UPLC (Ultra Performance Liquid Chromatography) analysis method for beclomethasone propionate related substances

Abstract

The invention provides an analysis method of beclomethasone propionate related substances. According to the method, ultra-high performance liquid chromatography is adopted, and chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a chromatographic column of a filler; a mobile phase A is an aqueous solution containing phosphoric acid, a mobile phase B is a mixed solution containing tetrahydrofuran, acetonitrile and methanol, and a specific gradient elution procedure is adopted. According to the method, seven impurities in beclomethasone propionate can be separated and detected. The method is good in specificity, good in separation effect, high in sensitivity and accurate in quantification, meanwhile, the mobile phase is simple to prepare, the steps of using buffer salt and adjusting the pH value are avoided, the analysis time can be completed only by 10 min, the time is shortened by 83% compared with the prior art, the analysis efficiency is greatly improved, the solvent dosage is reduced, and the analysis cost is reduced. According to the method, the separation degree of beclomethasone propionate and adjacent impurities is remarkably improved, the detection sensitivity is improved by more than 3 times, and the requirements of beclomethasone propionate raw material medicine and preparation quality control can be met.

Description

Technical Field

[0001] The present invention relates to the technical field of drug detection, and particularly relates to an analytical method for related substances of beclometasone dipropionate. Background Art

[0002] Any discussion of the prior art throughout the specification should not be construed as an admission that such prior art is well known or forms part of the common general knowledge in the art.

[0003] Beclometasone dipropionate, chemical name: 9-chloro-11β,17,21-trihydroxy-16β-methyl-1,4-pregnadiene-3,20-dione 17,21-dipropionate, CAS No.: 5534-09-8, molecular formula: C 28 H 37 ClO7, molecular weight: 521.05, chemical structure is as follows:

[0004]

[0005] Beclometasone dipropionate is a synthetic glucocorticoid with potent anti-inflammatory and anti-allergic effects and has various clinical uses, mainly including:

[0006] Bronchial asthma: It can effectively control airway inflammation, relieve asthma symptoms, and reduce the frequency and severity of asthma attacks. It can be used in both children and adult asthma patients. For example, for patients with moderate to severe persistent asthma, beclometasone dipropionate is often inhaled for maintenance treatment to improve lung function and quality of life.

[0007] Chronic obstructive pulmonary disease (COPD): It is applicable to COPD patients with airway spasm and airway inflammation, can reduce airway inflammation, relieve symptoms such as dyspnea, cough, and expectoration, and improve the exercise tolerance and quality of life of patients.

[0008] Skin diseases: It is used to treat various non-infectious skin diseases, such as eczema, contact dermatitis, neurodermatitis, seborrheic dermatitis, and psoriasis, etc. It can reduce skin inflammatory reactions and relieve symptoms such as itching, redness, and exudation. For example, for patients with chronic eczema, topical application of beclometasone dipropionate cream can effectively control the condition and promote the healing of skin lesions.

[0009] Otorhinolaryngological diseases: In nasal sprays, beclometasone dipropionate can be used for the prevention and treatment of perennial and seasonal allergic rhinitis, vasomotor rhinitis, reduce nasal mucosal inflammation, and relieve symptoms such as nasal congestion, runny nose, and sneezing.

[0010] Due to the extensive clinical application of beclomethasone dipropionate, its quality control is particularly important. However, there are many deficiencies in the current detection methods for its related substances. The detection method for related substances of beclomethasone dipropionate in the 2020 edition of the Chinese Pharmacopoeia is thin-layer chromatography, which is only a limit test method and the impurity content cannot be accurately quantified, resulting in ineffective control of related substances in beclomethasone dipropionate-related preparations.

[0011] Although the European Pharmacopoeia (EP) uses a high-performance liquid chromatography (HPLC) method, it has disadvantages such as complex mobile phase preparation (requiring the use of potassium dihydrogen phosphate buffer salt and adjusting the pH value), too long running time (about 59 minutes), and high analysis cost. In addition, the resolution between the main component and adjacent impurity D in the EP method is relatively low, only 1.79, close to the lower limit of the system suitability requirements; the detection sensitivity is insufficient, and the signal-to-noise ratios of various impurities are relatively low, which may lead to inaccurate detection results for low-concentration impurities.

[0012] Therefore, it is necessary to establish a set of related substance detection and analysis methods with strong specificity, good repeatability, high accuracy, high sensitivity, simplicity and rapidity, so as to conduct comprehensive and accurate quality control of beclomethasone dipropionate and ensure the safety and effectiveness of its clinical use. Summary of the Invention

[0013] The purpose of the present invention is to provide an ultra-high performance liquid chromatography (UPLC) analysis method for related substances of beclomethasone dipropionate to solve the technical problems existing in the prior art.

[0014] The existing detection methods for related substances of beclomethasone dipropionate mainly have the following defects: thin-layer chromatography can only perform qualitative analysis and cannot accurately quantify; traditional high-performance liquid chromatography has problems such as complex mobile phase preparation (requiring the use of buffer salts and adjusting the pH value), long analysis time (usually about 1 hour), unsatisfactory resolution between the main component and adjacent impurities, and insufficient detection sensitivity, resulting in low analysis efficiency and high cost.

[0015] The UPLC analysis method provided by the present invention solves the above technical problems through systematic optimization of chromatographic conditions, especially the simplified design of the mobile phase composition, reasonable column temperature control, and efficient gradient elution program. The method of the present invention can not only significantly shorten the analysis time (only 10 minutes), but also greatly improve the resolution between the main component and adjacent impurities and the detection sensitivity. At the same time, the mobile phase preparation is simple, without the use of buffer salts and pH adjustment, further reducing the analysis cost and extending the service life of the chromatographic column.

[0016] Specifically, the present invention is realized through the following technical solutions.

[0017] The present invention provides an analytical method for beclomethasone dipropionate related substances, which is detected by ultra-high performance liquid chromatography. The chromatographic conditions include: the chromatographic column is filled with octadecylsilyl silica gel; mobile phase A is an aqueous solution containing phosphoric acid; mobile phase B is a mixed solution containing tetrahydrofuran, acetonitrile and methanol; gradient elution is adopted.

[0018] In some embodiments, mobile phase A is an aqueous solution of 0.05-0.2% phosphoric acid, more preferably an aqueous solution of 0.1% phosphoric acid. By using a simple aqueous phosphoric acid solution to replace the traditional phosphate buffer solution, the method of the present invention not only simplifies the preparation process of the mobile phase, but also avoids the damage of the chromatographic column by the buffer salt, and at the same time can obtain good peak shapes and separation effects.

[0019] In some embodiments, the volume ratio of tetrahydrofuran, acetonitrile and methanol in mobile phase B is (3-7):(20-26):(22-28), more preferably 5:23:25. The present invention optimizes the composition of the organic phase, so that it can not only provide good dissolution ability and elution ability, but also ensure peak shapes and separation effects.

[0020] In some embodiments, the gradient elution program includes the following steps:

[0021] Within 0 to 3 minutes, maintain the volume ratio of mobile phase A to mobile phase B at 40-50:50-60. This stage is mainly used to separate beclomethasone and impurity A, and impurity B starts to elute.

[0022] Within 3 to 3.5 minutes, quickly adjust the volume ratio of mobile phase A to mobile phase B from 40-50:50-60 to 65-75:25-35. This stage completes the elution of impurity B and adjusts the elution intensity.

[0023] Within 3.5 to 4.8 minutes, maintain the volume ratio of mobile phase A to mobile phase B at 65-75:25-35. This stage is mainly used to elute the main peak of beclomethasone dipropionate.

[0024] Within 4.8 to 5.5 minutes, quickly adjust the volume ratio of mobile phase A to mobile phase B from 65-75:25-35 to 25-35:65-75. This stage is a transition stage, increasing the proportion of the organic phase to improve the elution intensity and accelerate the elution of subsequent impurities.

[0025] Within 5.5 to 10 minutes, adjust and maintain the volume ratio of mobile phase A to mobile phase B at 40-50:50-60. This stage is used to elute impurity D, impurity L, impurity C and impurity F, and return the system to the initial conditions.

[0026] In some preferred embodiments, the gradient elution program includes:

[0027] Within 0 to 3 minutes, maintain the volume ratio of mobile phase A to mobile phase B at 45:55;

[0028] Within 3 to 3.5 minutes, adjust the volume ratio of mobile phase A to mobile phase B from 45:55 to 70:30;

[0029] Within 3.5 to 4.8 minutes, maintain the volume ratio of mobile phase A to mobile phase B at 70:30;

[0030] Within 4.8 to 5.5 minutes, adjust the volume ratio of mobile phase A to mobile phase B from 70:30 to 30:70;

[0031] Within 5.5 to 6 minutes, adjust and maintain the volume ratio of mobile phase A to mobile phase B at 45:55;

[0032] Within 6 to 10 minutes, maintain the volume ratio of mobile phase A to mobile phase B at 45:55 until the end of the program.

[0033] This optimized gradient elution program can significantly shorten the analysis time while ensuring good separation performance. Through the carefully designed changes in elution strength, it ensures that each component is separated and detected within an appropriate retention time range, avoiding component overlap or excessive broadening.

[0034] In some embodiments, the detection wavelength of the chromatographic conditions is 240 - 270 nm, more preferably 254 nm. This wavelength range is selected based on the ultraviolet absorption characteristics of beclomethasone dipropionate and its related impurities, which can provide the best detection sensitivity and signal-to-noise ratio.

[0035] In some embodiments, the flow rate of the chromatographic conditions is 0.2 - 0.6 mL / min, more preferably 0.4 mL / min. This flow rate range takes into account the characteristics of the UPLC system and the specifications of the chromatographic column used, which can not only ensure sufficient column efficiency and resolution but also reduce solvent consumption and system backpressure.

[0036] In some embodiments, the column temperature of the chromatographic column is 25 - 40 °C, more preferably 33 °C. The present invention uses a lower column temperature (compared with 50 °C in the prior art), which can not only ensure good separation performance but also reduce the adverse effects of high temperature on the chromatographic column and the analyte, and extend the service life of the chromatographic column.

[0037] In some embodiments, the chromatographic column is a C18 chromatographic column with a column length of 50 - 150 mm, an inner diameter of 1.5 - 3.0 mm, and a packing particle size of 1.5 - 5 μm; more preferably, it is a Hypersil GOLD chromatographic column with a column length of 100 mm, an inner diameter of 2.1 mm, and a packing particle size of 3 μm. The present invention uses a shorter chromatographic column and packing with a smaller particle size, giving full play to the advantages of the UPLC system and improving the separation efficiency and analysis speed.

[0038] In some embodiments, the injection volume is 3 - 7 μL, more preferably 5 μL. This injection volume range is suitable for the UPLC system and the specifications of the chromatographic column used, which can not only provide sufficient detection sensitivity but also avoid peak shape deformation and decreased resolution caused by sample overload.

[0039] In some embodiments, the related substances of beclomethasone dipropionate include at least one of the following seven impurities: impurity A, impurity B, impurity C, impurity D, impurity F, impurity L, and beclomethasone.

[0040]

[0041]

[0042] The method of the present invention can simultaneously detect these 7 key impurities, covering more impurity types than the prior art, and can more comprehensively evaluate the quality of beclomethasone dipropionate.

[0043] In some embodiments of the present invention, the resolution between beclomethasone dipropionate and the adjacent impurity is not less than 1.5, especially the resolution with impurity D can reach 9.6 (while it is only 1.79 in the prior art), greatly improving the reliability and robustness of the analysis method.

[0044] In some embodiments of the present invention, the signal-to-noise ratio (S / N) of impurity detection in the analysis method is greater than 150, and this sensitivity is more than 3 times that of the prior art, which can accurately detect impurities at lower concentrations and meet more stringent quality control requirements.

[0045] In some embodiments of the present invention, the analysis method further includes using a diluent, which is a mixed solution of mobile phase A and mobile phase B, where the volume ratio of mobile phase A to mobile phase B is (40 - 50):(50 - 60), more preferably 45:55. This diluent composition is similar to the initial mobile phase, which can avoid peak shape deformation and retention time shift caused by sample solvent effect.

[0046] According to another aspect of the present invention, the present invention also provides the application of the above-mentioned analysis method in the quality control of beclomethasone dipropionate bulk drug or preparation.

[0047] Specifically, the present invention provides a method for quality control of beclomethasone dipropionate bulk drug or preparation by applying the above analysis method, which is characterized by including:

[0048] Preparing a sample solution to be tested;

[0049] Performing detection by using the described analysis method;

[0050] Determining the content of each impurity in beclomethasone dipropionate according to the detection result.

[0051] This application expands the practical value of the method of the present invention, making it applicable not only to the quality control of beclomethasone dipropionate bulk drug, but also to the quality evaluation of various preparations (such as inhalants, sprays, creams, etc.). By accurately determining the content of each impurity, the quality of the drug can be comprehensively evaluated to ensure the safety and effectiveness of clinical medication.

[0052] Compared with the prior art, the application method of the present invention can detect impurities in beclomethasone dipropionate more comprehensively and accurately. Especially, it has significant advantages in detecting low-content impurities, can meet the increasingly strict requirements for drug quality control, and provides reliable technical support for the quality control of beclomethasone dipropionate and its preparations.

[0053] Compared with the prior art, the advantages of the present invention include:

[0054] 1) Simplified mobile phase and efficient analysis process: The method of the present invention is simple in preparing the mobile phase, without using buffer salts such as potassium dihydrogen phosphate and adjusting the pH value, saving the time-consuming weighing and pH adjustment steps; at the same time, the analysis time is shortened from about 59 minutes to 10 minutes, shortening 83% of the analysis time, greatly improving the analysis efficiency; reducing the solvent consumption by about 70%, reducing the waste liquid discharge, and being more environmentally friendly and economical.

[0055] 2) Excellent separation performance and detection sensitivity: By using the analysis method of the present invention, 7 impurities of beclomethasone dipropionate can be effectively detected, and the separation effects between each impurity, between the main component and the adjacent impurity are all good. Especially, the resolution between beclomethasone dipropionate and impurity D is increased from 1.79 to 9.6, significantly improving the robustness of the method; the signal-to-noise ratios of each impurity at the 0.2% concentration level all exceed 150, far higher than 50 or less of the prior art, and the detection sensitivity is increased by more than 3 times, and impurities with a concentration of 0.02% or even lower can be accurately measured, far lower than the reporting limits required by various pharmacopoeias.

[0056] 3) Superior methodological characteristics: When using the method of the present invention to detect beclomethasone dipropionate, the method has good specificity, qualified precision, the blank solvent does not interfere with the detection of the main component and impurity content, and reliable analysis results can be provided for quality control.

[0057] 4) Gentler analysis conditions and extended equipment lifespan: When detecting beclomethasone dipropionate using the method of the present invention, the mobile phase does not contain inorganic buffer salts, reducing the damage to the chromatographic column by buffer salts; the operating temperature is reduced from 50 °C to 33 °C, reducing the adverse effects of long-term high temperature on the chromatographic column and the analyte; these optimization measures not only improve the reliability of the analysis results, but also significantly extend the service life of the chromatographic column and other equipment, further reducing the analysis cost.

[0058] 5) More comprehensive quality control ability: The method of the present invention can simultaneously detect 7 key impurities, including beclomethasone, covering more impurity types than the prior art, and can more comprehensively evaluate the quality status of beclomethasone dipropionate, providing stronger technical support for drug quality control and consistency evaluation.

[0059] In summary, the present invention provides an efficient, sensitive, simple and economical method for analyzing related substances of beclomethasone dipropionate, overcoming multiple drawbacks of the prior art, significantly improving the analysis efficiency and detection ability, and providing a better technical solution for the quality control of beclomethasone dipropionate and its preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] The accompanying drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments and descriptions thereof of the present invention are used to explain the present invention and do not constitute an improper limitation of the present invention. Hereinafter, the embodiments of the present invention will be described in detail in conjunction with the drawings, wherein:

[0061] Figure 1 It is the chromatogram of the system suitability solution for Comparative Example 1 (EP pharmacopoeia method) of the present invention.

[0062] Figure 2 It is the chromatogram of the system suitability solution for Example 1 of the present invention.

[0063] Figure 3 It is the localization chromatogram of impurity A for Example 1 of the present invention.

[0064] Figure 4 It is the localization chromatogram of impurity B for Example 1 of the present invention.

[0065] Figure 5 It is the localization chromatogram of impurity C for Example 1 of the present invention.

[0066] Figure 6 It is the localization chromatogram of impurity D for Example 1 of the present invention.

[0067] Figure 7 It is the localization chromatogram of impurity F for Example 1 of the present invention.

[0068] Figure 8 It is the localization chromatogram of impurity L for Example 1 of the present invention.

[0069] Figure 9 This is the impurity beclomethasone positioning chromatogram of Example 1 of the present invention.

[0070] Figure 10 This is the specificity overlay map of Example 1 of the present invention, showing the complete separation of each impurity and the main peak.

[0071] In the above figures, "BLMS" represents beclomethasone, "BSBLMS" represents beclomethasone propionate, and A - F represent the corresponding impurities. Detailed Embodiments

[0072] The present invention will be further described in combination with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit its scope. Experimental methods without specific conditions noted in the embodiments are usually carried out according to conventional conditions or conditions recommended by the manufacturer.

[0073] Unless otherwise defined, all professional and scientific terms used in the present invention should have the meanings familiar to those skilled in the art. In the field of chromatographic analysis, "resolution" refers to the degree of separation between adjacent chromatographic peaks, usually required to be not less than 1.5; "signal - to - noise ratio (S / N)" refers to the ratio of the chromatographic peak signal to the baseline noise, reflecting the detection sensitivity.

[0074] In the present invention, the numerical ranges of chromatographic conditions (such as flow rate, column temperature, concentration, etc.) represent the acceptable variation ranges confirmed during the validation of the analytical method. Within this range, the key performance indicators of the method can meet the analytical requirements.

[0075] Unless otherwise specified, the reagents or raw materials used in the present invention can be obtained through conventional channels and used according to the conventional methods in the art or the product instructions. The beclomethasone propionate reference substance and each related impurity reference substance used should meet the quality specifications required by the pharmacopoeia or relevant standards. In addition, any content similar or equivalent to the described method or material can be applied to the method of the present invention. The preferred embodiments and materials described in the present invention are only for illustrative purposes.

[0076] Without conflict, the embodiments in this article and the features in the embodiments can be combined with each other to obtain the best analytical effect.

[0077] Description of Main Substances and Impurities

[0078] The chemical information of beclomethasone propionate and its related impurities involved in the present invention is as follows:

[0079]

[0080] Instruments and Equipment: The main instruments and equipment used in the method of the present invention include: ultra-high performance liquid chromatograph (equipped with a diode array detector or ultraviolet detector); chromatographic data processing system; electronic balance (accuracy 0.01 mg); ultrasonic cleaner; pH meter (accuracy 0.01); microinjector; conventional laboratory glassware.

[0081] Reagents and Materials: Beclomethasone dipropionate reference substance (purity ≥ 99.0%); impurity A, impurity B, impurity C, impurity D, impurity F, impurity L, and beclomethasone reference substance (purity ≥ 95.0%); phosphoric acid (analytical pure, ≥ 85.0%); tetrahydrofuran (chromatographically pure, ≥ 99.9%); acetonitrile (chromatographically pure, ≥ 99.9%); methanol (chromatographically pure, ≥ 99.9%); ultrapure water (resistivity ≥ 18.2 MΩ·cm); potassium dihydrogen phosphate (analytical pure, ≥ 99.0%, for the comparative example).

[0082] General scheme of the analysis method of the present application

[0083] The method for analyzing related substances of beclomethasone dipropionate provided in this application is based on ultra-high performance liquid chromatography (UPLC) technology and uses the principle of reverse-phase chromatography for separation. This method is applicable to the detection and quantitative analysis of related substances in beclomethasone dipropionate raw materials and their preparations.

[0084] 1. Chromatographic Separation Principle

[0085] This method uses the principle of reverse-phase chromatography to achieve the separation of different polar compounds by the interaction between a non-polar stationary phase and a polar mobile phase. The chromatographic column uses octadecylsilane-bonded silica gel (C18) as the packing material, with a column length of 50 - 150 mm, an inner diameter of 1.5 - 3.0 mm, and a packing particle size of 1.5 - 5 μm. Such a stationary phase has moderate hydrophobicity and is particularly suitable for separating steroid structure drugs and their related impurities.

[0086] 2. Mobile Phase System Design:

[0087] The mobile phase uses a binary system:

[0088] Mobile phase A is an aqueous solution of 0.05 - 0.2% phosphoric acid, providing a moderate acidic environment to inhibit the dissociation of silanol groups and improve the peak shape.

[0089] Mobile phase B is a mixed organic solution with a volume ratio of tetrahydrofuran - acetonitrile - methanol of (3 - 7):(20 - 26):(22 - 28), comprehensively using the different selectivities of the three organic solvents to optimize the separation selectivity.

[0090] 3. Gradient Elution Strategy

[0091] A multi-stage gradient elution program is adopted: maintain a medium-strength elution condition (A:B = 40 - 50:50 - 60) at the beginning stage; then increase the proportion of the aqueous phase to 65 - 75% to optimize the separation of the main peak; in the later stage, increase the strength of the organic phase to 65 - 75% to accelerate the elution of the post-peak impurities; finally, restore the initial conditions to balance the system. This gradient design significantly shortens the analysis time while ensuring sufficient separation.

[0092] 4. Detection Techniques and Key Operating Parameters

[0093] Ultraviolet absorption detection is used, and the detection wavelength is 240 - 270 nm, corresponding to the characteristic absorption region of the α,β-unsaturated ketone structure in the steroid skeleton of beclomethasone propionate. Chromatographic analysis is carried out under the conditions of a column temperature of 25 - 40 °C and a flow rate of 0.2 - 0.6 mL / min, and the injection volume is 3 - 7 μL.

[0094] 5. General Operating Procedures

[0095] The implementation of this analytical method generally includes the following steps:

[0096] (1) Chromatographic system preparation: Install the chromatographic column into the UPLC system and balance the chromatographic column to a stable baseline according to the set conditions.

[0097] (2) Solution preparation:

[0098] Prepare mobile phase A and mobile phase B

[0099] Prepare the diluent by mixing mobile phase A and mobile phase B in proportion

[0100] Prepare the system suitability solution to contain 1 mg / mL of beclomethasone propionate and about 2 μg / mL of each impurity

[0101] Prepare the test solution according to the sample type so that the concentration of beclomethasone propionate is about 1 mg / mL.

[0102] (3) System suitability test: Inject the system suitability solution and confirm that the following key parameters meet the requirements:

[0103] The resolution between the main peak and the adjacent impurity is not less than 1.5, especially the resolution between beclomethasone propionate and impurity D;

[0104] The signal-to-noise ratio of the main impurity peaks should be high enough to ensure the detection sensitivity;

[0105] The peak shape is good, and the tailing factor is within the acceptable range.

[0106] (4) Sample analysis: Inject the test solution for analysis, record the chromatogram, and identify each impurity peak.

[0107] (5) Data processing: Calculate the content of each impurity based on the chromatographic analysis results. Those skilled in the art can calculate the impurity content according to conventional analysis methods, such as area normalization method or external standard method. For example, the relative content of impurities can be expressed as the percentage of the impurity peak area to the main peak area; or refer to the external standard method commonly used in the field of pharmaceutical analysis, and consider the differences in standard product concentration and response factor for more accurate quantitative calculation.

[0108] 6. Method robustness parameter range

[0109] Through systematic method validation experiments, it has been confirmed that this method can obtain reliable and consistent analysis results within the following narrower parameter variation ranges:

[0110] Flow rate: 0.38 - 0.42 mL / min

[0111] Column temperature: 31 - 35 °C

[0112] Concentration of phosphoric acid in mobile phase A: 0.08 - 0.12%

[0113] Initial mobile phase composition (A:B): 43:57 - 47:53

[0114] Detection wavelength: 252 - 256 nm

[0115] These parameter ranges are determined based on the results of the robustness tests during method validation and represent the acceptable parameter fluctuation ranges during actual analysis. Within this range, key performance indicators (such as resolution, peak shape, and detection sensitivity) can meet the analysis requirements, ensuring the accuracy and reliability of the results. In actual applications, the most suitable conditions should be selected within the above parameter ranges according to laboratory conditions and instrument characteristics, and the system performance should be confirmed through system suitability tests.

[0116] Comparative Example 1 EP Pharmacopoeia detection method

[0117] 1. Chromatographic conditions

[0118] Mobile phase A: 2.72 g / L potassium dihydrogen phosphate (adjusted to pH 2.35 with phosphoric acid)

[0119] Preparation method: Weigh 2.72 g of potassium dihydrogen phosphate, dissolve it in 1000 mL of water, and adjust the pH to 2.35 with phosphoric acid.

[0120] Mobile phase B: Tetrahydrofuran - acetonitrile - methanol (5:23:25)

[0121] Preparation method: Measure 5 parts of tetrahydrofuran, 23 parts of acetonitrile, and 25 parts of methanol (by volume ratio), and mix them evenly.

[0122] Diluent: Mobile phase A - mobile phase B (45:55, V / V)

[0123] Preparation method: Mobile phase A and mobile phase B are mixed evenly at a volume ratio of 45:55.

[0124] Chromatographic column: Caprisil C18-TH 250mm * 4.6mm, 5μm;

[0125] Detection wavelength is 254nm;

[0126] Flow rate is 1.4 mL / min;

[0127] Column temperature is 50°C;

[0128] Injection volume is 20 μL.

[0129] Elution method: Gradient elution, the program is as follows:

[0130]

[0131] 2. Solution preparation

[0132] Diluent: Mobile phase A - Mobile phase B (45:55, V / V)

[0133] Each impurity localization solution: Take appropriate amounts of beclomethasone dipropionate reference substance, impurity A, impurity B, impurity C, impurity D, impurity F, impurity L, and beclomethasone reference substance, place them in different volumetric flasks respectively, and dissolve them with the diluent to prepare solutions containing 0.1 mg / mL of beclomethasone dipropionate and other impurities.

[0134] System suitability solution: Take appropriate amounts of beclomethasone dipropionate reference substance, impurity A, impurity B, impurity C, impurity D, impurity F, impurity L, and beclomethasone reference substance, place them in the same volumetric flask, and dissolve them with the diluent to prepare a mixed solution containing 1 mg / mL of beclomethasone dipropionate and 2 μg / mL of other impurities.

[0135] Test solution: Take appropriate amount of beclomethasone dipropionate raw material, and dissolve it with the diluent to prepare a solution containing 1 mg / mL of beclomethasone dipropionate.

[0136] 3. Experimental results

[0137] The chromatographic results of the system suitability solution are shown in Table 1, and the chromatogram is shown in Figure 1 .

[0138] Table 1 Results of system suitability solution

[0139]

[0140] Note: " / " indicates not applicable. The resolution and signal-to-noise ratio (S / N) in Table 1 are calculated according to the standard method of the United States Pharmacopeia (USP).

[0141] From Figure 1 and the data in Table 1, the following key problems exist when using the EP Pharmacopoeia method for detection:

[0142] (1) Critical resolution: Although the resolution of each impurity meets the basic requirements (a resolution of ≥1.5 is considered complete peak separation), the resolution between the main component and the adjacent impurity D is only 1.79, close to the critical value. This means that when there are minor changes in the chromatographic conditions (such as fluctuations in the mobile phase composition, temperature changes, etc.), the resolution may drop below 1.5, resulting in insufficient method robustness.

[0143] (2) Insufficient detection sensitivity: The S / N values of impurities D, L, C, and F at the current concentration level (0.2%) are only 41.9, 39.6, 26.2, and 21.0 respectively, all of which do not exceed 50. This indicates that when the impurity content is below 0.2%, the detection sensitivity will be severely insufficient, which may lead to a significant reduction in quantitative accuracy and even the inability to detect extremely low levels of impurities.

[0144] (3) Poor peak shape: There are obvious peak shape problems for multiple impurity peaks. In particular, impurity D is located at the shoulder of the main peak, and its peak shape is affected by the main peak; the peak shapes of impurities L, C, and F are flat and have obvious tailing, with a low peak height / area ratio, indicating a wide and asymmetric peak. These peak shape problems will directly affect the quantitative accuracy, especially for low-concentration impurities.

[0145] (4) Excessive analysis time: According to Figure 1 shows that the complete analysis time requires approximately 43 minutes, with low efficiency. This not only reduces the sample detection throughput but also increases the solvent consumption and analysis cost.

[0146] In summary, although the EP Pharmacopoeia method can be used for the detection of related substances in beclomethasone dipropionate, there are obvious deficiencies in terms of resolution, detection sensitivity, peak shape, and analysis time, and it urgently needs to be optimized and improved.

[0147] Example 1 Analysis method of the present invention

[0148] 1. Chromatographic conditions

[0149] Mobile phase A: 0.1% phosphoric acid in water.

[0150] Preparation method: Measure 1.0 mL of phosphoric acid, dilute it to 1000 mL with ultrapure water, and shake well.

[0151] Mobile phase B: Tetrahydrofuran - acetonitrile - methanol (5:23:25, V / V / V)

[0152] Preparation method: Measure 5 parts of tetrahydrofuran, 23 parts of acetonitrile, and 25 parts of methanol (by volume ratio), and mix well.

[0153] Chromatographic column: Hypersil GOLD, 100 mm × 2.1 mm, 3 μm;

[0154] Elution method: gradient elution, the program is as follows:

[0155]

[0156]

[0157] The detection wavelength is 254 nm;

[0158] The flow rate is 0.40 mL / min;

[0159] The column temperature is 33 °C;

[0160] The injection volume is 5 μL.

[0161] 2. Solution preparation

[0162] Diluent: Mobile phase A - Mobile phase B (45:55, V / V)

[0163] Method for preparing diluent: Mix mobile phase A and mobile phase B evenly according to the volume ratio of 45:55.

[0164] Solutions for impurity localization: Use the solution prepared in Comparative Example 1.

[0165] System suitability solution: Use the solution prepared in Comparative Example 1.

[0166] Test solution: Use the solution prepared in Comparative Example 1.

[0167] 3. Experimental results

[0168] The chromatographic results of the system suitability solution are shown in Table 2, and the chromatogram is shown in Figure 2 , and the impurity localization chromatogram is shown in Figure 3 (Impurity A), Figure 4 (Impurity B), Figure 5 (Impurity C), Figure 6 (Impurity D), Figure 7 (Impurity F), Figure 8 (Impurity L), Figure 9 (Beclomethasone), and the specificity overlay map is shown in Figure 10 .

[0169] Table 2 System suitability and impurity localization results

[0170]

[0171]

[0172] Note: " / " indicates not applicable. The Resolution, Tailing, Plate Count, and Signal-to-Noise Ratio (S / N) in Table 2 were calculated according to the standard method of the United States Pharmacopeia (USP).

[0173] Figure 2 And Table 2 shows the analysis results of the system suitability solution of the UPLC method of the present invention, which are interpreted in detail as follows:

[0174] (1) Excellent separation effect: This method achieves complete separation of all components. Notably, the resolutions between the main peak (BSBLMS) and adjacent impurities B and D are as high as 6.1 and 9.6 respectively, far exceeding the USP requirement of 1.5 critical value, indicating that the method has extremely strong robustness. Even when the chromatographic conditions fluctuate, complete separation of each component can be ensured.

[0175] (2) Significantly improved detection sensitivity: The USP signal-to-noise ratios (S / N) of all impurities are significantly improved compared to the EP method: impurity D increased from 41.9 to 179.5 (increased by about 4 times), impurity L increased from 39.6 to 505.1 (increased by about 13 times), impurity C increased from 26.2 to 413.9 (increased by about 16 times), and impurity F increased from 21.0 to 361.7 (increased by about 17 times). This improvement means that the method of the present invention can accurately detect and quantify impurities with a concentration as low as 0.02%, far exceeding the detection capabilities of existing methods.

[0176] (3) Optimized peak shape quality: The data in Table 2 show that the USP tailing factors of all peaks are between 1.0 - 1.5, indicating good peak symmetry. Compared with Figure 1 the obvious tailing of multiple impurity peaks in [reference], the peak shape quality of this method has been significantly improved, which is crucial for accurate quantification.

[0177] (4) Efficient separation system: According to the USP Plate Count (the number of theoretical plates) data, multiple components show extremely high plate counts (such as impurity L reaching 132797 and impurity C reaching 136764), reflecting good separation efficiency. This is the result of combining UPLC technology with optimized chromatographic conditions.

[0178] (5) Significantly shortened analysis time: The complete analysis only takes about 8 minutes, which is about 83% shorter than the 43 minutes of the EP method. This improvement has significantly improved the analysis efficiency, reduced solvent consumption and analysis costs. All key impurities are separated and detected within 8 minutes while maintaining excellent separation effects.

[0179] (6) Similar component content distribution: The impurity content distribution (% Area) is similar to the EP method. The main peak accounts for 88.79%, and each impurity is between 1.37% - 2.03%, ensuring the consistency of the system suitability solution and making the results of the two methods comparable.

[0180] (7) More reasonable elution order: The relative retention time distribution of each component is more uniform, avoiding Figure 1 the serious tailing problem of late-eluting impurities (C, F) in the middle and late stages. The main impurities are concentrated within an appropriate retention time window, which not only ensures sufficient separation but also avoids excessive extension of the analysis time.

[0181] (8) Simplified mobile phase system: By using a simple 0.1% phosphoric acid aqueous solution to replace the complex potassium dihydrogen phosphate buffer solution (pH 2.35), it avoids the damage of the buffer salt to the chromatographic column and eliminates the pH adjustment step, significantly simplifying the operation process and improving the reliability and practicability of the method.

[0182] (9) Mild operating conditions: The operating temperature is reduced from 50°C to 33°C, reducing the adverse effects of long-term high temperature on the stationary phase of the chromatographic column and the analyte, extending the service life of the chromatographic column, and at the same time improving the stability and repeatability of the method.

[0183] (10) Resource conservation: Due to the significant reduction in analysis time and flow rate (from 1.4 mL / min to 0.4 mL / min), this method greatly reduces the solvent consumption, lowers the analysis cost, and is more environmentally friendly and economical. At the same time, the extended service life of the chromatographic column further reduces the analysis cost.

[0184] In summary, the UPLC method of this application has achieved an all-round technical improvement compared with the EP pharmacopoeia method. It not only has significant advantages in analytical performance (resolution, sensitivity, peak shape), but also has obvious improvements in practicability (simple operation, short analysis time) and economy (solvent conservation, extended column life). These advantages enable this method to analyze the related substances in beclomethasone dipropionate more accurately, quickly, and economically, providing more reliable technical support for drug quality control.

[0185] The above are only the preferred embodiments of the present invention and are not used to limit the scope of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still make various modifications to the technical solutions or equivalent replacements of some technical features after reading this specification. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall be regarded as falling within the protection scope of the present invention.

Claims

1. A method for analyzing related substances of beclomethasone dipropionate, characterized in that: The method adopts ultra-high performance liquid chromatography, and the chromatographic conditions include: Chromatographic column: Octadecylsilane bonded silica gel is used as filler; Mobile phase A: aqueous solution containing phosphoric acid; Mobile phase B: a mixed solution containing tetrahydrofuran, acetonitrile and methanol; Elution mode: gradient elution.

2. The analysis method according to claim 1, characterized in that The beclomethasone propionate related substances include at least one of the following impurities: impurity A, impurity B, impurity C, impurity D, impurity F, impurity L and beclomethasone; 3. The analysis method according to claim 1, characterized in that The mobile phase A is a 0.05-0.2% phosphoric acid aqueous solution; preferably, the mobile phase A is a 0.1% phosphoric acid aqueous solution.

4. The analysis method according to claim 1, characterized in that The volume ratio of tetrahydrofuran, acetonitrile and methanol in the mobile phase B is (3-7):(20-25):(22-28); preferably, the volume ratio of tetrahydrofuran, acetonitrile and methanol in the mobile phase B is 5:23:

25.

5. The analysis method according to any one of claims 1 to 4, characterized in that The gradient elution procedure includes: From 0 to 3 minutes, the volume ratio of mobile phase A to mobile phase B was maintained at 40-50:50-60; Adjust the volume ratio of mobile phase A to mobile phase B from 40-50:50-60 to 65-75:25-35 within 3 to 3.5 minutes; From 3.5 to 4.8 minutes, the volume ratio of mobile phase A to mobile phase B was maintained at 65-75:25-35; The volume ratio of mobile phase A to mobile phase B was adjusted from 65-75:25-35 to 25-35:65-75 within 4.8 to 5.5 minutes; Within 5.5 to 10 minutes, the volume ratio of mobile phase A to mobile phase B was adjusted and maintained at 40-50:50-60; Preferably, the gradient elution procedure comprises: From 0 to 3 minutes, the volume ratio of mobile phase A to mobile phase B was maintained at 45:55; The volume ratio of mobile phase A to mobile phase B was adjusted from 45:55 to 70:30 within 3 to 3.5 minutes; The volume ratio of mobile phase A to mobile phase B was maintained at 70:30 from 3.5 to 4.8 minutes; The volume ratio of mobile phase A to mobile phase B was adjusted from 70:30 to 30:70 within 4.8 to 5.5 minutes; The volume ratio of mobile phase A to mobile phase B was adjusted and maintained at 45:55 within 5.5 to 6 minutes; The volume ratio of mobile phase A to mobile phase B was maintained at 45:55 for 6 to 10 minutes until the end of the program.

6. The analysis method according to any one of claims 1 to 5, characterized in that The chromatographic column is a C18 chromatographic column with a column length of 50-150 mm, an inner diameter of 1.5-3.0 mm, and a filler particle size of 1.5-5 μm; Preferably, the chromatographic column is a Hypersil GOLD chromatographic column with a column length of 100 mm, an inner diameter of 2.1 mm, and a filler particle size of 3 μm; Preferably, the detection wavelength of the chromatographic conditions is 240-270 nm; Preferably, the detection wavelength of the chromatographic conditions is 254 nm.

7. The analysis method according to any one of claims 1 to 6, characterized in that The flow rate of the chromatographic conditions is 0.2-0.6 mL / min; Preferably, the flow rate of the chromatographic conditions is 0.4 mL / min; Preferably, the column temperature of the chromatographic column is 25-40°C, preferably 33°C.

8. The analysis method according to any one of claims 1 to 7, characterized in that The analysis method also includes using a diluent, wherein the diluent is a mixture of mobile phase A and mobile phase B, wherein the volume ratio of mobile phase A to mobile phase B is (40-50):(50-60); preferably, the volume ratio of mobile phase A to mobile phase B in the diluent is 45:

55.

9. The analysis method according to any one of claims 1 to 8, characterized in that: The analytical method allows the separation degree of impurity D and beclomethasone dipropionate to be no less than 1.5; Preferably, the impurity detection signal-to-noise ratio (S / N) of the analytical method is greater than 150.

10. Use of the analytical method according to any one of claims 1 to 9 in the quality control of beclomethasone dipropionate bulk drug or preparation.

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