A method for determining the dissolution of miconazole nitrate suppositories

Abstract

The invention discloses a dissolution instrument for measuring the dissolution rate of miconazole nitrate suppositories and a method for measuring the dissolution rate of miconazole nitrate suppositories. The invention uses a specific dissolution instrument, dissolution test conditions and chromatographic conditions to effectively dissolve miconazole nitrate in suppositories and completely separate them in a chromatogram, so that the dissolution rate of miconazole nitrate suppositories can be accurately measured, and the method can not only be used for in vitro consistency evaluation of miconazole nitrate suppositories, but also has important significance for evaluating the batch differences of miconazole nitrate suppositories, and has practical promotion and application value.

Description

Technical Field

[0001] The invention relates to a method for determining the dissolution of miconazole nitrate suppositories. Background Art

[0002] Vulvovaginal candidiasis (VVC) is an inflammation of the vulva and vagina caused by different Candida species (mainly Candida albicans), and is one of the most common vulvovaginal inflammations in women. Miconazole nitrate suppositories are currently the most commonly used vaginal suppositories for the clinical treatment of VVC.

[0003] The National Drug Evaluation Center's "Technical Requirements for the Evaluation of Generic Locally Acting Common Vaginal Preparations (Vaginal Tablets, Vaginal Suppositories) (Draft for Comments)" points out that the key quality attributes of vaginal suppositories include dissolution (or release), but there are currently no reports on the dissolution test method for miconazole nitrate suppositories.

[0004] In the dissolution test, an appropriate dissolution tester should be selected, and the test conditions, as well as the temperature, time, and dissolution medium of the dissolution test should be clarified to accurately measure the dissolution. However, the dissolution inspection items are not included in the domestic and international implementation standards. Therefore, it is necessary to establish a dissolution determination method for miconazole nitrate suppositories to control the quality of miconazole nitrate suppositories. Summary of the invention

[0005] To solve the above problems, the present invention provides a method for determining the dissolution rate of miconazole nitrate suppositories, which comprises the following steps:

[0006] 1) Preparation of reference solution: Take miconazole nitrate reference substance, add methanol to dissolve it, and dilute the dissolved solution with dissolution medium to obtain the reference solution;

[0007] 2) Preparation of test solution: Take miconazole nitrate suppositories, use acetic acid-sodium acetate buffer containing SDS as the dissolution medium, dissolve using a dissolution apparatus, and filter the dissolved solution to use as the test solution;

[0008] The dissolution apparatus comprises a stirring paddle (3), a sinker basket (4) and a dissolution cup (2), wherein the stirring paddle (3) is arranged in the dissolution cup (2), and the sinker basket (4) is located at the bottom of the dissolution cup (2);

[0009] The stirring paddle (2) is composed of a paddle rod and a paddle blade, wherein the upper end of the paddle rod is connected to the motor, and the lower end of the paddle rod is connected to the paddle blade;

[0010] The sinker (4) is cylindrical and is used for placing the miconazole nitrate suppository;

[0011] The dissolution cup (2) is a cup-shaped container with a hemispherical bottom, and is used to contain the dissolution medium.

[0012] 3) The content of miconazole nitrate in the reference solution and the test solution was determined by HPLC, and the solubility of miconazole nitrate suppositories was calculated.

[0013] Furthermore, in step 1), the mass volume ratio of the reference substance, methanol and dissolution medium is 10-20 mg: 10 ml: 40 ml.

[0014] Furthermore, in step 2), each miconazole nitrate suppository is dissolved with 1000 ml of dissolution medium; the dissolution medium is acetic acid-sodium acetate buffer containing 2.0% SDS; the pH value of the acetic acid-sodium acetate buffer is 4.5.

[0015] Furthermore, in step 2), the speed of the stirring paddle in the dissolution apparatus is 100 rpm and the time is 300 min.

[0016] Furthermore, the chromatographic conditions of the HPLC method in step 3) include: chromatographic column: octadecylsilane bonded silica gel as filler; mobile phase: 1.5% ammonium acetate solution-methanol-acetonitrile in a volume ratio of 20:40:40.

[0017] Furthermore, the chromatographic column is ZORBAX Eclipse Plus C18, 250×4.6 mm, 5 μm.

[0018] Furthermore, the chromatographic conditions also include: column temperature of 30°C, injection volume of 20 μL, flow rate of 1 ml / min, and detection wavelength of 230 nm.

[0019] Furthermore, the size of the sinker is 1.8*3cm.

[0020] Furthermore, the dissolution apparatus is a device in the dissolution and release determination method (the second method of 0931 of the fourth general rules of the 2020 edition of the Chinese Pharmacopoeia) with a cylindrical sinker of size 1.8*3cm added.

[0021] Furthermore, the dissolution cup is provided with a cup cover, and the cup cover has a hole.

[0022] Furthermore, the holes include a central hole; the central hole is the location of the stirring paddle, and the other holes are used for sampling or measuring temperature.

[0023] The invention discloses a method for determining the dissolution of a miconazole nitrate suppository. The method can effectively dissolve the miconazole nitrate in the suppository and completely separate the miconazole nitrate in the chromatogram through a specific dissolution instrument, dissolution test conditions and chromatographic conditions, so that the dissolution determination result of the miconazole nitrate suppository can accurately reflect the release characteristics of the miconazole nitrate, and effectively distinguish the quality difference of the miconazole nitrate suppository under different processes. The method can be used to evaluate the bioavailability of the active ingredient of the miconazole nitrate suppository and the uniformity of the preparation, realize the in vitro consistency evaluation of the miconazole nitrate suppository, and is also of great significance for evaluating the batch difference of the miconazole nitrate suppository. The method has practical promotion and application value.

[0024] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0025] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Basket dissolution

[0027] Figure 2 Paddle dissolution

[0028] Figure 3 Dissolution Method Investigation-Flow Cell Method (Ordinary Cell)

[0029] Figure 4 Schematic diagram of the sinker structure

[0030] Figure 5 Schematic diagram of the dissolution apparatus structure (1, sampling point, 2, dissolution cup, 3, stirring paddle, 4, sinker basket)

[0031] Figure 6 Dissolution curve-Company A

[0032] Figure 7 Dissolution curve - Company B

[0033] Figure 8 Dissolution curve - C enterprise

[0034] Fig. 9 Dissolution curve-D enterprise

[0035] Fig.10 Chromatogram of reference solution

[0036] Fig.11 Chromatogram of test solution DETAILED DESCRIPTION

[0037] The reagents, test drugs and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0038] Example 1 Determination of dissolution of miconazole nitrate suppositories by the present invention

[0039] 1) Preparation of reference solution: Take about 10 mg of miconazole nitrate reference substance, accurately weigh it, put it in a 50 ml volumetric flask, add 10 ml of methanol to dissolve it, dilute it to the scale with dissolution medium, shake well, and use it as the reference solution;

[0040] 2) Preparation of test solution: Take miconazole nitrate suppositories, use 1000 ml of pH 4.5 acetic acid-sodium acetate buffer (containing 2.0% SDS) as the dissolution medium, use a paddle-sedimentation blue device for dissolution, the speed is 100 rpm, the temperature is 37°C, the time is 300 min, filter, and take the filtrate as the test solution;

[0041] Paddle-sinking blue device ( Figure 5 ) comprises a stirring paddle (3), a cylindrical sinker basket (4) with a size of 1.8*3 cm and a dissolution cup (2), wherein the stirring paddle is composed of a paddle rod and a paddle blade, the upper end of the paddle rod is connected to the motor, and the lower end of the paddle rod is connected to the paddle blade; the sinker basket is used to place the miconazole nitrate suppository and is placed at the bottom of the dissolution cup; the dissolution cup is a cup-shaped container with a hemispherical bottom, used to hold the dissolution medium, and is equipped with a cup cover, the cup cover has holes, the holes include a central hole, the central hole is the position of the stirring paddle, and the other holes are used for sampling or measuring temperature.

[0042] 3) Pipette the reference solution and the test solution into the HPLC respectively; the chromatographic conditions are as follows:

[0043] Chromatographic column: Agilent, ZORBAX Eclipse Plus C18 (250×4.6 mm, 5 μm); mobile phase: 1.5% ammonium acetate-methanol-acetonitrile=20:40:40; detection wavelength: 230 nm; flow rate: 1 ml / min; injection volume: 20 μl; column temperature: 30°C.

[0044] 4) Calculate the dissolution rate of miconazole nitrate suppositories. The specific calculation formula is:

[0045] Dissolution (%) = W × reference substance content (%) × A r ×1000÷(D×A S ×B)×100%

[0046] W: weight of reference sample (g);

[0047] D: dilution volume of reference substance (ml);

[0048] A s: Peak area of ​​reference solution;

[0049] A r : Peak area of ​​test solution;

[0050] B: Labeled amount of miconazole nitrate suppository (g);

[0051] The beneficial effects of the present invention are described below by experimental examples:

[0052] Test Example 1 Study on the dissolution determination method of miconazole nitrate suppositories

[0053] 1. Determination of dissolution method

[0054] 1. Selection of dissolution medium

[0055] 1.1 pH of the dissolution medium

[0056] Miconazole nitrate suppository is a preparation for local treatment of vaginal bacterial infection. The pH value at the site of action is generally 3.8-4.4. Choosing acetic acid-sodium acetate buffer with pH 4.5 as the dissolution medium can simulate the acidic environment at the site of action.

[0057] 1.2 Selection of cosolvent

[0058] Since miconazole nitrate is insoluble in water, consider adding an appropriate amount of surfactant to the dissolution medium. In order to compare the solubilizing effects of different surfactants, 1000 ml of each of pH 4.5 acetic acid-sodium acetate buffer, pH 4.5 acetic acid-sodium acetate buffer (containing 1.0% Tween 80), and pH 4.5 acetic acid-sodium acetate buffer (containing 1.0% SDS) were used as dissolution media, respectively. The first method (basket method) of Part IV 0931 of the 2020 edition of the "Chinese Pharmacopoeia" was adopted, with a speed of 100 r / min and a temperature of 37°C. After 60 minutes, 2 ml of the solution was taken, filtered, and the filtrate was taken as the test solution. It was measured under the "Dissolution Sample Determination Method". The results are shown in Table 1. The specific method for dissolution sample determination is as follows:

[0059] Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Rules 0512).

[0060] Test solution Take the dissolved sample solution as the test solution.

[0061] Reference solution: Take about 10 mg of miconazole nitrate reference substance, weigh accurately, put it in a 50 ml volumetric flask, add 10 ml of methanol to dissolve it, dilute to the scale with dissolution medium, shake well, and use it as the reference solution.

[0062] The chromatographic conditions were as follows: octadecylsilane bonded silica gel was used as the filler (chromatographic column: Agilent, ZORBAX EclipsePlus C18 (250×4.6 mm, 5 μm)); 1.5% ammonium acetate-methanol-acetonitrile (20:40:40) was used as the mobile phase; the detection wavelength was 230 nm; the flow rate was 1 ml / min; the injection volume was 20 μl; the column temperature was 30°C, and the separation degree between the miconazole peak and other peaks should be greater than 10.

[0063] Determination method: Accurately measure the reference solution and the test solution and inject them into the liquid chromatograph, record the chromatogram, and calculate the dissolution amount (%) by the peak area according to the external standard method.

[0064] Table 1 Results of the test on the selection of cosolvent

[0065]

[0066]

[0067] As shown in Table 1, when SDS was added to the pH 4.5 buffer, the dissolution amount of miconazole nitrate suppositories was significantly increased, so SDS was selected as a cosolvent.

[0068] According to the test in the 2020 edition of the Chinese Pharmacopoeia, at 25°C, about 10 mg of miconazole nitrate can be completely dissolved in 14 ml of pH 4.5 acetic acid-sodium acetate buffer (containing 2.0% SDS). 1000 ml of pH 4.5 acetic acid-sodium acetate buffer (containing 2.0% SDS) can meet the sink conditions for the dissolution test of 0.2g miconazole nitrate suppositories.

[0069] In summary, 1000 ml of pH 4.5 acetic acid-sodium acetate buffer (containing 2.0% SDS) was selected as the dissolution medium.

[0070] 2. Dissolution method selection

[0071] Six batches of samples were randomly selected and tested according to Part IV 0931 of the 2020 edition of the "Chinese Pharmacopoeia". The dissolution method was investigated using 1000 ml of pH 4.5 acetic acid-sodium acetate buffer (containing 2.0% SDS) as the dissolution medium and a temperature of 37°C.

[0072] 2.1 Basket method and pulp method

[0073] The dissolution of miconazole nitrate suppositories within 5 hours was investigated using the basket method and the paddle method, both at a speed of 100 r / min. The results are as follows Figure 1 , Figure 2 .

[0074] Comparing the two methods, when the basket method was used, the matrix of miconazole nitrate suppository melted and blocked the basket holes, hindering the release of API into the dissolution medium, which was manifested as a slow sample dissolution rate and similar sample dissolution behaviors. When the paddle method was used, there were differences in the dissolution behaviors of the six batches of test samples, but during the experiment, there was a problem that the samples floated on the surface of the medium and rotated with the paddle during the dissolution process.

[0075] 2.2 Flow-through method

[0076] The European Pharmacopoeia and the British Pharmacopoeia recommend the use of the suppository pool in the flow cell method to test the dissolution of fatty matrix suppositories. The diameter of the standard suppository pool sample tube is 13mm. Since the appearance and size of miconazole nitrate suppositories produced by different manufacturers are different, samples from some manufacturers cannot be used with the standard suppository pool. In order to compare the dissolution behavior of miconazole nitrate suppositories from different manufacturers at the same time, an attempt was made to use the flow cell method-ordinary pool (diameter 22.6mm) for the experiment. The closed-loop mode was adopted, with 1000ml of pH 4.5 acetic acid-sodium acetate buffer (containing 2.0% SDS) as the dissolution medium, the temperature was 37°C, the medium flow rate was 16ml / min, the mass of the glass beads was 3g, and the results were as follows: Figure 3 Compared with the paddle method, the dissolution trends of the six batches of samples were similar, and the dissolution rates were reduced to varying degrees. The 300-min dissolution amounts of three batches of samples were approximately 100% (LCJ0V20, LDJ0H0H, LEJ4749), and the 300-min dissolution amounts of some batches of samples were abnormally low (21102002, the 5-hour dissolution amount was less than 10%).

[0077] When using the flow cell method-ordinary cell to investigate the dissolution behavior of adipose matrix plugs, due to the low density of the preparation (the preparation floats on the top of the sample cell) and the unidirectional flow of the dissolution medium, the melted matrix continues to accumulate on the filter membrane and clogs the filter membrane, which may affect the dissolution of the API. In order to verify whether there is a problem of low dissolution volume caused by membrane clogging in this flow cell method-ordinary cell dissolution test, miconazole nitrate suppositories (with the largest external diameter that can be used with standard suppository cells) produced by Hunan Fangsheng Pharmaceutical were selected for dissolution comparison tests in suppository cells (the structure of the suppository cell is different from that of the ordinary cell, and the melted matrix will not reach the filter membrane) and ordinary cells. The results are shown in Table 2.

[0078] Table 2 Comparison results of dissolution test in common pool (flow-through method) and suppository pool (flow-through method)

[0079]

[0080] As shown in Table 2, for the same batch of samples, the same experimental conditions, and the same time point, the dissolution amount in the suppository pool was significantly higher than that in the common pool. The flow cell method-common pool dissolution test of miconazole nitrate suppositories does have the problem of filter membrane clogging affecting API dissolution.

[0081] In summary, the basket method, paddle method and flow cell method are not suitable for investigating the dissolution behavior of miconazole nitrate suppositories.

[0082] In the final experiment, a sinker was added to the paddle method to improve the problems of sample floating and rotation with the paddle, and the dissolution behavior of miconazole nitrate suppositories was investigated by the paddle-sinker method. The size of the standard sinker in Part IV 0931 of the 2020 edition of the Chinese Pharmacopoeia does not match some of the miconazole nitrate suppositories studied in this study, so a self-made sinker (cylindrical sinker, stainless steel, size 1.8*3cm, Figure 4 ) to carry out experiments.

[0083] 3. Paddle-self-made sedimentation blue dissolution method speed selection

[0084] According to the dissolution and release test method (Method 2, Part 4, General Rules 0931, 2020 Edition of the Chinese Pharmacopoeia), 1000 ml of pH 4.5 acetic acid-sodium acetate buffer (containing 2.0% SDS) was used as the dissolution medium, and a homemade sinker was added to sink the suppository to the bottom of the dissolution cup. The dissolution test was carried out at 50 rpm, 75 rpm, and 100 rpm. The results showed that the dissolution rate of the sample increased as the speed increased. Finally, 100 rpm was selected to save experimental time while ensuring the method's ability to distinguish samples.

[0085] 2. Application of Dissolution Method

[0086] (1) Dissolution curve determination

[0087] The dissolution and release rate was determined according to the dissolution and release rate determination method (Method 2, 0931, General Rules of Part 4, 2020 Edition of the Chinese Pharmacopoeia).

[0088] The dissolution conditions were: 1000 ml of pH 4.5 acetic acid-sodium acetate buffer (containing 2.0% SDS) as the dissolution medium, a rotation speed of 100 revolutions per minute, a temperature of 37°C, and a homemade sinker basket to sink the suppository to the bottom of the dissolution cup. After 15 min, 30 min, 60 min, 90 min, 120 min, 150 min, 180 min, 210 min, 240 min, and 300 min, 1 ml of the solution was taken, filtered, and the filtrate was taken as the dissolution sample solution.

[0089] Determination method: Determine the dissolution rate (%) according to the dissolution sample determination method, and draw a dissolution curve with sampling time point (min) versus dissolution rate (%).

[0090] The dissolution test apparatus and method were used to investigate the in vitro dissolution behavior of commercially available miconazole nitrate suppositories. The dissolution curve test results were as follows: Figures 6 to 9 , where a representative chromatogram is shown in Figures 10-11. Combined with the clinical use time of miconazole nitrate suppositories and the differences in in vitro dissolution behavior of samples from different manufacturers, the dissolution test time was set at 300 minutes. The results showed that the dissolution behavior of samples from different manufacturers was significantly different within 5 hours, with small differences between batches of samples from company A and large differences between batches of samples from company D.

[0091] (2) Determination of quality standards for dissolution testing

[0092] If the dissolution mean of the same sample exceeds the content by more than 2.0%, it means that there is interference in the dissolution determination method, and at this time, unqualified samples will be misjudged as qualified. According to the method of the present invention, the dissolution mean of miconazole nitrate suppositories of different enterprises does not exceed the labeled amount of the main component of miconazole nitrate suppositories, and there is no misjudgment, which can accurately reflect the release characteristics of the active ingredient miconazole nitrate, and accurately evaluate its bioavailability and preparation uniformity. At the same time, it can also reflect the in vitro dissolution difference of miconazole nitrate suppositories of different enterprises, and realize the effective evaluation of the quality and process level of miconazole nitrate suppositories. The dissolution determination method of the present invention is used to detect miconazole nitrate suppositories of different enterprises, and the data are summarized and determined: the dissolution limit of miconazole nitrate suppositories using the method of the present invention is 60% (that is, the 300min dissolution should not be less than 60%).

[0093] 3. Research Significance

[0094] The dissolution determination method established in this method can not only be used for in vitro consistency evaluation of miconazole nitrate suppositories, but also reflect the batch-to-batch differences of miconazole nitrate suppositories, and has strong applicability and practical application value.

Claims

1. A method for determining the dissolution rate of miconazole nitrate suppositories, characterized in that: The method is used to investigate the dissolution behavior of adipose matrix plugs; it comprises the following steps: 1) Preparation of reference solution: Take miconazole nitrate reference substance, add methanol to dissolve it, and dilute the dissolved solution with dissolution medium to obtain the reference solution; 2) Preparation of test solution: Take miconazole nitrate suppositories, use acetic acid-sodium acetate buffer with pH value of 4.5 containing 2.0% SDS as dissolution medium, dissolve using a dissolution apparatus, take the dissolved solution and filter it to use as the test solution; The dissolution apparatus comprises a stirring paddle (3), a sinker basket (4) and a dissolution cup (2), wherein the stirring paddle (3) is arranged in the dissolution cup (2), and the sinker basket (4) is located at the bottom of the dissolution cup (2); The stirring paddle (3) is composed of a paddle rod and a paddle blade, wherein the upper end of the paddle rod is connected to the motor, and the lower end of the paddle rod is connected to the paddle blade; The sinker (4) is cylindrical, with a size of 1.8*3 cm, and is used to place the miconazole nitrate suppository; The dissolution cup (2) is a cup-shaped container with a hemispherical bottom, used for containing the dissolution medium; Each of the miconazole nitrate suppositories is dissolved with 1000 ml of dissolution medium; The speed of the stirring paddle in the dissolution apparatus is 100 rpm, and the time is 300 min; 3) Determine the content of miconazole nitrate in the reference solution and the test solution by HPLC, and calculate the solubility of miconazole nitrate suppository; The chromatographic conditions of the HPLC method include: chromatographic column: using octadecylsilane bonded silica gel as filler; Mobile phase: 1.5% ammonium acetate solution-methanol-acetonitrile in a volume ratio of 20:40:

40.

2. The method according to claim 1, characterized in that: Step 1) The mass volume ratio of the reference substance, methanol and dissolution medium is 10-20 mg: 10 ml: 40 ml.

3. The method according to claim 1, characterized in that: The chromatographic column is ZORBAX Eclipse Plus C18, 250×4.6 mm, 5 μm.

4. The method according to claim 3, characterized in that: The chromatographic conditions also include: column temperature of 30°C, injection volume of 20 μL, flow rate of 1 ml / min, and detection wavelength of 230 nm.

5. The method according to claim 1, characterized in that: The dissolution cup is provided with a cup cover, and the cup cover is provided with holes.

6. The method according to claim 5, characterized in that: The holes include a central hole; the central hole is the location of the stirring paddle, and the other holes are used for sampling or measuring temperature.

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