the test solution;

[0046] The preparation process of the control solution is as follows: Accurately measure an appropriate amount of the test solution and dilute it with a diluent to contain 10 μg of hydrocortisone acetate intermediate 2 per 1 mL of the solution as the control solution;

[0047] The diluent is a methanol-water-glacial acetic acid solution, in which the volume ratio of methanol, water and glacial acetic acid is 90:10:1;

[0048] 2) Set the liquid chromatography detection conditions:

[0049] Use an Agilent ZORBAX Eclipse Plus C18 chromatographic column with a specification of 4.6 mm × 250 mm and a packing particle size of 5 μm; The mobile phase consists of phase A and phase B, where phase A is water and phase B is acetonitrile; Flow rate: 1 mL / min; Column temperature: 30 °C;

[0050] Gradient elution program: 0 - 30 min, the volume percentage of mobile phase A is 75% and the volume percentage of mobile phase B is 25%; 30 - 45 min, the volume percentage of mobile phase A decreases from 75% to 60% and the volume percentage of mobile phase B increases from 25% to 40%; 45 min, the volume percentage of mobile phase A is 60% and the volume percentage of mobile phase B is 40%; 45 - 60 min, the volume percentage of mobile phase A is 60% and the volume percentage of mobile phase B is 40%; 60 - 65 min, the volume percentage of mobile phase A decreases from 60% to 50% and the volume percentage of mobile phase B increases from 40% to 50%; 65 min, the volume percentage of mobile phase A is 50% and the volume percentage of mobile phase B is 50%; 65 - 80 min, the volume percentage of mobile phase A is 50% and the volume percentage of mobile phase B is 50%.

[0051] 3) Accurately measure 20 μL of the reference solution and the test solution respectively, inject them into the liquid chromatograph, with the detection wavelength at 254 nm, detect and record the chromatogram; according to the chromatogram, use the peak area A as the ordinate and the concentration C as the abscissa to make a linear regression equation. The linear equation for impurity A is A = 0.7191C - 0.0122, the linear equation for impurity B is A = 0.7670C - 0.0096, the linear equation for impurity C is A = 0.7054C - 0.0125, the linear equation for impurity D is A = 0.5416C - 0.0081, the linear equation for impurity E is A = 0.6272C - 0.0660, the linear equation for impurity F is A = 0.6156C + 0.0019, the linear equation for impurity G is A = 0.5161C - 0.0098, the linear equation for impurity INT1 is A = 0.5559C - 0.0121, the linear equation for hydrocortisone acetate intermediate 2 is A = 0.6529C - 0.0188, the linear equation for impurity SM1-2 is A = 0.8779C - 0.0058, the linear equation for impurity SM-N is A = 0.7391C - 0.0071, the linear equation for impurity SM-N1-1 is A = 0.7750C - 0.0012, and the linear equation for cortisone is A = 0.6869C + 0.0084.

[0052] Example 2

[0053] 1. Establishment of impurity limits

[0054] After systematic and comprehensive research on the related substances of intermediate 2 in multiple batches of production, according to the research results, the quality standards for hydrocortisone acetate intermediate 2 are formulated as follows: the standard limit for impurity A (IMP-A) is ≤0.5%, the standard limit for impurity B (IMP-B) is ≤0.5%, the standard limit for impurity C (IMP-C) is ≤0.6%, the standard limit for impurity D (IMP-D) is ≤0.5%, the standard limit for impurity E (IMP-E) is ≤3.0%, the standard limit for impurity F (IMP-F) is ≤0.5%, the standard limit for impurity G (IMP-G) is ≤0.5%, the standard limit for impurity INT1 (INT1) is ≤1.0%, the standard limit for impurity SM1-2 is ≤0.2%, the standard limit for SM-N1-1 is ≤0.1%, the standard limit for impurity SM-N (SM-N) is ≤0.1%, the standard limit for cortisone (KDS) is ≤0.1%, the standard limit for the maximum unknown single impurity is ≤1.0%, and the standard limit for the total impurities is ≤5.0%.

[0055] 2. Comparison with the original impurity determination method

[0056] Table 3: Analytical method parameters

[0057]

[0058] The above-mentioned original impurity content determination method was formulated with reference to the related substance inspection method of hydrocortisone acetate in the pharmacopoeia. It can be seen from Figure 1 that in the original impurity content determination method, impurity A, impurity F and the unknown impurity could not be completely separated, which was not applicable to the detection of hydrocortisone acetate intermediate 2. In the method of the present invention, the solvent and other impurities do not interfere with the detection of known impurities, and the detection results are more accurate and reliable.

[0059] Example 3

[0060] The detection method of Example 1 was verified for method validation, and the verification was carried out respectively from system suitability, destructive test, quantitation limit, detection limit, linear relationship, precision, accuracy, and solution stability. The diluent used in this example was methanol-water-glacial acetic acid solution, in which the volume ratio of methanol, water and glacial acetic acid was 90:10:1.

[0061] 1. System suitability

[0062] Appropriately weigh the reference substances of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity INT1, impurity SM1-2, impurity SM-N, and impurity SM-N1-1 respectively, dissolve them with the diluent and dilute them respectively to prepare impurity stock solutions containing 100 μg of impurity A, 100 μg of impurity B, 120 μg of impurity C, 100 μg of impurity D, 200 μg of impurity E, 100 μg of impurity F, 100 μg of impurity G, 100 μg of impurity INT1, 100 μg of impurity SM1-2, 100 μg of impurity SM-N, and 100 μg of impurity SM-N1-1 in each 1 mL.

[0063] Take appropriate amounts of each impurity stock solution, and dilute them with the diluent respectively to prepare single-position solutions containing 5 μg of impurity A, 5 μg of impurity B, 6 μg of impurity C, 5 μg of impurity D, 30 μg of impurity E, 5 μg of impurity F, 5 μg of impurity G, 10 μg of impurity INT1, 2 μg of impurity SM1-2, 1 μg of impurity SM-N, and 1 μg of impurity SM-N1-1 in each 1 mL.

[0064] Take an appropriate amount of the test sample, weigh it precisely, and then add an appropriate amount of the impurity stock solution to the same volumetric flask, dissolve and dilute it with the diluent to prepare a mixed solution containing 1 mg of intermediate 2, 5 μg of impurity A, 5 μg of impurity B, 6 μg of impurity C, 5 μg of impurity D, 30 μg of impurity E, 5 μg of impurity F, 5 μg of impurity G, 10 μg of impurity INT1, 2 μg of impurity SM1-2, 1 μg of impurity SM-N, and 1 μg of impurity SM-N1-1 in each 1 mL as the system suitability solution.

[0065] 20 μL each of the blank solvent (methanol - water - glacial acetic acid solution, with a volume ratio of methanol, water, and glacial acetic acid of 90:10:1), each single impurity localization solution, and the system suitability solution were respectively injected into a high - performance liquid chromatograph, and the chromatograms were recorded to examine the separation of each impurity from the main component and adjacent impurities. The results are as Figure 2 shown in Table 4.

[0066] Table 4: Impurity Localization and Resolution Table

[0067]

[0068] As can be seen from Table 4, at a detection wavelength of 254 nm, the elution order of the mixed solution is IMP - A, IMP - B, IMP - F, IMP - C, Intermediate 2, IMP - D, INT1, SM1 - 2, SM - N1 - 1, IMP - G, IMP - E, SM - N. There is sufficient resolution between Intermediate 2 of hydrocortisone acetate and the impurities, and the blank solvent does not interfere with the determination of Intermediate 2 of hydrocortisone acetate and the impurities.

[0069] 2. Forced Degradation Tests

[0070] Undegraded solution: Take 10 mg of Intermediate 2 of hydrocortisone acetate, place it in a 10 - mL volumetric flask, dissolve and dilute to the mark with the diluent, and shake well to obtain the undegraded solution.

[0071] Acid - degraded solution: Take 10 mg of Intermediate 2 of hydrocortisone acetate, place it in a 10 - mL volumetric flask, add 1 mL of 1 mol / L hydrochloric acid solution, degrade at 60 °C for 2 hours, add 1 mL of 1 mol / L sodium hydroxide solution for neutralization, dissolve and dilute to the mark with the diluent, and shake well to obtain the acid - degraded solution.

[0072] Base - degraded solution: Take 10 mg of Intermediate 2 of hydrocortisone acetate, place it in a 10 - mL volumetric flask, add 1 mL of 0.5 mol / L sodium hydroxide solution, degrade at room temperature for 1 hour, add 1 mL of 0.5 mol / L hydrochloric acid solution for neutralization, dissolve and dilute to the mark with the diluent, and shake well to obtain the base - degraded solution.

[0073] Oxidation - degraded solution: Take 10 mg of Intermediate 2 of hydrocortisone acetate, place it in a 10 - mL volumetric flask, add 1 mL of 30% hydrogen peroxide solution, degrade at 60 °C for 2 hours, dissolve and dilute to the mark with the diluent, and shake well to obtain the oxidation - degraded solution.

[0074] Pyrolysis - degraded solution: Take 10 mg of Intermediate 2 of hydrocortisone acetate, place it in a 10 - mL volumetric flask, dissolve with an appropriate amount of diluent, heat and degrade at 60 °C for 3.5 hours, cool to room temperature, and then dissolve and dilute to the mark with the diluent, and shake well to obtain the pyrolysis - degraded solution.

[0075] High-temperature degradation solution: Take 10 mg of hydrocortisone acetate intermediate 2 and place it in a 10 mL volumetric flask. Heat it at 100 °C for 3.5 hours, cool it to room temperature, dissolve and dilute it to the mark with diluent, and shake well to obtain the high-temperature degradation solution.

[0076] Light degradation solution: Take the undegraded solution and place it under 4500 lux sunlight and 100 μW ultraviolet light for 20 hours to obtain the light degradation solution.

[0077] Acid blank solution: Take 1 mL of 1 mol / L hydrochloric acid and place it in a 10 mL volumetric flask. Add 1 mL of 1 mol / L sodium hydroxide to neutralize it, add diluent to dilute it to the mark, and shake well to obtain the acid blank solution.

[0078] Alkali blank solution: Take 1 mL of 0.5 mol / L hydrochloric acid and place it in a 10 mL volumetric flask. Add 1 mL of 0.5 mol / L sodium hydroxide to neutralize it, add diluent to dilute it to the mark, and shake well to obtain the alkali blank solution.

[0079] Oxidation blank solution: Take 1 mL of 30% hydrogen peroxide and place it in a 10 mL volumetric flask. Add diluent to dilute it to the mark, and shake well to obtain the oxidation blank solution.

[0080] Respectively take 20 μL of the above test solutions and inject them into the chromatograph, and record the chromatogram. Calculate the amounts of each impurity peak by the normalization method. The results are as Figure 3 shown.

[0081] 1) Peak purity

[0082] For the test solutions under various degradation conditions, diode array detection was used for peak purity determination. The peak purity results are shown in Tables 5 - 7 below.

[0083] Table 5: Peak purity results

[0084]

[0085] As can be seen from Table 5, after peak purity detection of the degraded samples under each condition, the peak purity is greater than 990.

[0086] 2) Material balance investigation

[0087] Table 6: Results of material balance investigation

[0088]

[0089] As can be seen from Table 6, through the analysis of the sample material balance data, the reduction in the main peak of hydrocortisone acetate intermediate 2 is basically consistent with the decrease in its content. The total peak areas detected for samples with the same concentration after degradation under different conditions are basically similar, indicating that this chromatographic condition can effectively detect all degradation impurities.

[0090] 3) Research on Degradation Impurities

[0091] Table 7: Impurity Profile of Hydrocortisone Acetate Intermediate 2 in the Forced Degradation Tests (Statistical by Normalization Method)

[0092]

[0093]

[0094]

[0095] Note: "-" indicates not detected.

[0096] As can be seen from Table 7, for Hydrocortisone Acetate Intermediate 2, after forced degradation under high temperature and light, no obvious changes were observed in each impurity; after acid degradation, Impurity A, Unknown Impurity 18, and Unknown Impurity 30 increased significantly, while Unknown Impurity 17 decreased significantly; after base degradation, Unknown Impurity 7, Unknown Impurity 8, Impurity A, and Unknown Impurity 30 increased significantly, while Unknown Impurity 17 and Impurity E decreased significantly; after pyrolysis degradation, Unknown Impurity 17 decreased significantly, and Unknown Impurity 30 increased significantly; after oxidation degradation, Impurity A and Impurity B increased significantly. The peak purity of the degraded samples under each condition was not less than 990.

[0097] 3. Quantitation Limit and Detection Limit

[0098] Weigh an appropriate amount of the reference substance accurately, dissolve and dilute it with the diluent to prepare an impurity stock solution. Pipette an appropriate amount of the above stock solution and dilute it with the diluent to prepare a mixed solution containing 5 μg of Impurity A, 5 μg of Impurity B, 5 μg of Impurity D, 5 μg of Impurity F, 5 μg of Impurity G, 6 μg of Impurity C, 30 μg of Impurity E, 10 μg of INT1, 10 μg of Intermediate 2, 2 μg of SM1-2, 1 μg of SM-N, and 1 μg of SM-N1-1 per 1 mL. Measure its quantitation limit (S / N≥10) and detection limit (S / N≥3) by the dilution method.

[0099] Weigh an appropriate amount of cortisone reference substance accurately, dissolve and dilute it with the diluent to prepare an impurity stock solution. Pipette an appropriate amount of the above stock solution and dilute it with the diluent to prepare an impurity solution containing 1 μg of cortisone impurity per 1 mL. Measure its quantitation limit (S / N≥10) and detection limit (S / N≥3) by the dilution method. The results are shown in Table 8 below.

[0100] Table 8: Results of Quantitation Limit

[0101]

[0102] As can be seen from Table 8, under this chromatographic condition, the quantitation limit and detection limit of Hydrocortisone Acetate Intermediate 2 and the impurities meet the requirements.

[0103] 4. Linearity and Range

[0104] Weigh appropriate amounts of the reference substances of each impurity and Intermediate 2, and dilute them with the diluent to prepare a mixed solution containing 25 μg of Impurity A, 25 μg of Impurity B, 25 μg of Impurity D, 25 μg of Impurity F, 25 μg of Impurity G, 30 μg of Impurity C, 150 μg of Impurity E, 50 μg of INT1, 50 μg of Intermediate 2, 10 μg of SM1-2, 5 μg of SM-N, and 5 μg of SM-N1-1 per 1 mL as the linear stock solution. Take the above solution and dilute it with the diluent according to Table 9-10 below to prepare linear solutions of various concentrations.

[0105] Table 9: Preparation of Linear Solutions

[0106]

[0107] Weigh an appropriate amount of the reference substance of Cortisone Impurity, and dilute it with the diluent to prepare a solution containing 5 μg of Cortisone Impurity per 1 mL as the linear stock solution. Take the above solution and dilute it with the diluent according to Table 10 below to prepare linear solutions of various concentrations.

[0108] Table 10: Preparation of Linear Solutions

[0109]

[0110] Precisely measure 20 μL of each of the above solutions and inject them into the high performance liquid chromatograph, record the chromatogram, measure the peak area, and perform linear regression with the peak area A as the ordinate and the concentration C as the abscissa. The results are as Figure 7 shown in Tables 11 to 22.

[0111] Table 11: Results of Linear Determination of IMP-A( Figure 7 a)

[0112]

[0113] Table 12: Results of Linear Determination of IMP-B( Figure 7 b)

[0114]

[0115] Table 13: Results of Linear Determination of IMP-C( Figure 7 c)

[0116]

[0117] Table 14: Results of Linear Determination of IMP-D( Figure 7 d)

[0118]

[0119]

[0120] Table 15: Results of the linearity determination of IMP-E ( Figure 7 e)

[0121]

[0122] Table 16: Results of the linearity determination of IMP-F ( Figure 7 f)

[0123]

[0124] Table 17: Results of the linearity determination of IMP-G ( Figure 7 g)

[0125]

[0126]

[0127] Table 17: Results of the linearity determination of INT1 ( Figure 7 h)

[0128]

[0129] Table 18: Results of the linearity determination of Intermediate 2 ( Figure 7 i)

[0130]

[0131] Table 20: Results of the linearity determination of SM1-2 ( Figure 7 j)

[0132]

[0133]

[0134] Table 19: Results of the linearity determination of SM-N ( Figure 7 k)

[0135]

[0136] Table 20: Results of the linearity determination of SM-N1-1 ( Figure 7 l)

[0137]

[0138] Table 21: Results of the linearity determination of Cortisone ( Figure 7 m)

[0139]

[0140] The calculation results of the correction factors for known impurities are shown in Table 24 below;

[0141] Calculation formula:

[0142]

[0143] In the formula: K 主成分 —Slope of the standard curve of Intermediate 2;

[0144] K 杂质 —Slope of the standard curve of the impurity.

[0145] Table 22: Known impurity correction factors

[0146] Name Slope Correction Factor IMP-A 0.7191 0.91 IMP-B 0.7670 0.85 IMP-C 0.7054 0.93 IMP-D 0.5416 1.21 IMP-E 0.6272 1.04 IMP-F 0.6156 1.06 IMP-G 0.5161 1.27 INT1 0.5559 1.17 Intermediate 2 0.6529 - SM1-2 0.8779 0.74 SM-N 0.7391 0.88 SM-N1-1 0.7750 0.84 Cortisone 0.6869 0.95

[0147] The relative correction factors of all known impurities are within the range of 0.2 - 5, and the impurity content can be calculated by the self - control method with correction factors.

[0148] From Figure 7 and Tables 11 - 23, it can be seen that for impurity A, it is in the range of 0.1040 - 10.3987 μg / mL; for impurity B, it is in the range of 0.2562 - 10.2474 μg / mL; for impurity C, it is in the range of 0.1266 - 12.6587 μg / mL; for impurity D, it is in the range of 0.1160 - 11.6046 μg / mL; for impurity E, it is in the range of 0.6415 - 64.1528 μg / mL; for impurity F, it is in the range of 0.2579 - 10.3163 μg / mL; for impurity G, it is in the range of 0.2723 - 10.8911 μg / mL; for INT1, it is in the range of 0.1767 - 17.6697 μg / mL; for Intermediate 2, it is in the range of 0.1964 - 19.6413 μg / mL; for SM1 - 2, it is in the range of 0.1010 - 4.0398 μg / mL; for SM - N, it is in the range of 0.0985 - 1.9709 μg / mL; for SM - N1 - 1, it is in the range of 0.0952 - 1.9050 μg / mL, and for cortisone, it is in the range of 0.0952 - 1.9050 μg / mL. The peak area has a good linear relationship with the measured concentration.

[0149] 5. Injection precision

[0150] Prepare a mixed solution containing 5 μg of impurity A, 5 μg of impurity B, 5 μg of impurity D, 5 μg of impurity F, 5 μg of impurity G, 6 μg of impurity C, 30 μg of impurity E, 10 μg of INT1, 10 μg of Intermediate 2, 2 μg of SM1 - 2, 1 μg of SM - N, 1 μg of SM - N1 - 1 per 1 mL, and an impurity solution containing 1 μg of cortisone per 1 mL. Take 20 μL and inject it into the liquid chromatograph respectively, and inject continuously for 6 times, and record the peak area. The results are shown in Table 25 below.

[0151] Table 23: Results of Injection Precision Test

[0152]

[0153] As can be seen from Table 25, after injecting continuously for 6 times, the injection precision of hydrocortisone acetate intermediate 2 and each impurity is good.

[0154] 6. Solution Stability

[0155] 1) Stability of Impurity Solution

[0156] Take the injection precision samples of impurities A, B, C, D, E, F, G, INT1, intermediate 2, SM1-2, SM-N, SM-N1-1 and inject them at 0, 3, 6, 9, 15, 21, 30 hours respectively; take the injection precision samples of cortisone and inject them at 0, 6, 12, 18, 21 hours respectively to investigate its within-day stability. The results are as Figure 4 shown in Table 26 and Table 27.

[0157] Table 24: Results of Impurity Solution Stability Test

[0158]

[0159] Table 25: Results of Impurity Solution Stability Test

[0160] 0h 6h 12h 18h 21h Average RSD Cortisone 0.69069 0.68174 0.69315 0.68633 0.67972 0.68633 0.83％

[0161] From Figure 4 Table 26 - 27, it can be seen that impurities A, B, C, D, E, F, G, INT1, intermediate 2, SM1-2, SM-N, SM-N1-1 have good stability in the solvent within 30 hours, and cortisone has good stability in the solvent within 21 hours.

[0162] 2) Stability of Test Solution

[0163] Take hydrocortisone acetate intermediate 2 and prepare a 1mg / mL test solution, and inject it at 0, 12, 24, 42, 62, 74, 92, 110 hours after preparation (calculated by area normalization method) to investigate the change of its impurities. The results are as Figure 5 shown in Table 28 - 29.

[0164] Table 26: Results of Test Solution Stability Test (Normalization Method)

[0165]

[0166] Table 27: Results of Test Solution Stability Test (Normalization Method)

[0167]

[0168] From Figure 5 and Table 28 - 29, it can be seen that there is no obvious change in the test solution within 110 hours, and the test solution is relatively stable.

[0169] 7. Repeatability

[0170] Take an appropriate amount of the test sample of Intermediate 2, dissolve it with a diluent and quantitatively dilute it to the mark to prepare a solution containing 1.0 mg per 1 mL as the test solution; transfer 1 mL of the test solution to a 100 - mL volumetric flask and dilute it to the mark with the diluent as the self - reference solution. Repeat the determination of 6 test samples. The results are shown in Table 30 below.

[0171] Table 30: Results of Repeatability Test

[0172]

[0173] It can be seen from Table 30 that the sample is repeatedly determined 6 times, and the repeatability is good.

[0174] 8. Recovery Rate of Spiked Samples

[0175] 1) Recovery Rate of Spiked Samples 1

[0176] Take appropriate amounts of each impurity reference substance, dissolve and dilute it with a diluent to prepare a mixed solution containing 25 μg of impurity A, 25 μg of impurity B, 25 μg of impurity D, 25 μg of impurity F, 25 μg of impurity G, 30 μg of impurity C, 150 μg of impurity E, 50 μg of INT1, 10 μg of SM1 - 2, 5 μg of SM - N, and 5 μg of SM - N1 - 1 per 1 mL as the recovery reserve solution. Precisely pipette the above - mentioned mixed reserve solution and dilute it according to Table 37 to prepare solutions at 50%, 100%, and 150% concentrations, with 3 replicates for each concentration. Prepare the recovery solutions according to Table 31.

[0177] Table 28: Preparation of Recovery Solutions

[0178]

[0179] Transfer 1 mL of each recovery solution to a 100 - mL volumetric flask and dilute it to the mark with the diluent as the self - reference solution. Pipette 20 μL of each of the above - mentioned samples for injection, record the peak areas of each known impurity, and calculate the recovery rates and RSDs of each known impurity. The results are shown in Tables 32 - 42 below.

[0180] Table 29: Experimental Results of Recovery Rate of IMP - A

[0181]

[0182] Table 30: Experimental Results of Recovery Rate of IMP - B

[0183]

[0184]

[0185] Table 31: Results of the spike recovery experiment for IMP-C

[0186]

[0187] Table 32: Results of the spike recovery experiment for IMP-D

[0188]

[0189] Table 33: Results of the spike recovery experiment for IMP-E

[0190]

[0191]

[0192] Table 34: Results of the spike recovery experiment for IMP-F

[0193]

[0194] Table 35: Results of the spike recovery experiment for IMP-G

[0195]

[0196]

[0197] Table 36: Results of the spike recovery experiment for INT1

[0198]

[0199] Table 40: Results of the spike recovery experiment for SM1-2

[0200]

[0201] Table 37: Results of the spike recovery experiment for SM-N

[0202]

[0203]

[0204] Table 38: Results of the spike recovery experiment for SM-N1-1

[0205]

[0206] Where: Original amount = Sampling weight × Impurity content; Measured added amount = Measured total amount - Original amount; Recovery rate = (Measured added amount / Added amount) × 100%;

[0207] As can be seen from Table 32 - 42, the average value of the recovery rate of impurity A by sample addition is 93.9%, and the RSD value is 1.0%; the average value of the recovery rate of impurity B by sample addition is 98.7%, and the RSD value is 0.7%; the average value of the recovery rate of impurity C by sample addition is 94.5%, and the RSD value is 1.1%; the average value of the recovery rate of impurity D by sample addition is 94.0%, and the RSD value is 0.9%; the average value of the recovery rate of impurity E by sample addition is 98.5%, and the RSD value is 2.7%; the average value of the recovery rate of impurity F by sample addition is 95.1%, and the RSD value is 1.1%; the average value of the recovery rate of impurity G by sample addition is 94.2%, and the RSD value is 1.0%; the average value of the recovery rate of INT1 by sample addition is 94.8%, and the RSD value is 1.1%; the average value of the recovery rate of SM1 - 2 by sample addition is 94.9%, and the RSD value is 0.9%; the average value of the recovery rate of SM - N by sample addition is 91.0%, and the RSD value is 0.6%; the average value of the recovery rate of SM - N1 - 1 by sample addition is 92.5%, and the RSD value is 1.2%. The recovery rates are within the range of 90% - 110%, and the RSDs are all less than 10%.

[0208] 2) Recovery rate by sample addition 2

[0209] Take appropriate amounts of each impurity reference substance, dissolve and dilute with a diluent to prepare a mixed solution containing 5 μg of impurity cortisone per 1 mL as the recovery stock solution. Precisely pipette the above - mentioned mixed stock solution and dilute it according to Table 43 below to prepare solutions at 50%, 100%, and 150%. Prepare 3 parallel portions for each concentration.

[0210] Table 39: Preparation of recovery solutions

[0211]

[0212] Pipette 1 mL of each recovery solution into a 100 - mL volumetric flask, dilute to the mark with a diluent as the self - reference solution. Pipette 20 μL of each of the above - mentioned samples for injection, record the peak areas of each known impurity, and calculate the recovery rates and RSDs of each known impurity. The results are shown in Table 44 below.

[0213] Table 40: Experimental results of the recovery rate of cortisone by sample addition

[0214]

[0215] Among them: Original amount = Sampling amount × Impurity content; Measured added amount = Measured total amount - Original amount; Recovery rate = (Measured added amount / Added amount) × 100%;

[0216] As can be seen from Table 44, the average value of the recovery rate of cortisone by sample addition is 104.2%, and the RSD value is 1.9%. The results are all within the acceptable range.

[0217] 9. Durability

[0218] The durability of the method for determining the impurity content in hydrocortisone acetate intermediate 2 was verified mainly from four aspects: different flow rates, different mobile phase ratios, different column temperatures, and different chromatographic columns. The results are as Figure 6 shown in Tables 45 - 46.

[0219] Table 41: Resolution results of the durability test

[0220]

[0221] Table 42: Sampling results of the durability test

[0222]

[0223]

[0224] From Figure 6 the durability test results in Tables 45 - 46, it can be seen that the chromatographic conditions of this method have good durability after fine-tuning of different mobile phase ratios, different flow rates, and different column temperatures.

[0225] 10. Intermediate precision

[0226] The related substance inspection of the same batch of samples was carried out by the same operator at different times according to the repeatability test method; by different operators at different times according to the repeatability test method; and by the same operator on different instruments at different times for the same batch of samples. The results are shown in Table 47 below.

[0227] Table 43: Results of the intermediate precision test

[0228]

[0229]

[0230] It can be seen from Table 47 that the intermediate precision is good under these chromatographic conditions.

[0231] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for determining the impurity content in hydrocortisone acetate intermediate 2 by HPLC, characterized in that, It includes the following steps: 1) Prepare the test solution and the reference solution; The preparation process of the test solution is as follows: Accurately measure the test sample of hydrocortisone acetate intermediate 2, and dilute it with a diluent to a solution containing 1 mg of hydrocortisone acetate intermediate 2 per 1 mL as the test solution; The preparation process of the reference solution is as follows: Accurately measure the test solution, and dilute it with a diluent to a solution containing 10 μg of hydrocortisone acetate intermediate 2 per 1 mL as the reference solution; 2) Set the high-performance liquid chromatography detection conditions: Use a chromatographic column filled with octadecylsilyl bonded silica gel. The mobile phase consists of phase A and phase B, where phase A is water and phase B is acetonitrile, and gradient elution is carried out; 3) Respectively and accurately measure the test solution and the reference solution, inject them into the liquid chromatograph, record the chromatogram, plot the standard curve, obtain the standard curve equation, and calculate the content of impurities in hydrocortisone acetate intermediate 2; The impurities in the hydrocortisone acetate intermediate 2 include impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity INT1, impurity SM1-2, cortisone, impurity SM-N, and impurity SM-N1-1; impurity A is C 21 H 30 O5, impurity B is C 21 H 30 O4, impurity C is C 23 H 30 O6, impurity D is C 23 H 30 O6, impurity E is C 23 H 30 O5, impurity F is C 23 H 32 O6, impurity G is C 25 H 34 O7, impurity INT1 is C 23 H 31 B r O6, impurity SM1-2 is C 19 H 24 O2, impurity SM-N is C 21 H 27 ClO3, impurity SM-N1-1 is C 21 H 28 O3, cortisone is C 21 H 28 O5.

2. The method for determining the impurity content in hydrocortisone acetate intermediate 2 by HPLC according to claim 1, characterized in that, In step 1), the diluent is a methanol-water-glacial acetic acid solution, where the volume ratio of methanol, water, and glacial acetic acid is 90:10:

1.

3. The method for determining the impurity content in hydrocortisone acetate intermediate 2 by HPLC according to claim 1, wherein In step 2), the gradient elution conditions are as follows: From 0 to 30 min, the volume percentage of mobile phase A is 75% and the volume percentage of mobile phase B is 25%; from 30 to 45 min, the volume percentage of mobile phase A decreases from 75% to 60% and the volume percentage of mobile phase B increases from 25% to 40%; at 45 min, the volume percentage of mobile phase A is 60% and the volume percentage of mobile phase B is 40%; from 45 to 60 min, the volume percentage of mobile phase A is 60% and the volume percentage of mobile phase B is 40%; from 60 to 65 min, the volume percentage of mobile phase A decreases from 60% to 50% and the volume percentage of mobile phase B increases from 40% to 50%; at 65 min, the volume percentage of mobile phase A is 50% and the volume percentage of mobile phase B is 50%; from 65 to 80 min, the volume percentage of mobile phase A is 50% and the volume percentage of mobile phase B is 50%.

4. The method for determining the impurity content in hydrocortisone acetate intermediate 2 by HPLC according to claim 1, wherein, In step 2), the chromatographic column is Agilent ZORBAX Eclipse Plus C18, with a specification of 250 nm × 4.6 mm, a packing particle size of 5 μm, and a column temperature of 30 °C.

5. The method for determining the impurity content in hydrocortisone acetate intermediate 2 by HPLC according to claim 1, characterized in that, In step 2), the gradient elution flow rate is 1.0 mL / min.

6. The method for determining the impurity content in hydrocortisone acetate intermediate 2 by HPLC according to claim 1, characterized in that, In step 3), the injection volume is 20 μL.

7. The method for determining the impurity content in hydrocortisone acetate intermediate 2 by HPLC according to claim 1, wherein In step 3), the detection wavelength of the liquid chromatograph is 254 nm.

8. The method for determining the impurity content in hydrocortisone acetate intermediate 2 by HPLC according to claim 1, characterized in that, In step 3), according to the chromatogram, a linear regression equation is made with the peak area A as the ordinate and the concentration C as the abscissa. When the detection wavelength is 254 nm, the linear equation of impurity A is A = 0.7191C - 0.0122, the linear equation of impurity B is A = 0.7670C - 0.0096, the linear equation of impurity C is A = 0.7054C - 0.0125, the linear equation of impurity D is A = 0.5416C - 0.0081, the linear equation of impurity E is A = 0.6272C - 0.0660, the linear equation of impurity F is A = 0.6156C + 0.0019, the linear equation of impurity G is A = 0.5161C - 0.0098, the linear equation of impurity INT1 is A = 0.5559C - 0.0121, the linear equation of hydrocortisone acetate intermediate 2 is A = 0.6529C - 0.0188, the linear equation of impurity SM1-2 is A = 0.8779C - 0.0058, the linear equation of impurity SM-N is A = 0.7391C - 0.0071, the linear equation of impurity SM-N1-1 is A = 0.7750C - 0.0012, and the linear equation of cortisone is A = 0.6869C + 0.0084.

9. The method for determining the impurity content in hydrocortisone acetate intermediate 2 by HPLC according to claim 8, wherein The linear concentration range of impurity A is 0.1040 - 10.3987 μg / mL, the quantitation limit is 0.063 μg / mL, and the detection limit is 0.016 μg / mL; the linear concentration range of impurity B is 0.2562 - 10.2474 μg / mL, the quantitation limit is 0.126 μg / mL, and the detection limit is 0.031 μg / mL; the linear concentration range of impurity C is 0.1266 - 12.6587 μg / mL, the quantitation limit is 0.072 μg / mL, and the detection limit is 0.018 μg / mL; the linear concentration range of impurity D is 0.1160 - 11.6046 μg / mL, the quantitation limit is 0.062 μg / mL, and the detection limit is 0.015 μg / mL; the linear concentration range of impurity E is 0.6415 - 64.1528 μg / mL, the quantitation limit is 0.094 μg / mL, and the detection limit is 0.047 μg / mL; the linear concentration range of impurity F is 0.2579 - 10.3163 μg / mL, the quantitation limit is 0.125 μg / mL, and the detection limit is 0.031 μg / mL; the linear concentration range of impurity G is 0.2723 - 10.8911 μg / mL, the quantitation limit is 0.126 μg / mL, and the detection limit is 0.016 μg / mL; the linear concentration range of impurity INT1 is 0.1767 - 17.6697 μg / mL, the quantitation limit is 0.058 μg / mL, and the detection limit is 0.029 μg / mL; the linear concentration range of hydrocortisone acetate intermediate 2 is 0.1964 - 19.6413 μg / mL, the quantitation limit is 0.069 μg / mL, and the detection limit is 0.017 μg / mL; the linear concentration range of impurity SM1-2 is 0.1010 - 4.0398 μg / mL, the quantitation limit is 0.052 μg / mL, and the detection limit is 0.026 μg / mL; the linear concentration range of impurity SM-N is 0.0985 - 1.9709 μg / mL, the quantitation limit is 0.051 μg / mL, and the detection limit is 0.026 μg / mL; the linear concentration range of impurity SM-N1-1 is 0.0952 - 1.9050 μg / mL, the quantitation limit is 0.049 μg / mL, and the detection limit is 0.025 μg / mL; the linear concentration range of cortisone is 0.0989 - 1.9790 μg / mL, the quantitation limit is 0.099 μg / mL, and the detection limit is 0.025 μg / mL.

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