CN116930365A - Detection method of single-ended EPOX modified PEG product - Google Patents

Detection method of single-ended EPOX modified PEG product Download PDF

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CN116930365A
CN116930365A CN202310919859.1A CN202310919859A CN116930365A CN 116930365 A CN116930365 A CN 116930365A CN 202310919859 A CN202310919859 A CN 202310919859A CN 116930365 A CN116930365 A CN 116930365A
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mobile phase
epox
solution
volume ratio
methanol
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李洋
李旭哲
何平
贺娇
赵宣
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Tianjin Jenkem Technology Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
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    • G01N2030/067—Preparation by reaction, e.g. derivatising the sample

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Abstract

The application provides a detection method of a single-ended EPOX modified PEG product, which comprises the following steps: (1) pretreatment: mixing the single-end EPOX modified PEG product with undecylamine, and carrying out a derivatization reaction to obtain a derivatization solution; (2) Detecting the derivative solution obtained in the step (1) by an HPLC-CAD method, wherein the main component of the single-ended EPOX modified PEG product is methoxy-polyethylene glycol-epoxy group. The detection method can effectively separate methoxy-polyethylene glycol-epoxy groups and active impurities in the single-ended EPOX modified PEG product, so that the quality of the single-ended EPOX modified PEG product is effectively controlled, and the method completely accords with the standard in the aspects of system applicability, specificity, detectability, quantification, linear range, recovery rate, repeatability and the like and has higher durability.

Description

Detection method of single-ended EPOX modified PEG product
Technical Field
The application belongs to the technical field of chemical analysis, and particularly relates to a detection method of a single-ended EPOX modified PEG product.
Background
Methoxy-polyethylene glycol-epoxy (Methoxy PEG Epoxide), abbreviated as mPEG-EPOX or M-EPOX, is a single-ended EPOX modified PEG product, which contains polyethylene glycol and epoxy in its structure. Methoxy-polyethylene glycol-epoxy is prepared by reacting Methoxy-polyethylene glycol (M-PEG) with epibromohydrin, and the Methoxy-polyethylene glycol-epoxy product may contain impurities including active impurities such as epoxy-polyethylene glycol-epoxy (Epoxide PEG Epoxide, EPOX-PEG-EPOX) and the like, and other impurities such as a small amount of unreacted Methoxy-polyethylene glycol, a small amount of fully ring-opened Methoxy-polyethylene glycol-glycerol (Methoxy PEG Glycerol, M-GLC) and the like. The epoxy structure in methoxy-polyethylene glycol-epoxy group can be connected with other compounds by ring opening under alkaline condition, so that it can be used as coupling agent. However, the active impurities generated during the preparation of methoxy-polyethylene glycol-epoxy groups, even if at low levels, still affect the quality of the coupled product, and therefore, a method for detecting the active impurities in methoxy-polyethylene glycol-epoxy groups is needed. However, methoxy-polyethylene glycol-epoxy groups are similar in chemical structure to methoxy-polyethylene glycol and methoxy-polyethylene glycol-glycerol, which are main impurities, and have similar molecular weights, are different only in end groups, have poor separation effect on a common C18 chromatographic column, and cannot be detected on an ultraviolet detector.
Zhang Wei et al (see HPLC-CAD determination of the free PEG content in the Nonoxynol Ether and Nonoxynol Ether plug [ J ]]Journal of pharmaceutical analysis, 2021,41 (08): 1417-1422.) discloses a method for determining the content of free polyethylene glycol in nonoxynol plugs by HPLC-CAD using HALO ES-C 18 Chromatographic column (4.6 mm. Times.250 mm,5 μm), mobile phase methanol-water (80:20), flow rate 1.0mL/min, column temperature 35 ℃, detector CAD. The results showed that PEG400 had a lower detection limit of 0.006. Mu.g, a lower quantification limit of 0.01. Mu.g, and a linear range of 1.05 to 5.25. Mu.g/mL. Wherein, the diluting solvent of the nonoxynol plug is changed from methanol to mobile phaseOn one hand, the interference of solvent peaks to PEG peaks is eliminated; on the other hand, the solvent contains a certain proportion of water, so that the volatilization of the solvent in the detection process can be reduced, and the measurement result is more accurate.
Patent document CN114778712a discloses a method for detecting the content of polyethylene glycol lipid and lipid nano particles containing the lipid, the method is characterized in that the method is used for detecting by high performance liquid chromatography, reverse phase chromatography is adopted, a detector is an electrospray detector, a mobile phase a is 0.01-0.5% trifluoroacetic acid aqueous solution, a mobile phase B is acetonitrile or methanol, and gradient elution is carried out.
At present, no report on separation analysis of main components and active impurities in methoxy-polyethylene glycol-epoxy products exists.
Disclosure of Invention
In order to overcome the defects of the prior art, the application aims to provide a detection method of a single-end EPOX modified PEG product, which can effectively separate main components and impurities, has good chromatographic peak shape, can effectively react the purity of the product, and has good separation degree and repeatability and high column efficiency.
In a first aspect of the present application, there is provided a method for detecting a single-ended EPOX-modified PEG product, the method comprising the steps of:
(1) Pretreatment: mixing the single-end EPOX modified PEG product with undecylamine, and carrying out a derivatization reaction to obtain a derivatization solution;
(2) Detecting the derivative solution obtained in the step (1) by an HPLC-CAD method;
preferably, the main component of the single-ended EPOX modified PEG product is methoxy-polyethylene glycol-epoxy group, and the structural formula isn is the degree of polymerization of the polyethylene glycol residues and has an average molecular weight of 1000 to 20000 daltons, preferably 5000 daltons.
Further, the undecylamine is dissolved in an organic solvent, and the organic solvent is one or more selected from methanol, ethanol, acetonitrile, ethyl acetate, acetone, dichloromethane, chloroform and cyclohexane, preferably methanol.
Further, the mass percentage concentration of the undecylamine is 10-40mg/mL, specifically 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40mg/mL, preferably 20-30mg/mL, more preferably 20mg/mL.
Further, the temperature of the derivatization reaction is 45 to 70 ℃, specifically 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 ℃, preferably 55 to 65 ℃, more preferably 60 ℃.
Further, in the HPLC-CAD method, the chromatographic conditions are as follows:
the stationary phase of the chromatographic column is octadecylsilane chemically bonded silica gel with end capping;
mobile phase a is an aqueous solution of trifluoroacetic acid having a concentration of 0.01-5% by volume, specifically such as 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5%, preferably 0.05-2%, more preferably 0.1%;
the mobile phase B is a mixed solution of acetonitrile, methanol and trifluoroacetic acid, wherein the volume ratio of acetonitrile to methanol is 1-10:1 (specifically, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1), the volume ratio of the trifluoroacetic acid to the mixed solution of acetonitrile and methanol is 0.01-5%:1 (concretely, 0.01 percent 1, 0.05 percent 1, 0.1 percent 1, 0.15 percent 1, 0.2 percent 1, 0.25 percent 1, 0.3 percent 1, 0.35 percent 1, 0.4 percent 1, 0.45 percent 1, 0.5 percent 1, 0.6 percent 1, 0.7 percent 1, 0.8 percent 1, 0.9 percent 1, 1 percent 1, 1.5 percent 1, 2 percent 1, 2.5 percent 1, 3 percent 1, 3.5 percent 1,4 percent 1, 4.5 percent 1, 5 percent 1).
Preferably, the volume ratio of acetonitrile to methanol is 1-5:1, more preferably 7:3.
preferably, the volume ratio of the trifluoroacetic acid to the mixed solution of acetonitrile and methanol is 0.05-2%:1, more preferably 0.1%:1.
a gradient elution procedure was used, which was as follows:
0min, the volume ratio of mobile phase A to mobile phase B is 80:20, a step of;
0-2min, the volume ratio of the mobile phase A to the mobile phase B is 80:20, performing isocratic elution;
2-5min, the volume ratio of the mobile phase A to the mobile phase B is gradually changed into 31 according to linearity: 69;
5-12min, the volume ratio of the mobile phase A to the mobile phase B is gradually changed into 30 according to linearity: 70;
and (3) for 12-15min, the volume ratio of the mobile phase A to the mobile phase B is gradually changed into 25 according to linearity: 75;
15-17min, the volume ratio of mobile phase A to mobile phase B is 25:75; performing isocratic elution;
17-20min, the volume ratio of the mobile phase A to the mobile phase B gradually changes to 0 according to linearity: 100;
20-25min, the volume ratio of the mobile phase A to the mobile phase B is 0:100, performing isocratic elution;
25-25.1min, the volume ratio of the mobile phase A to the mobile phase B is gradually changed into 80 according to linearity: 20, a step of;
25.1-32min, the volume ratio of mobile phase A to mobile phase B is 80:20, performing isocratic elution.
Further, the packing of the chromatographic column is silica with a modified surface, and the specification is 5 μm and 4.6x250mm.
Further, the flow rate of the mobile phase is 0.1-5mL/min, specifically, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5mL/min, preferably 1mL/min.
Further, in the HPLC-CAD method, the column temperature is 35 to 45 ℃, specifically 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 ℃, preferably 40 ℃.
Further, in the HPLC-CAD method, the sample introduction amount is 1 to 20. Mu.L, specifically, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20. Mu.L, preferably 10. Mu.L.
Further, parameters of the electrospray detector are as follows:
range of signal: 1-500pA, such as 100, 200, 500pA, preferably 500pA;
filtration constants: 0.1-10sec, specifically such as 0.1, 0.2, 0.5, 1, 2, 3.6, 5, 10sec, preferably 10sec;
temperature: 30-40 ℃, such as 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 ℃, preferably 35 ℃.
Further, in the detection method, the detection target comprises qualitative and quantitative detection of the main component methoxy-polyethylene glycol-epoxy group and qualitative and quantitative detection of impurities (including active impurities and other impurities).
Further, the active impurities include EPOX-PEG-EPOX and the like.
Further, the other impurities include M-PEG, M-GLC, etc.
Further, the calculation method of the purity of the methoxy-polyethylene glycol-epoxy group and the content of impurities (including active impurities and other impurities) comprises a main component self-comparison method (comprising a main component self-comparison method with correction factors and a main component self-comparison method without correction factors), an external standard method or an area normalization method.
Further, the purity of the main component (i.e., methoxy-polyethylene glycol-epoxy group) and the impurity content were calculated by a main component self-comparison method without adding correction factors, and the calculation formula is as follows:
impurity% = impurity peak area/total peak area 100
Principal component% = 1-SUM (impurity percentage)
In a second aspect of the application, there is provided the use of the detection method of the first aspect of the application in the quality assessment of single ended EPOX modified PEG products.
The application has the following beneficial effects:
the detection method can effectively separate methoxy-polyethylene glycol-epoxy groups and active impurities in the single-ended EPOX modified PEG product, so that the quality of the single-ended EPOX modified PEG product is effectively controlled, and the method completely accords with the standard in the aspects of system applicability, specificity, detectability, quantification, linear range, recovery rate, repeatability and the like and has higher durability.
Drawings
FIG. 1 shows chromatograms of sample derived solution 2 (black), dodecylamine solution (blue).
Fig. 2 shows a chromatogram of an undecylamine solution.
FIG. 3 shows chromatograms of sample-derived solution 1 (blue), sample-derived solution 3 (black), and sample solution (purple).
FIG. 4 shows chromatograms of sample derived solution 3 (black), sample derived solution 4 (blue), and sample solution (purple).
Fig. 5 shows a linear relationship diagram of a linear solution.
FIG. 6 shows a chromatogram of a test solution.
FIG. 7 shows a chromatogram of a 1% sample derived solution.
FIG. 8 shows a chromatogram of a 100% sample derived solution.
FIG. 9 shows a chromatogram of a mixed labeling solution of EPOX-5K-EPOX and EPOX-10K-EPOX derivative solutions.
Fig. 10 shows a mass spectrum of active impurity 1.
Fig. 11 shows a mass spectrum of active impurity 3.
Fig. 12 shows a mass spectrum of the active impurity 4.
Detailed Description
Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application relates.
In the present application, the term "M-EPOX (Methoxy PEG Epoxide)", also known as methoxy-polyethylene glycol-epoxy group, mPEG-EPOX, has the structural formulan is the degree of polymerization of the polyethylene glycol residues. It is a polyethylene glycol modifier, has good water solubility, and good compatibility with many organic solvents, and belongs to soluble polymer for supporting polypeptide synthesis or preparation of various polymersThe ethylene glycol conjugate has wide application in medicine research, medicine property slow release, new nano material research, cell culture and other fields. Typically, the average molecular weight of M-EPOX varies from thousands to tens of thousands, such as M-EPOX-1K, M-EPOX-2K, M-EPOX-5K, M-EPOX-10K, M-EPOX-20K, and the like.
In the application, the term M-PEG (Methoxy PEG), also known as methoxy-polyethylene glycol, polyethylene glycol monomethyl ether and mPEG, has the structural formulan is the degree of polymerization of the polyethylene glycol residues. It has good water solubility, wettability, lubricity, physiological inertia, no stimulation to human body, mildness, and wide application in cosmetics and pharmaceutical industry. Typically, the average molecular weight of M-PEG varies from several hundred to several tens of thousands, such as M-PEG-200, M-PEG-1K, M-PEG-2K, M-PEG-5K, M-PEG-10K, M-PEG-20K, and the like.
In the present application, the term "M-GLC (Methoxy PEG Glycerol)", also known as methoxy-polyethylene glycol-glycerol, has the structural formulan is the degree of polymerization of the polyethylene glycol residues. In general, the average molecular weight of M-GLC varies from thousands to tens of thousands, such as M-GLC-1K, M-GLC-2K, M-GLC-5K, M-GLC-10K, M-GLC-20K, and the like.
In the present application, the term "EPOX-PEG-EPOX (Epoxide PEG Epoxide, EPOX-PEG-EPOX)", also known as epoxy-polyethylene glycol-epoxy, ethylene oxide-polyethylene glycol-ethylene oxide, has the structural formulan is the degree of polymerization of the polyethylene glycol residues. Typically, the average molecular weight of the EPOX-PEG-EPOX varies from a few thousand to a few tens of millions, such as EPOX-PEG5K-EPOX, EPOX-PEG10K-EPOX, and the like.
In the application, the term PEG refers to polyethylene glycol, is a high molecular polymer, has no irritation, slightly bitter taste, good water solubility and good compatibility with a plurality of organic components, can be used as antistatic agents, softeners and the like, and has extremely wide application in industries such as cosmetics, pharmacy, chemical fiber, rubber, plastics, papermaking, paint, electroplating, pesticides, metal processing, food processing and the like.
In the present application, the term "stationary phase" is a phase that will be immobilized in chromatographic separation, resulting in retention of a sample.
In the present application, the term "mobile phase" refers to a substance that carries a component to be measured forward during chromatography, referred to as a mobile phase. And the other phase is in an equilibrium state with the stationary phase and drives the sample to move forward.
In the present application, the term "electrospray detector" is a universal detector that can detect any non-volatile and partially semi-volatile species and is gradient compatible for liquid chromatography qualitative and quantitative analysis of compounds without ultraviolet absorption, using the principle that the mass of the analyte in the sample is proportional to the signal current.
In the present application, the term "high-low concentration comparison method", also called main component self-comparison method, is used for measuring impurity content. The method can be classified into a principal component self-comparison method with correction factors added and a principal component self-comparison method without correction factors added. In the examples, the principal component self-alignment method without adding correction factors was used.
Principal component self-comparison with correction factor: precisely weighing appropriate amounts of the impurity reference substance and the reference substance to be measured, preparing a solution for measuring the impurity correction factor, injecting sample, recording a chromatogram, calculating the impurity correction factor according to a formula, namely, correction factor (f) = (As×Cr)/(Ar×Cs), wherein As is the peak area or peak height of the impurity reference substance, cr is the concentration of the reference substance to be measured, ar is the peak area or peak height of the reference substance to be measured, and Cs is the concentration of the component to be measured of the impurity reference substance. When the impurity content is measured, the solution to be tested is diluted to a solution equivalent to the specified medium limit according to the specified impurity limit under each variety item to be used as a control solution, and the sample is injected, so that the sensitivity of the instrument is adjusted, and the peak of the main component of the control solution reaches 10% -25% of the full scale range. And then taking proper amounts of the sample solution and the control solution, respectively sampling, wherein the recording time of the sample solution is 2 times of the retention time of the chromatographic peak of the main component except for the other stipulations, measuring the peak area of each impurity on the chromatogram of the sample solution, respectively multiplying the peak areas by corresponding correction factors, comparing the peak areas with the main component of the control solution, and then calculating the content of each impurity according to law.
Principal component self-comparison without correction factor: when there is no impurity reference substance, the main component self-reference method without correction factor can also be adopted. After preparing the control solution and adjusting the sensitivity of the instrument, taking a proper amount of the sample solution and the control solution, respectively injecting samples, wherein the recording time of the sample solution and the control solution is 2 times of the retention time of the chromatographic peak of the main component except for other regulations, measuring the peak area of each impurity on the chromatogram of the sample solution, comparing the peak area with the peak area of the main component of the control solution, and calculating the impurity content.
The structural formula of the polymer M-EPOX-5K in the embodiment of the application isThe average molecular weight was 5000 daltons, and n was the degree of polymerization of polyethylene glycol residues, supplied by Tianjin Kekai technology Co., ltd.
The polymers of the embodiment of the application, namely the EPOX-5K-EPOX and the EPOX-10K-EPOX, have the structural formula ofThe average molecular weight was 5000 daltons and 10000 daltons, respectively, and n was the degree of polymerization of polyethylene glycol residues, provided by Tianjin Kekai technology Co., ltd.
Embodiments of the present application will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present application and should not be construed as limiting the scope of the present application. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1: method optimization
1.1 chromatographic conditions
Instrument: an Shimadzu LC-40D high performance liquid chromatograph;
chromatographic column: the surface modified silica was filler (GL ScienceInertSustain C; 5 μm,4.6 x 250 mm);
mobile phase a: 0.1% (volume percent) aqueous trifluoroacetic acid solution, mobile phase B: 0.1% acetonitrile trifluoroacetate was used: methanol=7:3 (v/v), gradient elution was performed as in table 1;
detection by electrospray detector (CAD), the parameters are: range of Range or Range of signals: 500pA; filtration constants: 10sec; temperature: 35 ℃;
column temperature: 40 ℃;
flow rate: 1.0mL/min;
sample injection amount: 10. Mu.L;
work station: LC-Solution.
TABLE 1 gradient elution procedure
Time Mobile phase a Mobile phase B
0 80 20
2 80 20
5 31 69
12 30 70
15 25 75
17 25 75
20 0 100
25 0 100
25.1 80 20
32 80 20
1.2 preparation of solutions
Undecylamine solution: about 1g of the undecylamine sample is weighed and placed in a 50mL volumetric flask, a methanol solution is added for dissolution, and the volume is fixed to a scale, so that an undecylamine solution with the concentration of 20mg/mL is obtained.
Undecylamine solution (30 mg/mL): about 0.75g of the undecylamine sample is weighed and placed in a 25mL volumetric flask, a methanol solution is added for dissolution, and the volume is fixed to a scale, so that an undecylamine solution with the concentration of 30mg/mL is obtained.
Dodecylamine solution: about 1g of the dodecylamine sample is weighed and placed in a 50mL volumetric flask, a methanol solution is added for dissolution, and the volume is fixed to a scale, so that the dodecylamine solution with the concentration of 20mg/mL is obtained.
Sample derived solution 1: about 50mg of an M-EPOX-5K sample (supplied by Tianjin Kaiki technology Co., ltd.) is weighed and placed in a 5mL volumetric flask, an undecylamine solution with the concentration of 20mg/mL is added for dissolution, the volume is fixed to a scale, and the solution is placed in an air bath shaking table at the temperature of 60 ℃ and the rotating speed of 200rpm, and is subjected to overnight derivatization, so that a sample derivatization solution 1 is obtained.
Test article derivative solution 2: about 50mg of an M-EPOX-5K sample (supplied by Tianjin Kaiki technology Co., ltd.) is weighed and placed in a 5mL volumetric flask, a dodecylamine solution with the concentration of 20mg/mL is added for dissolution, the volume is fixed to a scale, and the mixture is placed in an air bath shaking table at the temperature of 60 ℃ and the rotating speed of 200rpm, and is subjected to overnight derivatization, so that a sample derivatization solution 2 is obtained.
Sample derived solution 3: about 50mg of an M-EPOX-5K sample (supplied by Tianjin Kaiki technology Co., ltd.) is weighed and placed in a 5mL volumetric flask, an undecylamine solution with the concentration of 20mg/mL is added for dissolution, the volume is fixed to a scale, and the mixture is placed in an air bath shaking table at the temperature of 45 ℃ and the rotating speed of 200rpm, and is subjected to overnight derivatization, so that a sample derivatization solution 3 is obtained.
Sample derived solution 4: about 50mg of an M-EPOX-5K sample (supplied by Tianjin Kaiki technology Co., ltd.) is weighed and placed in a 5mL volumetric flask, an undecylamine solution with a concentration of 30mg/mL is added for dissolution, the volume is fixed to a scale, and the mixture is placed in an air bath shaking table at a temperature of 45 ℃ and a rotating speed of 200rpm for overnight derivatization, so that a sample derivatization solution 4 is obtained.
Test solution: about 50mg of the M-EPOX-5K sample is weighed and placed in a 5mL volumetric flask, methanol is added for dissolution, and the volume is fixed to a scale, so that a sample solution with the concentration of 10mg/mL is obtained.
1.3 measurement
The detection was performed by HPLC-CAD, and the detection conditions were as described above.
Precisely measuring the undecylamine solution, dodecylamine solution, sample derived solution 1, sample derived solution 2, sample derived solution 3, sample derived solution 4 and 10 μl of sample solution, respectively, injecting into a liquid chromatograph, and recording 32min chromatogram.
1.4 selection of derivatizing reagents
As shown in fig. 1, black is the chromatogram of the sample-derived solution 2, and blue is the chromatogram of the dodecylamine solution. Twelve amine impurities interfere with the target peak before it emerges from the peak position, using twelve amine as a derivatizing agent.
As shown in fig. 2, the undecylamine solution did not interfere with the target peak.
Undecylamine was therefore chosen as the derivatizing agent in this experiment.
1.5 selection of the concentration of derivatizing reagent and reaction temperature
1.5.1 derivatization reagent concentration was 20mg/mL, reaction temperature 45℃compared to 60 ℃):
as shown in FIG. 3, purple is a sample solution, black is a sample derivative solution 3 (undecylamine concentration is 20mg/mL, reaction temperature is 45 ℃ C.), and blue is a sample derivative solution 1 (undecylamine concentration is 20mg/m L, reaction temperature is 60 ℃ C.), and it is seen that when reaction temperature is 45 ℃ C., some active impurities having EPOX groups are not reacted completely. Therefore, 60℃was chosen as the reaction temperature.
1.5.2 reaction temperature 45 ℃, concentration of derivatizing agent 20mg/mL compared to concentration of derivatizing agent 30 mg/mL:
as shown in FIG. 4, purple is a sample solution, black is a sample-derived solution 3 (undecylamine concentration is 20mg/mL, reaction temperature is 45 ℃ C.), and blue is a sample-derived solution 4 (undecylamine concentration is 30mg/m L, reaction temperature is 45 ℃ C.), and it is seen that when undecylamine concentration is 20mg/mL, some of the active impurities having EPOX groups are not reacted completely. From this, it was found that the reaction was completed by increasing the concentration of the undecylamine solution.
Finally, in this experiment, the undecylamine concentration was chosen to be 20mg/mL and the reaction temperature was 60 ℃.
Example 2: method verification
2.1 preparation of solutions
(1) Preparation of blank solution
About 1g of undecylamine sample is weighed and placed in a 50mL volumetric flask, methanol solution is added for dissolution, and the volume is fixed to a scale, so that a blank solution with the concentration of 20mg/mL is obtained.
(2) Preparation of sample derived solutions
About 50mg of the M-EPOX-5K sample is weighed and placed in a 5mL volumetric flask, a blank solution with the concentration of 20mg/mL is added for dissolution, and the volume is fixed to a scale, so that a sample derivative solution with the concentration of 10mg/mL is obtained. The sample-derived solution at a concentration of 10mg/mL was placed in an air-bath shaker at 60℃and a rotational speed of 200rpm, and was derived overnight. As a 100% sample derived solution.
Transferring 1mL of 100% sample derivative solution into a 100mL volumetric flask, adding a blank solution with the concentration of 20mg/mL, and fixing the volume to a scale to obtain the sample derivative solution with the concentration of 0.1mg/mL as the 1% sample derivative solution.
(3) Preparation of test sample solution
About 50mg of the M-EPOX-5K sample is weighed and placed in a 5mL volumetric flask, methanol is added for dissolution, and the volume is fixed to a scale, so that a sample solution with the concentration of 10mg/mL is obtained.
(4) Preparation of intermediate working solution
And transferring 1mL of the sample derivative solution with the concentration of 10mg/mL into a 10mL volumetric flask, adding a blank solution with the concentration of 20mg/mL, and fixing the volume to a scale to obtain an intermediate working solution with the concentration of 1 mg/mL.
(5) Preparation of a Linear solution
Linear solution 1: transferring 1mL of the intermediate working solution with the concentration of 1mg/mL into a 5mL volumetric flask, adding a blank solution with the concentration of 20mg/mL, and fixing the volume to a scale to obtain a linear solution 1 with the concentration of 0.2 mg/mL.
Linear solution 2: transferring 0.5mL of the intermediate working solution with the concentration of 1mg/mL into a 5mL volumetric flask, adding a blank solution with the concentration of 20mg/mL, and fixing the volume to a scale to obtain a linear solution 2 with the concentration of 0.1 mg/mL.
Linear solution 3: transferring 0.4mL of the intermediate working solution with the concentration of 1mg/mL into a 5mL volumetric flask, adding a blank solution with the concentration of 20mg/mL, and fixing the volume to a scale to obtain a linear solution 3 with the concentration of 0.08 mg/mL.
Linear solution 4: transferring 0.25mL of the intermediate working solution with the concentration of 1mg/mL into a 5mL volumetric flask, adding a blank solution with the concentration of 20mg/mL, and fixing the volume to a scale to obtain a linear solution 4 with the concentration of 0.05 mg/mL.
Linear solution 5: 1mL of linear solution with the concentration of 0.1mg/mL is removed, a blank solution with the concentration of 20mg/mL is added into a volumetric flask with the concentration of 2 to 5mL, and the volume is fixed to a scale, so that linear solution 5 with the concentration of 0.02mg/mL is obtained.
Linear solution 6: 1mL of linear solution with the concentration of 0.1mg/mL is removed, a blank solution with the concentration of 20mg/mL is added into a volumetric flask with the concentration of 2 to 10mL, and the volume is fixed to a scale, so that linear solution 6 with the concentration of 0.01mg/mL is obtained.
(6) Formulation of EPOX-5K-EPOX derivative solutions
About 50mg of the EPOX-5K-EPOX sample (supplied by Tianjin Kaiki technology Co., ltd.) was weighed into a 5mL volumetric flask, and a blank solution having a concentration of 20mg/mL was added for dissolution, and the volume was fixed to a scale, to obtain an EPOX-5K-EPOX derivative solution having a concentration of 10 mg/mL. The EPOX-5K-EPOX derivative solution at a concentration of 10mg/mL was placed in an air bath shaker at 60℃and a rotational speed of 200rpm for overnight derivatization.
(7) Formulation of EPOX-10K-EPOX derivative solutions
About 50mg of the EPOX-10K-EPOX sample (supplied by Tianjin Kaiki technology Co., ltd.) was weighed into a 5mL volumetric flask, and a blank solution having a concentration of 20mg/mL was added for dissolution, and the volume was fixed to a scale, to obtain an EPOX-10K-EPOX derivative solution having a concentration of 10 mg/mL. The EPOX-10K-EPOX derivative solution at a concentration of 10mg/mL was placed in an air bath shaker at 60℃and a rotational speed of 200rpm for overnight derivatization.
2.2 establishment of standard curve
The linear solution with each concentration is detected according to the chromatographic condition, the concentration X (mg/mL) is taken as an abscissa, the corresponding peak area Y is taken as an ordinate, linear regression is carried out, the linear result and the linear equation are shown in table 2 and fig. 5, the result shows that M-EPOX-5K has good linearity in the concentration range of 0.0102-0.2032mg/mL (0.1% -2%), the linear equation y=10732052.62762 x+91239.9807, R 2 ＝0.9977。
TABLE 2 Linear results for linear solutions
Concentration (mg/mL) 0.0102 0.0203 0.0508 0.0813 0.1016 0.2032
Peak area 154854 305372 637486 1015331 1211285 2238844
2.3 measurement
The detection was performed by HPLC-CAD under the conditions described in example 1.
Weighing the blank solution, the sample solution (figure 6), the 100% sample derivative solution (figure 8), the 1% sample derivative solution (figure 7), the mixed standard solution (figure 9) of the EPOX-5K-EPOX derivative solution and the EPOX-10K-EPOX derivative solution, injecting the mixed standard solution into a liquid chromatograph, recording the chromatogram, and calculating the purity and the active impurity content of the sample by adopting a self-contrast method.
FIG. 6 shows a chromatogram of a sample solution, M-EPOX-5K as a target peak, which cannot be separated from the PEG-related peak preceding it. FIG. 7 shows a chromatogram of a sample derived solution in which the active impurities having an EPOX group have been reacted with an undecylamine derivatizing agent with their peak positions shifted rearward to avoid the interference of the PEG-related peaks, and all the active impurities reacted with undecylamine have been also peaked and can be detected. The other peaks in fig. 7 after the undecylamine peak were presumed to be active impurities.
The test was performed on 6 batches of test pieces, and the test results are shown in table 3. The raw material of M-EPOX-5K is M-PEG-5K, wherein M-PEG-5K contains a small amount of diol, namely OH-PEG5K-OH and OH-PEG10K-OH. In the process of M-PEG-5K reaction to produce M-EPOX-5K, OH-PEG5K-OH and OH-PEG10K-OH become EPOX-PEG5K-EPOX and EPOX-PEG10K-EPOX, respectively, and other active impurities are also produced. As shown in Table 3, the sample-derived solution contained the main component M-EPOX-5K-derived product, active impurities 1 to 7, and EPOX-5K-EPOX and EPOX-10K-EPOX-derived products, and the active impurities were subjected to q-TOF screening, and the results are shown in Table 4.
The calculation formulas of the total peak area, the purity of the main component and the content of impurities (including active impurities and other impurities) are as follows:
total peak area=1% principal component area 100+sum (each impurity peak area)
Impurity% = impurity peak area/total peak area 100
Principal component% = 1-SUM (impurity percentage)
Table 3 test results
TABLE 4q-TOF screening results
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present application, and not for limiting the same; although the application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the application.

Claims (10)

1. A method for detecting a single-ended EPOX-modified PEG product, comprising the steps of:
(1) Pretreatment: mixing the single-end EPOX modified PEG product with undecylamine, and carrying out a derivatization reaction to obtain a derivatization solution;
(2) Detecting the derivative solution obtained in the step (1) by an HPLC-CAD method;
preferably, the main component of the single-ended EPOX modified PEG product is methoxy-polyethylene glycol-epoxy group, and the structural formula isn is the degree of polymerization of the polyethylene glycol residues and has an average molecular weight of 1000 to 20000 daltons, preferably 5000 daltons.
2. The method according to claim 1, wherein the undecylamine is dissolved in an organic solvent selected from one or more of methanol, ethanol, acetonitrile, ethyl acetate, acetone, dichloromethane, chloroform, cyclohexane, preferably methanol;
and/or, the mass percentage concentration of the undecylamine is 10-40mg/mL, preferably 20-30mg/mL, and more preferably 20mg/mL;
and/or the temperature of the derivatization reaction is 45-70 ℃, preferably 55-65 ℃, more preferably 60 ℃.
3. The method according to claim 1, wherein the stationary phase of the chromatographic column is octadecylsilane chemically bonded silica having a cap.
4. The method according to claim 1, wherein in the HPLC-CAD method, mobile phase a is an aqueous solution of trifluoroacetic acid with a volume percentage concentration of 0.01-5%, preferably 0.05-2%, more preferably 0.1%;
the mobile phase B is a mixed solution of acetonitrile, methanol and trifluoroacetic acid, wherein the volume ratio of acetonitrile to methanol is 1-10:1, the volume ratio of the trifluoroacetic acid to the mixed solution of acetonitrile and methanol is 0.01-5%:1, a step of;
preferably, the volume ratio of acetonitrile to methanol is 1-5:1, more preferably 7:3, a step of;
preferably, the volume ratio of the trifluoroacetic acid to the mixed solution of acetonitrile and methanol is 0.05-2%:1, more preferably 0.1%:1.
5. the method according to claim 1, wherein the HPLC-CAD method employs a gradient elution procedure as follows:
0min, the volume ratio of mobile phase A to mobile phase B is 80:20, a step of;
0-2min, the volume ratio of the mobile phase A to the mobile phase B is 80:20, performing isocratic elution;
2-5min, the volume ratio of the mobile phase A to the mobile phase B is gradually changed into 31 according to linearity: 69;
5-12min, the volume ratio of the mobile phase A to the mobile phase B is gradually changed into 30 according to linearity: 70;
and (3) for 12-15min, the volume ratio of the mobile phase A to the mobile phase B is gradually changed into 25 according to linearity: 75;
15-17min, the volume ratio of mobile phase A to mobile phase B is 25:75; performing isocratic elution;
17-20min, the volume ratio of the mobile phase A to the mobile phase B gradually changes to 0 according to linearity: 100;
20-25min, the volume ratio of the mobile phase A to the mobile phase B is 0:100, performing isocratic elution;
25-25.1min, the volume ratio of the mobile phase A to the mobile phase B is gradually changed into 80 according to linearity: 20, a step of;
25.1-32min, the volume ratio of mobile phase A to mobile phase B is 80:20, performing isocratic elution.
6. A method according to claim 3, wherein the packing of the chromatographic column is surface modified silica with a specification of 5 μm,4.6 x 250mm.
7. The method according to claim 4, wherein the flow rate of the mobile phase is 0.1-5mL/min, preferably 1mL/min.
8. The method according to claim 1, wherein the column temperature is 35-45 ℃, preferably 40 ℃, in the HPLC-CAD method;
and/or, in the HPLC-CAD method, the sample injection amount is 1-20 mu L, preferably 10 mu L.
9. The method according to claim 1, wherein the parameters of the electrospray detector are: filtration constants: 0.1-10sec, preferably 10sec; temperature: 30-40 ℃, preferably 35 ℃.
10. Use of the assay of claim 1 in the quality assessment of single ended EPOX modified PEG products.
CN202310919859.1A 2023-07-24 2023-07-24 Detection method of single-ended EPOX modified PEG product Pending CN116930365A (en)

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