Concentration detection method of limaprost tablet dissolution rate test solution

Abstract

According to the concentration detection method of the limaprost tablet dissolution rate test solution, the problems that limaprost tablet dissolution rate / dissolution curve detection concentration is low and specificity is poor are solved, and the established dissolution method is verified by a system and is accurate, reliable and good in precision. Establishment and application of the method can help research, development and marketing of limaprost tablets, fill the blank of clinical treatment of lumbar spinal stenosis, and bring new treatment gospel to vast patients.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical analysis, specifically to the concentration detection problem in the dissolution test of Limaprost tablets, and is widely applied in pharmaceutical preparations and pharmaceutical analysis quality control. Background Art

[0002] Limaprost is an orally administered prostaglandin E1 analogue, which has been proven to improve peripheral circulatory failure through vasodilatory and antithrombotic effects; it can also improve the poor blood flow of nerve tissues in cervical spondylosis and normalize nerve function. It was approved in 1988 for the treatment of ischemic symptoms such as skin ulcers, pain, and coldness associated with thromboangiitis obliterans. The structural formula of Limaprost is as follows:

[0003]

[0004] Limaprost tablets were jointly developed by Ono Pharmaceutical Co., Ltd. and Sumitomo Dainippon Pharma Co., Ltd. in Japan and were first launched in Japan in January 1988 under the trade names with a specification of 5 μg (highly active substance). After querying relevant patents and literature, no reports on the dissolution or dissolution curve of the original research drug product were found, nor were there any reports on the dissolution of low-dose products with the same specification as this product.

[0005] As an oral solid preparation, dissolution is a key quality attribute. However, during the development of generic drugs, many difficulties were found in the determination of the dissolution of this preparation. First, this product has the following characteristics: the main component is a highly active substance, the specification is only 5 μg / tablet, and the concentration of the dissolution test solution is extremely low. When the medium volume is 500 ml, the concentration of 100% dissolution solution is only 10 ng / ml, and when the medium is 900 ml, the concentration of 100% dissolution solution is only 5.56 ng / ml. Due to the low proportion of the main component and the high proportion of excipients, both method specificity and detection sensitivity are challenges.

[0006] Secondly, the second method (paddle method) in General Rule 0931 of the Chinese Pharmacopoeia (2020 Edition) is a commonly used method for determining the dissolution of drug tablets. This method uses a dissolution tester to place the tablets in a dissolution cup containing a suitable dissolution medium (such as water and buffer solution), with the medium temperature maintained at 37 ± 0.5 °C, and stirs with a paddle at a specified speed (usually 50 or 75 revolutions per minute). Samples are taken at specified time points (such as 5, 10, 15, 30, 45, 60 minutes), and an equal volume of medium is added to keep the volume constant. The samples taken are used to determine the drug concentration by methods such as ultraviolet-visible spectrophotometry or high-performance liquid chromatography to evaluate the rate and extent of drug release. The dissolution standard is set according to the drug characteristics, and usually, it is required that the dissolution amount reaches a certain percentage (such as 80%) within the specified time. This method is applicable to the dissolution determination of most tablets to ensure that the release and absorption of drugs in the body meet expectations. According to the requirements of the "Guidelines for the Determination and Comparison of Dissolution Curves of Oral Solid Dosage Forms", dissolution curve studies need to be carried out in dissolution media with pH values of 1.2, 4.5, and 6.8. However, it is currently known that the solution stability of the product of limaprost in the pH 1.2 medium is poor, and the dissolution solution in the pH 4.5 medium is close to pKa, with poor injection precision of the chromatographic system. Therefore, the applicability of the method is very poor.

[0007] The open text of Chinese Patent 202110670906.4, "A Method for Quantitative Determination of Limaprost in Biological Samples", records the ultra-high performance liquid chromatography-differential ion mobility-quadrupole time-of-flight high-resolution tandem mass spectrometry analysis technique; in the open text of Chinese Patent 202110692340.5, "A Method for Detecting Prostaglandin Substances in Biological Samples", it is recorded that the prostaglandin substances to be detected in biological samples are quantitatively converted into aminomethyl arylsulfonic acid derivatives by chemical derivatization method, and then detected by liquid chromatography-mass spectrometry online. However, the above methods may also be applicable to the trace detection of limaprost dissolution samples, but such methods have high requirements for personnel and instruments, high detection costs, poor applicability and operability, and are at least not suitable for a large number of analytical detections in the drug development process in terms of capital cost.

[0008] Common solutions for the dissolution of low-dose drugs mainly include: 1. Using the small-cup method included in the Chinese Pharmacopoeia, such as the fumarate formoterol tablets (specification: 40 μg, dissolution medium: 100 ml), repaglinide tablets (specification: 0.5 mg, dissolution medium: 100 ml), colchicine tablets (specifications: 0.5 mg and 1 mg, dissolution medium: 200 ml), etc. included in ChP 2020. 2. Selecting sensitive detection methods can also be used for the dissolution determination of low-dose drug preparations. For example, for the compound levonorgestrel tablets (specification: levonorgestrel 0.15 mg, ethinylestradiol 0.03 mg) included in ChP 2020, the fluorescence detector is used to determine the dissolution of ethinylestradiol. Although the small-cup method increases the concentration of the dissolved drug, compared with the paddle method in the ordinary cup, the shear stress generated during stirring and some physical parameters are quite different. The stirring degree is more intense than that of the ordinary cup paddle method, which actually relatively lowers the threshold of in vitro dissolution requirements, is not conducive to establishing the in vitro-in vivo correlation, and may not be able to distinguish the quality of drugs well. Fluorescence detectors usually require derivatization, and there are many experimental details to note during derivatization, and there may be by-products generated. Therefore, there are some technical defects.

[0009] Therefore, the current situation is that there is no simple and feasible dissolution detection method for limaprost tablets (5 μg / tablet), which is an urgent problem to be solved for the development of generic drugs. Summary of the Invention

[0010] Through in-depth research and continuous attempts by the inventors, the present invention provides a method for detecting the dissolution curve of limaprost tablets. This method can detect in pH 1.2 medium and pH 4.5 medium, establishes a control method for dissolution, and solves the problems of low concentration and poor specificity. The present invention also conducts systematic verification of each method, and the results show that the method has good specificity, and the sensitivity, accuracy, and precision indicators meet the requirements of the test. The established method is suitable for the control of limaprost tablets.

[0011] Specifically, the present invention provides a method for detecting the concentration of the dissolution test solution of limaprost tablets, which is detected based on the external standard method of liquid chromatography detection.

[0012] The liquid chromatography detection meets the following conditions:

[0013] An octadecylsilane-bonded silica gel packed chromatographic column is used, and the column temperature is 20°C to 31°C.

[0014] The injection volume is 500 μL to 2000 μL, and the injector is temperature-controlled at 15°C to 20°C.

[0015] The mobile phase composition is as follows: mobile phase A is a 0.02 mol / L potassium dihydrogen phosphate solution, and its pH is adjusted to 3.0 with 20% phosphoric acid; mobile phase B is a solution prepared by mixing 0.02 mol / L potassium dihydrogen phosphate solution, acetonitrile, and isopropanol in a volume ratio of (900 ± 50):(500 ± 30):(200 ± 10), with the preferred ratio being 900:530:200. The volume ratio of mobile phase A to mobile phase B is 10:90 to 12:88, with the preferred ratio being 10:90, and the flow rate is 0.8 ml / min to 1.0 ml / min.

[0016] In an embodiment of the present invention, in the concentration detection step of the dissolution test solution in a pH 1.2 medium, it is characterized in that a protective solution is added to the test solution, and the protective solution is a phosphate buffer solution containing sodium hydroxide.

[0017] In an embodiment of the present invention, in the dissolution test step in a pH 1.2 medium, after taking out the dissolution test solution from the dissolution tester, the following pretreatment is carried out: a phosphate buffer solution containing sodium hydroxide is added to the dissolution test solution to neutralize the hydrogen ions in the pH 1.2 medium to obtain a test sample solution.

[0018] In a preferred embodiment of the present invention, in the dissolution test step in a pH 1.2 medium, the phosphate buffer solution containing sodium hydroxide is prepared according to the following method:

[0019] Weigh an appropriate amount of potassium dihydrogen phosphate and sodium hydroxide, dissolve them in water and quantitatively dilute to prepare an aqueous solution with a mass content of 6.8% potassium dihydrogen phosphate and 3.6% sodium hydroxide, and that's it.

[0020] In the dissolution test in a pH 1.2 medium, the volume ratio of the test solution to the protective solution is 8:1 to 10:1, and the preferred volume ratio is 9:1.

[0021] In an embodiment of the present invention, in the dissolution test in a pH 4.5 medium, it is characterized in that the test sample and the reference solution are simultaneously added with a pH adjusting solution, and its pH is 6.8 ± 2.

[0022] In an embodiment of the present invention, in the dissolution test in a pH 4.5 medium, it is characterized in that the test sample and the reference solution are simultaneously added with a 6.8 ± 2 pH adjusting solution, and the final pH of the adjusted test sample and reference solution is 6.0 to 7.2.

[0023] In the concentration detection step of the dissolution test solution in a pH 4.5 medium, after taking out the dissolution test solution from the dissolution tester, the following pretreatment is carried out: Add a pH regulator to the dissolution test solution to adjust the pH of the solution to 6.0 - 7.2, and use this as the test solution. Also add the same pH regulator to the reference solution so that the pH of the reference solution is 6.0 - 7.2;

[0024] In a preferred embodiment of the present invention, in the concentration detection step of the dissolution test solution in a pH 4.5 medium, the pH regulator is prepared according to the following method: Appropriately take potassium dihydrogen phosphate and disodium hydrogen phosphate anhydrous, add an appropriate amount of water to dissolve them, and obtain a pH 6.8 medium solution, which is mixed with 1 mol / L NaOH solution in a volume ratio of 7:3 to obtain the pH regulator.

[0025] The volume ratio of the test solution or reference solution to the pH regulator is 8:1 to 10:1, and the preferred volume ratio is 9:1.

[0026] In a preferred embodiment of the present invention, in the concentration detection step of the dissolution test solution in a pH 4.5 medium, the reference solution is prepared according to the following method:

[0027] Mix the pH 4.5 medium and the pH adjustment solution in a ratio of 9:1, shake well to obtain a dilution solution.

[0028] Take about 20 mg of limaprost reference substance, weigh it accurately, place it in a 100 ml volumetric flask, dissolve it with anhydrous ethanol and dilute to the mark, shake well to obtain the stock solution of the reference solution; Accurately measure 1 mL of the stock solution of the reference solution, place it in a 100 ml volumetric flask, add anhydrous ethanol to dilute to the mark, shake well; Accurately measure 1 ml of this solution and place it in a 200 ml volumetric flask, first add 3 ml of anhydrous ethanol, and then add the dilution solution to dilute to the mark, shake well to obtain the reference solution.

[0029] The ethanol content of the limaprost reference solution is 2.0% - 3.5%.

[0030] The dissolution tester described in the present invention is a paddle method dissolution tester, generally referring to an instrument that meets the requirements of the second method in General Chapter 0931 of the Chinese Pharmacopoeia 2020 Edition.

[0031] In a preferred embodiment of the present invention, the method further includes a pH 6.8 medium dissolution curve detection step. In this step,

[0032] Take 1.7 g of potassium dihydrogen phosphate and 1.775 g of disodium hydrogen phosphate anhydrous, add an appropriate amount of water to dissolve them, and make up the volume to 1000 ml to obtain a pH 6.8 dissolution medium;

[0033] Take about 20 mg of the control substance of limaprost, weigh accurately, place it in a 100-ml volumetric flask, dissolve it with absolute ethanol and dilute to the mark, shake well to obtain the stock solution of the control solution. Then accurately measure 1 mL of the stock solution of the control solution, place it in a 200-ml volumetric flask, add absolute ethanol and dilute to the mark, shake well; accurately measure 1 ml and place it in a 100-ml volumetric flask, first add 1 ml of absolute ethanol, then add the dissolution medium and dilute to the mark, shake well to obtain the control solution with the final ethanol content of 2%;

[0034] The solution taken out from the dissolution tester is directly used as the test solution for the detection step of the dissolution curve in the pH 6.8 medium after filtration.

[0035] In a preferred embodiment of the present invention, the chromatographic conditions are as follows:

[0036] Flow rate: 0.8 ml / min;

[0037] Injection volume: 500 μl;

[0038] Column temperature: 30 °C;

[0039] Injector temperature: 15 °C;

[0040] Mobile phase A: Mobile phase B = 10:90.

[0041] In a preferred embodiment of the present invention, the chromatographic column is selected from the chromatographic columns of the TOSOH C18, Waters Atlantis T3, and Waters symmetry C18 series.

[0042] Compared with the prior art, the present invention has the following obvious advantages: The present invention solves the problems of low detection sensitivity and poor specificity of the dissolution / dissolution curve of limaprost tablets. The established dissolution method has been systematically verified, is accurate and reliable, and has good precision. The present invention specifically optimizes the concentration detection steps of the dissolution test solution in the pH 1.2 medium and the concentration detection steps of the dissolution test solution in the pH 4.5 medium, and specifically solves the problems of poor solution stability of the pH 1.2 medium product of limaprost and the dissolution solution of the pH 4.5 medium being close to pKa and poor chromatographic system applicability. It can be said that the invention fills the blank in the research of the dissolution / dissolution curve of this micro-dose preparation. The establishment and application of the method will contribute to the research and listing of limaprost tablets. Description of the Drawings

[0043] Figure 1 is the chromatogram of the pH 1.2 medium - blank solvent;

[0044] Figure 2 is the chromatogram of the pH 1.2 medium - control solution;

[0045] Figure 3 is the chromatogram of the blank excipient solution in pH 1.2 medium;

[0046] Figure 4 is the chromatogram of the test solution in pH 1.2 medium;

[0047] Figure 5 is the chromatogram of the blank solvent in pH 4.5 medium;

[0048] Figure 6 is the chromatogram of the reference solution in pH 4.5 medium;

[0049] Figure 7 is the chromatogram of the blank preparation solution in pH 4.5 medium;

[0050] Figure 8 is the chromatogram of the test solution in pH 4.5 medium;

[0051] Figure 9 is the chromatogram of the blank solvent in pH 6.8 medium;

[0052] Figure 10 is the chromatogram of the blank excipient solution in pH 6.8 medium;

[0053] Figure 11 is the chromatogram of the reference solution in pH 6.8 medium;

[0054] Figure 12 is the chromatogram of the test solution in pH 6.8 medium;

[0055] Figure 13 is the chromatogram of the blank solvent in water medium;

[0056] Figure 14 is the chromatogram of the reference solution in water medium;

[0057] Figure 15 is the chromatogram of the blank excipient solution in water medium;

[0058] Figure 16 is the chromatogram of the test solution in water medium;

[0059] Figure 17 is the chromatogram of the derivative typical chromatogram;

[0060] Figure 18 is the comparison chart of the chromatograms at different column temperatures;

[0061] Figure 19 is the chromatogram of the comparison chart of different flow rates (from top to bottom are 0.9 ml / min, 0.8 ml / min, 0.7 ml / min);

[0062] Figure 20It is a comparison chart of the chromatogram after adjusting the ratio of phase A and phase B of the eluent;

[0063] Figure 21 It is a typical chromatogram of different chromatographic columns;

[0064] Figure 22 It is an xy normalization chart of the linear range for different dissolution media. Detailed implementation manners

[0065] To further illustrate the elements of the present invention, the following will be described in conjunction with specific embodiments. However, the protection scope of the present invention is not limited to the following embodiments.

[0066] In the present invention, the phosphate buffer solution containing sodium hydroxide used in the dissolution test of limaprost tablets in pH 1.2 medium, sometimes also called the protective solution, and the liquid for adjusting the pH used in the dissolution test of limaprost tablets in pH 4.5 medium, sometimes also called the pH adjustment solution.

[0067] Example 1

[0068] Based on the following conventional methods, the dissolution behaviors of the dissolution medium at pH 6.8 and the dissolution behavior in water medium required by the "Guidelines for the Determination and Comparison of Dissolution Curves of Ordinary Oral Solid Preparations" were studied.

[0069] 1. Dissolution test method:

[0070] 1) Chromatographic conditions

[0071] Chromatographic column: Octadecylsilyl-bonded silica gel as the filler (Waters, 150 mm × 4.6 mm, 3.5 μm);

[0072] Mobile phase A: 0.02 mol / L potassium dihydrogen phosphate solution (adjusted to pH 3.0 with 20% phosphoric acid);

[0073] Mobile phase B: 0.02 mol / L potassium dihydrogen phosphate solution - acetonitrile - isopropanol (900:530:200);

[0074]

[0075] (2) Dissolution conditions

[0076] Dissolution medium: 500 ml of pH 6.8 dissolution medium;

[0077] Device: Second method (paddle method) in General Principles 0931 of the Chinese Pharmacopoeia 2020 edition;

[0078] Temperature: 37°C ± 0.5°C; Rotation speed: 50 rpm / min;

[0079] Sampling time: 30 min

[0080] (3) Preparation of solutions

[0081] Dissolution medium of pH 6.8: Take 1.7 g of potassium dihydrogen phosphate and 1.775 g of disodium hydrogen phosphate anhydrous, add appropriate amount of water to dissolve and then make up the volume to 1000 ml, and you will get it.

[0082] Stock solution of reference substance solution: Take about 20 mg of limaprost reference substance, accurately weigh it, place it in a 100-ml volumetric flask, dissolve it with absolute ethanol and dilute to the scale, shake well, and you will get it.

[0083] Reference substance solution: Accurately measure 1 ml of the stock solution of reference substance solution, place it in a 200-ml volumetric flask, add absolute ethanol to dilute to the scale, shake well; accurately measure 1 ml and place it in a 100-ml volumetric flask, first add 1 ml of absolute ethanol, and then add dissolution medium to dilute to the scale, shake well, and you will get it. (The final concentration of ethanol is 2%).

[0084] Test solution: Operate according to law. At 30 minutes, take 10 ml of the dissolution solution, filter it through a 0.45-μm PALL PVDF filter membrane, discard 6 ml of the initial filtrate, and take the subsequent filtrate.

[0085] (4) Determination

[0086] Accurately measure 500 μl each of the reference substance solution and the test solution, and inject them into the liquid chromatograph respectively, and record the chromatogram.

[0087] Inject the reference substance solution 1 continuously for 5 injections, and the RSD value of the peak area of the main peak should not be more than 5.0%, and the RSD value of the peak area of all the repeated injections of the reference substance solution 1 should not exceed 5.0%.

[0088] Inject the reference substance solution 2 for 1 injection, and the RD of the correction factor obtained from the average peak areas of the reference substance solution 1 and the reference substance solution 2 should not be more than 5.0%. (Note: Correction factor = concentration of reference substance solution / average peak area of reference substance solution).

[0089] 2. Detection method for dissolution curve in pH 6.8 medium:

[0090] Same as the dissolution method. Sampling time points: 5 min, 10 min, 15 min, 30 min, 45 min.

[0091] 3. Detection method for dissolution curve in water medium:

[0092] Except for the change of medium, other operations are the same as the dissolution method. Sampling time points: 5 min, 10 min, 15 min, 30 min, 45 min.

[0093] Example 2. Detection method for dissolution curve in pH 1.2 medium:

[0094] The dissolution curve determination results of limaprost tablets in pH 1.2 medium were extremely unstable. Therefore, after the sampling of the dissolution solution was completed, a phosphate buffer solution containing sodium hydroxide was added to neutralize the hydrogen ions in the pH 1.2 medium to ensure the accuracy of the detection results. To prevent excessive dilution of the dissolution solution and excessive destruction of the dissolution solution caused by local concentration, after the dissolution solution was taken out at each sampling time point, 1 ml of buffer solution was added for neutralization. That is, it was mixed according to the ratio of 9:1 of the dissolution test solution to the neutralization solution, shaken well, filtered through a 0.45 μm filter membrane, and then injected for analysis.

[0095] Protective solution (sodium hydroxide phosphate buffer solution): Weigh 34 g of potassium dihydrogen phosphate and 18 g of sodium hydroxide, place them in a beaker containing 500 mL of water, stir, dissolve, and mix well to obtain it.

[0096] The chromatographic conditions and sampling time points are the same as those in the pH 6.8 medium in Example 1.

[0097] Example 3. Detection method for dissolution curve in pH 4.5 medium:

[0098] The pKa of the bulk drug of this product is 4.78 ± 0.10 (Predicted). In the pH 4.5 medium, limaprost exists in both molecular and ionic forms, which may lead to poor injection precision and poor system suitability. Therefore, the test sample and the reference solution were both added with a pH adjusting solution to make the pH value of the final solution far from the pKa. The preparation method of the pH adjusting solution is as follows.

[0099] pH adjusting solution: Take the pH 6.8 medium (weigh 1.7 g of potassium dihydrogen phosphate and 1.775 g of anhydrous disodium hydrogen phosphate, add an appropriate amount of water to dissolve, and make up to 1000 ml to obtain it) and 1 mol / L NaOH solution and mix them according to the ratio of 7:3 to obtain it.

[0100] Diluent: Mix the pH 4.5 medium and the adjusting solution according to the ratio of 9:1, shake well to obtain it.

[0101] Stock solution of reference solution: Take about 20 mg of limaprost reference substance, accurately weigh it, place it in a 100 ml volumetric flask, dissolve it with absolute ethanol and dilute to the mark, shake well to obtain it.

[0102] Reference solution: Accurately measure 1 mL of the stock solution of the reference solution, place it in a 100 ml volumetric flask, add absolute ethanol to dilute to the mark, shake well; accurately measure 1 ml and place it in a 200 ml volumetric flask, first add 3 ml of absolute ethanol, and then add the diluent to dilute to the mark, shake well to obtain it.

[0103] Test solution: After sampling 9 ml according to the dissolution test method at the specified time, add 1 ml of the adjustment solution, shake well, filter, discard 6 ml, and take the subsequent filtrate (the concentration of limaprost is about 9 ng / ml).

[0104] The chromatographic conditions and sampling time points are the same as those in the pH 6.8 medium in Example 1.

[0105] In Examples 1 to 3, the conventional dissolution medium volume of 500 ml was selected as the medium volume of this product. To increase the response of the main component peak area, the injection volume was increased to 500 μl to improve the response. The dissolution and dissolution curves of multiple batches of products were detected according to the proposed method, and the results could meet the requirements. The system suitability of each medium method was good, and the linear range, accuracy, and precision results could all meet the detection requirements. The quantitative limit concentration level met the detection sensitivity requirements. The typical chromatograms of each medium in Examples 1 to 3 are shown in Figures 1 to 16 :

[0106] Comparative Example 1: Detection test of the concentration of the derivatization solvent

[0107] To investigate other chromatographic concentration detection methods, post-column derivatization with excessive alkali solution was attempted. The generated derivative was red-shifted to 280 nm, thereby avoiding the interference of the end absorption and enhancing its specificity and chromatographic peak response. We speculated that under alkaline conditions, the hydroxyl group on this compound dehydrated with the adjacent hydrogen to form a conjugated diene long-chain structure, causing changes in the ultraviolet spectrum and the absorption wavelength to shift towards the long-wave direction. The method is as follows:

[0108] Chromatographic column: Octadecylsilane-bonded silica gel as the filler (TOSOH, 150 mm × 4.6 mm, 5 μm);

[0109] Mobile phase: 0.02 mol / L potassium dihydrogen phosphate solution - acetonitrile - isopropanol (9:5:2);

[0110] Detection wavelength: 280 nm; Total flow rate: 0.8 ml / min;

[0111] Injection volume: 500 μl; Column temperature: 50 °C;

[0112] Injector temperature: 15 °C; Running time: 20 min.

[0113] Concentration of the derivatization solution: 1 mol / L sodium hydroxide solution.

[0114] The results are as follows Figure 17 , where 1. Blank - pH 1.0 medium; 2. Preparation - pH 1.0 medium; 3. Blank - pH 4.5 medium, 4. Preparation - pH 4.5 medium; 5. Blank - pH 6.8 medium; 6. Preparation - pH 6.8 medium.

[0115] Result analysis: For the derivative method, there are many chromatographic peaks in the blank medium, the baseline noise is high, and there is a risk of interfering with the determination of the main peak. During the experiment, it was found that when the same sample solution was injected continuously, the results were quite different. Different alkali solution concentrations had a certain impact on the size of the main peak area, and there were many influencing factors for the derivative. Therefore, this derivatization method is not suitable for the dissolution determination of this product.

[0116] Example 4: Comparison of effects at different column temperatures

[0117] Figure 18 The comparison chart of different column temperatures is shown. Since the concentration of the target substance is low, the influence of column temperature on the baseline is obvious. When the column temperature is 35 °C, the baseline bulges at the position of the main peak, interfering with the accurate determination; when the column temperature is 25 °C - 30 °C, there are no other impurity peaks interfering near the main peak, and the baseline is relatively stable, meeting the application requirements.

[0118] Example 5: Comparison of screening effects at different flow rates

[0119] Figure 19 The comparison chart of different flow rates is shown (from top to bottom are 0.9 ml / min, 0.8 ml / min, 0.7 ml / min in sequence). Comparing the chromatograms, when the flow rate is 0.7 ml / min, the baseline is not stable, affecting the accurate detection of the main peak. When the flow rate is 0.8 ml / min - 0.9 ml / min, there is no detection interference near the main peak, meeting the detection requirements.

[0120] Example 6: Study on different mobile phase ratios

[0121] Figure 20 The comparison chart of the effects after adjusting the ratio of eluent A:B is shown, where 1. 92% of phase B - reference solution; 2. 92% of phase B - preparation; 3. 90% of phase B - reference solution; 4. 90% of phase B - preparation; 5. 88% of phase B - reference solution; 6. 88% of phase B - preparation; 7. 86% of phase B - reference solution; 8. 86% of phase B - preparation.

[0122] From the results, it can be seen that when the volume ratio of phase B is 92% and 86%, the baseline bulges at the elution position of the main peak, with a risk of interfering with the detection; when the volume ratio of phase B is 88% - 90%, there are no other impurity peaks interfering at the elution position of the main peak, and the baseline is stable, meeting the detection requirements.

[0123] Example 7: Investigation of different injector temperatures

[0124] The reference solution was placed at different temperatures and injected at different time points. The analysis results are shown in the following table:

[0125] Table 1 Summary table of investigation of different injector temperatures

[0126]

[0127]

[0128] When the injector temperature is controlled at 5°C, 15°C, 20°C and without temperature control, the RSDs of the peak areas are 6.22%, 2.03%, 2.03% and 14.86% respectively. Among them, when the temperature is 5°C, 15°C and 20°C, the RSDs of the peak areas are all less than 5.0%. At 5°C, the RD values of the peak areas at some time points are relatively high compared with those at zero time, and there is no obvious change trend. When detecting at low concentrations, the injector temperature has a certain influence on the results. To obtain more parallel, stable and reliable results, it is applicable to control the injector temperature at 15°C - 20°C for dissolution determination, and the other two conditions are not applicable.

[0129] Example 8: Different organic phase ratios of the reference solution

[0130] The active ingredient of the target developed preparation is limaprost alfadextrin, which is soluble in water. The reference substance used is limaprost, which is more soluble in organic solvents. The results of the preliminary investigation on the system suitability when the reference solution contains ethanol concentrations of 2.0%, 3.5% and 5.5% are shown in the following table:

[0131] Table 2 Investigation of ethanol concentration

[0132]

[0133] When the reference solution contains ethanol concentrations of 2.0%, 3.5% and 5.5%, the RSDs of the peak areas for system suitability are 0.91%, 1.35% and 17.54% respectively. Among them, when the ethanol volume content is 2.0% - 5.0%, it can meet the detection requirements, preferably 2.0% - 3.5%. When the ethanol content is 5.5%, the method is not applicable.

[0134] Comparative Example 2: Comparative test between the method of directly detecting without adding a protective solution (phosphate buffer solution containing sodium hydroxide) in the pH 1.2 medium and the method of the present invention

[0135] Except that the source of the dissolution medium was replaced with the dissolution medium of pH 1.2, the concentration of the dissolution medium was measured in the same way as in Example 1, and the results are shown in Table 3.

[0136] Table 3 Investigation of no protective solution added to the dissolution medium

[0137]

[0138] The RSD of the peak area for system suitability of the reference solution without adding a protective solution is 9.18%, which cannot meet the detection requirements. The RSDs of the cumulative dissolution amounts at each time point are 44.3%, 19.3%, 9.9% and 21.8% respectively, which cannot meet the detection requirements.

[0139] Detected according to the pretreatment method of adding phosphate buffer solution containing sodium hydroxide in the present invention in Example 2, and the results are shown in Table 4 below.

[0140] Table 4 Investigation of adding protective solution to the dissolution medium

[0141]

[0142] After adding the phosphate neutralizing solution, the RSD of the peak area of the system suitability of the reference solution was 1.18%, and the RSDs of the cumulative dissolution amounts at each time point were 14.5%, 7.9%, 2.6%, and 5.1% respectively; the obtained dissolution results were relatively more stable and met the requirements of the guiding principles. Therefore, we chose to add the phosphate protective solution under the medium condition of pH 1.2.

[0143] Comparative Example 3: Comparison between the method of directly detecting without adding a pH regulator in the pH 4.5 medium and the method of the present invention

[0144] Except that the source of the dissolution medium was replaced with the dissolution medium of pH 4.5, when the concentration of the dissolution medium was measured in the same way as in Example 1, it was found that the system suitability failed directly many times (the RSD of the peak area was greater than 5.0% when the same solution was continuously injected several needles), and the change of the peak area of the system suitability was irregular. The typical data are shown in Table 5 below.

[0145] Table 5 System suitability in pH 4.5 medium (without adding pH regulator)

[0146]

[0147]

[0148] The reference solution 1 was continuously injected 10 times, and the RSD of the peak area was greater than 5.0%, and the injection precision was poor. After consulting the literature, it was found that the pKa of the API of this product was 4.78 ± 0.10 (Predicted), and limaprost existed in both molecular and ionic forms in the pH 4.5 medium, which might lead to poor injection precision and poor system suitability.

[0149] As in Example 3, adding a pH regulator to both the test sample and the reference solution to increase the pH value of the final solution and keep it away from pKa had a better effect. Furthermore, the present invention also investigated three different protective solutions, and the pH values of the final solutions were pH 5.98, pH 6.65, and pH 7.22 respectively. The data are shown in Table 6 below.

[0150] Table 6 Comparison table of the results of pH regulators

[0151]

[0152] For each sample solution with a specific pH value, two instruments were used for simultaneous injection to investigate the injection precision. It can be seen that after adjusting the pH value by adding the protective solution, the RSD of consecutive injections on both instruments was less than 5.0%, indicating good precision. The results for three different pH values all met the requirements.

[0153] Example 9 Methodological Verification of the Detection Method of the Present Invention

[0154] Method verification was carried out on the proposed method. For each investigation item, methodological verification was performed by repeating the experiments multiple times according to Examples 1 to 3.

[0155] 1. System Suitability

[0156] Table 7 Investigation of System Suitability

[0157]

[0158] 2. Specificity

[0159] Table 8 Investigation of Specificity

[0160]

[0161] 3. Linear Range

[0162] Table 9 Investigation of Linearity and Range

[0163]

[0164] 4. Limit of Quantification

[0165] Table 10 Limit of Quantification Data (in pH 6.8 Medium)

[0166]

[0167]

[0168] Table 11 Test Results of Limit of Quantification (in Water Medium)

[0169]

[0170] The limit of quantification of limaprost is approximately 2 ng, which is equivalent to 20% of the concentration of the test sample at 100% dissolution. Six parallel injections were performed, and the RSD value of the peak area was not greater than 10.0%. The signal-to-noise ratio S / N of six consecutive injections was not less than 10, meeting the requirements of the method test.

[0171] 5. Accuracy

[0172] Table 12 Test Results of Accuracy in pH 4.5 Medium

[0173]

[0174] Table 13 Results of Accuracy Test in pH 6.8 Medium

[0175]

[0176] Table 14 Results of Accuracy Test in pH 1.2 Medium

[0177]

[0178]

[0179] Table 15 Results of Accuracy Test in Aqueous Medium

[0180]

[0181] 6. Precision

[0182] Table 16 Precision Results (in pH 6.8 Medium)

[0183]

[0184] 7. Robustness

[0185] Table 17 Results of Robustness Test (in pH 6.8 Medium)

[0186]

[0187] Note: 1. Chromatographic columns 1, 2, and 3 are of the same brand and model but different batches.

[0188] 2. RSD%(n = 6) is the result calculated by summarizing the fine-tuned and normal conditions under the same chromatographic conditions.

[0189] The results show that the RSD(n = 6) values of the dissolution results determined by changing the column temperature (±2°C), flow rate (±0.05 ml / min), mobile phase ratio (±1%), mobile phase pH (±0.1), detection wavelength (±2 nm), and different batches of the same brand chromatographic columns compared with the dissolution results under normal conditions are 1.9%, 1.8%, 1.3%, 3.6%, 3.6%, and 2.5% respectively, all less than 10.0%. This method has good robustness to minor changes in chromatographic conditions.

[0190] 8. Investigation of Different Brands of Chromatographic Columns

[0191] The elution profiles of limaprost were compared on three types of chromatographic columns: TOSOH (4.6 mm × 150 mm, 5 μm), Waters Atlantis T3 (4.6 mm × 150 mm, 3 μm), and Waters symmetry C18 (4.6 mm × 150 mm, 3.5 μm), as Figure 21All three chromatographic columns can achieve good resolution. When using the Waters symmetry C18 (4.6 mm × 150 mm, 3.5 μm) chromatographic column, there are no other impurity peaks interfering near the main peak, the resolution with the front and rear peaks is the best, the peak shape is good, and the baseline nearby is relatively stable.

[0192] 9. Solution stability

[0193] The reference solution and the test solution were placed in the injector at 15 °C and detected at different time points, and the RSD% / RD% of the peak area was statistically analyzed to investigate the solution stability. The results are shown in the following table:

[0194] Table 18 Results of solution stability determination

[0195]

[0196]

[0197] The results show that when the samples are placed in the 15 °C injection tray, the reference solution in the pH 4.5 medium is stable within 44 h, and the test solution is stable within 40 h; the reference solution in the pH 6.8 medium is stable within 55.4 h, and the test solution is stable within 51.3 h; the reference solution in the pH 1.2 medium is stable within 31 h, and the test solution is stable within 24 h; the reference solution in the water medium is stable within 30 h, and the test solution is stable within 30 h.

[0198] In Example 10, the dissolution and dissolution curves of samples from different batches were detected. The dissolution of a large number of samples was detected using the established method, and the typical data are as follows:

[0199] Table 19 Results of dissolution determination at pH 6.8

[0200]

[0201] Table 19 Results of dissolution curve determination

[0202]

[0203] The present invention has been successfully used to detect the dissolution and dissolution curves of limaprost tablets, and the method has good adaptability.

[0204] The present invention solves the problems of low detection concentration and poor specificity in the detection of the dissolution / dissolution curve of limaprost tablets. The established dissolution method has been systematically verified, is accurate and reliable, and has good precision. The invention fills the blank in the research of the dissolution / dissolution curve of this micro preparation. The establishment and application of the method will contribute to the research and marketing of limaprost tablets, fill the clinical treatment blank of lumbar spinal stenosis, and bring new treatment gospel to the majority of patients.

[0205] The present invention illustrates the detailed method of the present invention through the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvement to the present invention, the equivalent substitution of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A method for detecting the concentration of a dissolution test solution of a limaprost tablet, based on an external standard method of liquid chromatography detection, wherein the liquid chromatography detection meets the following conditions: The chromatographic column was filled with octadecylsilane bonded silica gel and the column temperature was 20℃~31℃. The injection volume is 500μL~2000μL, and the temperature of the injector is controlled at 15℃~20℃. The mobile phase composition is as follows: mobile phase A is 0.02 mol / L potassium dihydrogen phosphate solution, the pH is adjusted to 3.0 with 20% phosphoric acid, mobile phase B is a solution of 0.02 mol / L potassium dihydrogen phosphate solution-acetonitrile-isopropanol in the ratio of (900±50):(500±30):(200±10), mobile phase A:mobile phase B is =10:90~12:88, and the flow rate is 0.8ml / min~1.0ml / min.

2. The detection method according to claim 1, characterized in that: The method comprises a concentration detection step of a pH 1.2 medium dissolution test solution. In the concentration detection step of the pH 1.2 medium dissolution test solution, a phosphate buffer solution containing sodium hydroxide is added to the dissolution test solution for pretreatment and then used as a test solution.

3. The detection method according to claim 1, characterized in that: The method comprises a concentration detection step of a pH 4.5 medium dissolution test solution, and a pre-treatment step of adding a 6.8±2 pH adjusting solution to the dissolution test solution and the reference substance solution at the same time, and adjusting the pH of the solution to 6.0-7.

2.

4. The detection method according to claim 2, characterized in that: In the concentration detection step of the pH 1.2 medium dissolution test solution, the phosphate buffer containing sodium hydroxide is prepared according to the following method: weigh appropriate amounts of potassium dihydrogen phosphate and sodium hydroxide, add water to dissolve and quantitatively dilute to prepare an aqueous solution with a mass content of 6.8% potassium dihydrogen phosphate and 3.6% sodium hydroxide, that is, The volume ratio of dissolution test solution: phosphate buffer containing sodium hydroxide is 8:1 to 10:

1.

5. The detection method according to claim 3, characterized in that: In the concentration detection step of the pH 4.5 medium dissolution test solution, the pH adjuster is prepared according to the following method: take appropriate amounts of potassium dihydrogen phosphate and anhydrous disodium hydrogen phosphate, add appropriate amounts of water to dissolve them, and the resulting pH 6.8 medium solution is mixed with 1 mol / L NaOH solution in a volume ratio of 7:

3.

6. The detection method according to claim 3, characterized in that: In the concentration test step of the pH 4.5 medium dissolution test solution, the reference solution is prepared as follows: The pH 4.5 medium and the pH adjusting solution are mixed in a volume ratio of 8:1 to 10:1, and shaken to obtain a diluted solution.

7. The detection method according to any one of claims 1 to 6, characterized in that: Take the limaprost reference substance, accurately weigh it, place it in a 100ml volumetric flask, add anhydrous ethanol to dissolve and dilute to the scale, shake well, and obtain the reference substance solution stock solution; accurately measure 1mL of the reference substance solution stock solution, place it in a 100ml volumetric flask, add anhydrous ethanol to dilute to the scale, and shake well; accurately measure 1ml of the solution, place it in a 200ml volumetric flask, first add anhydrous ethanol, then add the diluent to dilute to the scale, shake well, and obtain the reference substance solution. The ethanol content of the limaprost reference substance solution is 2.0% to 3.5%.

8. The detection method according to claim 1, characterized in that: The method also includes a pH 6.8 medium dissolution curve detection step, in which: Take appropriate amount of potassium dihydrogen phosphate and anhydrous disodium hydrogen phosphate, add appropriate amount of water to dissolve, and adjust the volume to prepare a dissolution medium with a pH of 6.8; Take about 20 mg of limaprost reference substance, accurately weigh it, put it in a 100 ml volumetric flask, add anhydrous ethanol to dissolve and dilute to the scale, shake well, and obtain the reference substance stock solution. Accurately measure 1 ml of the reference substance stock solution, put it in a 200 ml volumetric flask, add anhydrous ethanol to dilute to the scale, and shake well; accurately measure 1 ml and put it in a 100 ml volumetric flask, first add 1 ml of anhydrous ethanol, then add the dissolution medium to dilute to the scale, shake well, and obtain the reference substance solution with a final concentration of 2% ethanol content; The solution taken out from the dissolution tester was filtered and directly used as the test solution for the pH 6.8 medium dissolution curve detection step.

9. The detection method according to claim 1, characterized in that: The chromatographic column is selected from TOSOH C18, Waters Atlantis T3, and Waters symmetry C18 series columns.

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