Liquid chromatography-mass spectrometry analysis method for polyethylene glycol series auxiliary material components and key quality attributes thereof

Abstract

The invention belongs to the technical field of analysis, and provides a liquid chromatography-mass spectrometry analysis method for polyethylene glycol series auxiliary material components and key quality attributes thereof. In particular to a high performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) method for analyzing and identifying a polyethylene glycol component in an auxiliary material and a preparation and simultaneously evaluating four key quality attributes of polyethylene glycol, and the polyethylene glycol auxiliary material and a preparation sample thereof are analyzed by adopting the HPLC-HRMS analysis method. The polyethylene glycol component is identified based on high-resolution mass spectrum information and a linear relation between retention time and polymerization degree, and four key quality attributes of polyethylene glycol are evaluated based on a component identification result and an established mathematical calculation formula. According to the method, comprehensive, rapid and accurate analysis and identification of polyethylene glycol components in polyethylene glycol series auxiliary materials and preparations can be realized, simultaneous evaluation of four key quality attributes is realized, and the quality control efficiency and level of the polyethylene glycol auxiliary materials and the preparations thereof are effectively improved.

Description

Technical Field

[0001] This invention belongs to the field of analytical technology and relates to a method for analyzing and identifying polyethylene glycol (PEG) series excipient components and a method for evaluating their key quality attributes. In particular, it relates to a high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS) analytical method for PEG series excipient components, an identification method for PEG series excipient components based on HPLC-HRMS analysis of the colligative relationship between degree of polymerization and retention time, and a method for simultaneously evaluating four key quality attributes of PEG series excipients based on HPLC-HRMS identification and relative quantification of PEG components. Background Technology

[0002] Polyethylene glycol (PEG) is a mixture formed by the condensation polymerization of ethylene oxide and water, exhibiting good water solubility, biocompatibility, and stability. PEG is classified into different grades based on its average molecular weight or average number of oxyethylene groups (average degree of polymerization). PEG with different average degrees of polymerization exhibits different physicochemical properties, functions, and applications: PEG with a molecular weight less than 600 is a viscous, colorless liquid at room temperature and is commonly used in liquid formulations as a solvent, solubilizer, and stabilizer; PEG with a molecular weight greater than 1000 is a solid at room temperature and is widely used in semi-solid and solid formulations. Furthermore, PEG is widely used in biological agents such as protein and peptide drugs and mRNA vaccines. It can covalently modify proteins or peptides to improve their physicochemical properties and biological activity, and can participate in lipid modification to form novel delivery systems such as lipid nanoparticles and mRNA vaccines.

[0003] With the widespread application of polyethylene glycol (PEG) in pharmaceutical formulations, the importance of PEG quality control and evaluation is increasingly significant. Key quality attributes of PEG mainly include average degree of polymerization, average molecular weight, weight-average molecular weight, and molecular weight distribution coefficient. These key quality attributes reflect the molecular weight and molecular weight distribution characteristics of PEG. Changes in these attributes not only alter the physicochemical properties of the excipients themselves but may also affect their effectiveness and safety. Therefore, precise characterization of PEG components in excipients and formulations, and rapid and accurate evaluation of their key quality attributes, are crucial for improving the quality of excipients and formulations and ensuring their effectiveness and safety. The average degree of polymerization characterizes the average number of repeating oxyethylene structural units in polyethylene glycol (PEG), directly affecting the physicochemical properties and functions of excipients, and is one of the key quality attributes of PEG. Currently, no analytical method is specified in domestic or international pharmacopoeias. The average molecular weight is the statistical average of the molecular weights of all components of PEG, and domestic and international pharmacopoeias use end-group titration for analysis. The weight-average molecular weight is the average molecular weight calculated using mass as a weighting factor. The molecular weight distribution coefficient (MBC) is the ratio of the weight-average molecular weight to the average molecular weight, used to measure the breadth and dispersion of molecular weight distribution. Both weight-average molecular weight and MBC are analyzed using size exclusion chromatography in domestic and international pharmacopoeias. Currently, there is no single method that can simultaneously characterize and evaluate all four key quality attributes.

[0004] The challenge in analyzing polyethylene glycol (PEG) components and evaluating its critical quality attributes lies in the fact that PEG is polydisperse, with complex components, high structural similarity, and a wide distribution range. General physicochemical methods struggle to separate, identify, and quantify each component individually, making it difficult to calculate critical quality attributes such as the average degree of polymerization. Current methods for analyzing PEG critical quality attributes in pharmacopoeias, such as end-group titration and size exclusion chromatography, cannot separate and identify PEG components, provide degree of polymerization information, and can only evaluate one or two critical quality attributes separately. These methods suffer from drawbacks such as reliance on reference standards, cumbersome procedures, accuracy affected by various factors, and inability to evaluate critical quality attributes of PEG in formulations. Therefore, there is currently a lack of effective techniques for precisely characterizing PEG components and simultaneously evaluating multiple critical quality attributes.

[0005] HPLC-HRMS, characterized by high separation efficiency, high selectivity, high sensitivity, and fast scanning speed, is one of the ideal methods for analyzing complex samples. It can be used for the qualitative and quantitative analysis of complex compounds, playing a vital role in fields such as biomedicine. This invention applies this technology to the analysis and identification of polyethylene glycol (PEG) excipients. By establishing a mathematical model between component retention time and degree of polymerization, rapid discovery and identification of PEG components with different degrees of polymerization are achieved. The mass spectrometry discrimination effect is overcome by adding specific mobile phase additives. Based on this, a new method for simultaneously evaluating four key quality attributes of PEG is established based on the analytical, identification, and quantitative results of PEG components. Summary of the Invention

[0006] The technical problem solved by this invention is to provide a comprehensive, rapid, simple, and effective method for the analysis and identification of polyethylene glycol (PEG) components in PEG series excipients. This method completes the analysis of PEG series excipient components, overcomes the problems of complex sample composition and difficult structural identification of PEG series excipients, and improves the accuracy and sensitivity of PEG series excipient analysis. This invention also discovers the colligative relationship between the degree of polymerization and retention time of PEG series excipient components. Based on this relationship, a mathematical model can be established to quickly and accurately identify PEG series excipient components.

[0007] The technical problem solved by this invention also lies in providing an evaluation method for key quality attributes of polyethylene glycol (PEG) series excipients. Based on this method, four key quality attributes of PEG, such as average degree of polymerization, can be evaluated simultaneously without being affected by mass spectrometry quality discrimination effects, thereby improving the efficiency and level of evaluation of key quality attributes of PEG series excipients.

[0008] To solve the technical problem of this invention, the present invention provides the following technical solution:

[0009] In a first aspect of the present invention, a method for high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HDMS) coupled with other methods for the analysis and identification of polyethylene glycol (PEG) series excipients is provided, characterized in that the analysis and identification method comprises the following steps:

[0010] a. Chromatographic analysis conditions for polyethylene glycol series excipients:

[0011] A reversed-phase high-performance liquid chromatography (RP-HPLC) column was used as the HPLC column for analysis. The column length was selected from 20-250 mm. Mobile phase A was an aqueous solution of 0-10% formic acid or acetic acid containing 0-100 mmol / L ammonium formate or ammonium acetate. Mobile phase B was acetonitrile or methanol containing 0-10% formic acid or acetic acid. The mobile phase ratio was A+B = 100%. The analysis time was 5-100 min. During the analysis time, the elution was carried out from 0-40% mobile phase B to 10-100% mobile phase B. The flow rate was 0.1-1 ml / min. The column temperature was 10-60℃. The injection volume was 1-20 μL.

[0012] Preferably, the chromatographic analysis conditions for the above-mentioned polyethylene glycol series excipients are as follows: the reversed-phase high-performance liquid chromatography column has a column length of 30-200 mm; mobile phase A is an aqueous solution of 0-5% formic acid or acetic acid containing 0-50 mmol / L ammonium formate or ammonium acetate; mobile phase B is acetonitrile or methanol containing 0-5% formic acid or acetic acid; the mobile phase ratio is A+B=100%; the analysis time is 10-70 min; during the analysis time, elution is performed from 0-35% mobile phase B to 15-100% mobile phase B using a gradient elution; the flow rate is 0.2-0.8 ml / min; the column temperature is 20-50℃; and the injection volume is 1-15 μL.

[0013] Preferably, the chromatographic analysis conditions for the above-mentioned polyethylene glycol series excipients include: a reversed-phase high-performance liquid chromatography (HPLC) column with a length of 50-150 mm; mobile phase A being an aqueous solution of 0-2% formic acid or acetic acid containing 0-10 mmol / L ammonium formate or ammonium acetate; mobile phase B being acetonitrile or methanol containing 0-2% formic acid or acetic acid; the mobile phase ratio being A+B = 100%; the analysis time being 15-40 min; and elution during the analysis time from 5-30% mobile phase B to 25-60% mobile phase B via a gradient elution at a flow rate of 0.3-0.6 ml / min. The column temperature is 20-40℃, and the injection volume is 2-10 μL.

[0014] The optimal chromatographic analysis conditions for the above-mentioned polyethylene glycol series excipients are determined by adjusting the mobile phase composition, analysis time, and mobile phase elution gradient according to the different average molecular weights of the polyethylene glycols being analyzed and identified. The reversed-phase high-performance liquid chromatography column has a column length of 100-150 mm; mobile phase A is an aqueous solution of 0-1% formic acid or acetic acid containing 0-5 mmol / L ammonium formate or ammonium acetate; mobile phase B is acetonitrile or methanol containing 0-1% formic acid or acetic acid; the mobile phase ratio is A+B = 100%; the flow rate is 0.3-0.5 ml / min; the column temperature is 20-40℃; the injection volume is 2-10 μL; and the analysis time for polyethylene glycols with an average molecular weight of 300 to 600 is 10-20 min. For polyethylene glycol with an average molecular weight of 1000 to 2000, the analysis time is 15 to 25 minutes, during which the elution is performed from 5 to 15% of mobile phase B to 30 to 40% of mobile phase B. For polyethylene glycol with an average molecular weight of 4000 to 6000, the analysis time is 20 to 30 minutes, during which the elution is performed from 20 to 30% of mobile phase B to 30 to 40% of mobile phase B.

[0015] b. High-resolution mass spectrometry analysis conditions for polyethylene glycol series excipients:

[0016] The effluent from high performance liquid chromatography was analyzed by high-resolution mass spectrometry using an electrospray ionization source and positive ion detection mode.

[0017] Preferably, the high-resolution mass spectrometry analysis conditions for the above-mentioned polyethylene glycol series excipients include a full scan, a data-independent scan, or a data-dependent scan mode, with a scan range of 50-3200 Da.

[0018] More preferably, the high-resolution mass spectrometry analysis conditions for the above-mentioned polyethylene glycol series excipients include a full scan, a data-independent scan, or a data-dependent scan mode, with a scan range of 50-2000 Da.

[0019] The optimal high-resolution mass spectrometry (HMS) analysis conditions for the above-mentioned polyethylene glycol (PEG) series excipients are as follows: ion source parameters for HMS analysis are as follows: drying gas temperature: 150-350℃; drying gas flow rate: 5-20 L / min; spray gas flow rate: 10-50 psi; sheath gas temperature: 200-400℃; sheath gas flow rate: 5-20 L / min; capillary voltage: 2.0-5.0 kV; nozzle voltage: 0.1-1.0 kV.

[0020] c. Identification methods for components in polyethylene glycol series excipients:

[0021] (1) A method for identifying polyethylene glycol (PEG) series excipient components based on the colligative relationship between retention time and degree of polymerization, characterized in that the PEG components are polymers with the same structural units but different degrees of polymerization, and there is a colligative relationship between the retention time and degree of polymerization of the PEG. A mathematical model is established to determine the relationship between the retention time and degree of polymerization of the PEG series excipient components, and the theoretical retention time of each component in the PEG series excipients is calculated.

[0022] (2) Polyethylene glycol excipients and their formulations were analyzed using HPLC-HRMS. The colligative relationship between retention time and degree of polymerization was discovered and summarized. This colligative relationship is one of the bases for identifying polyethylene glycol components in polyethylene glycol excipients and their formulations. Candidate compounds were extracted using high-resolution quasi-molecular ion extraction for each component in the polyethylene glycol series of excipients. The measured and calculated retention times of the candidate compounds were matched. If the match was within the error range, the degree of polymerization of the compound was determined, thus achieving structural identification based on the colligative relationship between retention time and degree of polymerization.

[0023] In a second aspect of the present invention, a method for analyzing and evaluating key quality attributes of polyethylene glycol excipients based on high-performance liquid chromatography-high-resolution mass spectrometry is provided, characterized in that the analysis and evaluation method includes the following steps:

[0024] The samples were analyzed and identified using the high-performance liquid chromatography-high-resolution mass spectrometry method described in the first aspect of the invention. This method yielded information such as the degree of polymerization, molecular weight, and peak area of ​​each identified polyethylene glycol component. This information was then substituted into the established mathematical formulas for the key quality attributes of polyethylene glycol, enabling the simultaneous evaluation of four key quality attributes. The four key quality attributes refer to the average degree of polymerization N. Average Average molecular weight M n Weight-average molecular weight M w The formulas for calculating the molecular weight distribution coefficient D are as follows: In the formula, A i Represents the peak area of ​​the extracted ion chromatogram for each polyethylene glycol component, N i Represents the degree of polymerization of each polyethylene glycol component, M i This represents the molecular weight of each polyethylene glycol component.

[0025] Beneficial technical effects: The advantages of this invention are:

[0026] (1) Comprehensive, rapid and efficient HPLC-HRMS analysis of polyethylene glycol components in polyethylene glycol series excipients and their preparations can solve the problem of complex polyethylene glycol components and high structural similarity;

[0027] (2) Rapid identification of polyethylene glycol components in polyethylene glycol series excipients and their preparations based on a mathematical model of the colligative relationship between degree of polymerization and retention time;

[0028] (3) A colligative relationship between retention time and degree of polymerization was discovered in the HPLC-HRMS analysis of polyethylene glycol components.

[0029] (4) A method for simultaneously evaluating four key quality attributes of polyethylene glycol was established based on HPLC-HRMS analysis and identification of polyethylene glycol components, which is not affected by mass spectrometry quality discrimination response.

[0030] According to the present invention, comprehensive, rapid and efficient analysis and identification of each polyethylene glycol component in polyethylene glycol excipients and their formulations can be achieved. The polyethylene glycol component analysis of typical polyethylene glycol excipient samples and formulation samples is shown in Example 3.

[0031] According to the present invention, each polyethylene glycol component in polyethylene glycol excipients and their preparations can be identified, and information such as the degree of polymerization, molecular weight and chromatographic peak area of ​​each polyethylene glycol component in polyethylene glycol excipients and their preparations can be obtained.

[0032] According to the present invention, a colligative relationship between the retention time and the degree of polymerization of polyethylene glycol components in polyethylene glycol excipients and their preparations was discovered by HPLC-HRMS analysis.

[0033] According to the present invention, four key quality attributes of polyethylene glycol can be evaluated simultaneously based on the HPLC-HRMS analysis and identification method of polyethylene glycol components, without being affected by the mass spectrometry quality discrimination effect, and the evaluation results are accurate and reliable.

[0034] This invention has a very broad prospect for promotion and application. It can be applied to the analysis of polyethylene glycol components and the evaluation of key quality attributes in polyethylene glycol series excipients and pharmaceutical preparations containing polyethylene glycol series excipients. Attached Figure Description

[0035] Figure 1 Chemical structural formula of polyethylene glycol (A) and characteristic fragment structures and m / z values ​​(B) for mass spectrometry.

[0036] Figure 2 HPLC-HRMS total ion chromatogram (A) and extracted ion chromatogram (B) of polyethylene glycol mixed sample 1.

[0037] Figure 3 HPLC-HRMS total ion chromatogram (A) and extracted ion chromatogram (B) of polyethylene glycol mixed sample 2.

[0038] Figure 4HPLC-HRMS total ion chromatogram (A) and extracted ion chromatogram (B) of polyethylene glycol mixed sample 3.

[0039] Figure 5 Mathematical model of retention time and degree of polymerization of polyethylene glycol components

[0040] Figure 6 Mathematical formulas for four key quality properties of polyethylene glycol Detailed Implementation

[0041] The present invention will now be described in further detail with reference to the accompanying drawings and examples, so that those skilled in the art can implement it based on the description.

[0042] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0043] Example 1: Preparation, analysis, identification, and establishment of a mathematical model for polyethylene glycol mixed samples.

[0044] (1) Sample preparation

[0045] Polyethylene glycol 300, polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 1000, polyethylene glycol 1500, polyethylene glycol 2000, polyethylene glycol 4000 and polyethylene glycol 6000 were purchased from Aladdin Shanghai Biochemical Technology Co., Ltd.

[0046] Accurately weigh 10 mg each of PEG300, PEG400, PEG600, PEG1000, PEG1500, PEG2000, PEG4000, PEG6000 and PEG8000, add 10 ml of water, and vortex to prepare a solution of 1 mg / ml.

[0047] PEG was divided into three groups according to the average molecular weight of polyethylene glycol. The solutions were diluted with water and three mixed samples of polyethylene glycol were prepared for HPLC-HRMS analysis.

[0048] Polyethylene glycol mixed sample 1: 2 μg / ml PEG300, 2 μg / ml PEG400, 5 μg / ml PEG600, 10 μg / ml PEG1000.

[0049] Polyethylene glycol mixed sample 2: 2 μg / ml PEG300, 10 μg / ml PEG1000, 20 μg / ml PEG1500, 50 μg / ml PEG2000, 150 μg / ml PEG4000.

[0050] Polyethylene glycol mixed sample 3: 50 μg / ml PEG2000, 150 μg / ml PEG4000, 200 μg / ml PEG6000, 200 μg / ml PEG8000.

[0051] (2) Analytical methods

[0052] Polyethylene glycol mixed sample 1 was analyzed using method one, polyethylene glycol mixed sample 2 was analyzed using method two, and polyethylene glycol mixed sample 3 was analyzed using method three.

[0053] Analysis was performed using an Agilent 1290 / 6550 high-performance liquid chromatography-quadrupole time-of-flight mass spectrometer.

[0054] Analysis Method 1:

[0055] Chromatographic conditions 1: Waters Acquity UPLC BEH C8 column (2.1×100mm, 1.7μm), mobile phase A: water, mobile phase B: acetonitrile, mobile phase ratio: A+B=100%, analysis time: 15 min, gradient elution program: 0-15 min, 5-28% B. Flow rate: 0.3 ml / min; column temperature: 30℃; injection volume: 5 μl.

[0056] Mass spectrometry conditions: Agilent iFunnel 6550Q-TOF quadrupole time-of-flight mass spectrometer, using an AJS ESI ion source; sheath gas temperature: 300℃; dry gas temperature: 200℃; sheath gas flow rate: 12 L / min; dry gas flow rate: 14 L / min; capillary voltage: 4 kV; nozzle voltage: 500 V; collision energy: 40 eV; Full Scan mode; scan range: m / z: 50–1700.

[0057] Analysis Method Two:

[0058] Chromatographic conditions 2: Waters Acquity UPLC BEH C8 column (2.1×100mm, 1.7μm), mobile phase A: water, mobile phase B: acetonitrile, mobile phase ratio: A+B=100%, analysis time: 20 min, gradient elution program: 0-20 min, 10-36% B. Flow rate: 0.3 ml / min; column temperature: 30℃; injection volume: 5 μl.

[0059] The mass spectrometry conditions are the same as those for analytical method one.

[0060] Analysis Method 3:

[0061] Chromatographic conditions 3: Waters Acquity UPLC BEH C8 column (2.1×100mm, 1.7μm); mobile phase A was an aqueous solution containing 0.05mmol / L ammonium acetate; mobile phase B was acetonitrile; mobile phase ratio: A+B=100%; analysis time: 30min; gradient elution program: 0-30min, 27-38% B; flow rate: 0.3ml / min; column temperature: 30℃; injection volume: 5μl.

[0062] The mass spectrometry conditions are the same as those for analytical method one.

[0063] (3) Component identification methods

[0064] The first-order high-resolution mass spectrometry data, second-order high-resolution mass spectrometry data, and retention time of polyethylene glycol components in the polyethylene glycol mixed sample were analyzed and identified using Agilent Mass Hunter qualitative analysis software.

[0065] (4) Establishment of mathematical model

[0066] Based on the colligative relationship between the degree of polymerization and relative retention time of polyethylene glycol components, mathematical models were established for three polyethylene glycol analysis methods, with the degree of polymerization of the components as the abscissa and the relative retention time of the components as the ordinate. The logarithmic relationship formula of the model was obtained through regression analysis.

[0067] (5) Analysis and evaluation results

[0068] The chemical structural formula, mass spectrometry characteristic fragment structure formula, and m / z value of polyethylene glycol are as follows: Figure 1 As shown.

[0069] Using the established high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HSMS) method for analyzing polyethylene glycol components, 25 polyethylene glycol components with a degree of polymerization (n) ranging from 3 to 27 were identified in mixed sample 1. The TIC chromatogram and the EIC overlay chromatogram of each identified polyethylene glycol component are shown below. Figure 2 Using the established high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HSMS) method 2, 77 polyethylene glycol (PEG) components were identified in mixed sample 2, with a degree of polymerization (n) ranging from 3 to 81. The TIC chromatogram and EIC overlay plot of each identified PEG component for mixed sample 2 are shown below. Figure 3 Using the established high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HSMS) method 3, 159 polyethylene glycol components were identified in mixed sample 3, with a degree of polymerization (n) ranging from 11 to 171. The TIC chromatogram and EIC overlay plot of each identified polyethylene glycol component for mixed sample 3 are shown below. Figure 4 .

[0070] Based on the identification results of polyethylene glycol components, a colligative relationship was found between the relative retention times and degrees of polymerization of the polyethylene glycol components. The relative retention time of each component was calculated as the ratio of the component retention time to the internal standard retention time. In Method 1, which is applicable to the analysis of polyethylene glycol with molecular weights from 300 to 600, the internal standard is PEG (10), and the established mathematical model is as follows: Figure 5 As shown in A; in Method 2, which is applicable to the analysis of polyethylene glycol with molecular weights from 1000 to 2000, the internal standard is PEG(30), and the established mathematical model is as follows. Figure 5 As shown in B; in Method 3, which is applicable to the analysis of polyethylene glycol with molecular weights of 4000 to 6000, the internal standard is PEG(108), and the established mathematical model is as follows. Figure 5 As shown in Figure C, the established mathematical model can be used to rapidly discover, identify, and confirm polyethylene glycol components in excipients or formulations.

[0071] The degree of polymerization, molecular formula, molecular weight, quasi-molecular ion, measured retention time, theoretical retention time, and their deviations for the polyethylene glycol components identified in the three mixed samples are shown in Tables 1-3. It is evident that the established HPLC-HRMS analysis method for the three polyethylene glycol components can achieve fine characterization of each component in the sample. Furthermore, the deviations between the theoretical retention time calculated by the established mathematical model and the measured retention time are all less than 12%, enabling accurate prediction of component retention times.

[0072] Table 1. Information on polyethylene glycol components identified in mixed sample 1

[0073]

[0074]

[0075] Note: Relative percentage deviation of retention time = (Measured retention time - Theoretical retention time / Theoretical retention time) * 100%

[0076] Table 2 Information on polyethylene glycol components identified in mixed sample 2

[0077]

[0078]

[0079] Note: Relative percentage deviation of retention time = (Measured retention time - Theoretical retention time / Theoretical retention time) * 100%

[0080] Table 3. Information on the polyethylene glycol components identified in mixed sample 3.

[0081]

[0082]

[0083]

[0084] Note: Relative percentage deviation of retention time = (Measured retention time - Theoretical retention time / Theoretical retention time) * 100%

[0085] Example 2: Establishment of a method for component analysis and key quality attribute evaluation of polyethylene glycol reference standards.

[0086] (1) Sample preparation

[0087] The reference standards for polyethylene glycol 400, polyethylene glycol 1000, and polyethylene glycol 4000 were provided by the China National Institutes for Food and Drug Control.

[0088] Accurately weigh 10 mg of each polyethylene glycol reference standard, add 10 ml of water, vortex to mix and prepare a 1 mg / ml solution. Dilute the above solutions with water: polyethylene glycol 400 reference standard to 5 μg / ml, polyethylene glycol 1000 reference standard to 10 μg / ml, and polyethylene glycol 4000 reference standard to 150 μg / ml for HPLC-HRMS analysis.

[0089] (2) Analytical methods

[0090] Analysis was performed using an Agilent 1290 / 6550 high-performance liquid chromatography-quadrupole time-of-flight mass spectrometer.

[0091] The polyethylene glycol 400 reference standard was analyzed using method one of Example 1, the polyethylene glycol 1000 reference standard using method two of Example 1, and the polyethylene glycol 4000 reference standard using method three of Example 1. The specific methods were the same as in Example 1.

[0092] (3) Component identification methods

[0093] The first-order high-resolution mass spectrometry data, second-order high-resolution mass spectrometry data, and retention time of polyethylene glycol components in the polyethylene glycol reference standard were analyzed using Agilent Mass Hunter qualitative analysis software. The polyethylene glycol components were analyzed and identified, and information such as molecular weight, degree of polymerization, and chromatographic peak area of ​​the polyethylene glycol components in the reference standard were obtained.

[0094] (4) Key Quality Attribute Evaluation Methods

[0095] Based on the definitions of four key quality attributes of polyethylene glycol, establish mathematical calculation formulas for the evaluation of key attributes (e.g., Figure 6 As shown, by substituting the degree of polymerization, molecular weight, and chromatographic peak area information of the identified polyethylene glycol components into the formula, the average degree of polymerization, average molecular weight, weight-average molecular weight, and molecular weight distribution coefficient of the sample are calculated, thereby achieving the evaluation of four key quality attributes of polyethylene glycol.

[0096] (5) Analysis Results

[0097] Using the high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-MS) method for analyzing and identifying polyethylene glycol (PEG) components according to this invention, 10 PEG components were identified in PEG 400 reference standard, with a degree of polymerization (n) ranging from 4 to 13; 20 PEG components were identified in PEG 1000 reference standard, with a degree of polymerization (n) ranging from 13 to 32; and 106 PEG components were identified in PEG 4000 reference standard, with a degree of polymerization (n) ranging from 12 to 117. Relevant information is shown in Tables 4-6. The analytical results indicate that as the average molecular weight of the PEG excipients increases, the degree of polymerization of the contained PEG components increases, and the variety of PEG components also increases.

[0098] The labeled, calculated, and biased values ​​of the key quality attributes of polyethylene glycol (PEG) reference standards are shown in Table 7. The biases between the calculated and labeled average molecular weights of the three PEG reference standards were all less than 4.6%, the biases between the calculated and labeled weight-average molecular weights were less than 3.7%, and the biases between the calculated and labeled molecular weight distribution coefficients were less than 5.4%. These results demonstrate that the established key quality attribute evaluation method can accurately evaluate the four key quality attributes of PEG simultaneously.

[0099] Table 4. Information on polyethylene glycol components identified in polyethylene glycol 400 reference standard.

[0100]

[0101] Table 5. Information on polyethylene glycol components identified in polyethylene glycol 1000 reference standard.

[0102]

[0103]

[0104] Table 6. Information on polyethylene glycol components identified in polyethylene glycol 4000 reference standard.

[0105]

[0106]

[0107]

[0108] Table 7 Key Quality Attributes, Calculated Values, and Deviations of Polyethylene Glycol Reference Standard

[0109]

[0110] Example 3: Component analysis and evaluation of key quality attributes of polyethylene glycol excipients and polyethylene glycol-containing formulations.

[0111] (1) Sample preparation

[0112] Polyethylene glycol 400 excipient was purchased from Aladdin Shanghai Biochemical Technology Co., Ltd. 10 mg of polyethylene glycol 400 was accurately weighed, added to 10 ml of water, and vortexed to prepare a 1 mg / ml solution. The polyethylene glycol 400 was then diluted with water to a concentration of 5 μg / ml for HPLC-HRMS analysis.

[0113] Nimodipine injection (Bayer AG, Germany, batch number KV03Z0X) was purchased from the China-Japan Friendship Hospital. The nimodipine injection was diluted 1000 times with water for HPLC-HRMS analysis.

[0114] (2) Analytical methods

[0115] Analysis was performed using an Agilent 1290 / 6550 high-performance liquid chromatography-quadrupole time-of-flight mass spectrometer.

[0116] Both polyethylene glycol 400 excipient and nimodipine injection were analyzed using method one of Example 1.

[0117] (3) Component identification methods

[0118] The first-order high-resolution mass spectrometry data, second-order high-resolution mass spectrometry data, and retention time of polyethylene glycol components in excipient polyethylene glycol 400 and nimodipine injection were analyzed using Agilent Mass Hunter qualitative analysis software. Based on the mathematical model of the colligative relationship between degree of polymerization and retention time, the polyethylene glycol components were analyzed and identified.

[0119] (4) Key Quality Attribute Evaluation Methods

[0120] The evaluation method for key quality attributes is the same as in Example 2.

[0121] (5) Analysis Results

[0122] Using the high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-MS / MS) method for analyzing and identifying polyethylene glycol (PEG) components in this invention, 12 PEG components were identified in the excipient PEG 400, with a degree of polymerization (n) ranging from 4 to 15. Relevant information is shown in Table 8. Similarly, 12 PEG components were identified in the nimodipine injection formulation containing PEG 400, with a degree of polymerization (n) ranging from 4 to 15. Relevant information is shown in Table 9. The key quality attributes of PEG in the excipient PEG 400 and nimodipine injection are shown in Table 10, meeting the requirements of the Chinese Pharmacopoeia. Therefore, the established analytical method for PEG components and the key quality attribute evaluation method are suitable for the quality evaluation of PEG excipients and their formulations.

[0123] Table 8. Information on polyethylene glycol components identified in the excipient polyethylene glycol 400

[0124]

[0125] Table 9 Information on polyethylene glycol components identified in nimodipine injection.

[0126]

[0127] Table 10 Key Quality Attributes of Polyethylene Glycol in Excipients and Formulations

[0128] .

Claims

1. A method for analyzing and identifying polyethylene glycol series adjuvant components by high performance liquid chromatography-high resolution mass spectrometry, characterized by, The analysis and identification method comprises the following steps: 1) Analysis method of polyethylene glycol series auxiliary components by high performance liquid chromatography-high resolution mass spectrometry, comprising the following steps: a. Using a reversed-phase high performance liquid chromatography column as the liquid chromatography analysis column, the column length is selected from 20-250 mm, the mobile phase A is 0-10% formic acid or acetic acid aqueous solution containing 0-100 mmol / L ammonium formate or ammonium acetate, the mobile phase B is acetonitrile or methanol containing 0-10% formic acid or acetic acid, the mobile phase ratio is: A+B=100%, the analysis time is 5-100 min, elution from 0-40% of the mobile phase B to 10-100% of the mobile phase B within the analysis time, the flow rate is 0.1-1 ml / min, the column temperature is 10-60℃, and the injection volume is 1-20 μL, b. The effluent of step a is subjected to high resolution mass spectrometry analysis, and an electrospray ion source is used in positive ion detection mode; 2) Identification method of polyethylene glycol series auxiliary components based on the relationship between retention time and degree of polymerization, characterized in that the polyethylene glycol components are polymers with the same structural unit but different degrees of polymerization, and there is a relationship between the retention time and the degree of polymerization of the polyethylene glycol components, therefore, the components detected by the analysis method of step 1) can be identified according to the relationship, and the degree of polymerization of the components is characterized. The polyethylene glycol series auxiliary materials refer to polymers containing oxyethylene repeating structural units in the structure.

2. The method of analysis and authentication according to claim 1, characterized in that, The polyethylene glycol series auxiliary materials include polyethylene glycol 300, polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 1000, polyethylene glycol 1500, polyethylene glycol 2000, polyethylene glycol 4000 and polyethylene glycol 6000.

3. The method of analysis and identification according to claim 2, characterized in that, The reversed-phase high performance liquid chromatography column has a column length selected from 30-200 mm, the mobile phase A is 0-5% formic acid or acetic acid aqueous solution containing 0-50 mmol / L ammonium formate or ammonium acetate, the mobile phase B is acetonitrile or methanol containing 0-5% formic acid or acetic acid, the mobile phase ratio is: A+B=100%, the analysis time is 10-70 min, elution from 0-35% of the mobile phase B to 15-100% of the mobile phase B within the analysis time, the flow rate is 0.2-0.8 ml / min, the column temperature is 20-50℃, and the injection volume is 1-15 μL.

4. The method of analysis and authentication according to claim 1, characterized in that, The reversed-phase high performance liquid chromatography column has a column length selected from 50-150 mm, the mobile phase A is 0-2% formic acid or acetic acid aqueous solution containing 0-10 mmol / L ammonium formate or ammonium acetate, the mobile phase B is acetonitrile or methanol containing 0-2% formic acid or acetic acid, the mobile phase ratio is: A+B=100%, the analysis time is 15-40 min, elution from 5-30% of the mobile phase B to 25-60% of the mobile phase B within the analysis time, the flow rate is 0.3-0.6 ml / min, the column temperature is 20-40℃, and the injection volume is 2-10 μL.

5. The method of analysis and authentication according to claim 1, characterized in that, ​ 6. The method of analysis and authentication according to claim 1, characterized in that, According to the different average molecular weight of polyethylene glycol analyzed and identified, the composition of the mobile phase, analysis time and elution gradient of the mobile phase are adjusted; the reverse phase high performance liquid chromatography column has a column length selected from 100-150 mm, a mobile phase A being 0-1% formic acid or acetic acid aqueous solution containing 0-5 mmol / L ammonium formate or ammonium acetate, a mobile phase B being acetonitrile or methanol containing 0-1% formic acid or acetic acid, a mobile phase ratio: A+B=100%, a flow rate being 0.3-0.5 ml / min, a column temperature being 20-40℃, and an injection volume being 2-10 μL; the analysis time of polyethylene glycol with an average molecular weight of 300-600 is 10-20 min, and the gradient elution from 0-10% of the mobile phase B to 20-30% of the mobile phase B is performed within the analysis time; the analysis time of polyethylene glycol with an average molecular weight of 1000-2000 is 15-25 min, and the gradient elution from 5-15% of the mobile phase B to 30-40% of the mobile phase B is performed within the analysis time; the analysis time of polyethylene glycol with an average molecular weight of 4000-6000 is 20-30 min, and the gradient elution from 20-30% of the mobile phase B to 30-40% of the mobile phase B is performed within the analysis time.

7. The method of analysis and authentication according to claim 1, characterized in that, In the high resolution mass spectrometric analysis, the scanning mode is full scan, data non-dependent scan or data dependent scan mode, and the scanning range is 50-3200 Da.

8. The method of analysis and authentication according to claim 1, characterized in that, In the high resolution mass spectrometric analysis, the scanning mode is full scan, data non-dependent scan or data dependent scan mode, and the scanning range is 50-2000 Da.

9. The method of analysis and authentication according to claim 1, characterized in that, In the high resolution mass spectrometric analysis, the ion source parameters are as follows: drying gas temperature: 150-350℃; drying gas flow rate: 5-20 L / min; spray gas flow rate: 10-50 psi; sheath gas temperature: 200-400℃; sheath gas flow rate: 5-20 L / min; capillary voltage: 2.0-5.0 kV; nozzle voltage: 0.1-1.0 kV.

10. The method of analysis and authentication according to claim 1, characterized in that, In the polyethylene glycol series auxiliary material component identification method based on the relationship between the retention time and the degree of polymerization, the polyethylene glycol components are polymers with the same structural unit but different degrees of polymerization, and there is a relationship between the retention time and the degree of polymerization of the polyethylene glycol; a mathematical model of the relationship between the retention time and the degree of polymerization of the polyethylene glycol series auxiliary material components is established, and the theoretical retention time of each component of the polyethylene glycol series auxiliary material is calculated. After the sample is detected by the analysis method in step 1), the relative retention time of the candidate compound is matched with the calculated value according to the high resolution quasi-molecular ion extraction candidate compound in the polyethylene glycol series auxiliary material, and the degree of polymerization of the compound is determined within the error range, so as to realize the structure identification based on the relationship between the retention time and the degree of polymerization.

11. A method for analysis and evaluation of a key quality attribute of a polyethylene glycol series adjuvant by high performance liquid chromatography-high resolution mass spectrometry, characterized by, The method is based on high performance liquid chromatography-high resolution mass spectrometry analysis and identification method of polyethylene glycol components, analyzes and identifies polyethylene glycol components in polyethylene glycol adjuvants and preparations, obtains information such as the polymerization degree, molecular weight and chromatographic peak area of each identified polyethylene glycol component, and inputs the information into the established polyethylene glycol key quality attribute mathematical formula to simultaneously evaluate four key quality attributes of polyethylene glycol.

12. The analysis and evaluation method according to claim 11, characterized in that, The polyethylene glycol series adjuvants refer to polymers containing oxyethylene repeating structural units in the structure.

13. The analysis and evaluation method according to claim 12, characterized in that, The polyethylene glycol series adjuvants include polyethylene glycol 300, polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 1000, polyethylene glycol 1500, polyethylene glycol 2000, polyethylene glycol 4000 and polyethylene glycol 6000.

14. The analysis and evaluation method according to claim 11, characterized by, The four key quality attributes of the polyethylene glycol series adjuvants refer to average polymerization degree, average molecular weight, weight average molecular weight and molecular weight distribution coefficient.

15. The analysis and evaluation method according to claim 11, characterized by, The four key quality attributes of the polyethylene glycol series adjuvant are average degree of polymerization N Average , average molecular weight M n , weight average molecular weight M w , and molecular weight distribution coefficient D, the calculation formulas are respectively In the formula, A i represents the peak area of each polyethylene glycol component extracted ion chromatogram, N i represents the degree of polymerization of each polyethylene glycol component, M i represents the molecular weight of each polyethylene glycol component.

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