Method for detecting and quantifying impurity residues in polyethylene glycol-polycaprolactone triblock copolymer

Abstract

The invention discloses a method for detecting and quantifying impurity residues in a polyethylene glycol-polycaprolactone triblock copolymer. Impurities are a polyethylene glycol-polycaprolactone diblock copolymer and residual polyethylene glycol. The method comprises the following steps: derivatizing, separating and purifying a polymer and impurities, performing nuclear magnetic detection to detect impurities which are difficult to detect by a conventional analysis method, and calculating the content of the impurities according to a peak area ratio of a polymer derivatization functional group characteristic peak to an impurity derivatization functional group characteristic peak. The method is high in derivatization efficiency and simple in derivative separation, impurity residue information of the product can be effectively obtained through nuclear magnetic test calculation, and the method is a novel detection method which is simple to operate and high in practicability.

Description

Technical Field

[0001] The invention belongs to the field of analysis and detection, and specifically relates to a detection and quantitative method for residual impurities in a polyethylene glycol-polycaprolactone triblock copolymer. Background Art

[0002] Polyethylene glycol has good water solubility, lubricity, adhesion, solubilization and biocompatibility. At present, the polyethylene glycol-polycaprolactone triblock copolymer developed around polyethylene glycol is widely used in the field of medicine and medical devices. In the preparation process of polyethylene glycol-polycaprolactone triblock copolymer, there are problems of diblock polymer, polyethylene glycol and monomer residues; the analysis and detection of monomer residues currently have relatively mature testing methods, while the analysis and detection of diblock polymers and polyethylene glycol residues is still a difficult problem, especially in the preparation of low molecular weight polyethylene glycol-polycaprolactone triblock copolymers. The problem of impurity residues will not only affect the quality of the copolymer, but also introduce unknown risks to downstream products.

[0003] The detection of polymers usually uses gel permeation chromatography technology, which uses highly porous, non-ionic gel beads to separate the polydisperse polymers in the solution step by step. The molecular weight distribution can be obtained by using a molecular weight detector. It is currently the most widely used method for determining molecular weight distribution. However, in practical applications, this method is difficult to separate and characterize "polymer impurities" with similar molecular weights. This is because the separation and detection mechanism of this method is highly related to the molecular size / molecular weight of the polymer. Therefore, the polymer is often derivatized, and the "polymer impurities" cannot be detected.

[0004] Nuclear magnetic resonance spectroscopy is one of the most powerful tools for qualitative analysis of compound structure. Its advantages are that it does not require self-reference substances, the sample pretreatment process is relatively simple, the method is accurate, reliable, simple and fast. However, polymers with similar structures will also encounter the problem of characteristic peaks being masked in nuclear magnetic resonance testing. Therefore, it is impossible to detect the residues of diblock polymers and polyethylene glycol through nuclear magnetic resonance, and even the test of the structure of polyethylene glycol-polycaprolactone triblock copolymer will bring wrong results. Summary of the invention

[0005] In order to solve the technical problems mentioned in the prior art, the present invention has developed a nuclear magnetic resonance (NMR) quantitative method to detect and quantify impurities in polyethylene glycol-polycaprolactone triblock copolymers. Usually, even if a derivatization group is introduced into the polymer, the characteristic peaks of the derivatization group cannot be separated, because the "polymer to be tested after derivatization" and the "impurity polymer after derivatization" have similar structural properties. Therefore, the selection of the derivatization agent is crucial. The present invention uses benzoic acid derivatives to derivatize polyethylene glycol-polyester block copolymers, studies the nuclear magnetic resonance spectrum of the derivatized product, and realizes the quantitative detection of impurity residues in polyethylene glycol-polycaprolactone triblock copolymers.

[0006] The above-mentioned method for detecting and quantifying impurity residues in the polyethylene glycol-polycaprolactone triblock copolymer specifically comprises the following steps: (1) Using a benzoic acid derivative as a derivatization reagent, a polyethylene glycol-polycaprolactone triblock copolymer containing impurities is derivatized; the impurities are polyethylene glycol-polycaprolactone diblock polymer and polyethylene glycol; (2) the derivatized sample is dissolved in a deuterated reagent at a concentration of 10-30 mg / mL, and then the deuterated reagent containing the sample is subjected to nuclear magnetic resonance. 1 H NMR detection, the deuterated reagent is deuterated chloroform; (3) Calculate the impurity content in the polyethylene glycol-polycaprolactone triblock copolymer: ; Pr is the impurity content in the polyethylene glycol-polycaprolactone triblock copolymer, Ar is the sum of the characteristic peak areas of the impurities after derivatization treatment, and As is the characteristic peak area of ​​the polyethylene glycol-polycaprolactone triblock copolymer after derivatization treatment.

[0007] Furthermore, the structure of the polyethylene glycol-polycaprolactone triblock copolymer is: ; Among them, the molecular weight range of PEG is 600-2000; m1 and m2 are independently selected from integers of 0-28, and the sum of m1 and m2 is ≤56.

[0008] Furthermore, the derivatization reagent is 2,3,5-triiodobenzoic acid.

[0009] Furthermore, the molar ratio of the amount of the derivatization reagent to the hydroxyl functional group is (1-1.5):1.

[0010] Furthermore, the specific steps of the derivatization treatment are as follows: (1) dissolving the analyte in a solvent at a concentration of 0.05-0.5 g / ml; the solvent is one of dichloromethane, N,N-dimethylformamide, and tetrahydrofuran; the analyte comprises one or more of polyethylene glycol-polycaprolactone triblock copolymer, polyethylene glycol-polycaprolactone diblock polymer, and polyethylene glycol; (2) Add a derivatization reagent and DMAP, stir evenly, then add DCC and react at 0-35°C for 5-24 h; the molar ratio of the derivatization reagent to the hydroxyl functional group is (1-1.5):1, the molar ratio of the DCC to the hydroxyl functional group is (1-1.5):1, and the molar ratio of the DMAP to the hydroxyl functional group is (0.05-0.5):1; (3) Alkaline washing: After the derivatization reaction is completed, wash with saturated sodium carbonate aqueous solution 2-3 times; (4) The organic phase is dried over anhydrous sodium sulfate and then filtered; (5) The organic phase is precipitated with n-hexane to precipitate the solid, and the supernatant is filtered out; (6) The solid phase is dried in a vacuum drying oven to obtain benzoate of polyethylene glycol-polycaprolactone triblock copolymer.

[0011] In the present invention, the reaction formula of derivatization is as follows: .

[0012] Beneficial effects of the present invention: The problems of low derivatization conversion efficiency and difficult post-treatment of polymers were solved by selecting benzoic acid derivatives as derivatization reagents, which were grafted onto the reaction sites of the polymers to a "saturated state", thereby achieving successful detection of impurities in polyethylene glycol-polycaprolactone triblock copolymers. At the same time, the detection method is fast, the calculation is simple, and the test results are accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Shown are the GPC data of triblock polymers PCL-PEG-PCL with different molecular weights; Figure 2 Shown are the GPC-MALs data for the triblock polymer PCL-PEG-PCL; Figure 3 Shown are the NMR data of the triblock polymer PCL-PEG-PCL; Figure 4 is the NMR data of derivatized polyethylene glycol; Figure 5 The NMR data of the derivatized polyethylene glycol-polycaprolactone diblock polymer; Figure 6 is the NMR data of Example 1; Figure 7is the NMR data of Example 2; Figure 8 is the NMR data of Example 3; Fig. 9 is the NMR data of Example 4; Fig.10 is the NMR data of Example 5; Fig.11 is the NMR data of Example 6; Fig.12 This is the NMR data of Comparative Example 1. DETAILED DESCRIPTION

[0014] The following examples are used to further illustrate the present invention, and their purpose is to illustrate the present invention and should not be construed as limiting the scope of the present invention. Unless otherwise specified, all parts by weight and weight percentages are used below.

[0015] The raw materials used in the present invention, unless otherwise specified, are conventional commercially available products; the methods used in the present invention, unless otherwise specified, are conventional methods in the art.

[0016] The embodiments of the present invention are further described below with reference to a plurality of embodiments.

[0017] It should be clear that the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present application.

[0018] It should be noted that the molar ratio of the hydroxyl functional group in the present invention is calculated based on 100% polyethylene glycol-polycaprolactone triblock copolymer, because the molecular weights of polyethylene glycol-polycaprolactone diblock copolymer, polyethylene glycol-polycaprolactone triblock copolymer and polyethylene glycol are similar. In addition, the amount of benzoic acid derivative added in this reaction is excessive, and even if the amount of derivatization reagent, DMAP and DCC is calculated based on 100% polyethylene glycol-polycaprolactone triblock copolymer, the complete reaction of the polymer in the reaction can be ensured.

[0019] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0020] Example 1 Dissolve 1.0 g of weighed PEG1000 in 10 mL of analytical grade dichloromethane and add it to a 100 mL single-mouth bottle. Then add 1.1 g of 2,3,5-triiodobenzoic acid and 0.037 g of DMAP. After stirring for 5 min, add 0.45 g of DCC and react at room temperature for 12 h. After the reaction is completed, filter and wash twice with saturated sodium carbonate aqueous solution (10 mL×2). The organic phase is dried with anhydrous sodium sulfate and filtered. The derivatized product is collected by precipitation with n-hexane and dried in a vacuum drying oven to obtain 1.5 g. Take 12 mg of the sample, dissolve it in 1 ml of deuterated chloroform, and perform NMR testing to obtain the characteristic peak of PEG1000 derivatization ( Figure 4 ).

[0021] 1.6 g of weighed HO-PEG1000-PCL800 was dissolved in 20 mL of analytical grade dichloromethane and added to a 100 mL single-mouth bottle. Then, 1.25 g of 2,3,5-triiodobenzoic acid and 0.037 g of DMAP were added. After stirring for 5 min, 0.52 g of DCC was added and the mixture was reacted at room temperature for 12 h. After the reaction was completed, the mixture was filtered and washed twice with saturated sodium carbonate aqueous solution (20 mL×2). The organic phase was dried with anhydrous sodium sulfate and filtered. The derivatized product was collected by precipitation with n-hexane and dried in a vacuum drying oven to obtain 1.3 g. 15 mg of the sample was taken and dissolved in 1 ml of deuterated chloroform. The characteristic peaks of HO-PEG1000-PCL800 derivatization were obtained ( Figure 5 ).

[0022] Dissolve 2.5 g of weighed PCL800-PEG1000-PCL800 in 50 mL of analytical grade dichloromethane and add it to a 100 mL single-mouth bottle. Then add 1.25 g of 2,3,5-triiodobenzoic acid and 0.037 g of DMAP. After stirring for 5 min, add 0.52 g of DCC and react at 25°C for 12 h. After the reaction is completed, filter and wash twice with saturated sodium carbonate aqueous solution (50 mL×2). The organic phase is dried with anhydrous sodium sulfate and filtered. The derivatized product is collected by precipitation with n-hexane and dried in a vacuum drying oven to obtain 3.4 g. Take 15 mg of the sample, dissolve it in 1 ml of deuterated chloroform, and perform nuclear magnetic resonance testing to determine that the impurity content is 8.1% ( Figure 6 ).

[0023] In contrast, the impurities of the underivatized triblock polymer PCL-PEG-PCL were directly detected by GPC, GPC-MALs, and NMR, and no impurities were detected, such as Figure 1-3 shown.

[0024] Example 2 Dissolve 2.3 g of weighed PCL400-PEG1500-PCL400 in 30 mL of analytical grade dichloromethane and add it to a 100 mL single-mouth bottle. Then add 1.1 g of 2,3,5-triiodobenzoic acid and 0.061 g of DMAP. After stirring for 5 min, add 0.45 g of DCC and react at 20°C for 16 h. After the reaction is completed, filter and wash twice with saturated sodium carbonate aqueous solution (50 mL×2). The organic phase is dried over anhydrous sodium sulfate and filtered. The derivatized product is collected by precipitation with n-hexane and dried in a vacuum drying oven to obtain 3.0 g. Take 20 mg of the sample, dissolve it in 1 ml of deuterated chloroform, and perform nuclear magnetic resonance testing to determine that the impurity content is 28.5% ( Figure 7 ).

[0025] Example 3 3.4 g of weighed PCL1200-PEG1000-PCL1200 was dissolved in 34 mL of analytical grade dichloromethane and added to a 100 mL single-mouth bottle. Then, 1.5 g of 2,3,5-triiodobenzoic acid and 0.073 g of DMAP were added respectively. After stirring for 5 min, 0.62 g of DCC was added and reacted at 35°C for 8 h. After the reaction was completed, the mixture was filtered and washed twice with saturated sodium carbonate aqueous solution (50 mL×2). The organic phase was dried over anhydrous sodium sulfate and filtered. The derivatized product was collected by precipitation with n-hexane and dried in a vacuum drying oven to obtain 3.8 g. 10 mg of the sample was taken and dissolved in 1 ml of deuterated chloroform. Nuclear magnetic resonance testing was performed to determine that the impurity content was 6.7% ( Figure 8 ).

[0026] Example 4 Dissolve 2.7 g of weighed PCL600-PEG1500-PCL600 in 50 mL of analytical grade dichloromethane and add it to a 100 mL single-mouth bottle. Then add 1.15 g of 2,3,5-triiodobenzoic acid and 0.037 g of DMAP. After stirring for 5 min, add 0.52 g of DCC and react at 30°C for 5 h. After the reaction is completed, filter and wash twice with saturated sodium carbonate aqueous solution (50 mL×2). The organic phase is dried with anhydrous sodium sulfate and filtered. The derivatized product is collected by precipitation with n-hexane and dried in a vacuum drying oven to obtain 3.3 g. Take 30 mg of the sample, dissolve it in 1 ml of deuterated chloroform, and perform nuclear magnetic resonance testing to determine the impurity content of 8.7% ( Fig. 9 ).

[0027] Example 5 Dissolve 25 g of weighed PCL3200-PEG1000-PCL3200 in 50 mL of analytical grade dichloromethane and add it to a 500 mL single-mouth bottle. Then add 4.22 g of triiodobenzoic acid and 0.083 g of DMAP. After stirring for 5 min, add 1.74 g of DCC and react at 0°C for 24 h. After the reaction is completed, filter and wash three times with saturated sodium carbonate aqueous solution (50 mL×3). The organic phase is dried with anhydrous sodium sulfate and filtered. The derivatized product is collected by precipitation with n-hexane and dried in a vacuum drying oven to obtain 30.8 g. Take 12 mg of the sample, dissolve it in 1 ml of deuterated chloroform, and perform nuclear magnetic resonance testing to determine the impurity content of 2.7% ( Fig.10 ).

[0028] Example 6 The spike recovery experiment was carried out. The weighed PCL800-PEG1000-PCL800 5.0 g and PEG1000 0.25 g were dissolved in 100 mL of analytical pure dichloromethane and added to a 250 mL single-mouth bottle. Then 2,3,5-triiodobenzoic acid 2.4 g and DMAP 0.07 g were added respectively. After stirring for 5 min, DCC 0.99 g was added and reacted at 25 ° C for 12 h. After the reaction was completed, it was filtered and washed twice with saturated sodium carbonate aqueous solution (100 mL×2). The organic phase was dried with anhydrous sodium sulfate and filtered. The derivatized product was collected by precipitation with n-hexane and dried in a vacuum drying oven to obtain 7.4 g. Take 15 mg of the sample, dissolve it with 1 ml of deuterated chloroform, and perform nuclear magnetic resonance testing to determine the impurity content of 18.52% (the theoretical value of spike is 18.79%) ( Fig.11 ).

[0029] Comparative Example 1 The steps are the same as those in Example 1, except that 2,3,5-triiodobenzoic acid is replaced with 4-iodobenzoic acid. The NMR test is performed and no impurities are detected ( Fig.12 ).

[0030] Comparative Example 2 The steps are the same as those in Example 1, except that ethanol is used for precipitation during post-treatment, resulting in emulsification and failure to separate the polymer repeatedly.

[0031] The above embodiments describe in detail the structure, features and effects of the present invention. The above are only preferred embodiments of the present invention. Any changes made in accordance with the concept of the present invention, or modifications to equivalent embodiments with equivalent changes, which do not exceed the scope covered by the specification, should be within the protection scope of the present invention.

Claims

1. A method for detecting and quantifying residual impurities in a polyethylene glycol-polycaprolactone triblock copolymer, characterized in that: The following steps are involved: (1) Using benzoic acid derivatives as derivatization reagents, polyethylene glycol-polycaprolactone triblock copolymers containing impurities were derivatized; The impurities are polyethylene glycol-polycaprolactone diblock polymer and polyethylene glycol; (2) Dissolve the derivatized sample in a deuterated reagent at a concentration of 10-30 mg / mL, and then perform NMR on the deuterated reagent containing the sample. 1 H NMR detection, the deuterated reagent is deuterated chloroform; (3) Calculation of impurity content in polyethylene glycol-polycaprolactone triblock copolymer: ； Pr is the impurity content in the polyethylene glycol-polycaprolactone triblock copolymer, Ar is the sum of the characteristic peak areas of the impurities after derivatization treatment, and As is the characteristic peak area of ​​the polyethylene glycol-polycaprolactone triblock copolymer after derivatization treatment.

2. The method according to claim 1, characterized in that The benzoic acid derivative is 2,3,5-triiodobenzoic acid.

3. The method according to claim 1, characterized in that The molar ratio of the amount of the derivatization reagent to the hydroxyl functional group is (1-1.5):

1.

4. The method according to claim 1, characterized in that: The steps of the derivatization treatment are as follows: (1) dissolving the analyte in a solvent at a concentration of 0.05-0.5 g / ml; the solvent is one of dichloromethane, N,N-dimethylformamide, and tetrahydrofuran; the analyte comprises one or more of polyethylene glycol-polycaprolactone triblock copolymer, polyethylene glycol-polycaprolactone diblock polymer, and polyethylene glycol; (2) Add a derivatization reagent and 4-dimethylaminopyridine (DMAP), stir evenly, then add N,N'-dicyclohexylcarboximide (DCC), and react at 0-35°C for 5-24 h; the molar ratio of the derivatization reagent to the hydroxyl functional group is (1-1.5):1, the molar ratio of the DCC to the hydroxyl functional group is (1-1.5):1, and the molar ratio of the DMAP to the hydroxyl functional group is (0.05-0.5):1; (3) Alkaline washing: After the derivatization reaction is completed, wash with saturated sodium carbonate aqueous solution 2-3 times; (4) The organic phase is dried over anhydrous sodium sulfate and then filtered; (5) The organic phase is precipitated with n-hexane to precipitate the solid, and the supernatant is filtered out; (6) The solid phase is dried in a vacuum drying oven to obtain benzoate of polyethylene glycol-polycaprolactone triblock copolymer.

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