Method for detecting content of PEG (Polyethylene Glycol) in amphiphilic copolymer

Abstract

The invention provides a method for detecting the content of PEG in an amphiphilic copolymer, and relates to the technical field of compound detection analysis, and the method comprises the following steps: taking a PEG solution, adding a reagent D with gradient concentration and a buffer solution, diluting with deionized water to a constant volume, uniformly shaking, and developing; performing 200-800nm spectrum scanning by using an ultraviolet visible spectrophotometer, and determining the optimal dosage of the reagent D; determining the wavelength with the maximum absorption value of the complex by adopting the optimal dosage of the reagent D; drawing a standard curve, detecting the absorbance value of the sample and calculating the PEG content in the sample; wherein the reagent D comprises bismuth subnitrate, potassium iodide, glacial acetic acid and water. Linear regression is carried out by utilizing the absorbance value and the concentration value of the standard solution to obtain a linear equation, and the absorbance value of the sample solution is substituted for calculation. The analysis method has the advantages of low cost, good repeatability, rapidness, high efficiency and good precision, and is suitable for similar products in the market.

Description

Technical Field

[0001] The invention provides a method for detecting the PEG content in an amphiphilic copolymer, and relates to the technical field of compound detection and analysis. Background Art

[0002] Biodegradable materials, such as polylactic acid (PLA), poly(lactide-co-glycolide) (PLGA) and polycaprolactone (PCL), have shown extensive application potential in the biomedical field due to their excellent biodegradability, outstanding biocompatibility and good mechanical properties. Polyethylene glycol (PEG), as a hydrophilic polymer, has attracted widespread attention in the medical community due to its excellent biocompatibility, solubility in tissue fluid and excretion through the kidney filtration membrane. It has been certified by the US Food and Drug Administration (FDA) and is allowed to be used in the human body. Introducing PEG into the above polymer segments can combine the advantages of the two polymers and give the material excellent biocompatibility and water solubility, so that it is widely used in the biomedical field, especially in drug delivery systems, tissue engineering and gene delivery.

[0003] However, in the synthesis process of PEG copolymer, a certain amount of polyethylene glycol tends to remain, and this potential toxicity is particularly critical in pharmaceutical preparations, because the content and purity of polyethylene glycol are directly related to the safety and effectiveness of drugs. Therefore, the analysis and detection of polyethylene glycol is particularly important. At present, although a barium chloride method is provided in the 2015 edition of the three general rules of the Chinese Pharmacopoeia 3202 to detect the residual amount of polyethylene glycol in protein, the complex stability formed by the method is not good, the characteristic absorption peak is not significant, and the test result has a large contingency, so the method is not applicable. For the determination of polyethylene glycol content in amphiphilic block copolymers, there is currently a lack of effective methods. Although Chinese patent CN116297981A proposes a separation and detection method for copolymers, it focuses on the overall detection analysis of copolymer content, and does not go deep into the specific determination of free PEG content in copolymers. Similarly, Chinese patent CN101042351A and Chinese patent CN110243775A provide methods for detecting polyethylene glycol content in water and polyethylene glycol in copper foil electrolyte, respectively. However, the detection principles of these methods have been reflected in the prior art and are not suitable for detecting the free polyethylene glycol content in hydrophobic materials. In addition, although Chinese patent CN105067727B provides a method for determining the PEG content in polymer monomers, its specificity is too strong and it is difficult to be widely used in most fields including the materials of this study, and lacks universality. Summary of the invention

[0004] In order to overcome the difficulties of existing detection methods and fill the gap in the field of material detection, the purpose of the present invention is to provide a simple and accurate method for detecting the residual amount of PEG in PEG amphiphilic block copolymers. The specific scheme is as follows:

[0005] A method for detecting the PEG content in an amphiphilic copolymer comprises the following steps:

[0006] (2) Take the PEG solution, add reagent D and buffer solution of gradient concentration, dilute to volume with deionized water, shake well, and develop color; use a UV-visible spectrophotometer to perform spectral scanning at 200-800 nm to determine the optimal dosage of reagent D;

[0007] (3) Take the PEG solution, add the optimal amount of D reagent and buffer solution, dilute to volume with deionized water, shake well, and develop color; use a UV-visible spectrophotometer to perform a 200-800 nm spectrum scan to determine the wavelength with the maximum absorption value of the complex;

[0008] (4) preparing a PEG standard solution, diluting it to a gradient concentration, adding the optimal amount of D reagent and buffer solution, respectively, making up to volume with deionized water, shaking well, developing the color, detecting the absorbance at the wavelength determined in step (3) and drawing a standard curve;

[0009] (5) Take the test solution and add the optimal amount of D reagent and buffer in sequence, make up to volume with deionized water, shake well, develop color, detect the absorbance at the wavelength determined in step (3), and calculate the PEG content in the sample according to the standard curve;

[0010] Among them, reagent D includes bismuth subnitrate, potassium iodide, glacial acetic acid and water.

[0011] Preferably, the preparation method of the D reagent is: prepare a bismuth subnitrate solution and a potassium iodide solution respectively, and mix the bismuth subnitrate solution, the potassium iodide solution, glacial acetic acid and water in a volume ratio of 1:2:4:13.

[0012] Preferably, the concentration of the bismuth subnitrate solution is 0.01 mol / L, and the concentration of the potassium iodide solution is 2.4 mol / L.

[0013] Preferably, the buffer solution is an acetic acid-sodium acetate buffer solution with a pH of 4.8.

[0014] Preferably, the preparation method of the buffer solution is: weigh glacial acetic acid to prepare a 0.2 mol / L acetic acid solution; weigh sodium acetate to prepare a 0.2 mol / L sodium acetate solution; measure the sodium acetate solution and the acetic acid solution in a volume ratio of 50:32 to prepare an acetic acid-sodium acetate buffer solution with a pH of 4.8.

[0015] Preferably, the method for preparing the test solution in step (5) comprises: taking a sample to be tested, accurately weighing the mass, adding dichloromethane and then deionized water, shaking and extracting, and letting stand, and taking the aqueous phase as the test solution; the sample to be tested is a copolymer sample whose PEG content is to be determined.

[0016] Preferably, the color development time is 15 minutes.

[0017] Preferably, the gradient concentration of the dilution of the standard solution is in the range of 0-50 mg / L.

[0018] Preferably, the PEG amphiphilic copolymer includes one or more of polyethylene glycol-poly L-lactic acid (PEG-PLLA), polyethylene glycol-polycaprolactone (PEG-PCL), polyethylene glycol-polylactide (PEG-PLGA), polyethylene glycol monomethyl ether-poly L-lactic acid (mPEG-PLLA), polyethylene glycol monomethyl ether-polycaprolactone (mPEG-PCL), and polyethylene glycol monomethyl ether-polylactide (mPEG-PLGA).

[0019] Preferably, the standard comprises polyethylene glycol and polyethylene glycol monomethyl ether, and the molecular weight ranges from 1000 to 5000.

[0020] The beneficial effects of the present invention are as follows:

[0021] The invention discloses a method for determining the content of free polyethylene glycol in an amphiphilic copolymer by using an ultraviolet spectrophotometer, wherein the absorbance value of a standard solution and the numerical value of the concentration are used for linear regression to obtain a linear equation, and the absorbance value of a sample solution is brought into the equation for calculation. The analytical method of the invention has low cost, good repeatability, fast and high efficiency, good precision, and is applicable to similar products on the market. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the absorbance value and concentration curve of mPEG1000 standard solution (C = 5-30 mg / L).

[0023] Figure 2 This is the absorbance value and concentration curve of mPEG1000 standard solution (C = 0-10 mg / L).

[0024] Figure 3 It is the absorbance value and concentration curve of PEG1000 standard solution (C=0-30mg / L).

[0025] Figure 4 This is the absorbance value and concentration curve of mPEG5000 standard solution (C = 5-50 mg / L). DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0027] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only and are not intended to limit the content of this application.

[0028] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0029] Implementation Case 1

[0030] Establishment of mPEG1000 standard curve

[0031] The standard curve solution was prepared as follows: 0.25 g of mPEG10000 was accurately weighed into a 250 mL volumetric flask, and diluted with deionized water to make a 1000 mg / L polyethylene glycol standard stock solution. 0.25, 0.5, 0.75, 1.0, 1.25, and 1.5 mL were respectively taken into a 50 mL volumetric flask, and diluted with deionized water to make a concentration of 5, 10, 15, 20, 25, and 30 mg / L dilution solution as the standard curve solution.

[0032] The test solution was prepared as follows: 50 mg of mPEG1000-PCL was accurately weighed into a 10 mL centrifuge tube, 2 mL of dichloromethane was added to dissolve, and then 8 mL of deionized water was added, shaken, extracted, and allowed to stand. The aqueous phase was used as the test solution.

[0033] Colorimetric reagent dosage determination method: Take 5 mL of 20 mg / L mPEG1000 solution in a 10 mL colorimetric tube, add 1.0-2.0 mL of D reagent and 1 mL of pH = 4.8 acetic acid-sodium acetate buffer solution in sequence, dilute to the scale with deionized water, shake well, and color for 15 minutes; use a UV-visible spectrophotometer to perform a 200-800 nm spectrum scan to determine that the optimal dosage of D reagent is 1.8 mL.

[0034] mPEG1000 + Bi 4 -Maximum absorption wavelength determination method: Take 5 mL of 20 mg / L mPEG1000 standard solution in a 10 mL colorimetric tube, add 1.8 mL of D reagent and 1 mL of pH = 4.8 acetic acid-sodium acetate buffer solution in sequence, dilute to the scale with deionized water, shake well, and color for 15 minutes; use a UV-visible spectrophotometer to perform a 200-800 nm spectrum scan to determine that the complex has a maximum absorption value at 529 nm.

[0035] Calculation method of PEG residual amount: Linear regression is performed using the absorbance value of the standard solution and the concentration value to obtain linear equation 1: A = 0.0302C-0.0428, R 2 =0.9918, (C = 5-30 mg / L); Linear equation 2: A = 0.0192C + 0.0409, R 2 =0.9905, (C=0-10 mg / L), the free polyethylene glycol content can be calculated by substituting the absorbance value of the sample solution into the equation.

[0036] Implementation Case 2

[0037] Establishment of PEG1000 standard curve

[0038] The standard curve solution was prepared as follows: 0.25 g of PEG10000 was accurately weighed into a 250 mL volumetric flask, and diluted with deionized water to make a 1000 mg / L polyethylene glycol standard stock solution. 0.25, 0.5, 0.75, 1.0, 1.25, and 1.5 mL were respectively taken into a 50 mL volumetric flask, and diluted with deionized water to make a concentration of 5, 10, 15, 20, 25, and 30 mg / L dilution solution as the standard curve solution.

[0039] Color developer dosage determination method: Take 5 mL of 20 mg / L PEG1000 solution in a 10 mL colorimetric tube, add 1.0-2.0 mL of D reagent and 1 mL of pH = 4.8 acetic acid-sodium acetate buffer solution in sequence, dilute to the scale with deionized water, shake well, and color for 15 minutes; use a UV-visible spectrophotometer to perform a 200-800 nm spectrum scan to determine that the optimal dosage of D reagent is 1.6 mL.

[0040] PEG1000 + Bi 4 - Maximum absorption wavelength determination method: Take 5mL of 20mg / L PEG1000 aqueous solution in a 10mL colorimetric tube, add 1.6mL D reagent and 1mL pH=4.5 acetic acid-sodium acetate buffer solution in sequence, dilute to the scale with deionized water, shake well, and color for 15 minutes; use a UV-visible spectrophotometer to perform a 200-800nm ​​spectrum scan to determine that the complex has a maximum absorption value at 530nm.

[0041] Drawing of PEG1000 standard curve: Set the wavelength to 530nm, test the absorbance of 0, 5, 10, 15, 20, 25, 30mg / L PEG1000 solution mixed with the color developer to form a complex at 530nm, and draw a concentration-absorbance curve. A=0.0258C+0.0174,R 2 =0.9908 (C = 0-30 mg / L).

[0042] Table 1 Maximum absorption wavelength of 20 mg / L mPEG1000 standard solution at different dosages of reagent D.

[0043]

[0044] Table 2 Maximum absorption wavelength of 20mg / L PEG1000 standard solution at different dosages of reagent D.

[0045]

[0046] Implementation Case 3

[0047] Establishment of mPEG5000 standard curve

[0048] The standard curve solution was prepared as follows: 0.25 g of mPEG50000 was accurately weighed into a 250 mL volumetric flask, and diluted with deionized water to make a 1000 mg / L polyethylene glycol standard stock solution. 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, and 2.5 mL were respectively taken into a 50 mL volumetric flask, and diluted with deionized water to make a concentration of 5, 10, 15, 20, 25, 30, and 50 mg / L dilution solution as the standard curve solution.

[0049] mPEG5000 + Bi 4 - Maximum absorption wavelength determination method: Take 5mL of 20mg / L mPEG5000 aqueous solution in a 10mL colorimetric tube, add 1mL D reagent and 1mL pH = 4.8 acetic acid-sodium acetate buffer solution in sequence, dilute to the scale with deionized water, shake well, and color for 15 minutes; use a UV-visible spectrophotometer to perform a 200-800nm ​​spectrum scan to determine that the complex has a maximum absorption value at 509nm.

[0050] Drawing of mPEG5000 standard curve: Set the wavelength to 509nm, test the absorbance of 5, 10, 15, 20, 25, 30 and 50mg / L PEG solutions mixed with the color developer to form a complex at 509nm, and draw a concentration-absorbance curve; A=0.0157C+0.0149, R2 =0.9984 (C = 5-50 mg / L).

[0051] Implementation Case 4

[0052] The free PEG in the amphiphilic copolymer was detected using the standard detection method established in Example 1. Take the mPEG1000-PCL test solution with a molecular weight of 1.2w, add 1.8mL of D reagent and 1mL of pH = 4.8 acetic acid-sodium acetate buffer solution in sequence, dilute to the scale with deionized water, shake well, and color for 15 minutes. After testing and calculation, the concentration can be obtained to be 0.0021mg / mL.

[0053] Implementation Case 5

[0054] The free PEG in the amphiphilic copolymer was detected using the standard detection method established in Implementation Case 3. Take the mPEG1000-PLLA test solution with a molecular weight of 1.9w, add 1mL of D reagent and 1mL of pH=4.8 acetic acid-sodium acetate buffer solution in sequence, dilute to the scale with deionized water, shake well, and color for 15 minutes. After testing and calculation, the concentration can be obtained to be 0.0043mg / mL.

[0055] According to the above case implementation, the method of the present invention is simple to operate and has high accuracy in determining the free PEG content in amphiphilic polymers.

[0056] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

[0057] The present invention and its implementation methods are described above, which is not restrictive. The drawings are only one of the implementation methods of the present invention, and the actual application is not limited thereto. In short, if ordinary technicians in the field are inspired by it and design methods and embodiments similar to the technical solution without creativity without departing from the purpose of the invention, they should all fall within the protection scope of the present invention.

Claims

1. A method for detecting the PEG content in an amphiphilic copolymer, characterized in that: The following steps are involved: (1) Take PEG solution, add reagent D and buffer solution of gradient concentration, dilute to volume with deionized water, shake well, and develop color; use UV-visible spectrophotometer to perform spectral scanning at 200-800nm ​​to determine the optimal dosage of reagent D; (2) Take the PEG solution, add the optimal amount of D reagent and buffer solution, dilute to volume with deionized water, shake well, and develop color; use a UV-visible spectrophotometer to perform a 200-800 nm spectrum scan to determine the wavelength with the maximum absorption value of the complex; (3) preparing a PEG standard solution, diluting it to a gradient concentration, adding the optimal amount of D reagent and buffer solution, respectively, making up to volume with deionized water, shaking well, developing color, detecting the absorbance at the wavelength determined in step (3) and drawing a standard curve; (4) Take the test solution and add the optimal amount of D reagent and buffer in sequence, make up to volume with deionized water, shake well, develop color, detect the absorbance at the wavelength determined in step (3), and calculate the PEG content in the sample according to the standard curve; Among them, reagent D includes bismuth subnitrate, potassium iodide, glacial acetic acid and water.

2. The method for detecting the PEG content in the amphiphilic copolymer according to claim 1, characterized in that: The preparation method of the D reagent is: prepare a bismuth subnitrate solution and a potassium iodide solution respectively, and mix the bismuth subnitrate solution, the potassium iodide solution, glacial acetic acid and water in a volume ratio of 1:2:4:

13.

3. The method for detecting the PEG content in the amphiphilic copolymer according to claim 2, characterized in that: The concentration of the bismuth subnitrate solution is 0.01 mol / L, and the concentration of the potassium iodide solution is 2.4 mol / L.

4. The method for detecting the PEG content in the amphiphilic copolymer according to claim 1, characterized in that: The buffer solution is an acetic acid-sodium acetate buffer solution with a pH of 4.

8.

5. The method for detecting the PEG content in the amphiphilic copolymer according to claim 4, characterized in that: The preparation method of the buffer solution is as follows: weigh glacial acetic acid to prepare a 0.2 mol / L acetic acid solution; weigh sodium acetate to prepare a 0.2 mol / L sodium acetate solution; and measure the sodium acetate solution and the acetic acid solution in a volume ratio of 50:32 to prepare an acetic acid-sodium acetate buffer solution with a pH of 4.

8.

6. The method for detecting the PEG content in the amphiphilic copolymer according to claim 1, characterized in that: The preparation method of the test solution in step (4) comprises: taking a sample to be tested, accurately weighing the mass, adding dichloromethane and then deionized water, shaking and extracting, and letting stand, and taking the aqueous phase as the test solution; the sample to be tested is a copolymer sample whose PEG content is to be determined.

7. The method for detecting the PEG content in the amphiphilic copolymer according to claim 1, characterized in that: The color development time is 15 minutes; the gradient concentration of the standard solution dilution is in the range of 0-50 mg / L.

8. The method for detecting the PEG content in the amphiphilic copolymer according to claim 1, characterized in that: The PEG amphiphilic copolymer includes one or more of polyethylene glycol-poly L-lactic acid (PEG-PLLA), polyethylene glycol-polycaprolactone (PEG-PCL), polyethylene glycol-polylactide (PEG-PLGA), polyethylene glycol monomethyl ether-poly L-lactic acid (mPEG-PLLA), polyethylene glycol monomethyl ether-polycaprolactone (mPEG-PCL), and polyethylene glycol monomethyl ether-polylactide (mPEG-PLGA).

9. The method for detecting the PEG content in the amphiphilic copolymer according to claim 1, characterized in that: Standards include polyethylene glycol and polyethylene glycol monomethyl ether with molecular weights ranging from 1000 to 5000.

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