Pork lipidomics analysis method based on liquid chromatography-tandem mass spectrometry

Abstract

The invention belongs to the technical field of pork lipidome extraction and analysis, and discloses a pork lipidome analysis method based on liquid chromatography-tandem mass spectrometry, and the method comprises the following steps: S1, taking pig adipose tissue, and carrying out low-temperature grinding and refining treatment; s2, carrying out lipid extraction by adopting a two-phase solvent system; s3, performing centrifugal separation on the extracting solution, and collecting an upper-layer organic phase; s4, concentrating and drying the organic phase, and re-dissolving the organic phase in the chromatographic compatible solvent; and S5, carrying out separation and structure identification on the redissolved lipid by using an electron activation dissociation fragmentation liquid chromatography-tandem mass spectrometry system. A two-phase solvent system is adopted to replace chloroform, so that the risk of highly toxic solvent exposure is eliminated while the lipid recovery rate is guaranteed; an electron activation dissociation technology is integrated, chemical bonds are directionally broken through electron free radicals, and fine structure identification of pig characteristic lipid is achieved; and a support is provided for efficient extraction and fine analysis of the pig lipidome.

Description

Technical Field

[0001] This invention relates to the field of pork lipidome extraction and analysis technology, specifically a method for pork lipidome analysis based on liquid chromatography-tandem mass spectrometry. Background Technology

[0002] Tandem mass spectrometry (MS / MS) achieves compound structure resolution and quantification through collisional activated dissociation (CID), but it has inherent limitations when processing complex lipid molecules (such as glycerophospholipids and sphingolipids): insufficient fragment information—CID is based on thermodynamic equilibrium fragmentation, which easily leads to the loss of key structural information (such as acyl chain positional isomerism); and limited quantitative selectivity—isomers are difficult to generate diagnostic fragments, affecting the accurate quantification of trace lipids. Furthermore, while the traditional chloroform system (Folch / Bligh-Dyer method) has high extraction efficiency, it carries the risk of high toxicity (chloroform carcinogenicity) and lipid modification; emerging solvent solutions (such as isopropanol and MTBE) have lower toxicity, but their comprehensive extraction capabilities in porcine white adipose tissue lack systematic validation. Current technologies have not yet addressed the need for efficient extraction and precise analysis of porcine-specific lipid profiles. Summary of the Invention

[0003] This invention aims to provide a method for lipidomics analysis of pork based on liquid chromatography-tandem mass spectrometry (LC-MS / MS). It replaces chloroform with a methanol / MTBE / water / dichloromethane-isopropanol / methanol two-phase system, ensuring lipid recovery while eliminating the risk of exposure to highly toxic solvents. A breakthrough in intelligent fragmentation is achieved by integrating energy-tunable electron activated dissociation (EAD) technology, enabling the precise structural identification of characteristic porcine lipids (such as diglycerides) through the directional breaking of chemical bonds by electron free radicals. Analytical efficiency is significantly enhanced by establishing the first dual-mode LC-MS / MS whole-lipome analysis method for pork adipose tissue, combining lipid extraction with EAD structure verification. This provides support for the efficient extraction and precise analysis of porcine lipids, achieving comprehensive extraction and systematic validation from porcine adipose tissue.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

[0005] A method for lipidomics analysis of pork based on liquid chromatography-tandem mass spectrometry includes the following steps:

[0006] S1. Take a pig adipose tissue sample and perform low-temperature grinding and refining to obtain a homogeneous sample;

[0007] S2. Lipid extraction is performed on homogeneous samples using a two-phase solvent system, wherein the solvent system comprises MeOH, MTBE, H2O, CH2Cl2 and IPA / MeOH mixed solvent;

[0008] S3. Centrifuge the extract and collect the upper organic phase;

[0009] S4. After concentrating and drying the organic phase, redissolve it in a chromatographically compatible solvent.

[0010] S5. A unique electronically activated dissociation fragmentation liquid chromatography-tandem mass spectrometry system is used to separate and identify reconstituted lipids.

[0011] Furthermore, in S1, the low-temperature grinding and refining treatment is carried out under low-temperature conditions after being frozen with liquid nitrogen, with a temperature ≤ -40℃ and a grinding time of 3-10 minutes.

[0012] Further, in S2, the lipid extraction of the homogeneous sample using a two-phase solvent system includes the following steps:

[0013] S201. Add the solvents MeOH, CH2Cl2, MTBE and H2O to the homogeneous sample in a volume ratio of 6:4:20:5. Vortex for 10-15 minutes after each addition of solvent. After mixing, sonicate for 10-30 minutes and incubate overnight at -20°C.

[0014] After S202, centrifugation, and nitrogen blowing, add IPA / MeOH mixed solvent and vortex for 10-15 minutes; wherein the volume ratio of IPA to MeOH is 2:1.

[0015] Furthermore, the centrifugation conditions were: temperature -4℃, rotation speed 12000rpm, and time 20 minutes.

[0016] Furthermore, in S5, the electron activation dissociation fragmentation adopts the following parameters: electron energy range, 15 eV for positive ions and -5 eV for negative ions; data-dependent acquisition is performed in the m / z range of 100-1600 and 50-1600.

[0017] Furthermore, the method enables the simultaneous extraction of phospholipids, triglycerides, diglycerides, and sphingolipids from porcine subcutaneous adipose tissue.

[0018] The beneficial effects of the technical solution are:

[0019] 1. This invention employs a two-phase solvent system (methanol / methyl tert-butyl ether / water / dichloromethane-isopropanol / methanol) for simultaneous lipid extraction; and generates diagnostic fragment ions through a unique electron activated dissociation (EAD) fragmentation technology combined with LC-MS / MS analysis. This method achieves simultaneous and efficient extraction of phospholipids (PLs), diglycerides (DAGs), triglycerides (TAGs), and sphingolipids (SLs).

[0020] 2. This invention improves both extraction efficiency and safety. It uses a methanol / methyl tert-butyl ether / water / dichloromethane-isopropanol / methanol two-phase solvent system to completely replace the highly toxic chloroform, which facilitates recovery. While eliminating the risk of cancer to operators, it ensures high recovery rates of phospholipids and glycerides. In particular, the recovery rates of representative lipids (TG, DG, PC) in porcine adipose tissue are improved compared to the traditional single-phase method.

[0021] 3. This invention features a revolutionary structural analysis capability, a breakthrough in lipid structure analysis, and innovative integration of EAD fragmentation energy with selectable 15 eV and -15 eV. EAD technology precisely generates diagnostic lipid fragments (such as DAGs), solving the problem of isomerism that cannot be distinguished by collision-activated dissociation (CID).

[0022] 4. The integrated grinding → extraction → centrifugation → concentration process of this invention avoids complex purification steps, reduces the amount of organic solvent used and the cost of waste liquid treatment, and meets the standards of green laboratories.

[0023] 5. Promote the standardized application of pork lipidomics and establish the first lipid extraction and EAD mass spectrometry detection for pig adipose tissue, covering a variety of lipid molecules (PLs / DAGs / TAGs / SLs, etc.). Attached Figure Description

[0024] Figure 1 This is a comparison of the extraction rates of pork lipids in the pretreatment of pork using a method for pork lipidomics analysis based on liquid chromatography-tandem mass spectrometry (LC-MS / MS) according to the present invention.

[0025] Figure 2 This is a flowchart illustrating the advantages of combining electron activated dissociation (EAD) fragmentation technology with LC-MS / MS analysis in a method for analyzing pork lipidomics based on liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0026] Figure 3 The flowchart shows the structural identification and quantification of DG 36:2 via characteristic peaks 621 and 5320, which is part of a liquid chromatography-tandem mass spectrometry-based method for pork lipidomics analysis according to the present invention. Detailed Implementation

[0027] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments:

[0028] A method for whole-lipomics analysis of pork based on liquid chromatography-tandem mass spectrometry (LC-MS / MS) is proposed. This method employs electron activated dissociation (EAD) fragmentation technology and a two-phase extraction method (methanol / methyl tert-butyl ether / water / dichloromethane-isopropanol / methanol system). The combination of extraction method and detection technology ensures efficient and accurate analysis of lipid metabolites from porcine adipose tissue, thus providing strong technical support for lipidomics research. The specific method includes the following steps:

[0029] S1. Freeze the pork fat sample in liquid nitrogen (temperature ≤ -40℃) and grind it for 3-10 minutes until it is fine to obtain a homogeneous sample;

[0030] S2. Lipid extraction was performed on homogeneous samples using a two-phase solvent system.

[0031] S201. Weigh 10 mg of the homogenized fat sample, add 0.36 mL of pre-cooled (-20℃) methanol (MeOH), and vortex at 10 Hz for 20 minutes. Then add 0.24 mL of pre-cooled (-20℃) dichloromethane (CH2Cl2) and vortex at 10 Hz for 20 minutes. Next, add 1.2 mL of pre-cooled (-20℃) methyl tert-butyl ether (MTBE), and vortex the mixture again at 10 Hz for 20 minutes. Additionally, add 300 μL of pre-cooled (-4℃) ultrapure water to the sample, vortex the mixture again at 10 Hz for 10 minutes, and store at -20℃ overnight.

[0032] S202. Centrifuge the sample at 12000 rpm and 4℃ for 20 minutes, transfer the upper phase to a 2 mL Eppendorf tube, and dry at room temperature under a gentle nitrogen stream. Then, add 0.9 mL of pre-cooled (-20°C) reconstituted isopropanol / methanol (IPA / MeOH, 2:1 (v / v)) to the dried lipid extract, and sonicate the sample at 10 Hz for 15 minutes. The extraction rate comparison is shown in the figure below. Figure 1 As shown;

[0033] S3. Centrifuge the extract of S202 at 12000 rpm and 4℃ for 20 minutes; filter the obtained supernatant through a 0.20µm PVDF membrane, put it into a liquid chromatography vial, refrigerate at 4℃, and wait for LC-MS / MS analysis;

[0034] S4. After concentrating and drying the organic phase, redissolve it in a chromatographically compatible solvent.

[0035] S5. Separation and structural identification of reconstituted lipids using a unique electron activated dissociation (EAD) fragmentation LC-MS / MS system; the flowchart illustrating the advantages of combining EAD fragmentation technology with LC-MS / MS analysis is shown below. Figure 2 As shown, the flowchart for structural identification and quantification of DG 36:2 using the characteristic peak 621.5320 is as follows. Figure 3 As shown;

[0036] The chromatographic conditions were set as follows: a Phenomenex Kinetex C18 2.6 µm (100 × 2.1 mm) column, an autosampler temperature of 4 °C, a flow rate of 0.3 mL / min, a column temperature of 45 °C, and a gradient elution with an injection of 2 µL. The mobile phase was: A. Water:Methanol:Acetonitrile = 1:1:1 (5 mmol / L ammonium acetate), B. Isopropanol (5 mmol / L ammonium acetate). The gradient elution program was: 0.5–1.5 min, 20%–40%; 1.5–3 min, 40%–60%; 3–13 min, 60%–98%; 13–14 min, 98%; 14–14.1 min, 98%–20%; 14.1–17 min, 20%.

[0037] Mass spectrometry conditions were set as follows: positive and negative ion spray voltages of 5.5 kV and 4.5 kV, respectively; capillary temperature of 550 °C; sheath gas of 55 alb and curtain gas of 35 alb. The scanning range was MS1: 200–1200; MS2: 50–1200. Secondary fragmentation was performed in EAD mode, with positive and negative ion collision energies of 15 eV and -5 eV, respectively. Dynamic monitoring was used to exclude unnecessary MS / MS information.

[0038] The following parameters were used for electron-activated dissociation fragmentation: electron energy range, positive ion (ESI+) 15 eV, negative ion (ESI-) -5 eV; data-dependent acquisition (DDA) was performed in the m / z range of 100-1600 and 50-1600.

[0039] In summary, this invention employs a two-phase solvent system (methanol / methyl tert-butyl ether / water / dichloromethane-isopropanol / methanol) for simultaneous lipid extraction; and utilizes a unique electron activated dissociation (EAD) fragmentation technology combined with LC-MS / MS analysis to generate diagnostic fragment ions. This method achieves simultaneous and efficient extraction of phospholipids (PLs), diglycerides (DAGs), triglycerides (TAGs), and sphingolipids (SLs). Furthermore, a two-phase solvent system of methanol / methyl tert-butyl ether / water / dichloromethane-isopropanol / methanol is used to completely replace highly toxic chloroform, facilitating recovery and eliminating the risk of cancer to operators while ensuring high efficiency in the recovery of phospholipids and glycerides. Specifically, the recovery rates of representative lipids (TG, DG, PC) in porcine adipose tissue are improved compared to traditional single-phase methods. The method innovatively integrates EAD fragmentation energy, selectable at 15 eV and -15 eV, enabling EAD technology to accurately generate diagnostic lipid fragments (such as DAGs), solving the problem of isomerism that cannot be distinguished by collisional activated dissociation (CID). This method integrates a four-step process of grinding, extraction, centrifugation, and concentration, avoiding complex purification steps, reducing organic solvent usage and wastewater treatment costs, and meeting green laboratory standards. It promotes the standardized application of pork lipidomics, establishing the first lipid extraction and EAD mass spectrometry detection system specifically for porcine adipose tissue, covering various lipid molecules (PLs / DAGs / TAGs / SLs, etc.).

[0040] The above descriptions are merely embodiments of the present invention, and common knowledge regarding specific technical solutions or characteristics is not elaborated upon here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for porcine meat lipidomics analysis based on liquid chromatography-tandem mass spectrometry, characterized in that, The method comprises the following steps: S1, taking a pig fat tissue sample for low-temperature grinding and refining treatment to obtain a homogeneous sample; S2, using a two-phase solvent system to extract lipids from the homogeneous sample, wherein the solvent system comprises MeOH, MTBE, H2O, CH2Cl2 and an IPA / MeOH mixed solvent; S3, centrifuging the extraction liquid to collect the upper organic phase; S4, dissolving the organic phase in a chromatography-compatible solvent after concentration and drying; S5, using a unique electron-activated dissociation fragmentation liquid chromatography-tandem mass spectrometry system to separate and identify the re-dissolved lipids.

2. The method according to claim 1, wherein the method is a method for analyzing porcine lipidomics based on liquid chromatography-tandem mass spectrometry. In S1, the low-temperature grinding and refining treatment is carried out under low-temperature conditions after liquid nitrogen freezing, with a temperature of ≤-40℃ and a grinding time of 3-10 minutes.

3. The method according to claim 1, wherein the method is based on liquid chromatography-tandem mass spectrometry. In S2, the two-phase solvent system is used to extract lipids from the homogeneous sample, which comprises the following steps: S201, adding solvents MeOH, CH2Cl2, MTBE and H2O to the homogeneous sample in the order of volume ratio 6:4:20:5, vortexing for 10-15 minutes after each addition, ultrasonic treatment for 10-30 minutes after all mixing, and overnight treatment at-20℃; S202, after centrifugation and nitrogen blowing, adding an IPA / MeOH mixed solvent, vortexing for 10-15 minutes; wherein the volume ratio of IPA to MeOH is 2:

1.

4. The method according to claim 3, wherein the method is based on liquid chromatography-tandem mass spectrometry. The centrifugation conditions are: temperature of-4℃, rotation speed of 12000 rpm, and time of 20 minutes.

5. The method according to claim 1, wherein the method is based on liquid chromatography-tandem mass spectrometry. In S5, the electron-activated dissociation fragmentation uses the following parameters: electron energy range, positive ion 15 eV, negative ion-5 eV; data-dependent acquisition is performed in the range of m / z 100-1600 and 50-1600.

6. The method for porcine lipidomics analysis based on liquid chromatography-tandem mass spectrometry according to any one of claims 1-5, characterized in that: The method realizes synchronous extraction of phospholipids, triglycerides, diglycerides and sphingolipids in pig subcutaneous fat tissue.

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