Analytical method for peg-containing copolymers based on in-source cleavage and parallel reaction monitoring

Abstract

The application discloses an analysis method of PEG segment-containing copolymer based on in-source cleavage and parallel reaction monitoring, and comprises the following steps: testing a PEG segment-containing copolymer standard solution by using ultra-high performance liquid chromatography-tandem mass spectrometry, and recording the chromatographic peak area of a monitoring ion pair corresponding to each concentration; taking the concentration of the PEG segment-containing copolymer in the PEG segment-containing copolymer standard solution as the abscissa, taking the chromatographic peak area of the monitoring ion pair corresponding to the concentration as the ordinate, and performing regression analysis to obtain a standard curve; testing a sample to be measured by using ultra-high performance liquid chromatography-tandem mass spectrometry, recording the chromatographic peak area of the monitoring ion pair corresponding to the sample to be measured, and obtaining the concentration of the PEG segment-containing copolymer in the sample to be measured based on the chromatographic peak area of the monitoring ion pair corresponding to the sample to be measured and the standard curve. The application has the advantages of good specificity and reproducibility, high sensitivity, small sample requirement, simple sample preparation, low equipment cost and suitability for conventional analysis.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, and in particular to an analytical method for PEG-containing copolymers based on intrasource cleavage and parallel reaction monitoring. Background Technology

[0002] Soluplus is a novel amphiphilic nonionic pharmaceutical polymer developed by BASF (Germany). Its full chemical name is Polyvinylcaprolactam-polyvinyl acetate-polyethylene glycol graft copolymer. This polymer consists of 13% polyethylene glycol (PEG) 6000, 57% vinylcaprolactam, and 30% vinyl acetate, with an average molecular weight of approximately 118,000 g / mol (molecular weight range 90,000-140,000 g / mol). Soluplus possesses multiple functions including solubilization, crystal inhibition, steric stabilization, film formation, and thermosensitivity. It shows broad application prospects in drug delivery systems such as solid dispersions, supersaturated self-emulsifying drug delivery systems, nanosuspensions, polymer micelles, and soluble microneedles. It also demonstrates good application potential in antitumor, anti-inflammatory, anti-infective, and transdermal drug delivery fields. Current toxicological information indicates that Soluplus has good preliminary safety. However, current safety data for Soluplus primarily comes from oral administration studies, and systematic evaluations of its local exposure behavior after transdermal administration remain insufficient. Therefore, a more comprehensive and systematic testing and evaluation of the safety of Soluplus as a pharmaceutical excipient is necessary, which depends on establishing accurate quantitative analytical methods for Soluplus in biological matrices.

[0003] Existing methods for the quantitative detection of Soluplus have been reported in studies, including size exclusion chromatography-ultraviolet detection (SEC-UV), size exclusion chromatography-refractive index detection (SEC-RID), atmospheric pressure chemical ionization mass spectrometry (APCI-MS), and thermal pyrolysis-gas chromatography-mass spectrometry (Py-GC / MS). However, these methods have limitations such as poor specificity and reproducibility, low sensitivity, large sample requirements, and complex sample preparation. For example, the precision and accuracy of SEC-UV decrease significantly at sample concentrations below 0.1 mg / mL, making it difficult to meet the quantitative requirements of Soluplus in low-concentration biological samples. Although APCI-MS and Py-GC / MS can be used for polymer quantification, the equipment is expensive and the operation is complex, making them unsuitable for routine bioanalysis. Liquid chromatography-tandem mass spectrometry (LC-MS / MS), as the "gold standard" technology in modern bioanalysis, has significant advantages over the above methods in polymer quantification. However, Soluplus, as a multi-block graft copolymer, exhibits a polydisperse molecular weight distribution and contains multiple ionizable groups, resulting in a complex multi-charge distribution in mass spectrometry. This makes it difficult to directly select a single parent ion-daughter ion pair for quantification, unlike with small molecule drugs. This poses a core challenge for the quantitative analysis of Soluplus using LC-MS / MS. Although LC-MS / MS technology has demonstrated excellent performance in the quantitative analysis of polymers in biological matrices, its application in the quantitative analysis of Soluplus has not yet been reported.

[0004] In summary, there is an urgent need to establish a new analytical method to achieve accurate quantitative detection of Soluplus in biological matrices. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned technical deficiencies and propose an analytical method for PEG-containing copolymers based on intrasource pyrolysis and parallel reaction monitoring. This method solves the technical problems of the existing Soluplus quantitative analysis method, such as poor specificity and reproducibility, low sensitivity, large sample requirements, complex sample preparation, and expensive equipment, which makes it unsuitable for routine analysis.

[0006] This invention provides an analytical method for PEG-containing copolymers based on intrasource cleavage and parallel reaction monitoring, comprising the following steps: Prepare standard solutions containing PEG segment copolymers of different concentrations; The standard solution containing PEG segment copolymer was tested by ultra-high performance liquid chromatography-tandem mass spectrometry, and the chromatographic peak area of ​​the monitoring ion pair corresponding to each concentration was recorded. A standard curve was obtained by regression analysis with the concentration of PEG-containing copolymer in the standard solution as the x-axis and the chromatographic peak area of ​​the monitoring ion pair corresponding to that concentration as the y-axis. The sample was tested using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS), and the chromatographic peak area of ​​the corresponding monitoring ion pair was recorded. Based on the chromatographic peak area of ​​the corresponding monitoring ion pair and the standard curve, the concentration of PEG segment copolymer in the sample was obtained.

[0007] Compared with the prior art, the beneficial effects of the present invention include: This invention utilizes intrasource cleavage and parallel reaction monitoring to convert specific polymeric molecules into substituted ions for quantitative analysis. This enables highly sensitive and high-throughput detection of PEG-containing copolymers in biological matrices, providing a reliable technical means for studying their transdermal permeation behavior, formulation development, and systemic safety evaluation. It also offers a referable technical strategy for the quantitative analysis of similar polymers in other biological matrices. The method of this invention exhibits good specificity and reproducibility, high sensitivity, small sample requirements, simple sample preparation, and low equipment cost, making it suitable for routine analysis. Attached Figure Description

[0008] Figure 1 This is a diagram showing the cleavage pattern of the PEG chain segment in the structure of Soluplus after in-source cleavage in Embodiment 1 of the present invention; Figure 2 This is the first-order mass spectrum of Soluplus after in-source fragmentation in Example 1 of this invention, generating PEG-related characteristic ions. m / z 89.0605, 133.0860, 177.1124, 221.1383, etc.; Figure 3 This is a secondary mass spectrum of Soluplus after in-source fragmentation in Example 1 of this invention; wherein, the substituted parent ion is... m / z 221.1383; Figure 4 This is a Soluplus chromatographic peak area result diagram under different source collision-induced dissociation voltage values ​​in Example 1 of the present invention; among them, since the voltage value at 0~20eV is too low, no obvious characteristic peaks are seen in the chromatogram. Figure 5 This is a graph showing the chromatographic peak areas of Soluplus at CE values ​​of 30, 50, and 80 eV in Example 2 of the present invention. Figure 6 This is a graph showing the chromatographic peak areas of Soluplus at different flow rates in Example 3 of the present invention; Figure 7 This is a graph showing the chromatographic peak areas of Soluplus at different column temperatures in Example 4 of this invention; Figure 8 This is the chromatogram of Soluplus at a column temperature of 40 °C in Example 4 of the present invention; Figure 9 This is the standard curve of Soluplus in a blank matrix in Example 5 of the present invention; Figure 10 This refers to the amount of Soluplus retained in isolated skin of Bama pigs 24 hours after administration in Example 6 of the present invention; wherein, three replicates were performed for each concentration condition. Detailed Implementation

[0009] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0010] In the molecular structure of Soluplus, hydrophilic PEG6000 serves as the main chain, while lipophilic vinyl acetate and vinylcaprolactam are randomly copolymerized as side chains. The PEG segments are easily broken in mass spectrometry, generating a series of PEG-related characteristic fragment ions. Based on this structural characteristic, by controlling the voltage within the ion source, the high molecular weight precursor ion of Soluplus can be efficiently converted into low molecular weight PEG-related characteristic fragment ions. Simultaneously, these PEG-related characteristic fragment ions can further generate specific product ions in the collision chamber, potentially serving as alternative ions for Soluplus to achieve specific quantification in complex biological matrices. However, due to technical challenges such as the multi-charge distribution of the analyte in mass spectrometry making precursor ion identification impossible and the low selectivity of low-abundance fragment ion signals hindering quantitative analysis of Soluplus in biological matrices, there is currently a lack of mature technical solutions combining in-source fragmentation (CID) mode with parallel reaction monitoring (PRM) acquisition methods for Soluplus quantitative analysis, and no reports have been found of applying this strategy to studies of the concentration-effect relationship of Soluplus transdermal osmosis.

[0011] This invention employs ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) to provide an analytical method for PEG-containing copolymers based on in-source fragmentation and parallel reaction monitoring. Specifically, this invention utilizes in-source fragmentation technology to convert PEG-containing copolymers into PEG-related characteristic fragment ions. These PEG-related characteristic fragment ions further generate specific product ions in a collision chamber. The ions formed by these PEG-related fragment ions are used for quantitative analysis of the sample, overcoming the difficulty in screening stable and intact precursor ions for such PEG-containing copolymers. Optimized m / zThe quantitative analysis of PEG-containing copolymers using the 221.1383 / 89.0605 ion pair demonstrated good selectivity, linearity, accuracy, recovery, matrix effect, and stability, fully demonstrating the applicability of the method. When applied to IVPT experiments using Bama pig skin, this method revealed measurable drug retention in the skin, providing preliminary quantitative evidence for understanding the local exposure behavior after Soluplus transdermal administration. It can also support dosage selection in subsequent non-clinical evaluations of Soluplus-containing transdermal formulations. Based on these findings, this invention is proposed.

[0012] This invention provides an analytical method for PEG-containing copolymers based on intrasource cleavage and parallel reaction monitoring, comprising the following steps: S1. Prepare standard solutions containing PEG segment copolymers of different concentrations; S2. The standard solution containing PEG segment copolymer was tested by ultra-high performance liquid chromatography-tandem mass spectrometry, and the chromatographic peak area of ​​the monitoring ion pair corresponding to each concentration was recorded. S3. Using the concentration of PEG-containing copolymer in the standard solution as the x-axis and the chromatographic peak area of ​​the monitoring ion pair corresponding to that concentration as the y-axis, regression analysis was performed to obtain the standard curve. S4. The sample to be tested is tested by ultra-high performance liquid chromatography-tandem mass spectrometry, the chromatographic peak area of ​​the monitoring ion pair corresponding to the sample to be tested is recorded, and the concentration of PEG segment copolymer in the sample to be tested is obtained based on the chromatographic peak area of ​​the monitoring ion pair corresponding to the sample to be tested and the standard curve.

[0013] In this invention, in step S1, the PEG-containing copolymer is Soluplus.

[0014] In this invention, in step S1, the standard solution containing PEG segment copolymer is prepared using a blank matrix solution. The blank matrix solution is a biological matrix solution or solvent that does not contain PEG segment copolymer.

[0015] In some specific embodiments of the present invention, the blank matrix solution is a PBS solution containing a blank Bama pig skin-related matrix.

[0016] In this invention, in step S1, the concentration range of the standard solution containing PEG segment copolymer is 0.300~50.0μg / mL.

[0017] In this invention, in step S1, the standard solution containing PEG segment copolymer includes at least 5 concentration points.

[0018] In this invention, the chromatographic conditions in steps S2 and S4 are as follows: the chromatographic column is The XBridge Protein BEH C4 Column; mobile phase A is a 0.1% (v / v) formic acid aqueous solution, and mobile phase B is a 1:1 (v / v) acetonitrile-isopropanol mixed solvent; gradient elution is used; the column temperature is 30~50°C; the flow rate is 0.3~0.6 mL / min; and the injection volume is 5~20 μL.

[0019] In some specific embodiments of the present invention, the gradient elution conditions are as follows: 0.1~0.4 min, 80% mobile phase A; 0.4~2.5 min, 80%~60% mobile phase A; 2.5~3 min, 60%~40% mobile phase A; 3~3.5 min, 40%~10% mobile phase A; 3.5~4.5 min, 10% mobile phase A; 4.5~4.51 min, 10%~80% mobile phase A; 4.51~5.5 min, 80% mobile phase A.

[0020] In this invention, the mass spectrometry conditions in steps S2 and S4 are as follows: positive electrospray ionization mode is used; spray voltage is 3800V; capillary temperature is 320℃; resolution is 30000; AGC target value is 2e5; maximum injection time is 100 ms; source collision-induced dissociation voltage is 40~80 eV; collision energy is 30~80 eV.

[0021] In this invention, in steps S2 and S4, the monitored ion pairs are... m / z 221.1383 / 89.0605, and the mass-to-charge ratio matching tolerance is 20 ppm.

[0022] In this invention, in steps S2 and S4, the chromatographic peak area is the extracted chromatographic peak area of ​​the monitored ion pair daughter ions.

[0023] In this invention, in step S3, regression analysis is performed using the external standard method.

[0024] In this invention, in step S3, the regression analysis uses the weighted least squares method for regression calculation.

[0025] In some specific embodiments of the present invention, the weighting coefficient of the weighted least squares method is 1 / x. 2 .

[0026] The preferred determination conditions for the absolute quantitative analysis method of PEG-containing copolymers based on intrasource pyrolysis and parallel reaction monitoring in this invention embodiment are as follows: The chromatographic conditions were as follows: Shimadzu LC-40D X3 high-performance liquid chromatography system; column: The XBridgeProtein BEH C4 column (2.1 mm × 50 mm, 3.5 µm, 300 Å); mobile phase: A was 0.1% formic acid aqueous solution, B was acetonitrile-isopropanol (v / v = 1:1), gradient elution; column temperature: 40°C; flow rate: 0.4 mL / min; injection volume: 20 μL; the gradient elution program in the chromatographic conditions is shown in Table 1.

[0027] Table 1 Gradient elution program

[0028] Mass spectrometry conditions were as follows: a Q-Exactive HF-X Orbitrap mass spectrometer was used for detection in positive electrospray ionization mode, equipped with an ESI ionization source and Xcalibur data processing software; the full scan range was set to... m / z 80~1200; Spray voltage: 3800 V; Capillary temperature: 320 ℃; Resolution: 30000; AGC target value: 2e5; Maximum injection time: 100 ms; Intrasource collision-induced dissociation (CID): 60 eV; Collision energy (CE): 80 eV.

[0029] Example 1: Optimization of Intra-Source Collision-Induced Dissociation Voltage Dissolve 1 mg of Soluplus in 1 mL of methanol to prepare a 1 mg / mL stock solution, and dilute with acetonitrile-water (v / v=1 / 1) solution to 10 µg / mL. Take 200 μL of Soluplus solution into each vial for liquid chromatography-tandem mass spectrometry analysis. Set the CID voltage values ​​to 0, 20, 40, 60, 80, and 100 eV, respectively, and the other conditions are the same as the previously selected optimal determination conditions. Record the chromatogram and mass spectrometry response value.

[0030] Example 2: CE value optimization Dissolve 1 mg of Soluplus in 1 mL of methanol to prepare a 1 mg / mL stock solution, and dilute it to 10 µg / mL with acetonitrile-water (v / v=1 / 1) solution. Take 200 μL of Soluplus solution into each vial for liquid chromatography-tandem mass spectrometry analysis. Set the CE values ​​to 30, 50, and 80 eV, respectively, and the rest are the same as the optimized determination conditions mentioned above. Record the chromatogram and mass spectrometry response value.

[0031] Example 3: Flow Rate Optimization Dissolve 1 mg of Soluplus in 1 mL of methanol to prepare a 1 mg / mL stock solution, and dilute it to 10 µg / mL with acetonitrile-water (v / v=1 / 1) solution. Set the flow rates in the liquid phase conditions to 0.3, 0.4, 0.5, and 0.6 mL / min, and the rest to the same as the aforementioned preferred determination conditions. Record the chromatograms and mass spectrometry response values.

[0032] Example 4: Column Temperature Optimization Dissolve 1 mg of Soluplus in 1 mL of methanol to prepare a 1 mg / mL stock solution, and dilute it to 10 µg / mL with acetonitrile-water (v / v=1 / 1) solution; set the column temperature to 30, 40 and 50 °C, and the rest of the determination conditions are the same as those described above, and record the chromatogram and mass spectrometry response value.

[0033] Example 5: Establishment of the Soluplus Standard Curve 1 mg of Soluplus was dissolved in 1 mL of methanol to prepare a 1 mg / mL stock solution, which was then diluted with PBS solution containing blank Bama pig skin-related matrix to final concentrations of 0.300, 1.00, 2.00, 5.00, 10.0, 20.0, and 50.0 μg / mL to prepare calibration standards. The preparation of the PBS solution containing blank Bama pig skin-related matrix included: immobilizing Bama pig skin (Taizhou Taihe Biotechnology Co., Ltd., skin thickness 0.8–1.0 mm) on a Franz diffusion cell; adding PBS solution (pH 7.2–7.4) to the receiving chamber (initial volume 8.0 mL) and allowing it to stand in the transdermal diffusion apparatus; after the surface temperature of the Bama pig skin in the Franz diffusion cell stabilized, 500 μL of PBS solution was aspirated onto the skin surface; and the PBS medium sample in the receiving chamber was collected after 24 hours as the PBS solution containing blank Bama pig skin-related matrix.

[0034] Using the aforementioned preferred measurement conditions, ion pairs were recorded. m / z The chromatogram is 221.1383 / 89.0605; the external standard method was used, with the Soluplus solution concentration as the x-axis and the Soluplus peak area as the y-axis, and weighted average W=1 / x. 2 The least squares method is used to perform regression calculations, and the resulting linear regression equation is the standard curve.

[0035] Example 6: Transdermal permeation behavior of Soluplus on isolated Bama pig skin For the Soluplus in vitro transdermal assay, Soluplus solutions with dosages of 1.00, 10.0, and 20.0 mg / mL were prepared using PBS solution. Before the in vitro transdermal assay, PBS solution was added to the receiving chamber of the Franz diffusion cell, with an initial volume of 8.0 mL, and the chamber was left to stand in the transdermal diffusion apparatus. After the surface temperature of the Bama pig skin in the Franz diffusion cell stabilized, 300 μL of Soluplus solution was aspirated onto the skin surface, with three replicates for each concentration. At 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours, 500 μL of PBS medium samples were collected from the receiving chamber, and immediately, an equal volume of 500 μL of PBS solution was aspirated to replenish the liquid volume in the receiving chamber to 8.0 mL. To investigate the transdermal penetration behavior of Soluplus into isolated Bama pig skin, samples were aspirated from the receiving chamber at each sampling point. To calculate the retention of Soluplus in Bama pig skin at the end of the experiment, skin samples were collected from each diffusion cell and accurately weighed. The collected skin samples were cut into small pieces and extracted with 10 mL of methanol per gram of skin. The samples were homogenized, sonicated, centrifuged, and the supernatant was aspirated and analyzed to determine the amount of Soluplus retained in the skin.

[0036] Please see Figures 1-3 ,pass Figures 1-3 It can be seen that this invention, by employing a combined strategy of in-source cleavage and parallel reaction monitoring, successfully converted high molecular weight precursor ions into low molecular weight PEG-related characteristic fragment ions. m / z (89.0605, 133.0860, 177.1124, 221.1383, etc.), and further generate specific product ions as substitute ions.

[0037] Please see Figure 4 ,pass Figure 4 It can be seen that the source-in-source collision-induced dissociation voltage value of 60 eV is the highest, and 60 eV was finally selected for subsequent experiments.

[0038] Please see Figure 5 ,pass Figure 5 It can be seen that the source fragmentation efficiency is highest when the Soluplus CE value is 80 eV, and 80 eV was finally selected for subsequent experiments.

[0039] Please see Figure 6 ,pass Figure 6 It can be seen that the flow rate range of 0.3 to 0.6 mL / min has little impact on the detection response and chromatographic separation behavior of Soluplus, and 0.4 mL / min was finally selected for subsequent experiments.

[0040] Please see Figures 7-8 ,pass Figures 7-8 It can be seen that column temperature changes within the range of 30–50 °C have little impact on the detection response of Soluplus, and the chromatographic peak shapes remain well maintained under all column temperature conditions. Considering both column robustness and separation efficiency, this invention selects 40 °C as the optimal column temperature.

[0041] Please see Figure 9 ,pass Figure 9 As can be seen, the standard curve has a good linear relationship, indicating that the quantitative analysis method provided by this invention has a good linear relationship, high accuracy, good reproducibility, sensitivity and reliability, and can be used for the absolute quantitative analysis of PEG-containing copolymers represented by Soluplus.

[0042] Please see Figure 10 ,pass Figure 10 It can be seen that when the dosage concentration increases from 1.00 mg / mL to 20.0 mg / mL, the retention of Soluplus in the skin significantly increases, and a possible saturation trend is observed at the dosage concentration of 20.0 mg / mL. This method provides a practical analytical tool for the quantitative analysis of Soluplus and provides preliminary evidence for the local exposure assessment of transdermal drug delivery formulations containing Soluplus.

[0043] In summary, compared with the prior art, the present invention has the following beneficial effects: (1) Breakthrough in the technical bottleneck of direct quantitative analysis of block copolymers and / or graft copolymers by LC-MS / MS: This invention creatively adopts a strategy of combining source fragmentation with parallel reaction monitoring to address the complex mass spectrometry behavior of Soluplus with polydisperse and multicharge distribution of molecular weight. This is achieved by converting high molecular weight precursor ions into low molecular weight PEG-related characteristic fragment ions ( m / z (89.0605, 133.0860, 177.1124, 221.1383, etc.) and further generate specific product ions as substitute ions, cleverly circumventing the problem of not being able to select a single parent ion-daughter ion pair, successfully realizing the application of LC-MS / MS technology in Soluplus quantitative analysis, providing a new approach for the bioanalysis of this type of polymer.

[0044] (2) Significantly improved detection sensitivity and specificity: Compared to the existing SEC-UV method, which suffers from a significant decrease in precision and accuracy at concentrations below 0.1 mg / mL, the method of this invention employs an in-source lysis combined with a parallel reaction monitoring and acquisition mode, resulting in higher sensitivity (≤0.300 μg / mL), meeting the accurate quantitative requirements of Soluplus in low-concentration biological samples. Simultaneously, by employing a substitution ion strategy, the quantitative detection of PEG-containing copolymer segments is transformed into quantitative analysis using PEG substitution ions, achieving optimal specificity, sensitivity, and linearity. m / z Analysis of Soluplus using 221.1383 / 89.0605 effectively eliminated endogenous interference from complex biological matrices, ensuring the reliability of quantitative results.

[0045] (3) The first systematic quantification of Soluplus in transdermal drug delivery biomatrix was achieved: This invention applied the method to the in vitro transdermal experiment of Soluplus, and for the first time systematically studied the concentration-effect relationship of its transdermal penetration, filling the gap in quantitative analysis data in the systematic safety assessment of Soluplus after transdermal drug delivery, and providing key data support for subsequent formulation development and in vivo safety evaluation.

[0046] (4) It has wide applicability and high promotion value: The analytical strategy of the present invention is not only applicable to the quantitative detection of Soluplus, but also provides a direct technical reference and platform basis for the quantitative analysis of PEG-containing graft copolymers with similar structural features in other biological matrices.

[0047] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. An analytical method for PEG-containing copolymers based on intrasource pyrolysis and parallel reaction monitoring, characterized in that, Includes the following steps: Prepare standard solutions containing PEG segment copolymers of different concentrations; The standard solution containing PEG segment copolymer was tested by ultra-high performance liquid chromatography-tandem mass spectrometry, and the chromatographic peak area of ​​the monitoring ion pair corresponding to each concentration was recorded. A standard curve was obtained by performing regression analysis with the concentration of the PEG-containing copolymer in the standard solution as the x-axis and the chromatographic peak area of ​​the monitoring ion pair corresponding to the concentration as the y-axis. The sample to be tested was tested by ultra-high performance liquid chromatography-tandem mass spectrometry, and the chromatographic peak area of ​​the monitoring ion pair corresponding to the sample to be tested was recorded. Based on the chromatographic peak area of ​​the monitoring ion pair corresponding to the sample to be tested and the standard curve, the concentration of PEG segment copolymer in the sample to be tested was obtained.

2. The analytical method for PEG-containing copolymers based on intrasource pyrolysis and parallel reaction monitoring according to claim 1, characterized in that, The PEG-containing copolymer is Soluplus.

3. The analytical method for PEG-containing copolymers based on intrasource pyrolysis and parallel reaction monitoring according to claim 1, characterized in that, The standard solution containing PEG segment copolymer was prepared using a blank matrix solution; wherein... The blank matrix solution is a biological matrix solution or solvent that does not contain PEG-containing copolymer segments; and / or, The blank matrix solution is a PBS solution containing blank Bama pig skin-related matrix.

4. The analytical method for PEG-containing copolymers based on intrasource pyrolysis and parallel reaction monitoring according to claim 1, characterized in that, The concentration range of the PEG-containing copolymer standard solution is 0.300~50.0 μg / mL; and / or, The standard solution containing PEG segment copolymers includes at least 5 concentration points.

5. The analytical method for PEG-containing copolymers based on intrasource pyrolysis and parallel reaction monitoring according to claim 1, characterized in that, The chromatographic conditions were as follows: the column was The XBridge Protein BEH C4 Column; mobile phase A was a 0.1% (v / v) formic acid aqueous solution, and mobile phase B was a 1:1 (v / v) acetonitrile-isopropanol mixed solvent; gradient elution was used; the column temperature was 30–50°C; the flow rate was 0.3–0.6 mL / min; and the injection volume was 5–20 μL.

6. The analytical method for PEG-containing copolymers based on intrasource pyrolysis and parallel reaction monitoring according to claim 5, characterized in that, The gradient elution conditions are as follows: 0.1–0.4 min, 80% mobile phase A; 0.4–2.5 min, 80%–60% mobile phase A; 2.5–3 min, 60%–40% mobile phase A; 3–3.5 min, 40%–10% mobile phase A; 3.5–4.5 min, 10% mobile phase A; 4.5–4.51 min, 10%–80% mobile phase A; 4.51–5.5 min, 80% mobile phase A.

7. The analytical method for PEG-containing copolymers based on intrasource pyrolysis and parallel reaction monitoring according to claim 1, characterized in that, The mass spectrometry conditions were as follows: positive electrospray ionization mode was used; the spray voltage was 3800 V; the capillary temperature was 320 °C; the resolution was 30000; the AGC target value was 2e5; the maximum injection time was 100 ms; the source collision-induced dissociation voltage was 40~80 eV; and the collision energy was 30~80 eV.

8. The analytical method for PEG-containing copolymers based on intrasource pyrolysis and parallel reaction monitoring according to claim 1, characterized in that, The monitored ion pair is m / z 221.1383 / 89.0605, and the mass-to-charge ratio matching tolerance is 20 ppm.

9. The analytical method for PEG-containing copolymers based on intrasource pyrolysis and parallel reaction monitoring according to claim 1, characterized in that, The chromatographic peak area is the extracted chromatographic peak area of ​​the monitored ion pair sub-ions.

10. The analytical method for PEG-containing copolymers based on intrasource pyrolysis and parallel reaction monitoring according to claim 1, characterized in that, Regression analysis was performed using the external standard method; and / or, The regression analysis employs weighted least squares regression; the weighting coefficient for the weighted least squares method is 1 / x. 2 .

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