A method for quantitative analysis of DMG-PEG2000 in biological matrices

Abstract

This invention discloses a method for quantitative analysis of DMG-PEG2000 in biological matrices. The method employs ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry (UHPLC-MS / MS) in positive ion mode using an electrospray ionization source. A multi-channel selected reaction monitoring (CRM) method is established with m / z selected as 810.2±0.2 for Q1 and 495.5±0.2 for Q3 of DMG-PEG2000. The method provided by this invention avoids interference from DMG-PEG2000 in vivo degradation products and endogenous lipids in the biological matrix, solving the problem of biological matrix interference in the quantitative analysis of DMG-PEG2000. It exhibits high sensitivity and specificity.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for quantitative analysis of DMG-PEG2000 in biological matrices. Background Technology

[0002] DMG-PEG2000, short for 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000, is a nonionic surfactant that reduces surface tension and improves dispersibility. DMG-PEG2000 is widely used in the preparation of lipid nanoparticles (LNPs) to enhance their mucoid permeability and drug delivery efficiency. When DMG-PEG2000-containing LNPs encapsulating therapeutic drugs (such as mRNA or small molecule drugs) are administered intravenously or subcutaneously, the DMG-PEG2000 enters the circulation and distributes to various tissues along with the LNP. Therefore, quantitative analysis of the concentration of DMG-PEG2000 in biological matrices is crucial for studying the in vivo distribution, elimination mechanisms, and pharmacokinetics of LNP carrier materials.

[0003] However, quantitative analysis of DMG-PEG2000 faces two major challenges:

[0004] (1) Polydispersity interference: The PEG2000 chain in DMG-PEG2000 is composed of 34-54 ethylene glycol units (corresponding to m / z 600-1000), which is a multi-component mixture and is easily degraded in vivo into short-chain PEG fragments (m / z 400-700), which overlap with the target ions;

[0005] (2) Interference from biological matrix: Biological matrix (such as plasma) contains a large amount of endogenous lipids (such as phosphatidylcholine) and proteins, which can easily inhibit the ionization efficiency of DMG-PEG2000 and generate interference peaks, affecting the detection results.

[0006] In the existing technology, the analytical methods for DMG-PEG2000 mainly focus on the qualitative identification of LNP raw materials and finished products, such as high-resolution mass spectrometry full scan or evaporative light scattering detection. However, these methods have obvious defects: (1) Although high-resolution mass spectrometry (such as ACQUITY RDa) can identify the distribution of PEG units, it adopts full scan mode, which has weak resistance to biological matrix interference and low quantitative sensitivity (LOD ≥ 20 ng / mL in biological matrix); (2) The ELSD evaporative light scattering detector is a general-purpose detector with no specificity, which cannot distinguish DMG-PEG2000 from degradation products, and its sensitivity is extremely poor (LOQ ≥ 5 μg / mL), which is far from meeting the quantitative requirements of low concentration (5-5000 ng / mL) in vivo. In summary, the existing technical methods do not involve the pretreatment optimization of biological matrix, and cannot solve the problems of protein precipitation and removal of endogenous interference.

[0007] Therefore, there is an urgent need in this field to establish a highly sensitive and specific quantitative analysis method to avoid interference from DMG-PEG2000 in vivo degradation products and endogenous lipids in biological matrices, and to solve the problem of quantitative analysis of DMG-PEG2000 in biological matrices. Summary of the Invention

[0008] To address the aforementioned technical issues, this invention selects a specific molecular ion with a relatively high mass-to-charge ratio (M / C ratio) for DMG-PEG2000. This helps avoid potential interference from its in vivo degradation products in biological matrix samples. However, molecular ions with excessively high M / C ratios exhibit lower detection sensitivity. Therefore, molecular ions with M / C ratios in the range of 800 to 900 are more suitable. Regarding the selection of specific fragment ions, the characteristic spectral peak of the lipid fraction (m / z 495.5) theoretically better represents the analyte itself and avoids interference from PEG2000 in the sample. However, in complex biological matrix samples, endogenous interference is unknown and uncertain. Therefore, establishing a multi-channel selective reaction monitoring method and collecting biological matrix samples after lipid nanoparticle drug administration for method evaluation is indispensable. Ultimately, the optimal multi-channel selective reaction monitoring method is determined through evaluation and can be used for the quantitative analysis of DMG-PEG2000 in biological matrix samples after lipid nanoparticle drug administration.

[0009] In summary, this invention provides a method for quantitative analysis of DMG-PEG2000 in biological matrices. The method employs liquid chromatography-tandem quadrupole mass spectrometry for quantitative analysis and establishes a multi-channel selected reaction monitoring method. The method includes the following steps:

[0010] Step 1, Sample pretreatment: Take the biological matrix sample, add internal standard solution and mix well, then add protein precipitant, mix well and centrifuge, take the supernatant. The protein precipitant is a mixed solution of isopropanol and methanol, and the volume ratio of isopropanol to methanol in the mixed solution is 1:0.8~1:1.2.

[0011] Step 2, Liquid chromatography-tandem quadrupole mass spectrometry detection: Detection was performed using an electrospray ionization source in positive ion mode;

[0012] The liquid chromatography conditions were as follows: a C8 reversed-phase column with a particle size of 1.7–2.7 μm and a column length of 50–100 mm; mobile phase A was an acidic aqueous solution containing 5–10 mM ammonium acetate; mobile phase B was an acetonitrile solution containing 0.1%–0.5% formic acid; and gradient elution was used. The mass spectrometry conditions were as follows: the range of the Q1 molecular ion was selected to be 800–900 m / z; the Q3 fragment ion was fixed as the characteristic fragment ion of the DMG-PEG2000 lipid fraction, with an m / z of 495.5 ± 0.5; and a multi-channel selective reaction monitoring method for at least three groups of molecular ions → fragment ions was established.

[0013] Step 3, Method Validation and Quantification: The sensitivity, specificity, precision, and accuracy of the multi-channel selective reaction monitoring method were examined using standard curve samples. Combined with reproducibility verification using actual biological matrix samples, the optimal selective reaction monitoring channel was determined to be Q1 m / z 810.2±0.2→Q3 m / z 495.5±0.2. Then, a standard curve was established using the weighted least squares method with w=1 / x² to quantify DMG-PEG2000 in the biological matrix.

[0014] Specifically, the volume ratio of the protein precipitant to the biological matrix sample is 5:1 to 8:1, the centrifugation conditions are 4°C, 3000 to 3500 g for 8 to 12 minutes, and the internal standard solution is a 50% acetonitrile solution containing 100 to 500 ng / mL verapamil or propafenone, with a volume ratio of 1:1 between the internal standard solution and the biological matrix sample.

[0015] Specifically, the three molecular ion → fragment ion channels of the multichannel selected reaction monitoring method are: m / z 678.3 → 616.6, m / z 722.3 → 661.0 and m / z 810.2 → 495.5, wherein the mass spectrometry parameters of the m / z 810.2 → 495.5 channel are: declustering voltage 30~40 V and collision energy 30~40 eV.

[0016] Specifically, in step 2, the C8 reversed-phase column is a Waters ACQUITY UPLC BEH C8 column with a particle size of 1.7 μm, a column length of 50 mm, and an inner diameter of 2.1 mm; the gradient elution program is as follows: the initial mobile phase B ratio is 60%~70%, it is increased to 100% B phase within 1.2~1.8 minutes, maintained at 100% B phase for 3.2~3.8 minutes, and then restored to the initial B phase ratio for 3.51~3.81 minutes and maintained until the end of the run, with a total run time of 4~5 minutes.

[0017] Specifically, the tandem quadrupole mass spectrometer is a Sciex TRIPLEQUAD 6500 plus or equivalent mass spectrometer, and the electrospray ionization source parameters are: spray voltage 5000~6000V, ion source temperature 500~600℃, curtain gas pressure 25~35psi, nebulizing gas pressure 50~60psi, heating gas pressure 50~60psi, and collision gas pressure 8~10psi.

[0018] Specifically, in step 3, the preparation method of the standard curve sample is as follows: DMG-PEG2000 standard is dissolved in dimethyl sulfoxide to prepare a stock solution, diluted with 50% acetonitrile to prepare a series of working solutions, and added to blank biological matrix at a volume ratio of 1:19 to obtain standard curve samples with concentrations of 5, 10, 50, 500, 1000, 2000, 4000, and 5000 ng / mL.

[0019] Specifically, in step 3, the regression equation of the standard curve uses the theoretical concentration of DMG-PEG2000 as the abscissa and the peak area ratio of DMG-PEG2000 to the internal standard as the ordinate, with a regression coefficient R² ≥ 0.995.

[0020] Specifically, the biological matrix sample is an animal or human biological matrix after administration of lipid nanoparticle drugs, and the biological matrix sample includes rat plasma, mouse liver tissue homogenate, human urine or rat fecal homogenate.

[0021] Specifically, the sample volume of the biological matrix is ​​20~50μL. If the biological matrix is ​​a tissue sample, it needs to be homogenized first using a tissue homogenizer, and then protein precipitation treatment is performed according to step 1.

[0022] Specifically, the injection volume for the liquid chromatography-tandem quadrupole mass spectrometry detection is 1~2 μL, the column temperature is 35~45℃, and the flow rate is 0.5~0.7 mL / min; the needle washing solution is 50% acetonitrile solution.

[0023] The method for quantitative analysis of DMG-PEG2000 in biological matrices of the present invention has the following beneficial effects:

[0024] 1. The method for quantitative analysis of DMG-PEG2000 in biological matrices provided by this invention employs a dual-targeting strategy of "Q1 molecular ion screening (m / z 800-900 range) + Q3 immobilization of lipid core fragments (m / z 495.5)" to avoid interference from DMG-PEG2000 degradation products in vivo (such as short-chain PEG fragments at m / z 400-700) and endogenous lipids in biological matrices (such as phosphatidylcholine) at the source. No interference peaks are observed in rat blank plasma, solving the problem of interference in biological matrices. This method has good specificity and achieves interference-free quantification of DMG-PEG2000 in biological matrices.

[0025] 2. This method has a lower limit of quantification (LLOQ) as low as 10 ng / mL and an upper limit of quantification (ULOQ) of 2000 ng / mL. In addition to the conventional linear range of 10-2000 ng / mL, high-concentration samples can be diluted, and the accuracy of the diluted results still meets the requirements, perfectly matching the concentration changes of DMG-PEG2000 in the biological matrix after administration of lipid nanoparticles (LNPs). Compared with existing evaporative light scattering (ELSD, LOQ ≥ 5 μg / mL) detection methods, the sensitivity is improved by 500 times, solving the technical pain point of inaccurate quantification of low-concentration samples (such as tissues and excrement in the later stages of drug administration), and meeting the core needs of pharmacokinetic studies.

[0026] 3. The method provided by this invention has a highly efficient pretreatment method that is compatible with biological matrices. It uses an isopropanol-alcohol mixture as a protein precipitant, and the extraction recovery rate is greater than 85%. In addition, the pretreatment of this method only requires 15 minutes and does not require complicated purification steps. It can be directly adapted to various biological matrices such as plasma, tissue homogenate, and urine, taking into account both recovery rate and ease of operation, and reducing the time and labor costs of batch sample analysis. Attached Figure Description

[0027] Figure 1 This is the Q1 full scan spectrum of DMG-PEG2000 in Example 1.

[0028] Figure 2 This is the Q3 full scan spectrum of the DMG-PEG2000 molecular ion m / z 810.2 after CID in Example 1.

[0029] Figure 3 This is the standard curve of DMG-PEG2000 in rat plasma in Example 1.

[0030] Figure 4 This is the SRM mass chromatogram of the rat blank plasma sample in Example 1.

[0031] Figure 5This is the SRM mass chromatogram of the sample in rat plasma with the lower limit of quantification of DMG-PEG2000 in Example 1.

[0032] Figure 6 This is the m / z 678.3→616.6 channel SRM mass chromatogram of a rat blank plasma sample during the method development stage in Example 2.

[0033] Figure 7 This is the m / z 678.3→616.6 channel SRM mass chromatogram of a rat plasma sample containing DMG-PEG2000 (50 ng / mL) during the method development stage in Example 2.

[0034] Figure 8 This is the m / z 810.2→495.5 channel SRM mass chromatogram of a rat blank plasma sample during the method development stage in Example 2.

[0035] Figure 9 This is the m / z 810.2→495.5 channel SRM mass chromatogram of a rat plasma sample containing DMG-PEG2000 (50 ng / mL) during the method development stage in Example 2. Detailed Implementation

[0036] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] Unless otherwise specified, all reagents used in the following examples are commercially available reagents. Table 1 shows the reagents and consumables used in all the following examples, and Table 2 shows the instruments and models used in all the following examples.

[0038] Table 1. Reagent and Consumable Information

[0039]

[0040] Table 2 Instruments

[0041]

[0042] Example 1

[0043] This embodiment illustrates the quantitative analysis of DMG-PEG2000 in rat plasma using the method provided in this application, taking rat plasma as an example. The determination was performed using ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry (UPLC-MS / MS). The specific procedures are as follows:

[0044] A. Preparation of DMG-PEG2000 Standard Curve and Quality Control Samples: Accurately weigh 5 mg of DMG-PEG2000 standard and dissolve it in 1 mL of dimethyl sulfoxide to obtain a 5 mg / mL standard stock solution. Dilute the standard stock solution with 50% acetonitrile solution to prepare a series of standard curve working solutions. Add these solutions to rat blank plasma at a volume ratio of 1:19 and mix well to obtain standard curve samples with concentrations of 5, 10, 50, 500, 1000, 2000, 4000, and 5000 ng / mL, and quality control samples with concentrations of 5, 15, 200, 2500, and 3750 ng / mL, respectively.

[0045] B. Processing of rat plasma samples: Take 30 μL each of blank, standard curve, quality control and rat research samples, add 30 μL of internal standard solution (50% acetonitrile solution containing verapamil) and mix well. Add 180 μL of protein precipitant (isopropanol:methanol = 50:50) and mix well for 5 minutes. Then centrifuge at 4 °C and 3200 g for 10 minutes. Take 120 μL of supernatant and place it in an autosampler for injection.

[0046] C. Chromatographic conditions: The chromatographic column was a Waters ACQUITY UPLC BEH C8 1.7 μm 50 × 2.1 mm, the flow rate was 0.6 mL / min, mobile phase A was an aqueous solution containing 5 mM ammonium acetate, mobile phase B was a 95% acetonitrile solution containing 0.5% formic acid and 5 mM ammonium acetate, the injection solution was 50% acetonitrile solution, the column temperature was 40 °C, the injection volume was 1 μL, the run time was 4.2 minutes, and the gradient elution program was as follows: starting with 65% B phase, gradually increasing to 100% B phase at 1.5 minutes, maintaining 100% B phase for 3.5 minutes, and then returning to 65% B phase from 3.5 minutes until the end of the run.

[0047] D. Mass Spectrometry Conditions: A Sciex TRIPLEQUAD 6500 plus triple quadrupole mass spectrometer, equipped with an ESI ion source and Analyst data processing software. MRM detection in positive ion mode was used. The ion source spray voltage was 5500 V, the ion source temperature was 550 °C, the curtain gas nitrogen pressure was 30 psi, the nebulizer gas nitrogen pressure was 55 psi, the heating gas nitrogen pressure was 55 psi, and the collision gas nitrogen pressure was 9 psi. The DMG-PEG2000 detector showed Q1 at m / z 810.2, a declustering voltage of 35 V, and Q3 at m / z 495.5 with a collision energy of 35 eV.

[0048] Figure 1The Q1 full-scan spectrum of DMG-PEG2000 shows that the molecular ion peak of DMG-PEG2000 in positive ion mode of electrospray ionization is mainly distributed in the mass-to-charge ratio (m / z) range of 600–1000, corresponding to the PEG2000 chain structure containing 34–54 ethylene glycol units. Among them, the molecular ion peak at m / z 810.2 has the highest relative intensity, with an ion response value of 2.5 × 10⁻⁶. 4 The peaks are symmetrical and without obvious interference peaks. This invention selects molecular ions in the m / z range of 800~900, which avoids the PEG degradation product range of m / z 400~700, and selects the m / z 810.2 ion with the best response as the core detection target, laying the foundation for subsequent specific quantification and solving the problem of insufficient sensitivity in the prior art. Figure 2 This is the Q3 full-scan spectrum of the DMG-PEG2000 molecular ion (m / z 810.2) after collision-induced fragmentation (CID). It can be seen that after CID, multiple fragment ion peaks appear in the Q3 full-scan spectrum, with the fragment ion peak at m / z 495.5 exhibiting the highest relative intensity and an ion response value of 2.0 × 10⁻⁶. 4 The peak exhibits good separation from other fragment ions. Combined with fragment ion source analysis, m / z 495.5 is identified as the characteristic ion of the lipid core (1,2-dimyristic-rac-glycerol) remaining after the loss of the 3-oxy-polyethylene glycol 2000 group from DMG-PEG2000. This peak has no adjacent interfering peaks (peak area ratio exceeding 98%), demonstrating the rationality of selecting m / z 495.5 as the Q3 characteristic fragment ion in this invention. This completely avoids interference from PEG chain-related fragments (such as m / z 385). Compared to existing technologies using PEG fragment ions as the detection target, this method significantly improves specificity, ensuring the accuracy of quantitative results in biological matrices.

[0049] E. Standard Curve Regression: The mass chromatographic peaks detected by DMG-PEG2000 and the internal standard verapamil were integrated using Analyst software. The theoretical concentration of DMG-PEG2000 in the standard curve sample was plotted on the x-axis, and the ratio of the peak area of ​​DMG-PEG2000 to the peak area of ​​the internal standard was plotted on the y-axis. A weighted average was calculated using w=1 / x. 2 The least squares method is used to perform regression calculations, and the linear regression equation obtained is the standard curve (y=0.000302x+0.00122). Figure 3The standard curve of DMG-PEG2000 in rat plasma shows excellent linearity, with advantages such as a wide quantitative range (5~5000 ng / mL) and good linear stability. Compared with existing methods, this application can cover the full concentration range of DMG-PEG2000 in vivo after LNP administration, providing complete concentration-time data support for pharmacokinetic studies.

[0050] F. Quantitative Analysis and Method Validation: DMG-PEG2000 in quality control samples and rat plasma samples after administration of lipid nanoparticle drugs was quantitatively analyzed using the standard curve described in step E. Figure 4 The image shows the SRM quality chromatogram of a blank rat plasma sample. No obvious chromatographic peak was observed at the target retention time (2.1 ± 0.1 min) of DMG-PEG2000, with only baseline noise (peak area < 100 counts). This demonstrates that the sample pretreatment (isopropanol-methanol protein precipitation) and the detection channel (m / z 810.2 → 495.5) of the multi-channel selected reaction monitoring method of this invention can effectively remove interference from endogenous matrix in plasma, with no false positive signals. Figure 5 This is the SRM mass chromatogram of DMG-PEG2000 in rat plasma at the lower limit of quantification. It can be seen that a symmetrical and sharp chromatographic peak appears at the target retention time, and the peak shape has no tailing.

[0051] The sensitivity, specificity, recovery rate, precision, and accuracy of the method, as well as the accuracy and reproducibility of the actual sample determination, were investigated. The precision and accuracy results of the determination of DMG-PEG2000 in rat plasma samples are shown in Table 3, and the recovery rate results are shown in Table 4.

[0052] Table 3

[0053]

[0054] Table 4

[0055]

[0056] The above method was found to meet all the pre-set acceptance criteria and can be used for the quantitative analysis of DMG-PEG2000 in rat plasma after administration of lipid nanoparticle drugs.

[0057] Example 2

[0058] This example illustrates the screening process for quantitative analysis of SRM channels in rat plasma using DMG-PEG2000.

[0059] In positive ion mode of electrospray ionization, the molecular ion peak of DMG-PEG2000 is mainly distributed in the mass-to-charge ratio (m / z) range of 600–1000, corresponding to the PEG2000 chain structure containing 34–54 ethylene glycol units. Screening for SRM quantitative channels with strong mass spectrometry response and no endogenous interference is crucial for the sensitivity and specificity of the method. In the early stages of method development, this was mainly achieved by comparing the mass chromatograms of blank plasma samples and plasma samples containing DMG-PEG2000 collected by different SRM quantitative channels.

[0060] First, by comparing the Q1 full-scan chromatograms of blank plasma samples and plasma samples containing DMG-PEG2000 (50 ng / mL), DMG-PEG2000 molecular ions with strong responses in blank plasma were excluded. Using the remaining DMG-PEG2000 molecular ions as Q1 detection ions and the fragment ion at m / z 495.5 as Q3 detection ions, a multi-channel SRM method was established. Mass chromatograms of blank plasma samples and plasma samples containing DMG-PEG2000 (50 ng / mL) were acquired separately. By comparison, DMG-PEG2000 molecular ions with low responses in blank plasma samples and high signal-to-noise ratios in plasma samples containing DMG-PEG2000 (50 ng / mL) were selected as candidate ions. Through the above screening, three candidate molecular ions were obtained: m / z 678.3, m / z 722.3, and m / z 810.2.

[0061] A multichannel SRM method was established for the three candidate DMG-PEG2000 molecular ions selected through screening. The optimized detection channels were: m / z 678.3→616.6, m / z 722.3→661.0, and m / z 810.2→495.5. The recoveries of DMG-PEG2000 at low (150 ng / mL) and high (7500 ng / mL) concentrations in plasma control samples were investigated using these three quantitative channels, and the results met the acceptance criteria. In the comparison of mass chromatograms of blank plasma samples and plasma samples containing DMG-PEG2000 (50 ng / mL), it was found that the detection interference of the m / z 810.2→495.5 channel in the blank plasma sample was less than that of the other two channels.

[0062] Taking the comparison of m / z 678.3→616.6 and m / z 810.2→495.5 channels as an example, Table 5 shows the recovery rate results of DMG-PEG2000 quality control samples in plasma detected by the m / z 678.3→616.6 channel, and Table 6 shows the recovery rate results of DMG-PEG2000 quality control samples in plasma detected by the m / z 810.2→495.5 channel.

[0063] Table 5

[0064]

[0065] Table 6

[0066]

[0067] As can be seen from the results in Tables 5 and 6, the recovery rates of the two channels showed little variation and met the acceptance criteria. Figure 6 and Figure 7 Mass chromatograms of blank plasma samples and plasma samples containing DMG-PEG2000 (50 ng / mL) were collected from channels 678.3 to 616.6 of m / z, respectively. It can be seen that the response intensity (cps) of DMG-PEG2000 at the retention time (1.73 min) in the blank sample is about 800, while the response intensity (cps) of the plasma sample containing DMG-PEG2000 (50 ng / mL) is about 4600, which is equivalent to 6 times that of the blank sample. Figure 8 and Figure 9 Mass chromatograms of blank plasma samples and plasma samples containing DMG-PEG2000 (50 ng / mL) were collected using the m / z 810.2→495.5 channel. The results show that the response intensity (cps) at the retention time (1.73 min) of DMG-PEG2000 in the blank sample is approximately 100, while the response intensity (cps) in the plasma sample containing DMG-PEG2000 (50 ng / mL) is approximately 2200, equivalent to 22 times that of the blank sample. In summary, it can be predicted that the m / z 810.2→495.5 channel will have better detection specificity when the limit of quantitation is reduced from 50 ng / mL to 10 ng / mL.

[0068] Therefore, m / z 810.2→495.5 was selected as the quantitative channel, and the other two SRM channels were selected as qualitative channels. After further optimization of the liquid chromatography conditions, the lower limit of quantification of DMG-PEG2000 in plasma was 10 ng / mL.

[0069] In summary, the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for quantitatively analyzing DMG-PEG 2000 in a biological matrix, characterized by, The method adopts liquid chromatography tandem quadrupole mass spectrometry for quantitative analysis, establishes a multi-channel selected reaction monitoring method, and comprises the following steps: Step 1, sample pretreatment: take a biological matrix sample, add an internal standard solution and mix, then add a protein precipitant, mix and centrifuge, take the supernatant, the protein precipitant is a mixed solution of isopropanol and methanol, the volume ratio of isopropanol to methanol in the mixed solution is 1:0.8~1:1.2; Step 2, liquid chromatography tandem quadrupole mass spectrometry detection: detection is carried out in positive ion mode by electrospray ion source; Among them, the liquid chromatography conditions are: C8 reversed phase chromatographic column, particle size 1.7~2.7 μm, column length 50~100 mm, mobile phase A is acidic aqueous solution containing 5~10 mM ammonium acetate, mobile phase B is acetonitrile solution containing 0.1%~0.5% formic acid, and the elution mode is gradient elution; the gradient elution program is: the initial mobile phase B ratio is 60%~70%, increased to 100% B phase within 1.2~1.8 minutes, maintained at 100% B phase for 3.2~3.8 minutes, restored to the initial B phase ratio for 3.51~3.81 minutes and maintained until the end of the run, the total running time is 4~5 minutes; the mass spectrometry conditions are: the Q1 molecular ion is selected in the range of 800~900 m / z, the Q3 fragment ion is fixed as the characteristic fragment ion of the DMG-PEG2000 lipid moiety, m / z 495.5±0.5, and at least 3 groups of molecular ion→fragment ion multi-channel selected reaction monitoring methods are established; Step 3, method verification and quantification: the sensitivity, specificity, precision and accuracy of the multi-channel selected reaction monitoring method are investigated by standard curve samples, combined with the reproducibility verification of actual biological matrix samples, to determine the optimal selected reaction monitoring channel, which is Q1 m / z 810.2±0.2→Q3 m / z 495.5±0.2, then the weighted w=1 / x² least squares method is used to establish a standard curve for the quantification of DMG-PEG2000 in biological matrix; Among them, the biological matrix is all rat plasma.

2. The method for quantitatively analyzing DMG-PEG2000 in a biological matrix according to claim 1, characterized by, The volume ratio of the protein precipitant to the biological matrix sample is 5:1~8:1, the centrifugation condition is 4℃, 3000~3500 g centrifugation for 8~12 minutes, the internal standard solution is 50% acetonitrile solution containing 100~500 ng / mL verapamil or propafenone, and the volume ratio of the internal standard solution to the biological matrix sample is 1:

1.

3. The method of quantitatively analyzing DMG-PEG2000 in a biological matrix according to claim 1, characterized by, The 3 groups of molecular ion→fragment ion channels of the multi-channel selected reaction monitoring method are: m / z 678.3→616.6, m / z 722.3→661.0 and m / z 810.2→495.5, wherein the mass spectrometry parameters of the m / z 810.2→495.5 channel are: declustering voltage 30~40 V, collision energy 30~40 eV.

4. The method of quantitatively analyzing DMG-PEG2000 in a biological matrix according to claim 1, characterized by, In step 2, the C8 reverse phase chromatographic column is a Waters ACQUITY UPLC BEH C8 chromatographic column with a particle size of 1.7 μm, a column length of 50 mm, and an inner diameter of 2.1 mm.

5. The method of quantitatively analyzing DMG-PEG2000 in a biological matrix according to claim 1, characterized by, The tandem quadrupole mass spectrometer is a Sciex TRIPLEQUAD 6500 plus mass spectrometer, and the parameters of the electrospray ion source are as follows: a spray voltage of 5000-6000 V, an ion source temperature of 500-600 °C, a gas curtain pressure of 25-35 psi, an atomization gas pressure of 50-60 psi, a heating gas pressure of 50-60 psi, and a collision gas pressure of 8-10 psi.

6. The method of quantitatively analyzing DMG-PEG2000 in a biological matrix according to claim 1, characterized by, In step 3, the preparation method of the standard curve sample is as follows: the DMG-PEG2000 standard is dissolved in dimethyl sulfoxide to prepare a stock solution, which is diluted with 50% acetonitrile to prepare a series of working solutions, and then added to a blank biological matrix at a volume ratio of 1:19 to obtain standard curve samples with concentrations of 5, 10, 50, 500, 1000, 2000, 4000, and 5000 ng / mL.

7. The method of quantitatively analyzing DMG-PEG2000 in a biological matrix according to claim 1, characterized by, In step 3, the regression equation of the standard curve is obtained by taking the theoretical concentration of DMG-PEG2000 as the abscissa and the peak area ratio of DMG-PEG2000 to the internal standard as the ordinate, and the regression coefficient R² is ≥0.

995.

8. The method of quantitatively analyzing DMG-PEG2000 in a biological matrix according to claim 1, wherein, The sampling volume of the biological matrix sample is 20-50 μL. If the biological matrix is a tissue sample, it needs to be prepared into a homogenate by a tissue homogenizer first, and then subjected to protein precipitation treatment according to step 1.

9. The method of quantitatively analyzing DMG-PEG 2000 in a biological matrix according to claim 1, wherein, The injection volume for liquid chromatography tandem quadrupole mass spectrometry detection is 1-2 μL, the column temperature is 35-45 °C, and the flow rate is 0.5-0.7 mL / min. The needle washing liquid is 50% acetonitrile solution.

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