Method for determining dissolution curve of atorvastatin calcium in acidic medium

Abstract

The invention discloses a method for determining a dissolution curve of atorvastatin calcium in an acidic medium, and belongs to the technical field of pharmaceutical analysis. The determination method provided by the invention comprises the following steps: dissolving an atorvastatin calcium drug to be determined in an acidic dissolution medium under a stirring condition, taking out a dissolution solution at different dissolution times, centrifuging, taking a supernatant, adding the supernatant into a stabilizer, and uniformly mixing to obtain a test solution; detecting the content of atorvastatin calcium in the test solution by adopting high performance liquid chromatography to obtain dissolution rates at different time; establishing a dissolution curve according to the dissolution rate and the dissolution time; wherein the stabilizer is a mixed solution of an organic solvent and an alkali metal hydroxide water solution. According to the method, through the solubilizing effect of the organic solvent and the dual synergistic mechanism of the alkaline environment, the degradation reaction and drug precipitation process continuously occurring after the sample is taken out is effectively inhibited, and the accuracy of the dissolution rate detection result is remarkably improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical analysis, and particularly relates to a method for determining the dissolution curve of atorvastatin calcium in an acidic medium. BACKGROUND

[0002] The information disclosed in the background of the present application is only intended to increase the understanding of the overall background of the present application and should not necessarily be regarded as acknowledging or implicitly suggesting that this information constitutes prior art known to those of ordinary skill in the art.

[0003] Atorvastatin calcium, as a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, has become the largest prescription cholesterol-lowering drug and the core variety of cardiovascular and cerebrovascular disease treatment worldwide. Since its launch, the drug has continued to occupy an important position in the global pharmaceutical market due to its significant clinical efficacy. Especially in the context of the increasing incidence and mortality of cardiovascular and cerebrovascular diseases year by year, the demand for generic research and development of atorvastatin calcium formulations has shown a significant growth trend, and the quality control research of related oral solid preparations has become the focus of the industry.

[0004] In the solid preparation research and development system, dissolution research as a core indicator for evaluating the in-vitro release characteristics of drugs has important guiding value for prescription process optimization and bioequivalence prediction. However, the current atorvastatin calcium dissolution determination method has exposed significant technical defects in the acidic medium. Atorvastatin calcium has chemical stability problems in the acidic environment of pH=1.0~2.0, not only will it undergo degradation, but also the dissolved substances will appear secondary precipitation with the extension of the test time. The conventional immediate termination reaction strategy cannot block the continuous degradation and precipitation process after the sample is taken out, resulting in systematic deviation of the test results, so that the test data cannot truly reflect the inherent dissolution characteristics of the preparation, which seriously affects the correlation study of in-vitro dissolution curve and in-vivo absorption. This technical defect not only increases the risk of failure of generic drug bioequivalence tests, but also forms a substantial obstacle to the scientific establishment of drug quality standards, objectively restricting the research and development process of high-quality generic drugs. At present, there is no effective stabilization method for the dissolution detection of such unstable compounds. SUMMARY

[0005] Therefore, the present application provides a method for determining the dissolution curve of atorvastatin calcium in an acidic medium, which adds a stabilizer to the atorvastatin calcium dissolution sample taken out from the acidic medium to avoid degradation and precipitation of the sample in the acidic environment, and ensures the accuracy of the dissolution test.

[0006] The present application provides a method for determining the dissolution curve of atorvastatin calcium in an acidic medium, comprising the following steps: Dissolve atorvastatin calcium drug under test in the acidic dissolution medium under stirring, take out the dissolution liquid at different dissolution time, take the supernatant after centrifugation and add to the stabilizer, and mix after adding to the stabilizer to be used as the test solution; Detect the atorvastatin calcium content in the test solution by high performance liquid chromatography to obtain the dissolution at different time; Establish the dissolution curve according to the dissolution and dissolution time; The stabilizer is a mixed solution of an organic solvent and an aqueous alkali metal hydroxide solution.

[0007] Compared with the prior art, the present application has the following beneficial effects: (1) The present application solves the chemical stability problem of atorvastatin calcium in the acidic dissolution medium by introducing a stabilizer system containing an organic solvent and an alkali metal hydroxide, effectively inhibits the degradation reaction and drug precipitation process that continues to occur after the sample is taken out through the dual synergistic mechanism of the solubilization of the organic solvent and the alkaline environment, so that the actual concentration of atorvastatin calcium in the dissolution liquid is truly retained, and the accuracy of the dissolution detection result is significantly improved, laying a technical foundation for establishing a reliable in-vitro dissolution-in-vivo absorption correlation model.

[0008] (2) The present application breaks through the technical bottleneck of distorted bioequivalence prediction caused by poor drug stability in the traditional acidic dissolution medium, significantly reduces the risk of bioequivalence test failure caused by dissolution data deviation in generic drug research and development through standardized operation process; through scientific and reliable dissolution curve data support, accelerates the quality consistency evaluation process of generic drugs, provides key technical support for the accessibility improvement of cardiovascular and cerebrovascular disease treatment drugs, and has significant industry promotion value. DETAILED DESCRIPTION

[0009] The drawings accompanying the specification of the present application serve to provide further understanding of the present application, the illustrative embodiments of the present application and the description thereof serve to explain the present application, and do not constitute an improper limitation on the present application. Obviously, other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0010] Figure 1 is the dissolution curve graph of the cumulative dissolution and dissolution time of examples 1-4 and comparative examples of the present application. DETAILED DESCRIPTION

[0011] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the present application belongs.

[0012] The application provides a method for determining the dissolution curve of atorvastatin calcium in an acid medium, comprising the following steps: The atorvastatin calcium to be tested is dissolved in an acid dissolution medium under stirring, and dissolution liquid is taken out at different dissolution times, and the supernatant is taken out after centrifugation and added to a stabilizer, and after mixing, the mixture is used as a test solution; The content of atorvastatin calcium in the test solution is detected by high performance liquid chromatography (HPLC), and the dissolution rate at different times is obtained. The dissolution curve is established according to the dissolution rate and the dissolution time. The stabilizer is a mixed solution of an organic solvent and an aqueous alkali metal hydroxide solution.

[0013] The acid medium simulates the gastric juice environment and can reflect the dissolution behavior of the drug in the stomach of the human body, which is crucial for evaluating the bioavailability of the drug. The dissolution curve directly reflects the in vitro release rate and degree of the drug, and is an important basis for prescription screening, process optimization and bioequivalence (BE) test of generic drugs. When atorvastatin calcium is dissolved in an acid dissolution medium, the drug will be dissolved due to the enhanced protonation, but at the same time, it will face the dual risks of chemical degradation and precipitation. Therefore, the stabilizer is added to the supernatant obtained after centrifugation after sampling, the organic solvent in the stabilizer is used to quickly disperse the drug molecules, the polarity of the solution is reduced, the solubility of the drug molecules is enhanced, and the concentration is prevented from being reduced due to precipitation; at the same time, the alkali metal hydroxide quickly neutralizes the acid environment, blocks the hydrolysis reaction catalyzed by H⁺, and thus maintains the stability of the drug in the solution. The supernatant obtained after centrifugation of the dissolution liquid is used for subsequent determination, which can remove the undissolved excipients or particulate matter, and ensures the accuracy of the detection.

[0014] The application does not make special restrictions on the specific dissolution method, and the operation can be performed according to the Chinese Pharmacopoeia 2020 edition four-part general rule 0931 “dissolution and release determination method”, and the paddle method is preferably used for dissolution. The dosage ratio of atorvastatin calcium to be tested to the acid dissolution medium is (60-100) mg: 900 mL.

[0015] The application does not make special restrictions on the specific sampling time, and the commonly used sampling time in the field can be used, the sampling time needs to cover the whole dissolution process (such as 5, 10, 15, 20, 30, 45, 60 min), and the dissolution amount needs to reach the platform period.

[0016] The application detects the content of atorvastatin calcium in the test solution by high performance liquid chromatography (HPLC), and compared with the ultraviolet spectrophotometry (UV method), HPLC has higher specificity and can distinguish the main peak of the drug from the degradation product.

[0017] In the present application, the organic solvent is selected from N,N-dimethylformamide, dimethyl sulfoxide, N-methyl pyrrolidone; more preferably N,N-dimethylformamide (DMF), the selected organic solvent can effectively dissolve atorvastatin calcium, and has good compatibility with HPLC mobile phase. The alkali metal hydroxide is selected from sodium hydroxide, potassium hydroxide or lithium hydroxide; more preferably sodium hydroxide, which is low in cost and high in solubility.

[0018] In the present application, the concentration of the aqueous alkali metal hydroxide solution is 0.1-10 mol / L, more preferably 0.2-2 mol / L, which can be selected according to the acidic dissolution medium, and the amount needs to be controlled to prevent local over-alkalization.

[0019] In the present application, the volume fraction of the organic solvent in the stabilizer is 30-60%, more preferably 40-55%. The above range can not only avoid drug precipitation, but also has good compatibility with the chromatographic column to avoid interference with the separation effect of the chromatographic column.

[0020] In the present application, the pH of the acidic dissolution medium is 1.0-2.0, more preferably 1.0-1.5. This acidic environment simulates the pH environment of human gastric juice to evaluate the release behavior of the drug in the gastrointestinal tract.

[0021] In the present application, the volume ratio of the supernatant to the stabilizer is (1-4):1. The specific volume ratio can be determined according to the experimental conditions to ensure a faster neutralization rate while avoiding excessive dilution that leads to insufficient HPLC detection limit.

[0022] In the present application, the chromatographic conditions of the high performance liquid chromatography are as follows: the column temperature is 25-45℃, which can improve the chromatographic separation efficiency, reduce the viscosity of the mobile phase, and shorten the analysis time. The flow rate is 0.8-2.0 mL / min to balance the separation speed and column pressure and avoid loss of column efficiency. The detection wavelength is 240-250 nm, which matches the maximum absorption wavelength (about 244 nm) of atorvastatin calcium to ensure the detection sensitivity. The injection volume is 5-20 μL, which is small, so the presence of the stabilizer does not have a significant impact on the chromatographic column. The chromatographic column uses octadecylsilane-bonded silica gel as the filler to realize the separation of atorvastatin calcium and other components by reverse phase chromatography.

[0023] In the present application, the mobile phase of the high performance liquid chromatography includes acetonitrile, tetrahydrofuran and citric acid buffer. Acetonitrile as a strong eluent can adjust the elution strength to shorten the retention time. Tetrahydrofuran helps to adjust the polarity to improve the separation selectivity of hydrophobic impurities. Citric acid buffer can inhibit the ionization of atorvastatin calcium to improve the chromatographic stability.

[0024] Further, the concentration of the citric acid buffer is 0.04-0.06 mol / L, and the pH of the citric acid buffer is 4.2-4.8, so that the weakly acidic environment can stabilize the ionized state of the drug, reduce peak tailing caused by ionization, and improve separation efficiency. Further, the volume ratio of acetonitrile, tetrahydrofuran and citric acid buffer is (20-30) : (15-25) : (50-60), so as to balance the elution capacity and separation effect.

[0025] The technical solutions of the present application will be further described below in combination with specific examples. The reagents used in the following examples are not specially limited, and commercially available products known to those skilled in the art can be used.

[0026] Example 1 The present example provides a method for determining the dissolution curve of atorvastatin calcium in an acidic medium. The specific steps are as follows: 1. Dissolution: 80 mg of atorvastatin calcium to be tested was placed in 900 mL of an acidic dissolution medium (hydrochloric acid solution with pH = 1.2), and dissolution was carried out by paddle method with stirring speed of 50 rpm. The sampling time was 5, 10, 15, 20, 30, 45 and 60 min.

[0027] 2. Preparation of test solution: The removed dissolution solution was centrifuged at 10 mL, and 1 mL of supernatant was taken, then mixed with stabilizer at a volume ratio of 1:1 to obtain the test solution, wherein the stabilizer was DMF and 1 mol / L NaOH solution at a volume ratio of 1:1.

[0028] 3. HPLC detection: HPLC chromatographic conditions: The column temperature was 30℃, the flow rate was 1.5 mL / min, the detection wavelength was 244 nm, the injection volume was 10 μL, and the chromatographic column was filled with octadecylsilane bonded silica gel (C18 4.6 mm x 150 mm, 5 μm or a chromatographic column with equivalent performance).

[0029] The mobile phase was acetonitrile-tetrahydrofuran-citric acid buffer, wherein the volume ratio of acetonitrile, tetrahydrofuran and citric acid buffer was 27:20:53; the concentration of the citric acid buffer was 0.05 mol / L, and the preparation method was as follows: taking citric acid 9.62 g, adding water 1000 mL to dissolve, and adjusting the pH value to 4.5 with ammonia water.

[0030] The content of atorvastatin calcium in the test solution was detected by HPLC to obtain the dissolution rate, and the dissolution curve was established according to the dissolution rate and dissolution time.

[0031] Example 2 The difference between this example and Example 1 is that the volume ratio of supernatant to stabilizer in the preparation of the test sample solution is 2:1. The test sample solution is prepared as follows: The centrifuged 10 mL of the dissolution solution is taken, 2 mL of the supernatant is taken, and then mixed with the stabilizer at a volume ratio of 2:1 to obtain the test sample solution, wherein the stabilizer is a DMF and 1 mol / L NaOH solution at a volume ratio of 1:1.

[0032] Example 3 The difference between this example and Example 1 is that the volume ratio of supernatant to stabilizer in the preparation of the test sample solution is 3:1. The test sample solution is prepared as follows: The centrifuged 10 mL of the dissolution solution is taken, 3 mL of the supernatant is taken, and then mixed with the stabilizer at a volume ratio of 3:1 to obtain the test sample solution, wherein the stabilizer is a DMF and 1 mol / L NaOH solution at a volume ratio of 1:1.

[0033] Example 4 The difference between this example and Example 1 is that the volume ratio of supernatant to stabilizer in the preparation of the test sample solution is 4:1. The test sample solution is prepared as follows: The centrifuged 10 mL of the dissolution solution is taken, 4 mL of the supernatant is taken, and then mixed with the stabilizer at a volume ratio of 4:1 to obtain the test sample solution, wherein the stabilizer is a DMF and 1 mol / L NaOH solution at a volume ratio of 1:1.

[0034] Comparative Example The difference between this example and Example 1 is that the stabilizer is not added in the comparative example. The test sample solution is prepared as follows: The centrifuged 10 mL of the dissolution solution is taken, 5 mL of the supernatant is taken as the test sample solution.

[0035] Test Example 1. Dissolution curve The cumulative dissolution data of different sampling times of Examples 1-4 and the comparative example are shown in Table 1, and the dissolution curve of the cumulative dissolution and dissolution time is shown in Figure 1 .

[0036] Table 1 Cumulative dissolution (unit: %) of different sampling times of Examples 1-4 and the comparative example

[0037] From Table 1 and Figure 1As can be seen from the table 2, the atorvastatin calcium reached the maximum dissolution in 3-5 minutes in 900 ml of acidic medium, but the drug solution was in a continuous degradation state in the acidic environment of the dissolution cup at the subsequent sampling points, so the cumulative dissolution was in a continuous decreasing trend. The detection values of the sample of the comparative example at the same dissolution time points were always low, which was mainly because the sample was still in an acidic environment before detection, and the drug was continuously degraded (such as hydrolysis) or precipitated due to supersaturation, which caused the dissolution to be abnormally low. The abnormal fluctuation may be caused by the temporary redissolution of the precipitated particles. The examples neutralize the acidity of the sample by NaOH (pH rises to neutral) and inhibit the aggregation of drug molecules by DMF, which blocks the continuous degradation reaction and precipitation after sampling, so as to more truly retain the drug concentration in the dissolution solution. The dissolution curve shows a gentle decrease after reaching the peak at 5 min.

[0038] 2. Stability test: The solution stability of the test solution of examples 1-4 and the comparative example at 0 h and after standing for 2 h was investigated, and the results are summarized in table 2.

[0039] Table 2 Solution stability data of the test solution of examples 1-4 and the comparative example at 0 h and after standing for 2 h

[0040] *Note: Impurity IV is atorvastatin lactone, and its structural formula is as follows: .

[0041] Impurity IV is generated because the structure of atorvastatin calcium contains two key groups: Carboxyl group (-COOH): located at the end of the side chain, hydroxyl group (-OH): located at the δ position of the carboxyl group, under acidic conditions: the oxygen atom of the carboxyl group is protonated to form a more easily leaving group (-COOH2), and the intramolecular hydroxyl group (-OH) acts as a nucleophile to attack the carboxyl carbon atom. One molecule of water (H2O) is lost to form a six-membered ring lactone structure (δ-lactone). +

[0042] As can be seen from table 2, the test solution without stabilizer will change from a transparent and clear solution to a flocculent solution after standing for 2 h, and the peak of impurity IV can be detected at 0 h and 2 h, and the peak area of the main component at 2 h decreases more. However, the test solutions with stabilizers (examples 1-4) all exhibit excellent stability.

[0043] ​The above merely provides the preferred embodiments of the present application, and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the principles and technical scope of the present application shall fall into the scope of the present application.

Claims

1. A method for determining the dissolution curve of atorvastatin calcium in an acidic medium, characterized in that, Includes the following steps: Atorvastatin calcium, the test drug, was dissolved in an acidic dissolution medium under stirring. The dissolution solution was taken out at different dissolution times, centrifuged, and the supernatant was added to the stabilizer. After mixing, it was used as the test solution. The atorvastatin calcium content in the test solution was detected by high performance liquid chromatography, and the dissolution rate at different times was obtained. Establish a dissolution profile based on the dissolution rate and dissolution time; The stabilizer is a mixed solution of an organic solvent and an aqueous solution of an alkali metal hydroxide.

2. The method for determining the dissolution curve of atorvastatin calcium in acidic medium as described in claim 1, characterized in that, The organic solvent is selected from N,N-dimethylformamide, dimethyl sulfoxide, or N-methylpyrrolidone; the alkali metal hydroxide is selected from sodium hydroxide, potassium hydroxide, or lithium hydroxide.

3. The method for determining the dissolution curve of atorvastatin calcium in acidic medium as described in claim 1, characterized in that, The concentration of the alkali metal hydroxide aqueous solution is 0.1~10 mol / L.

4. The method for determining the dissolution curve of atorvastatin calcium in acidic medium as described in claim 1, characterized in that, The volume fraction of the organic solvent in the stabilizer is 30-60%.

5. The method for determining the dissolution curve of atorvastatin calcium in acidic medium as described in claim 1, characterized in that, The pH of the acidic leaching medium is 1.0~2.

0.

6. The method for determining the dissolution curve of atorvastatin calcium in acidic medium as described in claim 1, characterized in that, The volume ratio of the supernatant to the stabilizer is (1~4):

1.

7. The method for determining the dissolution curve of atorvastatin calcium in acidic medium as described in claim 1, characterized in that, The chromatographic conditions for the high performance liquid chromatography are as follows: column temperature 25~45℃, flow rate 0.8~2mL / min, detection wavelength 240~250nm, injection volume 5~20μL, and the column is packed with octadecylsilane-bonded silica gel.

8. The method for determining the dissolution curve of atorvastatin calcium in acidic medium as described in claim 1, characterized in that, The mobile phase of the high-performance liquid chromatography includes acetonitrile, tetrahydrofuran, and citrate buffer.

9. The method for determining the dissolution curve of atorvastatin calcium in acidic medium as described in any one of claims 8, characterized in that, The concentration of the citrate buffer solution is 0.04~0.06 mol / L, and the pH of the citrate buffer solution is 4.2~4.

8.

10. The method for determining the dissolution curve of atorvastatin calcium in acidic medium as described in claim 9, characterized in that, The volume ratio of acetonitrile, tetrahydrofuran, and citrate buffer is (20~30): (15~25): (50~60).

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