Method for determining content of glucose in star-shaped PLGA (poly (lactic-co-glycolic acid)) and pretreatment method

Abstract

The invention discloses a method for determining the content of glucose in star-shaped PLGA and a pretreatment method, a mixed solution of triethylamine and methanol is adopted as a hydrolysis solvent to perform pretreatment on PLGA or PLA in a polyester compound so as to form a stable hydrolysis solution, and the hydrolysis method is adopted to rapidly extract the bonded glucose in the star-shaped PLGA, so that the content of the glucose in the star-shaped PLGA is determined. Then, HPLC-ELSD is used for detection, and the content of glucose is calculated according to the linear regression equation and the logarithm value of the glucose peak area in the chromatogram of the sample to be detected. The detection method is easy and convenient to operate, rapid in detection and high in sensitivity, quantitative detection of trace glucose in the star-shaped PLGA caused by glucose can be achieved, characterization methods and quality control methods of the quality attribute of the star-shaped PLGA are enriched, research on performance parameters of a sustained and controlled release preparation with the star-shaped PLGA as a carrier is facilitated, and the application prospect is wide. And a reference is provided for evaluating process change and quality difference of the sustained and controlled release preparation.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a method for determining the glucose content in star-shaped PLGA and a pretreatment method. Background Art

[0002] At present, the polyester compounds commonly used are polylactic acid (PLA) and lactide-co-glycolide copolymer (PLGA). Different structural units or proportions in polymers of the same molecular weight correspond to different biodegradation rates. According to the difference in polymerization units, PLA is divided into PLLA formed by L-lactic acid, PDLA formed by D-lactic acid, and PDLLA formed by D-lactic acid and L-lactic acid; PLGA is usually classified according to the molar percentage of lactide and glycolide. For example, the common PLGA (5050) and PLGA (7525) refer to the molar percentages of lactide and glycolide in the polymer of 50:50 and 75:25 respectively. In addition, with the in-depth study of PLGA, it can be further divided into star-shaped, tree-shaped, comb-shaped, ring-shaped, and multi-arm-shaped according to the different initiators or branching degrees. These polymers are all biodegradable materials and can be used in drug delivery systems, tissue engineering, medical devices and other fields.

[0003] Among them, PLGA has the advantages of low immunity, good biocompatibility and controllable degradability, and has a good application prospect in the field of biomedicine, and is often used as a skeleton material for sustained-release drug delivery systems. At present, PLGA has been approved by the FDA as a sustained-release carrier for microspheres and implants, and is therefore widely studied in the field of sustained-release of drugs (including proteins, peptides, genes, etc.).

[0004] PLGA is usually made of glycolide and lactide as starting materials, and is formed by ring-opening polymerization under the action of an initiator. The properties of PLGA are not only related to the ratio and molecular weight of glycolide and lactide, but also different initiators will cause changes in the branching structure of PLGA, resulting in great differences in chemical, mechanical, diffusion, biological and other properties, which ultimately affect the performance of the prepared sustained-release preparations in terms of hydrophilicity, degradation rate, drug release rate, etc. Compared with linear PLGA with a single structure, star-shaped, tree-shaped, comb-shaped, ring-shaped, and multi-arm PLGA with higher branching degree are increasingly attracting attention. Star-shaped PLGA is usually synthesized with glycerol, pentaerythritol, glucose, etc. as initiators, and it shows superiority in biocompatibility, drug loading, degradation performance and processability. For example, the carrier of Sanostatin (octreotide acetate microspheres for injection) launched by Novartis is a star-shaped PLGA prepared with glucose as an initiator.

[0005] PLGA, as a carrier of microsphere preparation, plays a leading role in release kinetics. Therefore, the characterization and study of PLGA performance parameters are conducive to further controlling the release behavior of microspheres. In the preparation process of star-shaped PLGA using glucose as an initiator, the amount of initiator affects the branching degree and molecular weight of the polymer, and thus affects its degradation rate. Currently, the characterization of glucose in this type of PLGA mostly uses glucose identification reaction or nuclear magnetic resonance carbon spectroscopy (13CNMR) for qualitative detection. In addition, although patent application CN112578067A discloses that glucose in star-shaped PLGA can be quantitatively detected by LC-MS / MS, the operation is complicated and time-consuming. When the star-shaped PLGA is hydrolyzed in a sodium hydroxide solution, glucose is easily converted into isomers such as fructose in a strong alkaline environment. Since its LC-MS / MS detection involves atomization and ionization steps, it is generally not suitable for non-volatile salts. The pretreatment may use a low-concentration sodium hydroxide solution, through a longer hydrolysis time and a smaller injection volume, thereby avoiding a significant impact on the test results and the mass spectrometer detector. Similarly, inorganic acids and bases will form high-boiling point salts that are difficult to volatilize. The pretreatment is also not suitable for the application of other detection equipment such as evaporative light detectors; further, in its detection process, 0.1% ammonia water and 75% acetonitrile water are used as mobile phases. The solvent acetonitrile is highly toxic, and multiple peaks are obtained in the test results. The quantitative calculation of its detection is complex, which is not conducive to repeatable detection. Therefore, there is an urgent need to provide a new and ideal pretreatment method for polyester compounds, which can achieve efficient and rapid hydrolysis while significantly reducing the limitations of subsequent detection methods, and provide a simple, rapid, environmentally safe and green method for quantitative detection of covalently bonded glucose in star-shaped PLGA. Summary of the invention

[0006] The purpose of the present invention is to provide a method for determining the content of glucose in star-shaped PLGA, aiming to solve the problem of accurate quantitative detection of trace glucose in star-shaped PLGA triggered by glucose. The detection method is simple to operate, fast to detect, and highly sensitive. It can realize the quantitative detection of trace glucose in star-shaped PLGA triggered by glucose, enrich the characterization method and quality control method of the quality attributes of star-shaped PLGA, contribute to the study of performance parameters of sustained-release preparations with star-shaped PLGA as a carrier, and provide a reference for evaluating process changes and quality differences of sustained-release preparations.

[0007] The present invention also provides a pretreatment method for polyester compounds, aiming to solve the problem of rapid hydrolysis of PLGA or PLA in the polyester compounds.

[0008] The technical solution adopted by the present invention to solve its technical problem is: A method for measuring the glucose content in star-shaped PLGA comprises the following steps: (1) Pretreatment The star-shaped PLGA prepared with glucose as an initiator is used as a test sample. The test sample is placed in a hydrolysis solvent, hydrolyzed into a clear solution, and then a volatile organic acid is added to neutralize it, and then the solution is fixed to volume with water to obtain a test solution with a concentration of 25±2.5mg / ml; a hydrolysis solvent is added to a glucose reference substance, and the volatile organic acid is neutralized, and finally the solution is fixed to volume with water to obtain a glucose standard curve solution; (2) HPLC-ELSD detection The sample solution to be tested and the glucose standard curve solution are subjected to high performance liquid chromatography to obtain chromatograms of the sample solution to be tested and the glucose standard curve, respectively; (3) Content calculation Establishing a linear regression equation based on the logarithmic value of the concentration of the glucose standard curve solution and the logarithmic value of the glucose peak area in the chromatogram of the glucose standard curve; The glucose content in the sample to be tested is calculated according to the linear regression equation and the logarithmic value of the glucose peak area in the chromatogram of the sample to be tested.

[0009] At present, the pretreatment methods for rapid hydrolysis of polyester compounds are extremely limited. The prior art discloses the use of sodium hydroxide to pre-treat and hydrolyze star-shaped PLGA, but it is not suitable for the detection of glucose content in star-shaped PLGA using an evaporative light detector in the present invention, because after treatment with a non-volatile acid or base such as hydrochloric acid, nitric acid or sodium hydroxide, it is easy to form high-boiling products with other components including PLGA hydrolysate, interfering with the detection. In order to be suitable for evaporative light detector detection, the present invention uses volatile organic acids or bases to effectively avoid their interference, greatly simplify the detection method, easy to operate and ensure detection accuracy. If the pretreatment method of the present invention is used, the subsequent conventional LC-MS / MS is not applicable and must be further improved before it can be applicable.

[0010] The research team of the present invention conducted a large number of exploratory tests on the hydrolysis of PLGA and PLA in polyester compounds and found that when solvents such as acetone, tetrahydrofuran, acetonitrile, etc. that can dissolve such polyester compounds are used, the hydrolysis process is extremely slow. When solvents such as methanol, ethanol, and isopropanol are used, the hydrolysis process will also be relatively slow, but when triethylamine is selected as a solvent, the hydrolysis process is also relatively slow, which is not conducive to the accuracy of subsequent experimental operations and results. Based on the above experiments, it was explored and found that when the hydrolysis solvent is acetone, tetrahydrofuran, ethanol, and isopropanol, respectively, mixed with triethylamine, the polyester compounds in the present invention, especially the star-shaped PLGA, cannot be quickly hydrolyzed, and when acetonitrile and triethylamine are used as hydrolysis solvents, the color of the solution changes, and the hydrolysis product cannot be completely soluble in water. However, when methanol and triethylamine are mixed in a specific ratio as a hydrolysis solvent, it is unexpected that the polyester compounds of the present invention can be quickly hydrolyzed, and the hydrolysis product is easily soluble in water, which is beneficial to the subsequent detection steps and ensures the accuracy of the detection results.

[0011] According to this accidental discovery, methanol and triethylamine can hydrolyze the polymer in the present invention quickly and form a clear solution in a specific volume ratio range of (1 to 6): 1, and the solution state remains unchanged after adding water to the volume, and it is still a stable clear solution without turbidity. According to research, when the volume ratio of methanol to triethylamine is greater than 6: 1, the test result is significantly lower, which may be because the more methanol, the more it tends to the hydrolysis effect of methanol as a single solvent; and when the volume ratio of methanol to triethylamine is less than 6: 1, for example, when the amount of methanol added is 6ml and the amount of triethylamine added is 2-3ml, although the water bath for 2h has no effect on the test result, the slight increase of triethylamine leads to a stronger alkalinity of the solution, which significantly affects the life of the chromatographic column, and more volatile acid needs to be added during neutralization, introducing more solvents, increasing the detection cost, and at the same time, it is not conducive to the reuse of the chromatographic column, nor is it conducive to the repeatability of the detection method. Therefore, the ideal hydrolysis mixed solvent should be added with as little volatile acid or base as possible, which can not only achieve rapid hydrolysis of the polyester compound in the present invention, but also be suitable for subsequent detection steps.

[0012] Based on the results of the aforementioned exploratory studies, the present invention team speculated on the principle of rapid hydrolysis when triethylamine and methanol coexist in a specific ratio: methanol or triethylamine, as nucleophilic reagents, can attack the ester bonds on the polyester polymer chains to hydrolyze, but the process is extremely slow; the triethylamine with a lower volume ratio in the present invention also has a base catalytic effect, and under the appropriate alkaline conditions formed by it, it is beneficial for the lone electron pairs on the oxygen atoms in methanol to attack the ester bonds in the polyester compounds, accelerating the nucleophilic reaction, and the appropriate alkaline environment can neutralize the acid in the hydrolysis product, which is conducive to the continued hydrolysis reaction. Therefore, polyester compounds can achieve rapid hydrolysis under the synergistic effect of a mixed solution of methanol and triethylamine. However, other alcohol reagents such as ethanol are weaker in nucleophilicity than methanol, so they react more slowly with ester bonds than methanol, thereby significantly reducing the hydrolysis rate of polyester compounds and failing to achieve rapid hydrolysis.

[0013] Furthermore, based on the above hydrolysis principle, the hydrolysis step of the present invention is universal for polyester polymers, and is suitable for commercially purchased or prepared PLGA or PLA, wherein the molar ratio of lactide to glycolide in the PLGA is 50-75:50-25, and the molecular weight is generally within 10kDa to 110kDa. The hydrolysis time can be appropriately adjusted according to the molecular weight, and the rapid hydrolysis of the present invention is hydrolysis within 24 hours to form a clear solution; generally, according to the pretreatment method of the present invention, it is further preferred that the hydrolysis is performed within 2 hours to form a clear solution. The polyester compounds of the present invention with a larger molecular weight can be hydrolyzed to a clear state by appropriately extending the hydrolysis time. The hydrolysis process of different molecular weights can be referred to in Example 20.

[0014] As an embodiment, in step (1), the hydrolysis solvent is a mixed solvent of methanol and triethylamine in a volume ratio of 1 to 6:1, the hydrolysis temperature is 30 to 60°C, and the hydrolysis time is within 24 hours. As a preferred embodiment, the hydrolysis temperature is 50°C and the hydrolysis time is 2 hours.

[0015] As an embodiment, in step (1), the volatile organic acid is a mixture of one or both of formic acid and glacial acetic acid; the volume ratio of methanol: triethylamine: volatile organic acid: water is 1 to 6: 1: 1: 7 to 2. As a preferred embodiment, the volume ratio of methanol: triethylamine: volatile organic acid: water is 2: 1: 1: 6.

[0016] As an embodiment, in step (2), the HPLC detection parameters are set as follows: Gradient elution was performed with water and methanol as the mobile phase; The flow rate of the mobile phase is 0.8-1.2 ml / min; The injection volume is 20μl~100μl; The column temperature is 35-45°C, and the column is a C18 column, including a Waters Atlantis T3 column; The carrier gas of the evaporative light scattering detector was nitrogen at a pressure of 40 psi; the drift tube temperature was 50-90°C, and the nebulizer mode was cooling.

[0017] As a preferred embodiment, in step (2), the HPLC detection parameters are set to: Gradient elution was performed with water and methanol as the mobile phase; The flow rate of the mobile phase was 1.0 ml / min; The injection volume was 50 μl; The chromatographic column was Waters Atlantis T3 (4.6 × 250 mm, 5 μm), and the column temperature was 40 °C; The carrier gas for the evaporative light scattering detector was nitrogen at 40 psi; the drift tube temperature was 70 °C and the nebulizer mode was refrigerated.

[0018] For the analysis of sugar compounds, gel columns, ion chromatography columns, amino columns and other sugar analysis columns are often used to separate different types of sugar substances. The fillers of gel columns and ion chromatography columns are prone to swelling in organic solvents. Based on the characteristics of the fillers, water or sodium chloride solution is generally used as the mobile phase, which is not suitable for a high proportion of organic phase as the mobile phase or solvent. The amino column is prone to hydrolysis and loss when bonded to the stationary phase using aminopropyl, resulting in the chromatographic column being not durable. For the detection of glucose in star-shaped PLGA, the PLGA must be hydrolyzed first, and organic reagents are required for hydrolysis and the hydrolysis products are relatively complex. The use of the above-mentioned sugar analysis columns can easily damage the chromatographic column. The C18 column used in the present invention has no special requirements on the selection of mobile phase and solvent, and can ideally avoid various problems of the above-mentioned commonly used gel column, ion chromatography column, amino column and other sugar analysis chromatographic columns; at the same time, considering that glucose may be isomerized under strong alkaline conditions, the present invention effectively utilizes the C18 column to avoid the high discrimination between glucose and its various isomers by the various chromatographic columns of the above-mentioned sugar analysis, and combines the gradient elution of the specific mobile phase during chromatographic separation, so that no matter whether there are multiple glucose isomers in the sample solution, they all present a total characteristic peak together with glucose in the chromatogram, avoiding the above-mentioned prior art that does not consider the C18 column when quantifying glucose, but often considers high discrimination in order to improve the detection accuracy, and confirms multiple peaks separately and calculates them by summing, which causes troubles in achieving its accurate detection and increases the detection burden and the complexity of quantitative calculation.

[0019] As an implementation scheme, the scheme for gradient elution with water and methanol as mobile phase is selected from the following: Solution 1: 0 min, water 100%, methanol 0%; 4.5 min, water 100%, methanol 0%; 5 min, water 15%, methanol 85%; 15 min, water 15%, methanol 85%; Option 2: 0 min, water 95%, methanol 5%; 4.5 min, water 95%, methanol 5%; 5 min, water 15%, methanol 85%; 15 min, water 15%, methanol 85%.

[0020] As an embodiment, the concentration range of the glucose standard curve solution is 1 μg / ml to 40 μg / ml, and the concentration gradient is at least 5.

[0021] As a preferred embodiment, the concentration range of the glucose standard curve solution is 2 μg / ml to 20 μg / ml.

[0022] A pretreatment method for polyester compounds comprises placing the polyester compounds in a hydrolysis solvent at a hydrolysis temperature of 30 to 60° C. for a hydrolysis time of less than 24 hours; the hydrolysis solvent is a mixed solvent of methanol and triethylamine at a volume ratio of 1 to 6:1.

[0023] For the pretreatment of the polyester compound of the present invention, it is further applicable to the pretreatment of the star-shaped PLGA described therein, but the quantification of glucose in the star-shaped PLGA needs to be combined with the corresponding detection method. Through the pretreatment of the polyester compound of the present invention, the problem of rapid hydrolysis of the star-shaped PLGA can be ideally solved, but for the detection of glucose in the star-shaped PLGA, the separation of the hydrolysis product and glucose should also be considered to avoid the interference of multiple hydrolysis products of the star-shaped PLGA, and ensure the accuracy of the detection result. Based on the difference in the response of the evaporative light detector to substances with different boiling points in the present invention and combined with the subsequent specific detection conditions, thus, through the hydrolysis solvent mixed in a specific proportion in the pretreatment of the present invention, and the adaptive adjustment of the hydrolysis temperature and hydrolysis time within the scope of the present invention, the ideal control of the hydrolysis degree of the star-shaped PLGA is achieved, not only to achieve its rapid hydrolysis to release bonded glucose, but also to be able to ideally control the interference of multiple products after hydrolysis on the detection, that is, to ensure that the small molecule fragment hydrolysis products generated after the hydrolysis of the star-shaped PLGA are all at a lower boiling point, and basically have no response in the evaporative light detector, and to ensure that the hydrolysis products of the macromolecular fragments can be ideally separated from glucose through gradient elution.

[0024] As an embodiment, the polyester compound is PLGA or PLA, wherein the molar ratio of lactide to glycolide in the PLGA is 50-75:50-25.

[0025] As a preferred embodiment, the PLGA is a star-shaped PLGA prepared using glucose as an initiator.

[0026] The beneficial effects of the present invention are: (1) The present invention provides a pretreatment method for PLGA or PLA in polyester compounds. Based on the research results, the relevant principles of methanol and triethylamine as specific hydrolysis solvents in the present invention are explored, and it has been verified through experiments that it is universal for PLGA and PLA of various types and molecular weights, and can ideally achieve rapid hydrolysis within 24 hours, more preferably rapid hydrolysis within 2 hours, and can continuously form a stable clear solution, which is beneficial to the safe and rapid hydrolysis pretreatment application of polyester compound related detection methods.

[0027] (2) The present invention also provides a treatment method based on the aforementioned (1) for application to the hydrolysis of star-shaped PLGA, which effectively ensures the smooth implementation of the subsequent specific detection method for glucose in the star-shaped PLGA, can ideally control the degree of hydrolysis of the star-shaped PLGA, quickly release glucose bonded to the polymer, and effectively avoid interference with the detection by other various hydrolysis products after hydrolysis, or no response or effective separation, thereby ensuring the accuracy of the detection results.

[0028] (3) The present invention also provides a new HPLC-ELSD liquid chromatography method for detecting the glucose content in star-shaped PLGA, which uses water and methanol as mobile phases and realizes quantitative detection of glucose through gradient elution, significantly simplifies the operation steps and the accurate acquisition of results, and ideally realizes a wider range of multiple parameters in the detection steps, effectively reduces the impact of human operation errors on the detection results, and further effectively guarantees the accuracy of the detection results.

[0029] (4) The pretreatment combined with detection method of the present invention uses few types of solvents in the whole process, and they are commonly available and only involve a small amount of conventional non-toxic methanol and triethylamine. It is environmentally friendly and highly safe, and does not require special conditions or special equipment. It is easy to operate, fast to detect, highly accurate, and low in cost, which is conducive to the subsequent processing of the detection. It can realize the quantitative detection of glucose in the star-shaped PLGA triggered by glucose, and is suitable for repetitive detection applications in various scenarios of experiments and industrialization. It is of great significance to the characterization of this type of PLGA and the study of the performance parameters of microspheres using it as a carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a standard curve diagram of the glucose content determination method provided in Example 1 of the present application; Figure 2 is a specific chromatogram of the sample solution to be tested provided in Example 1 of the present application; Figure 3 The sample solution diagram after hydrolysis and volume adjustment using triethylamine and acetonitrile; Figure 4 The sample solution is hydrolyzed using triethylamine and isopropanol (or acetone, tetrahydrofuran); Figure 5 The sample solution was hydrolyzed using triethylamine (or methanol). DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described in detail below through specific embodiments.

[0032] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following embodiments, unless otherwise specified, are all conventional methods in the art.

[0033] In the embodiment of the present invention, the star-shaped PLGA is prepared by using glucose as an initiator, which can be obtained by commercial purchase (such as the star-shaped PLGA of Evonic) or by existing technology, such as US5922338 (the star-shaped PLGA disclosed has a molecular weight of 20,000 to 200,000, preferably a molecular weight of 20,000 to 80,000). The star-shaped PLGA of the embodiment is prepared according to US5922338, taking a molecular weight of 60 kDa and a molar ratio of lactide to glycolide of 55:45 as an example.

[0034] Example 1 A method for determining the glucose content in star-shaped PLGA comprises the following steps: Preparation of the sample solution to be tested: Take about 250 mg of star-shaped PLGA, weigh it accurately, put it in a 10 ml volumetric flask, add 1 ml of triethylamine and 2 ml of methanol, seal it, and place it in a 50°C water bath for 2 hours until the PLGA is hydrolyzed to clarity. Take it out and cool it to room temperature, add 1 ml of glacial acetic acid, make up to volume with water, and mix well.

[0035] Preparation of standard curve solution: Take an appropriate amount of glucose reference substance, weigh accurately, add triethylamine, methanol, glacial acetic acid, and finally make up to volume with water. The ratio of methanol, triethylamine, glacial acetic acid and water is 2:1:1:6. Prepare solutions containing 2, 5, 10, 15, and 40 μg / ml of glucose respectively. (The concentration of the reference substance solution is about 10 μg / ml) Accurately measure 50 μl of the above standard curve solution and the sample solution to be tested, inject them into the high performance liquid chromatograph, and perform the test under the following chromatographic conditions:

[0036] Standard curve drawing: Take the standard curve solution, inject it into HPLC-ELSD for measurement, and record the corresponding chromatogram (see Figure 1 ) With the logarithm of glucose concentration as the abscissa X and the logarithm of the peak area as the ordinate Y, a standard curve was drawn and the regression equation was calculated.

[0037] Result calculation: Take the obtained sample solution to be tested, inject it into HPLC-ELSD for determination, and substitute the obtained peak area into the linear regression equation of the obtained standard curve to obtain the content of glucose in the sample to be tested. The linear parameters and glucose content results measured in Example 1 are shown in Table 1.

[0038] Table 1. Linear parameters and glucose content results measured in Example 1

[0039] Example 2 Example 2 provides a method for determining the glucose content in star-shaped PLGA. The detection method is basically the same as the detection method in Example 1, except that the starting ratio of the mobile phase is different. The water:methanol ratio in the starting gradient is 95:5, and the other conditions are the same as in Example 1. The specific gradient elution process is shown in Table 2 below, and the test results are shown in Table 3 below.

[0040] Table 2. Gradient elution process in Example 2 Time / min water% Methanol% 0 95 5 4.5 95 5 5 15 85 15 15 85 Table 3. Glucose content test results in Example 1-2

[0041] Embodiment 3-4 Examples 3-4 respectively provide a method for determining the glucose content in star-shaped PLGA. The detection method is basically the same as the detection method in Example 1, except that the temperature of the chromatographic column is different. The other conditions are the same as Example 1. The specific detection conditions and results are shown in Table 4 below.

[0042] Embodiment 5-6 Examples 5-6 respectively provide a method for determining the glucose content in star-shaped PLGA. The detection method is basically the same as the detection method in Example 1, except that the temperature of the drift tube is different. The other conditions are the same as Example 1. The specific detection conditions and results are shown in Table 4 below.

[0043] Embodiment 7-8 Examples 7-8 respectively provide a method for determining the glucose content in star-shaped PLGA. The detection method is basically the same as the detection method in Example 1, except that the flow rate used is different. The other conditions are the same as Example 1. The specific detection conditions and results are shown in Table 4 below.

[0044] Examples 9-10 Examples 9-10 respectively provide a method for determining the glucose content in star-shaped PLGA. The detection method is basically the same as the detection method in Example 1, except that the injection volume used is different. The other conditions are the same as Example 1. The specific detection conditions and results are shown in Table 4 below.

[0045] Table 4. Glucose content test results in Examples 3-10 Example Changing conditions Glucose retention time / min Glucose content% Compared with RD% of Example 1 Example 3 Column temperature 35℃ 3.152 0.116 1.4 Example 4 Column temperature 45℃ 3.135 0.109 1.8 Example 5 Drift tube temperature 50°C 3.147 0.114 0.3 Example 6 Drift tube temperature 90°C 3.137 0.114 0.3 Example 7 Flow rate: 0.8ml / min 3.900 0.118 2.0 Example 8 Flow rate: 1.2 ml / min 2.641 0.108 2.0 Example 9 Injection volume 20 μl 3.146 0.111 0.8 Example 10 Injection volume 100 μl 3.130 0.113 0.1 When the column temperature of the chromatographic column changes by ±5°C, the drift tube temperature changes by ±20°C, and the injection volume is 20μl and 100μl, the RD value of the test result is less than 2% compared with the test result of Example 1; when the flow rate changes by ±0.2ml / min, the glucose retention time changes, but the RD value of the test result is less than 2% compared with the test result of Example 1, which is within the durability range. In the present invention, within the above-mentioned detection parameter range, the changes in column temperature, drift tube temperature and flow rate are all controlled within the durability range of the liquid chromatography instrument, and changing the parameter data range corresponding to the conditions has no obvious effect on the test results. The present invention does not have excessively high requirements for the detection parameters, and can achieve ideal detection within a relatively wide range. It allows the fluctuation of condition parameters in a certain range during the detection process due to the influence of human operation or environmental factors, effectively avoiding the significant impact of the detection results caused by the overly harsh high-precision requirements of the detection method, and further ensuring the accuracy of the detection results.

[0046] Embodiment 11 Example 11 provides a method for determining the glucose content in star-shaped PLGA, which is consistent with the detection method in Example 1, except that formic acid is used instead of glacial acetic acid in the preparation of the sample solution to be tested, and the rest is the same as in Example 1. The specific configuration process is as follows, and the test results are shown in Table 5 below.

[0047] Preparation of the sample solution to be tested: Take about 250 mg of star-shaped PLGA, weigh accurately, place in a 10 ml volumetric flask, add 1 ml of triethylamine and 2 ml of methanol, seal, and place in a 50°C water bath for 2 hours until the PLGA is hydrolyzed to clarity, take out and cool to room temperature, add 1 ml of formic acid, make up to volume with water, and mix well.

[0048] Examples 12-19 Examples 12-17 respectively provide a method for determining the glucose content in star-shaped PLGA. The detection method is consistent with the detection method in Example 1, except that the water bath temperature and time during the hydrolysis process of the sample solution to be tested are different. The specific detection conditions and results are shown in Table 5 below.

[0049] Examples 18-19 Examples 18-19 respectively provide a method for determining the glucose content in star-shaped PLGA. The detection method is consistent with the detection method in Example 1, except that different amounts of methanol are added during the preparation of the sample solution to be tested. The specific detection conditions and results are shown in Table 5 below.

[0050] Table 5. Glucose content test results in Examples 11-19 Example Sample preparation changes Glucose retention time / min Glucose content% Compared with RD% of Example 1 Embodiment 11 Formic acid instead of glacial acetic acid 3.149 0.114 0.5 Example 12 Water bath temperature 30℃+1 hour 3.153 0.110 1.3 Example 13 Water bath temperature 30℃+24 hours 3.150 0.113 0.2 Embodiment 14 Water bath temperature 50℃+1 hour 3.151 0.115 1.0 Embodiment 15 Water bath temperature 50℃+24 hours 3.147 0.114 0.3 Example 16 Water bath temperature 60℃+1 hour 3.157 0.115 0.7 Embodiment 17 Water bath temperature 60℃+24 hours 3.152 0.116 1.1 Embodiment 18 Methanol volume 1ml 3.150 0.109 1.7 Embodiment 19 Methanol volume 6ml 3.150 0.108 2.0 When the sample preparation and water bath conditions are changed, the RD value of the test results is no more than 2% of the test results of Example 1. The present invention has no significant effect on the test results within the above conditions.

[0051] Embodiment 20 The hydrolysis solvent of Example 1 was used to hydrolyze PLGA or PLA of different polyester compounds. The hydrolysis parameters are shown in Tables 6 and 7.

[0052] Preparation of the sample solution to be tested: Take about 250 mg of PLGA of different types and molecular weights, weigh accurately, place in a 10 ml volumetric flask, add 1 ml of triethylamine and 2 ml of methanol, seal, and observe the hydrolysis of PLGA at different times in a 50°C water bath. After PLGA is hydrolyzed into a clear solution, take it out and cool it to room temperature, add 1 ml of glacial acetic acid, make up to volume with water, mix well, and observe the phenomenon.

[0053] Table 6. Hydrolysis results of PLGA of different types and molecular weights in Example 20 Table 7 Hydrolysis results of PLA with different molecular weights in Example 20 Results: PLGA or PLA of different types and molecular weights can be rapidly hydrolyzed to a clear solution within about 2 hours, and more ideally within 1 hour, or even in about 0.5 hours. After dilution and constant volume, the solution can still be stably maintained as a clear solution, which can effectively avoid interference with subsequent detection.

[0054] Comparative Example The comparative example provides a method for determining the glucose content in star-shaped PLGA. The detection method is consistent with the detection method in Example 1, except that only triethylamine or methanol is added during the preparation of the sample solution to be tested, or ethanol, acetonitrile, isopropanol, acetone, and tetrahydrofuran are used instead of methanol. The rest is the same as in Example 1. The specific configuration process is as follows, and the specific results are shown in Table 8 and Figures 3 to 5 shown.

[0055] Comparative Example 1 Preparation of the sample solution to be tested: Take about 250 mg of star-shaped PLGA, weigh accurately, place in a 10 ml volumetric flask, add 1 ml of triethylamine and 2 ml of acetonitrile, seal, and place in a 50 ° C water bath for about 2 hours until the PLGA is hydrolyzed to a clear solution, take out and cool to room temperature, add 1 ml of glacial acetic acid and dilute with water to obtain solid precipitate. For specific phenomena, see Figure 3 , which obviously affects the test results, so the mixed solvent of triethylamine and acetonitrile is not applicable.

[0056] Comparative Example 2 Preparation of the sample solution to be tested: Take about 250 mg of star-shaped PLGA, weigh accurately, place in a 10 ml volumetric flask, add 1 ml of triethylamine and 2 ml of ethanol, seal, and place in a 50°C water bath for about 10 hours until the PLGA is hydrolyzed to clarity, take out and cool to room temperature, add 1 ml of glacial acetic acid, dilute with water, and mix well. The specific test results are shown in Table 8.

[0057] Comparative Example 3 Preparation of the sample solution to be tested: Take about 250 mg of star-shaped PLGA, weigh it accurately, put it in a 10 ml volumetric flask, add 1 ml of triethylamine and 2 ml of isopropanol (or acetone, tetrahydrofuran), seal it, and place it in a 50°C water bath for more than 24 hours. PLGA is still not completely hydrolyzed. The specific phenomenon is shown in Figure 4 , which obviously affects the test results, so the mixed solvent of triethylamine and isopropanol (or acetone, tetrahydrofuran) is not applicable.

[0058] Comparative Example 4 Preparation of the sample solution to be tested: Take about 250 mg of star-shaped PLGA, weigh accurately, place in a 10 ml volumetric flask, add 3 ml of triethylamine (or methanol), seal, and place in a 50°C water bath for more than 24 hours. PLGA is still not completely hydrolyzed. For specific phenomena, see Figure 5 , which obviously affects the test results, so the single solvent of triethylamine (or methanol) is not applicable.

[0059] Table 8. Glucose content test results in Comparative Example 2 Comparative Example Sample preparation changes Glucose retention time / min Glucose content% Comparative Example 2 2ml ethanol 3.155 0.064 Results: When methanol in the hydrolysis solvent was replaced with acetonitrile, after 2 hours in a 50°C water bath, the color of the solution changed to a brown clear solution. When glacial acetic acid and water were added to the volume, a light yellow solid substance precipitated. In the embodiment, the solution after hydrolysis was a colorless clear solution, and the solution did not change after the volume was fixed. This shows that when triethylamine and acetonitrile and triethylamine and methanol are used as hydrolysis solvents, different by-products are produced in the hydrolysis process of the polymer, and part of the former product is insoluble in water, resulting in significantly different phenomena.

[0060] The nucleophilicity of reagents such as ethanol, isopropanol, acetone, tetrahydrofuran, and triethylamine is weaker than that of methanol. Although PLGA can be dissolved in acetone and tetrahydrofuran solvents, its hydrolysis reaction is relatively slow compared to methanol and triethylamine as hydrolysis solvents. When methanol is used alone, there is no base catalytic effect of triethylamine, and the hydrolysis reaction slows down accordingly. When the sample to be tested is hydrolyzed with a mixed solvent of ethanol and triethylamine in Comparative Example 2, it takes 10 hours to be placed in a 50°C water bath to hydrolyze the solution to a clear state, and the detection result is significantly lower than that in Example 1, and the detection result is inaccurate. In addition, when methanol in the hydrolysis solvent is replaced with isopropanol, acetone, tetrahydrofuran and other solvents or only triethylamine and methanol are added, the sample to be tested cannot be completely hydrolyzed within 24 hours under 50°C water bath conditions. The above treatment method is not conducive to detection because the solution is not completely clear in the end, and it is impossible to judge whether the bonded glucose in PLGA is completely extracted, which significantly affects the detection accuracy.

[0061] Verification Example 1—Specificity: Preparation of unhydrolyzed sample solution: Take about 250 mg of the star-shaped PLGA of Example 1, weigh accurately, place in a 10 ml volumetric flask, add 3 ml of purified water, seal, leave at room temperature for about 2 hours, dilute to scale with purified water, mix, centrifuge, and take the supernatant. Detection was performed using the same detection method as in Example 1. No peak appeared at the glucose retention time. The detection spectrum is shown in FIG. Figure 2 .

[0062] The unhydrolyzed sample solution was tested, and no peak appeared at the glucose retention time, indicating that free glucose was not detected. This result shows that there is no residual glucose added during the synthesis process in the star-shaped PLGA, which will not interfere with the quantitative experiment. The method has good specificity and ensures the accuracy of the quantitative results. The test results before and after the hydrolysis of the sample solution showed that the glucose detected by this method was all glucose obtained after the hydrolysis of PLGA.

[0063] Verification Example 2—Accuracy Test Take about 250 mg of the star-shaped PLGA of Example 1, weigh it accurately, put it in a 10 ml volumetric flask, accurately add glucose solution (2 μg / ml), add 1 ml of triethylamine, and 2 ml of methanol, seal it, and place it in a 50°C water bath for 2 hours until the PLGA is hydrolyzed to clarity, take it out and cool it to room temperature, add 1 ml of glacial acetic acid, and then make up to volume with water and mix well. (3 replicates, quantitative limit spiked sample solution A1-3) Take about 250 mg of the star-shaped PLGA of Example 1, weigh it accurately, put it in a 10 ml volumetric flask, accurately add glucose solution (5 μg / ml), add 1 ml of triethylamine, and 2 ml of methanol, seal it, and place it in a 50°C water bath for 2 hours until the PLGA is hydrolyzed to clarity, take it out and cool it to room temperature, add 1 ml of glacial acetic acid, and then make up to volume with water and mix well. (3 replicates, 50% spiked sample solutions B1-3) Take about 250 mg of the star-shaped PLGA of Example 1, weigh it accurately, put it in a 10 ml volumetric flask, accurately add glucose solution (10 μg / ml), triethylamine 1 ml, and methanol 2 ml, seal it, and place it in a 50°C water bath for 2 hours until the PLGA is hydrolyzed to clarity, take it out and cool it to room temperature, add 1 ml of glacial acetic acid, and then make up to volume with water and mix well. (3 replicates, 100% spiked sample solutions C1-3) Take about 250 mg of the star-shaped PLGA of Example 1, weigh it accurately, put it in a 10 ml volumetric flask, accurately add glucose solution (15 μg / ml), add 1 ml of triethylamine, and 2 ml of methanol, seal it, put it in a 50°C water bath for 2 hours until the PLGA is hydrolyzed to clarity, take it out and cool it to room temperature, add 1 ml of glacial acetic acid, and then make up to volume with water and mix it. (3 parallel parts, 150% spiked sample solutions D1-3) Inject 50 μl of the above solution for HPLC-ELSD analysis, and the results are shown in Table 9.

[0064] Table 9 Accuracy of glucose content in the tested samples According to Table 9, the accuracy was calculated based on the measured content of glucose added to the sample to be tested. The results showed that the recovery rate of the glucose content in the sample to be tested was 97.8% to 104.7%, which meets the accuracy limit of 92% to 105% specified in the General Rules 9101 of Part IV of the 2020 edition of the "Chinese Pharmacopoeia". The accuracy of this method is good.

[0065] Verification Example 3—Repeatability Test The star-shaped PLGA of Example 1 was prepared in parallel in 6 portions according to the preparation method of the test sample solution in Example 1, and 50 μl of each portion was injected for HPLC-ELSD analysis. The results are shown in Table 10.

[0066] Table 10 Repeatability results of glucose content in the sample solution to be tested According to Table 10, the relative standard deviation of the content was calculated based on the measured glucose content in the sample to be tested. The result showed that the relative standard deviation of the glucose content was 1.8%, which was in line with the requirement of not exceeding 2% stipulated in the General Rules 9101 of Part IV of the 2020 edition of the Chinese Pharmacopoeia. This method has good repeatability.

[0067] The above-described embodiment is only a preferred solution of the present invention and does not limit the present invention in any form. There are other variations and modifications without exceeding the technical solution described in the claims.

Claims

1. A method for determining the glucose content in star-shaped PLGA, characterized in that: The following steps are involved: (1) Pretreatment The star-shaped PLGA prepared with glucose as an initiator is used as a test sample. The test sample is placed in a hydrolysis solvent, hydrolyzed into a clear solution, and then a volatile organic acid is added to neutralize it, and then the solution is fixed to volume with water to obtain a test solution with a concentration of 25±2.5mg / ml; a hydrolysis solvent is added to a glucose reference substance, and the volatile organic acid is neutralized, and finally the solution is fixed to volume with water to obtain a glucose standard curve solution; (2) HPLC-ELSD detection The sample solution to be tested and the glucose standard curve solution are subjected to high performance liquid chromatography to obtain chromatograms of the sample solution to be tested and the glucose standard curve, respectively; (3) Content calculation Establishing a linear regression equation based on the logarithmic value of the concentration of the glucose standard curve solution and the logarithmic value of the glucose peak area in the chromatogram of the glucose standard curve; The glucose content in the sample to be tested is calculated according to the linear regression equation and the logarithmic value of the glucose peak area in the chromatogram of the sample to be tested.

2. The method according to claim 1, characterized in that In step (1), the hydrolysis solvent is a mixed solvent of methanol and triethylamine in a volume ratio of 1 to 6:1, the hydrolysis temperature is 30 to 60° C., and the hydrolysis time is within 24 hours.

3. The method according to claim 2, characterized in that In step (1), the volatile organic acid is formic acid, glacial acetic acid or a mixture of both; the volume ratio of methanol: triethylamine: volatile organic acid: water is 1-6:1:1:7-2.

4. The method according to claim 1, characterized in that: In step (2), the HPLC detection parameters are set as follows: gradient elution with water and methanol as mobile phases; The flow rate of the mobile phase is 0.8-1.2 ml / min; The injection volume is 20μl~100μl; The column temperature is 35-45°C, and the column is a C18 column, including a Waters Atlantis T3 column; The carrier gas of the evaporative light scattering detector was nitrogen at a pressure of 40 psi; the drift tube temperature was 50-90°C, and the nebulizer mode was cooling.

5. The method according to claim 4, characterized in that For gradient elution with water and methanol as mobile phase, choose one of the following: Solution 1: 0 min, water 100%, methanol 0%; 4.5 min, water 100%, methanol 0%; 5 min, water 15%, methanol 85%; 15 min, water 15%, methanol 85%; Option 2: 0 min, water 95%, methanol 5%; 4.5 min, water 95%, methanol 5%; 5 min, water 15%, methanol 85%; 15 min, water 15%, methanol 85%.

6. The method according to claim 1, characterized in that The concentration range of the glucose standard curve solution is 1 μg / ml to 40 μg / ml, and the concentration gradient is at least 5.

7. The method according to claim 6, characterized in that The concentration range of the glucose standard curve solution is 2 μg / ml to 20 μg / ml.

8. A pretreatment method for polyester compounds, characterized in that: The polyester compound is placed in a hydrolysis solvent at a hydrolysis temperature of 30 to 60° C. for a hydrolysis time of less than 24 hours; the hydrolysis solvent is a mixed solvent of methanol and triethylamine in a volume ratio of 1 to 6:

1.

9. The pre-treatment method according to claim 8, characterized in that: The polyester compound is PLGA or PLA.

10. The pre-treatment method according to claim 9, characterized in that: The PLGA is a star-shaped PLGA prepared by using glucose as an initiator.

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