CN113176358A

CN113176358A - Pretreatment method and detection method of micro sample for metabonomics and lipidomics research

Info

Publication number: CN113176358A
Application number: CN202110512265.XA
Authority: CN
Inventors: 徐英; 任建洪; 张永霞; 谷从顺; 戴贞丽; 顾梦南
Original assignee: Suzhou Bionovogene Biomedical Technology Co ltd
Current assignee: Suzhou Bionovogene Biomedical Technology Co ltd
Priority date: 2021-05-11
Filing date: 2021-05-11
Publication date: 2021-07-27
Anticipated expiration: 2041-05-11
Also published as: CN113176358B

Abstract

The invention belongs to the technical field of omics analysis methods, and relates to a pretreatment method and a detection method of a micro sample for metabonomics and lipidomics research. Specifically, the pretreatment method comprises the steps of carrying out single-phase extraction on a trace sample to obtain a metabonomics sample, and then carrying out two-phase extraction to obtain a lipidomics sample; the detection method comprises the steps of preparing a mixed solution of metabonomics and lipidomics internal standards, preprocessing the mixed solution and a trace sample at the same time, and then analyzing the metabonomics and the lipidomics. The method sequentially introduces metabonomics internal standard and lipidomics internal standard, selects internal standard with the same property or the same type as reference basis by identifying the obtained substance types, respectively calculates the concentration of a target substance in a sample by utilizing the peak area and the concentration of the internal standard substance, and can simultaneously obtain the qualitative results of metabolites and lipids.

Description

Pretreatment method and detection method of micro sample for metabonomics and lipidomics research

Technical Field

The invention belongs to the technical field of omics analysis methods, and relates to a pretreatment method of a micro sample for metabonomics and lipidomics research and a detection method of the micro sample constructed based on the pretreatment method.

Background

Metabolomics (metabolomics) is one of important components of system biology, and is mainly studied for polar metabolites (i.e., hydrophilic substances, usually polar small molecular compounds), and is roughly classified into amino acids, energy metabolites, nucleotides, carbohydrates, coenzyme factors, and the like. Lipidomics (lipidomics) are independent subjects derived from metabolomics, and the study of lipids (i.e. lipophilic substances) is focused on, and generally includes 8 major classes such as fatty acids, glycerolipids, glycerophospholipids, sphingolipids, sterols (or steroids), polyprenols, glycolipids and polyketides^[1,2]。

Because the chemical properties of polar metabolites and lipids are greatly different, the extraction reagents and extraction methods of samples in metabolomics and lipidomics are different in the sample pretreatment process of omics research. For example, the polar metabolite is extracted with methanol (MeOH), Acetonitrile (ACN), and water (H)₂O) and the like, and the extraction solvent of the lipid mainly comprises Dichloromethane (DCM), chloroform (TCM), methyl tert-butyl ether (MTBE) and other weak polar organic solvents^[3,4](ii) a For another example, the polar metabolite extraction method is biased towards direct ultrasound or oscillation, while the lipid extraction method is based on liquid-liquid extraction, and the target lipid is extracted by the difference of partition coefficients of the lipid in the aqueous phase and the organic phase. Although the polar metabolites and lipids are chemically different, there is often a need to integrate them orComplement to clarify the associated metabolic mechanism, thereby more comprehensively characterizing the metabolic profile of the whole biological system^[5]。

However, obtaining target substances from micro-samples (such as tumor tissues, cerebral cortex, hippocampus, plant samples cultured by arabidopsis thaliana, thallus, silkworm blood or shrimp blood, exosomes and the like) has been a bottleneck and difficulty in omics research. Because of the sampling limitation of biological samples, it is difficult to obtain satisfactory samples (e.g., richer sample size, and sufficient sample weight or volume), the conventional practice mostly increases the sample size by physical mixing of samples of the same group, but such operation cannot observe biological differences among individual samples. Moreover, it is more difficult to satisfy the requirement of the simultaneous study of metabonomics and lipidomics in the limited micro-samples.

Based on the distribution coefficient difference of the polar metabolites and the lipids in the extraction solvent, the extraction parts of the polar metabolites and the lipids can be respectively obtained through a proper extraction method or steps, and the research of two omics can be effectively developed by using the same sample. For example, chinese patent application CN108593821A discloses a sample extraction method for tissue sample metabonomics and lipidomics research, which comprises adding a methanol-water mixed solvent to a tissue sample, extracting, then taking all the supernatant (hydrophilic extract) for metabonomics research, and adding methyl tert-butyl ether to the extraction residue to extract lipid, which is used as a lipophilic extract for lipidomics research. The method can complete extraction by using two solvents with different polarities step by step, and can not only satisfy the full coverage of polar metabolites and lipids, but also avoid the problem that polar metabolites are distributed to a lipid extraction layer when a single mixed solvent is used for extraction. Joran Villaret-Cazadamont et al disclose a biphasic extraction method for simultaneous and accurate analysis of polar metabolites and lipids, which is carried out in a single biological sample, but differs from the specific steps of the stepwise extraction method described above in that the extraction solvent system is a mixed solution of acetonitrile/methanol/0.1% formic acid (2/2/1; v/v/v) and dichloromethane, the upper layer is taken, concentrated and used for metabonomic studies, the lower layer is taken, and concentratedThe method is used for lipidomics research after shrinkage, breaks through the limitation of insufficient sample amount and is beneficial to the research of functional metabolome^[6]. Jelena Sostar et al compared the original Matyash process (MTBE/MeOH/H)₂O; 10/3/2.5; v/v/v), modified Matyash Process (MTBE/MeOH/H)₂O; 2.6/2.0/2.4; v/v/v) and stepwise Bligh-Dyer method (chloroform/methanol/water; 2.0/2.0/1.8; v/v/v) and found that the number of metabolites obtained by the modified Matyash method is 1-30% more than the original Matyash method and 1-40% more than the Bligh-Dyer method^[7]. In addition, the reproducibility evaluation index RSD value calculated according to the peak signal response is lower than that of the other two methods, which shows that the method has certain advantages.

Although the above-mentioned methods (either stepwise extraction using two single-phase solvents in sequence or simultaneous extraction using a two-phase solvent) enable trace samples to meet the basic requirements of simultaneous metabolomics and lipidomics studies, the extraction efficiency of the target substance is greatly affected because these methods ignore the subtle differences in the chemical properties of polar metabolites and lipids and the important role of the extraction solvent (especially methanol used in most methods) in two-phase extraction.

At present, the following problems mainly exist:

(1) in the extraction process of polar metabolites and lipids, methanol can dissolve most polar metabolites, even some slightly polar lipids, besides the function of protein precipitation, so that the method of taking all methanol supernatant (such as the method in CN 108593821A) will inevitably affect the coverage of the polar lipids in lipidomics samples;

(2) although the synchronous extraction method using a biphasic mixed solvent can effectively simplify the process, some polar metabolites or lipids can be dispersed in both the aqueous phase and the organic phase, resulting in loss of the content of the target substance during the respective detection (e.g., the method of Joran Villaret al-Cazadamont et al);

(3) in the process of lipid extraction, factors such as the acid-base property of the extraction solvent and the extraction temperature also affect the extraction efficiency of some lipids, for example, Phosphatidic Acid (PA) and Phosphatidylserine (PS) have poor solubility in a methanol-chloroform solvent system, and the extraction efficiency is not high.

In addition, the simultaneous detection of metabonomics and lipidomics is not realized at present, and the detection method is limited to the research range of single metabonomics or single lipidomics.

Non-patent document

1.Wilmanski T.,Rappaport N.,Earls J.C.,et al.,Blood metabolome predicts gut microbiomeα-diversity in humans[J],Nat.Biotechnol.,2019,37(10):1217-1228.

2.Holcapek M.,Liebisch G.,Ekroos K.,Lipidomic analysis[J],Anal.Chem.,2018,90(7):4249-4257.

3.Vuckovic D.,Current trends and challenges in sample preparation for global metabolomics using liquid chromatography-mass spectrometry[J],Anal.Bioanal.Chem.,2012,403:1523-1248.

4.Tsugawa H.,Ikeda K.,Takahashi M.,et al.,A lipidome atlas in MS-DIAL 4[J],Nat.Biotechnol.,2020,38(10):1159-1163.

5.Treves H.,Siemiatkowska B.,Luzarowska U.,et al.,Multi-omics reveals mechanisms of total resistance to extreme illumination of a desert alga[J],Nat.Plants,2020,6(8):1031-1043.

6.Villaret-Cazadamont J.,Poupin N.,Tournadre A.,et al.,An optimized dual extraction method for the simultaneous and accurate analysis of polar metabolites and lipids carried out on single biological samples[J],Metabolites,2020,10(9):338.

7.Sostare J.,Di Guida R.,Kirwan J.,et al.,Comparison of modified Matyash method to conventional solvent systems for polar metabolite and lipid extractions[J],Anal.Chim.Acta.,2018,1037:301-315.

Disclosure of Invention

Problems to be solved by the invention

Aiming at the defects in the prior art, the invention provides a pretreatment method of a micro sample for metabonomics and lipidomics research. In addition, on the basis of a sample pretreatment method, the invention also provides a metabonomics and lipidomics detection method to make up for the blank of the prior art.

Means for solving the problems

In one aspect, the present invention provides a method for pretreating a micro sample for metabonomics and lipidomics research, comprising the following steps:

1) adding a single-phase extraction solvent into the micro sample, oscillating, performing single-phase extraction at room temperature, centrifuging at low temperature, and taking out part of supernatant, namely the metabonomics sample;

2) and adding a two-phase extraction solvent into the residual supernatant and residues, oscillating, performing two-phase extraction in an ice bath, centrifuging at low temperature, taking out an organic phase, concentrating and drying in vacuum, adding a redissolution solvent into the concentrate, uniformly mixing by vortex, centrifuging at low temperature, and taking out the supernatant, namely the lipidomics sample.

Further, the trace amount in step 1) is 1/2 sample amount which is lower than the detection requirement of the conventional method sample amount.

Further, the single-phase extraction solvent in the step 1) is C_1-4An aliphatic alcohol or an aqueous solution thereof, preferably methanol or an aqueous solution thereof, more preferably methanol.

Further, the dosage ratio of the micro sample to the single-phase extraction solvent in the step 1) is 1mg:4 μ L-1 mg:10 μ L, preferably 1mg:5 μ L-1 mg:8 μ L, and more preferably 1mg:6 μ L.

Further, the volume ratio of the supernatant to the single-phase extraction solvent in the step 1) is 1: 1.5-1: 10, preferably 1: 2-1: 5, and more preferably 1: 3.

Further, the biphasic extraction solvent in step 2) is composed of C_1-4Aliphatic alcohol, aqueous acid solution (aqueous formic acid solution, aqueous acetic acid solution or hydrochloric acid) and C_1-4The halogenated hydrocarbon is preferably composed of methanol, hydrochloric acid (preferably 0.1-1M hydrochloric acid, more preferably 0.5-1M hydrochloric acid, most preferably 1M hydrochloric acid) and chloroform, and more preferably composed of methanol, hydrochloric acid (preferably 0.1-1M hydrochloric acid, more preferably 0.5-1M hydrochloric acid, most preferably 1M hydrochloric acid) and chloroform in a volume ratio of 5:1: 10.

Further, the dosage ratio of the micro sample to the biphasic extraction solvent in the step 2) is 1mg:10 μ L-1 mg:20 μ L, preferably 1mg:15 μ L-1 mg:18 μ L, and more preferably 1mg:16 μ L.

Further, the redissolving solvent in the step 2) is C_1-4One or more of aliphatic alcohols, preferably methanol and/or isopropanol, more preferably a methanol/isopropanol mixed solvent in a volume ratio of 1: 4.

Further, the dosage ratio of the micro sample to the redissolution solvent in the step 2) is 1mg:1 μ L-1 mg:5 μ L, preferably 1mg:1.5 μ L-1 mg:3 μ L, and more preferably 1mg:2 μ L.

Preferably, step 2) also comprises a supplementary biphasic extraction between the removal of the organic phase and the vacuum concentration drying, comprising: adding supplementary biphase extraction solvent into residue after taking out organic phase, mixing uniformly by vortex, extracting in ice bath, centrifuging at low temperature, taking out organic phase, and mixing with organic phase taken out after biphase extraction.

Further, supplementing the biphasic extraction solvent in step 2) with C_1-4Fatty alcohols and C_1-4The halogenated hydrocarbon is preferably composed of methanol and chloroform, and more preferably composed of methanol and chloroform in a volume ratio of 1: 2.

Further, the dosage ratio of the micro sample to the supplementary biphasic extraction solvent in the step 2) is 1mg:5 μ L-1 mg:15 μ L, preferably 1mg:8 μ L-1 mg:12 μ L, and more preferably 1mg:10 μ L.

In another aspect, the present invention provides a method for metabolomics and lipidomics detection of a micro sample, comprising the steps of:

1) taking a proper amount of various metabonomics internal standards, and respectively adding water or C_1-4Preparing fatty alcohol into multiple single metabonomics internal standard mother liquor, respectively taking appropriate amount of the single metabonomics internal standard mother liquor, mixing, and adding water or C_1-4Diluting fatty alcohol to prepare a metabonomics internal standard mixed solution; taking a proper amount of multiple lipidomics internal standards, and respectively adding C_1-4Preparing fatty alcohol into multiple single lipidomics internal standard mother liquor, then respectively taking appropriate amount of various single lipidomics internal standard mother liquor, mixing and using C_1-4Diluting fatty alcohol to prepare a lipidomics internal standard mixed solution;

2) adding a metabonomics internal standard mixed solution and C into a micro sample_1-4Oscillating fatty alcohol or water solution thereof, performing single-phase extraction at room temperature, centrifuging at low temperature, and taking out partial supernatant to obtain a metabonomics sample for later use; adding lipidomics internal standard mixed solution, acid water solution and C into the residual supernatant and residues_1-4Halogenated hydrocarbons and C_1-4Oscillating fatty alcohol, performing two-phase extraction in an ice bath, centrifuging at low temperature, taking out an organic phase, concentrating and drying in vacuum, adding a redissolving solvent into a concentrate, uniformly mixing by vortex, centrifuging at low temperature, and taking out a supernatant, namely a lipidomics sample for later use;

3) and respectively carrying out metabonomic analysis and lipidomic analysis on the metabonomic sample and the lipidomic sample, and respectively completing the detection of the polar metabolites and the lipids in the micro-sample based on the metabonomic internal standard and the lipidomic internal standard.

Further, the various metabonomic internal standards in step 1) include succinic acid-2,2,3,3-d4, cholic acid-2,2,3,4,4-d5, L-phenylalanine-d5, DL-methionine-3,3,4,4-d4, DL-tryptophan-2,3,3-d3 and choline chloride-trimethyl-d 9; the concentration of the metabonomics internal standard in the single metabonomics internal standard mother liquor is 1-15 mg/mL, preferably 1-5 mg/mL, and more preferably 1-2 mg/mL; the concentration of each metabonomics internal standard in the metabonomics internal standard mixed solution is 5-50 mug/mL, preferably 5-30 mug/mL, and more preferably 5-15 mug/mL.

Further, the various internal lipidomics standards in step 1) comprise phosphatidylcholine 16:0-d31-18:1, phosphatidylethanolamine 14:0-14:0, phosphatidylglycerol 16:0-d31-18:1, phosphatidylserine 14:0-14:0 and phosphatidic acid 17:0-17: 0; the concentration of the lipidomic internal standard in the single lipidomic internal standard mother liquor is 1-5 mg/mL, preferably 1-3 mg/mL, and more preferably 1-2 mg/mL; the concentration of each lipidomic internal standard in the lipidomic internal standard mixed solution is 10-50 mug/mL, preferably 10-30 mug/mL, and more preferably 20-30 mug/mL.

Further, the trace amount in step 2) is 1/2 sample amount which is lower than the detection requirement of the conventional method sample amount.

Further, the solutions are mixed in step 2) by metabonomics internal standard toAnd C_1-4The fatty alcohol or its water solution (preferably in a volume ratio of 2:1) constitutes a single-phase extraction solvent, and the dosage ratio of the micro-sample to the single-phase extraction solvent is 1mg:4 μ L-1 mg:10 μ L, preferably 1mg:5 μ L-1 mg:8 μ L, more preferably 1mg:6 μ L.

Further, the volume ratio of the supernatant to the single-phase extraction solvent in the step 2) is 1: 1.5-1: 10, preferably 1: 2-1: 5, and more preferably 1: 3.

Further, in the step 2), a mixed solution, an acid aqueous solution and C are prepared from an internal standard of lipidomics_1-4Halogenated hydrocarbons and C_1-4The two-phase extraction solvent is composed of fatty alcohol (preferably four in a volume ratio of 2:1:10:3), and the dosage ratio of the micro-sample to the two-phase extraction solvent is 1mg:10 muL-1 mg:20 muL, preferably 1mg:15 muL-1 mg:18 muL, and more preferably 1mg:16 muL.

Further, the redissolving solvent in the step 2) is C_1-4The dosage ratio of the micro sample to the redissolving solvent is 1mg:1 muL-1 mg:5 muL, preferably 1mg:1.5 muL-1 mg:3 muL, and more preferably 1mg:2 muL.

Further, step 2) is performed by C_1-4Fatty alcohols and C_1-4The halogenated hydrocarbon (preferably the volume ratio of the halogenated hydrocarbon to the complementary biphasic extraction solvent is 1:2), and the dosage ratio of the trace sample to the complementary biphasic extraction solvent in the step 2) is 1mg:5 μ L-1 mg:15 μ L, preferably 1mg:8 μ L-1 mg:12 μ L, and more preferably 1mg:10 μ L.

Further, in the above-mentioned detection method, C_1-4The aliphatic alcohol is methanol and/or isopropanol, preferably methanol; the acid aqueous solution is formic acid aqueous solution, acetic acid aqueous solution or hydrochloric acid, preferably 0.1-1M hydrochloric acid, more preferably 0.5-1M hydrochloric acid, most preferably 1M hydrochloric acid; c_1-4The halogenated hydrocarbon is chloroform and/or dichloromethane, preferably chloroform.

ADVANTAGEOUS EFFECTS OF INVENTION

The pretreatment method can simultaneously obtain the hydrophilic metabolite and the lipophilic lipid from the same trace biological sample, and is more suitable for the trace biological sample with high sampling difficulty and/or limited sampling amount compared with the traditional method of respectively obtaining the polar metabolite and the lipid from two samples.

Firstly, compared with a single two-phase extraction mode adopting a mixed extraction solvent, the pretreatment method adopts an extraction mode divided into two steps, wherein a single-phase solvent is used for extracting polar metabolites in the first step, and the mixed extraction solvent is used for extracting lipid in the second step, so that the extraction efficiency is obviously improved, and the loss of concentration caused by different distribution of hydrophilic/lipophilic substances in two phases in the single extraction process is avoided.

Secondly, the two-step extraction mode adopted by the pretreatment method is different from the two-step extraction mode in the conventional method. In the case of extracting hydrophilic metabolites, the conventional method requires the extraction liquid (supernatant) containing polar metabolites to be completely removed, whereas the pretreatment method of the present invention takes only a part thereof, and the remaining part is subjected to biphasic extraction of lipids together with the sample residue. Can meet the detection requirement of metabolites and can also meet the effective extraction of substances with stronger polarity in the lipid, so that the amount of the detected lipid is higher than that of the conventional two-step extraction.

Finally, the pretreatment method of the invention also adds an acidic solute to the biphasic extraction solvent during lipid extraction, and from the viewpoint of detection results, the operation is beneficial to the dissolution of acidic lipids from a sample and/or the extraction of the acidic lipids by an organic solvent, and is particularly suitable for the high-efficiency extraction of lipids such as phosphatidic acid and the like.

In addition, based on the pretreatment method, the invention also develops a detection method suitable for simultaneously carrying out metabonomics and lipidomics researches on trace biological samples. The method sequentially introduces metabonomics internal standard and lipidomics internal standard, selects internal standard with the same property or the same type as reference basis by identifying the obtained substance types, respectively calculates the concentration of a target substance in a sample by utilizing the peak area and the concentration of the internal standard substance, and can simultaneously obtain the qualitative results of metabolites and lipids.

Drawings

FIG. 1 shows a method for pre-processing samples for various metabolomics and lipidomics studies (A: separate extraction with two biological samples; B: single biphasic extraction with one biological sample; C: stepwise extraction with one biological sample, wherein the extraction solution containing polar metabolites is completely removed; D: stepwise extraction with one biological sample, wherein the extraction solution containing polar metabolites is partially removed and the extraction solvent is supplemented with acid when lipids are extracted).

FIG. 2 shows a UPLC-MS chromatogram of lipid group base peaks after a pretreatment method of single extraction with a biphasic solvent and fractional extraction of the invention on rat cerebral cortex (A: chromatogram of base peaks after single extraction with a biphasic solvent in a positive ion mode; B: chromatogram of base peaks after fractional extraction in a positive ion mode; C: chromatogram of base peaks after single extraction with a biphasic solvent in a negative ion mode; and D: chromatogram of base peaks after fractional extraction in a negative ion mode).

FIG. 3 shows UPLC-MS lipidosome base-peak chromatograms of mouse brain tissues after pretreatment of all or part of the taken metabolite extract (A: the all-taken base-peak chromatogram in positive ion mode; B: the partially-taken base-peak chromatogram in positive ion mode; C: the all-taken base-peak chromatogram in negative ion mode; and D: the partially-taken base-peak chromatogram in negative ion mode).

FIG. 4 shows a UPLC-MS chromatogram of lipid group base peaks after pretreatment of rat cerebral cortex by acid-free and acid-free lipid extraction (A: chromatogram of base peaks of lipid extracted in positive ion mode, B: chromatogram of base peaks of lipid extracted in positive ion mode, C: chromatogram of base peaks of lipid extracted in negative ion mode, and D: chromatogram of base peaks of lipid extracted in negative ion mode).

FIG. 5 shows the comparison of lipid levels of rat cerebral cortex and mouse brain tissue after different pretreatment methods (A: primary annotation in Parent mode; B: secondary annotation in Product mode).

FIG. 6 shows the comparison of the amount of lipids in mouse brain tissue after pretreatment with different kinds of acids and different concentrations of hydrochloric acid (A: primary annotation in Parent mode; B: secondary annotation in Product mode).

Detailed Description

[ definition of terms ]

As used herein, unless otherwise indicated, the term "sample" refers to a biological sample used in omics studies, including, but not limited to, animal samples (e.g., human or animal blood, urine, feces, saliva, hair, cells, tissue, organs, etc.), plant samples (e.g., plant roots, stems, leaves, flowers, fruits, seeds, etc.), microbial samples (e.g., microbial cells, spores, fermentation broth, culture fluid, etc.), subcellular structural samples (e.g., organelle mitochondria, exosomes, vesicles, etc.), and the like. The term "trace" as used herein refers to a sample size of 1/2 that is lower than the detection requirement for a sample size of conventional methods. For example, when the conventional method is to collect samples for metabolomics and lipidomics studies separately and the detection requirement for the sample amount is 100mg (actually, 200mg samples are required in total), the "trace amount" at this time means a sample amount of less than 50 mg. The term "microsample" as used herein refers to a biological sample used in omics studies and having a sample size on the order of a micro, such as brain tissue, which is generally small but common in experiments.

As used herein, unless otherwise indicated, the term "extraction solvent" refers to a solvent used to extract a desired component of a substance from a sample, "single-phase extraction solvent" refers to a single solvent used as an extraction solvent or a combination of two or more solvents in homogeneous form, "biphasic extraction solvent" refers to a combination of two or more solvents in partially or completely immiscible two phases used as an extraction solvent.

As used herein, unless otherwise indicated, the term "C" is used_1-4Fatty alcohol "refers to an aliphatic alcohol substance having 1 to 4 carbon atoms, generally having a certain hydrophilicity, for extracting or dissolving polar small molecular compounds. Common C_1-4Aliphatic alcohols include, but are not limited to, methanol (or industrial alcohol), ethanol (or industrial alcohol), n-propanol, and mixtures thereof,Isopropanol, tert-butanol, and the like.

As used herein, unless otherwise indicated, the term "acid" means an acid capable of liberating a proton to form H when dissolved in water₃O⁺The substance of (1). Common acids include, but are not limited to, inorganic acids, for example, oxygen-containing inorganic acids such as sulfuric acid, nitric acid, phosphoric acid, carbonic acid, and the like, oxygen-free inorganic acids such as hydrogen fluoride, hydrogen chloride, hydrogen bromide, hydrogen iodide, and the like; and organic acids, for example, carboxylic acids such as formic acid (or formic acid), acetic acid (or acetic acid), propionic acid, etc., sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc.

As used herein, unless otherwise indicated, the term "C" is used_1-4Halogenated hydrocarbon "means a hydrocarbon material having from 1 to 4 carbon atoms and substituted by at least 1 halogen atom, generally having a certain lipophilicity, for extracting or solubilizing non-polar or less polar compounds. Common C_1-4Halogenated hydrocarbons include, but are not limited to, methyl chloride, methylene chloride, chloroform, tetrachloromethane, 1, 2-dichloroethane, and the like.

The term "internal standard" (or "internal standard") as used herein, unless otherwise indicated, refers to a suitable pure product that is added to a test sample during analysis to calculate the amount of the component to be tested.

The term "isotope" as used herein, unless otherwise specified, refers to an interaction between different species belonging to the same element, having the same number of protons and different numbers of neutrons. For example, the hydrogen element comprises protium (H or¹H) Deuterium (D or²H) And tritium (T or³H) Three isotopes, carbon element, including carbon 12: (¹²C) Carbon 13 (C)¹³C) And carbon 14 (C)¹⁴C) Three isotopes, nitrogen element comprises nitrogen 14: (¹⁴N) and nitrogen 15: (¹⁵N) two isotopes, the oxygen element comprising oxygen 16: (¹⁶O), oxygen 17(¹⁷O) and oxygen 18: (¹⁸O) three isotopes.

As used herein, unless otherwise indicated, the term "isotopic internal standard" refers to a stable internal standard substance formed after at least one more abundant isotope contained in a structure has been replaced with a less abundant isotope. For example, in the molecular structure of choline chloride as an internal standard, if nine hydrogen atoms (or protium atoms) contained in three methyl groups are all replaced by deuterium atoms, the corresponding isotopic internal standard, choline chloride-trimethyl-d9, can be obtained.

[ method for pretreating sample ]

The invention provides a pretreatment method of a micro sample for metabonomics and lipidomics research. The method may comprise the steps of:

In one embodiment of the invention, the minor amount in step 1) may be 1/2 sample size lower than the detection requirement of conventional methods.

In one embodiment of the present invention, the micro-sample in step 1) may be a biological sample used for omics research and the sample size is of micro-scale, such as brain tissue.

In one embodiment of the invention, the monophasic extraction solvent, which may be C, is used in step 1) to extract the target substance (e.g., polar metabolite) in the sample_1-4A fatty alcohol or an aqueous solution thereof.

In a preferred embodiment of the present invention, the single-phase extraction solvent in step 1) may be methanol, or may be an aqueous methanol solution.

In a more preferred embodiment of the present invention, the single-phase extraction solvent in step 1) may be methanol.

In one embodiment of the present invention, the volume of the single-phase extraction solvent in step 1) is determined based on the weight of the micro-sample, and the ratio of the micro-sample to the single-phase extraction solvent may be 1mg:4 μ L to 1mg:10 μ L.

In a preferred embodiment of the present invention, the ratio of the amount of the micro-sample to the monophasic extraction solvent in step 1) may be 1mg: 5. mu.L to 1mg: 8. mu.L.

In a more preferred embodiment of the present invention, the ratio of the amount of the micro-sample to the monophasic extraction solvent in step 1) may be 1mg: 6. mu.L.

In one embodiment of the invention, the single phase extraction in step 1) is carried out under shaking conditions, the shaking frequency may be 40Hz and the shaking time may be 30 min.

The supernatant obtained after single-phase extraction in step 1) contains both polar metabolites serving as a metabonomics research target substance and slightly polar lipids serving as a lipidomics research target substance, and the coverage of lipidomic samples can be influenced if the supernatant is taken completely, so that only part of the supernatant can be taken on the premise of meeting the requirement of the quantity of metabonomics samples. For example, in one embodiment of the present invention, the volume ratio of the portion of supernatant removed in step 1) to the previously added single-phase extraction solvent may be 1:1.5 to 1: 10.

In a preferred embodiment of the present invention, the volume ratio of the portion of supernatant removed in step 1) to the single-phase extraction solvent may be 1:2 to 1: 5.

In a more preferred embodiment of the present invention, the volume ratio of the portion of supernatant removed in step 1) to the single-phase extraction solvent may be 1: 3.

In one embodiment of the present invention, the target substance (e.g., lipid) in the sample is extracted in step 2) using a biphasic extraction solvent, which may consist of one or more hydrophilic solvents and one or more lipophilic solvents, wherein: the hydrophilic solvent may be water, an aqueous acid solution or a lower/middle aliphatic alcohol, and the lipophilic solvent may be an aliphatic ether or a halogenated hydrocarbon. From the viewpoint of solvent density, it is possible to use either a hydrophilic solvent larger than a lipophilic solvent or a lipophilic solvent larger than a hydrophilic solvent. For example, the biphasic extraction solvent in step 2) may be prepared from C_1-4Fatty alcohol, aqueous acid solution (formic acid aqueous solution, acetic acid aqueous solution or hydrochloric acid aqueous solution)) And C_1-4A halogenated hydrocarbon.

In a preferred embodiment of the present invention, the biphasic extraction solvent in step 2) may consist of methanol, hydrochloric acid (preferably 0.1-1M hydrochloric acid, more preferably 0.5-1M hydrochloric acid, most preferably 1M hydrochloric acid) and chloroform.

In a more preferred embodiment of the present invention, the biphasic extraction solvent in step 2) may consist of methanol, hydrochloric acid (preferably 0.1-1M hydrochloric acid, more preferably 0.5-1M hydrochloric acid, most preferably 1M hydrochloric acid) and chloroform in a volume ratio of 5:1: 10.

In one embodiment of the present invention, the volume of the biphasic extraction solvent in step 2) also needs to be determined based on the weight of the microsample, and the ratio of the microsample to the biphasic extraction solvent can be 1mg:10 μ L to 1mg:20 μ L.

In a preferred embodiment of the present invention, the ratio of the amount of the microsample to the biphasic extraction solvent in step 2) may be in the range of 1mg: 15. mu.L to 1mg: 18. mu.L.

In a more preferred embodiment of the present invention, the ratio of the amount of the microsample to the biphasic extraction solvent in step 2) may be 1mg: 16. mu.L.

In one embodiment of the invention, the time for biphasic extraction in step 2) may be 40 min.

In one embodiment of the invention, step 2) may include a supplemental biphasic extraction step in addition to utilizing a biphasic extraction step to obtain the target substance (e.g., lipids) in the sample. This supplementary biphasic extraction step may be carried out after the biphasic extraction and removal of the organic phase, and before the removal of the organic phase by vacuum concentration and drying, and the specific operations may be as follows: adding supplementary biphase extraction solvent into residue after taking out organic phase, mixing uniformly by vortex, extracting in ice bath, centrifuging at low temperature, taking out organic phase, and mixing with organic phase taken out after biphase extraction.

In one embodiment of the present invention, the make-up biphasic extraction solvent in step 2) may be either substantially identical in composition or may differ from the biphasic extraction solvent. For example, when the biphasic extraction solvent is composed of C_1-4Fatty alcohol, aqueous acid solution and C_1-4HalogenatedThe supplemental biphasic extraction solvent may consist of C when composed of hydrocarbons_1-4Fatty alcohols and C_1-4A halogenated hydrocarbon.

In a preferred embodiment of the present invention, in step 2), when the biphasic extraction solvent consists of methanol, hydrochloric acid and chloroform, the supplemental biphasic extraction solvent may consist of methanol and chloroform.

In a more preferred embodiment of the present invention, in step 2), when the biphasic extraction solvent consists of methanol, hydrochloric acid and chloroform in a volume ratio of 2:1:4, the supplemental biphasic extraction solvent may consist of methanol and chloroform in a volume ratio of 1: 2.

In one embodiment of the present invention, the volume of the supplemented biphasic extraction solvent in step 2) also needs to be determined based on the weight of the microsample, and the ratio of the microsample to the supplemented biphasic extraction solvent can be from 1mg:5 μ L to 1mg:15 μ L.

In a preferred embodiment of the present invention, the ratio of the amount of the micro-sample to the amount of the complementary biphasic extraction solvent in step 2) may be in the range of 1mg:8 μ L to 1mg:12 μ L.

In a more preferred embodiment of the present invention, the ratio of the amount of microsample to make-up biphasic extraction solvent in step 2) may be 1mg:10 μ L.

In one embodiment of the invention, the time for the supplementary biphasic extraction in step 2) may be 10 min.

In one embodiment of the present invention, in step 2), either the organic phase obtained by only two-phase extraction or the combined organic phase obtained by two-phase extraction and complementary two-phase extraction, is subjected to vacuum concentration and drying, and then to reconstitution with a suitable reconstitution solvent, so as to obtain the lipidomic sample. For example, the reconstitution solvent may be C_1-4One or more of fatty alcohols.

In a preferred embodiment of the present invention, the redissolving solvent in step 2) may be methanol and/or isopropanol.

In a more preferred embodiment of the present invention, the redissolving solvent in step 2) may be a methanol/isopropanol mixed solvent in a volume ratio of 1: 4.

In the present invention, the volume of the redissolving solvent in step 2) can be adjusted according to specific needs. For example, in one embodiment of the present invention, the ratio of the amount of the micro sample to the amount of the reconstitution solvent in step 2) may be 1mg:1 μ L to 1mg:5 μ L.

In a preferred embodiment of the present invention, the ratio of the amount of the micro-sample to the amount of the reconstitution solvent in step 2) may be 1mg:1.5 μ L to 1mg:3 μ L.

In a more preferred embodiment of the present invention, the ratio of the amount of the micro sample to the amount of the reconstitution solvent in step 2) may be 1mg: 2. mu.L.

In one embodiment of the invention, the low temperature centrifugation may be performed at a temperature of 4 ℃, a rotation speed of 12000rpm, and a time of 10 min.

[ Metabolic and lipidomic detection methods ]

The invention provides a metabonomics and lipidomics detection method of a micro sample. The method may comprise the steps of:

2) adding a metabonomics internal standard mixed solution and C into a micro sample_1-4Oscillating fatty alcohol or water solution thereof, performing single-phase extraction at room temperature, centrifuging at low temperature, and taking out partial supernatant to obtain a metabonomics sample for later use; adding lipidomics internal standard mixed solution, acid water solution and C into the residual supernatant and residues_1-4Halogenated hydrocarbons and C_1-4Oscillating fatty alcohol, performing two-phase extraction in ice bath, centrifuging at low temperature, collecting organic phase, vacuum concentrating, drying, and adding into concentrateRedissolving the solvent, uniformly mixing by vortex, centrifuging at low temperature, and taking out supernatant fluid to obtain a lipidomics sample for later use;

In the detection methods of metabonomics and lipidomics, the introduction of an internal standard is crucial, and the internal standard can be used for correcting the retention time of chromatographic peaks corresponding to each component in a sample to be detected, and can also be used for normalizing peak areas and calculating the relative content of each component.

In the present invention, the internal standard is selected mainly according to the following conditions:

1. when a plurality of internal standards are used, chromatographic peaks corresponding to each internal standard component need to be distributed in a chromatogram more uniformly, in other words, retention time corresponding to each internal standard component needs to be distributed in elution time more uniformly;

2. the detection of the object to be detected cannot be interfered by the corresponding internal standard, in other words, the chromatographic separation degree of the object to be detected and the corresponding internal standard needs to meet the detection requirement;

3. the types of the internal standards are diversified as much as possible so as to be suitable for the correction requirements (including positive and negative ion modes) of different objects to be detected;

4. the internal standard needs to withstand the sample pre-treatment method, especially the extraction process, and needs to maintain stable properties after the sample is extracted.

In one embodiment of the present invention, the plurality of metabonomic internal standards in step 1) may include polar small molecule compounds such as carboxylic acids, steroids, amino acids, alkaloids (especially quaternary ammonium alkaloid) and/or isotopic labels thereof, for example, succinic acid-2,2,3,3-d 4(succinic acid-2,2,3,3-d4), cholic acid-2,2,3,4,4-d 5(cholic acid-2,2,3,4,4-d5), L-phenylalanine-d 5(L-phenylalanine-d5), DL-methionine-3,3,4,4-d 4(DL-methionine-3,3,4,4-d4), DL-tryptophan-2,3,3-d3 (DL-typhan-2, 3,3-d3) and choline chloride-trimethyl-d 9(choline chloride-trimetyl-d 9).

In one embodiment of the invention, the concentration of the single metabonomic internal standard mother liquor in step 1) needs to be maintained, for example, the concentration can be 1-15 mg/mL, preferably 1-5 mg/mL, and more preferably 1-2 mg/mL based on the metabonomic internal standard.

In one embodiment of the present invention, the metabonomics internal standard mixed solution in step 1) also needs to be maintained at a certain concentration, for example, the concentration can be 5-50 μ g/mL, preferably 5-30 μ g/mL, and more preferably 5-15 μ g/mL based on various metabonomics internal standards.

In one embodiment of the invention, the various internal lipidomic standards in step 1) may comprise phospholipid derivatives of polar small-molecule compounds such as alkaloids (in particular quaternary ammonium alkaloid type alkaloids), ethanolamines, polyols, amino acids and/or isotopic labels thereof, for example, phosphatidylcholine 16:0-d31-18:1(PC 16:0-d31-18:1), phosphatidylethanolamine 14:0-14:0(PE 14:0-14:0), phosphatidylglycerol 16:0-d31-18:1(PG 16:0-d31-18:1), phosphatidylserine 14:0-14:0(PS 14:0-14:0) and phosphatidic acid 17:0-17:0(PA 17:0-17: 0).

In one embodiment of the invention, the concentration of the single lipidomic internal standard mother liquor in step 1) needs to be maintained, for example, the concentration can be 1-5 mg/mL, preferably 1-3 mg/mL, and more preferably 1-2 mg/mL based on the lipidomic internal standard.

In one embodiment of the invention, the mixed solution of lipidomic internal standards in step 1) needs to be maintained at a certain concentration, for example, the concentration can be 10-50 μ g/mL, preferably 10-30 μ g/mL, and more preferably 20-30 μ g/mL based on various lipidomic internal standards.

In one embodiment of the present invention, the trace amount in step 2) may be 1/2 sample size lower than the detection requirement of conventional method sample size.

In one embodiment of the present invention, the micro sample in step 2) may be a biological sample used for omics research and the sample size is of micro-scale, such as brain tissue.

In one embodiment of the invention, the solution and C can be mixed in step 2) from a metabolomic internal standard_1-4Fatty alcohol or its aqueous solution composition single-phase extractionThe solvent is preferably used in a volume ratio of 2: 1.

In one embodiment of the present invention, the ratio of the amount of the micro-sample to the monophasic extraction solvent in step 2) may be 1mg: 4. mu.L to 1mg: 10. mu.L, preferably 1mg: 5. mu.L to 1mg: 8. mu.L, and more preferably 1mg: 6. mu.L.

In one embodiment of the present invention, the volume ratio of the supernatant to the single-phase extraction solvent in step 2) may be 1:1.5 to 1:10, preferably 1:2 to 1:5, more preferably 1: 3.

In one embodiment of the invention, the mixed solution, the aqueous acid solution, C, can be defined in step 2) from an internal lipidomics standard_1-4Halogenated hydrocarbons and C_1-4The fatty alcohol constitutes the biphasic extraction solvent, preferably the volume ratio of the four may be 2:1:10: 3.

In one embodiment of the present invention, the ratio of the amount of the microsample to the biphasic extraction solvent in step 2) may be 1mg: 10. mu.L to 1mg: 20. mu.L, preferably 1mg: 15. mu.L to 1mg: 18. mu.L, more preferably 1mg: 16. mu.L.

In one embodiment of the invention, the redissolving solvent in step 2) may be C_1-4A fatty alcohol.

In one embodiment of the present invention, the ratio of the amount of the micro-sample to the amount of the reconstitution solvent in step 2) may be 1mg:1. mu.L to 1mg: 5. mu.L, preferably 1mg: 1.5. mu.L to 1mg: 3. mu.L, and more preferably 1mg: 2. mu.L.

In a preferred embodiment of the invention, step 2) may also comprise a supplementary biphasic extraction step. The supplemental biphasic extraction step may comprise: adding supplementary biphase extraction solvent into residue after taking out organic phase, mixing uniformly by vortex, extracting in ice bath, centrifuging at low temperature, taking out organic phase, and mixing with organic phase taken out after biphase extraction.

In one embodiment of the invention, step 2) may be performed by C_1-4Fatty alcohols and C_1-4The halogenated hydrocarbon composition supplements the biphasic extraction solvent, preferably in a volume ratio of 1: 2.

In one embodiment of the present invention, the ratio of the amount of the micro-sample to the amount of the complementary biphasic extraction solvent in step 2) may be 1mg: 5. mu.L to 1mg: 15. mu.L, preferably 1mg: 8. mu.L to 1mg: 12. mu.L, more preferably 1mg: 10. mu.L.

In one embodiment of the invention, C_1-4The aliphatic alcohol may be methanol and/or isopropanol, preferably methanol; the acid aqueous solution can be formic acid aqueous solution, acetic acid aqueous solution or hydrochloric acid, preferably 0.1-1M hydrochloric acid, more preferably 0.5-1M hydrochloric acid, and most preferably 1M hydrochloric acid; c_1-4The halogenated hydrocarbon may be chloroform and/or dichloromethane, preferably chloroform.

The invention will be further illustrated by the figures and specific examples. Unless otherwise indicated, materials, reagents, instruments, software and the like used in the following examples are all available by conventional commercial means.

The first embodiment is as follows: result comparison of pretreatment method of rat cerebral cortex sample through single extraction of biphasic solvent and pretreatment method of the invention

1. Preparing a metabonomics internal standard mixed solution and a lipidomics internal standard mixed solution:

1.1 preparation of metabonomics internal standard mixed solution:

taking appropriate 6 metabonomics internal standards (all isotope marker internal standards) such as succinic acid-2,2,3,3-d4, cholic acid-2,2,3,4,4-d5, L-phenylalanine-d5, DL-methionine-3,3,4,4-d4, DL-tryptophan-2,3,3-d3, choline chloride-trimethyl-d9 and the like, respectively adding water or methanol to prepare 6 metabonomics internal standard mother solutions (wherein, the DL-methionine-3,3,4,4-d4 and DL-tryptophan-2,3,3-d3 are dissolved by water, the rest is dissolved by methanol, the concentration of the 6 internal standards is about 1mg/mL), respectively taking appropriate 6 single metabonomics mother solutions, mixing and correspondingly adding appropriate water (or methanol), mixed solutions of metabonomic internal standards were prepared (6 internal standards were used at concentrations of about 15. mu.g/mL cholic acid-2,2,3,4,4-d5, about 10. mu.g/mL succinic acid-2,2,3,3-d4 and DL-methionine-3,3,4,4-d4, and about 5. mu.g/mL L-phenylalanine-d5, DL-tryptophan-2,3,3-d3 and choline chloride-trimethyl-d 9).

1.2 preparation of lipidomics internal standard mixed solution:

taking appropriate amount of 5 lipidomic internal standards (comprising 3 internal standards of prototype compounds and 2 internal standards of isotope markers) such as phosphatidylcholine 16:0-d31-18:1, phosphatidylethanolamine 14:0-14:0, phosphatidylglycerol 16:0-d31-18:1, phosphatidylserine 14:0-14:0 and phosphatidic acid 17:0-17:0, and respectively adding methanol (or isopropanol) to prepare 5 single lipidomic internal standard mother liquor (wherein the concentration of the 5 internal standards is that phosphatidylcholine 16:0-d31-18:1 and phosphatidylethanolamine 14:0-14:0 is about 2mg/mL, phosphatidylglycerol 16:0-d31-18:1, phosphatidylserine 14:0-14:0 and phosphatidic acid 17:0-17:0 is about 1mg/mL), then respectively taking a proper amount of 5 single lipidomic internal standard mother solutions, mixing and correspondingly adding a proper amount of methanol (or isopropanol) to prepare lipidomic internal standard mixed solution (wherein the concentration of the 5 internal standards is as follows: phosphatidylglycerol 16:0-d31-18:1, phosphatidylserine 14:0-14:0 and phosphatidic acid 17:0-17:0 is about 30 mu g/mL, phosphatidylcholine 16:0-d31-18:1 and phosphatidylethanolamine 14:0-14:0 is about 20 mu g/mL).

2. Extracting trace biological samples by different pretreatment methods:

2.1 pretreatment method for single extraction of biphasic solvent (sample No. 6):

weighing about 50mg of rat cerebral cortex, placing the rat cerebral cortex into a clean centrifuge tube (2mL), adding 200 mu L of metabonomics internal standard mixed solution prepared under item 1.1 and 100 mu L of lipidomics internal standard mixed solution prepared under item 1.2, adding 200 mu L of chloroform and 450 mu L of chloroform/methanol mixed solvent (2:1, v/v), placing 2 steel balls, oscillating at the frequency of 40Hz for 30s, and oscillating for 2 times; after the sample is crushed, oscillating for 5min again, and placing on ice to continue extracting for 40 min; after extraction, centrifuging in a centrifuge at 4 ℃ for 10min at 12000 rpm; and taking out 400 mu L of all the supernatant, concentrating and drying in vacuum, adding 100 mu L of methanol into the residue for redissolving, uniformly mixing in a vortex manner, centrifuging in a centrifuge at 4 ℃ for 10min at 12000rpm, and taking out the supernatant for metabonomics research.

And (3) placing 500 mu L of the lower-layer organic phase left after all the supernatant is taken out in another clean centrifuge tube (2mL), adding 50 mu L of 1M hydrochloric acid and 450 mu L of chloroform/methanol mixed solvent (2:1, v/v) into the residue, performing vortex mixing, placing on ice for further extraction for 10min, taking out all the lower-layer organic phase, combining with the previously taken out lower-layer organic phase, performing vacuum concentration and drying, adding 100 mu L of isopropanol/methanol mixed solvent (4:1, v/v) into the residue for redissolution, performing vortex mixing, centrifuging in a centrifuge at 4 ℃ for 10min at 12000rpm, and taking out the supernatant for lipidomics research.

The flow of the method is similar to the method B in FIG. 1.

2.2 pretreatment method of the invention (sample No. 3):

weighing about 50mg of rat cerebral cortex, placing in a clean centrifuge tube (2mL), adding 200 μ L of metabonomics internal standard mixed solution prepared under item 1.1 and 100 μ L of methanol, placing 2 steel balls, oscillating at 40Hz frequency for 30s, and oscillating for 2 times; after the sample is crushed, keeping the room temperature environment for extraction and oscillation for 30 min; after extraction, centrifuging in a centrifuge at 4 ℃ for 10min at 12000 rpm; a portion of the supernatant (approximately 100 μ L) was removed for metabonomic studies.

Adding 100 μ L of lipidomics internal standard mixed solution prepared under item 1.2, 50 μ L of 1M hydrochloric acid and 200 μ L of chloroform into the residual supernatant and residue, adding 450 μ L of chloroform/methanol mixed solvent (2:1, v/v), oscillating at 40Hz frequency for 10s, and oscillating for 2 times; placing on ice, and extracting for 40 min; after extraction, centrifuging in a centrifuge at 4 ℃ for 10min at 12000 rpm; and taking 500 mu L of the lower organic phase, placing the lower organic phase in another clean centrifugal tube (2mL), adding 500 mu L of chloroform/methanol mixed solvent (2:1, v/v) into the residue, performing vortex mixing, placing the mixture on ice for continuous extraction for 10min, taking out all the lower organic phase, combining the lower organic phase with the lower organic phase taken out previously, performing vacuum concentration and drying, adding 100 mu L of isopropanol/methanol mixed solvent (4:1, v/v) into the residue for redissolution, performing vortex mixing, centrifuging the mixture in a 4 ℃ centrifuge for 10min at 12000rpm, and taking out the supernatant for lipidomic research.

The flow of the method is similar to the method D in fig. 1.

3. Selecting an instrument and analyzing conditions:

3.1 selecting the instrument:

the Thermo Scientific Vanquish liquid chromatograph is connected with the Thermo Scientific Q-active mass spectrometer in series, and the metabonomics and the lipidomics adopt the same set of equipment.

3.2 selecting liquid chromatography conditions:

3.2.1 liquid chromatography conditions for metabolomics:

a chromatographic column: waters ACQUITY UPLC HST 3 column (150 mm. times.2.1 mm,1.8 μm);

mobile phase: in a positive ion mode, 0.1% formic acid aqueous solution (1 mL formic acid in every 1L aqueous solution) is used as a mobile phase A, and 0.1% formic acid acetonitrile solution (1 mL formic acid in every 1L acetonitrile solution) is used as a mobile phase B; in the negative ion mode, 5mM ammonium formate aqueous solution (5 mmol ammonium formate per 1L aqueous solution) is used as a mobile phase A, and acetonitrile is used as a mobile phase B;

and (3) an elution mode: gradient elution was used, the specific procedure was as follows: 0-1 min, 2% of mobile phase A; 1-9 min, 2% -50% of mobile phase A; 9-12 min, 50% -98% of mobile phase A; 12-13.5 min, 98% of mobile phase A; 13.5-14 min, 98-2% of mobile phase A; 14-20 min, 2% of mobile phase A;

flow rate: 0.25 mL/min;

sample introduction amount: 2 mu L of the solution;

column temperature: at 40 ℃.

3.2.2 liquid chromatography conditions of lipidomics:

a chromatographic column: waters ACQUITY UPLC BEH C18 column (100 mm. times.2.1 mm,1.7 μm);

mobile phase: in a positive ion mode, an acetonitrile/water mixed solvent (64:40, v: v) as a mobile phase A containing 0.1% of formic acid and 10mM of ammonium formate (i.e., 1mL of formic acid and 10mmol of ammonium formate per 1L of the acetonitrile/water mixed solvent) and an isopropanol/acetonitrile mixed solvent (90:10, v: v) as a mobile phase B containing 0.1% of formic acid and 10mM of ammonium formate (i.e., 1mL of formic acid and 10mM of ammonium formate per 1L of the isopropanol/acetonitrile mixed solvent) are used;

and (3) an elution mode: gradient elution was used, the specific procedure was as follows: 0-2 min, 85% -70% of mobile phase A; for 2-2.5 min, 70-52% of mobile phase A; 2.5-11 min, 52% -18% of mobile phase A; 11-11.5 min, 18% -1% of mobile phase A; 11.5-12 min, 1% of mobile phase A; 12-12.1 min, 1% -85%; 12.1-15 min, 85% of mobile phase A;

flow rate: 0.35 mL/min;

sample introduction amount: 2 mu L of the solution;

column temperature: at 50 ℃.

3.3 Mass Spectrometry conditions were selected:

3.3.1 mass spectrometric conditions of metabolomics:

the data acquisition mode is as follows: performing measurement by adopting a primary mass spectrum full-scan and data-dependent secondary mass spectrum scan (full MS-ddMS2) mode, and simultaneously obtaining primary and secondary mass spectrum data;

an ion source: electrospray ion source (ESI);

spraying voltage: 3.5kV in the positive ion mode and 2.5kV in the negative ion mode;

sheath gas pressure: 30 arb;

auxiliary gas pressure: 10 arb;

capillary temperature: 325 ℃;

the full-scanning resolution is 70000, the secondary resolution is 17500, and the mass spectrum scanning range is 81-1000 m/z;

and (3) performing secondary cracking by adopting a high-energy induced cracking technology (HCD), wherein the collision voltage is 30eV, and unnecessary secondary mass spectrum information is dynamically excluded.

3.3.2 mass spectrometric conditions of lipidomics:

an ion source: electrospray ion source (ESI);

sheath gas pressure: 30 arb;

auxiliary gas pressure: 10 arb;

capillary temperature: 325 ℃;

the full scanning resolution is 35000, the secondary resolution is 17500, and the mass spectrum scanning range is 150-2000 m/z;

4. Metabolomics and lipidomics data processing:

4.1 Metabonomics data processing:

transposition of original data: and respectively obtaining 4 raw data files of the No. 6 sample and the No. 3 sample in a positive ion mode and a negative ion mode, and performing data transposition by ProteWizard software (open source software, which can be obtained by http:// proteowzerd. sourceform. net) to form an mzXML file respectively comprising primary mass spectrum transposed data and secondary mass spectrum transposed data in the positive ion mode and the negative ion mode.

And (3) deconvoluting transposed data: the method comprises the following steps that (1) deconvolution is carried out on an mzXML file of primary mass spectrum transposition data in a positive ion mode and a negative ion mode by means of a biowork cloud platform (corresponding open source software and scripts can also be downloaded through http:// www.bioconductor.org), 7297 primary variables of a No. 6 sample are obtained in the positive ion mode, and 5505 primary variables are obtained in the negative ion mode; the No. 3 sample obtains 9602 primary variables in a positive ion mode, obtains 7721 primary variables in a negative ion mode, and correspondingly outputs peak _ pos.xlsx and peak _ neg.xlsx files serving as primary deconvolution results, wherein the two files respectively comprise primary variable information such as xc _ ms id (primary variable number), mz (parent ion mass-to-charge ratio), rt (retention time), intensity (peak area) and the like.

Secondary mass spectrometric data analysis-metabolite identification: combining the deconvolved primary variable with the second-level mass spectrum transposed data to identify the metabolites, wherein 481 metabolites are obtained from the No. 6 sample in a positive ion mode, and 184 metabolites are obtained in a negative ion mode; whereas sample No. 3 obtained 470 metabolites in positive ion mode and 181 metabolites in negative ion mode. As shown in table 1, the amounts of the substances from which metabolomics were obtained by the two methods differed little.

TABLE 1.6 and 3 metabolite counts from non-targeted metabolomic testing of samples

4.2 lipidomics data processing:

sample No. 6 and sample No. 3 are subjected to positive and negative ion modes to obtain lipidomics raw data files 4 x raw, and the base peak ion chromatograms of the two samples are shown in FIG. 2. Intuitively, in a positive ion mode, the chromatographic peaks of the sample No. 3 and the sample No. 6 in a 5-7.7 min time period are compared to know that the number of the obtained peak chromatograms of the sample No. 3 is more than that of the sample No. 6; in the negative ion mode, when the chromatographic peak at the time point of 3.61min is compared, the chromatographic response in the sample No. 3 is higher than that in the sample No. 6.

The primary annotation was annotated by Thermo lipidsearch 4.2 commercial software, where the parameters selected in the primary annotation were mainly partenserch _ QEX mode, ExpType (experimental type) was LC-MS mode, partent tolerance (Parent ion bias) was 3ppm, and large Range was 0.1min, the primary annotation was lipid based on fatty acyl and other lipids, and the additive plus ion mode selected + H, + NH₄, + Na, addition anion mode selection-H, + HCOO^-，+CH₃COO^-. The parameters selected in the secondary annotation mainly include Product search _ QEX, both of which are 5ppm, m-Score threshold of 2 (coenzyme Q is generally selected to be more than 2), m-Score of 3 (representing lipid result with m-Score value of more than 3 only), and ID Quality filter of A, B, C and D (accuracy discrimination, accuracy is A from high to low>B>C>D) Lipid annotation results the results of the first (Parent) and second (Product) annotations are A and B, respectively, as shown in FIG. 5 for pos/neg-3 and pos/neg-6 for sample No. 3 and sample No. 6, respectively.

5. The results of the two pretreatment methods were compared:

as shown in fig. 5, in the Parent mode, the number of lipids in the positive ion (pos) and negative ion (neg) modes of sample No. 3 is 1018 and 888, respectively, and the number of lipids in sample No. 6 is 983 and 839, respectively; in Product mode, the number of lipids in positive ion (pos) and negative ion (neg) modes for sample No. 3 was 622 and 536, respectively, and 614 and 522 for sample No. 6. The above results show that the amount of lipid identified in sample No. 3 is significantly better than that of sample No. 6 (especially in Parent mode), which indicates that the pretreatment method of sample No. 3 (i.e. the pretreatment method of the present invention) can significantly improve the extraction efficiency of lipid components in a trace amount of sample.

Example two: result comparison of pretreatment method for taking out all metabolite extracting solutions from mouse brain tissue samples and pretreatment method of the invention

the same as the first embodiment.

2. Extracting trace biological samples by different pretreatment methods:

2.1 pretreatment method of the present invention (sample No. A1):

weighing about 50mg of mouse brain tissue, placing the mouse brain tissue into a clean centrifuge tube (2mL), adding 200 mu L of metabonomics internal standard mixed solution prepared under item 1.1 and 100 mu L of methanol, placing 2 steel balls, oscillating for 30s at the frequency of 40Hz, and oscillating for 2 times; after the sample is crushed, keeping the room temperature environment for extraction and oscillation for 30 min; after extraction, centrifuging in a centrifuge at 4 ℃ for 10min at 12000 rpm; a portion of the supernatant (approximately 150 μ L) was removed for metabonomic studies.

Adding 100 μ L of lipidomics internal standard mixed solution prepared under item 1.2, 50 μ L of 1M hydrochloric acid, 150 μ L of water and 200 μ L of chloroform into the residual supernatant and residue, adding 450 μ L of chloroform/methanol mixed solvent (2:1, v/v), oscillating for 10s at 40Hz frequency, and oscillating for 2 times; placing on ice, and extracting for 40 min; after extraction, centrifuging in a centrifuge at 4 ℃ for 10min at 12000 rpm; taking 550 mu L of the lower organic phase, placing the lower organic phase in another clean centrifugal tube (2mL), adding 500 mu L of chloroform/methanol mixed solvent (2:1, v/v) into the residue, performing vortex mixing, placing the mixture on ice for continuous extraction for 10min, taking out all the lower organic phase, combining the lower organic phase with the lower organic phase taken out previously, performing vacuum concentration and drying, adding 100 mu L of isopropanol/methanol mixed solvent (4:1, v/v) into the residue for redissolution, performing vortex mixing, centrifuging the mixture in a 4 ℃ centrifuge for 10min at 12000rpm, and taking out the supernatant for lipidomics research.

The flow of the method is similar to the method D in fig. 1.

2.2 pretreatment method by removing all supernatants (sample No. A2):

weighing about 50mg of mouse brain tissue, placing the mouse brain tissue into a clean centrifuge tube (2mL), adding 200 mu L of metabonomics internal standard mixed solution prepared under item 1.1 and 100 mu L of methanol, placing 2 steel balls, oscillating for 30s at the frequency of 40Hz, and oscillating for 2 times; after the sample is crushed, keeping the room temperature environment for extraction and oscillation for 30 min; after extraction, centrifuging in a centrifuge at 4 ℃ for 10min at 12000 rpm; all supernatants were removed for metabonomics studies.

To the residue were added 100. mu.L of the lipidomics internal standard mixed solution prepared under item 1.2, 50. mu.L of 1M hydrochloric acid, 150. mu.L of water and 200. mu.L of chloroform, and 450. mu.L of a chloroform/methanol mixed solvent (2:1, v/v) was added, followed by shaking at a frequency of 40Hz for 10 seconds and 2 times; placing on ice, and extracting for 40 min; after extraction, centrifuging in a centrifuge at 4 ℃ for 10min at 12000 rpm; taking 550 mu L of the lower organic phase, placing the lower organic phase in another clean centrifugal tube (2mL), adding 500 mu L of chloroform/methanol mixed solvent (2:1, v/v) into the residue, performing vortex mixing, placing the mixture on ice for continuous extraction for 10min, taking out all the lower organic phase, combining the lower organic phase with the lower organic phase taken out previously, performing vacuum concentration and drying, adding 100 mu L of isopropanol/methanol mixed solvent (4:1, v/v) into the residue for redissolution, performing vortex mixing, centrifuging the mixture in a 4 ℃ centrifuge for 10min at 12000rpm, and taking out the supernatant for lipidomics research.

The flow of the process is similar to the process C in fig. 1.

3. Selecting an instrument and analyzing conditions:

the same as the first embodiment.

4. Metabolomics and lipidomics data processing:

4.1 Metabonomics data processing:

deconvolution results after metabonomics raw data conversion: the method comprises the following steps that (1) deconvolution is carried out on an mzXML file of primary mass spectrum transposed data in a positive ion mode and a negative ion mode by means of a biowork cloud platform (corresponding open source software and scripts can also be downloaded through http:// www.bioconductor.org), 10648 primary variables are obtained from an A1 sample in the positive ion mode, and 13919 primary variables are obtained in the negative ion mode; a2 sample obtains 10673 primary variables in a positive ion mode, obtains 14002 primary variables in a negative ion mode, and correspondingly outputs peak _ pos.xlsx and peak _ neg.xlsx files serving as primary deconvolution results, wherein the two files respectively comprise primary variable information such as xc _ ms id (primary variable number), mz (parent ion mass-to-charge ratio), rt (retention time), intensity (peak area) and the like.

Secondary mass spectrometric data analysis-metabolite identification: combining the deconvoluted primary variable with the transposed data of the secondary mass spectrum to identify the metabolites, wherein 478 metabolites are obtained from the sample A1 in a positive ion mode, and 187 metabolites are obtained in a negative ion mode; while sample number a2 obtained 504 metabolites in positive ion mode and 166 metabolites in negative ion mode. As shown in table 2, the amounts of the substances from which metabolomics were obtained by the two methods differed little.

TABLE 2 metabolite numbers obtained from sample A1 and sample A2 by non-targeted metabonomics testing

4.2 lipidomics data processing:

referring to the first example, the base peak ion chromatogram is shown in fig. 3, since the high signal response lipid covers some information of the low signal response lipid, the spectrum of the positive and negative ion modes of the sample a1 and the sample a2 in the base peak ion chromatogram is relatively similar.

5. The results of the two pretreatment methods were compared:

as shown in fig. 5, in the Parent mode, the lipid numbers of the sample a1 in the positive ion (pos) and negative ion (neg) modes are 1713 and 1022 respectively, and the sample a2 corresponds to 1683 and 1002; in the Product mode, the lipid numbers of the sample a1 in the positive ion (pos) and negative ion (neg) modes were 843 and 627, and the sample a2 was 811 and 596, respectively. The above results show that the amount of lipid identified in sample a2 is significantly better than that of sample a1, which indicates that the pretreatment method of sample a1 (i.e. the pretreatment method of the present invention) can significantly improve the extraction efficiency of lipid components in a trace amount of sample, and also reflects that the supernatant after a part of extracted polar metabolites is retained before lipid extraction, which will contribute to high coverage of subsequent lipid extraction.

Example three: results of pretreatment method of rat cerebral cortex sample without adding acid in water phase are compared with results of pretreatment method of the invention

the same as the first embodiment.

2. Extracting trace biological samples by different pretreatment methods:

2.1 pretreatment method without adding acid to the aqueous phase (sample No. D-M-1):

weighing about 50mg of rat cerebral cortex, placing in a clean centrifuge tube (2mL), adding 200 μ L of metabonomics internal standard mixed solution prepared under item 1.1 and 100 μ L of methanol, placing 2 steel balls, oscillating at 40Hz frequency for 30s, and oscillating for 2 times; after the sample is crushed, keeping the room temperature environment for extraction and oscillation for 30 min; after extraction, centrifuging in a centrifuge at 4 ℃ for 10min at 12000 rpm; a portion of the supernatant (approximately 150 μ L) was removed for metabonomic studies.

Adding 100 μ L of lipidomics internal standard mixed solution prepared under item 1.2, 200 μ L of water and 200 μ L of chloroform into the residual supernatant and residue, adding 450 μ L of chloroform/methanol mixed solvent (2:1, v/v), oscillating for 10s at 40Hz, and oscillating for 2 times; placing on ice, and extracting for 40 min; after extraction, centrifuging in a centrifuge at 4 ℃ for 10min at 12000 rpm; taking 550 mu L of the lower organic phase, placing the lower organic phase in another clean centrifugal tube (2mL), adding 500 mu L of chloroform/methanol mixed solvent (2:1, v/v) into the residue, performing vortex mixing, placing the mixture on ice for continuous extraction for 10min, taking out all the lower organic phase, combining the lower organic phase with the lower organic phase taken out previously, performing vacuum concentration and drying, adding 100 mu L of isopropanol/methanol mixed solvent (4:1, v/v) into the residue for redissolution, performing vortex mixing, centrifuging the mixture in a 4 ℃ centrifuge for 10min at 12000rpm, and taking out the supernatant for lipidomics research.

The flow of the process is similar to the process D in fig. 1, only water is used instead of acid water.

2.2 pretreatment method of the invention (sample No. D-M-2):

The flow of the method is similar to the method D in fig. 1.

3. Selecting an instrument and analyzing conditions:

the same as the first embodiment.

4. Metabolomics and lipidomics data processing:

4.1 Metabonomics data processing:

deconvolution results after metabonomics raw data conversion: the method comprises the following steps that (1) deconvolution is carried out on an mzXML file of primary mass spectrum transposition data in a positive ion mode and a negative ion mode by means of a biowork cloud platform (corresponding open source software and scripts can also be downloaded through http:// www.bioconductor.org), a sample D-M-1 obtains 10011 primary variables in the positive ion mode, and 11700 primary variables in the negative ion mode; the D-M-2 sample obtains 9960 primary variables in a positive ion mode, obtains 11970 primary variables in a negative ion mode, correspondingly outputs peak _ pos.xlsx and peak _ neg.xlsx files serving as primary deconvolution results, and the two files respectively comprise primary variable information such as xc _ ms id (primary variable number), mz (parent ion mass-to-charge ratio), rt (retention time), intensity (peak area) and the like.

Secondary mass spectrometric data analysis-metabolite identification: combining the deconvolved primary variable with the transposed data of the secondary mass spectrum to identify the metabolites, wherein 419 metabolites are obtained from the sample D-M-1 in a positive ion mode, and 171 metabolites are obtained in a negative ion mode; while sample D-M-2 obtained 451 metabolites in positive ion mode and 171 metabolites in negative ion mode. As shown in Table 3, the amount of the identified metabolite in the sample D-M-2 is slightly larger than that in the sample D-M-1, but theoretically the amounts should be slightly different.

TABLE 3 metabolite counts from non-targeted metabonomics testing of samples D-M-1 and D-M-2

4.2 lipidomics data processing:

referring to example one, the basic peak ion chromatogram is shown in fig. 4, in positive ion mode, the number of substance peaks (B) obtained by acid extraction is greater than the number of substance peaks (a) obtained by non-acid extraction within a time period of 1min to 5 min; in the negative ion mode, the number (D) of substance peaks obtained by acid extraction is more than the number (C) of substance peaks obtained by non-acid extraction in a time period of 2min-5min, and the signal response of the whole ion flow diagram in D is 1.73 multiplied by 10⁹Whereas the signal response in C is only 1.05X 10⁹The ionic strength obtained in the acid-added system is also superior to that obtained in the extraction without acid.

5. The results of the two pretreatment methods were compared:

as shown in FIG. 5, in the Parent mode, the lipid numbers of the D-M-1 sample in the positive ion (pos) and negative ion (neg) modes are 1098 and 782, respectively, and the D-M-2 sample corresponds to 1283 and 942; in the Product mode, the number of lipids in the positive ion (pos) and negative ion (neg) modes of the sample D-M-1 was 778 and 450, respectively, and the number of lipids in the sample D-M-2 was 852 and 576. The results show that the quantity of the lipid identified in the sample D-M-2 is obviously superior to that of the sample D-M-1, and the pretreatment method of the sample D-M-2 (namely the pretreatment method of the invention) can obviously improve the extraction efficiency of lipid components in a trace sample, and also reflects that the introduction of acid in the lipid extraction process can better extract certain lipid.

Example four: results of pretreatment methods of mouse brain tissue samples with different acid types and different hydrochloric acid concentrations are compared

the same as the first embodiment.

2. Extracting trace biological samples by different pretreatment methods:

2.1 pretreatment method of the present invention (sample No. T1):

2.2 pretreatment method using different types of acid extraction in lipid extraction (sample nos. T2 and T3):

To the residual supernatant and the residue were added 100. mu.L of the lipidomics internal standard mixed solution prepared under item 1.2, 50. mu.L of 3% formic acid (mass concentration, m/v, the same shall apply hereinafter), 150. mu.L of water and 200. mu.L of chloroform, followed by addition of 450. mu.L of chloroform/methanol mixed solvent (2:1, v/v) and shaking at 40Hz for 10s for 2 times; placing on ice, and extracting for 40 min; after extraction, centrifuging in a centrifuge at 4 ℃ for 10min at 12000 rpm; taking 550 mu L of the lower organic phase, placing the lower organic phase in another clean centrifugal tube (2mL), adding 500 mu L of chloroform/methanol mixed solvent (2:1, v/v) into the residue, performing vortex mixing, placing the mixture on ice for continuous extraction for 10min, taking out all the lower organic phase, combining the lower organic phase with the lower organic phase taken out previously, performing vacuum concentration and drying, adding 100 mu L of isopropanol/methanol mixed solvent (4:1, v/v) into the residue for redissolution, performing vortex mixing, centrifuging the mixture in a 4 ℃ centrifuge for 10min at 12000rpm, and taking out supernatant for lipidomic research, wherein the sample is marked as T2.

Another pretreatment was carried out in the same manner as in item 2.2, except that the acid added was acetic acid at a mass concentration of 3% (M/v), and the acid was designated as sample T3 (note: when the molar concentration of hydrochloric acid was 1M (mol/L), the corresponding concentration of hydrochloric acid was about 3% by mass, M/v).

2.3 pretreatment methods for different hydrochloric acid concentrations (sample Nos. T4 and T5)

The pretreatment was carried out in the same manner as in item 2.1, and hydrochloric acid was added at concentrations of 0.1M and 0.5M, respectively, as indicated by samples T4 and T5.

3. Selecting an instrument and analyzing conditions:

the same as the first embodiment.

4. Metabolomics and lipidomics data processing:

4.1 Metabonomics data processing:

deconvolution results after metabonomics raw data conversion: the mzXML file of the primary mass spectrum transposed data in the positive and negative ion modes is deconvoluted by means of a biodeep cloud platform (http:// www.bioconductor.org/downloading corresponding open source software and scripts can also be used), and the annotation result of each sample is shown in table 4. The metabonomics experiment operation method is the same, so that the parallelism of the metabolite identification result is better.

TABLE 4 metabolite numbers obtained from T1-T5 samples by non-targeted metabonomics detection

4.2 lipid data processing results:

as shown in fig. 6, in the Parent mode, the numbers of lipids of the T1 sample in the positive ion (pos) mode and the negative ion (neg) mode are 1244 and 868, the T2 sample is 1048 and 772, the T3 sample is 1152 and 814, the T4 sample is 921 and 669, and the T5 sample is 1158 and 779, respectively; in the Product mode, the numbers of lipids in the positive ion (pos) and negative ion (neg) modes of the T1 sample are 546 and 600, the T2 sample is 511 and 530, the T3 sample is 597 and 548, the T4 sample is 413 and 420, and the T5 sample is 529 and 554, respectively. The above results show that, from the Product model, the extraction efficiency of hydrochloric acid and acetic acid is almost the same, and the total number of lipids is about 1145; however, according to the Parent model, the extraction efficiency of hydrochloric acid is superior to acetic acid and other treatment methods. Combining the results of the part and Product, it is known that the efficiency of hydrochloric acid for lipid extraction is better than other acids (formic acid or acetic acid), and the optimal hydrochloric acid concentration during extraction is 1M.

Claims

1. A pretreatment method of micro samples for metabonomics and lipidomics research comprises the following steps:

2. The pretreatment method according to claim 1, wherein in step 1),

the single-phase extraction solvent is C_1-4An aliphatic alcohol or an aqueous solution thereof, preferably methanol or an aqueous solution thereof, more preferably methanol;

the dosage ratio of the micro sample to the single-phase extraction solvent is 1mg:4 muL-1 mg:10 muL, preferably 1mg:5 muL-1 mg:8 muL, and more preferably 1mg:6 muL;

the volume ratio of the partial supernatant to the single-phase extraction solvent is 1: 1.5-1: 10, preferably 1: 2-1: 5, and more preferably 1: 3.

3. The pretreatment method according to claim 1 or 2, wherein in step 2),

the biphasic extraction solvent consists of C_1-4Fatty alcohol, aqueous acid solution and C_1-4Halogenated hydrocarbons, preferably consisting of C in a volume ratio of 5:1:10_1-4Fatty alcohol, aqueous acid solution and C_1-4A halogenated hydrocarbon composition; wherein the acid aqueous solution is formic acid aqueous solution, acetic acid aqueous solution or hydrochloric acid, preferably 0.1-1M hydrochloric acid, more preferably 0.5-1M hydrochloric acid, and most preferably 1M hydrochloric acid; said C is_1-4The aliphatic alcohol is preferably methanol; said C is_1-4Halogenated hydrocarbons are preferably chloroform;

the dosage ratio of the micro sample to the two-phase extraction solvent is 1mg:10 muL-1 mg:20 muL, preferably 1mg:15 muL-1 mg:18 muL, and more preferably 1mg:16 muL;

the redissolving solvent is C_1-4One or more of fatty alcohols, preferably methanol and/or isopropanol, more preferablyA methanol/isopropanol mixed solvent with the volume ratio of 1:4 is preferred;

the dosage ratio of the micro sample to the redissolution solvent is 1mg:1 muL-1 mg:5 muL, preferably 1mg:1.5 muL-1 mg:3 muL, and more preferably 1mg:2 muL.

4. The pretreatment method according to any one of claims 1 to 3,

step 2) also comprises a supplementary biphasic extraction between the removal of the organic phase and the vacuum concentration drying, comprising: adding a supplementary two-phase extraction solvent into the residue left after the organic phase is taken out, uniformly mixing by vortex, extracting in an ice bath, centrifuging at low temperature, taking out the organic phase, and combining with the organic phase taken out after the two-phase extraction; wherein

Said additional biphasic extraction solvent consists of C_1-4Fatty alcohols and C_1-4A halogenated hydrocarbon, preferably consisting of methanol and chloroform, more preferably consisting of methanol and chloroform in a volume ratio of 1: 2;

the dosage ratio of the micro sample to the supplementary biphasic extraction solvent is 1mg:5 muL-1 mg:15 muL, preferably 1mg:8 muL-1 mg:12 muL, and more preferably 1mg:10 muL.

5. A method for metabolomics and lipidomics detection of micro-samples, comprising the following steps:

2) adding a metabonomics internal standard mixed solution and C into a micro sample_1-4Shaking fatty alcohol or its water solution, extracting at room temperature with single phase, and separating at low temperatureTaking out part of supernatant from the heart to obtain a metabonomics sample for later use; adding lipidomics internal standard mixed solution, acid water solution and C into the residual supernatant and residues_1-4Halogenated hydrocarbons and C_1-4Oscillating fatty alcohol, performing two-phase extraction in an ice bath, centrifuging at low temperature, taking out an organic phase, concentrating and drying in vacuum, adding a redissolving solvent into a concentrate, uniformly mixing by vortex, centrifuging at low temperature, and taking out a supernatant, namely a lipidomics sample for later use;

6. The detection method according to claim 5, wherein, in step 1),

the plurality of metabonomic internal standards include succinate-2, 2,3,3-d4, cholic acid-2,2,3,4,4-d5, L-phenylalanine-d5, DL-methionine-3,3,4,4-d4, DL-tryptophan-2,3,3-d3 and choline chloride-trimethyl-d 9;

the concentration of the metabonomics internal standard in the single metabonomics internal standard mother liquor is 1-15 mg/mL, preferably 1-5 mg/mL, and more preferably 1-2 mg/mL;

the concentration of each metabonomics internal standard in the metabonomics internal standard mixed solution is 5-50 mug/mL, preferably 5-30 mug/mL, and more preferably 5-15 mug/mL;

the multiple lipidomic internal standards comprise phosphatidylcholine 16:0-d31-18:1, phosphatidylethanolamine 14:0-14:0, phosphatidylglycerol 16:0-d31-18:1, phosphatidylserine 14:0-14:0 and phosphatidic acid 17:0-17: 0;

the concentration of the lipidomic internal standard in the single lipidomic internal standard mother liquor is 1-5 mg/mL, preferably 1-3 mg/mL, and more preferably 1-2 mg/mL;

the concentration of each lipidomic internal standard in the lipidomic internal standard mixed solution is 10-50 mug/mL, preferably 10-30 mug/mL, and more preferably 20-30 mug/mL.

7. The detection method according to claim 5 or 6, wherein, in step 2),

mixing solutions from the metabolomics internal standards and the C_1-4The fatty alcohol or its water solution constitutes a single-phase extraction solvent, preferably in a volume ratio of 2: 1;

the volume ratio of the partial supernatant to the single-phase extraction solvent is 1: 1.5-1: 10, preferably 1: 2-1: 5, more preferably 1: 3;

mixing the solution with the lipidomics internal standard, the acid aqueous solution and the C_1-4Halogenated hydrocarbons and said C_1-4The fatty alcohol constitutes a two-phase extraction solvent, and the volume ratio of the fatty alcohol to the fatty alcohol is 2:1:10: 3;

the redissolving solvent is C_1-4A fatty alcohol;

8. The detection method according to any one of claims 5 to 7,

Said additional biphasic extraction solvent consists of C_1-4Fatty alcohols and C_1-4Halogenated hydrocarbon, preferably in a volume ratio of 1: 2;

9. The detection method according to any one of claims 5 to 8,

said C is_1-4The aliphatic alcohol is methanol and/or isopropanol, preferably methanol;

the acid water solution is formic acid water solution, acetic acid water solution or hydrochloric acid, preferably 0.1-1M hydrochloric acid, more preferably 0.5-1M hydrochloric acid, and most preferably 1M hydrochloric acid;

said C is_1-4The halogenated hydrocarbon is chloroform and/or dichloromethane, preferably chloroform.