In-vitro release method of glucocorticoid compound ointment

Abstract

The invention relates to the technical field of drug performance testing, and discloses an in-vitro release method of glucocorticoid compound ointment, which comprises the step of performing experiments by adopting a liquid chromatograph and a diffuser. According to the in-vitro release method of the glucocorticoid compound ointment, the same in-vitro release method is adopted to research a compound preparation, so that the operation process is simplified, the time and data problems caused by method difference are reduced, and the analysis efficiency is effectively improved; a set of liquid phase detection method is used for analyzing the compound preparation, so that repeated debugging and frequent switching of detection programs are avoided, the equipment performance is fully excavated, and the liquid phase analysis efficiency is improved; and the method can meet the requirements on sensitivity and distinguishing power of the two components, can accurately discriminate subtle changes of different components, and provides reliable data support for quality control, efficacy evaluation and the like of the compound preparation.

Description

Technical Field

[0001] The present invention relates to the technical field of drug performance testing, and specifically provides an in vitro release method for a compound glucocorticoid ointment. Background Art

[0002] The in vitro release test is one of several important means to evaluate the dosage forms and formulations of semi-solid preparations (such as creams, ointments, gels, etc.). The degree and rate of in vitro release are comprehensive manifestations of drug performance (including the solubility of active ingredients, particle size, and rheology of the dosage form, etc.), and it is one of the standard methods to characterize the preparation characteristics of topical drugs, which can reflect the physical and chemical changes in semi-solid drugs. The in vitro release has the characteristics of strong operability, high reproducibility, and relatively high sensitivity, and can be used to determine the comparison of the in vitro release rates of the topical drug to be tested and the reference preparation.

[0003] Calcipotriol and betamethasone ointment is a compound preparation mainly used for the treatment of psoriasis, and its main components are calcipotriol and betamethasone. Among them, calcipotriol is a vitamin D analogue, which can inhibit the excessive proliferation of skin cells (keratinocytes) and induce their differentiation, thereby reducing the abnormal hyperplasia of keratinocytes in psoriatic lesions. Betamethasone belongs to glucocorticoid drugs, and glucocorticoids have powerful anti-inflammatory, anti-allergic, immunosuppressive and other effects. In calcipotriol and betamethasone ointment, betamethasone can reduce skin inflammatory reactions. The in vitro release test method is helpful for the prescription screening and process confirmation of calcipotriol and betamethasone cream projects. Summary of the Invention

[0004] The purpose of the present invention is to provide an in vitro release method for a compound glucocorticoid ointment to solve the problems raised in the above background art.

[0005] To solve the above technical problems, the present invention provides the following technical solutions:

[0006] An in vitro release method for a compound glucocorticoid ointment, which combines a liquid chromatograph experiment and a diffusion apparatus experiment. The mobile phase in the liquid chromatograph experiment is disodium hydrogen phosphate buffer salt - acetonitrile; the receiving liquid in the diffusion apparatus experiment is water - tetrahydrofuran.

[0007] Preferably, the mobile phase in the liquid chromatograph experiment is disodium hydrogen phosphate buffer salt - acetonitrile 45 - 55:45 - 55; the disodium hydrogen phosphate buffer salt - acetonitrile is 50:50.

[0008] Preferably, the receiving liquid in the diffusion apparatus experiment is water - tetrahydrofuran 65 - 75:25 - 35; the receiving liquid in the diffusion apparatus experiment is water - tetrahydrofuran 70:30.

[0009] Preferably, the injection volume for the liquid chromatography experiment is 35 - 45 μl; preferably, the injection volume is 40 μl.

[0010] Preferably, the chromatographic conditions for the in vitro release experiment using the liquid chromatograph include:

[0011] The chromatographic column is ChromCore C18, 4.6 mm × 150 mm, 5 μm;

[0012] The column temperature is 30 °C;

[0013] The detection wavelength is UV, 240 nm;

[0014] The injection volume is 40 μl;

[0015] The mobile phase is 5 mmol / L disodium hydrogen phosphate buffer - acetonitrile (50:50);

[0016] The flow rate is 1.5 ml / min;

[0017] The running time is 10 min;

[0018] The reference solution is prepared by accurately weighing 10 mg of calcipotriol and betamethasone raw materials, placing them in a 50 ml volumetric flask, adding acetonitrile and sonicating to dissolve, then diluting to the mark and shaking well to obtain the reference stock solution. Accurately pipette 1 ml of the reference stock solution into a 25 ml volumetric flask, add the mobile phase to dilute to the mark and shake well.

[0019] The in vitro release conditions of the diffusion apparatus include:

[0020] The filter membrane uses 0.45 μm PTFE;

[0021] The receiving solution is water - tetrahydrofuran;

[0022] The temperature is 32 °C;

[0023] The rotation speed is 600 rpm;

[0024] The sampling / replenishment volume is 5 ml;

[0025] The sampling time points are 1 h, 2 h, 3 h, 4 h, 5 h, 6 h.

[0026] Preferably, the method for determination using the liquid chromatograph includes:

[0027] Step 1: Prepare a suitable mobile phase and prepare the sample to be analyzed into a suitable solution to ensure that the sample can be dissolved and the concentration is within the detectable range of the instrument;

[0028] Step 2: Turn on the power of the liquid chromatograph and perform preliminary work, such as flushing the pump head and degassing the mobile phase;

[0029] Step 3: Injection can be performed using a manual injector or an automatic injection device. After injection, immediately turn the injection valve back to the Inject state. At this time, the liquid phase system starts to enter the sampling state;

[0030] Step 4: The mobile phase flows through the injection valve by the infusion pump. After mixing with the sample solution, it is loaded into the chromatographic column;

[0031] Step 5: Each component flowing out of the chromatographic column passes through the detector respectively. The detector converts the sample concentration into an electrical signal. The electrical signal generated by the detector is transmitted to the data processing system. The data processing system records and processes these signals and presents them in the form of a chromatogram on the chromatographic workstation. Qualitative and quantitative analyses are performed based on the sample peaks in the chromatogram.

[0032] Preferably, the method for determination by the diffusion instrument includes:

[0033] Step 1: Prepare the drug to be tested into a suitable dosage form such as a solution or gel of a certain concentration. Select a suitable release model and process it to a state that meets the experimental requirements, such as cutting it into a suitable size, cleaning, and soaking;

[0034] Step 2: Set relevant parameters through the operation panel or supporting software of the diffusion instrument, such as release area, temperature, humidity, stirring speed, and test time;

[0035] Step 3: Use tools to accurately load the prepared drug sample onto one side of the release membrane, and then add an appropriate amount of receiving liquid to the receiving pool on the other side. The receiving liquid is used to receive the drug permeating through the membrane;

[0036] Step 4: Start the diffusion instrument, and the instrument starts to run according to the set parameters. During the experiment, the temperature control system will keep the set temperature stable, and the stirring device will stir at the set speed to make the drug in the receiving liquid evenly distributed. At the same time, the diffusion instrument will automatically collect data, and through detection devices such as sensors, it will monitor and record relevant data such as the rate and total amount of the drug passing through the skin in real time;

[0037] Step 5: After the experiment, transfer the data collected by the diffusion instrument to a computer or other data processing device, and use the corresponding data analysis software to process and analyze the data.

[0038] Compared with the prior art, the beneficial effects achieved by the present invention are:

[0039] First, the present invention simplifies the operation process by using the same set of in vitro release methods to study compound preparations, reduces time and data problems caused by method differences, and effectively improves the analysis efficiency. Second, a set of liquid chromatography detection methods is used to analyze compound preparations, avoiding multiple debugging and frequent switching of detection procedures, fully exploiting the performance of the equipment, and enhancing the liquid chromatography analysis efficiency. Third, this method can meet the requirements for the sensitivity and resolution of two components, accurately identify subtle changes in different components, and provide reliable data support for the quality control and efficacy evaluation of compound preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is the isocratic elution chromatogram of calcipotriol and betamethasone of the present invention;

[0041] Figure 2 It is the release curve of calcipotriol of the present invention (50%, 100%, 150% main component intensity);

[0042] Figure 3 It is the release curve of betamethasone of the present invention (50%, 100%, 150% main component intensity);

[0043] Figure 4 It is the chromatogram of Liquid Chromatography Method ① of the present invention;

[0044] Figure 5 It is the chromatogram of Liquid Chromatography Method ② of the present invention;

[0045] Figure 6 It is the chromatogram of Liquid Chromatography Method ③ of the present invention;

[0046] Figure 7 It is the chromatogram of Liquid Chromatography Method ④ of the present invention;

[0047] Figure 8 It is the precision of 6 injections of STD (betamethasone dipropionate) prepared according to Liquid Chromatography Method ④ of the present invention;

[0048] Figure 9 It is the precision of 6 injections of STD (calcipotriol monohydrate) prepared according to Liquid Chromatography Method ④ of the present invention;

[0049] Figure 10 It is Specificity 1 of Table 4 of the present invention;

[0050] Figure 11 It is Specificity 2 of Table 4 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0051] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0052] Example 1:

[0053] 1.1 The chromatographic conditions for the in vitro release experiment using a liquid chromatograph are as follows:

[0054]

[0055] 1.2 The in vitro release conditions of the diffusion apparatus are as follows:

[0056] Filter membrane: 0.45μm PTFE

[0057] Receiving liquid: water - tetrahydrofuran (70:30)

[0058] Temperature: 32°C

[0059] Rotation speed: 600 rpm

[0060] Sampling / liquid replenishment volume: 5 ml

[0061] Sampling time points: 1 h, 2 h, 3 h, 4 h, 5 h, 6 h

[0062] 1.3 Experimental procedure

[0063] Cut a 0.45μm PTFE filter membrane to a diameter of 28 mm, place it in the receiving liquid for activation for 30 min, take it out, dry the moisture with non-woven fabric, lay the pre-treated filter membrane flat, weigh the sample, remove the excess sample with a scraper, transfer the filter membrane and the sample to the diffusion cell, fix it, add the receiving liquid to the receiving cell so that there are no bubbles between it and the filter membrane, and add it to the scale line. The temperature is 32°C, the rotation speed is 600 revolutions per minute, the sampling time points are 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, take 5 ml of the solution at the specified time points, and replenish the receiving medium at the same temperature and the same volume after sampling at each time point. Take out the solution at each sampling point as the test solution.

[0064] Precisely measure the blank solution, the reference solution, and the test solution, inject them into the liquid chromatograph, record the chromatogram, and calculate according to the external standard method with the peak area.

[0065] 1.4 Experimental results

[0066] As shown in the appendix Figure 1 , 2 and Figure 3As shown, in this experiment, the peak emergence times of calcipotriol and betamethasone were approximately 6.087 min and 7.697 min respectively, with normal peak emergence. There were no other peak interferences before and after the emergence of the main peak, and the asymmetry degrees were 1.0 and 1.0 respectively. The theoretical plate numbers were 9348 and 9900 (see Figure 1 ), which could meet the detection requirements.

[0067] Example 2: Regarding the high performance liquid chromatography conditions

[0068] 2.1 Preparation of reference solution:

[0069] STD (calcipotriol monohydrate): 20 mg - 100 ml (dissolved in acetonitrile and diluted to the mark with purified water);

[0070] STD (betamethasone dipropionate): 20 mg - 100 ml (dissolved in acetonitrile and diluted to the mark with purified water);

[0071] STD: Mix 5 ml of STD (betamethasone dipropionate) and STD (calcipotriol monohydrate).

[0072] 2.2 Conduct an experiment using the following HPLC chromatographic conditions:

[0073]

[0074]

[0075] The above mobile phase is complex to prepare, and calcipotriol elutes at around 15 min. The running time of this liquid phase method is about 20 min. To improve the detection efficiency, it is necessary to further explore the reasons and improve the chromatographic conditions.

[0076] 2.3 Exploratory experiment on the mobile phase

[0077] Mobile phase A: 5 mmol / L Na 2 HPO 4 (pH 6.8)

[0078] Mobile phase B: Acetonitrile

[0079] Mobile phase C: Purified water

[0080] Liquid phase method ①: Mobile phase A - mobile phase B (50:50), injection volume 10 μl, flow rate 1 ml / min, running time: 10 min, column temperature 30 °C. The results are as shown in the attached Figure 4 chromatogram.

[0081] It can be seen that when collecting using liquid phase method 1, betamethasone did not elute within 10 min and remained for the next injection, resulting in an experimental failure.

[0082] Liquid phase method ②: Mobile phase A - Mobile phase B (30:70), injection volume is 40 μl, and other conditions remain unchanged as in liquid phase method ①. Collection is carried out, and the results are as attached Figure 5 .

[0083] It can be seen that: The retention times of betamethasone and calcipotriol are close, and the two cannot achieve baseline separation, affecting the accuracy of the detection results.

[0084] Liquid phase method ③: Mobile phase B - Mobile phase C (50:50), injection volume is 40 μl, running time is 15 min, and other conditions remain unchanged as in liquid phase method ①. Collection is carried out, and the results are as attached Figure 6 .

[0085] It can be seen that: The peak of calcipotriol has severe tailing, interfering with the elution of betamethasone and affecting the detection accuracy of the two main peaks.

[0086] Liquid phase method ④: Mobile phase A - Mobile phase B (50:50), injection volume 40 μl, flow rate 1.5 ml / min, and other conditions remain unchanged as in liquid phase method ①. Collection is carried out, and the results are as attached Figure 7 .

[0087] It can be seen that: There are no impurity peaks interfering with the elution positions of calcipotriol and betamethasone, the specificity is good, and the running time is short, with high detection efficiency.

[0088] Through further comprehensive exploration of conditions such as injection volume, running time, types of mobile phases, ratios, and column temperature, it is finally found that when the mobile phase is A - mobile phase B (45 - 55:45 - 55) and the injection volume is 35 - 45 μl, the experimental requirements are met. Further, when the mobile phase is A - mobile phase B (50:50), the flow rate is 1.5 ml / min, the running time is 10 min, and the injection volume is 40 μl, the experimental requirements are met optimally.

[0089] To investigate the stability and precision of this solution, 3 solutions prepared according to liquid phase method ④ are collected. Among them, STD (calcipotriol monohydrate) is collected 6 times, and the results are as follows: STD (betamethasone dipropionate), the precision of 6 times is good, and the results are as attached Figure 8 ; STD (calcipotriol monohydrate), the precision of 6 times is good, and the results are as attached Figure 9 .

[0090] The precision of STD (betamethasone dipropionate) for 6 times is good

[0091] The precision of STD (calcipotriol monohydrate) for 6 times is good;

[0092] Example 3: Regarding the in vitro release test conditions:

[0093] Selection of test device: There are various measuring devices for the in vitro release test of semi-solid preparations. The Franz diffusion cell method is the simplest and most practical. The technical solution of the present invention is based on the Franz diffusion cell method for method development.

[0094] 3.1 Diffusion cell system: A diffusion cell system with a standard open-cap ground glass-like surface, having an inner diameter of 20 mm and a volume of 18 ml. The effective release area is 3.14 cm 2 .

[0095] 3.2 Selection of rotation speed and receiving liquid temperature: Referring to USP<1724>, 600 rpm and 32 °C are selected as the in vitro release conditions.

[0096] 3.3 IVRT experiment time and sampling time points:

[0097] The in vitro release time is usually 4 - 6 hours, and the number of sampling time points is generally not less than 5. Therefore, the test time is 6 hours, and the sampling time points are 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h.

[0098] 3.5 Selection of sample loading amount:

[0099] In vitro release needs to meet pseudo-infinite sample loading, that is, to ensure that the concentration at the boundary is relatively constant within a certain time, a certain amount of drug needs to be given and a certain thickness needs to be maintained. Therefore, the sample loading amount is 550 mg (±5%).

[0100] 3.6 Investigation of medium and filter membrane

[0101] Perform a release experiment on the diffusion instrument, and take the replenishing liquid volume as 5 ml. The inventor investigated the release of calcipotriol and betamethasone cream in many media, including water-ethanol-phosphoric acid (50:50:1), water-ethanol-phosphoric acid (30:70:1), tetrahydrofuran-water (10:90), tetrahydrofuran-water (30:70), etc. And at the same time, many filter membranes were investigated, including 0.4 μm polycarbonate membrane, 0.45 μm nylon membrane, 0.22 μm nylon 6 membrane, 0.45 μm PTFE membrane, 0.45 μm polyethersulfone membrane, 0.45 μm mixed cellulose membrane, 0.45 μm cellulose acetate membrane, 0.45 μm nitrocellulose membrane for in vitro release. Among them, when using 0.45 μm mixed cellulose membrane, 0.45 μm cellulose acetate membrane, and 0.45 μm nitrocellulose membrane for in vitro release test, the filter membranes all showed damage and were not applicable. Make a regression curve of the amount of drug released per unit area (μg / cm 2 ) against the square root of relative time, and compare the release rates (see Table 2). It is found that tetrahydrofuran-water (30:70) is the best release medium, and at the same time, combined with 0.45 μm PTFE membrane, the release result of calcipotriol and betamethasone cream can be optimized.

[0102] Table 2 Results of Medium Investigation

[0103]

[0104]

[0105] 3.7 Investigation on the Saturated Solubility of the Receiving Medium:

[0106] The receiving medium should be appropriate. For example, aqueous buffer solution is used for water-soluble drugs, or hydroalcoholic medium is used for drugs with poor water solubility, or other appropriately adjusted media. The solubilities of calcipotriol and betamethasone raw materials shaken for 24 h in media such as tetrahydrofuran-water (10:90), isopropanol-water (50:50), and acetonitrile-PBS (50:50) were investigated respectively (see Table 3). Approximately 10 mg of each of the two raw materials was added to 200 ml of the medium. In both media, calcipotriol and betamethasone raw materials could be completely dissolved. Therefore, the solubility requirements of the receiving medium for in vitro release were met.

[0107] Table 3 Solubility Results of Calcipotriol and Betamethasone Raw Materials in Different Media

[0108]

[0109] Example 4 Investigation on the Discrimination Ability of the IVRT Method:

[0110] To investigate the discrimination ability of the in vitro release method for the main components of the formulation, with a sample loading of approximately 550 mg, at 600 rpm, 32 °C, using tetrahydrofuran-water (30:70) as the medium, a 0.45 μm PTFE filter membrane, a replenishing fluid volume of 5 ml, and sampling time points of 1, 2, 3, 4, 5, and 6 h, the in vitro release of calcipotriol and betamethasone ointments with 50%, 100%, and 150% main component strengths was investigated respectively. The average release curves of each main component strength are shown in Figure 2 and Figure 3 . The results showed that the average release rate of calcipotriol V(50%): 1.157 (μg / cm 2 / h 1 / 2 ) < V(100%): 1.953 (μg / cm 2 / h 1 / 2 ) < V(150%): 2.986 (μg / cm 2 / h 1 / 2 ), and the average release rate of betamethasone V(50%): 10.106 (μg / cm 2 / h 1 / 2 ) < V(100%): 17.169 (μg / cm 2 / h 1 / 2)<V(150%):26.773(μg / cm 2 / h 1 / 2 ), indicating that this method has good sensitivity. Taking 50%, 100%, and 150% as the abscissa and the average release rate as the ordinate to plot the regression curve, the linear correlation coefficient R 2 > 0.99, indicating that this method has good specificity. The 50% main component intensity, the 150% main component intensity, and the 90% confidence interval of 100% are not within the range of 75% - 133.33% (see Table 4), indicating that this method has good selectivity. In summary: This method has good discrimination ability for the intensities of calcipotriol and betamethasone at 50%, 100%, and 150%.

[0111] Table 4 Investigation Results

[0112]

[0113]

[0114] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present application. The scope of the present application is defined by the appended claims and their equivalents.

Claims

1. An in vitro release method of a glucocorticoid compound ointment, the method adopts a combination of a liquid chromatography experiment and a diffusion instrument experiment, characterized in that: The mobile phase in the liquid chromatograph experiment is disodium hydrogen phosphate buffer salt-acetonitrile; the receiving liquid in the diffusion instrument experiment is water-tetrahydrofuran.

2. The in vitro release method of a glucocorticoid compound ointment according to claim 1, characterized in that: The mobile phase of the liquid chromatograph experiment is a disodium hydrogen phosphate buffer salt-acetonitrile with a volume ratio of 45-55:45-55; the volume ratio of the disodium hydrogen phosphate buffer salt-acetonitrile is 50:

50.

3. The in vitro release method of a glucocorticoid compound ointment according to claim 1, characterized in that: The volume ratio of water to tetrahydrofuran in the diffusion instrument experiment is 65-75:25-35; the volume ratio of water to tetrahydrofuran in the diffusion instrument experiment is 70:

30.

4. The in vitro release method of a glucocorticoid compound ointment according to claim 1, characterized in that: The injection volume of the liquid chromatography experiment is 35-45 μl; the injection volume is 40 μl.

5. The in vitro release method of a glucocorticoid compound ointment according to claim 1, characterized in that: (1) The chromatographic conditions for the in vitro release experiment using the liquid chromatograph include: The chromatographic column was ChromCore C18, 4.6 mm × 150 mm, 5 μm; The column temperature is 30℃ The detection wavelength is UV, 240nm; The injection volume was 40 μl; The mobile phase was sodium hydrogen phosphate buffer-acetonitrile 50:50; Flow rate: 1.5 ml / min; The running time is 10 minutes; (2) The in vitro release conditions of the diffusion instrument include: The filter membrane uses 0.45μm PTFE; The receiving liquid was water-tetrahydrofuran 70:30; Temperature is 32℃ The rotation speed is 600 rpm; The sampling / refilling volume is 5 ml; The sampling time points are 1h, 2h, 3h, 4h, 5h, and 6h.

6. The in vitro release method of a glucocorticoid compound ointment according to claim 1, characterized in that: The method of the liquid chromatograph determination comprises: Step 1: Prepare a suitable mobile phase to prepare the sample to be analyzed into a suitable solution, ensuring that the sample can be dissolved and the concentration is within the detectable range of the instrument; Step 2: Turn on the power of the liquid chromatograph and perform preliminary work, such as flushing the pump head and exhausting the mobile phase; Step 3: Use a manual injector or automatic injector to inject samples. After injecting samples, immediately return the injection valve to the Inject state. At this time, the liquid phase system begins to enter the sampling state. Step 4: The mobile phase flows through the injection valve via the infusion pump, mixes with the sample solution, and is loaded into the chromatographic column; Step 5: Each component flowing out of the chromatographic column passes through the detector respectively. The detector converts the sample concentration into an electrical signal. The electrical signal generated by the detector is transmitted to the data processing system. The data processing system records and processes these signals and presents them in the form of a spectrum on the chromatographic workstation. Qualitative and quantitative analysis is performed based on the sample peaks in the spectrum.

7. The in vitro release method of a glucocorticoid compound ointment according to claim 1, characterized in that: The method of the diffusometer measurement comprises: Step 1: Prepare the drug to be tested into a suitable dosage form such as a solution or gel of a certain concentration, select a suitable release model, and process it to a state that meets the experimental requirements, such as cutting it into a suitable size, washing, and soaking; Step 2: Set relevant parameters such as release area, temperature, humidity, stirring speed, and test time through the operation panel of the diffuser or the supporting software; Step 3: Use a tool to accurately load the prepared drug sample onto one side of the release membrane, and then add an appropriate amount of receiving solution into the receiving pool on the other side. The receiving solution is used to receive the drug that permeates the membrane. Step 4: Start the diffusion instrument, and the instrument starts to operate according to the set parameters. During the experiment, the temperature control system will keep the set temperature stable, and the stirring device will stir at the set speed to make the drug in the receiving solution evenly distributed. At the same time, the diffusion instrument will automatically collect data, and monitor and record the rate and total amount of drugs passing through the skin in real time through sensors and other detection devices; Step 5: After the experiment, the data collected by the diffusion instrument is transferred to a computer or other data processing equipment, and the data is processed and analyzed using the corresponding data analysis software.

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