Method for improving LC / MS analysis sensitivity

Abstract

The invention relates to the technical field of liquid chromatography / mass spectrometry analysis, and discloses a method for improving LC / MS analysis sensitivity, which is suitable for detecting trace lipid metabolites (containing isomeride) of a biological sample. The core technical scheme is as follows: a magnetic molecularly imprinted polymer-solid phase support liquid-liquid extraction secondary purification system is adopted to remove matrix impurities, a gradient mobile phase containing a composite additive is matched to optimize separation, and the detection specificity is improved by an ion mobility spectrometry-segmented selective ion monitoring combined mode; and a stable isotope internal standard is synchronously introduced to correct quantitative deviation. Experiments show that the LOD of the method is as low as 0.12-0.25 ng / mL, the matrix effect is smaller than or equal to 5%, the separation degree and the recovery rate are excellent, low-abundance metabolic marker differences can be accurately captured, and a support is provided for disease diagnosis and metabonomics research.

Description

Technical Field

[0001] This invention relates to the field of liquid chromatography / mass spectrometry (LC / MS) analysis technology, and more specifically, to a method for improving the sensitivity of LC / MS analysis. Background Technology

[0002] The accurate detection of lipid metabolites in biological samples (such as plasma, serum, and liver tissue homogenates) is a core foundation for metabolomics research, early disease diagnosis, and the elucidation of pathological mechanisms. Especially in studies on the correlation between metabolic diseases (such as diabetes and obesity) and traditional Chinese medicine constitution, the detection of low-abundance lipid biomarkers (such as PCs, PEs, LysoPCs, and their isomers) directly determines the reliability of research conclusions. However, current LC / MS analytical techniques for lipid detection in biological samples have long faced four interrelated technical bottlenecks, severely limiting detection sensitivity and accuracy: First, strong matrix interference: The large amount of proteins, salts, and endogenous small molecule impurities in biological samples can adsorb target substances or inhibit ionization, resulting in a matrix effect that generally exceeds 30%, severely reducing detection sensitivity. Second, difficulty in detecting low-abundance substances: Disease-related characteristic metabolic markers are often in trace amounts (ng / mL or even pg / mL), and the limits of detection (LOD) of conventional extraction and detection methods are high, making it difficult to capture the differences in such substances. Third, poor separation of isomers: Lipid isomers (such as phosphatidylcholine with different double bond positions) have similar chromatographic retention behaviors, which traditional LC / MS cannot effectively separate, leading to deviations in quantitative results and the omission of key biological information. Fourth, low quantitative accuracy: Loss of target substances during sample pretreatment (such as extraction, elution, and drying) and the lack of effective correction methods result in poor repeatability of quantitative results.

[0003] While existing technologies attempt to improve performance by optimizing individual steps such as mobile phase pH and adjusting extraction solvent ratios, they have failed to provide a systematic solution and cannot simultaneously address the four major challenges: matrix interference, low abundance detection, isomer separation, and quantitative correction. Therefore, developing an integrated, highly sensitive LC / MS analytical method that combines efficient purification, targeted enrichment, precise separation, and accurate quantification has become a critical technological need for breakthroughs in the field of lipid metabolomics research of biological samples. Summary of the Invention

[0004] In view of this, the present invention proposes a method to improve the sensitivity of LC / MS analysis, aiming to solve the problems in the current technology that cannot simultaneously solve matrix interference, cannot achieve low abundance detection, and cannot achieve isomer separation.

[0005] This invention provides a method for improving the sensitivity of LC / MS analysis, comprising the following steps: (1) Sample pretreatment: S101: Take the biological sample to be tested, add CHCl3 / MeOH (1:1, v / v) extraction solution at a volume ratio of 1:6, and add 0.6%~1.5% of the total mass of the extraction solution of composite modifier and 8 ng / mL stable isotope internal standard. After vortexing and mixing, centrifuge at 4℃ and 13000rpm for 12min, and collect the lower CHCl3 phase. S102: Pass the CHCl3 phase through a solid-supported liquid-liquid extraction (SLE) column (the packing material is modified diatomaceous earth), and elute twice with 4 column volumes of CHCl3 / MeOH (3:1, v / v), allowing each eluent to stand for 3 min, and then combine the eluents; S103: Add magnetic molecularly imprinted polymer (MMIP) to the eluent, and enrich by shaking under a 0.3T magnetic field for 18-25 min. After magnetic separation of MMIP, elute with CHCl3 / MeOH. Dry the eluent with nitrogen gas and resuspend it in 80 μL of CHCl3 / MeOH (1:1, v / v). Filter through a 0.22 μm organic phase membrane and store at -20℃. (2) LC chromatography separation: S201: A Waters ACQUITY UPLC HSS T3 column was used, with a column temperature of 42℃, an autosampler temperature of 4℃, and an injection volume of 4μL. S202: Mobile phase A is ultrapure water + 0.06% formic acid + 0.03% trifluoroacetic acid + 0.012% ammonium fluoride, mobile phase B is acetonitrile-isopropanol-propylene carbonate, flow rate 0.28 mL / min; S203: Gradient elution program: 0 min Phase A 95%, Phase B 5%; 1-4 min Phase A decreases to 75%, Phase B increases to 25%; 4-9 min Phase A decreases to 35%, Phase B increases to 65%; 9-12 min Phase A decreases to 3%, Phase B increases to 97%; 12-13.5 min Phase A is maintained at 3%, Phase B at 97%; 13.5-16 min Phase A recovers to 95%, Phase B recovers to 5%. (3) IMS-MS detection: S301: The ion source is an electrospray ion source, with 6% isopropanol-acetonitrile mixed vapor added to the sheath gas; Positive mode: heater 330℃, sheath gas flow rate 50arb, auxiliary gas flow rate 20arb, electrospray voltage 3.3KV, capillary temperature 370℃; Negative mode: heater 330℃, sheath gas flow rate 50arb, auxiliary gas flow rate 20arb, electrospray voltage 3.5KV, capillary temperature 370℃; S302: IMS parameters: Drift gas is high-purity nitrogen, flow rate is 55 mL / min, drift voltage is 850 V, separation voltage is 2200 V, drift tube temperature is 42 ℃, and transfer interface temperature is 160 ℃. S303: The scanning mode is segmented selective ion monitoring, which divides the m / z 60~1100 into 4 segments, with a first-level mass spectrometry resolution of 75000 and a second-level mass spectrometry resolution of 18000. The collision energy of each segment is independently optimized. S304: The stable isotope internal standard method was used for quantification. The content was calculated by substituting the peak area ratio of the target lipid to the internal standard into the standard curve.

[0006] In this invention, the composite modifier functions as follows: The composite modifier works synergistically with the CHCl3 / MeOH extract; 1-butyl-3-methylimidazolium hexafluorophosphate reduces the interfacial tension between lipids and solvent, enhancing the solubility of low-abundance lipids; SDS inhibits lipid residue in the aqueous phase. The two form a complementary "solubilization-prevention" effect, significantly improving extraction efficiency. If the addition amount is less than 0.6%, lipid solubility is insufficient, extraction efficiency decreases significantly, and LOD increases significantly; while if the addition amount is greater than 1.5%, it easily leads to increased viscosity of the extract, difficulty in centrifugation and separation, increased impurity entrainment, and enhanced matrix effect.

[0007] In this invention, the SLE column can utilize the porous structure of diatomaceous earth to adsorb protein and salt impurities in plasma, achieving a protein removal rate of over 98% and avoiding impurity inhibition of ionization.

[0008] In this invention, MMIP has a targeted enrichment function: by using molecularly imprinted polymers to specifically recognize target lipids (PC, PE, etc.) at their specific recognition sites, it achieves "capture-enrichment" while rejecting non-target impurities, forming a "broad-spectrum purification-precise enrichment" complement to SLE, and reducing the matrix effect to an extremely low level.

[0009] In this invention, a 0.3T magnetic field can quickly adsorb MMIP, avoid lipid loss caused by centrifugation, shorten the separation time after enrichment, and improve operational efficiency; if the magnetic field strength deviates (e.g., 0.2T or 0.4T), MMIP separation is incomplete and the recovery rate decreases.

[0010] In this invention, formic acid in mobile phase A improves lipid ionization efficiency, trifluoroacetic acid inhibits adsorption of silanol on the chromatographic column, and ammonium fluoride enhances phospholipid dissociation. The three form a synergistic effect of "ionization-anti-adsorption-promoting dissociation". Acetonitrile in mobile phase B provides moderate elution capacity, isopropanol enhances the solubility of highly hydrophobic lipids, and propylene carbonate reduces non-specific binding of lipids to the stationary phase. The three complement each other to avoid peak tailing.

[0011] In this invention, gradient elution is performed in an orderly manner according to lipid polarity to avoid peak overlap; the flow rate is increased to 0.32 mL / min in the 9-12 min stage to quickly elute highly hydrophobic lipids (such as PA and PI) and reduce peak broadening; when the flow rate is below 0.28 mL / min, the retention time of highly hydrophobic lipids is too long (>15 min), resulting in peak tailing; when the flow rate is above 0.32 mL / min, the separation decreases and the risk of lipid peak overlap increases.

[0012] In this invention, the Waters ACQUITY UPLC HSS T3 column is adapted to both polar and nonpolar lipids. A column temperature of 42°C ensures stable lipid retention time (fluctuation ±0.1 min). Below 38°C, the binding of lipids to immobilized molecules is enhanced, and the retention time drift is >0.5 min. Above 45°C, the retention time of low-polar lipids is too short, and the resolution drops below 1.2.

[0013] In this invention, IMS utilizes the difference in collision cross-sections of lipid molecules to separate isomers (e.g., PC36:2Δ6,9 / Δ9,12), while SSIM focuses on target ions in m / z segments to avoid interference from non-target ions. The two work synergistically to improve sensitivity and specificity. Without IMS: isomers cannot be separated (resolution < 1.0); without SSIM: the proportion of non-target ion signals increases, and the signal-to-noise ratio decreases by 50%.

[0014] In this invention, the ion source parameters are set as follows: adding 6% isopropanol-acetonitrile mixed vapor to the sheath gas reduces the surface tension of lipids and increases ionization efficiency by 40%; a heater temperature of 330°C and a capillary temperature of 370°C ensure rapid lipid vaporization and avoid condensation; below 300°C: lipid vaporization is incomplete, and ionization efficiency decreases by 30%; above 380°C: lipid molecules break down, and the detection signal is distorted.

[0015] In this invention, in step S101, the composite modifier is preferably a mixture of 1-butyl-3-methylimidazolium hexafluorophosphate and sodium dodecyl sulfate, with a preferred mass ratio of 3:1, and the total amount added is preferably 1.0% of the total mass of the extract; the stable isotope internal standard is preferably... 2 The H6-labeled phosphatidylcholine is preferably ≥98% pure.

[0016] In this invention, the complementary effect is optimal when the mass ratio of 1-butyl-3-methylimidazolium hexafluorophosphate (solubilizer) to SDS (anti-loss) is 3:1. If the ratio deviates (e.g., 2:2), the balance between solubilization and anti-loss is broken, and the extraction efficiency decreases significantly.

[0017] In this invention, PC 36:2-d6 is selected as an internal standard. Its physicochemical properties are consistent with the target lipid, which can correct for losses in the entire sample pretreatment (extraction, elution, drying) and detection process, and reduce quantitative deviation. If a non-isotopic internal standard (such as ordinary PC36:2) is selected, the quantitative RSD will increase and the accuracy will decrease significantly.

[0018] In this invention, in step S102, the modified diatomaceous earth packing of the SLE column is preferably treated with 0.5% trimethylchlorosilane, the particle size is preferably 60~120μm, and the column volume is preferably 5mL; the eluent CHCl3 / MeOH is preferably added at 2.5 times the column volume each time, and the eluent is combined after two elutions, preferably after preliminary filtration through a 0.45μm filter membrane.

[0019] In this invention, diatomaceous earth is modified with 0.5% trimethylchlorosilane to enhance hydrophobicity and reduce lipid adsorption; the particle size is 60~120μm: ensuring a suitable pore structure for the packing material, a protein adsorption rate >98%, and a stable eluent flow rate (1 drop / 2~3s). A particle size <60μm results in too small a pore size, too slow an eluent flow rate (<1 drop / 5s), and low operating efficiency; a particle size >120μm results in too large a pore size, reducing the protein adsorption rate to below 85% and enhancing the matrix effect.

[0020] In this invention, elution is performed twice (2.5 column volumes each time) and allowed to stand for 3 minutes to ensure that lipids are fully eluted and to avoid residues. If elution is performed only once, the lipid recovery rate will decrease by 10% to 15%. If the standing time is less than 2 minutes, the elution will be insufficient and the loss of low-abundance lipids will be significant.

[0021] In this invention, the preparation process of the magnetic molecularly imprinted polymer in step S103 is as follows: using a lipid mixture as the template molecule, methacrylic acid as the functional monomer, ethylene glycol dimethacrylate as the crosslinking agent, azobisisobutyronitrile as the initiator, and Fe3O4 magnetic nanoparticles as the carrier; the preferred molar ratio of template molecule: methacrylic acid: ethylene glycol dimethacrylate: Fe3O4 is 1:5:25:0.8, polymerized in a chloroform-methanol mixed solvent at 65°C for 10 hours, ground and passed through a 200-mesh sieve, the template molecule is removed by elution with CHCl3 / MeOH, and then vacuum dried for later use.

[0022] In this invention, the lipid mixture is preferably composed of PC 36:2, PE 38:4, and LysoPC 18:1, with a molar ratio preferably of 1:1:1.

[0023] In this invention, the template molecule provides a specific recognition site, the functional monomer MAA forms hydrogen bonds with the template molecule, the cross-linking agent EDMA constructs a three-dimensional network structure, and Fe3O4 provides magnetic separation functionality. When the molar ratio of these four components is 1:5:25:0.8, the MMIP adsorption capacity and specificity are optimal. However, when the template molecule ratio deviates, MMIP exhibits adsorption bias towards certain lipid types, and the enrichment balance decreases.

[0024] In this invention, the mobile phase B is composed of acetonitrile, isopropanol and propylene carbonate in a volume ratio preferably of 65:30:5.

[0025] In this invention, in step S202, the pH value of mobile phase A is preferably adjusted to pH 3.0~3.3 by 1 mol / L hydrochloric acid or sodium hydroxide; the amount of propylene carbonate added in mobile phase B is preferably 5% to enhance the solubility of highly hydrophobic lipids and reduce peak broadening; the mobile phase is preferably filtered through a 0.22 μm filter membrane and ultrasonically degassed for 30 min before use.

[0026] In this invention, 5% propylene carbonate can reduce the van der Waals forces between highly hydrophobic lipids (such as PE and PI) and the stationary phase, thereby reducing peak broadening; when the addition amount is less than 3%, the peak tailing factor of highly hydrophobic lipids is >1.5; when it is greater than 7%, the polarity of the mobile phase decreases, the retention time of low-polarity lipids is too short, and the separation is insufficient.

[0027] In this invention, in step S203, the preferred flow rate for gradient elution is: increasing from 0.28 mL / min to 0.32 mL / min in the 9-12 min stage, and decreasing back to 0.28 mL / min in the 13.5-16 min stage; in the elution program, the preferred flow rate change rate in the 1-4 min stage is 5% / min, and the preferred flow rate in the 4-9 min stage is 7% / min, to ensure that lipids are eluted in an orderly manner according to polarity.

[0028] In this invention, the flow rate is adjusted in stages: 0-9 min at 0.28 mL / min: ensuring sufficient separation of moderately polar lipids (such as LysoPC and PC); 9-12 min at 0.32 mL / min: rapidly eluting highly hydrophobic lipids and avoiding peak broadening; 13.5-16 min at 0.28 mL / min: equilibrating the column and preparing for the next sample detection. Deviations in the flow rate can lead to disordered lipid elution order and increased risk of peak overlap.

[0029] In this invention, in step S301, the addition of isopropanol-acetonitrile mixed vapor in the sheath gas is preferably precisely controlled by a gas mixer, the mixing ratio is preferably 1:1, and the total proportion is preferably 6%; the position of the spray needle of the ESI ion source is adjusted to preferably form a 15° angle with the capillary axis to improve ionization efficiency.

[0030] In this invention, isopropanol-acetonitrile (1:1) mixed vapor: isopropanol reduces the surface tension of lipids, and acetonitrile enhances volatility. The two complement each other to improve ionization efficiency. If the ratio deviates (e.g., 2:1): the ionization efficiency decreases by 20% to 25%. If the addition amount is less than 4%, the improvement effect is not significant. If it is more than 8%, the risk of ion source contamination increases.

[0031] In this invention, a 15° included angle aligns the spray direction with the capillary axis, reducing ion collision loss and improving ion transport efficiency by 30%; angle deviation (e.g., 0° or 30°) increases ion loss and reduces the signal-to-noise ratio by 40%.

[0032] In this invention, in step S303, the four segments and corresponding collision energies of the segment-selective ion monitoring are preferably: Segment 1: m / z 60~220, collision energy 18eV, for detecting short-chain lipid fragments; Segment 2: m / z 220~480, collision energy 22eV, for detecting lysophospholipids; Segment 3: m / z 480~750, collision energy 26eV, for detecting medium molecular weight phospholipids; Segment 4: m / z 750~1100, collision energy 30eV, for detecting high molecular weight phospholipids.

[0033] In this invention, the molecules are segmented according to m / z 60~220, 220~480, 480~750, and 750~1100, corresponding to short-chain fragments, lysophospholipids, medium-molecular-weight phospholipids, and high-molecular-weight phospholipids, to achieve targeted detection. If the molecules are not segmented, non-target ion signals will interfere, and the signal-to-noise ratio of low-abundance lipids will decrease significantly.

[0034] In this invention, the collision energy increases with increasing m / z (18eV→30eV) and is positively correlated with the lipid molecular weight: low energy is used for low m / z lipids (short chain fragments) to avoid excessive fragmentation, while high energy is used for high m / z lipids (high molecular weight phospholipids) to ensure sufficient fragmentation; if the energy is below the set value, the lipid fragmentation is incomplete and the ion abundance decreases; if the energy is above the set value, the fragment ions are disordered and the qualitative accuracy decreases.

[0035] In this invention, the preferred method for preparing the standard curve in step S304 is as follows: the target lipid standard is diluted with CHCl3 / MeOH to a series of concentrations of 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, and 50 ng / mL. An internal standard of 8 ng / mL PC 36:2-d6 is added to each concentration. The standard curve is then analyzed according to the LC chromatography separation method in step (2) and the IMS-MS detection method in step (3). A standard curve is plotted with the target lipid concentration as the abscissa and the peak area ratio of the target lipid to the internal standard as the ordinate. R 2 ≥0.9995.

[0036] In this invention, 0.5~50 ng / mL covers the actual content range of the target lipids in the sample (ng / mL level), ensuring linear correlation of the standard curve (R0). 2 ≥0.9995); concentrations below 0.5 ng / mL: linear deviation at the low concentration end; concentrations above 50 ng / mL: saturation at the high concentration end, disrupting the linear relationship.

[0037] In this invention, 8 ng / mL PC 36:2-d6 is added to each concentration standard to eliminate instrument response fluctuations and operational errors. If no internal standard is added, the RSD of the standard curve increases, and the quantitative deviation increases.

[0038] Another object of the present invention is to provide an application of the method for improving the sensitivity of LC / MS analysis, characterized in that the method is applied to the LC / MS analysis of lipid metabolites in mammalian plasma, serum, liver tissue homogenate, and adipose tissue homogenate samples; the lipid metabolites include PC, PE, LysoPC, PA, PG, PI and their corresponding isomers.

[0039] Compared with the prior art, the beneficial effects of the present invention are as follows: 1) This invention innovatively constructs a “SLE-MMIP two-stage purification system”. SLE adsorbs more than 98% of the protein (residual 0.022~0.028 mg / mL), and MMIP targets and captures the target lipids. The two work together to reduce the matrix effect to ≤5%, which is 85% lower than the traditional method, and completely solves the problem of impurity inhibition ionization. The composite mobile phase further reduces non-specific binding and avoids peak tailing.

[0040] 2) This invention achieves a breakthrough in the detection of low-abundance lipids through multiple synergistic processes of composite modifiers, MMIP enrichment, and SSIM targeted scanning, with LOD as low as 0.12~0.25ng / mL (70% lower than traditional methods) and signal-to-noise ratio of 17.9~29.1, successfully capturing key metabolic markers at the ng / mL level.

[0041] 3) This invention introduces the combination of IMS and LC / MS to separate isomers by utilizing the difference in molecular collision cross sections, achieving a separation degree of 1.9~2.5, filling the gap in traditional methods that cannot distinguish structurally similar lipids, and providing accurate structural information for mechanism research.

[0042] 4) This invention employs PC 36:2-d6 stable isotope internal standard for full-process calibration, eliminating pretreatment and instrument fluctuation errors. The recovery rate is stable at 96.8%~99.5%, the precision RSD≤0.8%, and the standard curve R 2 ≥0.9995; Precise parameter optimization ensures symmetrical peak shape and stable retention time, meeting the quantitative requirements of trace analysis.

[0043] 5) The analytical method of the present invention is adaptable to various samples such as plasma and liver tissue homogenate. It maintains low protein residue and low impurity peaks even for complex matrix samples. It can detect multiple lipids and isomers such as PC and PE, and provides a unified high-sensitivity analytical platform. Detailed Implementation

[0044] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0045] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0046] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0047] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0048] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0049] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. 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.

[0050] Preparation of experimental materials (applicable to all examples and comparative examples) Sample sources: 60 plasma samples from healthy adults (fasting venous blood for 12 hours, plasma separated by centrifugation at 3000 rpm for 10 minutes, stored at -80℃); 20 liver tissue homogenate samples from SD rats (rats were sacrificed after fasting for 24 hours, liver tissue was taken and added to physiological saline at a mass-to-volume ratio of 1:4, homogenized using a tissue homogenizer at 12000 rpm for 3 minutes, and stored at -80℃).

[0051] Reagent specifications: 1-Butyl-3-methylimidazolium hexafluorophosphate (purity ≥99%, Sigma), SDS (purity ≥99%, Sinopharm Group), PC 36:2-d6 standard (purity ≥98%, Cambridge Isotope Laboratories), modified diatomaceous earth (treated with 0.5% trimethylchlorosilane, particle size 60μm / 90μm / 120μm, Aladdin), formic acid / trifluoroacetic acid / ammonium fluoride (chromatographic grade, Merck), propylene carbonate (chromatographic grade, Tokyo Chemicals), chloroform / methanol / acetonitrile / isopropanol (chromatographic grade, Fisher), BCA protein assay kit (Thermo).

[0052] Instruments and equipment: Waters ACQUITY UPLC H-Class system, Thermo Q Exactive HF-X mass spectrometer (with IMS module), 5m LSL E column (self-made, column material is polypropylene, packing material is modified diatomaceous earth, packing height is 3cm), 0.3T permanent magnet (gaussmeter calibrated, magnetic field uniformity ±5%), high-speed refrigerated centrifuge (Thermo, model ST16R, maximum speed 15000rpm), vortex mixer (IKA, model Vortex Genius 3, speed adjustable from 0 to 3000rpm), pH meter (Mettler, model FE28, accuracy ±0.01), nitrogen blowing device (Organomation, model N-EVAP 112, temperature adjustable from 0 to 99℃), tissue homogenizer (IKA, model T18, maximum speed 30000rpm).

[0053] General process for MMIP preparation: The template molecule (PC 36:2 0.1mmol + PE 38:4 0.1mmol + LysoPC18:1 0.1mmol):MAA (functional monomer):EDMA (crosslinking agent):Fe3O4 (carrier) = 1:5:25:0.8 (molar ratio), with 0.02g AIBN (initiator) added, was dissolved in 12mL of chloroform-methanol mixed solvent (4:1, v / v); polymerization was carried out in a water bath at 65℃ for 10h under nitrogen protection; the polymerized product was ground and passed through a 200-mesh sieve, eluted with CHCl3 / MeOH (1:2, v / v) until no template molecule peak was detected by LC / MS, and then vacuum dried (60℃, 2h) for later use; the prepared MMIP had a particle size of 100~200μm and an adsorption capacity of 8.5±0.3mg / g for the target lipid.

[0054] Example 1 1) Sample pretreatment S101: Extraction and Internal Standard Addition. Take 1 mL of plasma sample from a healthy person (taken from -80℃ freezer, thawed at 4℃ for 1 hour, and gently inverted 3 times to mix), and place it in a 15 mL stoppered polypropylene centrifuge tube; accurately add 6 mL of CHCl3 / MeOH mixed extraction solution (1:1, v / v, pre-cooled at 4℃); add composite modifier (0.024 g 1-butyl-3-methylimidazolium hexafluorophosphate + 0.008 g SDS, mass ratio 3:1, dissolved in 100 μL CHCl3 beforehand) based on 0.6% of the total extract mass; add 8 μL of 1 μg / mL PC. 36:2-d6 internal standard solution (final concentration 8 ng / mL, internal standard prepared with methanol); place the centrifuge tube in a vortex mixer and vortex at 3000 rpm for 3 min; then place it in a high-speed refrigerated centrifuge and centrifuge at 4℃ and 13000 rpm for 12 min; after centrifugation, carefully aspirate the lower CHCl3 phase (about 3 mL, the upper MeOH- aqueous phase containing proteins and salts, which should be discarded) with a 1 mL pipette (with a quartz tip) and transfer it to a new 15 mL centrifuge tube.

[0055] S102: SLE purification: Take a 5 mL SLE column (60 μm modified diatomaceous earth packing material, pre-activated with 2 mL CHCl3, with a flow rate of 1 drop / 2 s during activation); slowly load the CHCl3 phase collected in S101 onto the column (loading flow rate 1 drop / 3 s), and let it stand for 5 min after loading to ensure sufficient contact between the sample and the packing material; add eluent (CHCl3 / MeOH = 3:1, v / v) twice, 2.5 mL each time (2.5 column volumes), and let it stand for 3 min after each addition, controlling the elution flow rate at 1 drop / 2 s; collect the two eluents (approximately 5 mL), filter them through a 0.45 μm organic phase filter membrane (0.45 μm pore size, PTFE material) to remove fine particulate impurities; take 100 μL of the eluent and detect it using a BCA protein detection kit, the protein concentration is 0.025 mg / mL.

[0056] S103: MMIP Enrichment and Magnetic Separation. Add 80 μL of MMIP suspension (100 mg / mL CHCl3 solution, pre-dispersed by sonication for 5 min) to the eluent after filtration in S102; place the centrifuge tube in a 0.3T permanent magnet-assisted shaker and enrich at 2000 rpm for 18 min at room temperature (25℃); after turning off the shaker, use the permanent magnet to adhere to the tube wall for 1 min to ensure complete MMIP adhesion, and discard the supernatant; add 1.5 mL of MMIP solution to the centrifuge tube. Eluent with CHCl3 / MeOH (1:1, v / v), vortex at 3000 rpm for 1.5 min; adsorb again with a permanent magnet for 1 min, collect the eluent; transfer the eluent to a 1.5 mL centrifuge tube, place it in a nitrogen blower, and dry to near dryness at 45 °C and 0.1 MPa nitrogen flow (approximately 10 min, residual volume ≤ 5 μL); resuspend in 80 μL CHCl3 / MeOH (1:1, v / v), filter with a 0.22 μm organic phase filter membrane (PTFE material), transfer to a vial (with inner tube), and store at -20 °C for analysis.

[0057] 2) LC chromatography separation S201: Chromatographic system parameters: Column: Waters ACQUITY UPLC HSS T3 (2.1×100mm, 1.8μm, column efficiency ≥10000 plates / m); Column temperature: 42℃ (column oven temperature control accuracy ±0.1℃); Autosampler temperature: 4℃; Injection volume: 4μL (using full loop injection mode, injection loop volume 10μL, needle washing solvent is CHCl3 / MeOH=1:1, v / v, needle washing 3 times, each needle washing volume 50μL).

[0058] S202: Mobile Phase Preparation Mobile Phase A: Ultrapure water (resistivity 18.2 MΩ·cm) + 0.06% formic acid (v / v) + 0.03% trifluoroacetic acid (v / v) + 0.012% ammonium fluoride (w / v); adjust pH to 3.0 with 1 mol / L hydrochloric acid solution (pH meter calibrated on-site); ultrasonically degas for 30 min (300W power, nitrogen protection during degassing); filter through a 0.22 μm filter membrane (aqueous, PES material). Mobile Phase B: Acetonitrile-isopropanol-propylene carbonate = 65:30:5 (v / v / v); ultrasonically degas for 30 min (300W power); filter through a 0.22 μm filter membrane (organic phase, PTFE material). Flow Rate: Initial flow rate 0.28 mL / min (flow rate accuracy ±0.01 mL / min).

[0059] S203: Gradient elution procedure The specific gradient elution procedure is shown in Table 1: Table 1. Gradient elution program for S203

[0060] 3) IMS-MS detection S301: ESI ion source parameters Ion source type: Electrospray ionization (ESI); Spray needle position: 15° angle with capillary axis (2mm from capillary inlet); Sheath gas: High-purity nitrogen (purity ≥99.999%), with 6% (v / v) isopropanol-acetonitrile mixed vapor (1:1, v / v, precisely controlled by a gas mixer), flow rate 50 arb (1 arb ≈ 1 mL / min); Auxiliary gas: High-purity nitrogen, flow rate 20 arb; Tail gas flow rate: 1 arb; Heater temperature: 330℃; Electrospray voltage (positive mode): 3.3KV, (negative mode): 3.5KV; Capillary temperature: 370℃; S-Lens RF Level (positive mode): 38%, (negative mode): 68%; Ion transmission tube temperature: 370℃ (synchronously controlled with capillary temperature).

[0061] S302: IMS parameters: Drift gas: High-purity nitrogen (purity ≥99.999%), flow rate 55 mL / min (controlled by mass flow controller, accuracy ±0.1 mL / min); Drift voltage: 850 V (high-voltage power supply accuracy ±1%); Separation voltage: 2200 V; Drift tube temperature: 42℃ (temperature control accuracy ±0.5℃); Transfer interface temperature: 160℃ (interface material is quartz to avoid lipid condensation); Migration time resolution: 0.01 ms; Ion delay time: 0.1 ms.

[0062] S303: SSIM scan parameters The specific SSIM scanning parameters are shown in Table 2: Table 2. SSIM Scanning Parameters

[0063] S304: Preparation of Standard Curve for Quantitative Calculation: Six target lipid standards were diluted with CHCl3 / MeOH (1:1, v / v) to a series of concentrations of 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, and 50 ng / mL. An internal standard of 8 ng / mL PC 36:2-d6 was added to each concentration. LC-IMS-MS was performed according to the above parameters. A standard curve was plotted with the target lipid concentration as the abscissa (x) and the peak area ratio of the target lipid to the internal standard as the ordinate (y). The linear regression equation and R0 were calculated. 2 as follows: PC 36:2: y = 0.021x + 0.003, R 2 =0.9996 PE 38:4: y = 0.019x + 0.002, R 2 =0.9995 LysoPC 18:1:y=0.023x+0.004,R 2 =0.9997 PA 34:2: y = 0.017x + 0.001, R 2 =0.9995 PG 36:4: y=0.018x+0.002, R 2 =0.9996 PC 36:2 Δ6,9: y=0.020x+0.003, R 2 =0.9995 Sample quantification: Thermo Lipid Search 5.0 software was used to extract the chromatographic peak areas of the target lipid and the internal standard, calculate the peak area ratio, and substitute it into the corresponding standard curve to calculate the content.

[0064] 4) Test results: The test results are shown in Table 3: Table 3. Summary of test results for Example 1

[0065] Example 2 1) Sample pretreatment S101: Extraction and Addition of Internal Standard. Take 1 mL of plasma sample from a healthy person (processed as in Example 1) and place it in a 15 mL stoppered polypropylene centrifuge tube; add 6 mL of CHCl3 / MeOH mixed extraction solution (1:1, v / v, pre-cooled at 4℃); add composite modifier (0.04 g 1-butyl-3-methylimidazolium hexafluorophosphate + 0.013 g SDS, mass ratio 3:1, dissolved in 100 μL CHCl3 before adding) based on 1.0% of the total mass of the extraction solution; add 8 μL of 1 μg / mL PC 36:2-d6 internal standard solution; vortex at 3000 rpm for 3 min; centrifuge at 4℃ and 13000 rpm for 12 min; aspirate the lower CHCl3 phase (about 3 mL) into a new centrifuge tube.

[0066] S102: SLE purification: Take 5 mL of SLE column (packing material is 90 μm modified diatomaceous earth, pre-activated with 2 mL CHCl3); load sample at a flow rate of 1 drop / 3 s, let stand for 5 min; add eluent (CHCl3 / MeOH=3:1, v / v) twice, 2.5 mL each time, let stand for 3 min each time; collect the eluent (about 5 mL), filter through a 0.45 μm PTFE membrane; BCA detection protein concentration is 0.022 mg / mL.

[0067] S103: MMIP enrichment and magnetic separation: Add 80 μL of MMIP suspension (100 mg / mL CHCl3, ultrasonically dispersed for 5 min) to the eluent; enrich by shaking at 2000 rpm for 22 min under a 0.3T magnetic field; separate MMIP by magnetic adsorption and discard the supernatant; add 1.5 mL of CHCl3 / MeOH (1:1, v / v) and vortex for 1.5 min; collect the eluent by magnetic adsorption again; dry to dryness under nitrogen at 45℃, resuspend in 80 μL of CHCl3 / MeOH (1:1, v / v), filter through a 0.22 μm PTFE membrane, and store at -20℃.

[0068] 2) LC chromatography separation S201: The chromatographic column, column temperature, injector temperature, and injection volume are the same as in Example 1; the injection loop washing needle parameters are the same as in Example 1.

[0069] S202: Mobile phase A was prepared in the same manner as in Example 1, with pH adjusted to 3.1 using 1 mol / L hydrochloric acid; mobile phase B was prepared in the same manner as in Example 1; ultrasonic degassing and filtration were performed in the same manner as in Example 1; initial flow rate was 0.28 mL / min.

[0070] S203: The gradient elution procedure is the same as in Example 1 (the time, mobile phase ratio, and flow rate changes are consistent).

[0071] 3) IMS-MS detection S301: The ESI ion source parameters are the same as in Example 1 (spray needle angle, sheath gas vapor ratio, temperature, and voltage are all the same).

[0072] S302: IMS parameters are the same as in Example 1 (drift gas flow rate, voltage, and temperature are all the same).

[0073] S303: The SSIM scanning parameters are the same as in Example 1 (segmentation, collision energy, and resolution are all the same).

[0074] S304: Standard curve preparation is the same as in Example 1 (linear regression equation and R²) 2 (Consistent); the sample quantification method is the same as in Example 1.

[0075] 4) Test results The test results are shown in Table 4: Table 4. Summary of test results for Example 2

[0076] Example 3 1) Sample pretreatment S101: Extraction and Addition of Internal Standard. Take 1 mL of plasma sample from a healthy person (processed as in Example 1) and place it in a 15 mL stoppered polypropylene centrifuge tube; add 6 mL of CHCl3 / MeOH mixed extraction solution (1:1, v / v, pre-cooled at 4℃); add a composite modifier (0.06 g 1-butyl-3-methylimidazolium hexafluorophosphate + 0.02 g SDS, mass ratio 3:1, dissolved in 100 μL CHCl3 before adding) based on 1.5% of the total mass of the extraction solution; add 8 μL of 1 μg / mL PC 36:2-d6 internal standard solution; vortex at 3000 rpm for 3 min; centrifuge at 4℃ and 13000 rpm for 12 min; transfer the lower CHCl3 phase (about 3 mL) to a new centrifuge tube.

[0077] S102: SLE purification: Take 5 mL of SLE column (filled with 120 μm modified diatomaceous earth, pre-activated with 2 mL of CHCl3); load sample at a flow rate of 1 drop / 3 s, let stand for 5 min; add eluent (CHCl3 / MeOH=3:1, v / v) twice, 2.5 mL each time, let stand for 3 min each time; collect the eluent (about 5 mL), filter through a 0.45 μm PTFE membrane; BCA detection protein concentration is 0.026 mg / mL.

[0078] S103: MMIP enrichment and magnetic separation: Add 80 μL of MMIP suspension (100 mg / mL CHCl3, ultrasonically dispersed for 5 min) to the eluent; enrich by shaking at 2000 rpm for 25 min under a 0.3T magnetic field; separate MMIP by magnetic adsorption and discard the supernatant; add 1.5 mL of CHCl3 / MeOH (1:1, v / v) and vortex for 1.5 min; collect the eluent by magnetic adsorption again; dry to dryness under nitrogen at 45℃, resuspend in 80 μL of CHCl3 / MeOH (1:1, v / v), filter through a 0.22 μm PTFE membrane, and store at -20℃.

[0079] 2) LC chromatography separation S201: The chromatographic column, column temperature, injector temperature, and injection volume are the same as in Example 1; the injection loop washing needle parameters are the same as in Example 1.

[0080] S202: Mobile phase A was prepared in the same manner as in Example 1, with pH adjusted to 3.3 using 1 mol / L hydrochloric acid; mobile phase B was prepared in the same manner as in Example 1; ultrasonic degassing and filtration were performed in the same manner as in Example 1; initial flow rate was 0.28 mL / min.

[0081] S203: The gradient elution procedure is the same as in Example 1 (the time, mobile phase ratio, and flow rate changes are consistent).

[0082] 3) IMS-MS detection S301~S304: The parameters and standard curves are the same as in Example 1.

[0083] 4) Test results The test results are shown in Table 5: Table 5. Summary of test results for Example 3

[0084] Example 4 1) Sample pretreatment S101: Extraction and Addition of Internal Standard. Take 1 mL of plasma sample from a healthy person (processed as in Example 1) and place it in a 15 mL stoppered polypropylene centrifuge tube; add 6 mL of CHCl3 / MeOH mixed extraction solution (1:1, v / v, pre-cooled at 4℃); add composite modifier (0.04 g 1-butyl-3-methylimidazolium hexafluorophosphate + 0.013 g SDS, mass ratio 3:1, 1.0% addition); add 8 μL of 1 μg / mL PC 36:2-d6 internal standard; vortex at 3000 rpm for 3 min; centrifuge at 4℃ and 13000 rpm for 12 min; aspirate the CHCl3 phase (approximately 3 mL).

[0085] S102: SLE purification: Take 5 mL of SLE column (packing material is 90 μm modified diatomaceous earth); activation, loading, and elution are the same as in Example 2; BCA detection protein concentration is 0.023 mg / mL.

[0086] S103: MMIP enrichment and magnetic separation oscillation enrichment time 22 min, the rest is the same as in Example 2; the eluent treatment is the same as in Example 2.

[0087] 2) LC chromatography separation S201: The chromatographic column, column temperature, injector temperature, and injection volume are the same as in Example 1; the injection loop washing needle parameters are the same as in Example 1.

[0088] S202: Adjust the pH of mobile phase A to 3.1; prepare mobile phase B as in Example 1; perform ultrasonic degassing and filtration as in Example 1; initial flow rate 0.29 mL / min.

[0089] S203: Gradient elution procedure The specific gradient elution procedure is shown in Table 6: Table 6. Gradient Elution Program Table

[0090] 3) IMS-MS detection S301~S304: The parameters and standard curves are the same as in Example 1.

[0091] 4) Test results The test results are shown in Table 7: Table 7. Summary of test results for Example 4

[0092] Example 5 Sample pretreatment S101: Extraction and Addition of Internal Standard. Take 2 mL of rat liver tissue homogenate sample (taken from -80℃ freezer, thawed at 4℃ for 1 h, centrifuged at 1000 rpm for 5 min to remove precipitate), and place it in a 15 mL stoppered polypropylene centrifuge tube; add 12 mL of CHCl3 / MeOH mixed extraction solution (1:1, v / v, pre-cooled at 4℃) at a volume ratio of 1:6; add composite modifier (0.08 g 1-butyl-3-methylimidazolium hexafluorophosphate + 0.027 g SDS, mass ratio 3:1, total mass of extraction solution 1.0%); add 16 μL of 1 μg / mL PC 36:2-d6 internal standard solution (final concentration 8 ng / mL); vortex at 3000 rpm for 5 min (tissue homogenate has high viscosity, extend vortex time); centrifuge at 4℃ and 13000 rpm for 15 min (extend centrifugation time to ensure complete phase separation); aspirate the lower CHCl3 phase (about 6 mL) into a new 15 mL centrifuge tube.

[0093] S102: SLE purification: Take 5 mL of SLE column (the packing material is 90 μm modified diatomaceous earth, pre-activated with 4 mL of CHCl3; due to the increased sample volume, the activation volume is doubled); load the CHCl3 phase onto the column twice (3 mL each time), with a flow rate of 1 drop / 4 s; let stand for 5 min after each loading; add eluent twice (CHCl3 / MeOH=3:1, v / v), 5 mL each time (2.5 times the column volume), and let stand for 3 min each time; collect the eluent (about 10 mL), filter through a 0.45 μm PTFE membrane; take 100 μL of the eluent, and the protein concentration detected by BCA is 0.028 mg / mL.

[0094] S103: MMIP enrichment and magnetic separation: Add 160 μL of MMIP suspension (100 mg / mL CHCl3, ultrasonically dispersed for 5 min, with the amount increasing proportionally to the volume of the eluent) to the eluent; enrich by shaking at 2000 rpm for 22 min under a 0.3T magnetic field; separate MMIP by magnetic adsorption and discard the supernatant; add 3 mL of CHCl3 / MeOH (1:1, v / v) and vortex for 1.5 min; collect the eluent by magnetic adsorption again; dry to dryness under nitrogen at 45℃ (approximately 15 min, due to the increase in eluent volume); resuspend in 80 μL of CHCl3 / MeOH (1:1, v / v), filter through a 0.22 μm PTFE membrane, and store at -20℃.

[0095] 2) LC chromatography separation S201: The chromatographic column, column temperature (42℃), injector temperature (4℃), and injection volume (4μL) are the same as in Example 1; the parameters for the injection loop washing needle are the same as in Example 1.

[0096] S202: Adjust the pH of mobile phase A to 3.1; prepare mobile phase B as in Example 1; perform ultrasonic degassing and filtration as in Example 1; initial flow rate 0.28 mL / min.

[0097] S203: The gradient elution procedure is the same as in Example 1.

[0098] 3) IMS-MS detection S301~S304: The parameters and standard curves are the same as in Example 1; because the tissue homogenate matrix contains a small amount of pigment, an ion source cleaning step is added before MS detection (rinse the spray needle with 50% methanol aqueous solution for 10 min).

[0099] 4) Test results The test results are shown in Table 8: Table 8. Summary of test results for Example 5

[0100] 5) Widespread practicality verification Example 5 validated the applicability of this method in liver tissue homogenates. Even with tissue samples exhibiting higher viscosity and more complex matrices, it maintained low protein residue (0.028 mg / mL) and a low number of impurity peaks (12), with no significant differences in LOD, recovery rate, and precision compared to plasma samples. This demonstrates that this method is adaptable to various biological samples, including plasma, serum, and liver / adipose tissue homogenates, providing a unified, highly sensitive analytical platform for metabolomics research, disease diagnosis, and drug development, and possesses significant industrial and clinical application value.

[0101] Comparative Example 1 1) Only the amount of composite modifier added in S101 was changed to 0.5% (total mass of extract), that is, 0.02g 1-butyl-3-methylimidazolium hexafluorophosphate + 0.007g SDS (mass ratio 3:1); the remaining steps and parameters were completely consistent with those in Example 2.

[0102] 2) Test results The test results are shown in Table 9: Table 9. Summary of Test Results for Comparative Example 1

[0103] 3) Conclusion When the amount of composite modifier added is less than 0.6%, the solubility of lipids in the CHCl3 phase decreases, the extraction efficiency decreases, the LOD increases by 196%~275%, the matrix effect is enhanced (ME% decreases to 65%~73%), and the recovery rate decreases by 15%~19%, proving that the addition range of 0.6%~1.5% is the key to ensuring extraction efficiency.

[0104] Comparative Example 2 1) The sample pretreatment step S103 was removed. The eluent collected in S102 was directly filtered through a 0.45 μm filter membrane, and 5 mL of the eluent was blown dry with nitrogen at 45 °C. It was then resuspended in 80 μL of CHCl3 / MeOH (1:1, v / v). All other steps and parameters were completely consistent with those in Example 2.

[0105] 2) Test results The test results are shown in Table 10: Table 10. Summary of test results for Comparative Example 2

[0106] 3) Conclusion Omitting the MMIP step prevents the targeted enrichment of lipids, and the small molecule impurities remaining in the matrix are not removed, resulting in a significantly enhanced matrix effect (ME% < 70%), an increase in LOD of 200% to 250%, and the inability to separate isomers. This demonstrates that MMIP is the core step for achieving matrix purification and sensitivity enhancement.

[0107] Comparative Example 3 1) Only the pH of mobile phase A in the LC separation was changed to 2.8 (adjusted with 1 mol / L hydrochloric acid); the rest of the steps and parameters were completely consistent with those in Example 2.

[0108] 2) Test results The test results are shown in Table 11: Table 11. Summary of test results for Comparative Example 3

[0109] 3) Conclusion When the mobile phase pH is below 3.0, the ionization of silanol groups on the surface of the silica matrix of the column is inhibited, and the non-specific adsorption of lipids is enhanced, resulting in severe peak tailing (tailing factor > 1.7), decreased ionization efficiency, increased LOD by 120%~125%, and reduced isomer resolution to 1.2 (partial overlap). This proves that the pH range of 3.0~3.3 is the key to ensuring peak shape and resolution.

[0110] Comparative Example 4 1) Only the drift tube temperature in the IMS detection was changed to 38°C; the rest of the steps and parameters were completely consistent with those in Example 2.

[0111] 2) Test results The test results are shown in Table 12: Table 12. Summary of test results for Comparative Example 4

[0112] 3) Conclusion When the IMS drift tube temperature deviates from 42°C to 38°C, the thermal motion rate of lipid molecules within the drift tube decreases. Some highly hydrophobic lipid molecules undergo slight condensation due to the lower temperature, leading to decreased ion migration efficiency and poorer migration time repeatability (RSD > 4%). Particularly for lipid isomers (PC 36:2 Δ6,9 and Δ9,12), the difference in their collision cross-sections cannot be effectively reflected at low temperatures, resulting in a separation degree of 1.0 (complete overlap) and a LOD increase of over 50% compared to Example 2. This demonstrates that 42°C is a key parameter for ensuring IMS separation performance, migration time stability, and isomer differentiation ability.

[0113] Comparative Example 5 1) The sample pretreatment step S102 was removed. The CHCl3 phase collected in S101 was directly added to MMIP for enrichment (take 3 mL of CHCl3 phase, add 80 μL of MMIP suspension, and follow up with shaking, magnetic adsorption, elution and other operations as in Example 2); the remaining steps and parameters are completely consistent with Example 2.

[0114] 2) Test results The test results are shown in Table 13: Table 13. Summary of test results for Comparative Example 5

[0115] 3) Conclusion Omitting the SLE step resulted in a significant increase in protein residue in the sample from 0.022 mg / mL in Example 2 to 0.35 mg / mL. A large amount of protein entered the MMIP enrichment stage, competing for MMIP's target adsorption sites and severely inhibiting the ionization efficiency of the ESI ion source in subsequent MS analysis (ME% < 60%). The LOD increased by more than 250% compared to Example 2, and the recovery rate decreased by 15%–19%. Furthermore, the non-specific binding of proteins to lipids led to complete overlap of isomers. This demonstrates that SLE is a crucial step for removing protein impurities from plasma and reducing matrix effects, and cannot be omitted.

[0116] Comparative Example 6 1) Only the collision energy of SSIM segment 1 (m / z 60~220) in IMS-MS detection was changed to 15eV; the rest of the steps and parameters were completely consistent with those in Example 2.

[0117] 2) Test results

[0118] 3) Conclusion When the collision energy of SSIM segment 1 is below 18 eV, the fragmentation efficiency of short-chain lipid fragments (m / z 60~220) is insufficient, and the ion abundance is significantly reduced to 1×10⁻⁶ compared with Example 2. 5 Below cps, the signal-to-noise ratio dropped from over 20 to below 8; the LOD increased by more than 30% compared to Example 2, making it unable to effectively capture low-abundance short-chain lipid fragment signals. This demonstrates that the collision energy range of 18–30 eV is crucial for ensuring sufficient ionization of lipid fragments in each segment and achieving the required signal intensity.

[0119] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for improving the sensitivity of LC / MS analysis, characterized in that, Includes the following steps: (1) Sample pretreatment: S101: Take the biological sample to be tested, add CHCl3 / MeOH extraction solution at a volume ratio of 1:6, and add 0.6%~1.5% of the total mass of the extraction solution of composite modifier and 8ng / mL stable isotope internal standard. After vortexing and mixing, centrifuge at 4℃ and 13000rpm for 12min, and collect the lower CHCl3 phase. S102: Pass the CHCl3 phase through a solid-supported liquid-liquid extraction column, elute twice with 4 column volumes of CHCl3 / MeOH, let stand for 3 min each time, and combine the eluents; S103: Add the magnetic molecularly imprinted polymer to the eluent, and enrich it by shaking under a 0.3T magnetic field for 18-25 min. After separating the magnetic molecularly imprinted polymer by magnetic adsorption, elute with CHCl3 / MeOH. After drying the eluent with nitrogen gas, resuspend it in 80 μL CHCl3 / MeOH, filter it through a 0.22 μm organic phase membrane, and store it at -20℃. (2) LC chromatography separation: S201: A Waters ACQUITY UPLC HSS T3 column was used, with a column temperature of 42℃, an autosampler temperature of 4℃, and an injection volume of 4μL. S202: Mobile phase A is ultrapure water + 0.06% formic acid + 0.03% trifluoroacetic acid + 0.012% ammonium fluoride, mobile phase B is acetonitrile-isopropanol-propylene carbonate, flow rate 0.28 mL / min; S203: Gradient elution program: 0 min Phase A 95%, Phase B 5%; 1-4 min Phase A decreases to 75%, Phase B increases to 25%; 4-9 min Phase A decreases to 35%, Phase B increases to 65%; 9-12 min Phase A decreases to 3%, Phase B increases to 97%; 12-13.5 min Phase A is maintained at 3%, Phase B at 97%; 13.5-16 min Phase A recovers to 95%, Phase B recovers to 5%. (3) IMS-MS detection: S301: The ion source is an electrospray ion source, with 6% isopropanol-acetonitrile mixed vapor added to the sheath gas; Positive mode: heater 330℃, sheath gas flow rate 50arb, auxiliary gas flow rate 20arb, electrospray voltage 3.3KV, capillary temperature 370℃; Negative mode: heater 330℃, sheath gas flow rate 50arb, auxiliary gas flow rate 20arb, electrospray voltage 3.5KV, capillary temperature 370℃; S302: IMS parameters: Drift gas is high-purity nitrogen, flow rate is 55 mL / min, drift voltage is 850 V, separation voltage is 2200 V, drift tube temperature is 42 ℃, and transfer interface temperature is 160 ℃. S303: The scanning mode is segmented selective ion monitoring, which divides the m / z 60~1100 into 4 segments, with a first-level mass spectrometry resolution of 75000 and a second-level mass spectrometry resolution of 18000. The collision energy of each segment is independently optimized. S304: The stable isotope internal standard method was used for quantification. The content was calculated by substituting the peak area ratio of the target lipid to the internal standard into the standard curve.

2. The method for improving LC / MS analytical sensitivity according to claim 1, characterized in that, In step S101, the composite modifier is a mixture of 1-butyl-3-methylimidazolium hexafluorophosphate and sodium dodecyl sulfate, with a mass ratio of 3:1, and the total amount added is 1.0% of the total mass of the extract; the stable isotope internal standard is... 2 H6-labeled phosphatidylcholine, purity ≥98%.

3. The method for improving LC / MS analytical sensitivity according to claim 1 or 2, characterized in that, In step S102, the modified diatomaceous earth packing of the SLE column is treated with 0.5% trimethylchlorosilane, with a particle size of 60~120μm and a column volume of 5mL; the eluent CHCl3 / MeOH is added at 2.5 times the column volume each time, and the eluents are combined after two elutions and preliminarily filtered through a 0.45μm filter membrane.

4. The method for improving LC / MS analytical sensitivity according to claim 3, characterized in that, In step S103, the preparation process of the magnetic molecularly imprinted polymer is as follows: using a lipid mixture as the template molecule, methacrylic acid as the functional monomer, ethylene glycol dimethacrylate as the crosslinking agent, azobisisobutyronitrile as the initiator, and Fe3O4 magnetic nanoparticles as the carrier; the molar ratio of template molecule: methacrylic acid: ethylene glycol dimethacrylate: Fe3O4 is 1:5:25:0.8, polymerized in a chloroform-methanol mixed solvent at 65℃ for 10h, ground and passed through a 200-mesh sieve, the template molecule is removed by elution with CHCl3 / MeOH, and vacuum dried for later use; The lipid mixture consists of PC 36:2, PE 38:4, and LysoPC 18:1 in a molar ratio of 1:1:

1. The mobile phase B consists of acetonitrile, isopropanol and propylene carbonate in a volume ratio of 65:30:

5.

5. The method for improving LC / MS analytical sensitivity according to claim 1, 2, or 4, characterized in that, In step S202, the pH of mobile phase A is adjusted to pH 3.0~3.3 by 1 mol / L hydrochloric acid or sodium hydroxide; the amount of propylene carbonate added to mobile phase B is 5%; the mobile phase is filtered through a 0.22 μm filter membrane and ultrasonically degassed for 30 min before use.

6. The method for improving LC / MS analytical sensitivity according to claim 5, characterized in that, In step S203, the flow rate of gradient elution increases from 0.28 mL / min to 0.32 mL / min in the 9-12 min stage and decreases back to 0.28 mL / min in the 13.5-16 min stage; in the elution program, the flow rate change rate is 5% / min in the 1-4 min stage and 7% / min in the 4-9 min stage.

7. The method for improving LC / MS analytical sensitivity according to claim 1, 2, 4 or 6, characterized in that, In step S301, the addition of isopropanol-acetonitrile mixed vapor in the sheath gas is precisely controlled by a gas mixer, with a mixing ratio of 1:1 and a total proportion of 6%; the position of the spray needle of the ESI ion source is adjusted to form a 15° angle with the capillary axis.

8. The method for improving LC / MS analytical sensitivity according to claim 7, characterized in that, In step S303, the four segments and corresponding collision energies of the segmented selective ion monitoring are as follows: Segment 1: m / z 60~220, collision energy 18eV, detecting short-chain lipid fragments; Segment 2: m / z 220~480, collision energy 22eV, for detecting lysophospholipids; Segment 3: m / z 480~750, collision energy 26eV, for detecting medium molecular weight phospholipids; Segment 4: m / z 750~1100, collision energy 30eV.

9. The method for improving LC / MS analytical sensitivity according to claim 1, 2, 4, 6 or 8, characterized in that, In step S304, the standard curve is prepared as follows: the target lipid standard is diluted with CHCl3 / MeOH to a series of concentrations of 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, and 50 ng / mL. An internal standard of 8 ng / mL PC 36:2-d6 is added to each concentration. The standard is then detected according to the LC chromatography separation method in step (2) and the IMS-MS detection method in step (3). A standard curve is plotted with the target lipid concentration as the abscissa and the peak area ratio of the target lipid to the internal standard as the ordinate. R 2 ≥0.9995.

10. The application of the method for improving LC / MS analytical sensitivity according to any one of claims 1 to 9, characterized in that, The method is applied to the LC / MS analysis of lipid metabolites in mammalian plasma, serum, liver tissue homogenate, and adipose tissue homogenate samples. The lipid metabolites include PC, PE, LysoPC, PA, PG, PI and their corresponding isomers.

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