In-vitro release test method of diazepam rectum gel

Abstract

The invention discloses an in-vitro release test method of diazepam rectum gel, which comprises the following steps: preparing a test solution: smearing the diazepam rectum gel on an artificial membrane, placing the artificial membrane on an automatic transdermal diffusion instrument, carrying out a release experiment, and collecting a solution in a diffusion cell as the test solution according to a sampling time point; a proper amount of a diazepam reference substance is weighed, a methanol solution is added to prepare a reference substance stock solution with the concentration being 1 mg / mL, and the reference substance stock solution is diluted to prepare a reference substance solution with the concentration being 0.1 mg / mL; and testing the test solution and the reference solution by adopting a high performance liquid chromatography. The in-vitro release method provided by the invention has good discrimination, linearity, precision and durability. By comparing the release amounts of the diazepam self-developed preparation and the reference preparation, the consistent release rate of each batch of the self-developed preparation and the reference preparation is ensured, so that the quality of the self-developed preparation is ensured.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceutical chemistry, and in particular to an in vitro release test method of diazepam rectal gel. Background Art

[0002] Diazepam is a commonly used anti-anxiety, anticonvulsant and anti-epileptic drug in clinical practice. Rectal gel can be used to control the condition during acute attacks. It is easy to operate and can effectively control the speed of medication, minimize the irritation to the anus and rectum of the child, and can effectively avoid the first-pass elimination effect. The rectum has abundant veins, thin mucosa, and special blood circulation channels, so the drugs injected into the rectum can be quickly absorbed.

[0003] In vitro release is to examine the rate and extent of drug release from the system. It is an important indicator for quality research and stability research. It can reflect the physical and chemical changes in semi-solid drugs. It is also an important parameter for determining drug efficacy and dosage. Therefore, developing a suitable in vitro release test method will help in the prescription screening and process confirmation of the diazepam rectal gel project.

[0004] Currently, neither the Chinese Pharmacopoeia nor the United States Pharmacopoeia records the in vitro release method of diazepam rectal gel, making it impossible to ensure the consistency between homemade preparations and reference preparations. Summary of the invention

[0005] The object of the present invention is to provide an in vitro release test method for diazepam rectal gel, which fills the above-mentioned deficiencies in the prior art and realizes effective detection of the in vitro release rate of diazepam rectal gel.

[0006] An in vitro release test method for diazepam rectal gel comprises the following steps:

[0007] Prepare the test solution: apply diazepam rectal gel on the artificial membrane, and place the artificial membrane on the automatic transdermal diffusion instrument to conduct a release experiment. Collect the solution in the diffusion cell according to the sampling time point as the test solution;

[0008] Prepare the reference solution: weigh an appropriate amount of diazepam reference substance, add methanol solution to prepare a reference substance stock solution with a concentration of 1 mg / mL, and dilute it to prepare a reference substance solution with a concentration of 0.1 mg / mL;

[0009] Detection: Use high performance liquid chromatography to detect the test solution and the reference solution to obtain the release amount of the homemade sample and the reference preparation. Compare the release amount of the homemade sample with that of the reference preparation to judge the quality of the homemade sample.

[0010] In some embodiments of the present invention, the artificial membrane is one of a polyvinylidene fluoride membrane, a polyethersulfone membrane, a polytetrafluoroethylene membrane, and a nylon membrane.

[0011] In some embodiments of the present invention, the pore size of the artificial membrane is 0.45 μm.

[0012] In some embodiments of the present invention, the receiving medium in the automatic transdermal diffusion instrument is a mixed solution of buffer and ethanol, wherein the volume ratio of buffer to ethanol is 50:50 to 80:20; preferably, the volume ratio of buffer to ethanol is 60:40 to 80:20.

[0013] In some embodiments of the present invention, in the step of preparing the test solution, the sample loading method is 4 mm quantitative loop loading, and the sample loading amount is 0.6 g:

[0014] In some embodiments of the present invention, in the step of preparing the test solution, the sampling method is to completely remove and replenish an equal amount of receiving medium, and the sampling time points include 0.5h, 1h, 2h, 3h, 4h, 5h, and 6h.

[0015] In some embodiments of the present invention, in the step of preparing the test solution, the volume of the Franz diffusion cell is 12.5 ml, and the injection hole area is 1.77 cm 2 The stirring rate in the receiving chamber is 300-500 rpm, and the temperature in the receiving chamber is maintained at 37° C. by a water bath in the interlayer.

[0016] Preferably, the stirring rate in the receiving chamber is 400 rpm.

[0017] In some embodiments of the present invention, in the step of preparing the test solution, the stirring rate in the receiving chamber is 300-500 rpm.

[0018] In some embodiments of the present invention, in the detection step, the chromatographic conditions of the high performance liquid chromatography method include a chromatographic column of Agilent ZORBAX SB-C8 3.0×150mm 5um, a column temperature of 30°C, a UV detector, a detection wavelength of 254nm, an injection volume of 10μl, a mobile phase of 0.025mol / L potassium dihydrogen phosphate solution-methanol-acetonitrile (44:34:22), and a flow rate of 1.0ml / min.

[0019] Description: The buffer in the present invention is a phosphate buffer (pH 6.8), and the preparation method is as follows: take 250 ml of 0.2 mol / L potassium dihydrogen phosphate solution, add 118 ml of 0.2 mol / L sodium hydroxide solution, dilute with water to 1000 ml, and shake well to obtain the solution.

[0020] The beneficial effects of the present invention are as follows: the present invention utilizes a Franz diffusion cell to simulate the in vitro release of a diazepam rectal gel, thereby obtaining a sample solution containing diazepam, and then accurately detects the content in the sample solution by a liquid chromatograph, with high detection accuracy. Methodological verification shows that the in vitro release method of the present invention has good discrimination, linearity, precision and durability. The present invention ensures that each batch of the self-developed diazepam preparation and the reference preparation have a consistent release rate by comparing the release amount of the self-developed diazepam preparation and the reference preparation, so as to ensure the quality of the self-developed preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a comparison chart of the release rates of three specifications for distinguishing force investigation in Example 1 of the present invention.

[0022] Figure 2 It is a linear trend diagram of the average release rate of IVRT and the three specifications and concentrations of samples in Example 1 of the present invention.

[0023] Figure 3 This is a comparison chart of the release rates of the self-developed preparation and the reference preparation in Example 2 of the present invention.

[0024] Figure 4 This is a comparison chart of the release rates of the self-developed preparation and the reference preparation in Example 3 of the present invention. DETAILED DESCRIPTION

[0025] The present invention will be further described below in conjunction with the following embodiments and drawings, but the protection scope of the present invention is not limited thereto.

[0026] Example 1

[0027] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0028] Instruments used in the embodiments of the present invention:

[0029] Automatic transdermal diffusion system, TD-12AT, Shenzhen Huarong Analytical Instrument Co., Ltd.;

[0030] High performance liquid chromatograph, LC-2030C 3D, Shimadzu, Japan;

[0031] Analytical balance, XS205, METTLER TOLEDO;

[0032] Analytical balance, ME204, METTLER TOLEDO;

[0033] Analytical balance, MS304TS, METTLER TOLEDO;

[0034] Ultrasonic cleaner, KQ5200E, Kunshan Ultrasonic Instrument Co., Ltd.;

[0035] Pure water and ultrapure water all-in-one machine, MING-CHE 24UV, Millipore.

[0036] Reagents used in the embodiments of the present invention:

[0037] Potassium dihydrogen phosphate, analytical grade, Sinopharm Chemical Reagent Co., Ltd.;

[0038] Sodium hydroxide, analytical grade, Tianjin Xinbote Chemical Co., Ltd.;

[0039] Anhydrous ethanol, analytical grade, Tianjin Fuchen Chemical Reagent Co., Ltd.;

[0040] Methanol, chromatography grade, SIGMA;

[0041] Acetonitrile, chromatography grade, SIGMA;

[0042] Ultrapure water, homemade in the laboratory.

[0043] Reference substances used in the embodiments of the present invention:

[0044] Diazepam (purity: 99.9%; manufacturer: China Food and Drug Inspection Institute).

[0045] The test samples used in the embodiments of the present invention are:

[0046] Reference preparation: diazepam rectal gel (batch number: WKBW-3; specification: 20 mg: 4 ml; manufacturer: ValeuntPharma);

[0047] Self-prepared preparation: 50% diazepam rectal gel sample (batch number: 240319-1; specification: 2.5 mg / ml);

[0048] 100% diazepam rectal gel sample (batch number: 240319-2; specification: 5.0 mg / ml;);

[0049] 150% diazepam rectal gel sample (batch number: 240319-3; specification: 7.5 mg / ml;);

[0050] Diazepam rectal gel (batch number: 20210202; specification: 20mg: 4ml);

[0051] Diazepam rectal gel (batch number: 20210203; specification: 20mg: 4ml);

[0052] Diazepam rectal gel (batch number: 20210204; specification: 20 mg: 4 ml).

[0053] The liquid chromatography conditions in the embodiments of the present invention, the specific parameters are as follows:

[0054] Instrument: HPLC

[0055] Chromatographic column: Agilent ZORBAX SB-C8 3.0×150mm 5um

[0056] Mobile phase: 0.025 mol / L potassium dihydrogen phosphate solution (adjust pH to 5.0 with 0.1 mol / L sodium hydroxide solution)-methanol-acetonitrile (44:34:22)

[0057] Flow rate: 1.0mL / min

[0058] Injection volume: 10 μL

[0059] Column temperature: 30°C

[0060] Detection wavelength: 254nm

[0061] Example 1

[0062] In this example, the in vitro release of diazepam rectal gel was investigated, and the method was as follows:

[0063] (1) Preparation of test solution:

[0064] To ensure pseudo-infinite loading, 0.6 g was accurately weighed and placed in a quantitative loop, and an in vitro release test was performed under the conditions of pH 6.8 buffer-ethanol (80:20) as the receiving medium, polyethersulfone membrane (0.45 μm) as the isolation membrane, temperature of 37°C, stirring speed of 400 rpm, complete sampling, rehydration volume of 12.5 ml, and sampling time of 0.5, 1, 2, 3, 4, 5, and 6 h. The subsequent filtrate was sampled and analyzed.

[0065] (2) Preparation of reference solution:

[0066] Weigh an appropriate amount of diazepam reference substance, add methanol solution to prepare a reference substance stock solution with a concentration of 1 mg / mL, and dilute it to prepare a reference substance solution with a concentration of 0.1 mg / mL.

[0067] (3) Membrane confirmation

[0068] The isolation membrane was placed in a receiving medium at 37℃±1℃ (the receiving medium was low concentration-release amount 10% and high concentration-release amount 40%) for incubation for 6 hours, and the determination was repeated 3 times; at least 3 receiving media without membrane were simultaneously examined to examine whether the filter membrane had a large adsorption effect on the drug, thereby affecting the results. The results showed that the recovery rate of the drug in high and low concentration solutions was 95% to 105%, and there was no obvious adsorption effect, as shown in Table 1.

[0069] Note: Low concentration - 10% release receptor medium preparation: Add diazepam drug substance to the blank receptor medium so that the concentration of diazepam in the receptor medium is equivalent to the 10% release of diazepam rectal gel.

[0070] High concentration - 40% release receptor medium preparation: Add diazepam drug substance to the blank receptor medium so that the concentration of diazepam in the receptor medium is equivalent to 40% release of diazepam rectal gel.

[0071] The screening results of different isolation membrane materials are shown in Table 2, and the recovery rates are all between 95% and 105%.

[0072] Table 1 Membrane confirmation test results

[0073]

[0074]

[0075] Table 2 Isolation film material screening

[0076] name Recovery rate (%) Polyethersulfone membrane 99.4 Nylon membrane 97.3 Polytetrafluoroethylene membrane 99.8 Polyvinylidene fluoride film 97.6

[0077] The adsorption effects of the above four membranes are all low and they are all used as isolation membranes.

[0078] (4) Linear range of different media

[0079] In view of the solubility of diazepam, different ratios of pH 6.8 buffer and ethanol were selected for medium screening. The release of diazepam in pH 6.8 buffer, pH 6.8 buffer-ethanol (90:10), and pH 6.8 buffer-ethanol (80:20) media was investigated under the conditions of polyethersulfone membrane (0.45 μm), temperature of 37°C, and stirring speed of 400 rpm.

[0080] The results are shown in Table 3. The linear correlation coefficient of diazepam in pH 6.8 buffer-ethanol (80:20) medium is greater than 0.97, which meets the requirements.

[0081] Table 3 Linear investigation of different media

[0082]

[0083] (5) Precision

[0084] According to the preparation method of the test solution, different personnel operated at different times. The coefficient of variation of the release rate was investigated to be ≤15%. The results are shown in Table 4. The coefficient of variation of the release rate was 3.9% <15%, and the method had good precision.

[0085] Table 4 Precision test results

[0086]

[0087]

[0088] (6) Discrimination

[0089] In order to investigate the ability of the in vitro release method to distinguish diazepam, the in vitro release of diazepam rectal gel samples with a main component of 50%, 100%, and 150% was investigated under the conditions of a sample load of 0.6 g, a receiving medium of pH 6.8 buffer-ethanol (80:20), a separation membrane of polyethersulfone membrane (0.45 μm), a temperature of 37°C, and a stirring speed of 400 rpm. The release rate comparison diagram is shown in FIG. Figure 1 .

[0090] The results showed that the release rates of the 50%, 100%, and 150% formulations were 426.22, 869.87, and 1179.57, increasing in sequence, indicating that the method had good sensitivity, as shown in Table 5; the lower and upper limits of the 90% confidence intervals of the release rate of the 100% formulation and the 50% formulation were 198.14% and 210.97%; the linear correlation coefficient R between the formulation concentration and the average release rate of IVRT was 2 is 0.9896>0.95, indicating that the method has good specificity. Figure 2 The lower and upper limits of the 90% confidence intervals for the release rates of the 100% formulation and the 150% formulation were 68.72% and 71.84% respectively; both were outside the limit range of 75% to 133.33%, i.e., the 50% and 150% formulations were not equivalent to the 100% formulation, and the method had discriminatory power, as shown in Table 6.

[0091] Table 5 Differentiation force-release rate results

[0092] Specification Pool 1 Pool 2 Pool 3 Pool 4 Pool 5 Pool 6 average value 50％ 429.34 431.77 405.99 426.74 436.25 427.25 426.22 100％ 801.66 879.49 903.00 878.20 855.52 901.37 869.87 150％ 1103.84 1187.19 1204.58 1200.62 1171.9 1209.26 1179.57

[0093] Table 6 Discrimination-Confidence Interval Results

[0094] Sample Specifications Upper limit of 90% confidence interval (%) Lower limit of 90% confidence interval (%) 50% and 100% 210.97 198.14 150% and 100% 71.84 68.72

[0095] The results of all survey projects were verified by comprehensive methodology and passed.

[0096] Example 2

[0097] (1) Preparation of test solution:

[0098] Weigh 0.6 g accurately and place it in a quantitative loop. Perform an in vitro release test under the following conditions: the receiving medium is pH 6.8 buffer-ethanol (80:20), the isolation membrane is a polytetrafluoroethylene membrane (0.45 μm), the temperature is 37°C, the stirring speed is 400 rpm, the sampling method is complete sampling, the rehydration volume is 12.5 ml, and the sampling time is 0.5, 1, 2, 3, 4, 5, 6 h. Take the filtrate for sampling and analysis.

[0099] (2) Preparation of reference solution: The method and determination are the same as those in Example 1.

[0100] Take the reference preparation of diazepam rectal gel and the self-made preparation, prepare according to the test solution, inject and analyze, measure and calculate the confidence interval of the ratio of the in vitro release rate of the self-made preparation (T) to the in vitro release rate of the reference preparation (R), see Table 7, Figure 3 .

[0101] The results showed that the lower and upper limits of the 90% confidence interval of the release rate of the self-developed formulation and the reference formulation were 98.18% and 102.64%, which were within the limit of 90% to 111%, that is, the self-developed formulation was equivalent to the reference formulation.

[0102] Table 7 Confidence interval results

[0103]

[0104] Example 3

[0105] (1) Preparation of test solution:

[0106] Weigh 0.6 g accurately and place it in a quantitative loop. Perform an in vitro release test under the following conditions: the receiving medium is pH 6.8 buffer-ethanol (60:40), the isolation membrane is a polyethersulfone membrane (0.45 μm), the temperature is 37°C, the stirring speed is 500 rpm, the sampling method is complete sampling, the rehydration volume is 12.5 ml, and the sampling time is 0.5, 1, 2, 3, 4, 5, 6 h. Take the subsequent filtrate for sampling and analysis.

[0107] (2) Preparation of mixed reference solution:

[0108] Same as Example 1

[0109] The confidence interval of the ratio of the in vitro release rate of the self-developed preparation (T) to the in vitro release rate of the reference preparation (R) was measured and calculated, see Table 8. Figure 4 .

[0110] The results showed that the lower and upper limits of the 90% confidence interval of the release rate of the self-developed formulation and the reference formulation were 100.97% and 109.06%, which were within the limit of 90% to 111%, that is, the self-developed formulation was equivalent to the reference formulation.

[0111] Table 8 Confidence interval results

[0112]

[0113] In summary, the present invention has good discrimination ability, and the in vitro release behaviors of the self-developed preparation and the reference preparation are consistent.

[0114] Of course, it should be understood that the above is merely illustrative of the application of the principles of the present invention. A variety of modifications and alternatives may be devised by those skilled in the art without departing from the spirit and scope of the present invention, and the appended claims are intended to encompass such modifications and alternatives. Thus, although the features and details of the present invention are described above in conjunction with what are currently considered to be the most practical and preferred embodiments of the present invention, it is apparent to those skilled in the art that many modifications may be made without departing from the principles and concepts set forth herein, including but not limited to changes in size, material, shape, morphology, operating function and mode of operation, components, and uses.

Claims

1. An in vitro release test method for diazepam rectal gel, characterized in that: The following steps are involved: Prepare the test solution: Apply diazepam rectal gel on the artificial membrane, and place the artificial membrane on the automatic transdermal diffusion instrument to perform a release experiment. Collect the solution in the diffusion cell according to the sampling time point as the test solution.

2. An in vitro release test method for diazepam rectal gel as claimed in claim 1, characterized in that: The method also includes the following steps: preparing a reference substance solution: weighing an appropriate amount of a diazepam reference substance, adding a methanol solution to prepare a reference substance stock solution with a concentration of 1 mg / mL, and diluting the solution to prepare a reference substance solution with a concentration of 0.1 mg / mL; Detection: Use high performance liquid chromatography to detect the test solution and the reference solution to obtain the release amount of the homemade sample and the reference preparation. Compare the release amount of the homemade sample with that of the reference preparation to judge the quality of the homemade sample.

3. An in vitro release test method for the diazepam rectal gel according to claim 1 or 2, characterized in that: The artificial membrane is one of polyvinylidene fluoride membrane, polyethersulfone membrane, polytetrafluoroethylene membrane and nylon membrane.

4. An in vitro release test method for the diazepam rectal gel according to claim 1 or 3, characterized in that: The pore size of the artificial membrane is 0.45 μm.

5. An in vitro release test method for diazepam rectal gel as claimed in claim 1, characterized in that: The receiving medium in the automatic transdermal diffusion instrument is a mixed solution of buffer and ethanol, wherein the volume ratio of the buffer to the ethanol is 50:50 to 80:20; preferably, the volume ratio of the buffer to the ethanol is 60:40 to 80:

20.

6. An in vitro release test method for diazepam rectal gel as claimed in claim 1, characterized in that: In the step of preparing the test solution, the sample loading method is 4 mm quantitative loop loading, and the sample loading amount is 0.6 g.

7. An in vitro release test method for diazepam rectal gel as claimed in claim 1, characterized in that: In the step of preparing the test solution, the sampling method is to completely remove and replenish an equal amount of receiving medium. The sampling time points include 0.5h, 1h, 2h, 3h, 4h, 5h, and 6h.

8. An in vitro release test method for diazepam rectal gel as claimed in claim 1, characterized in that: In the step of preparing the test solution, the volume of the Franz diffusion cell is 12.5 ml and the injection hole area is 1.77 cm 2 The stirring rate in the receiving chamber is 300-500 rpm, and the temperature in the receiving chamber is maintained at 37° C. by a water bath in the interlayer.

9. An in vitro release test method for diazepam rectal gel as claimed in claim 8, characterized in that: In the step of preparing the test solution, the stirring rate in the receiving chamber is 300-500 rpm; preferably, the stirring rate in the receiving chamber is 400 rpm.

10. An in vitro release test method for diazepam rectal gel according to claim 1, characterized in that: In the detection step, the chromatographic conditions of the high performance liquid chromatography method include an Agilent ZORBAX SB-C8 3.0×150mm 5um chromatographic column, a column temperature of 30° C., a UV detector, a detection wavelength of 254 nm, an injection volume of 10 μl, a mobile phase of 0.025 mol / L potassium dihydrogen phosphate solution-methanol-acetonitrile (44:34:22), and a flow rate of 1.0 ml / min.

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