Determination method for release degree of vitamin E compound sustained-release microspheres

Abstract

The invention relates to the field of pharmaceutical analytical chemistry, and provides a method for measuring the release degree of vitamin E compound sustained-release microspheres, which comprises the following steps: 1) adding the vitamin E compound sustained-release microspheres into a release medium under a dissolution condition to prepare a test solution; 2) dissolving vitamin E by using alcohol and the release medium to prepare a reference solution; (3) respectively detecting the test solution and the reference solution by using a liquid chromatography, and determining the release rate; wherein the dissolution condition is 37 DEG C + / -0.5 DEG C to 45 DEG C + / -0.5 DEG C, and the release medium is a 70v / v% propylene glycol phosphate buffer salt solution containing 0.5 w / v%-1.0 w / v% Tween 80. The in-vitro release analysis method suitable for the vitamin E compound sustained-release preparation is successfully constructed, the accuracy is high, the repeatability is good, the operation is simple and convenient, and efficient application and quality control of the vitamin E sustained-release preparation in different scenes can be realized.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical analytical chemistry, and more particularly to a method for determining the release rate of sustained-release microspheres of vitamin E compounds. Background Art

[0002] Vitamin E compounds, as important fat-soluble vitamins, are chemically composed of two major components: tocopherols and tocotrienols. α-Tocopherol, a representative antioxidant due to its high biological activity, plays a central role in regulating metabolic processes. In addition to its remarkable antioxidant properties, vitamin E compounds possess multiple physiological activities: they promote normal development and function of the reproductive system by regulating hormone secretion; they act as a key protective factor for T lymphocytes, enhancing cellular immune responses and maintaining immune homeostasis; and they significantly reduce the incidence of cardiovascular and cerebrovascular diseases such as myocardial infarction and cerebral infarction by inhibiting platelet aggregation. They also demonstrate significant therapeutic effects in tissue repair and antioxidant damage in the repair of wounds such as burns and frostbite, as well as in everyday health management applications such as beauty and skincare. This multifunctionality has made vitamin E compounds key active ingredients in the pharmaceutical, nutritional, and aesthetic fields, and their mechanisms of action and application development continue to garner significant attention.

[0003] Vitamin E compound products currently on the market primarily come in conventional dosage forms, including oral preparations, injections, topical preparations, and enteric-coated capsules. However, these dosage forms generally suffer from the limitation of short duration of action both in vivo and in vitro. In contrast, vitamin E compound sustained-release microspheres, as an innovative drug delivery system, are gaining increasing attention. Through a unique sustained-release mechanism, they precisely regulate the release rate of vitamin E compounds in vivo, maintaining a stable and effective concentration in the body, thereby achieving the goal of long-term antioxidant therapy or aesthetic repair.

[0004] In the quality control system for sustained-release preparations, release rate testing is a core evaluation indicator, directly related to the product's effectiveness and safety. However, there is currently little research in both domestic and international academic and industry fields on release rate analysis methods for sustained-release preparations of vitamin E compounds. Existing methods also suffer from incomplete release of sustained-release microspheres of vitamin E compounds. Therefore, new detection methods are needed to promote the industrialization and clinical application of sustained-release dosage forms of vitamin E compounds. Summary of the Invention

[0005] To address the above technical issues, the present invention provides a method for determining the release rate of sustained-release microspheres of vitamin E compounds with high accuracy, high precision, and strong discrimination. The method of the present invention can effectively control the release behavior of vitamin E compounds in sustained-release preparations and monitor release quality indicators. In the fields of drug development and medical aesthetics, it not only provides reliable data support for in vitro release kinetics research, but also can become a key bridge connecting in vitro release characteristics with in vivo biological effects by simulating the in vivo absorption environment. It can also achieve efficient application and quality control of sustained-release preparations of vitamin E compounds in different scenarios.

[0006] The assay method of the present invention comprises: 1) adding the vitamin E compound sustained-release microspheres to a release medium under dissolution conditions to prepare a test solution; 2) dissolving the vitamin E compound using alcohol and the release medium to prepare a reference solution; 3) using liquid chromatography to test the test solution and the reference solution to determine the release rate; The dissolution conditions are 37°C±0.5°C~45°C±0.5°C, and the release medium is 70v / v% propylene glycol phosphate buffered saline solution containing 0.5w / v%~1.0w / v% Tween 80.

[0007] Preferably, the step 1) comprises adding the vitamin E compound microspheres to the release medium preheated to 37°C±0.5°C~45°C±0.5°C, allowing to stand under the dissolution conditions, and centrifuging the dissolution solution at the sampling time to obtain the test solution.

[0008] More preferably, the sampling time includes 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 32 h, 48 h, 56 h, 72 h, 96 h and 120 h.

[0009] Preferably, in step 2), the alcohol is methanol.

[0010] Preferably, in step 3), the liquid chromatography conditions include: Mobile phase: 97v / v% methanol in water; Chromatographic column: Waters Symmetry Shield RP18 (3.5 μm, 4.6 mm × 100 mm); Detector: UV detector.

[0011] More preferably, the liquid chromatography conditions further include: Flow rate: 1.0 mL / min; elution time: 10 min; column temperature: 30°C; detection wavelength: 292 nm; injection volume: 20 μL.

[0012] Preferably, the release rate of the vitamin E compound sustained-release microspheres is calculated by the external standard method.

[0013] More preferably, the release rate of the vitamin E compound sustained-release microspheres at the nth sampling time is calculated by the following formula (I) and formula (II), where n≥1: Formula (I) Formula (II) in, C is the concentration of vitamin E compounds in the test solution, mg / mL; Cs is the concentration of the reference solution, mg / mL; and are the main peak areas of the test solution and the reference solution, respectively; Cn is the concentration of vitamin E compounds in the test solution at the nth sampling time calculated according to formula (I), mg / mL; Vn is the total volume of the test solution at the nth sampling, mL; Ci is the concentration of vitamin E compounds in the test solution before the nth time calculated according to formula (I), mg / mL; 1 is the sampling volume each time, mL; m s is the mass of vitamin E compound in vitamin E compound sustained-release microspheres, mg; D is the degree of release.

[0014] Preferably, the vitamin E compound sustained-release microspheres are microspheres obtained by encapsulating the vitamin E compound with a hydrophobic polymer.

[0015] More preferably, the hydrophobic polymer is one or more of polycaprolactone and its copolymer poly-L-lactide-caprolactone, poly-L-lactic acid, poly-D-lactic acid and its copolymer poly-L-lactic acid / poly-D-lactic acid-glycolic acid copolymer.

[0016] The beneficial effects of the present invention are: The present invention successfully constructs an in vitro release analysis method suitable for sustained-release preparations of vitamin E compounds. The method has significant technical advantages and practical value: the use of a simple-structured release device and a combination of conventional laboratory reagents significantly reduces experimental operation skills, greatly improves the reproducibility and scalability of the method between different laboratories, and provides a standardized solution for the quality control of sustained-release preparations of vitamin E compounds. By selecting specific organic solvent types and proportions and surfactant types and proportions to form a composite release medium, the water solubility defect of vitamin E compounds is improved by the solubilizing effect of propylene glycol, and a pH-stable neutral environment is constructed in combination with a buffer salt system, effectively avoiding the degradation risk of sustained-release materials caused by fluctuations in acid-base conditions during the dissolution process; surfactants promote the diffusion and release of vitamin E compounds from sustained-release microspheres by reducing liquid-solid interfacial tension, thereby forming a triple regulatory mechanism of "stable pH environment-efficient dissolution-interfacial mass transfer optimization", which improves the problem of incomplete release of vitamin E compounds.

[0017] After methodological verification, the concentration of vitamin E compounds was in the range of about 6µg / mL~160µg / mL, and there was a good linear relationship between the concentration of vitamin E compounds and the chromatographic peak area. 2 ≥0.9999; the spiked recoveries at different concentration levels were in the range of 95%~105%, with RSD less than 5.0%, indicating good accuracy; and the detection solution was stored at room temperature for 7 days with a recovery in the range of 95%~105%, indicating excellent stability.

[0018] The method of the present invention fills the technical gap in the field of release rate analysis of sustained-release preparations of vitamin E compounds at home and abroad, provides key technical support for the research and development, production and quality evaluation of such preparations, and promotes the standardized application of sustained-release dosage forms in the fields of medicine and medical aesthetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the blank solution chromatogram of Example 1; Figure 2 This is the chromatogram of the reference substance solution in Example 1; Figure 3 This is a chromatogram of the test solution of Example 1; Figure 4 This is the release curve of Example 1; Figure 5 It is the linear curve of Example 1; Figure 6 This is the blank solution chromatogram of Example 2; Figure 7 This is the chromatogram of the reference substance solution in Example 2; Figure 8 This is a chromatogram of the test solution of Example 2; Figure 9 This is the release curve of Example 2; Figure 10 It is the linear curve of Example 2; Figure 11 This is the blank solution chromatogram of Example 3; Figure 12 This is the chromatogram of the reference substance solution in Example 3; Figure 13 This is a chromatogram of the test solution of Example 3; Figure 14 This is the release curve of Example 3; Figure 15 This is the linear curve of Example 3; Figure 16 This is the release curve of Comparative Example 1; Figure 17 This is the release curve of Comparative Example 2; Figure 18 This is the release curve of Comparative Example 3; Figure 19 This is the release curve of Comparative Example 4; Figure 20 This is the release curve of Comparative Example 5; Figure 21 This is the release curve of Comparative Example 6; Figure 22 This is the release curve of Comparative Example 7. DETAILED DESCRIPTION

[0020] The present invention will be further described below in conjunction with the accompanying drawings and Examples, but these embodiments are exemplary, only to facilitate those skilled in the art to further understand the characteristics of the present invention, and do not constitute any limitation to the scope of the present invention. The reagents used in the present invention, unless otherwise specified, are conventional commercially available products. The percentages occurring in the present invention, unless otherwise specified, are all mass percentages.

[0021] In the present invention, "sustained-release vitamin E compound microspheres" refer to tiny spherical particles with a particle size of 1 to 250 μm, formed by dispersing, adsorbing, or encapsulating vitamin E compounds in a polymer matrix or macromolecule. These particles slowly release vitamin E compounds in vivo or control the release of vitamin E compounds at a certain rate. The polymer matrix or macromolecule may include chitosan, cyclodextrin, polycaprolactone (PCL) and its copolymer poly-L-lactide-caprolactone (PLCL), poly-L-lactic acid, poly-D-lactic acid, and its copolymer poly-L-lactic acid / poly-D-lactic acid-co-glycolic acid, etc. In a preferred embodiment of the present invention, the sustained-release vitamin E compound microspheres are microspheres obtained by encapsulating vitamin E compounds in a hydrophobic polymer. More preferably, the hydrophobic polymer is one or more of polycaprolactone and its copolymer poly-L-lactide-caprolactone, poly-L-lactic acid, poly-D-lactic acid, and its copolymer poly-L-lactic acid / poly-D-lactic acid-co-glycolic acid. The method of the present invention is particularly suitable for microspheres containing hydrophobic polymers through the selection of the release medium, and can make the vitamin E compounds in the microspheres release completely, and the detection results are accurate and reliable. In addition to the polymer and the vitamin E compounds, the vitamin E compound sustained-release microspheres in the present invention can also contain other components, such as vitamin C, collagen, amino acids, small molecule active peptides, etc. The vitamin E compound sustained-release microspheres of the present invention can be prepared using methods known in the present invention, such as emulsification solvent evaporation method, spray drying method, physical ball milling method, high temperature melting spheroidization method and solvent-non-solvent method, etc., and the preparation method will not affect the assay method of the present invention. The vitamin E compound sustained-release microspheres in the embodiment of the present invention are prepared by emulsification solvent evaporation method.

[0022] In the present invention, "vitamin E compounds" is used as a general term, and vitamin E compounds and their esters and salts are collectively referred to as vitamin E compounds. The vitamin E compounds can be selected from at least one of vitamin E, vitamin E acetate, vitamin E succinate, vitamin E palmitate, vitamin E succinate, vitamin E nicotinate, vitamin E phosphate, vitamin E linoleate, vitamin E laurate, and vitamin E retinoic acid ester, but are not limited thereto.

[0023] The determination method of the present invention comprises: 1) adding the vitamin E compound sustained-release microspheres to a release medium under dissolution conditions to prepare a test solution; 2) using alcohol and the release medium to dissolve the vitamin E to prepare a reference solution; 3) using liquid chromatography to detect the test solution and the reference solution respectively to determine the release rate; wherein the dissolution conditions are 37°C±0.5°C~45°C±0.5°C, and the release medium is a 70v / v% propylene glycol phosphate buffered saline solution containing 0.5w / v%~1.0w / v% Tween 80. The present invention uses a composite release medium to improve the poor water solubility of vitamin E through the solubility-promoting effect of propylene glycol. Combined with a buffer salt system, it creates a pH-stable environment, effectively avoiding the risk of degradation of the sustained-release material due to fluctuations in acid-base conditions during the dissolution process. The surfactant reduces the liquid-solid interfacial tension, promoting the diffusion and release of vitamin E from the sustained-release microspheres. This creates a triple regulatory mechanism of "stable pH environment - efficient dissolution - optimized interfacial mass transfer," enabling accurate and reliable detection of the release rate of vitamin E compound sustained-release microspheres by liquid chromatography. In a preferred embodiment, the pH of the release medium is 7.4±0.1.

[0024] The release medium of the present invention can be prepared as follows: 1.36g of potassium dihydrogen phosphate and 1.42g of sodium dihydrogen phosphate are added to 1000mL of ultrapure water, and the pH is adjusted to 7.4±0.1 with sodium hydroxide. To 300mL of this solution, 700mL of propylene glycol and 5g-10g of Tween 80 are added and mixed thoroughly. The release medium must be heated and degassed before use. The above preparation method is for example only, and those skilled in the art can adjust the ratios of the reagents as needed to obtain the desired volume of release medium.

[0025] The release medium of the present invention can support the vitamin E compound microspheres to release vitamin E compounds over a longer period of time and can avoid the problem of incomplete release of vitamin E compounds. In a preferred embodiment of the present invention, step 1) includes adding the vitamin E compound microspheres to the release medium that has been preheated to 37°C ± 0.5°C ~ 45°C ± 0.5°C, standing under the dissolution conditions, and taking the dissolution solution at the sampling time and centrifuging to obtain the test solution. Preferably, the sampling time can be 15min, 30min, 1h, 2h, 4h, 8h, 12h, 24h, 32h, 48h, 56h, 72h, 96h and 120h. By testing the test solution at different sampling time points, the release behavior of the microspheres at different time points can be analyzed as needed, providing reliable data support for in vitro release kinetics studies.

[0026] Using alcohol to dissolve vitamin E can ensure sufficient dissolution of vitamin E and prepare a reference solution with accurate content, thereby ensuring the accuracy of the detection method of the present invention. The type of alcohol is not particularly limited, such as methanol, ethanol, etc., as long as it can fully dissolve vitamin E and does not affect the liquid chromatography column. In a preferred embodiment of the present invention, the alcohol is methanol.

[0027] In a preferred embodiment of the present invention, the liquid chromatography conditions include: mobile phase: 97 v / v% methanol in water; chromatographic column: Waters Symmetry Shield RP18 (3.5 μm, 4.6 mm × 100 mm); and detector: UV detector. More preferably, the liquid chromatography conditions further include: flow rate: 1.0 mL / min; elution time: 10 min; column temperature: 30°C; detection wavelength: 292 nm; and injection volume: 20 μL. By optimizing the liquid chromatography conditions, the detection results can be more accurate and reproducible.

[0028] In a preferred embodiment of the present invention, the release rate of the vitamin E compound sustained-release microspheres is calculated by the external standard method. More preferably, the release rate of the vitamin E compound sustained-release microspheres at the nth sampling time is calculated by the following formula (I) and formula (II), where n≥1: Formula (I) Formula (II) in, C is the concentration of vitamin E compounds in the test solution, mg / mL; Cs is the concentration of the reference solution, mg / mL; and are the main peak areas of the test solution and the reference solution, respectively; Cn is the concentration of vitamin E compounds in the test solution at the nth sampling time calculated according to formula (I), mg / mL; Vn is the total volume of the test solution at the nth sampling, mL; Ci is the concentration of vitamin E compounds in the test solution before the nth time calculated according to formula (I), mg / mL; 1 is the sampling volume each time, mL; m s is the mass of vitamin E compound in vitamin E compound sustained-release microspheres, mg; D is the degree of release.

[0029] Among them, the mass of vitamin E compound in vitamin E compound sustained-release microspheres m s The determination can be carried out in accordance with the inspection items of vitamin E-related substances in Part II of the Chinese Pharmacopoeia <2020 Edition>. For example, n-hexane is used as a diluent to prepare the vitamin E reference substance and the vitamin E compound sustained-release microsphere test solution respectively, and the Shimadzu 2030 gas chromatograph is used to set the chromatographic conditions and calculate the content in accordance with the above provisions of the Chinese Pharmacopoeia. Example 1

[0030] 1.1 Solution preparation Preparation of release medium: Add 1.36g of potassium dihydrogen phosphate and 1.42g of sodium dihydrogen phosphate to 1000mL of ultrapure water and adjust the pH to 7.4±0.1 with sodium hydroxide. Add 700mL of propylene glycol and 10g of Tween 80 to 300mL of this solution and mix thoroughly to obtain a 70v / v% propylene glycol phosphate buffer solution with 1.0w / v% Tween 80 as the release medium. Heat and degas before use.

[0031] Preparation of Reference Solution: Accurately weigh 20.77 mg of Vitamin E Reference (McLean, D832569) into a 50 mL volumetric flask. Dissolve and dilute to volume with methanol. Shake well to prepare the reference stock solution. Accurately measure 1 mL of the reference stock solution into a 10 mL volumetric flask. Add release medium and dilute to volume. Shake well to prepare the reference solution.

[0032] Preparation of sustained-release microspheres containing vitamin E compounds: Dissolve 1.3 g of poly-L-lactic acid and 0.7 g of vitamin E in 25 mL of dichloromethane to form an oil phase. Add the oil phase to a 1% (w / v) aqueous solution of polyvinyl alcohol, homogenize, and emulsify to obtain an emulsion. Stir and evaporate to remove the organic solvent. Filter through a mesh, rinse with ultrapure water, and lyophilize to obtain powdered microspheres.

[0033] Preparation of test solution: Accurately weigh approximately 50 mg of vitamin E sustained-release microspheres prepared according to the above method and place them into a stoppered ground-mouth glass vial. Add 150 mL of release medium preheated to 45°C and place the stoppered ground-mouth glass vial in a constant temperature water bath at 45°C ± 0.5°C. Remove 1 mL of the dissolution solution from the vial at 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 32 h, 48 h, 56 h, 72 h, 96 h, and 120 h. Centrifuge the dissolution sample solution at 10,000 rpm for 10 min to obtain the test solution. Prepare six replicate samples using the same method (weighing 51.74 mg, 50.65 mg, 53.15 mg, 51.81 mg, 52.50 mg, and 50.28 mg, respectively).

[0034] 1.2 Chromatographic conditions High-performance liquid chromatography (HPLC) was performed on a Waters Alliance E2695 instrument. The chromatographic column was a Waters Symmetry Shield RP18 (3.5 μm, 4.6 mm × 100 mm). The mobile phase was a 97 v / v% methanol solution. The elution time was 10 min at a flow rate of 1.0 mL / min and an isocratic elution method was used. The column temperature was 30 °C. The detector was an ultraviolet detector. The detection wavelength was 292 nm. The injection volume was 20 μL.

[0035] 1.3 Detection Take the blank solution (release medium), reference solution, and test solution at each time point and analyze them in turn using high performance liquid chromatography. The chromatogram of the blank solution is as follows: Figure 1 The chromatogram of the reference solution is shown in Figure 2 The chromatogram of the test solution is shown in Figure 3 As shown. According to the external standard method, the peak area was calculated (Formula (I) and (II)), and the release rate at different sampling times was calculated. The release curve is shown in Figure 4 The release data are shown in Table 1.

[0036] Table 1

[0037] From Table 1 and Figure 4 It can be seen that with 15 min as the first sampling point, the release reaches a plateau at 48 h. The RSD of the release at the initial sampling time point is less than 10%, and the RSD of the release at the subsequent sampling time points is less than 5%, indicating that the method of the present invention has good repeatability and stable release behavior.

[0038] 1.4 Methodological Validation Exclusivity Depend on Figure 1 and 2 It can be seen that compared with the blank solution, the reference solution has a peak at 5.118min, which indicates that the peak corresponds to vitamin E. Figure 2 and 3 It can be seen that a peak appears at 5.010 min in the test solution, which is basically consistent with the retention time of vitamin E in the reference solution. It can be seen that the peak here corresponds to the vitamin E in the test solution. From the above results, it can be seen that the blank solution has no interference at the retention time of vitamin E and will not affect the determination of the test sample. The method of the present invention has good specificity.

[0039] Repeatability As can be seen from Table 1, the RSDs of the six parallel sample measurements at the three sampling time points of 15 min, 8 h, and 48 h were as follows: the RSD of the release rate of the 15 min sample was 8.4%, less than 10%; the RSD of the 8 h sample was 3.1%, less than 5%; the RSD of the 48 h sample was 0.7%, less than 5%. The repeatability results were good.

[0040] Accuracy Preparation of blank microspheres: Weigh 1.3 g of poly-L-lactic acid (PLLA) and dissolve it in 25 mL of dichloromethane to form an oil phase. Add the oil phase to a 1% (w / v) aqueous solution of polyvinyl alcohol (PVA), homogenize, and emulsify to obtain an emulsion. Stir and evaporate to remove the organic solvent. Filter through a mesh, rinse with ultrapure water, and lyophilize to obtain powdered microspheres.

[0041] According to the preparation method of the test solution, a blank microsphere solution was prepared, and different masses of vitamin E reference substances were added to the blank microsphere solution to prepare spiked solutions of low, medium, and high concentrations. Three parallel samples were prepared for each concentration. The concentration of each spiked solution was calculated according to formula (I) and multiplied by the volume of the blank microsphere solution to obtain the measured amount. The spiked recovery was calculated by dividing the measured amount by the spiked amount. The results are shown in Table 2.

[0042] Table 2

[0043] As can be seen from Table 2, at low, medium and high concentrations, the recovery results were all in the range of 95% to 105%, and the RSD was less than 5.0%, demonstrating good accuracy results.

[0044] Linear Add different amounts of vitamin E reference stock solution to the blank solution to prepare 7 linear solutions with different concentrations. Detect according to the chromatographic conditions in Section 1.2 and plot the linear relationship between vitamin E concentration and peak area. The linear graph is shown in Figure 5 The measurement results are shown in Table 3.

[0045] Table 3

[0046] ,Depend on Figure 5 As can be seen from Table 3, when the vitamin E concentration is in the range of 6.2 μg / mL to 156.1 μg / mL, the linear equation is y=2023.9x+28.963, R 2 =0.9999, and there is a good linear relationship between concentration and peak area.

[0047] stability The reference solution, 15 min test solution, 8 h test solution, and 48 h test solution were placed at room temperature for 7 days, and then tested according to the chromatographic conditions in Section 1.2 above to examine the recovery rate of the stability solution concentration. The test results are shown in Table 4.

[0048] Table 4

[0049] As shown in Table 4, the recovery rates of the four solutions were between 95% and 105%. The reference solution and the test solution used in the method of the present invention were stable at room temperature for at least 7 days. Example 2

[0050] 2.1 Solution preparation Preparation of release medium: Add 1.36g of potassium dihydrogen phosphate and 1.42g of sodium dihydrogen phosphate to 1000mL of ultrapure water and adjust the pH to 7.4±0.1 with sodium hydroxide. Add 700mL of propylene glycol and 5g of Tween 80 to 300mL of this solution and mix thoroughly to obtain a 70v / v% propylene glycol phosphate buffered saline solution containing 0.5w / v% Tween 80 as the release medium. Heat and degas before use.

[0051] Preparation of Reference Solution: Accurately weigh 20.82 mg of Vitamin E Reference Solution into a 50 mL volumetric flask. Dissolve and dilute to volume with methanol and shake well to prepare the reference solution. Accurately weigh 1 mL of the reference solution into a 10 mL volumetric flask. Dilute to volume with release medium and shake well to prepare the reference solution.

[0052] Preparation of test solution: Accurately weigh approximately 50 mg of the vitamin E compound sustained-release microspheres prepared according to the method of Example 1 and place them into a stoppered ground-mouth glass vial. Add 150 mL of release medium preheated to 45°C and place the stoppered ground-mouth glass vial in a constant temperature water bath at 45°C ± 0.5°C. Remove 1 mL of the dissolution solution from the vial at 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 32 h, 48 h, 56 h, 72 h, 96 h, and 120 h, respectively. Centrifuge the dissolution sample solution at 10,000 rpm for 10 min to obtain the test solution. Prepare six replicate samples in the same manner (weighing 52.64 mg, 50.41 mg, 50.95 mg, 50.37 mg, 50.65 mg, and 50.81 mg, respectively).

[0053] 2.2 Chromatographic conditions Same as Example 1.

[0054] 2.3 Detection Take the blank solution (release medium), reference solution, and test solution at each time point and analyze them in turn using high performance liquid chromatography. The chromatogram of the blank solution is as follows: Figure 6 The chromatogram of the reference solution is shown in Figure 7 The chromatogram of the test solution is shown in Figure 8 As shown. According to the external standard method, the peak area was calculated (Formula (I) and (II)), and the release rate at different sampling times was calculated. The release curve is shown in Figure 9 The release data are shown in Table 5.

[0055] Table 5

[0056] From Table 5 and Figure 9 It can be seen that with 15 min as the first sampling point, the release reaches a plateau at 72 h. The RSD of the release at the initial sampling time point is less than 10%, and the RSD of the release at subsequent sampling time points is less than 5%, indicating that the method of the present invention has good repeatability and stable release behavior.

[0057] 2.4 Methodological Validation Exclusivity Depend on Figure 6 and 7 It can be seen that compared with the blank solution, the reference solution has a peak at 5.151min, which indicates that the peak corresponds to vitamin E. Figure 7 and 8 It can be seen that a peak appears at 5.053 min in the test solution, which is basically consistent with the retention time of vitamin E in the reference solution. It can be seen that the peak here corresponds to the vitamin E in the test solution. From the above results, it can be seen that the blank solution has no interference at the retention time of vitamin E and has no effect on the determination of the test sample. The method of the present invention has good specificity.

[0058] Repeatability As can be seen from Table 5, the RSDs of the 6 parallel sample measurements at the three sampling time points of 15 min, 12 h, and 72 h were as follows: the RSD of the release rate of the 15 min sample was 9.0%, which was less than 10%; the RSD of the 12 h sample was 2.8%, which was less than 5%; and the RSD of the 72 h sample was 1.9%, which was less than 5%. The repeatability results were good.

[0059] Accuracy According to the method of Example 1, three spiked solutions of low, medium and high concentrations were prepared, and the spiked recoveries were measured. The results are shown in Table 6.

[0060] Table 6

[0061] As can be seen from Table 6, at low, medium and high concentrations, the recovery results were all in the range of 95% to 105%, and the RSD was less than 5.0%, demonstrating good accuracy results.

[0062] Linear Add different amounts of vitamin E reference stock solution to the blank solution to prepare 7 linear solutions with different concentrations. Detect according to the chromatographic conditions in Section 2.2 and plot the linear relationship between vitamin E concentration and peak area. The linear graph is shown in Figure 10 The measurement results are shown in Table 7.

[0063] Table 7

[0064] Depend on Figure 10 As can be seen from Table 7, when the vitamin E concentration is in the range of 6.3 μg / mL to 158.4 μg / mL, the linear equation is y=1960.3x+43.23, R 2 =1, and there is a good linear relationship between concentration and peak area.

[0065] stability The reference solution, 15 min test solution, 12 h test solution, and 72 h test solution were placed at room temperature for 7 days, and then tested according to the chromatographic conditions in Section 2.2 above to examine the recovery rate of the stability solution concentration. The test results are shown in Table 8.

[0066] Table 8

[0067] As shown in Table 8, the recovery rates of the four solutions were between 95% and 105%. The reference solution and the test solution used in the method of the present invention were stable at room temperature for at least 7 days. Example 3

[0068] 3.1 Solution preparation Preparation of release medium: Add 1.36g of potassium dihydrogen phosphate and 1.42g of sodium dihydrogen phosphate to 1000mL of ultrapure water and adjust the pH to 7.4±0.1 with sodium hydroxide. Add 700mL of propylene glycol and 10g of Tween 80 to 300mL of this solution and mix thoroughly to obtain a 70v / v% propylene glycol phosphate buffer solution with 1.0w / v% Tween 80 as the release medium. Heat and degas before use.

[0069] Preparation of Reference Solution: Accurately weigh 20.93 mg of Vitamin E Reference Solution into a 50 mL volumetric flask. Dissolve and dilute to volume with methanol and shake well to prepare the reference solution. Accurately weigh 1 mL of the reference solution into a 10 mL volumetric flask. Dilute to volume with release medium and shake well to prepare the reference solution.

[0070] Preparation of test solution: Accurately weigh approximately 50 mg of the vitamin E compound sustained-release microspheres prepared according to the method of Example 1 and place them into a stoppered ground-mouth glass vial. Add 150 mL of release medium preheated to 37°C and place the stoppered ground-mouth glass vial in a constant temperature water bath at 37°C ± 0.5°C. Remove 1 mL of the dissolution solution from the vial at 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 32 h, 48 h, 56 h, 72 h, 96 h, and 120 h, respectively. Centrifuge the dissolved sample solution at 10,000 rpm for 10 min to obtain the test solution. Prepare six replicate samples in the same manner (weighing 50.85 mg, 51.39 mg, 50.41 mg, 52.61 mg, 51.54 mg, and 52.46 mg, respectively).

[0071] 3.2 Chromatographic conditions Same as Example 1.

[0072] 3.3 Detection Take the blank solution (release medium), reference solution, and test solution at each time point and analyze them in turn using high performance liquid chromatography. The chromatogram of the blank solution is as follows: Figure 11 The chromatogram of the reference solution is shown in Figure 12 The chromatogram of the test solution is shown in Figure 13 As shown. According to the external standard method, the peak area was calculated (Formula (I) and (II)), and the release rate at different sampling times was calculated. The release curve is shown in Figure 14 , the release data are shown in Table 9.

[0073] Table 9

[0074] From Table 9 and Figure 14 It can be seen that with 15 min as the first sampling point, the release reaches a plateau after 120 h. The RSD of the release at the initial sampling time point is less than 10%, and the RSD of the release at the subsequent sampling time points is less than 5%, indicating that the method of the present invention has good repeatability and stable release behavior.

[0075] 3.4 Methodological Validation Exclusivity Depend on Figure 11 and 12It can be seen that compared with the blank solution, the reference solution has a peak at 5.105min, which indicates that the peak corresponds to vitamin E. Figure 12 and 13 It can be seen that a peak appears at 4.993 min in the test solution, which is basically consistent with the retention time of vitamin E in the reference solution. It can be seen that the peak here corresponds to the vitamin E in the test solution. From the above results, it can be seen that the blank solution has no interference at the retention time of vitamin E and will not affect the determination of the test sample. The method of the present invention has good specificity.

[0076] Repeatability As can be seen from Table 9, the RSDs of the six parallel sample measurements at the three sampling time points of 15 min, 24 h, and 120 h were as follows: the RSD of the release rate of the 15 min sample was 7.1%, less than 10%; the RSD of the 24 h sample was 2.7%, less than 5%; and the RSD of the 120 h sample was 1.3%, less than 5%. The repeatability results were good.

[0077] Accuracy According to the method of Example 1, three spiked solutions of low, medium and high concentrations were prepared, and the spiked recovery was measured. The results are shown in Table 10.

[0078] Table 10

[0079] As can be seen from Table 10, at low, medium and high concentrations, the recovery results were all in the range of 95% to 105%, and the RSD was less than 5.0%, demonstrating good accuracy results.

[0080] Linear Add different amounts of vitamin E reference stock solution to the blank solution to prepare 7 linear solutions with different concentrations. Detect according to the chromatographic conditions in Section 3.2 and plot the linear relationship between vitamin E concentration and peak area. The linear graph is shown in Figure 15 The measurement results are shown in Table 11.

[0081] Table 11

[0082] Depend on Figure 15 As can be seen from Table 11, when the vitamin E concentration is in the range of 6.4 μg / mL to 159.1 μg / mL, the linear equation is y=1997.8x-84.434, R 2 =0.9999, and there is a good linear relationship between concentration and peak area.

[0083] stability The reference solution, 15 min test solution, 24 h test solution, and 120 h test solution were placed at room temperature for 7 days. They were then tested according to the chromatographic conditions in Section 3.2 above to examine the recovery of the stability solution concentration. The test results are shown in Table 12.

[0084] Table 12

[0085] As shown in Table 12, the recovery rates of the four solutions were between 95% and 105%. The reference solution and the test solution used in the method of the present invention were stable at room temperature for at least 7 days.

[0086] Comparative Example 1 1.1 Solution Preparation Preparation of release medium: Combine 1.36g of potassium dihydrogen phosphate and 1.42g of sodium dihydrogen phosphate in 1000mL of ultrapure water and adjust the pH to 7.4±0.1 with sodium hydroxide. Add 10g of Tween 80 to 1000mL of this solution and mix thoroughly to obtain a 1.0 w / v% Tween 80 phosphate buffered saline solution as the release medium. Heat and degas before use.

[0087] Preparation of Reference Solution: Accurately weigh 20.19 mg of Vitamin E Reference Solution into a 50 mL volumetric flask. Dissolve and dilute to volume with methanol and shake well to prepare the reference solution. Accurately measure 1 mL of the reference solution into a 10 mL volumetric flask. Dilute to volume with release medium and shake well to prepare the reference solution.

[0088] Preparation of test solution: Accurately weigh approximately 50 mg of the vitamin E compound sustained-release microspheres prepared according to the method of Example 1 and place them into a stoppered ground-mouth glass vial. Add 150 mL of release medium preheated to 45°C and place the stoppered ground-mouth glass vial in a constant temperature water bath at 45°C ± 0.5°C. Remove 1 mL of the dissolution solution from the vial at 1 h, 4 h, 8 h, 12 h, 24 h, 32 h, 48 h, 56 h, 72 h, 96 h, and 120 h, respectively. Centrifuge the dissolution sample solution at 10,000 rpm for 10 min to obtain the test solution. Prepare six replicate samples using the same method (weighing 52.35 mg, 50.95 mg, 53.51 mg, 50.56 mg, 53.12 mg, and 50.48 mg, respectively).

[0089] 1.2 Chromatographic conditions Same as Example 1.

[0090] 1.3' Detection The blank solution (release medium), reference solution, and test solution at each time point were analyzed sequentially using a high performance liquid chromatograph. The release rates at different sampling times were calculated using the external standard method using peak area calculation (Formulas (I) and (II)). The release curves are shown in Table 1. Figure 16 , the release data are shown in Table 13.

[0091] Table 13

[0092] From Table 13 and Figure 16 It can be seen that with 1 hour as the first sampling point, the release rate in 120 hours is 18.29%, and the release behavior is extremely slow. The RSDs of the release rates at 1 hour and 4 hours are both greater than 10%, and the RSDs of the release rates at 8 hours and 12 hours are both greater than 5%. The repeatability of the release method between samples in the initial stage of release is relatively poor, and the release behaviors vary greatly.

[0093] Comparative Example 2 2.1 Solution Preparation Preparation of release medium: Add 1.36g of potassium dihydrogen phosphate and 1.42g of sodium dihydrogen phosphate to 1000mL of ultrapure water and adjust the pH to 7.4±0.1 with sodium hydroxide. Add 700mL of propylene glycol to 300mL of this solution and mix thoroughly to obtain a 70v / v% propylene glycol phosphate buffered saline solution as the release medium. Heat and degas before use.

[0094] Preparation of Reference Solution: Accurately weigh 20.61 mg of Vitamin E Reference Solution into a 50 mL volumetric flask. Dissolve and dilute to volume with methanol and shake well to prepare the reference solution. Accurately measure 1 mL of the reference solution into a 10 mL volumetric flask. Dilute to volume with release medium and shake well to prepare the reference solution.

[0095] Preparation of test solution: Accurately weigh approximately 50 mg of the vitamin E compound sustained-release microspheres prepared according to the method of Example 1 and place them into a stoppered ground-mouth glass vial. Add 150 mL of release medium preheated to 45°C and place the stoppered ground-mouth glass vial in a constant temperature water bath at 45°C ± 0.5°C. Remove 1 mL of the dissolution solution from the vial at 1 h, 4 h, 8 h, 12 h, 24 h, 32 h, 48 h, 56 h, 72 h, 96 h, and 120 h, respectively. Centrifuge the dissolved sample solution at 10,000 rpm for 10 min to obtain the test solution. Prepare six replicate samples using the same method (weighing 53.21 mg, 52.14 mg, 50.84 mg, 51.34 mg, 51.84 mg, and 52.48 mg, respectively).

[0096] 2.2 Chromatographic conditions Same as Example 1.

[0097] 2.3' Detection The blank solution (release medium), reference solution, and test solution at each time point were analyzed sequentially using a high performance liquid chromatograph. The release rates at different sampling times were calculated using the external standard method using peak area calculation (Formulas (I) and (II)). The release curves are shown in Table 1. Figure 17 , the release data are shown in Table 14.

[0098] Table 14

[0099] From Table 14 and Figure 17 It can be seen that with 1 hour as the first sampling point, the release rate in 120 hours is 43.89%, the release behavior is extremely slow, and the RSD of the release rate at 1 hour and 4 hours are both greater than 10%. The repeatability of the release method between samples in the early stage of release is relatively poor, and the release behavior varies greatly.

[0100] Comparative Example 3 3.1 Solution preparation Preparation of release medium: Add 1.36g of potassium dihydrogen phosphate and 1.42g of sodium dihydrogen phosphate to 1000mL of ultrapure water and adjust the pH to 7.4±0.1 with sodium hydroxide. Add 700mL of propylene glycol and 2.5g of Tween 80 to 300mL of this solution and mix thoroughly to obtain a 70v / v% propylene glycol phosphate buffer solution with 0.25w / v% Tween 80 as the release medium. Heat and degas before use.

[0101] Preparation of Reference Solution: Accurately weigh 21.05 mg of Vitamin E Reference Solution into a 50 mL volumetric flask. Dissolve and dilute to volume with methanol and shake well to prepare the reference solution. Accurately measure 1 mL of the reference solution into a 10 mL volumetric flask. Dilute to volume with release medium and shake well to prepare the reference solution.

[0102] Preparation of test solution: Accurately weigh approximately 50 mg of the vitamin E compound sustained-release microspheres prepared according to the method of Example 1 and place them into a stoppered ground-mouth glass vial. Add 150 mL of release medium preheated to 45°C and place the stoppered ground-mouth glass vial in a constant temperature water bath at 45°C ± 0.5°C. Remove 1 mL of the dissolution solution from the vial at 1 h, 4 h, 8 h, 12 h, 24 h, 32 h, 48 h, 56 h, 72 h, 96 h, and 120 h, respectively. Centrifuge the dissolved sample solution at 10,000 rpm for 10 min to obtain the test solution. Prepare six replicate samples using the same method (weighing 50.78 mg, 51.65 mg, 52.31 mg, 50.86 mg, 53.42 mg, and 51.20 mg, respectively).

[0103] 3.2 Chromatographic conditions Same as Example 1.

[0104] 3.3' Detection The blank solution (release medium), reference solution, and test solution at each time point were analyzed sequentially using a high performance liquid chromatograph. The release rates at different sampling times were calculated using the external standard method using peak area calculation (Formulas (I) and (II)). The release curves are shown in Table 1. Figure 18 , the release data are shown in Table 15.

[0105] Table 15

[0106] From Table 15 and Figure 18 It can be seen that with 1 hour as the first sampling point, the release rate in 120 hours is 64.26%, the release behavior is slow, and the RSD of the release rates at 1 hour, 4 hours and 12 hours are all greater than 10%. The release behavior between samples in the initial stage of release is quite different.

[0107] Comparative Example 4 4.1 Solution preparation To prepare the release medium: Add 1.36g of potassium dihydrogen phosphate and 1.42g of sodium dihydrogen phosphate to 1000mL of ultrapure water and adjust the pH to 7.4±0.1 with sodium hydroxide. To 300mL of this solution, add 700mL of propylene glycol and 10g of sodium dodecyl sulfate (SDS) to obtain a 70v / v% propylene glycol phosphate buffered saline solution with 1.0w / v% SDS as the release medium. Heat and degas before use.

[0108] Preparation of Reference Solution: Accurately weigh 20.61 mg of Vitamin E Reference Solution into a 50 mL volumetric flask. Dissolve and dilute to volume with methanol and shake well to prepare the reference solution. Accurately measure 1 mL of the reference solution into a 10 mL volumetric flask. Dilute to volume with release medium and shake well to prepare the reference solution.

[0109] Preparation of test solution: Accurately weigh approximately 50 mg of the vitamin E compound sustained-release microspheres prepared according to the method of Example 1 and place them into a stoppered ground-mouth glass vial. Add 150 mL of release medium preheated to 45°C and place the stoppered ground-mouth glass vial in a constant temperature water bath at 45°C ± 0.5°C. Remove 1 mL of the dissolution solution from the vial at 1 h, 4 h, 8 h, 12 h, 24 h, 32 h, 48 h, 56 h, 72 h, 96 h, and 120 h, respectively. Centrifuge the dissolved sample solution at 10,000 rpm for 10 min to obtain the test solution. Prepare six replicate samples using the same method (weighing 50.64 mg, 51.65 mg, 50.15 mg, 52.31 mg, 53.70 mg, and 50.98 mg, respectively).

[0110] 4.2 Chromatographic conditions Same as Example 1.

[0111] 4.3' Detection The blank solution (release medium), reference solution, and test solution at each time point were analyzed sequentially using a high performance liquid chromatograph. The release rates at different sampling times were calculated using the external standard method using peak area calculation (Formulas (I) and (II)). The release curves are shown in Table 1. Figure 19 , the release data are shown in Table 16.

[0112] Table 16

[0113] From Table 16 and Figure 19 It can be seen that with 1h as the first sampling point, the release rate at 120h is 50.84%, the release behavior is slow, and the RSD of the release rates at 1h, 4h, 8h and 12h are all greater than 10%, and the release behavior between samples in the initial stage of release is quite different; the RSD of the release rates at 24h and 32h is greater than 5%, and the release behavior between samples is quite different.

[0114] Comparative Example 5: 5.1 Solution Preparation Preparation of release medium: Add 1.36g of potassium dihydrogen phosphate and 1.42g of sodium dihydrogen phosphate to 1000mL of ultrapure water and adjust the pH to 7.4±0.1 with sodium hydroxide. Add 500mL of this solution to 500mL of propylene glycol and 10g of Tween 80, and mix thoroughly to obtain a 50v / v% propylene glycol phosphate buffered saline solution with 1.0w / v% Tween 80 as the release medium. Heat and degas before use.

[0115] Preparation of Reference Solution: Accurately weigh 22.45 mg of Vitamin E Reference Solution into a 50 mL volumetric flask. Dissolve and dilute to volume with methanol and shake well to prepare the reference solution. Accurately weigh 1 mL of the reference solution into a 10 mL volumetric flask. Dilute to volume with release medium and shake well to prepare the reference solution.

[0116] Preparation of test solution: Accurately weigh approximately 50 mg of the vitamin E compound sustained-release microspheres prepared according to the method of Example 1 and place them into a stoppered ground-mouth glass vial. Add 150 mL of release medium preheated to 45°C and place the stoppered ground-mouth glass vial in a constant temperature water bath at 45°C ± 0.5°C. Remove 1 mL of the dissolution solution from the vial at 1 h, 4 h, 8 h, 12 h, 24 h, 32 h, 48 h, 56 h, 72 h, 96 h, and 120 h, respectively. Centrifuge the dissolved sample solution at 10,000 rpm for 10 min to obtain the test solution. Prepare six replicate samples using the same method (weighing 51.68 mg, 50.62 mg, 52.89 mg, 53.15 mg, 51.63 mg, and 53.30 mg, respectively).

[0117] 5.2 Chromatographic conditions Same as Example 1.

[0118] 5.3' Detection The blank solution (release medium), reference solution, and test solution at each time point were analyzed sequentially using a high performance liquid chromatograph. The release rates at different sampling times were calculated using the external standard method using peak area calculation (Formulas (I) and (II)). The release curves are shown in Table 1. Figure 20 , the release data are shown in Table 17.

[0119] Table 17

[0120] From Table 17 and Figure 20 It can be seen that with 1h as the first sampling point, the release rate in 120h is 75.33%, the release behavior is relatively slow, and the RSD of the release rates at 1h, 4h and 12h are all greater than 10%. The release behavior differences between samples in the initial stage of release are large; the RSD of the release rates at 24h, 32h, 48h and 56h are all not less than 5%, indicating that the release conditions are unstable and easily increase the differences between samples.

[0121] Comparative Example 6 6.1 Solution Preparation Preparation of release medium: Add 1.36g of potassium dihydrogen phosphate and 1.42g of sodium dihydrogen phosphate to 1000mL of ultrapure water and adjust the pH to 7.4±0.1 with sodium hydroxide. Add 700mL of methanol and 10g of Tween 80 to 300mL of this solution and mix thoroughly to obtain a 1.0w / v% Tween 80 in 70v / v% methanolic phosphate buffer solution as the release medium. Heat and degas before use.

[0122] Preparation of Reference Solution: Accurately weigh 21.95 mg of Vitamin E Reference Solution into a 50 mL volumetric flask. Dissolve and dilute to volume with methanol and shake well to prepare the reference solution. Accurately measure 1 mL of the reference solution into a 10 mL volumetric flask. Dilute to volume with release medium and shake well to prepare the reference solution.

[0123] Preparation of test solution: Accurately weigh approximately 50 mg of the vitamin E compound sustained-release microspheres prepared according to the method of Example 1 and place them into a stoppered ground-mouth glass vial. Add 150 mL of release medium preheated to 45°C and place the stoppered ground-mouth glass vial in a constant temperature water bath at 45°C ± 0.5°C. Remove 1 mL of the dissolution solution from the vial at 1 h, 4 h, 8 h, 12 h, 24 h, 32 h, 48 h, 56 h, 72 h, 96 h, and 120 h, respectively. Centrifuge the dissolved sample solution at 10,000 rpm for 10 min to obtain the test solution. Prepare six replicate samples using the same method (weighing 50.96 mg, 50.72 mg, 51.49 mg, 51.15 mg, 52.67 mg, and 53.02 mg, respectively).

[0124] 6.2 Chromatographic conditions Same as Example 1.

[0125] 6.3' Detection The blank solution (release medium), reference solution, and test solution at each time point were analyzed sequentially using a high performance liquid chromatograph. The release rates at different sampling times were calculated using the external standard method using peak area calculation (Formulas (I) and (II)). The release curves are shown in Table 1. Figure 21 , the release data are shown in Table 18.

[0126] Table 18

[0127] From Table 18 and Figure 21 It can be seen that with 1h as the first sampling point, the release rate in 120h is 23.39%, and the release behavior is extremely slow. The RSD of the release rates at 1h and 4h are both greater than 10%, and the release behavior between samples in the initial stage of release is quite different; the RSD of the release rates at other multiple sampling points is greater than 5%, and the repeatability of the release conditions is poor. In addition, the boiling point of methanol is 64.7℃. Using 45℃ as the release medium will cause methanol to volatilize, and the relative proportion will decrease in the later stage of release, resulting in inconsistent release conditions.

[0128] Comparative Example 7 7.1 Solution Preparation Preparation of release medium: Add 1.36g of potassium dihydrogen phosphate and 1.42g of sodium dihydrogen phosphate to 1000mL of ultrapure water and adjust the pH to 7.4±0.1 with sodium hydroxide. Add 700mL of ethanol and 10g of Tween 80 to 300mL of this solution and mix thoroughly to obtain a 1.0w / v% Tween 80 in 70v / v% ethanolic phosphate buffered saline solution as the release medium. Heat and degas before use.

[0129] Preparation of Reference Solution: Accurately weigh 21.36 mg of Vitamin E Reference Solution into a 50 mL volumetric flask. Dissolve and dilute to volume with methanol and shake well to prepare the reference solution. Accurately measure 1 mL of the reference solution into a 10 mL volumetric flask. Dilute to volume with release medium and shake well to prepare the reference solution.

[0130] Preparation of test solution: Accurately weigh approximately 50 mg of the vitamin E compound sustained-release microspheres prepared according to the method of Example 1 and place them into a stoppered ground-mouth glass vial. Add 150 mL of release medium preheated to 45°C and place the stoppered ground-mouth glass vial in a constant temperature water bath at 45°C ± 0.5°C. Remove 1 mL of the dissolution solution from the vial at 1 h, 4 h, 8 h, 12 h, 24 h, 32 h, 48 h, 56 h, 72 h, 96 h, and 120 h, respectively. Centrifuge the dissolved sample solution at 10,000 rpm for 10 min to obtain the test solution. Prepare six replicate samples using the same method (weighing 52.55 mg, 51.98 mg, 51.56 mg, 52.31 mg, 50.58 mg, and 50.54 mg, respectively).

[0131] 7.2 Chromatographic conditions Same as Example 1.

[0132] 7.3' Detection The blank solution (release medium), reference solution, and test solution at each time point were analyzed sequentially using a high performance liquid chromatograph. The release rates at different sampling times were calculated using the external standard method using peak area calculation (Formulas (I) and (II)). The release curves are shown in Table 1. Figure 22 , the release data are shown in Table 19.

[0133] Table 19

[0134] From Table 19 and Figure 22As can be seen, the release rate after 120 hours was 25.30%, indicating extremely slow release behavior. The RSD of the release rate after 1 hour was greater than 10%, indicating significant variability between samples in the initial release period. The RSD of the release rates at multiple other sampling points was greater than 5%, indicating poor reproducibility of these release conditions. Furthermore, ethanol has a boiling point of 78.4°C and is highly volatile as a release medium at 45°C. The relative proportion of ethanol decreased in the later stages of release, indicating inconsistent release conditions.

[0135] It can be seen from the above embodiments and comparative examples that the present invention enhances the solubility of vitamin E by using a surfactant, Tween 80, by selecting a specific release medium, and simultaneously utilizing the synergistic effect of propylene glycol and buffer salts to achieve precise control of the release rate of the microspheres. The constant temperature release environment of 37°C to 45°C ± 0.5°C is used to simulate the physiological temperature range of the human body, thereby constructing a scientific experimental basis for in vitro and in vivo correlation studies. Compared with the comparative example, the present invention shortens the release experimental period of the sustained-release microspheres of vitamin E compounds to within 1 week, solves the problem of incomplete release in the prior art, and significantly improves the research throughput. At the same time, the optimized conditions enable vitamin E to be slowly released at a controllable rate, effectively avoiding instability problems such as oxidation and degradation caused by long-term exposure. The detection data of the present invention has small deviation and high accuracy, providing a reliable theoretical basis and technical support for research and development.

[0136] The above description is merely a preferred embodiment of the present invention and does not limit the present invention in any way. Any person skilled in the art who, without departing from the scope of the present invention, makes any equivalent substitution, modification, or other changes to the technical solution and technical content disclosed in the present invention shall be deemed to be within the scope of the present invention and still fall within the scope of protection of the present invention.

Claims

1. A method for determining the release rate of vitamin E compound sustained-release microspheres, characterized in that: The method comprises: 1) adding the vitamin E compound sustained-release microspheres to a release medium under dissolution conditions to prepare a test solution; 2) dissolving vitamin E in alcohol and the release medium to prepare a reference solution; 3) using liquid chromatography to test the test solution and the reference solution to determine the release rate; The dissolution conditions are 37°C±0.5°C~45°C±0.5°C, and the release medium is 70v / v% propylene glycol phosphate buffered saline solution containing 0.5w / v%~1.0w / v% Tween 80.

2. The measuring method according to claim 1, wherein The step 1) includes adding the vitamin E compound microspheres to the release medium preheated to 37°C±0.5°C~45°C±0.5°C, allowing it to stand under the dissolution conditions, and centrifuging the dissolution solution at the sampling time to obtain the test solution.

3. The measuring method according to claim 2, wherein The sampling times include 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 32 h, 48 h, 56 h, 72 h, 96 h and 120 h.

4. The measuring method according to claim 1, wherein In the step 2), the alcohol is methanol.

5. The measuring method according to claim 1, wherein In step 3), the liquid chromatography conditions include: Mobile phase: 97v / v% methanol in water; Chromatographic column: Waters Symmetry Shield RP18 (3.5 μm, 4.6 mm × 100 mm); Detector: UV detector.

6. The measuring method according to claim 5, characterized in that The liquid chromatography conditions further include: Flow rate: 1.0 mL / min; elution time: 10 min; column temperature: 30°C; detection wavelength: 292 nm; injection volume: 20 μL.

7. The measuring method according to claim 1, wherein The release rate of the vitamin E compound sustained-release microspheres was calculated by the external standard method.

8. The measuring method according to claim 7, wherein The release rate of the vitamin E compound sustained-release microspheres at the nth sampling time is calculated by the following formulas (I) and (II), where n≥1: Formula (I) Formula (II) in, C is the concentration of vitamin E compounds in the test solution, mg / mL; Cs is the concentration of the reference solution, mg / mL; and are the main peak areas of the test solution and the reference solution, respectively; Cn is the concentration of vitamin E compounds in the test solution at the nth sampling time calculated according to formula (I), mg / mL; Vn is the total volume of the test solution at the nth sampling, mL; Ci is the concentration of vitamin E compounds in the test solution before the nth time calculated according to formula (I), mg / mL; 1 is the sampling volume each time, mL; m s is the mass of vitamin E compound in vitamin E compound sustained-release microspheres, mg; D is the degree of release.

9. The assay method according to any one of claims 1 to 8, wherein The vitamin E compound sustained-release microspheres are microspheres obtained by encapsulating the vitamin E compound in a hydrophobic polymer.

10. The measuring method according to claim 9, characterized in that The hydrophobic polymer is one or more of polycaprolactone and its copolymer poly L-lactide-caprolactone, poly L-lactic acid, poly D-lactic acid and its copolymer poly L-lactic acid / poly D-lactic acid-glycolic acid copolymer.

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