CN113237982A

CN113237982A - Derivatization method of hydroxyl metabolites and efficient non-targeted metabonomics analysis method

Info

Publication number: CN113237982A
Application number: CN202110480323.5A
Authority: CN
Inventors: 赵爽
Original assignee: Xiamen Mailio Technology Co ltd
Current assignee: Xiamen Mailio Technology Co ltd
Priority date: 2021-04-30
Filing date: 2021-04-30
Publication date: 2021-08-10

Abstract

The invention relates to a derivatization method of hydroxyl metabolites and a non-targeted metabonomics high-efficiency analysis method, wherein the derivatization method of the hydroxyl metabolites comprises the following steps: step 1: extracting hydroxyl 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: mixing the dried sample with a second solvent to dissolve the dried sample, adding 4-dimethylaminopyridine and dansyl chloride, uniformly mixing, and heating for incubation; and adding sodium hydroxide after the incubation is finished, then heating for incubation, and finally adding formic acid 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 metabolite detected is oneThe peak pair reduces the influence caused by instrument drift and matrix, so that the quantification is more accurate.

Description

Derivatization method of hydroxyl 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 hydroxyl 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.

Hydroxyl 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. The Chinese patent application CN1480726A discloses a method for pre-column derivatization of sulfonyl isocyanate of hydroxyl compounds and mercapto compounds, which comprises the steps of taking sulfonyl isocyanate compounds as derivatization reagents in a solvent without active hydrogen, adding water to terminate the reaction after the pre-column derivatization of the hydroxyl compounds or the mercapto compounds, and adopting sulfonyl isocyanate in the method, wherein the detection sensitivity of the marked substances is to be improved.

Disclosure of Invention

The invention aims to overcome the problems of the existing metabonomics technology and provide a derivatization method of hydroxyl metabolites, which can perform derivatization labeling on the hydroxyl-containing metabolites so as to facilitate 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 hydroxyl metabolite comprises a step 1, which is mainly to extract the metabolite from a sample by using a first solvent, and then to dry the solvent containing the metabolite. The extraction method comprises protein precipitation in plasma sample analysis, tissue homogenization treatment and the like. The first solvent used for extraction may be water, methanol, etc. Drying hydroxyl metabolites to remove solvent interference to obtain dry sample without solvent, precipitating protein in sample with high protein content, such as plasma or serum, and collecting supernatant and drying 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 hydroxyl metabolite comprises a step 2, and for derivatization treatment, a dried sample needs to be redissolved, and a second solvent is adopted, preferably the same as an eluent for subsequent liquid chromatographic separation. And 2, reacting the labeled reagent with the metabolite to obtain a derivative sample. Wherein, the reaction principle is as follows:

wherein R represents any chemical group, dansyl chloride acts as a labeling reagent and reacts with a metabolite to generate a derivatized metabolite; 4-dimethylamino pyridine is used as a reaction activating reagent and an excellent leaving group, so that the marking capability of dansyl chloride can be further improved, and the reaction activity is enhanced; NaOH is a quenching reagent that quenches excess of the labeling reagent, and excess labeling reagent interferes with and inhibits the signal for metabolite detection if excess labeling reagent is not quenched. After quenching, the pH is preferably adjusted to 2.5-3.5 by the addition of a pH adjusting reagent. If pH adjustment is not performed, the pH of the solution becomes high due to the addition of sodium hydroxide, and if pH adjustment is not performed, the column and the apparatus are damaged. The pH regulating reagent is preferably formic acid, which has the advantages of weak acidity, no interference to metabolite detection and no damage to detection instruments.

Compared with the conventional labeling reagent such as sulfonyl isocyanate, the dansyl chloride is more suitable for liquid chromatography-mass spectrometry. The aromatic ring structure in the dansyl chloride can obviously improve the hydrophobicity of the derivatized metabolite and is more suitable for reverse chromatographic analysis; n (CH) in dansyl chloride₃)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 labeling, on one hand, combining an LC-UV technology to blend different samples, avoiding the characteristic peak intensity difference caused by the concentration difference of metabolites, and reducing the misjudgment rate; on the other hand, for low-concentration metabolites, different samples were mixed according to the peak area ratio N of 1: after N is mixed, the characteristic peak intensity of low-concentration metabolites can be enhanced, missing detection is avoided, and the sensitivity is improved; finally, the process is carried out in a batch,¹²c mark +¹³The C mark provides isotope internal standards for all hydroxyl metabolites, the detected metabolites are presented in a peak pair mode, the impurity interference is an independent peak, the identification is convenient, the quantitative accuracy is greatly improved,thousands to tens of thousands of metabolites can be detected, so that the detection result is more reliable.

The specific scheme is as follows:

a derivatization method of hydroxyl metabolites comprises the following steps:

step 1: extracting hydroxyl 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: mixing the dried sample with a second solvent to dissolve the dried sample, adding 4-dimethylaminopyridine and dansyl chloride, uniformly mixing, and heating for incubation; and adding sodium hydroxide after the incubation is finished, then heating for incubation, and finally adding a pH regulator to obtain a derivatization 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 an aqueous acetonitrile solution.

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; 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.

Furthermore, in the step 2, 4-dimethylamino pyridine is used as a reaction activating reagent and is used as a leaving group, so that the labeling capacity of dansyl chloride is improved, and the reaction activity is enhanced; adding 4-dimethylaminopyridine and dansyl chloride into dansyl chloride serving as a labeling reagent, uniformly mixing the dansyl chloride and the 4-dimethylaminopyridine by vortex, and incubating the mixture at the temperature of between 50 and 60 ℃ for 60 to 70 minutes; and sodium hydroxide is used as a quenching reagent for quenching the excessive labeling reagent, after the sodium hydroxide is added, the mixed solution is incubated for 10-15 minutes at the temperature of 50-60 ℃, and finally, a pH regulator is added to obtain a derivatization sample.

Further, after adding sodium hydroxide, incubation is performed by heating, and after the incubation is completed, a pH adjusting reagent formic acid is added to adjust the pH to 2.5 to 3.5, thereby obtaining a derivatized sample.

The invention also provides a non-targeted metabonomics high-efficiency analysis method, 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 hydroxyl metabolites to perform derivatization, wherein the labeling reagent adopted by the derivatization sample a is¹²C labeling reagent, i.e.¹²C-dansyl chloride; the labeling reagent adopted by the derivative sample b is¹³C labeling reagent, i.e.¹³C-dansyl chloride;

and B: respectively carrying out liquid chromatography serial ultraviolet detection on the derivatization sample a and the derivatization sample b by adopting an LC-UV device to obtain a map, defining the area ratio of characteristic peaks of the derivatization sample a and the derivatization sample b in the map as N, and then carrying out the following steps of (1): mixing the N in a volume ratio to obtain 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, in the step B, the liquid chromatography is connected in series with ultraviolet detection, and the conditions of the liquid chromatography are as follows: liquid chromatography using C₁₈The chromatographic column comprises a mobile phase A which is an aqueous solution of formic acid, a mobile phase B which is a mixed solution of formic acid and acetonitrile, and ultraviolet detection conditions of: the detection wavelength was 338 nm.

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 hydroxyl metabolites, provided by the invention, after the sample is subjected to derivatization, 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, after the non-targeted metabonomics high-efficiency analysis method is processed by adopting the derivatization method of the hydroxyl 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 urine plasma sample provided in example 1 of the present invention;

FIG. 2 is a characteristic peak diagram of a urine plasma sample provided in comparative example 1 according to 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.

Example 1

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 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.

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 parts of 1 part of dried sample, 25. mu.L of acetonitrile/water (3:1, v/v) for mass spectrum reconstitution, 25. mu.L of reaction activating reagent (4-dimethylaminopyridine), and 40. mu.L of reaction activating reagent¹²And C, labeling a reagent (dansyl chloride), uniformly mixing by vortex, and then incubating for 60 minutes at the temperature of 60 ℃. After incubation was complete, 5. mu.L of reagent (sodium hydroxide) was added for quenchingExcess labeling reagent was quenched and the mixed solution was incubated at 60 ℃ for 10 minutes. Finally, 25. mu.L of a pH adjusting reagent (formic acid solution) was added to adjust the pH to about 3, to obtain a derivatized sample a.

Adding 25 μ L acetonitrile/water (3:1, v/v) to 1 part of dried sample to redissolve the sample, and adding 25 μ L reaction activating reagent (4-dimethylaminopyridine) and 40 μ L¹³And C, labeling a reagent (dansyl chloride), uniformly mixing by vortex, and then incubating for 60 minutes at the temperature of 60 ℃. After completion of the incubation, 5. mu.L of a reagent (sodium hydroxide) for quenching the excess labeled reagent was added, and the mixed solution was incubated at 60 ℃ for 10 minutes. Finally, 25 μ L of a pH adjusting reagent (formic acid solution) was added to adjust the pH to about 3, thereby obtaining a derivative sample b.

Respectively carrying out liquid chromatography serial ultraviolet detection on the derivatization sample a and the derivatization sample B by adopting an LC-UV device, wherein the liquid chromatography adopts a C18 chromatographic column, the mobile phase A is a formic acid aqueous solution, the mobile phase B is a formic acid and acetonitrile mixed solution, and the ultraviolet detection conditions are as follows: the detection wavelength was 338 nm. Obtaining a map through LC-UV detection, wherein the area ratio of characteristic peaks of the derivative sample a to the derivative sample b in the map is 1:1, and then, the ratio of the area of the characteristic peaks of the derivative sample a to the area of the characteristic peaks of the derivative sample b in the map is 1:1, and obtaining a detection sample.

It should be noted that, for a urine sample of the same person on the same day, the area ratio of the characteristic peaks in the map of the derivatization sample a and the derivatization sample b is generally 1:1, and at this time, the derivatization sample a and the derivatization sample b are mixed in equal volume.

For urine samples sampled by the same person at different times or urine samples of different persons, due to individual differences, even the same metabolite may have a difference in characteristic peak intensity, and at this time, the area ratio N of the characteristic peak in the map of the derivative sample a to the characteristic peak in the derivative sample b is not equal to 1. In the prior art, misjudgment is often caused by inconsistent conditions of metabolite detection due to differences of individuals in space and time, namely, two persons A and B who are in the same health are marked by a conventional method and then subjected to liquid quality analysis, the obtained maps have obvious differences, tumor factors carried by the persons A may appear, and the persons B do not suffer from tumors.

Aiming at the problem, the invention adopts an LC-UV device to respectively carry out liquid chromatography serial ultraviolet detection on the derivatization sample a and the derivatization sample b to obtain a map, the area ratio of characteristic peaks of the derivatization sample a and the derivatization sample b in the map is defined as N, and then the derivatization sample a and the derivatization sample b are subjected to liquid chromatography serial ultraviolet detection according to the ratio of 1: and the volume ratio of N is mixed, so that misjudgment caused by metabolite concentration difference can be avoided.

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.

TABLE 1 liquid quality analysis main parameter table

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 hydroxyl derivative metabolites, and the internal standards can be detected unless the metabolites do not contain the hydroxyl metabolites, so that the non-preset detection target is realized, 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.

Derivatization of dried samples and efficient non-targeted metabolomics analysis are described in example 1.

Example 3

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 as 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 sample in a refrigerator at the temperature of-80 ℃ to serve as a dried sample.

Example 4

Taking a sample of mammalian cells, a minimum of 10 is required⁶And (4) cells. The culture medium of the dish was removed by a suction operation, and precooled PBS (4 ℃) was gently added to the dish, and washed 3 times to remove the residual culture medium 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.

Comparative example 1

Comparative example 1 the same urine sample as in example 1 was analyzed in substantially the same manner as in example 1 except that the sample was not labeled with dansyl chloride and, since no dansyl chloride was labeled, the sample did not have ultraviolet absorption and fluorescence characteristics, and thus LC-UV quantification and subsequent sample mixing adjustment operations could not be performed.

Specifically, in comparative example 1, a dried sample obtained from a urine sample was aliquoted and used as a sample, 25. mu.L of acetonitrile/water (3:1, v/v) was added to redissolve the sample, and the redissolved sample was directly sent to an LC-MS apparatus for liquid mass analysis, and the characteristic peak pattern obtained is shown in FIG. 2. As can be seen from fig. 2, the number of confirmed metabolite species is reduced dramatically, and the intensity of the characteristic peak is low, so that many low-intensity interference peaks occur, and it is difficult to obtain accurate detection results.

Comparative example 2

The urine sample in example 1 was collected and processed as follows: 50 μ L urine, 10 μ L sodium chloride (saturated NaCl), 5 μ L hydrochloric acid (6M), 150 μ L ethyl acetate for extraction, 2 times extraction, separating ethyl acetate, mixing the two separated ethyl acetates, blowing with nitrogen, dissolving with 50 μ L acetonitrile, 25 μ L for use¹²C label, 25 μ L for¹³And C, marking.

Compared with the example 1, the characteristic peaks obtained by the marking method and the subsequent detection method are found in the same way as in the example 1, the number of the peak pairs detected in the comparative example 2 is less, which shows that the recovery rate of the sample is reduced by adopting the pretreatment method in the comparative example 2, and although the operation is increased by adding the ethyl acetate for extraction, the detection accuracy is reduced.

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

1. A derivatization method of hydroxyl metabolites is characterized in that: the method comprises the following steps:

step 2: mixing the dried sample with a second solvent to dissolve the dried sample, and then adding 4-dimethylamino pyridine and dansyl chloride, wherein the dansyl chloride is used as a labeling reagent, and heating and incubating the mixture after uniformly mixing; and adding sodium hydroxide after the incubation is finished, then heating for incubation, and finally adding a pH regulator to obtain a derivatization sample.

2. The method for derivatizing a hydroxyl metabolite 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;

3. The method for derivatizing a hydroxyl 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;

4. The method for derivatizing a hydroxyl metabolite according to any one of claims 1 to 3, wherein: in the step 2, 4-dimethylamino pyridine is used as a reaction activating reagent and a leaving group, so that the marking capability of dansyl chloride is improved, and the reaction activity is enhanced; adding 4-dimethylaminopyridine and dansyl chloride into dansyl chloride serving as a labeling reagent, uniformly mixing the dansyl chloride and the 4-dimethylaminopyridine by vortex, and incubating the mixture at the temperature of between 50 and 60 ℃ for 60 to 70 minutes; and sodium hydroxide is used as a quenching reagent for quenching the excessive labeling reagent, after the sodium hydroxide is added, the mixed solution is incubated for 10-15 minutes at the temperature of 50-60 ℃, and finally, a pH regulator is added to obtain a derivatization sample.

5. The method for derivatizing a hydroxyl metabolite according to any one of claims 1 to 3, wherein: after adding sodium hydroxide, heating and incubating, adding a pH adjusting reagent formic acid after the incubation is finished, and adjusting the pH to 2.5-3.5 to obtain a derivatization sample.

6. A non-targeted metabonomics high-efficiency analysis method is characterized in that: the method comprises the following steps:

step A: equally dividing the object to be tested into at least 2 parts, and performing derivatization by the derivatization method of the hydroxyl metabolite according to any one of claims 1 to 5, wherein the labeling reagent adopted by the derivatized sample a is¹²C labeling reagent, i.e.¹²C-dansyl chloride; the labeling reagent adopted by the derivative sample b is¹³C labeling reagent, i.e.¹³C-dansyl chloride;

7. The non-targeted metabolomics-based high-efficiency analysis method of claim 6, which comprises: and B, serially connecting the liquid chromatogram with ultraviolet detection, wherein the liquid chromatogram conditions are as follows: liquid chromatography using C₁₈The chromatographic column comprises a mobile phase A which is an aqueous solution of formic acid, a mobile phase B which is a mixed solution of formic acid and acetonitrile, and ultraviolet detection conditions of: the detection wavelength was 338 nm.

8. 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.

9. The non-targeted metabolomics-based high-efficiency analysis method of claim 8, wherein: the conditions for the liquid mass analysis in step C include a mass spectrum scanning range m/z of 220-1000.

10. The non-targeted metabolomics-efficient analysis method according to any of claims 6-9, 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.