Method for measuring average molecular weight of poloxamer by using nuclear magnetic resonance technology
The average molecular weight of poloxamer is measured by nuclear magnetic resonance technology, which solves the problems of few types of measurement methods and complex operations in the existing technology, and achieves a fast, accurate and convenient measurement effect.
Patent Information
- Application Number
- CN202411712751.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-27
- Publication Date
- 2025-03-07
AI Technical Summary
In the prior art, there are few types of measurement methods for poloxamer average molecular weight and complex operations, making it difficult to quickly and accurately measure.
The average molecular weight of poloxamer is measured by nuclear magnetic resonance technology, and the liquid to be tested is obtained by reacting with halogenated benzene isocyanate in deuterated chloroform, and then a nuclear magnetic test is performed to calculate the average molecular weight based on the specific nuclear magnetic peak integral.
It realizes the average molecular weight measurement of poloxamer with convenient operation, no need for reference and internal standard substances, fast detection speed, high accuracy, good reproducibility and strong specificity.
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Abstract
Description
Technical Field
[0001] The invention relates to a method for measuring the average molecular weight of poloxamer by utilizing nuclear magnetic resonance technology. Background Art
[0002] Poloxamer (PEO-PPO-PEO) is a triblock copolymer of polyoxypropylene (PPO) and polyoxyethylene (PEO). The trade name of this substance is Pluronic. Due to the different ratios of PPO and PEO and the average molecular weight, poloxamer has a variety of specifications and models, and the properties it exhibits are also different.
[0003] The naming rule for different models is to add three digits after "Poloxamer". The first two digits represent the average molecular weight of the polyoxypropylene segment, and the last digit represents the proportion of the average molecular weight of the polyoxyethylene segment in the copolymer. For example, the first two digits "18" in Poloxamer 188 represent that the average molecular weight of the polyoxypropylene chain is 18×100=1800 (the actual value is 1750, which is rounded to 1800); and the last digit "8" represents that the average molecular weight of the polyoxyethylene chain accounts for 80% of the total average molecular weight.
[0004] Common methods for determining the average molecular weight of polymers include mass spectrometry, gel chromatography, end group titration, and osmotic pressure. According to the 2020 edition of the Chinese Pharmacopoeia, the average molecular weight of poloxamer 188 and poloxamer 407 was determined by titration. However, the end group titration method has some disadvantages, such as time-consuming and labor-intensive, high reagent cost, serious environmental pollution, high technical requirements for operators, and poor parallelism of results.
[0005] It can be seen that it is particularly important to develop a method to quickly and accurately measure the average molecular weight of poloxamer. Summary of the invention
[0006] The technical problem to be solved by the present invention is to overcome the defects of the prior art that the methods for measuring the average molecular weight of poloxamer are few in variety and complicated in operation. Therefore, the present invention provides a method for measuring the average molecular weight of poloxamer using nuclear magnetic resonance technology. The measuring method provided by the present invention has good operability, good accuracy, high stability and good application prospects.
[0007] In order to achieve the above object, the present invention adopts the following technical solution: The present invention provides a method for measuring the average molecular weight of poloxamer, which comprises the following steps: Step (1): reacting poloxamer with halogenated phenyl isocyanate in deuterated chloroform to obtain a test solution; Step (2): subjecting the test solution to a nuclear magnetic resonance test, and obtaining the average molecular weight Mr of the poloxamer according to the following formula; ; Wherein, I1 is the integral of the peak at 3.8~3.1 of the mixture of poloxamer and deuterated chloroform, and I2 is the integral of the peak at 1.1 of the mixture of poloxamer and deuterated chloroform; I3 is the integral of the peak at 4.3 of the test solution, and I4 is the integral of the peak at 3.8-3.2 of the test solution.
[0008] In one embodiment, the poloxamer and deuterated chloroform mixture further comprises heavy water.
[0009] In one embodiment, the deuterated deuterated chloroform is an anhydrous solvent (e.g., dried). In one embodiment, the poloxamer and deuterated chloroform mixed solution is prepared by the following method, which comprises the following steps: mixing the poloxamer, the deuterated chloroform and heavy water. Preferably, the method comprises the following steps: mixing and dissolving the poloxamer and the deuterated chloroform, and adding heavy water (e.g., shaking after adding).
[0010] Preferably, the mass volume ratio of the poloxamer to the deuterated chloroform may be (1-300):1 g / L, for example, 100:1 g / L.
[0011] The amount of heavy water added is conventional in the art, such as one drop of heavy water.
[0012] In one embodiment, in step (1), the reaction is carried out in the presence of an organic tin catalyst, preferably in the presence of a dibutyltin dilaurate catalyst.
[0013] In one embodiment, in step (1), the mass volume ratio of the poloxamer to the deuterated chloroform is (50-150):1 g / L, for example, 100:1 g / L.
[0014] In one embodiment, in step (1), the mass volume ratio of the poloxamer to the halogenated phenyl isocyanate is (1-3): 1 mg / μL, for example 2: 1 mg / μL.
[0015] In one embodiment, in step (1), the volume ratio of the organotin catalyst to the halogenated phenyl isocyanate is 1:(2-8):1, for example, 1:5.
[0016] In one embodiment, in step (1), the halogenated phenyl isocyanate is 2-fluorophenyl isocyanate.
[0017] In step (1), the reaction progress can be monitored by conventional methods in the art, such as nuclear magnetic resonance method. The measurement conditions of the nuclear magnetic resonance method can be as described in any embodiment of the present invention. Preferably, the reaction endpoint is when the poloxamer reaction is complete or no longer proceeds, for example, the peak area ratio of I3 to I4 is stable or substantially stable. The reaction time can be 60 min.
[0018] In one embodiment, step (1) comprises the following reaction steps: reacting poloxamer with halogenated phenyl isocyanate I-1 in deuterated chloroform in the presence of a catalyst (e.g., an organotin catalyst, preferably a dibutyltin dilaurate catalyst) to obtain a compound of formula I; ; Wherein, a and c are the polymerization degree of polyoxyethylene (C2H4O) in poloxamer, and b is the polymerization degree of polyoxypropylene (C2H4O) in poloxamer; R1 is halogen (e.g., F, Cl or Br), e.g., F; i is 0, 1, 2 or 3.
[0019] In some embodiments, a, b, and c are conventional in the art, for example, the sum of a and c is 80, and b is 27; or, the sum of a and c is 95-105, and b is 54-60; preferably, the poloxamer is poloxamer 188 or poloxamer 407.
[0020] In the present invention, the NMR test conditions are set as conventional conditions in the art, as long as the requirements for molecular NMR tests of this type in the art are met. Preferably, the NMR test uses a zg30 pulse sequence.
[0021] In some embodiments, the test temperature of the NMR test is 290-310K, for example, 298K.
[0022] In some embodiments, the radio frequency center O1P of the NMR test is set to 4-7 ppm, such as 6.2 ppm.
[0023] In some embodiments, the spectrum width of the NMR test is set to 15-22 ppm, such as 19.8 ppm.
[0024] In some embodiments, the relaxation delay time of the NMR test is set to 7.5-14 s, such as 8 s, 10 s and 12 s.
[0025] In some embodiments, the sampling time of the NMR test is 1.5-3.5 s, such as 2.75 s.
[0026] In some embodiments, the gain RG of the NMR test is set to 8-11, such as 9.8.
[0027] In some embodiments, the number of sampling points of the nuclear magnetic resonance test is set to 14-18, for example, 16.
[0028] In some embodiments, the number of empty scans of the NMR test is 1-5, for example, 2.
[0029] Preferably, the nuclear magnetic resonance test (1H-NMR) detection conditions are: zg30 pulse sequence, temperature 298K, RF center: 6.2ppm, spectrum width SWH: 19.8ppm, relaxation delay time 10s, sampling time: 2.75s, gain: 9.8, sampling points: 64k, sampling times: 16, empty scan times: 2. Further preferably, after the test is completed, automatic baseline calibration and manual phase correction are performed to obtain I1, I2, I3 and I4.
[0030] In one embodiment, the determination method comprises the following steps: Step (1): mixing loxamer and deuterated chloroform, adding heavy water, and obtaining a mixture of loxamer and deuterated chloroform; Step (2): Poloxamer is mixed with deuterated chloroform, and 2-fluorophenyl isocyanate is added in the presence of dibutyltin dilaurate to react to obtain a test solution; steps (1) and (2) are performed in no particular order; Step (3): subjecting the test solution to a nuclear magnetic resonance test, and obtaining the average molecular weight Mr of the poloxamer according to the following formula; ; Wherein, I1 is the integral of the peak of the mixture of loxamer and deuterated chloroform in the range of 3.8-3.1 (δ), and I2 is the integral of the peak at 1.1 (δ) of the mixture of loxamer and deuterated chloroform; I3 is the integral of the peak at 4.3 (δ) of the test solution, and I4 is the integral of the peak in the interval of 3.8-3.2 (δ) of the test solution; The detection conditions are as follows: zg30 pulse sequence, temperature 298K, RF center: 6.2ppm, spectrum width SWH: 19.8ppm, relaxation delay time 10s, sampling time: 2.75s, gain: 9.8, sampling points: 64k, sampling times: 16, and empty scan times: 2.
[0031] The present invention provides a compound represented by formula I; ; wherein R1, i, a, b and c are as defined in any one of the present invention.
[0032] In one embodiment, the compound represented by formula I is Preferably, a and c are the polymerization degrees of polyoxyethylene (C2H4O) in poloxamer 188 or 407, and b is the polymerization degree of polyoxypropylene (C2H4O) in poloxamer 188 or 407.
[0033] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.
[0034] The reagents and raw materials used in the present invention are commercially available.
[0035] The positive progress effect of the present invention is that the method for measuring the average molecular weight of poloxamer provided by the present invention is easy to operate, does not require reference substances and internal standards, has a fast detection speed, good accuracy, good reproducibility, strong specificity, and has a good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS
[0037] Figure 1 Quantitative hydrogen spectra of test solution 1 (A) and test solution 2 (B). DETAILED DESCRIPTION
[0038] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.
[0039] Instruments and test chemicals : Bruker 600MHz NMR spectrometer, equipped with AVANCE NEO cabinet and PABBO double resonance broadband probe; Mettler Toledo XP205 100,000th electronic balance.
[0040] Deuterated chloroform (CDCl3, 99.8% D, Shanghai Adamas Reagent Co., Ltd.), deuterated water (D2O, 99.9% D, Shanghai Adamas Reagent Co., Ltd.), dibutyltin dilaurate (95%, Shanghai MacLean Biochemical Technology Co., Ltd.), 2-fluorophenyl isocyanate (97%, Shanghai MacLean Biochemical Technology Co., Ltd.), phthalic anhydride (98%, Shanghai MacLean Biochemical Technology Co., Ltd.), pyridine (55.0%-58.0%, Shanghai Aladdin Biochemical Technology Co., Ltd.), 0.5 mol·L-1 sodium hydroxide titration solution (99.5%, Shanghai Anaiji Chemical Co., Ltd.), poloxamer (Shanghai Adamas Reagent Co., Ltd., batch numbers P2870201, P2739652, P2893805, P2893803, P2546786). Example 1
[0041] Prepare two test solution (test solution 1 and test solution 2).
[0042] Test solution 1: Take about 100 mg of the poloxamer sample with batch number P2870201, add 1 mL of deuterated chloroform to dissolve and clarify, then add one drop of heavy water, shake and transfer to a nuclear magnetic resonance tube for testing. Test solution 2: Take about 100 mg of the poloxamer sample with batch number P2870201, add 1 mL of deuterated chloroform to dissolve and clarify, use a pipette to accurately add 10 μL of dibutyltin dilaurate and 50 μL of 2-fluorophenyl isocyanate, shake thoroughly to ensure uniform mixing, leave for one hour to ensure sufficient reaction, and then transfer the solution to a nuclear magnetic resonance tube for testing; The detection conditions were tested using the zg30 pulse sequence at a probe temperature of 298K. The key instrument parameters were RF center O1P: 6.2ppm, spectrum width SWH: 19.8ppm, relaxation delay time D1: 10s, sampling time AQ: 2.75s, gain RG: 9.8, sampling points TD: 64k, sampling times NS: 16, and empty scan times DS: 2. After data acquisition, topspin4.0.5 software was used for data processing, automatic baseline calibration and manual phase correction were performed, and the spectrum was obtained as shown below. Figure 1 The average molecular weight of poloxamer is calculated according to the following formula.
[0043] The analysis of the NMR signal peaks of poloxamer is shown in Table 1 below, and the average molecular weight is calculated by the formula: Poloxamer is a block copolymer composed of polyoxyethylene (PEO) and polyoxypropylene (PPO), and its average molecular weight is equal to the sum of the average molecular weights of polyoxyethylene and polyoxypropylene. Among them, the molecular weight of the PEO segment is 44.05, and the molecular weight of the PPO segment is 58.08. The average chain length is represented by DP, so the average molecular weight of poloxamer is calculated as: Mr=DP PEO ×44.05+DP PPO ×58.08; like Figure 1Described, wherein, A is the test spectrum of the test solution 1, B is the test spectrum of the test solution 2, I1 represents the integral of the 4.20~3.20 interval of the test solution 1, I2 is the integral of the 1.10~0.80 interval of the test solution 1, I3 is the integral of the 4.50~4.40 interval of the test solution 2, and I4 is the integral of the 4.20~3.20 interval of the test solution 2. Among them, I3 corresponds to the terminal methylene proton of poloxamer. The average chain length is calculated by the ratio of the integral value of PEO and PPO to I3, respectively, and the total integral value (I4) of the methylene of PEO and PPO in the test sample 2 can be converted by the ratio of I2 to I1 in the test sample 1. The following formula can be obtained: ; Right now: ; After simplification, we can get: ; Table 1 Attribution of hydrogen spectrum signal peaks of poloxamer .
[0044] In Example 1, the conditions are selected as follows: (1) 2-Fluorophenyl isocyanate and dibutyltin dilaurate used in the pretreatment process are both soluble in deuterated chloroform but insoluble in deuterated water. Deuterated chloroform was selected as the solvent for NMR measurement.
[0045] (2) Determination of reaction time In order to ensure that the terminal hydroxyl group of poloxamer reacts completely with 2-fluorophenyl isocyanate, so as to accurately calculate its true molecular weight, the test solution is subjected to a timed nuclear magnetic resonance test. Samples are taken every 10 minutes, and the ratio of I4 to I3 peak areas is observed to determine the reaction process. The results show that the reaction stabilizes after 40 minutes, indicating that the reaction is substantially completed at this time. Therefore, Example 1 selects 60 minutes as the reaction time of poloxamer and 2-fluorophenyl isocyanate.
[0046] ⑦ Selection of instrument parameters Relaxation delay time: By measuring the longitudinal relaxation time T1, the time required for the nuclear spin system to return from the excited state to the equilibrium state can be determined. The T1 values of the test solutions I1, I2, I3 and I4 were measured to be 1.48 seconds, 0.76 seconds, 0.79 seconds and 0.86 seconds, respectively. D1 was selected to be 10 seconds to meet the test requirements.
[0047] Scan times The number of acquisitions has a direct impact on the signal-to-noise ratio (S / N) of the NMR test. 1H qNMR usually requires a signal-to-noise ratio of 250 or more. When the number of scans was set to 16, the signal-to-noise ratios of the test solutions I1, I2, I3, and I4 were 313814, 27526, 1604, and 347421, respectively, all meeting the test criteria. Therefore, the number of scans was finally determined to be 16. Embodiment 2:
[0048] The method of the present invention comprises the following steps: Prepare two test solution (test solution 1 and test solution 2).
[0049] Test solution 1: Take about 100 mg of the poloxamer sample with batch number P2739652, add 1 mL of deuterated chloroform to dissolve and clarify, then add a drop of heavy water, shake and transfer to the NMR tube for testing. Test solution 2: Take about 100 mg of the poloxamer sample with batch number P2739652, add 1 mL of deuterated chloroform to dissolve and clarify, use a pipette to accurately add 10 μL of dibutyltin dilaurate and 50 μL of 2-fluorophenyl isocyanate, shake thoroughly to ensure uniform mixing, leave for one hour to ensure sufficient reaction, and then transfer the solution to the NMR tube for testing; the detection conditions use the zg30 pulse sequence to test at a probe temperature of 298K. The key instrument parameters are radio frequency center O1P: 6.2ppm, spectrum width SWH: 19.8ppm, relaxation delay time D1: 10s, sampling time AQ: 2.75s, gain RG: 9.8, sampling points TD: 64k, sampling times NS: 16, and empty scan times DS: 2. The average molecular weight of poloxamer is calculated according to the formula in Example 1. Embodiment 3:
[0050] Prepare two test solution (test solution 1 and test solution 2).
[0051] Test solution 1: Take about 100 mg of the poloxamer sample with batch number P2893805, add 1 mL of deuterated chloroform to dissolve and clarify, then add a drop of heavy water, shake and transfer to the NMR tube for testing. Test solution 2: Take about 100 mg of the poloxamer sample with batch number P2893805, add 1 mL of deuterated chloroform to dissolve and clarify, use a pipette to accurately add 10 μL of dibutyltin dilaurate and 50 μL of 2-fluorophenyl isocyanate, shake thoroughly to ensure uniform mixing, leave for one hour to ensure full reaction, and then transfer the solution to the NMR tube for testing; the detection conditions use the zg30 pulse sequence and the probe temperature is 298K for testing. The key instrument parameters are radio frequency center O1P: 6.2ppm, spectrum width SWH: 19.8ppm, relaxation delay time D1: 10s, sampling time AQ: 2.75s, gain RG: 9.8, sampling points TD: 64k, sampling times NS: 16, and empty scan times DS: 2. The average molecular weight of poloxamer is calculated according to the formula in Example 1. Embodiment 4:
[0052] Prepare two test solution (test solution 1 and test solution 2).
[0053] Test solution 1: Take about 100 mg of the poloxamer sample with batch number P2893803, add 1 mL of deuterated chloroform to dissolve and clarify, then add a drop of heavy water, shake and transfer to the NMR tube for testing. Test solution 2: Take about 100 mg of the poloxamer sample with batch number P2893803, add 1 mL of deuterated chloroform to dissolve and clarify, use a pipette to accurately add 10 μL of dibutyltin dilaurate and 50 μL of 2-fluorophenyl isocyanate, shake thoroughly to ensure uniform mixing, leave for one hour to ensure full reaction, and then transfer the solution to the NMR tube for testing; the detection conditions use the zg30 pulse sequence to test at a probe temperature of 298K. The key instrument parameters are radio frequency center O1P: 6.2ppm, spectrum width SWH: 19.8ppm, relaxation delay time D1: 10s, sampling time AQ: 2.75s, gain RG: 9.8, sampling points TD: 64k, sampling times NS: 16, and empty scan times DS: 2. The average molecular weight of poloxamer is calculated according to the formula in Example 1. Embodiment 5:
[0054] Prepare two test solution (test solution 1 and test solution 2).
[0055] Test solution 1: Take about 100 mg of the poloxamer sample with batch number P2546786, add 1 mL of deuterated chloroform to dissolve and clarify, then add a drop of heavy water, shake and transfer to the NMR tube for testing. Test solution 2: Take about 100 mg of the poloxamer sample with batch number P2546786, add 1 mL of deuterated chloroform to dissolve and clarify, use a pipette to accurately add 10 μL of dibutyltin dilaurate and 50 μL of 2-fluorophenyl isocyanate, shake thoroughly to ensure uniform mixing, leave for one hour to ensure full reaction, and then transfer the solution to the NMR tube for testing; the detection conditions use the zg30 pulse sequence to test at a probe temperature of 298K. The key instrument parameters are radio frequency center O1P: 6.2ppm, spectrum width SWH: 19.8ppm, relaxation delay time D1: 10s, sampling time AQ: 2.75s, gain RG: 9.8, sampling points TD: 64k, sampling times NS: 16, and empty scan times DS: 2. The average molecular weight of poloxamer is calculated according to the formula in Example 1.
[0056] The comparison of the content results of the above examples 1-5 with the results of the pharmacopoeia method is shown in Table 2 Table 2 Comparison of poloxamer methods
[0057] The results between different batches and different methods showed that the test results of the NMR method were consistent with those of the pharmacopoeia method (end group titration method).
[0058] The method of the present invention is stable and reliable and can accurately determine the average molecular weight of poloxamer. The methodological validation test data are as follows: ① Test solution 1: Take about 100 mg of poloxamer test sample, add 1 mL of deuterated chloroform to dissolve and clarify, then add one drop of heavy water, shake and transfer to a nuclear magnetic resonance tube for testing. Test solution 2: Take about 100 mg of poloxamer test sample with batch number P2739652, add 1 mL of deuterated chloroform to dissolve and clarify, use a pipette to accurately add 10 μL of dibutyltin dilaurate and 50 μL of 2-fluorophenyl isocyanate, shake thoroughly to ensure uniform mixing, leave for one hour to ensure sufficient reaction, and then transfer the solution to a nuclear magnetic resonance tube for testing.
[0059] ②The zg30 pulse sequence was used for testing at a probe temperature of 298 K. The key instrument parameters are RF center O1P: 6.2 ppm, spectrum width SWH: 19.8 ppm, relaxation delay time D1: 10 s, sampling time AQ: 2.75 s, gain RG: 9.8, sampling points TD: 64 k, sampling times NS: 16, and empty scan times DS: 2.
[0060] Example 6 Precision Experiment Take poloxamer with batch number P2870201, prepare 6 portions of test sample 1 and test sample 2 according to method ①, and measure according to conditions in ②. After adjusting the baseline and phase, integrate and calculate the average molecular weight of poloxamer. The results show that the average molecular weights of poloxamer in the test solution are 7886.1, 7914.3, 7799.3, 7851.9, 7838.9 and 7899.4, respectively, with an RSD of 0.54%, indicating that this method has good precision.
[0061] Example 7 Stability Test A portion of each of the random sample 1 and the sample 2 in Example 6 was taken and measured at 0, 4, 12, 24, and 48 h according to the conditions in ②. The results showed that the average molecular weight of the poloxamer in the test solution was 7 772.4, 7 812.0, 7 825.8, 7 744.4, and 7 819.5, respectively, and the RSD was 0.45%, indicating that the test solution 1 and the test solution 2 were stable within 48 h.
[0062] Example 8 Durability Test Take one sample 1 and one sample 2 randomly selected from Example 7, change the temperature and relaxation time in the conditions, and compare them with the results of the initial test conditions. The test results are shown in Table 3. The ratio of the results after slight changes in the test conditions to the results under normal conditions is between 98% and 102%, indicating that the method has good durability.
[0063] Table 3 Method robustness
[0064] The above experiments can clearly show that the present invention discloses a method for determining the average molecular weight of poloxamer. The quantitative hydrogen nuclear magnetic resonance spectroscopy method can quickly and accurately determine the average molecular weight of poloxamer, does not require reference substances and internal standards, is simple to prepare, has a fast detection speed, good reproducibility, and strong specificity. It makes a creative contribution to the inspection of pharmaceutical excipients and has good economic and social benefits.
[0065] Comparative Example 1 Referring to Example 1, deuterated chloroform was replaced with heavy water in the preparation method of test solution 2, and it was observed that it was immiscible with halogenated phenyl isocyanate.
[0066] Comparative Example 2 Referring to Example 1, deuterated chloroform was replaced with deuterated dimethyl sulfoxide in the preparation method of test solution 2, and it was observed that the water peak interfered with the calculation results.
[0067] Comparative Example 3 Referring to Example 1, deuterated chloroform was replaced with deuterated methanol in the preparation method of test solution 2. As a result, it was observed that the deuterated methanol solvent peak interfered with the calculation results.
[0068] Comparative Example 4 Referring to Example 1, deuterated chloroform was replaced with deuterated acetone in the preparation method of test solution 2. It was observed that deuterated acetone and poloxamer were immiscible.
[0069] Comparative Example 5 Referring to Example 1, deuterated chloroform was replaced with deuterated acetonitrile in the preparation method of test solution 2. It was observed that deuterated acetonitrile and poloxamer were immiscible.
[0070] Although the specific embodiments of the present invention are described above, it should be understood by those skilled in the art that this is only for illustration and the protection scope of the present invention is defined by the appended claims. Those skilled in the art may make various changes or modifications to these embodiments without departing from the principles and essence of the present invention, but these changes and modifications all fall within the protection scope of the present invention.
Claims
1. A method for measuring the average molecular weight of poloxamer, characterized in that: It includes the following steps: Step (1): reacting poloxamer with halogenated phenyl isocyanate in deuterated chloroform to obtain a test solution; Step (2): subjecting the test solution to a nuclear magnetic resonance test, and obtaining the average molecular weight Mr of the poloxamer according to the following formula; Mr=(I3+I4)*(44.05*I1+33.39*I2) / ( I1*I3); Wherein, I1 is the integral of the peak at 3.8~3.1 of the mixture of poloxamer and deuterated chloroform, and I2 is the integral of the peak at 1.1 of the mixture of poloxamer and deuterated chloroform; I3 is the integral of the peak at 4.3 of the test solution, and I4 is the integral of the peak at 3.8-3.2 of the test solution.
2. The method for measuring the average molecular weight of poloxamer according to claim 1, wherein: It meets one or more of the following conditions: (1) The poloxamer and deuterated chloroform mixture further comprises heavy water; (2) The deuterated chloroform is an anhydrous solvent; (3) The poloxamer and deuterated chloroform mixed solution is prepared by the following method, which comprises the following steps: mixing the poloxamer, the deuterated chloroform and heavy water; preferably comprises the following steps: mixing and dissolving the poloxamer and the deuterated chloroform, and adding heavy water; (4) In step (1), the reaction is carried out in the presence of an organic tin catalyst, preferably in the presence of dibutyltin dilaurate catalyst; (5) In step (1), the halogenated phenyl isocyanate is 2-fluorophenyl isocyanate.
3. The method for measuring the average molecular weight of poloxamer according to claim 2, wherein: It meets one or more of the following conditions: (1) In the method for preparing the mixed solution of poloxamer and deuterated chloroform, the mass volume ratio of the poloxamer to the deuterated chloroform is (1-300):1 g / L, preferably (50-150):1 g / L, for example 100:1 g / L; (2) In the method for preparing the mixed solution of poloxamer and deuterated chloroform, the amount of heavy water added is one drop.
4. The method for measuring the average molecular weight of poloxamer according to claim 1 or 2, characterized in that: It meets one or more of the following conditions: (1) In step (1), the mass volume ratio of the poloxamer to the deuterated chloroform is (50-150):1 g / L, for example, 100:1 g / L; (2) In step (1), the mass volume ratio of the poloxamer to the halogenated phenyl isocyanate is (1-3): 1 mg / μL, for example 2: 1 mg / μL; (3) In step (1), the volume ratio of the organotin catalyst to the halogenated phenyl isocyanate is 1:(2-8):1, for example 1:
5.
5. The method for measuring the average molecular weight of poloxamer according to claim 1 or 2, characterized in that: Step (1) comprises the following reaction steps: reacting poloxamer with halogenated phenyl isocyanate I-1 in deuterated chloroform in the presence of a catalyst to obtain a compound represented by formula I; ； Wherein, a and c are the polymerization degree of polyoxyethylene in poloxamer, and b is the polymerization degree of polyoxypropylene in poloxamer; R1 is halogen; i is 0, 1, 2 or 3.
6. The method for measuring the average molecular weight of poloxamer according to claim 5, characterized in that: It meets one or more of the following conditions: (1) The halogen is F, Cl or Br, for example F; (2) The sum of a and c is 80, and b is 27; or, the sum of a and c is 95-105, and b is 54-60; preferably, the poloxamer is poloxamer 188 or poloxamer 407.
7. The method for measuring the average molecular weight of poloxamer according to claim 1, characterized in that: It meets one or more of the following conditions: (1) The nuclear magnetic resonance test uses the zg30 pulse sequence; (2) The test temperature of the nuclear magnetic resonance test is 290-310K, for example, 298K; (3) The radio frequency center O1P of the NMR test is set to 4-7 ppm, for example 6.2 ppm; (4) The spectrum width of the NMR test is set to 15-22 ppm, for example, 19.8 ppm; (5) The relaxation delay time of the NMR test is set to 7.5-14 s, such as 8 s, 10 s and 12 s; (6) The sampling time of the nuclear magnetic resonance test is 1.5-3.5 s, for example 2.75 s; (7) The gain RG of the NMR test is set to 8-11, for example 9.8; (8) The number of sampling points of the nuclear magnetic resonance test is set to 14-18, for example, 16; (9) The number of empty scans of the NMR test is 1-5, for example 2.
8. The method for measuring the average molecular weight of poloxamer according to claim 1, wherein: The nuclear magnetic resonance test detection conditions are: using zg30 pulse sequence, temperature 298K, RF center: 6.2ppm, spectrum width SWH: 19.8ppm, relaxation delay time 10s, sampling time: 2.75s, gain: 9.8, number of sampling points: 64k, number of sampling times: 16, number of empty scans: 2; further preferably, after the test is completed, automatic baseline calibration and manual phase correction are performed, and the I1, I2, I3 and I4 are obtained by integration.
9. The method for measuring the average molecular weight of poloxamer according to claim 1, wherein: It includes the following steps: Step (1): mixing loxamer, deuterated chloroform and heavy water to obtain a mixture of loxamer and deuterated chloroform; Step (2): Poloxamer is mixed with deuterated chloroform, and 2-fluorophenyl isocyanate is added in the presence of dibutyltin dilaurate to react to obtain a test solution; steps (1) and (2) are performed in no particular order; Step (3): subjecting the test solution to a nuclear magnetic resonance test, and obtaining the average molecular weight Mr of the poloxamer according to the following formula; Mr=(I3+I4)*(44.05*I1+33.39*I2) / ( I1*I3); Wherein, I1 is the integral of the peak at 3.8-3.1 of the mixture of loxamer and deuterated chloroform, and I2 is the integral of the peak at 1.1 of the mixture of loxamer and deuterated chloroform; I3 is the peak integral at 4.3 of the test solution, and I4 is the peak integral at 3.8-3.2 of the test solution; The detection conditions are as follows: zg30 pulse sequence, temperature 298K, RF center: 6.2ppm, spectrum width SWH: 19.8ppm, relaxation delay time 10s, sampling time: 2.75s, gain: 9.8, sampling points: 64k, sampling times: 16, and empty scan times:
2.
10. A compound of formula I; ； in, R1, i, a, b and c are as described in claim 5 or 6; Preferably, the compound represented by formula I is .