CN114813998B - Polymer detection method - Google Patents
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- CN114813998B CN114813998B CN202210296693.8A CN202210296693A CN114813998B CN 114813998 B CN114813998 B CN 114813998B CN 202210296693 A CN202210296693 A CN 202210296693A CN 114813998 B CN114813998 B CN 114813998B
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Abstract
The invention discloses a method for detecting the content of polymers Azide to (Amide to linker) to PEG to b to PLA, which is gel exclusion chromatography, and uses a high performance liquid chromatograph to detect, wherein the method can rapidly and accurately separate products and impurities through a porous gel chromatographic column, accords with the standard in the aspects of quantification, linear range and the like, can accurately detect the purity of the products in the linear range through an area normalization method, is convenient and fast to operate, is well separated, and has higher durability.
Description
Technical Field
The invention relates to the technical field of chromatography, in particular to a method for detecting polymer purity.
Background
Polylactic acid is a non-toxic, non-irritating biological material with good biological safety and degradability, and has been widely used in the biomedical material fields such as bone fixation repair materials, assembly engineering bracket materials, drug controlled release carriers and the like. However, polylactic acid materials are highly hydrophobic, which reduces biocompatibility with other substances.
An Azide-linker-PEG-b-PLA polymer with the structural formula of
The biological composite material not only maintains the original good properties of polylactic acid, such as easy degradation, no toxicity and no stimulation, but also introduces PEG groups into the linker, thereby greatly improving the hydrophilicity of the polylactic acid polymer, and the introduction of azide groups further widens the application fields of the polymer, such as surface modification, adhesion, drug delivery and the like.
However, in the prior art, there is no suitable detection method for the above-mentioned polymers.
If the reverse phase liquid phase detection is carried out by adopting a conventional chromatographic column, the phenomenon of poor main peak type can occur, and the aim of effectively separating other impurities can not be achieved, so that the purity of the polymer can not be effectively determined.
Exclusion chromatography is also known as space exclusion chromatography or gel permeation chromatography. Is a chromatographic technique for separation according to the size of the sample molecule. The separation mechanism of exclusion chromatography is three-dimensional exclusion, and no interaction exists between the sample components and the stationary phase. The packing material of the column is a gel, which is a surface inert, containing many pores or steric network of different sizes. The pore size of the gel was comparable to the size of the sample being separated. Only constituent molecules having a diameter smaller than the opening of the pores, which are relatively large for mobile phase molecules, are allowed to enter, so that mobile phase molecules can freely diffuse out of the person. For the component molecules with different sizes, the component molecules can respectively permeate into the gel pores to different depths, and a large number of component molecules can permeate into the gel macropores, but cannot enter the micropores, and are even completely repelled. Small component molecules can permeate into the large pores and the small pores, even enter deeply, and are not easy to elute at once. Thus, the large component molecules stay in the column for a short period of time, are quickly eluted, and their elution volumes (i.e., retention times) are small. The retention time of small component molecules in the chromatographic column is longer, the elution volume is kept at the bottom of the column, and the elution process of separating the small component molecules according to the size of the molecules is not completed until the smallest molecules in all the holes reach the outlet of the column.
However, in the prior art, no related report on detection of polymers Azide to (Amide to linker) to PEG to b to PLA by gel permeation chromatography has been found yet.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a method for detecting the content of polymers Azide to (Amide to linker) to PEG to b to PLA, which can effectively reflect the purity of the polymers, is beneficial to controlling the purity of the polymers, and has good separation degree and repeatability and high column efficiency.
The invention provides a method for detecting the content of a polymer, which is Azide (Amide-linker) to PEG-b-PLA, and has the structural formula of
Wherein the molecular weight of the PEG moiety is 4500-5500 daltons, preferably the PEG moietyHas a molecular weight of 5000 daltons; the molecular weight of the PLA moiety is 10000-20000 daltons, preferably the molecular weight of said PLA moiety is 16000 daltons; n is the degree of polymerization of polyethylene glycol residues and m is the degree of polymerization of polylactic acid residues.
The method is gel exclusion chromatography, and uses high performance liquid chromatograph to detect, and the chromatographic conditions are:
the high performance liquid chromatograph adopts porous gel as filler, the gel is one of resin-based gel, sephadex, polyacrylamide gel, agarose gel, polystyrene gel, sephadex LH-20 and silica gel, preferably the gel is high performance resin-based gel.
Further, the mobile phase in the gel exclusion chromatography is one of acetonitrile aqueous solution, methanol aqueous solution and tetrahydrofuran aqueous solution, preferably acetonitrile aqueous solution.
Further, the volume concentration of the mobile phase is 5 to 30% (v/v, e.g., 5%, 10%, 11%, 12%, 13%, 14%, 15%, 15.5%, 16%, 16.5%, 17%, 17.1%, 17.2%, 17.3%, 17.4%, 17.5%, 17.6%, 17.7%, 17.8%, 17.9%, 18%, 18.5%, 19%, 19.5%, 20%, 25%, 30%), preferably, the volume concentration of the mobile phase is 10 to 20%.
In one embodiment of the invention, the mobile phase has a volume concentration of 15%.
Further, the detection adopts an isocratic elution program.
Further, the elution time is 20 to 60 minutes (e.g., 20, 25, 30, 35, 40, 45, 50, 55, 60 minutes), preferably 30 to 50 minutes.
In one embodiment of the invention, the elution time is 40min.
Further, the exclusion molecular weight of the gel chromatographic column in the gel exclusion chromatography is 100-50000 daltons.
Further, the length of the gel chromatographic column is 30-350 mm (such as 33, 150, 300 mm), preferably the length of the gel chromatographic column is 300mm.
Further, the diameter of the gel chromatographic column is 4-8 mm (e.g. 4.6, 7.8 mm), preferably the diameter of the gel chromatographic column is 7.8mm.
Further, the pore diameter of the filler is
(e.g.)>
) Preferably, the pore size of the filler is +.>
Further, the particle size of the filler is 3 to 7 μm (e.g., 3 μm, 4 μm, 5 μm, 6 μm, 7 μm), preferably, the particle size of the filler is 5 μm.
Further, the flow rate of the high performance liquid chromatograph is 0.1-1.0 mL/min (such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 mL/min), preferably, the flow rate of the high performance liquid chromatograph is 0.5-0.8 mL/min.
In one embodiment of the invention, the flow rate of the high performance liquid chromatograph is 0.5mL/min.
Further, the detector of the high performance liquid chromatograph may be an electrospray detector (CAD), and the parameters are:
acquisition frequency: 5Hz;
filtration constants: 3.6s;
temperature: 35 ℃.
Further, the column temperature of the high performance liquid chromatograph is 25 to 45 ℃ (e.g., 25, 28, 30, 32, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 ℃), preferably 38 to 42 ℃.
In one embodiment of the invention, the column temperature of the high performance liquid chromatograph is 40 ℃;
further, the sample injection amount of the high performance liquid chromatograph is 10 to 30 μl (such as 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 μl), preferably 15 to 25 μl.
In one embodiment of the present invention, the sample injection amount of the high performance liquid chromatograph is 20 μl.
The method for measuring the content of the polymer Azide (Amide-linker) to PEG-b-PLA is an external standard method, and specifically comprises the following steps:
(1) And (3) making a standard curve: taking polymers Azide to (Amide to linker) to PEG to b to PLA with known content as a reference, carrying out gradient dilution by using a solvent to obtain a linear reference solution, detecting by using a gel exclusion chromatography, sucking the linear reference solution, injecting the linear reference solution into a high performance liquid chromatograph, passing through a gel chromatographic column, detecting by using an electrospray detector (CAD), recording a chromatogram, and obtaining parameters such as retention time, peak height, peak area and the like to obtain a standard curve;
(2) The measuring method comprises the following steps: placing samples to be detected, namely, the Azide (Amide-linker) to the PEG-b-PLA into a volumetric flask, adding a solvent into the volumetric flask to perform constant volume, obtaining a sample solution, detecting by using a gel exclusion chromatography, sucking the sample solution, injecting the sample solution into a high performance liquid chromatograph, passing through a gel column chromatography column, detecting by using an electrospray detector (CAD), recording the chromatograms, obtaining parameters such as peak height, peak area and the like, substituting relevant parameters corresponding to the polymers Azide (Amide-linker) to the PEG-b-PLA into a standard curve, and calculating to obtain the content of the polymers Azide (Amide-linker) to the PEG-b-PLA.
Further, the step (1) of preparing a control stock solution is further included before the linear control solution is obtained.
Further, the preparation of the reference substance stock solution comprises the steps of weighing polymers Azide to (Amide to linker) to PEG to b to PLA with known content, putting the polymers into a volumetric flask, and adding a solvent into the volumetric flask to fix the volume to prepare the reference substance stock solution.
Further, the method for detecting the polymer further comprises the steps of preparing a blank solution and preparing a quantitative limiting solution.
Further, the preparing of the blank solution includes a step of selecting a solvent as the blank solution.
Further, the preparation of the quantitative limiting solution comprises the steps of weighing polymers Azide to (Amide to linker) to PEG to b to PLA with known content, putting the polymers into a volumetric flask, and adding a mobile phase into the volumetric flask to perform constant volume to prepare the quantitative limiting solution.
Further, the solvent in the steps (1) and (2) may be one of acetonitrile, methanol and tetrahydrofuran, preferably acetonitrile.
Further, the high performance liquid chromatograph may be a Thermo U3000 high performance liquid chromatograph.
Further, the chromatographic column of the high performance liquid chromatograph can be Acclaim SEC-300,7.8mm×300mm;5 μm;
size exclusion chromatography column.
On the other hand, the invention provides the application of the detection method in the quality evaluation of the polymers Azide- (Amide-linker) -PEG5000-b-PLA 16K.
On the other hand, the invention provides application of the detection method in detection of high molecular polymers.
The detection method can rapidly and accurately separate products and impurities through the porous gel chromatographic column, accords with the standard in quantitative, linear range and other aspects, can accurately determine the purity of the products in the linear range through an area normalization method, is convenient to operate, is good in separation, and has higher durability.
Drawings
FIG. 1 is a nuclear magnetic spectrum of polymers Azide to (Amide to linker) to PEG5000 to b to PLA 16K.
FIG. 2 is a liquid chromatogram of a test performed using a SEC-1000 column.
FIG. 3 is a liquid chromatogram of the detection using a flow rate of 0.4mL/min.
Fig. 4 is a liquid chromatogram of a sample solution.
Fig. 5 is a liquid chromatogram of a blank solution.
FIG. 6 is a liquid chromatogram of a quantitative limiting solution.
FIG. 7 is a graph of the linear relationship of the linear control solution.
FIG. 8 is a liquid chromatogram of polymer Azide- (Amide-linker) -PEG5000-b-PLA16K for reverse phase liquid detection using octadecylsilane chemically bonded silica column.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The structural formula of the polymer Azide- (Amide-linker) -PEG5000-b-PLA16K in the embodiment of the application is that
Wherein n is the degree of polymerization of polyethylene glycol residues, m is the degree of polymerization of polylactic acid residues, and the molecular weight of the PEG moiety is 5000 daltons; the molecular weight of the PLA fraction is 16000 daltons.
The characterization results of the polymers Azide to (Amide to linker) to PEG5000 to b to PLA16K are as follows:
1. nuclear magnetic characterization:
the nuclear magnetic spectrum of the polymer Azide to (Amide to linker) to PEG5000 to b to PLA16K is shown in figure 1.
2. And (3) viscosity characterization:
and (3) carrying out viscosity characterization on the polymers Azide to (Amide to linker) to PEG5000 to b to PLA16K by adopting a capillary method, and measuring the viscosity to be 0.32dL/g.
Example 1
Screening of chromatographic columns:
1. chromatographic conditions:
instrument: thermo U3000 high performance liquid chromatograph
Chromatographic column: molecular exclusion chromatography column with porous gel as stationary phase (Thermo, acclaim SEC-1000,7.8 mm. Times.300 mm, 5 μm; 1000A)
Mobile phase: the volume ratio of acetonitrile to water is 15:85.
elution procedure: isocratic elution, 40min.
Flow rate: 0.4mL/min.
Column temperature: 40 ℃.
Sample injection amount: 20. Mu.L.
Work station: chromeleon.
2. Preparing a solution:
sample solution: precisely weighing about 40mg of Azide- (Amide-linker) -PEG5000-b-PLA16K, placing in a 10mL volumetric flask, adding a proper amount of acetonitrile for dissolution, adding acetonitrile for volume fixation to a scale, and shaking uniformly to obtain a 4mg/mL sample solution.
3. And (3) measuring: and (3) adopting a high performance liquid chromatograph, precisely sucking the prepared solution for detection by using a gel exclusion chromatography, passing through a gel column chromatography column, detecting by using an electrospray detector (CAD), recording a chromatogram, and obtaining parameters such as peak height, peak area and the like.
As shown in fig. 2, the peak shape of the model chromatographic column is wider.
Example 2
Screening of chromatographic condition flow rate:
1. chromatographic conditions:
instrument: thermo U3000 high performance liquid chromatograph
Chromatographic column: molecular exclusion chromatography column with porous gel as stationary phase (Thermo, acclaim SEC-300,7.8 mm. Times.300 mm, 5 μm; 300A)
Mobile phase: the volume ratio of acetonitrile to water is 15:85.
elution procedure: isocratic elution, 40min.
Flow rate: 0.4mL/min.
Column temperature: 40 ℃.
Sample injection amount: 20. Mu.L.
Work station: chromeleon.
2. Preparing a solution:
sample solution: precisely weighing about 40mg of Azide- (Amide-linker) -PEG5000-b-PLA16K, placing in a 10mL volumetric flask, adding a proper amount of acetonitrile for dissolution, adding acetonitrile for volume fixation to a scale, and shaking uniformly to obtain a 4mg/mL sample solution.
3. Measurement
And (3) adopting a high performance liquid chromatograph, precisely sucking the prepared solution for detection by using a gel exclusion chromatography, passing through a gel column chromatography column, detecting by using an electrospray detector (CAD), recording a chromatogram, and obtaining parameters such as peak height, peak area and the like. As shown in fig. 3, the flow rate separation degree is poor.
Example 3:
1. chromatographic conditions:
instrument: thermo U3000 high performance liquid chromatograph
Chromatographic column: molecular exclusion chromatography column with porous gel as stationary phase (Thermo, acclaim SEC-300,7.8 mm. Times.300 mm, 5 μm; 300A)
Mobile phase: the volume ratio of acetonitrile to water was 15:85.
Elution procedure: isocratic elution, 40min.
Flow rate: 0.5mL/min.
Column temperature: 40 ℃.
Sample injection amount: 20. Mu.L.
Work station: chromeleon.
Blank solution: acetonitrile.
2. Preparation of the solution
Control stock solution: about 200mg of Azide- (Amide-linker) -PEG5000-b-PLA16K is precisely weighed, placed in a 10mL volumetric flask, dissolved by adding a proper amount of acetonitrile, then added with acetonitrile to a scale, and shaken well.
Sample solution: precisely weighing about 40mg of Azide- (Amide-linker) -PEG5000-b-PLA16K, placing in a 10mL volumetric flask, adding a proper amount of acetonitrile for dissolution, adding acetonitrile for volume fixation to a scale, and shaking uniformly to obtain a 4mg/mL sample solution.
Limit of quantitation (LOQ) solution: precisely weighing about 10mg of Azide- (Amide-linker) -PEG5000-b-PLA16K, placing in a 10mL volumetric flask, adding a proper amount of acetonitrile for dissolution, adding acetonitrile for volume fixation to a scale, shaking uniformly to obtain 1mg/mL solution, accurately sucking 0.4mL of the solution in the 10mL volumetric flask, and diluting with acetonitrile to the scale.
Linear control solution: accurately transferring the control stock solution (20 mg/mL) into volumetric flasks of 0.02mL, 0.04mL, 0.05mL, 0.1mL, 0.25mL, 1.0mL, 1.5mL and 2.0mL to 10mL respectively, and adding acetonitrile to fix the volume to the scale to obtain solutions of 0.04mg/mL, 0.08mg/mL, 0.1mg/mL, 0.2mg/mL, 0.5mg/mL, 2mg/mL, 3mg/mL and 4 mg/mL.
3. Measurement
And (3) adopting a high performance liquid chromatograph, precisely sucking the various prepared solutions for detection by using a gel exclusion chromatograph, passing through a gel column chromatographic column, detecting by using an electrospray detector (CAD), recording a chromatogram, and obtaining parameters such as peak height, peak area and the like.
Fig. 4 to 5 are liquid chromatograms of a sample solution, a blank solution, and a quantitative limiting solution, respectively.
Table 1 shows the linear results of the polymer Azide- (Amide-linker) -PEG5000-b-PLA16K linear control solution.
TABLE 1
The standard curve is plotted according to the results of Table 1 as shown in FIG. 7, and the regression coefficient R corresponding to the linear equation 2 The fitting degree of the linear equation is better as 0.9970.
Comparative example 1:
the polymer Azide- (Amide-linker) -PEG5000-b-PLA16K was subjected to reversed phase liquid phase detection using octadecylsilane chemically bonded silica column.
1. Chromatographic conditions:
instrument: SHIMADZU high performance liquid chromatograph
Chromatographic column: chromatography column with octadecylsilane chemically bonded silica gel as stationary phase (Thermo, hypersil GOLD C18,4.6 mm. Times.250 mm, 5 μm)
Mobile phase: mobile phase a was water and mobile phase B was acetonitrile.
Elution procedure: gradient elution was performed for 20min.0min, the volume ratio of the mobile phase A to the mobile phase B is 90:10; performing linear elution after 0-2min, wherein the volume ratio of the mobile phase A to the mobile phase B is linearly gradually changed to 30:70; 2-10min, linearly gradually changing the volume ratio of the mobile phase A to the mobile phase B to 5:95, and performing linear elution; performing isocratic elution for 10-16min with the volume ratio of mobile phase A to mobile phase B of 5:95; the volume ratio of the mobile phase A to the mobile phase B is gradually changed into 90:10 linearly for 16-17min, and linear elution is carried out; performing isocratic elution after 17-20min with the volume ratio of mobile phase A to mobile phase B of 90:10;
flow rate: 1mL/min.
Column temperature: 40 ℃.
Sample injection amount: 10 mu L.
Work station: labsolution.
2. Preparing a solution:
sample solution: about 20mg of Azide- (Amide-linker) -PEG5000-b-PLA16K is precisely weighed, placed in a 10mL volumetric flask, dissolved by adding a proper amount of acetonitrile, then added with acetonitrile to fix the volume to a scale, and uniformly shaken to obtain 2mg/mL sample solution.
3. Measurement
Adopting a high performance liquid chromatograph, using an octadecylsilane chemically bonded silica column, precisely sucking the prepared solution for detection, using an electrospray detector (CAD) for detection, recording a chromatogram, and obtaining parameters such as peak height, peak area and the like. As shown in FIG. 8, when the polymer Azide- (Amide-linker) -PEG5000-b-PLA16K was subjected to reverse phase liquid phase detection by using an octadecylsilane chemically bonded silica column, the obtained chromatogram had poor peak shape and poor effect.
Claims (16)
1. A method for detecting content of a polymer is characterized in that the polymer is Azide (Amide-linker) to PEG-b-PLA, and the structural formula of the polymer is as follows
Wherein n is the polymerization degree of polyethylene glycol residue, m is the polymerization degree of polylactic acid residue, and the molecular weight of the PEG part is 4500-5500 daltons; the molecular weight of the PLA part is 10000-20000 daltons, the method is gel exclusion chromatography, the detection is carried out by using a high performance liquid chromatograph, and the chromatographic conditions are as follows:
the chromatographic column of the high performance liquid chromatograph adopts porous gel as filler, and the gel is resin-based gel;
the detection adopts an isocratic elution program;
the mobile phase in the gel exclusion chromatography is acetonitrile aqueous solution, and the mobile phase is: the volume ratio of acetonitrile to water is 15:85.
2. the method for detecting the content of the polymer according to claim 1, wherein the elution time is 20-60 min.
3. The method for detecting the polymer content according to claim 2, wherein the elution time is 30-50 min.
4. The method of claim 2, wherein the elution time is 40 minutes.
5. The method for detecting the polymer content according to claim 1, wherein the exclusion molecular weight of the gel column in the gel exclusion chromatography is 100-50000 daltons.
6. The method for detecting the polymer content according to claim 1, wherein the pore diameter of the filler is 100-300 a.
7. The method of claim 6, wherein the filler has a pore size of 300 a.
8. The method for detecting the content of the polymer according to claim 1, wherein the particle size of the filler is 3-7 μm.
9. The method for detecting the content of a polymer according to claim 8, wherein the filler has a particle size of 5 μm.
10. The method for detecting the polymer content according to claim 1, wherein the flow rate of the high performance liquid chromatograph is 0.1-1.0 ml/min.
11. The method for detecting the content of the polymer according to claim 10, wherein the flow rate of the high performance liquid chromatograph is 0.5-0.8 ml/min.
12. The method for detecting the content of the polymer according to claim 10, wherein the flow rate of the high performance liquid chromatograph is 0.5mL/min.
13. The method for detecting the polymer content according to claim 1, wherein the column temperature of the high performance liquid chromatograph is 25-45 ℃.
14. The method for detecting the polymer content according to claim 1, wherein the column temperature of the high performance liquid chromatograph is 38-42 ℃.
15. The method for detecting the content of the polymer according to claim 1, wherein the column temperature of the high performance liquid chromatograph is 40 ℃.
16. The method for detecting the content of the polymer according to claim 1, wherein the molecular weight of the PEG part is 5000 daltons, the molecular weight of the PLA part is 16000 daltons, and the method for detecting the content of the polymer is an external standard method, which specifically comprises the following steps:
(1) And (3) making a standard curve: taking polymers Azide to (Amide to linker) to PEG to b to PLA with known content as a reference, carrying out gradient dilution by using a solvent to obtain a linear reference solution, detecting by using a gel exclusion chromatography, sucking the linear reference solution, injecting the linear reference solution into a high performance liquid chromatograph, passing through a gel chromatographic column, detecting under a detector, recording a chromatogram, and obtaining retention time, peak height and peak area to obtain a standard curve;
(2) The measuring method comprises the following steps: placing samples to be tested, namely, amide-linker, PEG-b-PLA into a volumetric flask, adding a solvent into the volumetric flask to fix the volume to obtain a sample solution, detecting by using a gel exclusion chromatography, sucking the sample solution, injecting the sample solution into a high performance liquid chromatograph, passing through the gel column chromatography, detecting under a detector, recording the chromatograms, obtaining peak heights and peak areas, substituting relevant parameters corresponding to the polymers, namely, amide-linker, PEG-b-PLA into a standard curve, and calculating to obtain the content of the polymers, namely, amide-linker, PEG-b-PLA.
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| EP2634179A1 (en) * | 2012-02-28 | 2013-09-04 | Sanofi | Functional PLA-PEG copolymers, the nanoparticles thereof, their preparation and use for targeted drug delivery and imaging |
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| EP2634179A1 (en) * | 2012-02-28 | 2013-09-04 | Sanofi | Functional PLA-PEG copolymers, the nanoparticles thereof, their preparation and use for targeted drug delivery and imaging |
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