CN113156028A - Derivatization method of carbonyl metabolites and efficient non-targeted metabonomics analysis method - Google Patents

Derivatization method of carbonyl metabolites and efficient non-targeted metabonomics analysis method Download PDF

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CN113156028A
CN113156028A CN202110480349.XA CN202110480349A CN113156028A CN 113156028 A CN113156028 A CN 113156028A CN 202110480349 A CN202110480349 A CN 202110480349A CN 113156028 A CN113156028 A CN 113156028A
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赵爽
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Xiamen Mailio Technology Co ltd
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Abstract

The invention relates to a derivatization method of carbonyl metabolites and a non-targeted metabonomics high-efficiency analysis method, wherein the derivatization method of the carbonyl metabolites comprises the following steps: step 1: extracting carbonyl metabolites of a sample to be detected by adopting a first solvent, and then drying to remove the first solvent to obtain a dried sample; step 2: dissolving the dried sample, adding a pH adjusting reagent to adjust the pH, then adding dansyl hydrazine, uniformly mixing, heating for incubation, then cooling to terminate the reaction, drying to obtain a solid, and redissolving the solid to obtain a derivatization sample. The non-target metabonomics high-efficiency analysis method is synchronously carried out by adopting the derivatization method12C mark +13The C label improves the metabolite detection sensitivity, can detect more metabolites simultaneously, and has higher metabolite coverage rate; the detected metabolite is a peak pair, and the instrument drift and matrix band are reducedThe influence of (2) eliminates noise interference, so that the quantification is more accurate.

Description

Derivatization method of carbonyl metabolites and efficient non-targeted metabonomics analysis method
Technical Field
The invention relates to a metabonomics analysis technology, in particular to a derivatization method of carbonyl metabolites and a non-targeted metabonomics high-efficiency analysis method.
Background
Metabonomics is an important component of system biology and has wide application prospects in the field of clinical medicine. Metabolomics analysis techniques include: NMR, GC-MS, CE-MS and LC-MS, which can detect metabolites to a certain extent, but still have a plurality of difficulties, such as low ionization efficiency of the metabolites, weak mass spectrum signals, lack of isotope internal standard for quantification, small amount of detected metabolites, high interference and the like.
For example, LC-MS is analyzed by liquid chromatography-mass spectrometry, and the respective metabolites in different samples are compared to determine all metabolites therein. Essentially, metabolic fingerprinting involves comparing the mass spectrum peaks of metabolites in different individuals, eventually understanding the structures of different compounds, and establishing a complete set of analysis methods for identifying the characteristics of these different compounds. For metabolites, there is not only the characteristic of mass spectrum peaks. Moreover, Mass Spectrometry (MS) cannot detect all metabolites, not because of insufficient sensitivity of mass spectrometry, but because mass spectrometry can only detect ionized species, but some metabolites cannot be ionized in a mass spectrometer, thus causing inaccuracy.
In order to solve the above problems, the prior art adopts a targeted metabonomics technology to analyze a specific target. Targeted metabolomics refers to: aiming at several target compounds or all or part of metabolites related to a certain path, a detection method with strong specificity, high sensitivity and good repeatability is constructed by using a standard substance to carry out quantification and analysis on the target compounds. Targeted metabolomics focuses only on the study of several or several classes of metabolites known to have a possible biological effect, and therefore its data processing is simpler and more convenient than non-targeted metabolomics. For example, patent application CN107085032A discloses an analysis method of chemical derivatization matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), comprising the following steps: (1) small molecule compounds: the dialdehyde compound is used as a connecting arm and is modified on the amino compound through condensation reaction, so that the structure of the drug metabolite derives an aldehyde group. (2) Polypeptide-derivatized small molecule compounds: adding aldehyde compound and polypeptide into phosphate buffer solution with pH of 4-6 at a molar ratio of 50-100:1, performing condensation reaction at 5-37 deg.C, and modifying N-terminal cysteine of amino acid sequence of polypeptide to obtain derivative reagent of aldehyde compound. (3) MALDI-TOF-MS analysis: and (3) performing sample derivatization according to a 1 mu L sample mixed matrix alpha-cyano-4-hydroxycinnamic acid CHCA point plate, performing matrix assisted laser desorption ionization-time-of-flight mass spectrometry after air drying at room temperature, and adopting a positive ion reflection mode. The method uses dialdehyde compounds to modify the drug metabolites with amino structures, is limited to the case of the amino metabolites in drugs, and provides corresponding isotope internal standards for all detected metabolites although the isotope internal standards exist, so that the inaccuracy problem still exists.
In contrast, non-targeted metabonomics refers to unbiased detection of dynamic changes of all small molecule metabolites (mainly endogenous small molecule compounds with a relative molecular weight within 1000 Da) before and after stimulation or disturbance in cells, tissues, organs or organisms by using technologies such as LC-MS, GC-MS, NMR and the like, differential metabolites are screened by biological information analysis, pathway analysis is carried out on the differential metabolites, and the physiological mechanism of the changes is revealed. Since the non-targeted metabonomics have no definite target, the original data have large difference and large data quantity, and how to obtain ideal analysis results from the data are one of the problems to be solved by the non-targeted metabonomics.
Carbonyl metabolites are generally present in most biological samples, such as various animal body fluids (blood, urine, cerebrospinal fluid, etc.), animal and plant tissue samples, cells, etc. Chinese patent application CN112125827A discloses a synthesis method for a derivatization reagent of a steroid compound containing carbonyl, which comprises the steps of firstly utilizing 1, 3-dibromopropane and N-hydroxyphthalimide to react and synthesize 2- (3-bromopropoxy) isoindoline-1, 3-diketone; preparing 3- ((1, 3-dioxo-2-isoindolinyl) -oxy) -N, N, N-trimethylpropyl ammonium bromide by using 2- (3-bromopropoxy) isoindoline-1, 3-dione; and finally, performing hydrazinolysis on the 3- ((1, 3-dioxo-2-isoindolinyl) -oxy) -N, N, N-trimethylpropyl ammonium bromide, wherein the detection sensitivity of the method needs to be improved.
Disclosure of Invention
The invention aims to overcome the problems of the existing metabonomics technology, and provides a derivatization method of carbonyl metabolites, which can perform derivatization labeling on the carbonyl-containing metabolites, thereby facilitating the identification of the metabolites by the subsequent liquid chromatography-mass spectrometry. The sample types detectable by the method are rich, such as plasma, serum, tissues, cells, urine, hair and the like, and almost cover the whole sample to be detected, so the method has wide application range.
In the invention, the derivatization method of the carbonyl metabolite comprises a step 1, mainly extracting the metabolite from a sample by using a first solvent, wherein the first solvent can be water, methanol and the like; the solvent containing the metabolite is then dried. The extraction method comprises protein precipitation in plasma sample analysis, tissue homogenization treatment and the like. The carbonyl metabolite is dried in order to remove the first solvent interference, resulting in a dried sample without the first solvent. For samples with high protein content, such as plasma or serum, tissue should be precipitated with protein, and the supernatant should be dried to obtain dry sample. For samples with low protein content, such as urine, cells, protein precipitation can be omitted. For urine, if turbidity is present, filtration operations may be added, for example: centrifuging urine sample at 3-5 deg.C and centrifugal force of 15000g for 10-20 min, collecting supernatant, filtering the supernatant with 0.2-0.3 μm filter, filtering, collecting filtrate, completely drying the filtrate with vacuum centrifugal concentrator, and stopping drying when there is no solvent in the product.
It should be noted that step 1 of the present invention lays the foundation of the subsequent steps, and the interference of solvents in various metabolites is removed through step 1, so that the interference of proteins in the metabolites is reduced, and the direction of derivatization and detection of soluble metabolites is established.
In the invention, the derivatization method of the carbonyl metabolite comprises a step 2 of reacting a labeled reagent with the metabolite for derivatization treatment to obtain a derivatization sample. Wherein, the reaction principle is as follows:
Figure BDA0003048332050000041
in the above formula R1、R2Represents any chemical group.
Wherein the dansyl hydrazide is used as a reaction labeling reagent to be derived from the metabolite so as to generate the derivatized metabolite. First, a pH adjusting agent was added to adjust the pH to 2.0. If pH adjustment is not performed, the reaction activity is weak and the labeling reaction cannot be completed. The pH adjusting reagent is preferably hydrochloric acid, which has the advantage of being volatile and can be easily removed by drying, thereby not affecting the mass spectrometric detection and protecting the liquid chromatographic column. After the dansyl hydrazide labeling reaction, physical cooling quenching reaction is adopted, and the advantage is that no interference is introduced.
The inventor thinks that the dansyl hydrazide label is more suitable for liquid chromatography-mass spectrometry analysis, because the aromatic ring structure in the dansyl hydrazide can obviously improve the hydrophobicity of the metabolite after derivatization and is more suitable for reverse chromatographic analysis; -N (CH) in dansyl hydrazide3)2The structure can remarkably improve the detection sensitivity of the derivatized metabolite in a mass spectrum positive ion mode; at the same time, an isotope atom may be introduced at the methyl group13And C, the isotope internal standard is used for improving the quantitative performance of mass spectrum.
The invention also provides a non-targeted metabonomics high-efficiency analysis method based on the derivatization treatment, which comprises the following steps of12C mark +13C marks and provides isotope internal standards for all carbonyl metabolites, detected metabolites are presented in a peak pair mode, and impurities are interfered into single peaks, so that the identification is facilitated, the quantitative accuracy is greatly improved, thousands of metabolites can be detected, and the detection result is more reliable.
The specific scheme is as follows:
a derivatization method of carbonyl metabolites comprises the following steps:
step 1: extracting carbonyl metabolites of a sample to be detected by adopting a first solvent, and then drying to remove the first solvent to obtain a dried sample;
step 2: and mixing the dried sample with a second solvent to dissolve the dried sample, adding a pH regulating reagent to regulate the pH, then adding dansyl hydrazide, uniformly mixing, heating for incubation, then cooling to terminate the reaction, drying to obtain a solid, and adding the solid into the second solvent for redissolution to obtain a derivative sample.
Further, in step 1, after protein precipitation treatment is carried out on a sample to be detected, centrifugation treatment is carried out to obtain a supernatant, and the supernatant is dried to obtain the dried sample;
optionally, the first solvent is methanol, water or methanol-water solution;
optionally, the second solvent is an eluent for liquid chromatographic separation of the derivatized sample, preferably water or aqueous acetonitrile.
Further, in step 1, the sample to be detected is a plasma or serum sample, the sample is centrifuged for 10-20 minutes at the temperature of 3-5 ℃ and the centrifugal force of more than 15000g, the supernatant is taken after centrifugation, methanol is added, vortex treatment is carried out to completely precipitate protein, then centrifugation is carried out, the supernatant is taken, the obtained supernatant is completely dried by a vacuum centrifugal concentrator, and the drying is stopped until no solvent exists in the product, so that the dried sample is obtained;
optionally, the sample to be detected is a urine sample, the sample is centrifuged for 10 to 20 minutes under the conditions that the temperature is 3 to 5 ℃ and the centrifugal force is more than 15000g, and supernatant is taken after centrifugation; completely drying the obtained supernatant by using a vacuum centrifugal concentrator until no solvent exists in the product, and stopping drying to obtain the dried sample;
optionally, the sample to be detected is a tissue sample, the tissue sample is mixed with methanol, homogenization treatment is carried out, and then incubation is carried out for 10-15 minutes at-20 ℃; after incubation, centrifuging for 10-20 minutes at 3-5 ℃ under the condition that the centrifugal force is more than 15000g, and taking supernatant after centrifugation; completely drying the obtained supernatant by using a vacuum centrifugal concentrator until no solvent exists in the product, and stopping drying to obtain the dried sample;
optionally, the sample to be tested is a cell sample, and a minimum of 10 is required6Subjecting the cell sample to cell lysis treatment to obtain lysate at 3-5 deg.C under centrifugal force>Centrifuging at 15000g for 10-20 min, and collecting supernatant; the supernatant obtained was completely dried by vacuum centrifugation concentrator untilAnd stopping drying when no solvent exists in the product to obtain the dried sample.
Further, in step 2, hydrochloric acid is used as a pH adjusting reagent to adjust the pH to 2.0, dansyl hydrazide is used as a labeling reagent, the incubation temperature is 30-45 ℃, the incubation time is 50-100 minutes, after the incubation is completed, the mixture is transferred to an environment with a temperature of-85 to-70 ℃ for 10-15 minutes to terminate the reaction, a solid is obtained after drying treatment, and the solid is added with the second solvent for redissolution to obtain the derivatization sample.
Further, the drying treatment is drying by using a nitrogen blowing concentrator to obtain the solid, and adding acetonitrile water solution to redissolve to obtain the derivatization sample.
The invention provides a high-efficiency analysis method of non-targeted metabonomics, which comprises the following steps:
step A: equally dividing the object to be detected into at least 2 parts, and respectively adopting the derivatization method of the carbonyl metabolites to perform derivatization, wherein the labeling reagent adopted by the derivatization sample a is12C labeling reagent, i.e.12C-dansyl hydrazide; the labeling reagent adopted by the derivative sample b is13C labeling reagent, i.e.13C-dansyl hydrazide;
and B: and (c) subjecting the derivatized sample a and the derivatized sample b to a reaction in a ratio of 1:1, obtaining a detection sample;
and C: and B, sending the detection sample obtained in the step B into an LC-MS device, carrying out liquid mass analysis, and obtaining a characteristic peak diagram of the metabolite, wherein the metabolite is presented in the characteristic peak diagram in the form of a peak pair, and the metabolite is identified through the characteristic peak diagram.
Further, the conditions for the liquid mass analysis in step C include that the liquid chromatography adopts C18And (3) a chromatographic column, wherein the mobile phase A is an aqueous solution of formic acid, and the mobile phase B is a mixed solution of formic acid and acetonitrile.
Further, the conditions of the liquid mass analysis in step C include a mass spectrum scanning range m/z of 220-.
Further, in step C, the substance existing in the form of a single peak in the characteristic peak diagram is interference noise and is not a true metabolite.
Has the advantages that:
according to the derivatization method of the carbonyl metabolites, after the sample is derivatized, the detection sensitivity of the metabolites is improved, more metabolites can be detected at the same time, and the metabolite coverage rate is higher.
Furthermore, the non-targeted metabonomics high-efficiency analysis method is processed by the derivatization method of the carbonyl metabolites, the detected metabolites are a peak pair, isotope internal standards are generated for all the derivatized metabolites, the influence caused by instrument drift and matrix is reduced, noise interference is eliminated, and the quantification is more accurate.
Drawings
In order to illustrate the technical solution of the present invention more clearly, the drawings will be briefly described below, and it is apparent that the drawings in the following description relate only to some embodiments of the present invention and are not intended to limit the present invention.
FIG. 1 is a characteristic peak diagram of a sample provided in accordance with an embodiment 1 of the present invention;
FIG. 2 is a characteristic peak diagram of a sample provided in accordance with an embodiment 2 of the present invention.
Detailed Description
Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the present invention, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available. In the following examples, "%" means weight percent, unless otherwise specified.
Example 1
Tissue samples were taken, weighed and placed in centrifuge tubes. If the sample is too large, the original sample is cut off so that the weight of the sample is around 150-200 mg. Add pre-cooled methanol/water solution to centrifuge tube, following 500 μ L methanol/water solution: 100mg of tissue, 4:1 in methanol/water solution, v/v. The tissue sample was homogenized using a homogenizer for 15 seconds. The homogenization speed and time can be adjusted as necessary depending on the type of sample (e.g., harder tissue requires a higher rate and longer time). After homogenization, the samples were incubated in a refrigerator at-20 ℃ for 10 minutes. After incubation, centrifugation was carried out at 4 ℃ for 10 minutes under conditions of centrifugal force >10000 g. Taking the supernatant into centrifuge tubes, and centrifuging each centrifuge tube at low speed to ensure that the sample liquid drops on the tube wall are deposited at the bottom of the centrifuge tube. And completely drying the supernatant by using a vacuum centrifugal concentrator, stopping drying when no solvent is at the bottom of a centrifugal tube, and storing the obtained dried sample in a refrigerator at the temperature of-80 ℃ for subsequent derivatization treatment.
Description of the drawings: the above 25. mu.L of supernatant was dried for about 1 hour. Any residual solvent will severely interfere with the chemical labeling reaction, but too long a drying time (e.g., drying for more than 3 hours) will also result in a loss of metabolite content.
The method is introduced by combining a non-targeted metabonomics high-efficiency analysis method, the dry sample is equally divided into 3 parts, 2 parts of the dry sample are taken for derivatization treatment, and 1 part of the dry sample is reserved for future reference. 2, 1 part of the dried sample was taken out, 25. mu.L of mass spectrum-grade water was added to reconstitute the sample, 25. mu.L of a pH adjusting reagent (hydrochloric acid) was added to the sample to adjust pH 2, and 25. mu.L of the pH adjusting reagent was added12C a labeling reagent: (12C-dansyl hydrazide) was added, vortexed, and incubated at 40 ℃ for 60 minutes. After completion of incubation, the samples were transferred to-80 ℃ and left for 10 minutes to terminate the reaction. After the sample is dried by using a nitrogen blowing concentrator, 100 mu L of acetonitrile/water mixed solution (50:50, v/v) is added for redissolution, and a derivatization sample a is obtained.
To another 1 part of the dried sample was added 25. mu.L of a pH adjusting reagent (hydrochloric acid) to adjust the pH to 2, followed by addition of 25. mu.L of the reagent13C a labeling reagent: (13C-dansyl hydrazide) was added, vortexed, and incubated at 40 ℃ for 60 minutes. After completion of incubation, the samples were transferred to-80 ℃ and left for 10 minutes to terminate the reaction. After the sample is dried by using a nitrogen blowing concentrator, 100 mu L of acetonitrile/water mixed solution (50:50, v/v) is added for redissolution, and a derivative sample b is obtained.
And (c) subjecting the derivatized sample a and the derivatized sample b to a reaction in a ratio of 1:1, and obtaining a detection sample.
The detection sample is sent into an LC-MS device for liquid mass analysis, the specific conditions are shown in Table 1, and the characteristic peak diagram of the metabolite is obtained and shown in FIG. 1. The elution method in Table 1 greatly shortens the analysis time of the sample, improves the analysis efficiency and improves the analysis flux.
TABLE 1 liquid quality analysis main parameter table
Figure BDA0003048332050000091
In the elution gradient in Table 1, MPB represents mobile phase B, and the percentages are volume percentages. By adopting the elution method, the analysis time of the sample is greatly shortened, the analysis efficiency is improved, and the analysis flux is improved.
As can be seen from FIG. 1, the metabolites detected are present as pairs of peaks, which is the appearance of one in the mass spectrum12Peak of C label and one13C mark peak, but the two peaks refer to the same substance, and the interference noise is a single peak, thereby filtering some interference noise, reducing the influence caused by instrument drift and matrix, and leading the detected metabolite to be more accurate. And labeled metabolites have improved sensitivity, and metabolites with low original concentration or low response can be detected, so that the detected metabolite quantity is improved, and about 2000 metabolites can be detected by one urine sample.
In addition, the method generates internal standards for all carbonyl derivative metabolites, and the internal standards can be detected unless the metabolites do not contain the carbonyl metabolites, so that the detection target is not preset, and compared with the conventional targeted metabonomics detection method, the method provided by the invention has the advantages that the comprehensiveness of the obtained data is better, and the accuracy of further analysis through the data is higher.
Example 2
Taking a plasma sample, centrifuging for 15 minutes under the conditions of 4 ℃ and centrifugal force of more than 15000g (12000rpm), and taking a supernatant to a 0.5mL centrifuge tube after centrifugation; and (3) transferring 30 mu L of sample supernatant into a corresponding microcentrifuge tube, adding 90 mu L of precooled LC-MS or HPLC-grade methanol into the microcentrifuge tube containing 30 mu L of sample, fully whirling the microcentrifuge tube containing the sample and the methanol to completely precipitate the protein, and then carrying out low-speed centrifugation on the microcentrifuge tube to precipitate the mixture in the tube at the bottom of the microcentrifuge tube. Placing the vortexed mixture in a refrigerator at-20 deg.C for at least 1 hour; after 1 hour, the centrifuge tube was removed from the refrigerator, centrifuged at >10000g for 15 minutes and 90 μ L of the supernatant was pipetted into a new microfuge tube. And (3) completely drying 90 mu L of supernatant by using a vacuum centrifugal concentrator, stopping drying when no solvent is at the bottom of a centrifugal tube, and storing the obtained dried sample in a refrigerator at the temperature of-80 ℃ for subsequent derivatization treatment. Description of the drawings: the above 90. mu.L of supernatant takes about 1 to 2 hours to dry. Any residual solvent will severely interfere with the chemical labeling reaction, but too long a drying time (e.g., drying for more than 3 hours) will also result in a loss of metabolite content.
The derivatization method and the non-targeted metabonomics high-efficiency analysis method of the dried sample are shown in example 1, and the obtained characteristic peak diagram is shown in figure 2.
Example 3
A urine sample was collected, centrifuged at 4 ℃ at a centrifugal force of more than 15000g (12000rpm) for 15 minutes, and the supernatant was collected to a 1.5mL centrifuge tube. And completely drying the obtained supernatant by using a vacuum centrifugal concentrator, and stopping drying when no solvent is at the bottom of a centrifugal tube to obtain a dry sample.
If the urine sample has not been filtered before, the supernatant needs to be filtered using a 0.2-0.3 μm filter. After filtration, the filtrate was taken to a centrifuge tube. And then drying by using a vacuum centrifugal concentrator, and stopping drying when the bottom of a centrifugal tube has no solvent to obtain a dry sample.
Derivatization of dried samples and efficient non-targeted metabolomics analysis are described in example 1.
Example 4
Taking a sample of mammalian cells, a minimum of 10 is required6And (4) cells. Removing the culture medium from the culture dish by aspiration and gently adding the culture medium to the culture dishPre-cooled PBS (4 ℃) was added and washed 3 times to remove residual media and extracellular metabolites. Pre-cooled (-20 ℃) methanol was added to stop cell metabolism. Cells were scraped from the culture dish with a cell scraper. The scraper should be thoroughly cleaned before scraping off each culture dish/well. The scraped cells and methanol solvent were transferred to a 2mL microcentrifuge tube. An equal amount of pre-cooled (-20 ℃) methanol was added again to the petri dish, and the remaining cells and solvent were scraped off and transferred to the same microcentrifuge tube. The microcentrifuge tube containing the cells and solvent was dried using a centrifugal vacuum concentrator. 400 μ L of pre-cooled methanol-water (1:1, v/v) was added to the dried centrifuge tube. Fully whirling the centrifugal tube to redissolve the cells, and performing cell lysis by using a repeated freeze-thaw method, wherein the steps are as follows: placing the test tube in liquid nitrogen for 2 min with tweezers to ensure complete freezing of liquid, taking out the test tube, placing in ice water bath, thawing the sample by shaking in water for 2 min, repeating freezing and thawing operation for more than 4 times, centrifuging the tube at 4 deg.C under centrifugal force>Centrifugation was carried out at 15000g for 10min, the supernatant was carefully transferred to a new tube and the supernatant was completely dried using a centrifugal vacuum concentrator to obtain a dried sample.
Derivatization of dried samples and efficient non-targeted metabolomics analysis are described in example 1.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various features described in the above embodiments may be combined in any suitable manner without departing from the scope of the invention. The invention is not described in detail in order to avoid unnecessary repetition.
In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.

Claims (9)

1. A derivatization method of carbonyl metabolites is characterized in that: the method comprises the following steps:
step 1: extracting carbonyl metabolites of a sample to be detected by adopting a first solvent, and then drying to remove the first solvent to obtain a dried sample;
step 2: and mixing the dried sample with a second solvent to dissolve the dried sample, adding a pH adjusting reagent to adjust the pH, then adding dansyl hydrazide, wherein the dansyl hydrazide is a labeling reagent, uniformly mixing, heating for incubation, then cooling to terminate the reaction, drying to obtain a solid, and adding the solid into the second solvent to redissolve to obtain a derivatized sample.
2. The method for derivatizing carbonyl metabolites according to claim 1, wherein: step 1, after protein precipitation treatment is carried out on a sample to be detected, centrifugation treatment is carried out to obtain supernatant, and the supernatant is dried to obtain a dried sample;
optionally, the first solvent is methanol, water or methanol-water solution;
optionally, the second solvent is an eluent for liquid chromatographic separation of the derivatized sample, preferably water or aqueous acetonitrile.
3. The method for derivatizing a carbonyl metabolite according to claim 2, wherein: in the step 1, the sample to be detected is a plasma or serum sample, the sample is centrifuged for 10 to 20 minutes under the conditions that the temperature is 3 to 5 ℃ and the centrifugal force is more than 15000g, the supernatant is taken after centrifugation, methanol is added, vortex treatment is carried out to completely precipitate protein, then centrifugation is carried out, the supernatant is taken, the obtained supernatant is completely dried by a vacuum centrifugal concentrator, and the drying is stopped until no solvent exists in the product, so that the dried sample is obtained;
optionally, the sample to be detected is a urine sample, the sample is centrifuged for 10 to 20 minutes under the conditions that the temperature is 3 to 5 ℃ and the centrifugal force is more than 15000g, and supernatant is taken after centrifugation; completely drying the obtained supernatant by using a vacuum centrifugal concentrator until no solvent exists in the product, and stopping drying to obtain the dried sample;
optionally, the sample to be detected is a tissue sample, the tissue sample is mixed with methanol, homogenization treatment is carried out, and then incubation is carried out for 10-15 minutes at-20 ℃; after incubation, centrifuging for 10-20 minutes at 3-5 ℃ under the condition that the centrifugal force is more than 15000g, and taking supernatant after centrifugation; completely drying the obtained supernatant by using a vacuum centrifugal concentrator until no solvent exists in the product, and stopping drying to obtain the dried sample;
optionally, the sample to be tested is a cell sample, and a minimum of 10 is required6Subjecting the cell sample to cell lysis treatment to obtain lysate at 3-5 deg.C under centrifugal force>Centrifuging at 15000g for 10-20 min, and collecting supernatant; and completely drying the obtained supernatant by using a vacuum centrifugal concentrator until the product is free of the solvent, and stopping drying to obtain the dried sample.
4. The method for derivatizing carbonyl metabolites according to any one of claims 1 to 3, wherein: in the step 2, hydrochloric acid is used as a pH adjusting reagent to adjust the pH to 2.0, dansyl hydrazine is used as a labeling reagent, the incubation temperature is 30-45 ℃, the incubation time is 50-100 minutes, the incubation is transferred to an environment with the temperature of-85 to-70 ℃ after the incubation is finished, the incubation is placed for 10-15 minutes to terminate the reaction, a solid is obtained after drying treatment, and the solid is added with the second solvent for redissolution to obtain the derivatization sample.
5. The method for derivatizing a carbonyl metabolite according to claim 4, wherein: and the drying treatment is to blow and dry the solid by using a nitrogen blowing concentrator to obtain the solid, and add acetonitrile water solution to redissolve to obtain the derivatization sample.
6. A non-targeted metabonomics high-efficiency analysis method is characterized in that: the method comprises the following steps:
step A: the method comprising dividing the test object into at least 2 parts, each using the carbonyl-based metabolite of any one of claims 1 to 5A derivatization method, wherein the derivatization sample a adopts the labeling reagent12C labeling reagent, i.e.12C-dansyl hydrazide; the labeling reagent adopted by the derivative sample b is13C labeling reagent, i.e.13C-dansyl hydrazide;
and B: and (c) subjecting the derivatized sample a and the derivatized sample b to a reaction in a ratio of 1:1, obtaining a detection sample;
and C: and B, sending the detection sample obtained in the step B into an LC-MS device, carrying out liquid mass analysis, and obtaining a characteristic peak diagram of the metabolite, wherein the metabolite is presented in the characteristic peak diagram in the form of a peak pair, and the metabolite is identified through the characteristic peak diagram.
7. The non-targeted metabolomics-based high-efficiency analysis method of claim 6, which comprises: the conditions for the liquid mass analysis in step C include that the liquid chromatography adopts C18And (3) a chromatographic column, wherein the mobile phase A is an aqueous solution of formic acid, and the mobile phase B is a mixed solution of formic acid and acetonitrile.
8. The non-targeted metabolomics-based high-efficiency analysis method of claim 7, wherein: the conditions for the liquid mass analysis in step C include a mass spectrum scanning range m/z of 220-1000.
9. The non-targeted metabolomics-efficient analysis method according to any of claims 6-8, wherein: in step C, the substance existing in the form of a single peak in the characteristic peak diagram is interference noise and is not a true metabolite.
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