Method for quantitatively analyzing DMG-PEG2000 in biological matrix

Abstract

The invention discloses a method for quantitative analysis of DMG-PEG2000 in a biological matrix, which adopts ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry for quantitative analysis, selects an electrospray ion source positive ion mode, selects m / z 810.2 + / -0.2 for Q1 of DMG-PEG2000, selects m / z 495.5 + / -0.2 for Q3 of DMG-PEG2000, and establishes a multi-channel selective reaction monitoring method. According to the method provided by the invention, interference of DMG-PEG2000 in-vivo degradation products and biological matrix endogenous lipid is avoided, the problem of biological matrix interference in DMG-PEG2000 quantitative analysis is solved, and the method is high in sensitivity and good in specificity.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a method for quantitatively analyzing DMG-PEG2000 in a biological matrix. BACKGROUND

[0002] DMG-PEG2000, also known as 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000, is a non-ionic surfactant that can reduce surface tension and improve substance dispersibility. DMG-PEG2000 is widely used in the preparation of lipid nanoparticles (LNP) to enhance the mucus permeability and drug delivery efficiency of LNP. When DMG-PEG2000-containing LNP encapsulating therapeutic drugs (such as mRNA and small molecule drugs) is administered intravenously or subcutaneously, DMG-PEG2000 will enter the body circulation with LNP and distribute to various tissues. Therefore, quantitative analysis of the concentration of DMG-PEG2000 in a biological matrix is crucial for studying the in vivo distribution, elimination pattern, and pharmacokinetics of LNP carrier materials.

[0003] However, quantitative analysis of DMG-PEG2000 faces two major challenges: (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 into short-chain PEG fragments (m / z 400-700) in vivo, overlapping with the target ion; (2) Biological matrix interference: biological matrices (such as plasma) contain a large amount of endogenous lipids (such as phosphatidylcholine) and proteins, which can easily inhibit the ionization efficiency of DMG-PEG2000 and produce interference peaks, affecting the detection results.

[0004] In the prior art, the analysis 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 method or evaporative light scattering detection method. However, these methods have obvious defects: (1) High-resolution mass spectrometry (such as ACQUITY RDa) can identify PEG unit distribution, but uses full scan mode, which has weak resistance to biological matrix interference and low quantitative sensitivity (LOD≥20 ng / mL in biological matrix); (2) ELSD evaporative light scattering detector is a general detector without specificity, which cannot distinguish DMG-PEG2000 from degradation products, and has extremely poor sensitivity (LOQ≥5 μg / mL), which cannot meet the quantitative requirements of low concentration (5-5000 ng / mL) in vivo. In summary, the prior art methods do not involve optimization of biological matrix pretreatment, and cannot solve the problems of protein precipitation and endogenous interference removal.

[0005] Therefore, the technical field urgently needs to establish a high-sensitivity and high-specificity quantitative analysis method to avoid the interference of DMG-PEG2000 in vivo degradation products and endogenous lipids in biological matrix, and solve the problem of quantitative analysis of DMG-PEG2000 in biological matrix. SUMMARY

[0006] To solve the above technical problems, the present application selects specific molecular ions with relatively large mass-to-charge ratio of DMG-PEG2000, which helps to avoid potential interference of in vivo degradation products in biological matrix samples. However, molecular ions with too large mass-to-charge ratio have lower detection sensitivity, so it is more appropriate to investigate molecular ions with mass-to-charge ratio in the range of 800 to 900. In the selection of specific fragment ions, the characteristic mass spectrum peak (m / z 495.5) of the lipid part is more representative of the measured substance itself in theory, and avoids the interference of PEG2000 in the sample. However, in complex biological matrix samples, endogenous interference is unknown and uncertain, so it is indispensable to establish a multiple channel selected reaction monitoring method and collect biological matrix samples after administration of lipid nanoparticle drugs for method investigation. Finally, the optimal multiple channel selected reaction monitoring method is determined by investigation, which can be used for quantitative analysis of DMG-PEG2000 in biological matrix samples after administration of lipid nanoparticle drugs.

[0007] In summary, the present application provides a method for quantitative analysis of DMG-PEG2000 in biological matrix, which uses liquid chromatography tandem quadrupole mass spectrometry for quantitative analysis, and establishes a multiple channel selected reaction monitoring method, which comprises the following steps: Step 1, sample pretreatment: take biological matrix sample, add internal standard solution and mix, then add protein precipitant, mix and centrifuge, take supernatant, the protein precipitant is a mixed solution of isopropyl alcohol and methanol, the volume ratio of isopropyl alcohol to methanol in the mixed solution is 1:0.8~1:1.2; Step 2, liquid chromatography tandem quadrupole mass spectrometry detection: detection by electrospray ion source in positive ion mode; Wherein, 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, elution mode is gradient elution; the mass spectrometry conditions are: the investigation range of Q1 molecular ion is 800~900 m / z, Q3 fragment ion is fixed as the characteristic fragment ion of DMG-PEG2000 lipid part, m / z 495.5±0.5, at least 3 groups of molecular ion→fragment ion multiple channel selected reaction monitoring method are established; Step 3, method validation and quantification: the sensitivity, specificity, precision and accuracy of the multi-channel selected reaction monitoring method were investigated by standard curve samples, combined with the reproducibility verification of the actual biological matrix samples, to determine the optimal selected reaction monitoring channel, which was Q1 m / z 810.2±0.2→Q3 m / z 495.5±0.2, and then the weighted w=1 / x2 least squares method was used to establish a standard curve to quantify DMG-PEG2000 in biological matrix.

[0008] Specifically, the volume ratio of the protein precipitation agent 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.

[0009] Specifically, 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.

[0010] Specifically, in step 2, the C8 reversed-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; the gradient elution program is: the initial mobile phase B ratio is 60%~70%, which is increased to 100% B within 1.2~1.8 minutes, maintained at 100% B for 3.2~3.8 minutes, restored to the initial B phase ratio at 3.51~3.81 minutes and maintained until the end of the run, and the total run time is 4~5 minutes.

[0011] Specifically, the tandem quadrupole mass spectrometer is a Sciex TRIPLEQUAD 6500 plus type or equivalent performance mass spectrometer, and the electrospray ion source parameters are: spray voltage 5000~6000 V, ion source temperature 500~600℃, gas curtain gas pressure 25~35 psi, atomization gas pressure 50~60 psi, heating gas pressure 50~60 psi, and collision gas pressure 8~10 psi.

[0012] Specifically, 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, diluted with 50% acetonitrile to prepare a series of working solutions, and 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.

[0013] Specifically, in step 3, the regression equation of the standard curve takes 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 greater than or equal to 0.995.

[0014] Specifically, the biological matrix sample is an animal or human biological matrix after administration of a lipid nanoparticle drug, and the biological matrix sample includes rat plasma, mouse liver tissue homogenate, human urine, or rat fecal homogenate.

[0015] Specifically, the sampling amount of the biological matrix sample is 20-50 μL, and 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.

[0016] Specifically, the injection volume of the liquid chromatography tandem quadrupole mass spectrometry detection is 1-2 μL, the column temperature is 35-45°C, the flow rate is 0.5-0.7 mL / min, and the needle washing liquid is 50% acetonitrile solution.

[0017] The method for quantitatively analyzing DMG-PEG2000 in a biological matrix provided by the present application has the following beneficial effects: 1. The method for quantitatively analyzing DMG-PEG2000 in a biological matrix provided by the present application avoids the interference of DMG-PEG2000 in vivo degradation products (such as m / z 400-700 short-chain PEG fragments) and endogenous lipids (such as phosphatidylcholine) in the biological matrix through a double-targeting strategy of "Q1 molecular ion selection (m / z 800-900 range) + Q3 fixed lipid core fragment (m / z 495.5)", there is no interference peak in rat blank plasma, the problem of biological matrix interference is solved, the specificity is good, and the non-interference quantitative analysis of DMG-PEG2000 in the biological matrix is realized.

[0018] 2、The lower limit of quantification (LLOQ) of the method is as low as 10 ng / mL, and the upper limit of quantification (ULOQ) is 2000 ng / mL. In addition to the conventional linear range of 10-2000 ng / mL, for high-concentration samples, dilution can be performed, and the accuracy of the results after dilution still meets the requirements, fully matching the concentration changes of DMG-PEG2000 in the biological matrix after LNP administration. Compared with the existing evaporative light scattering detection (ELSD, LOQ≥5 μg / mL), the sensitivity is improved by 500 times, solving the technical pain point that low-concentration samples (such as tissues and excreta at the late stage of administration) cannot be accurately quantified, and meeting the core needs of pharmacokinetic research.

[0019] 3、The method provided by the application is efficient and suitable for biological matrix, and isopropanol-alcohol mixed solution is used as a protein precipitant, and the extraction recovery rate is greater than 85%. In addition, the pretreatment method only needs 15 min, and does not need complex purification steps, and can be directly adapted to various biological matrices such as plasma, tissue homogenate and urine, and the recovery rate and operation convenience are considered, and the time and labor cost of batch sample analysis is reduced. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is the Q1 full scan spectrum of DMG-PEG2000 in Example 1.

[0021] Figure 2 is the Q3 full scan spectrum of DMG-PEG2000 molecular ion m / z 810.2 after CID in Example 1.

[0022] Figure 3 is the standard curve of DMG-PEG2000 in rat plasma in Example 1.

[0023] Figure 4 is the SRM mass spectrum of a blank rat plasma sample in Example 1.

[0024] Figure 5 is the SRM mass spectrum of the lower limit of quantification sample of DMG-PEG2000 in rat plasma in Example 1.

[0025] Figure 6 is the m / z 678.3→616.6 channel SRM mass spectrum of a blank rat plasma sample in the method development stage in Example 2.

[0026] Figure 7 is the m / z 678.3→616.6 channel SRM mass spectrum of a rat plasma sample containing DMG-PEG2000 (50 ng / mL) in the method development stage in Example 2.

[0027] Figure 8is the m / z 810.2→495.5 channel SRM mass chromatogram of the rat blank plasma sample in the method development stage in Example 2.

[0028] Figure 9 is the m / z 810.2→495.5 channel SRM mass chromatogram of the rat plasma sample containing DMG-PEG2000 (50 ng / mL) in the method development stage in Example 2. DETAILED DESCRIPTION

[0029] The technical solutions of the present application will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0030] The reagents used in the following examples are all commercial reagents unless otherwise specified. Table 1 is the reagent and consumable information used in all the following examples, and Table 2 is the instrument and model information used in all the following examples.

[0031] Table 1 Reagent and Consumable Information

[0032] Table 2 Instruments

[0033] Example 1 This example shows that using the method provided in the present application, DMG-PEG2000 in rat plasma is quantitatively analyzed using rat plasma as an example, and ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry (UPLC-MS / MS) is used for determination. The specific operation is as follows: A. Preparation of DMG-PEG2000 standard curve and quality control sample: accurately weigh 5 mg of DMG-PEG2000 standard, dissolve 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 a series of standard curve working solutions, add 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, 5000 ng / mL and quality control samples with concentrations of 5, 15, 200, 2500, 3750 ng / mL.

[0034] B. Rat plasma sample processing: Take 30 μL of each of the blank, standard curve, quality control and rat study samples, add 30 μL of internal standard solution (50% acetonitrile solution containing verapamil) and mix well, then add 180 μL of protein precipitant (isopropyl alcohol:methanol = 50:50) and mix well for 5 minutes, then centrifuge at 3200 g at 4 °C for 10 minutes, and take 120 μL of supernatant and place it in the autosampler for injection.

[0035] C. Chromatographic conditions: The chromatographic column is Waters ACQUITY UPLC BEH C8 1.7 μm 50 × 2.1mm, the flow rate is 0.6 mL / min, the mobile phase A is a water solution containing 5mM ammonium acetate, the mobile phase B is a 95% acetonitrile solution containing 0.5% formic acid and 5mM ammonium acetate, the needle washing liquid is 50% acetonitrile solution, the column temperature is 40 °C, the injection volume is 1 μL, the running time is 4.2 minutes, and the gradient elution program is: starting with 65% B phase, gradually increasing to 100% B phase at 1.5 minutes, maintaining 100% B phase elution to 3.5 minutes, and returning to 65% B phase from 3.51 minutes until the end of the run.

[0036] D. Mass spectrometry conditions: 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, ion source spray voltage 5500 V, ion source temperature 550 °C, gas curtain gas nitrogen pressure 30 psi, atomization gas nitrogen pressure 55 psi, heating gas nitrogen pressure 55 psi, collision gas nitrogen pressure 9 psi, Q1 of DMG-PEG2000 detection m / z 810.2, declustering voltage 35 V, Q3 m / z 495.5, collision energy 35 eV.

[0037] Figure 1 The Q1 full scan spectrum of DMG-PEG2000 can be seen that the molecular ion peak of DMG-PEG2000 in the positive ion mode of the electrospray ion source is mainly distributed in the mass-to-charge ratio (m / z) 600~1000 range, corresponding to the PEG2000 chain structure containing 34~54 ethylene glycol units in the molecule; among them, the molecular ion peak at m / z 810.2 has the highest relative intensity, the ion response value reaches 2.5×10 4 counts, and the peak shape is symmetrical and has no obvious interference peak. The present application selects the molecular ion in the range of m / z 800~900 to avoid the PEG degradation product interval of m / z 400~700, and selects the m / z 810.2 ion with the optimal response as the core detection object, which lays a foundation for subsequent specific quantification and solves the problem of insufficient sensitivity in the prior art.Figure 2 is the Q3 full scan spectrum after CID of DMG-PEG2000 molecular ion m / z 810.2, it can be seen that the molecular ion m / z 810.2 after collision-induced fragmentation (CID) appears multiple fragment ion peaks in the Q3 full scan spectrum, among which the fragment ion peak at m / z 495.5 has the highest relative intensity, and the ion response value reaches 2.0 x 10 4 counts, and has good separation from other fragment ions. Combined with fragment ion source analysis, m / z 495.5 is the characteristic ion of the lipid core (1,2-dimyristoyl-rac-glycerol) remaining after the loss of 3-oxypolyethylene glycol 2000 group from DMG-PEG2000, and there is no adjacent interfering peak (peak area ratio is more than 98%), which proves the rationality of selecting m / z 495.5 as the Q3 characteristic fragment ion, which can completely avoid the interference of PEG chain related fragments (such as m / z 385), compared with the prior art using PEG fragment ions as detection objects, the specificity of the method is significantly improved, ensuring the accuracy of the quantitative results in the biological matrix.

[0038] E. Standard curve regression: using Analyst software to integrate the mass chromatographic peaks of DMG-PEG2000 and internal standard verapamil, using the theoretical concentration of DMG-PEG2000 in the standard curve sample as the abscissa, and the ratio of DMG-PEG2000 peak area and internal standard peak area as the ordinate, using the weighted w=1 / x 2 least squares method for regression operation, the straight line regression equation is the standard curve (y=0.000302x+0.00122), Figure 3 is the standard curve of DMG-PEG2000 in rat plasma, it can be seen that the linear relationship is excellent, and has the advantages of wide quantitative range (5~5000 ng / mL) and good linear stability, compared with the existing method, the present application can cover the whole concentration change interval of DMG-PEG2000 in vivo after LNP administration, and provide complete concentration-time data support for pharmacokinetic research.

[0039] F. Quantitative analysis and method investigation: using the standard curve described in step E to quantitatively analyze DMG-PEG2000 in the quality control sample and rat plasma sample after administration of the lipid nanoparticle drug. Figure 4The 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.

[0040] 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.

[0041] Table 3

[0042] Table 4

[0043] 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.

[0044] Example 2 This example illustrates the screening process for quantitative analysis of SRM channels in rat plasma using DMG-PEG2000.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] Table 5

[0050] Table 6

[0051] 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 7Mass 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, respectively. It can be seen 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) of 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.

[0052] 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.

[0053] 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 quantitative analysis of DMG-PEG2000 in biological matrices, characterized in that, The method employs liquid chromatography-tandem quadrupole mass spectrometry for quantitative analysis, establishing a multi-channel selective reaction monitoring method. The method includes the following steps: 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. Step 2, Liquid chromatography-tandem quadrupole mass spectrometry detection: Detection was performed using an electrospray ionization source in positive ion mode; 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. 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.

2. The method for quantitative analysis of DMG-PEG2000 in biological matrices according to claim 1, characterized in that, The volume ratio of the protein precipitant to the biological matrix sample is 5:1 to 8:1, and the centrifugation conditions are 4°C, 3000 to 3500 g for 8 to 12 minutes. The internal standard solution is a 50% acetonitrile solution containing 100 to 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 for quantitative analysis of DMG-PEG2000 in biological matrices according to claim 1, characterized in that, 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. Among them, 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.

4. The method for quantitative analysis of DMG-PEG2000 in biological matrices according to claim 1, characterized in that, 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%, which 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. The total run time is 4~5 minutes.

5. The method for quantitative analysis of DMG-PEG2000 in biological matrices according to claim 1, characterized in that, The tandem quadrupole mass spectrometer was a Sciex TRIPLEQUAD 6500 plus or equivalent mass spectrometer. The electrospray ionization source parameters were: spray voltage 5000~6000V, ion source temperature 500~600℃, curtain gas pressure 25~35psi, nebulizer gas pressure 50~60psi, heating gas pressure 50~60psi, and collision gas pressure 8~10psi.

6. The method for quantitative analysis of DMG-PEG2000 in biological matrices according to claim 1, characterized in that, In step 3, the standard curve sample is prepared by dissolving DMG-PEG2000 standard in dimethyl sulfoxide to prepare a stock solution, diluting it with 50% acetonitrile to prepare a series of working solutions, and adding them 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 for quantitative analysis of DMG-PEG2000 in biological matrices according to claim 1, characterized in that, 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.

8. The method for quantitative analysis of DMG-PEG2000 in biological matrices according to claim 1, characterized in that, 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.

9. The method for quantitative analysis of DMG-PEG2000 in biological matrices according to claim 1, characterized in that, 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.

10. The method for quantitative analysis of DMG-PEG2000 in biological matrices according to claim 1, characterized in that, 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 a 50% acetonitrile solution.

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