Method for determining content of polylactic acid and / or polylactic acid-glycolic acid copolymer

Abstract

The invention provides a method for determining the content of polylactic acid and / or a polylactic acid-glycolic acid copolymer. The method comprises the following steps: step 1, preparing a reference solution of the polylactic acid or the polylactic acid-glycolic acid copolymer and a test solution; step 2, detecting the self reference substance solution and the test solution through gel permeation chromatography, wherein the detection chromatographic conditions of the gel permeation chromatography comprise at least one of the following items: (1) the flow rate is 0.3 mL / min-1. 0 mL / min; (2) a detector is a differential refraction detector; (3) the column temperature is 33-40 DEG C; (4) a chromatographic column is a fat-soluble volume exclusion chromatographic column; (5) the mobile phase is tetrahydrofuran or dichloromethane; and step 3, calculating through an external standard method formula to obtain the content of the test solution. The content determination method can accurately determine the content of the polylactic acid and / or the polylactic acid-glycolic acid copolymer, and is simple to operate, convenient to calculate, accurate and rapid.

Description

Technical Field

[0001] This application relates to the field of detection technology, and particularly to a method for determining the content of polylactic acid and / or poly(lactic-co-glycolic acid). Background Art

[0002] Polylactic acid (PLA) is a synthetic biopolymer with good biocompatibility, biodegradability, and renewability, and is non-toxic. Polylactic acids with different molecular weight distributions can be applied to the drug delivery system of pharmaceutical preparations. Poly(lactic-co-glycolic acid) (PLGA) is randomly polymerized from two monomers, lactic acid and glycolic acid, and is a degradable functional polymer organic compound with good biocompatibility, non-toxicity, and good properties of forming capsules and films. It is widely used in the fields of pharmaceuticals, medical engineering materials, and modern industry. During the process of researching the pharmaceutical preparation technology containing polylactic acid and / or poly(lactic-co-glycolic acid), accurately determining the content of polylactic acid and / or poly(lactic-co-glycolic acid), or detecting the residual amount of polylactic acid and / or poly(lactic-co-glycolic acid) after the production line is cleaned during the production process of pharmaceutical preparations containing polylactic acid and / or poly(lactic-co-glycolic acid) all require a content determination and analysis method for polylactic acid and / or poly(lactic-co-glycolic acid) that can meet the requirements. Currently, the main methods for determining and analyzing the content of polylactic acid and / or poly(lactic-co-glycolic acid) are ion chromatography and nuclear magnetic resonance. However, the sample treatment operation of ion chromatography is complex, and the calculation process is cumbersome; the nuclear magnetic resonance method has a high cost and complex data processing. Summary of the Invention

[0003] Based on this, this application provides a method for determining the content of polylactic acid and / or poly(lactic-co-glycolic acid) with simple operation, convenient calculation, accurate and fast speed, and low cost.

[0004] This application provides a method for determining the content of polylactic acid and / or poly(lactic-co-glycolic acid), including the following steps:

[0005] Step 1: Prepare a self-control solution of polylactic acid or poly(lactic-co-glycolic acid), and a test solution;

[0006] Step 2: Detect the self-control solution and the test solution by gel permeation chromatography. The detection chromatographic conditions of the gel permeation chromatography include at least one of the following items:

[0007] (1) The flow rate is 0.3 mL / min - 1.0 mL / min;

[0008] (2) The detector is a differential refractive index detector;

[0009] (3) The column temperature is 33°C - 40°C;

[0010] (4) The chromatographic column is a lipophilic size exclusion chromatographic column;

[0011] (5) The mobile phase is tetrahydrofuran or dichloromethane;

[0012] Step 3: Calculate the content of the test solution through the external standard method formula.

[0013] In some embodiments, the chromatographic column is one of MonoGPC-100, MonoGPC-300, and MonoGPC-500.

[0014] In some embodiments, the preparation method of the self-control sample solution of polylactic acid or poly(lactic acid-glycolic acid) copolymer includes:

[0015] Mix the self-control sample of polylactic acid or poly(lactic acid-glycolic acid) copolymer with a solvent, and the solvent is selected from one of tetrahydrofuran and dichloromethane.

[0016] In some embodiments, the concentration of polylactic acid or poly(lactic acid-glycolic acid) copolymer in the test solution is 1.2 mg / ml - 1.9 mg / ml.

[0017] In some embodiments, the preparation method of the test solution includes:

[0018] Mix the test sample with a solvent to prepare the test solution; the solvent is selected from one of tetrahydrofuran and dichloromethane.

[0019] In some embodiments, the weight average molecular weight of the poly(lactic acid-glycolic acid) copolymer is 10,000 - 50,000.

[0020] In some embodiments, the weight average molecular weight of the polylactic acid is 50,000 - 190,000.

[0021] For the above-provided content determination method of polylactic acid and / or poly(lactic acid-glycolic acid) copolymer, the sample does not require pretreatment, and the determination is carried out by gel permeation chromatography, with low cost; and the self-control external standard method is used to calculate the content of polylactic acid and / or poly(lactic acid-glycolic acid) copolymer, which has good specificity, a simple calculation process, high accuracy, good repeatability, and high sensitivity. Description of the Drawings

[0022] To more clearly illustrate the technical solutions in the embodiments of the present application and more comprehensively understand the present application and its beneficial effects, the following will briefly introduce the attached drawings required for the description of the embodiments. Obviously, the attached drawings in the following description are only some embodiments of the present application, and those skilled in the art can obtain other attached drawings based on these drawings without creative labor.

[0023] Figure 1 It is the chromatogram of the blank solvent in Example 1;

[0024] Figure 2 It is the chromatogram of the control solution in Example 1;

[0025] Figure 3 It is the linear standard curve in Example 1;

[0026] Figure 4 It is the chromatogram of the blank solvent in Example 2;

[0027] Figure 5 It is the chromatogram of the control solution in Example 2;

[0028] Figure 6 It is the linear standard curve in Example 2;

[0029] Figure 7 It is the chromatogram of the blank solvent in Example 3;

[0030] Figure 8 It is the chromatogram of the control solution in Example 3;

[0031] Figure 9 It is the linear standard curve in Example 3;

[0032] Figure 10 It is the chromatogram of the blank solvent in Example 4;

[0033] Figure 11 It is the chromatogram of the control solution in Example 4;

[0034] Figure 12 It is the linear standard curve in Example 4;

[0035] Figure 13 It is the chromatogram of the blank solvent in Example 5;

[0036] Figure 14 It is the chromatogram of the control solution in Example 5;

[0037] Figure 15 It is the linear standard curve in Example 5. Detailed implementation manners

[0038] For ease of understanding the present application, the present application will be described more comprehensively below with reference to related embodiments. The following gives preferred embodiments of the present application. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosed content of the present application more thorough and comprehensive.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs. The terms used in the description of this application herein are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0040] The terms "and / or", "or / and", "and / or" used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. The said any and all combinations include any two of the related listed items, any more of the related listed items, or the combination of all the related listed items. It should be noted that when connecting at least three items with at least two conjunctions selected from "and / or", "or / and", "and / or", it should be understood that in this application, this technical solution undoubtedly includes the technical solution connected by "logical AND", and also undoubtedly includes the technical solution connected by "logical OR".

[0041] In the present application, among the technical features described in an open-ended manner, there are included closed technical solutions composed of the listed features, as well as open technical solutions including the listed features.

[0042] In the present application, regarding numerical ranges, unless otherwise specified, the above numerical ranges are considered continuous and include the minimum and maximum values of the range, as well as each value between such minimum and maximum values. Further, when the range refers to integers, it includes each integer between the minimum and maximum values of the range. In addition, when providing multiple ranges to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0043] Only some numerical ranges are specifically disclosed herein. However, any lower limit can be combined with any upper limit to form a range not explicitly recited; and any lower limit can be combined with other lower limits to form a range not explicitly recited, and similarly any upper limit can be combined with any other upper limit to form a range not explicitly recited. In addition, each individually disclosed point or single numerical value itself can be used as a lower limit or an upper limit and combined with any other point or single numerical value or combined with other lower limits or upper limits to form a range not explicitly recited.

[0044] The temperature parameters in this application, unless otherwise specified, allow for either constant temperature treatment or treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, ±1°C are allowed.

[0045] In this text, the "suitable combination method", "suitable method", "any suitable method", etc. The "suitable" mentioned is subject to being able to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0046] In this application, "further", "even further", "especially", etc. are used for descriptive purposes, indicating differences in content, but should not be construed as limiting the protection scope of this application.

[0047] In this application, "optionally", "optional", "option", mean having or not having, that is, either one of the two alternative schemes of "having" or "not having". If "optional" appears multiple times in a technical solution, unless otherwise specified and there are no contradictions or mutual restrictions, each "optional" is independent.

[0048] In the description of the application, the meaning of "multiple" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0049] If there is no special instruction, all embodiments and optional embodiments of this application can be combined with each other to form new technical solutions. If there is no special instruction, all technical features and optional technical features of this application can be combined with each other to form new technical solutions.

[0050] If there is no special instruction, all steps of this application can be carried out sequentially or randomly, and preferably sequentially.

[0051] In the related art, the main methods for the determination and analysis of the content of polylactic acid are ion chromatography and nuclear magnetic resonance. However, there are problems such as complex sample handling operations, cumbersome calculation processes, high costs, and complex data processing.

[0052] This application provides a method for determining the content of polylactic acid and / or poly(lactic acid - glycolic acid) copolymer, including the following steps:

[0053] Step 1: Prepare a self - reference solution of polylactic acid or poly(lactic acid - glycolic acid) copolymer, and a test solution;

[0054] Step 2: Detect the self - reference solution and the test solution by gel permeation chromatography. The detection chromatographic conditions of the gel permeation chromatography include at least one of the following items:

[0055] (1) The flow rate is 0.3 mL / min - 1.0 mL / min;

[0056] (2) The detector is a refractive index detector;

[0057] (3) The column temperature is 33°C - 40°C;

[0058] (4) The chromatographic column is a lipophilic size exclusion chromatography column;

[0059] (5) The mobile phase is tetrahydrofuran or dichloromethane;

[0060] Step 3: Calculate the content of the test solution through the external standard method formula.

[0061] Understandably, for the content determination method provided in this application, the sample does not need to be treated, and gel permeation chromatography is used for determination, with low cost; and the external standard method with self-control is used to calculate the content of polylactic acid and / or poly(lactic acid - glycolic acid) copolymer, which has good specificity, a simple calculation process, high accuracy, good repeatability, and high sensitivity.

[0062] The content detection method provided in this application can accurately detect the content of polylactic acid and / or poly(lactic acid - glycolic acid) copolymer in pharmaceutical preparations, providing a basis for the safety and effectiveness of pharmaceutical preparations. And this content detection method can accurately detect the residual amount of polylactic acid in the cleaning and verification of the production line of pharmaceutical preparations containing polylactic acid, providing a basis for effective cleaning methods and cleaning effects.

[0063] As an example, the flow rate during gel permeation chromatography can be, but is not limited to, 0.3 mL / min, 0.4 mL / min, 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min, 1 mL / min, or the range between any two of the above flow rates, etc.

[0064] In some alternative embodiments, the flow rate during gel permeation chromatography is 0.3 mL / min - 0.7 mL / min. Optionally, the flow rate during gel permeation chromatography is 0.5 mL / min.

[0065] As an example, the column temperature during gel permeation chromatography can be, but is not limited to, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, or the range between any two of the above temperatures, etc.

[0066] In some alternative embodiments, the column temperature during gel permeation chromatography is 35°C - 40°C. Optionally, the column temperature during gel permeation chromatography is 35°C.

[0067] In some embodiments, the chromatographic column used in gel permeation chromatography is one of MonoGPC-100, MonoGPC-300, and MonoGPC-500.

[0068] In some embodiments, the method for preparing the self-control reference substance solution of polylactic acid or poly(lactic-co-glycolic) acid copolymer comprises:

[0069] Mixing the self-control reference substance of polylactic acid or poly(lactic-co-glycolic) acid copolymer with a solvent selected from one of tetrahydrofuran and dichloromethane.

[0070] In some embodiments, the concentration of polylactic acid or poly(lactic-co-glycolic) acid copolymer in the self-control reference substance solution is 1.2 mg / ml - 1.9 mg / ml. For example, it can be, but is not limited to, 1.2 mg / ml, 1.3 mg / ml, 1.4 mg / ml, 1.5 mg / ml, 1.6 mg / ml, 1.7 mg / ml, 1.8 mg / ml, 1.9 mg / ml, or the range between any two of the above values.

[0071] In some embodiments, the method for preparing the test sample solution comprises: mixing the test sample with a solvent to prepare the test sample solution; the solvent is selected from one of tetrahydrofuran and dichloromethane.

[0072] As an example, the method for preparing the reference substance solution comprises: taking about 30 mg of polylactic acid, weighing accurately, placing it in a 20-ml volumetric flask, dissolving and diluting to the mark with tetrahydrofuran to prepare a solution with a concentration of about 1.5 mg / ml, standing until the solution is clear, shaking well, and then obtaining the solution.

[0073] As an example, the method for preparing the test sample solution comprises: taking an appropriate amount of the sample, weighing accurately, dissolving and diluting to the mark with tetrahydrofuran to prepare a solution with a concentration of about 1.5 mg / ml, standing until the solution is clear, shaking well, and then obtaining the solution.

[0074] In some embodiments, the weight-average molecular weight of the poly(lactic-co-glycolic) acid copolymer is 10,000 - 50,000; for example, it can be, but is not limited to, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, or the range between any two of the above weight-average molecular weights.

[0075] In some embodiments, the weight-average molecular weight of polylactic acid is 50,000 - 190,000; for example, it can be, but is not limited to, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 190,000, or the range between any two of the above weight-average molecular weights.

[0076] The technical solution of the present application will be described in detail below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present application and not to limit the scope of the present application. For the experimental methods without specific conditions in the following embodiments, the guidance given in the present application is preferably referred to, and it can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions recommended by the manufacturer, or by referring to the experimental methods known in the art.

[0077] In the following specific embodiments, for the measurement parameters of raw material components, if there is no special explanation, there may be slight deviations within the weighing accuracy range. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0078] When determining the content of the analyte in the test sample in Examples 1-5, the peak area corresponding to the test sample solution was determined by gel chromatography, and the content of the analyte in the test sample was calculated by the external standard method based on the formula in Table 1. The analytes, analyte molecular weights, extraction solvents used when preparing the test sample solution, chromatographic columns, column temperatures, and flow rates in Examples 1-5 are shown in Table 2 respectively.

[0079] Table 1

[0080]

[0081] Table 2

[0082]

[0083] Example 1: Chromatographic condition 1 (PLGA (Mw 10,000) + THF + MonoGPC-500 + 0.3 ml / min + 33 °C)

[0084] 1. Solution preparation

[0085] Blank solvent: Tetrahydrofuran

[0086] Self-control solution: Take about 30 mg of PLGA (weight-average molecular weight of 10,000), weigh accurately, place it in a 20 ml volumetric flask respectively, add an appropriate amount of tetrahydrofuran and let it stand for at least 4 hours, dissolve and dilute to the scale to prepare a solution with a concentration of about 1.5 mg / ml, shake well, and obtain it (prepare 2 portions in parallel).

[0087] Repeatability solution: Take about 30 mg of PLGA (weight-average molecular weight of 10,000), weigh accurately, place it in a 20 ml volumetric flask, add tetrahydrofuran and let it stand for at least 4 hours, dissolve and dilute to the scale to prepare a solution with a concentration of about 1.5 mg / ml, shake well, and obtain it (prepare 6 portions in parallel).

[0088] Linear solution: Take 24.42 mg (24 mg), 27.22 mg (27 mg), 30.53 mg (30 mg), 33.16 mg (33 mg), and 36.36 mg (36 mg) of PLGA (weight-average molecular weight of 10,000), accurately weigh, place them in 20-ml volumetric flasks respectively, add an appropriate amount of tetrahydrofuran, let stand for at least 4 hours, dissolve and dilute to the mark. Shake well to obtain the solution.

[0089] Test solution: Take 44 mg of PLGA (weight-average molecular weight of 10,000) microspheres (drug loading amount is about 31%), accurately weigh, place it in a 20-ml volumetric flask, add tetrahydrofuran, let stand for at least 4 hours, dissolve and dilute to the mark to prepare a solution with a PLGA concentration of about 1.5 mg / ml, shake well to obtain (prepare 2 portions in parallel).

[0090] 2. Chromatographic conditions: The mobile phase is tetrahydrofuran, flow rate: 0.3 ml / min; detector: differential refractive index detector; column temperature: 33 °C; chromatographic column: MonoGPC-500 (7.8 mm × 300 mm) 5 μm.

[0091] 3. Results: The blank solvent has no interference. The RSD (n = 6) of the continuous 6 injections of the system suitability solution is 0.44%, and the recovery rate of the reference solution is 100.63%. The RSD (n = 6) of the repeatability test is 0.63%, the linear correlation coefficient r is 0.9996, the ratio of the Y-axis intercept to the corresponding value of 100% is 5.55%, and the ratio of the residual standard deviation to the 100% response value is 0.53%. See Figures 1-3 , Table 3-5. The results show that the system suitability, repeatability, and linearity of this method for measuring PLGA with a weight-average molecular weight of 10,000 are all good.

[0092] 4. Determination of PLGA (weight-average molecular weight of 10,000) in the test solution:

[0093] Accurately measure 20 μL of the test solution to be measured and inject it into the above chromatographic condition system. The peak areas of the test samples obtained are: 286980 and 256322. Through the calculation formula of the external standard method in Table 1, Qx (content percentage of the test sample): The contents of the test samples are 68.34% and 64.34%, that is: the average content of PLGA in this test sample is 66.34%, see Table 6.

[0094] Table 3: System suitability results (chromatographic condition 1)

[0095]

[0096] Table 4: Repeatability test results (chromatographic condition 1)

[0097]

[0098] Table 5: Linear Results (Chromatographic Conditions 1)

[0099]

[0100] Table 6: Test Sample Detection Results (Chromatographic Conditions 1)

[0101]

[0102] Example 2: Chromatographic Conditions 2 (PLGA (Mw 50,000) + THF + MonoGPC-500 + 1.0 ml / min + 40 °C)

[0103] 1. Solution Preparation

[0104] Blank Solvent: Tetrahydrofuran

[0105] Self-Control Solution: Take about 30 mg of PLGA (weight-average molecular weight of 50,000), accurately weigh it, place it in a 20 ml volumetric flask respectively, add an appropriate amount of tetrahydrofuran, let it stand for at least 4 hours, dissolve and dilute to the mark to prepare a solution with a concentration of about 1.5 mg / ml, shake well, and you will get (prepare 2 portions in parallel).

[0106] Repeatability Solution: Take about 30 mg of PLGA (weight-average molecular weight of 50,000), accurately weigh it, place it in a 20 ml volumetric flask, add tetrahydrofuran and let it stand for at least 4 hours, dissolve and dilute to the mark to prepare a solution with a concentration of about 1.5 mg / ml, shake well, and you will get (prepare 6 portions in parallel).

[0107] Linear Solution: Take 24.61 mg (24 mg), 27.78 mg (27 mg), 30.28 mg (30 mg), 33.64 mg (33 mg), 36.78 mg (36 mg) of PLGA (weight-average molecular weight of 50,000), accurately weigh them, place them in 20 ml volumetric flasks respectively, add an appropriate amount of tetrahydrofuran, let it stand for at least 4 hours, dissolve and dilute to the mark. Shake well, and you will get.

[0108] Test Sample Solution: Take 34 mg of PLGA (weight-average molecular weight of 50,000) microspheres (drug loading amount about 12%), accurately weigh it, place it in a 20 ml volumetric flask, add tetrahydrofuran and let it stand for at least 4 hours, dissolve and dilute to the mark to prepare a solution containing PLGA with a concentration of about 1.5 mg / ml, shake well, and you will get (prepare 2 portions in parallel).

[0109] 2. Chromatographic Conditions: The mobile phase is tetrahydrofuran, flow rate: 1.0 ml / min; detector: refractive index detector; column temperature: 40 °C; chromatographic column: MonoGPC-500 (7.8 mm × 300 mm) 5 μm.

[0110] 3. Results: The blank solvent showed no interference. The RSD (n = 6) of the system suitability solution for 6 consecutive injections was 0.79%, and the recovery rate of the reference solution was 100.06%. The RSD (n = 6) of the repeatability test was 0.80%, the linear correlation coefficient r was 0.9980, the ratio of the Y-axis intercept to the corresponding value of 100% was 2.08%, and the ratio of the residual standard deviation to the 100% response value was 1.10%. See Figures 4-6 , Table 7 - 9. The results indicate that the system suitability, repeatability, and linearity of this method for measuring PLGA with a weight-average molecular weight of 50,000 are all good.

[0111] 4. Determination of PLGA (weight-average molecular weight of 50,000) in the test solution:

[0112] Precisely measure 20 μL of the test solution to be measured and inject it into the above chromatographic conditions system. The peak areas of the test samples obtained are: 80520 and 87625. Through the calculation formula of the external standard method in Table 1, Qx (content of the test sample %) is obtained: The contents of the test samples are 85.36% and 87.79%, that is: The average content of PLGA in this test sample is 86.57%, as shown in Table 10.

[0113] Table 7: System Suitability Results (Chromatographic Conditions 2)

[0114]

[0115] Table 8: Repeatability Test Results (Chromatographic Conditions 2)

[0116]

[0117] Table 9: Linearity Results (Chromatographic Conditions 2)

[0118]

[0119] Table 10: Test Results of the Test Sample (Chromatographic Conditions 2)

[0120]

[0121] Example 3: Chromatographic Conditions 3 (PLA (Mw 190,000) + THF + MonoGPC - 100 + 1.0 ml / min + 33 °C)

[0122] 1. Solution Preparation

[0123] Blank Solvent: Tetrahydrofuran

[0124] Self-Control Solution: Take about 30 mg of PLA (weight-average molecular weight of 190,000), accurately weigh it, place it in a 20 ml volumetric flask respectively, add an appropriate amount of tetrahydrofuran, let it stand for at least 4 hours, dissolve and dilute to the mark to prepare a solution with a concentration of about 1.5 mg / ml, shake well, and that's it (prepare 2 portions in parallel).

[0125] Repeated solution: Take about 30 mg of PLA (weight-average molecular weight is 190,000), accurately weigh, place in a 20-ml volumetric flask, add tetrahydrofuran, let stand for at least 4 hours, dissolve and dilute to the mark to prepare a solution with a concentration of about 1.5 mg / ml, shake well, and you will get it. (Prepare 6 parallel portions).

[0126] Linear solution: Take 24.71 mg (24 mg), 27.80 mg (27 mg), 30.44 mg (30 mg), 33.62 mg (33 mg), 36.30 mg (36 mg) of PLA (weight-average molecular weight is 190,000), accurately weigh, respectively place in 20-ml volumetric flasks, add appropriate amount of tetrahydrofuran, let stand for at least 4 hours, dissolve and dilute to the mark. Shake well, and you will get it.

[0127] Test solution: Take 38 mg of PLA (weight-average molecular weight is 190,000) microspheres (drug loading is about 21%), accurately weigh, place in a 20-ml volumetric flask, add tetrahydrofuran, let stand for at least 4 hours, dissolve and dilute to the mark to prepare a solution with a concentration of about 1.5 mg / ml, shake well, and you will get it (prepare 2 parallel portions).

[0128] 2. Chromatographic conditions: The mobile phase is tetrahydrofuran, flow rate: 1.0 ml / min; detector: differential refractive index detector; column temperature: 33 °C; chromatographic column: MonoGPC-100(7.8 mm×150 mm) 5 μm.

[0129] 3. Results: The blank solvent has no interference. The RSD (n = 6) of the continuous 6 injections of the system suitability solution is 0.32%, and the recovery rate of the reference solution is 100.46%. The RSD (n = 6) of the repeatability test is 0.33%, the linear correlation coefficient r is 0.9994, the ratio of the Y-axis intercept to the corresponding value of 100% is 1.93%, and the ratio of the residual standard deviation to the 100% response value is 0.60%. See Figures 7-9 , Table 11 - 13. The results show that the system suitability, repeatability and linearity of this method for measuring PLA with a weight-average molecular weight of 190,000 are all good.

[0130] 4. Determination of PLA (weight-average molecular weight is 190,000) in the test solution:

[0131] Accurately measure 20 μL of the test solution to be measured and inject it into the above chromatographic condition system. The peak areas of the test samples obtained are: 72523 and 79236. Through the calculation formula of the external standard method in Table 1, Qx (content of the test sample %) is obtained: the contents of the test samples are 74.53% and 77.93%, that is: the average content of PLA in this test sample is 76.23%, see Table 14.

[0132] Table 11: System suitability results (chromatographic condition 3)

[0133]

[0134] Table 12: Results of Repeatability Test (Chromatographic Conditions 3)

[0135]

[0136] Table 13: Linear Results (Chromatographic Conditions 3)

[0137]

[0138] Table 14: Test Results of Test Samples (Chromatographic Conditions 3)

[0139]

[0140] Example 4: Chromatographic Conditions 4 (PLA (Mw 110,000) + Dichloromethane + MonoGPC-300 + 0.3 ml / min + 35°C)

[0141] 1. Solution Preparation

[0142] Blank Solvent: Dichloromethane

[0143] Self-Control Solution: Take about 30 mg of PLA (weight-average molecular weight of 110,000), accurately weigh, place in a 20 ml volumetric flask respectively, add an appropriate amount of dichloromethane, let stand for at least 4 hours, dissolve and dilute to the scale to prepare a solution with a concentration of about 1.5 mg / ml, shake well, and obtain (prepare 2 portions in parallel).

[0144] Repeatability Solution: Take about 30 mg of PLA (weight-average molecular weight of 110,000), accurately weigh, place in a 20 ml volumetric flask, add dichloromethane, let stand for at least 4 hours, dissolve and dilute to the scale to prepare a solution with a concentration of about 1.5 mg / ml, shake well, and obtain. (Prepare 6 portions in parallel)

[0145] Linear Solution: Take 24.41 mg (24 mg), 27.52 mg (27 mg), 30.83 mg (30 mg), 33.42 mg (33 mg), 36.49 mg (36 mg) of PLA (weight-average molecular weight of 110,000), accurately weigh, place in 20 ml volumetric flasks respectively, add an appropriate amount of dichloromethane, let stand for at least 4 hours, dissolve and dilute to the scale. Shake well, and obtain.

[0146] Test Sample Solution: Take 35 mg of PLA (weight-average molecular weight of 110,000) microspheres (drug loading about 15%), accurately weigh, place in a 20 ml volumetric flask, add tetrahydrofuran, let stand for at least 4 hours, dissolve and dilute to the scale to prepare a solution with a concentration of about 1.5 mg / ml, shake well, and obtain (prepare 2 portions in parallel).

[0147] 2. Chromatographic conditions: The mobile phase is dichloromethane, flow rate: 0.3 ml / min; detector: differential refractive index detector; column temperature: 35 °C; chromatographic column: MonoGPC-300 (7.8 mm × 300 mm) 5 μm.

[0148] 3. Results: The blank solvent had no interference. The RSD (n = 6) of the system suitability solution for 6 consecutive injections was 1.34%, and the recovery rate of the reference solution was 99.75%. The RSD (n = 6) of the repeatability test was 0.97%, the linear correlation coefficient r was 0.9964, the ratio of the Y-axis intercept to the 100% corresponding value was 3.53%, and the ratio of the residual standard deviation to the 100% response value was 1.54%. See Figures 10-12 , Tables 15 - 17. The results showed that the system suitability, repeatability, and linearity of this method for measuring PLA with a weight-average molecular weight of 110,000 were all good.

[0149] 4. Determination of PLA (weight-average molecular weight of 110,000) in the test solution:

[0150] Precisely measure 20 μL of the test solution to be measured and inject it into the above chromatographic condition system. The peak areas of the test samples obtained were: 135980 and 150112. Through the calculation formula of the external standard method in Table 1, Qx (content of the test sample %) was obtained: the contents of the test samples were 80.95% and 83.02%, that is: the average content of PLA in this test sample was 81.98%, as shown in Table 18.

[0151] Table 15: System suitability results (chromatographic condition 4)

[0152]

[0153] Table 16: Repeatability test results (chromatographic condition 4)

[0154]

[0155] Table 17: Linearity results (chromatographic condition 4)

[0156]

[0157] Table 18: Test results of the test sample (chromatographic condition 4)

[0158]

[0159] Example 5: Chromatographic condition 5 (PLA (Mw 50,000) + dichloromethane + MonoGPC-300 + 0.5 ml / min + 35 °C)

[0160] 1. Solution preparation

[0161] Blank solvent: dichloromethane

[0162] Self-control solution: Weigh accurately about 30 mg of PLA (weight-average molecular weight is 50,000), place it in a 20-ml volumetric flask respectively, add an appropriate amount of dichloromethane, let it stand for at least 4 hours, dissolve and dilute to the scale to prepare a solution with a concentration of about 1.5 mg / ml, shake well, and you will get it (prepare 2 portions in parallel).

[0163] Repeatability solution: Weigh accurately about 30 mg of PLA (weight-average molecular weight is 50,000), place it in a 20-ml volumetric flask, add dichloromethane and let it stand for at least 4 hours, dissolve and dilute to the scale to prepare a solution with a concentration of about 1.5 mg / ml, shake well, and you will get it. (Prepare 6 portions in parallel)

[0164] Linear solution: Take 24.08 mg (24 mg), 27.71 mg (27 mg), 30.75 mg (30 mg), 33.21 mg (33 mg), 36.67 mg (36 mg) of PLA (weight-average molecular weight is 50,000), weigh accurately, place them in 20-ml volumetric flasks respectively, add an appropriate amount of dichloromethane, let it stand for at least 4 hours, dissolve and dilute to the scale. Shake well, and you will get it.

[0165] Test solution: Weigh accurately 33 mg of PLA (weight-average molecular weight is 50,000) microspheres (drug loading is about 11%), place it in a 20-ml volumetric flask, add tetrahydrofuran and let it stand for at least 4 hours, dissolve and dilute to the scale to prepare a solution with a concentration of about 1.5 mg / ml, shake well, and you will get it. (Prepare 2 portions in parallel) 2. Chromatographic conditions: The mobile phase is dichloromethane, flow rate: 0.5 ml / min; detector: differential refractive index detector; column temperature: 35 °C; chromatographic column: MonoGPC-300(7.8 mm × 300 mm) 5 μm.

[0166] 3. Results: The blank solvent has no interference. The RSD (n = 6) of the continuous 6 injections of the system suitability solution is 1.37%, and the recovery rate of the reference solution is 101.57%. The RSD (n = 6) of the repeatability test is 0.81%, the linear correlation coefficient r is 0.9978, the ratio of the Y-axis intercept to the corresponding value of 100% is 3.30%, and the ratio of the residual standard deviation to the 100% response value is 1.24%. See Figures 13-15 , Table 19 - 21. The results show that the system suitability, repeatability and linearity of this method for measuring PLA with a weight-average molecular weight of 50,000 are all good.

[0167] 4. Determination of PLA (weight-average molecular weight is 50,000) in the test solution:

[0168] Precisely measure 20 μL of the test solution to be measured and inject it into the above chromatographic conditions system. The peak areas of the test samples obtained are: 100365 and 93236. Through the calculation formula of the external standard method in Table 1, Qx is obtained: The contents of the test samples are 87.49% and 85.02%, that is: The average content of PLA in this test sample is 86.26%, see Table 22.

[0169] Table 19: System Suitability Results (Chromatographic Conditions 5)

[0170]

[0171] Table 20: Repeatability Test Results (Chromatographic Conditions 5)

[0172]

[0173] Table 21: Linearity Results (Chromatographic Conditions 5)

[0174]

[0175] Table 22: Test Results of Test Samples (Chromatographic Conditions 5)

[0176]

[0177] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as falling within the scope described in this specification.

[0178] The above-described embodiments merely represent several implementation manners of the present application. The description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the appended claims.

Claims

1. A method for determining the content of polylactic acid and / or poly(lactic-co-glycolic acid), characterized in that, It includes the following steps: Step 1: Prepare a self - reference solution of polylactic acid or poly(lactic - co - glycolic acid) and a test solution; Step 2: Detect the self - reference solution and the test solution by gel permeation chromatography. The detection chromatographic conditions of the gel permeation chromatography include at least one of the following items: (1) The flow rate is 0.3 mL / min - 1.0 mL / min; (2) The detector is a differential refractive index detector; (3) The column temperature is 33°C - 40°C; (4) The chromatographic column is a lipophilic size - exclusion chromatographic column; (5) The mobile phase is tetrahydrofuran or dichloromethane; Step 3: Calculate the content of the test solution by the external standard method formula.

2. The assay method according to claim 1, characterized in that, The chromatographic column is one of MonoGPC - 100, MonoGPC - 300 and MonoGPC - 500.

3. The content determination method according to claim 1, characterized in that, The preparation method of the self - reference solution of polylactic acid or poly(lactic - co - glycolic acid) includes: Mix the self - reference of polylactic acid or poly(lactic - co - glycolic acid) with a solvent, and the solvent is selected from one of tetrahydrofuran and dichloromethane.

4. The assay method according to claim 1, wherein The concentration of polylactic acid or poly(lactic - co - glycolic acid) in the test solution is 1.2 mg / ml - 1.9 mg / ml.

5. The content determination method according to claim 1, characterized in that, The preparation method of the test solution includes: Mix the test sample with a solvent to prepare the test solution; the solvent is selected from one of tetrahydrofuran and dichloromethane.

6. The content determination method according to any one of claims 1 to 5, characterized in that, The weight - average molecular weight of the poly(lactic - co - glycolic acid) is 10,000 - 50,000.

7. The content determination method according to any one of claims 1 to 5, characterized in that The weight - average molecular weight of the polylactic acid is 50,000 - 190,000.

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