CN107340337B

CN107340337B - Method and kit for detecting metabolites in urine

Info

Publication number: CN107340337B
Application number: CN201610286816.4A
Authority: CN
Inventors: 张必良; 胡俊杰; 吕小侠; 叶奕栋
Original assignee: Guangzhou Ribobio Co ltd
Current assignee: Guangzhou Ribobio Co ltd
Priority date: 2016-04-29
Filing date: 2016-04-29
Publication date: 2020-07-14
Anticipated expiration: 2036-04-29
Also published as: CN107340337A

Abstract

The invention relates to a method for detecting metabolites in urine, which comprises the following steps of (1) removing impurities in a urine sample, namely removing urea, sulfur and phosphorus, and precipitating protein to obtain urine C, (2) drying, namely drying the urine C to obtain urine residue D, (3) oximation, namely oximation of β -carbonyl in acid or alcohol metabolites by using an oximation reagent to obtain urine E, (4) derivatization, namely derivatization of the urine E by using a derivatization reagent to obtain a urine sample F to be tested, wherein the derivatization reagent is a mixed reagent consisting of a silylation reagent, a catalyst and a catalyst stabilizer, and the catalyst stabilizer is selected from dithioerythritol, dithiothreitol, 1-propanethiol, ethanethiol and 2-mercaptoethanol, and (5) detection, namely detecting the urine sample F to be tested by using a gas chromatography-mass spectrometry combined method.

Description

Method and kit for detecting metabolites in urine

Technical Field

The invention relates to the technical field of biochemical detection, in particular to a gas chromatography-mass spectrometry detection method and a kit for metabolites in urine.

Background

The urine contains a large amount of metabolic compounds related to the life activities of human bodies, and the metabolic condition and disease information of the human bodies can be reflected by detecting the content of the metabolic compounds, so that the urine provides favorable help for the research and diagnosis of diseases, particularly the diagnosis, treatment and intervention of metabolic diseases. However, in the field of urine metabolite detection, the prior art has the following disadvantages:

firstly, the existing methods detect more organic acids (see chinese patent, CN 102621262 a, 2012.08.01), and the methods detect a single kind of metabolites, and cannot meet the requirement of screening multiple kinds of metabolites by one-time analysis.

Secondly, the urine sample pretreatment process takes a long time (see chinese patent, CN 102621249B, 2014.01.08), and although a plurality of metabolic compounds can be detected by one-time analysis, the pretreatment cycle is long, and it is difficult to meet the requirement of batch or high-throughput detection.

Thirdly, most of the existing qualitative and quantitative modes need to add standard substances, the operation is complicated, and some standard substances are difficult to obtain or have hidden pollution hazards.

In conclusion, a rapid, accurate and high-throughput urine metabolite detection method is in urgent need of development.

Disclosure of Invention

Based on the above, the invention provides an accurate, convenient, efficient and high-flux method for detecting metabolites in urine, aiming at the defects of single metabolite detection species, long time consumption, need of standard substances and the like in the existing urine metabolite detection method.

The specific technical scheme is as follows:

a method for detecting metabolites in urine, comprising the steps of:

(1) removing impurities in the sample:

(1.1) removing urea: adding a urease aqueous solution into a urine sample, incubating, and removing urea in the urine sample to obtain urine A;

(1.2) removal of sulfur and phosphorus: adding an active adsorbent into the urine A, and removing sulfur and phosphorus in the urine A by ultrasonic treatment to obtain urine B;

(1.3) precipitation of proteins: adding a protein precipitation reagent into the urine B, uniformly mixing, centrifuging to precipitate the protein in the urine B, and taking supernatant to obtain urine C;

(2) and (3) drying: drying the urine C to obtain a urine residue D;

(3) oximation, namely adding an oximation reagent into the urine residue D to oximate β -carbonyl in the acid or alcohol metabolites to obtain urine E;

(4) derivatization: adding a derivatization reagent into the urine E for derivatization reaction to obtain a urine sample F to be tested; the derivatization reagent is a mixed reagent consisting of a silanization reagent, a catalyst and a catalyst stabilizer; the catalyst stabilizer is selected from dithioerythritol, dithiothreitol, 1-propanethiol, ethanethiol and 2-mercaptoethanol;

(5) and (3) detection: and (3) taking the urine sample F to be tested, and detecting by using a gas chromatography-mass spectrometry combined method.

In some embodiments, the detection conditions of the gas chromatography-mass spectrometry combination method are that the sample injection amount is 0.9-1.1 mu L, the split-flow mode is adopted for sample injection, the temperature of a sample injection port is 245-255 ℃, the carrier gas is helium, the flow rate is 1.1-1.3m L/min, the initial temperature of a column box is 55-65 ℃, the temperature of 17 ℃/min is increased to 315-325 ℃, the operation temperature is 315-325 ℃, the temperature is maintained for 7-9min, the temperature of a transmission pipe is 295-305 ℃, the ion source mode is an EI source, and the mass spectrometry detection mode is a full scan mode.

In some embodiments, the detection conditions of the gas chromatography-mass spectrometry combined method are that the sample injection amount is 1 mu L, the split-flow mode is adopted for sample injection, the temperature of a sample injection port is 250 ℃, the carrier gas is helium, the flow rate is 1.2m L/min, the initial temperature of a column box is 60 ℃, the temperature of 17 ℃/min is increased to 320 ℃, the operation temperature is kept for 8min, the temperature of a transmission pipe is 300 ℃, the ion source mode is an EI source, and the mass spectrometry detection mode is a full-scan mode.

In some of the embodiments, the silylation reagent in step (4) is selected from N-methyl-N- (trimethylsilane) -trifluoroacetamide, bis (trimethylsilane) trifluoroacetamide, N- (tert-butyldimethylsilane) -N-methyltrifluoroacetamide, tert-butyldimethylsilane or N, N-diethylaminotrimethylsilane, the catalyst is selected from trimethylchlorosilane, trimethyliodosilane or trimethylsilylimidazole, the mass ratio of the silylation reagent, the catalyst and the catalyst stabilizer is 100 μ L: 1 μ L: 0.2-0.5mg, and the volume ratio of the derivatization reagent to the urine sample is 0.25-1: 1.

In some embodiments, the ratio of the active adsorbent in step (1.2) to the urine sample is 0.5-25 mg: 1m L, and the active adsorbent is activated alumina.

In some of these embodiments, the time of the sonication in step (1.2) is 1-10 min.

In some of these embodiments, the protein precipitation reagent in step (1.3) is selected from one or more of methanol, ethanol, propanol, butanol, isopropanol, isobutanol, pyruvic acid, acetone, acetonitrile, pyridine; the volume ratio of the protein precipitation reagent to the urine sample is 2-15: 1.

In some of these embodiments, the drying of step (2) is a step drying selected from two of desiccant drying, nitrogen blower drying, or freeze drying.

In some embodiments, the drying in step (2) is drying with a desiccant and then blow-drying with a nitrogen blower or freeze-drying.

In some of these embodiments, the desiccant is selected from the group consisting of magnesium oxide, calcium sulfate, anhydrous copper sulfate, anhydrous magnesium sulfate, potassium carbonate, anhydrous sodium sulfate.

In some embodiments, the oximation reagent in step (3) is selected from pyridine, acetonitrile or triethylamine solution of methoxyamine hydrochloride or ethoxyamine hydrochloride, wherein the content of methoxyamine hydrochloride or ethoxyamine hydrochloride is 1-10 wt%; the volume ratio of the oximation reagent to the urine sample is 0.1-1: 1.

In some embodiments, the oximation and/or derivatization is performed under microwave conditions, the power of the microwave is 500-900W, and the reaction time of the oximation or derivatization is 1-15 min.

In some of these embodiments, the urine sample is selected from fresh urine, frozen urine, a urine filter paper sheet.

In some of these embodiments, the metabolites include bases, nucleosides, organic acids, ketones, aldehydes, and sugars.

The invention relates to a method for detecting metabolites in urine, which is based on the principle that human urine contains hundreds of human metabolic intermediate or terminal products, but simultaneously contains a large amount of substances interfering detection, such as urea, phosphate, sulfate, protein and the like, and the interference is firstly eliminated when accurate detection of the metabolites is required.

The invention also provides a kit for detecting metabolites in urine.

The specific technical scheme is as follows:

a kit for detecting metabolites in urine comprises a derivatization reagent, wherein the derivatization reagent is a mixed reagent of a silylation reagent, a catalyst and a catalyst stabilizer, and the catalyst stabilizer is selected from dithioerythritol, dithiothreitol, 1-propanethiol, ethanethiol and 2-mercaptoethanol.

In some of these embodiments, the silylating agent is selected from N-methyl-N- (trimethylsilane) -trifluoroacetamide, bis (trimethylsilane) trifluoroacetamide, N- (tert-butyldimethylsilane) -N-methyltrifluoroacetamide, tert-butyldimethylsilane or N, N-diethylaminotrimethylsilane, the catalyst is selected from trimethylchlorosilane, trimethyliodosilane or trimethylsilylimidazole, and the mass ratio of the silylating agent, the catalyst and the catalyst stabilizer is 100 μ L: 1 μ L: 0.2-0.5 mg.

In some of these embodiments, the kit further comprises an active sorbent for the removal of sulfur and phosphorus, the active sorbent being activated alumina.

In some of these embodiments, the kit further comprises the following components: (1) a reagent for removing urea, (2) a protein precipitating agent for removing protein, (3) a drying agent, and (4) an oximation reagent.

In some of these embodiments, the protein precipitation reagent is selected from one or more of methanol, ethanol, propanol, butanol, isopropanol, isobutanol, pyruvic acid, acetone, acetonitrile, pyridine.

In some embodiments, the oximation agent is selected from pyridine, acetonitrile or triethylamine solution of methoxyamine hydrochloride or ethoxyamine hydrochloride, wherein the content of methoxyamine hydrochloride or ethoxyamine hydrochloride is 1-10%.

The method for detecting the metabolites in the urine has the following advantages and beneficial effects:

the detection method provided by the invention is simple and rapid to operate, causes little loss to a target substance to be detected, has high accuracy and sensitivity of detection results, can be used for batch processing, can detect hundreds of metabolites in a urine sample at one time, has high analysis efficiency, uses few reagents, does not need standard substances, and has low cost and little pollution.

According to the detection method, the catalyst stabilizer is added into the traditional derivatization reagent in the derivatization step, so that the derivatization reaction is more thorough, and the detection result is more accurate.

The detection method further preferably selects the activated alumina as the active absorbent for removing sulfur and phosphorus, achieves good sulfur and phosphorus removal effect, reduces the loss of target metabolites, improves the accuracy of detection results, and is beneficial to the accurate detection of low-content metabolites.

The detection method of the invention further prefers to dry step by step in the drying step, and compared with one-step nitrogen blow drying, the detection method can remove moisture to the maximum extent on the basis of no loss of the target, is beneficial to the proceeding of oximation and derivatization reaction, and improves the accuracy of the detection result.

The detection method further preferably adopts microwave-assisted oximation and/or derivatization, so that the pretreatment time of the sample is greatly shortened, and oximation and derivatization can be completed within 1-15 min; the oximation reaction is carried out by using the traditional heating method, the same effect can be achieved only by reacting for 2 hours at 60 ℃, and the same effect can be achieved only by reacting for 1 hour at 70 ℃ when the traditional heating method is used for carrying out the derivatization reaction, which shows that the reaction time can be greatly shortened and the analysis efficiency can be improved by microwave-assisted oximation and/or derivatization.

The detection method of the invention uses the deconvolution function and the standard spectrum library retrieval function equipped by the Masshunter workstation, and can rapidly determine the nature of the compound; the ratio of the peak area of the target compound to the peak area of creatinine is adopted for quantification, the treatment is simple, and errors caused by operation or instruments can be reduced.

Drawings

FIG. 1 is a chromatogram of a fresh urine sample of example 1;

FIG. 2 is a chromatogram of a fresh urine sample of example 2;

FIG. 3 is a chromatogram of a fresh urine sample of example 3;

FIG. 4 is a chromatogram of a fresh urine sample of example 4;

FIG. 5 is a chromatogram of a fresh urine sample of example 5;

FIG. 6 is a chromatogram of a fresh urine sample of example 6;

FIG. 7 is a chromatogram of a fresh urine sample of example 7;

FIG. 8 is a chromatogram of a fresh urine sample of example 8;

FIG. 9 is a chromatogram of a fresh urine sample of example 9;

FIG. 10 is a chromatogram of a fresh urine sample of example 10;

FIG. 11 is a chromatogram of a fresh urine sample of example 11;

FIG. 12 is a chromatogram of a fresh urine sample of example 12;

FIG. 13 is a chromatogram of a fresh urine sample of example 13;

FIG. 14 is a chromatogram of a fresh urine sample of example 14;

FIG. 15 is a chromatogram of a fresh urine sample of example 15;

FIG. 16 is a chromatogram of a fresh urine sample of example 16;

FIG. 17 is a chromatogram of a fresh urine sample of example 17;

FIG. 18 is a chromatogram of a fresh urine sample of example 18;

FIG. 19 is a chromatogram of a fresh urine sample of example 19;

FIG. 20 is a chromatogram of a fresh urine sample of example 20;

FIG. 21 is a chromatogram of a fresh urine sample of example 21;

FIG. 22 is a chromatogram of a fresh urine sample of example 22;

FIG. 23 is a chromatogram of a fresh urine sample of example 23;

FIG. 24 is a chromatogram of a fresh urine sample of example 24.

Detailed Description

The method for detecting metabolites in urine according to the present invention will be further described with reference to the following examples.

Urease: purchased from Sigma-Aldrich, cat #: u1500-100 KU;

activated alumina: purchased from mcelin, cat # s: a820850;

example 1

The method for detecting metabolites in urine of the present embodiment includes the following steps:

(1) sampling, taking 200 mu L fresh urine as a urine sample to be tested.

(2) And (3) urea removal, namely adding 20 mu L of urease aqueous solution containing 100U of urease into the urine sample to be detected, and incubating for 10min at 37 ℃ to obtain urine A.

(3) Removing sulfur and phosphorus: adding 0.1mg of active adsorbent into the urine A, and performing ultrasonic treatment for 2min to obtain urine B; wherein the active adsorbent is active alumina.

(4) Precipitating protein by adding 1.7m L protein precipitation reagent (frozen methanol) into urine B, mixing, centrifuging to precipitate protein, and collecting supernatant, i.e. urine C.

(5) Drying step by step: and adding 5mg of anhydrous sodium sulfate into the urine C, uniformly mixing, centrifuging, taking supernate, and drying by using a nitrogen blowing instrument to obtain a urine residue D.

(6) And (3) microwave-assisted oximation, namely adding 50 mu L oximation reagent into the urine residue D, sealing the sample, placing the sample in a microwave reaction furnace with the power of 700W for reaction for 5min, and oximating β -carbonyl in acid or alcohol metabolites to obtain urine E, wherein the oximation reagent is pyridine solution containing 2 wt% of methoxyamine hydrochloride.

(7) And (3) microwave-assisted derivatization, namely adding 100 mu L of a derivatization reagent into the urine E, sealing the sample, and placing the sealed sample in a microwave reaction furnace with the power of 700W for reaction for 2min to obtain a test urine sample F, wherein the derivatization reagent is a mixed reagent of N-methyl-N- (trimethylsilane) -trifluoroacetamide, trimethylchlorosilane and dithioerythritol, and the mixed reagent is 1000 mu L, 10 mu L and 4 mg.

(8) And (3) detecting by taking a urine sample F to be tested on a machine and using an Agilent 7890B-5977A type gas mass spectrometer with an HP-5ms (30m × 250 μm × 0.25 μm) chromatographic column.

The method comprises the specific parameters of sample introduction amount of 1 mu L, split-flow mode sample introduction, sample introduction port temperature of 250 ℃, helium gas as carrier gas, flow rate of 1.2m L/min, solvent delay of 4.5min, column box initial temperature of 60 ℃, temperature rise of 17 ℃/min to 320 ℃, operation temperature of 320 ℃, holding time of 8min, transmission pipe temperature of 300 ℃, ion source mode of EI source, mass spectrum detection mode of full scanning mode, scanning period of 0.4s and sample analysis time of 23 min.

(9) And (4) analyzing results: performing spectrum analysis by using an Agilent Masshunter workstation, performing qualitative analysis on a target by using a standard spectrum library retrieval function, performing quantitative analysis by using a peak area ratio of a target characteristic ion to a creatinine characteristic ion, and measuring 248 compounds, wherein a chromatogram is shown in figure 1, and a quantitative result is shown in figure 1.

Examples 2 to 4

Example 2-4A method for detecting metabolites in urine, which uses the same urine sample as in example 1,

the procedure is as in example 1 except for the differences indicated in the table below. The chromatograms are shown in FIGS. 2-4, and the quantitative results are shown in Table 1.

Examples	Example 2	Example 3	Example 4
				Active adsorbent	2mg activated alumina	5mg activated alumina	100μLBaCl₂0.1 mol/L aqueous solution

TABLE 1 comparison of analytical results of examples 1-4

As can be seen from Table 1, compared with example 4, the loss of sulfur and phosphorus to the target metabolites is small in examples 1 to 3 by using activated alumina, especially the determination result of the target metabolites with low content of metabolites such as reference interval <5 is more accurate, and the barium agent may cause that the low content of metabolites cannot be detected; a large number of experiments show that 0.1-5mg of active alumina can achieve the effects of removing phosphorus and sulfur without influencing the detection of target metabolites.

Examples 5 to 8

Example 5-8A method for measuring metabolites in urine, which uses a urine sample identical to that of example 1, was performed in the same manner as in example 1 except that the amount of activated alumina added in step (3) was 1mg and the differences shown in the following table. The chromatograms are shown in FIGS. 5-8, and the quantitative results are shown in Table 2.

Examples	Example 5	Example 6	Example 7	Example 8
					Protein precipitation reagent	Frozen ethanol	Frozen propanol	Freezing butanol	Freezing methanol: acetonitrile (V/V, 4/1)

Table 2 examples 5-8 analysis results (only 40 data are shown randomly as an illustration)

As can be seen from Table 2, the effect of using ethanol, propanol, butanol, methanol/acetonitrile (V/V, 4/1) and the like as the protein-precipitating agent was not very different. The inventor finds out through a large number of experiments that the purpose of accurately measuring the target metabolite can be achieved by selecting one or more of methanol, ethanol, propanol, butanol, isopropanol, isobutanol, pyruvic acid, acetone, acetonitrile and pyridine as the precipitation reagent.

Examples 9 to 12

Examples 9-12A method for detecting metabolites in urine using the same urine sample as in example 1 was performed in the same manner as in example 5 except for the differences shown in the following table. The chromatograms are shown in FIGS. 9-12, and the quantitative results are shown in Table 3.

Table 3 comparison of analytical results for examples 9-12 (only 40 data are shown at random as an illustration).

As can be seen from Table 3, any one of pyridine solution containing 1% of ethoxyamine hydrochloride, acetonitrile solution containing 1% of methoxyamine hydrochloride, triethylamine solution containing 1% of methoxyamine hydrochloride and pyridine solution containing 10% of methoxyamine hydrochloride can be used as an oximation reagent to achieve the purpose of detecting metabolites in urine samples, and the effect difference is small. The inventor finds through a large number of experiments that the oximation reagent can meet the requirements of a pyridine solution containing 1-10% of methoxyamine hydrochloride or ethoxyamine hydrochloride, an acetonitrile solution containing 1-10% of methoxyamine hydrochloride or ethoxyamine hydrochloride and a triethylamine solution containing 1-10% of methoxyamine hydrochloride or ethoxyamine hydrochloride, wherein the pyridine solution containing 2% of methoxyamine hydrochloride is the best choice because the use amount is the least under the condition of complete oximation.

Examples 13 to 16

Example 13-16A method for detecting metabolites in urine, which uses a urine sample identical to that of example 1, was performed in the same manner as in example 1 except that the amount of activated alumina added in step (3) was 1mg and the differences shown in the following table. The chromatograms are shown in FIGS. 13-16, and the quantitative results are shown in Table 4.

Table 4 comparison of analytical results of examples 13 to 16 (only 40 data are shown at random as an illustration)

As can be seen from Table 4, the microwave-assisted oximation in examples 13-15 compared with the conventional heating method in example 16 can greatly shorten the reaction time and improve the analysis efficiency while achieving the same effect. The inventor finds that microwave-assisted oximation can meet the requirement of detecting metabolites in urine samples within 1-15min through a large number of experiments, and the effect difference is not obvious.

Examples 17 to 20

Examples 17-20A method for detecting metabolites in urine, which uses a urine sample identical to that of example 1, was performed in the same manner as in example 1 except that the amount of activated alumina added in step (3) was 1mg and the differences shown in the following table. The chromatograms are shown in FIGS. 17-20, and the quantitative results are shown in Table 5.

TABLE 5 comparison of analytical results of examples 17 to 20 (only 40 data are shown at random as an illustration)

As can be seen from Table 5, examples 17 to 19, although the compositions of the derivatization reagents were different, could achieve the purpose of accurately detecting the target metabolite in the urine sample, and the results were not very different, and the inventors found through a large number of experiments that the target metabolite was not detected when the silylation reagent was selected from N-methyl-N- (trimethylsilane) -trifluoroacetamide, bis (trimethylsilane) trifluoroacetamide, N- (t-butyldimethylsilane) -N-methyltrifluoroacetamide, t-butyldimethylchlorosilane or N, N-diethylaminotrimethylsilane, the catalyst was selected from trimethylchlorosilane, trimethyliodosilane or trimethylsilylimidazole, the catalyst stabilizer was selected from dithioerythritol, dithiothreitol, 1-propanethiol, ethanethiol and 2-mercaptoethanol, and the mass ratio of the silylation reagent, the catalyst and the catalyst stabilizer was 1000. mu. L: 10. mu. L: 2-5mg, the derivatization reagent in example 20 was not completely derivatized with the conventional derivatization reagent without the catalyst stabilizer, and the quantitative result of the target metabolite was lower than that in general and part of the low metabolite was not detected.

Examples 21 to 24

Example 21-24A method for detecting metabolites in urine using the same urine sample as in example 1, the same procedure as in example 1 was repeated except that 1mg of activated alumina was added in step (3), the derivatizing agent in step (7) was a mixed reagent of N-methyl-N- (trimethylsilane) -trifluoroacetamide, trimethylchlorosilane, dithiothreitol (1000. mu. L: 10. mu. L: 4mg) and the differences shown in the following table, the chromatograms were as shown in FIG. 21-FIG. 24, and the quantitative results were as shown in Table 6.

TABLE 6 comparison of analytical results of examples 21 to 24 (only 40 data are shown at random as an illustration)

Note: the reference interval in tables 1-6 refers to the range of metabolite concentrations in normal human urine measured using the present protocol, measured as the metabolite concentration of the urine sample measured using the present protocol, in units of mmol/mol creatinine.

As can be seen from Table 6, the microwave-assisted derivatization in examples 21 to 23 can greatly shorten the reaction time and improve the analysis efficiency while achieving the same detection effect as the derivatization in example 24 by the conventional heating method. The inventor finds that the microwave-assisted derivatization can meet the requirement of detecting the metabolites in the urine sample within 1-15min through a large number of experiments, and the effect is not greatly different.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A method for detecting metabolites in urine, comprising the steps of:

(1) removing impurities in the sample:

(1.2) removal of sulfur and phosphorus: adding an active adsorbent into the urine A, and removing sulfur and phosphorus in the urine A by ultrasonic treatment to obtain urine B; the active adsorbent is active alumina;

(2) and (3) drying: drying the urine C to obtain a urine residue D;

(3) oximation, namely adding an oximation reagent into the urine residue D to oximate β -carbonyl in the acid or alcohol metabolites to obtain urine E, wherein the oximation is carried out under the microwave condition, the power of the microwave is 500-900W, and the reaction time is 1-15 min;

(4) derivatization: adding a derivatization reagent into the urine E for derivatization reaction to obtain a urine sample F to be tested; the derivatization reagent is a mixed reagent consisting of a silanization reagent, a catalyst and a catalyst stabilizer; the catalyst stabilizer is selected from dithioerythritol, dithiothreitol, 1-propanethiol, ethanethiol and 2-mercaptoethanol; the derivatization is carried out under the microwave condition, the power of the microwave is 500-900W, and the reaction time is 1-15 min;

2. The method for detecting the metabolites in the urine according to claim 1, wherein the silylation reagent in the step (4) is selected from N-methyl-N- (trimethylsilane) -trifluoroacetamide, bis (trimethylsilane) trifluoroacetamide, N- (tert-butyldimethylsilane) -N-methyltrifluoroacetamide, tert-butyldimethylchlorosilane or N, N-diethylaminotrimethylsilane, the catalyst is selected from trimethylchlorosilane, trimethyliodosilane or trimethylsilylimidazole, the ratio of the silylation reagent to the catalyst stabilizer is 100 μ L: 1 μ L: 0.2-0.5mg, and the volume ratio of the derivatization reagent to the urine sample is 0.25-1: 1.

3. The method for detecting the metabolites in the urine according to claim 1 or 2, wherein the ratio of the active adsorbent in the step (1.2) to the urine sample is 0.5-25 mg: 1m L.

4. The method for detecting the metabolites in the urine according to claim 1 or 2, wherein the protein precipitation reagent in step (1.3) is selected from one or more of methanol, ethanol, propanol, butanol, isopropanol, isobutanol, pyruvic acid, acetone, acetonitrile, pyridine; the volume ratio of the protein precipitation reagent to the urine sample is 2-15: 1.

5. The method for detecting metabolites in urine according to claim 1 or 2, wherein the drying in step (2) is a step drying, and the drying is selected from two of drying agent drying, nitrogen blowing or freeze drying.

6. The method for detecting metabolites in urine according to claim 1 or 2, wherein the oximation reagent in step (3) is selected from pyridine, acetonitrile or triethylamine solution of methoxyamine hydrochloride or ethoxyamine hydrochloride, wherein the content of methoxyamine hydrochloride or ethoxyamine hydrochloride is 1-10 wt%; the volume ratio of the oximation reagent to the urine sample is 0.1-1: 1.

7. The method for detecting metabolites in urine according to claim 1 or 2, wherein the oximation and/or derivatization is performed under microwave conditions, wherein the power of the microwave is 700W, and the reaction time of the oximation or derivatization is 1-15 min.

8. The method according to claim 1 or 2, wherein the metabolites include bases, nucleosides, organic acids, ketones, aldehydes, and sugar metabolites.

9. The kit for detecting the metabolites in urine is characterized by comprising a derivatization reagent and an active adsorbent for removing sulfur and phosphorus, wherein the derivatization reagent is a mixed reagent of a silylation reagent, a catalyst and a catalyst stabilizer, the catalyst stabilizer is selected from dithioerythritol, dithiothreitol, 1-propanethiol, ethanethiol and 2-mercaptoethanol, and the active adsorbent is active alumina.