CN112098534A

CN112098534A - Method for detecting metabolite and kit thereof

Info

Publication number: CN112098534A
Application number: CN202010807231.9A
Authority: CN
Inventors: 赵伟军; 周伟; 史夏青; 章旭日; 汪小知; 张厚德; 郝雷; 段兴斌
Original assignee: Biruisi Hangzhou Technology Co ltd
Current assignee: Biruisi Hangzhou Technology Co ltd
Priority date: 2020-11-18
Filing date: 2020-11-18
Publication date: 2020-12-18

Abstract

The invention provides a method for detecting metabolites and a kit thereof, belonging to the technical field of metabolic detection. The method solves the problems that the existing metabolite generated by human body metabolism is trace, the existing detection method is difficult to accurately and quantitatively detect the components in exhaled breath or body fluid, and the like, and the method for detecting the human metabolite comprises the following steps: s01: the booster is ingested by the subject and then the body fluid or exhaled breath of the subject is collected; s02: converting the exhaled gas or body fluid into an object to be tested; s03: and detecting the content of the target detection object in the body fluid or the exhaled breath. The invention has the advantages of high detection sensitivity and the like.

Description

Method for detecting metabolite and kit thereof

Technical Field

The invention belongs to the technical field of metabolic detection, and particularly relates to a method for detecting a metabolic product and a kit thereof.

Background

The exhaled air, body fluid and the living metabolism of the human body and the living metabolism of bacteria are closely related to each other, while the variety of bacteria in the human body is various, and the composition of flora can greatly influence the health condition of the human body. The gas or body fluid exhaled by the human body is analyzed by a specific detection means, so that the difference of numerical values is distinguished, the metabolic condition in the human body can be indirectly reflected, and the examination direction of subsequent diseases is provided for medical personnel.

However, because the existing metabolic products generated by human body metabolism are trace, the existing detection method is difficult to accurately and quantitatively detect the components in exhaled breath or body fluid.

Disclosure of Invention

The first object of the present invention is to solve the above problems in the prior art, and to provide a method for detecting metabolites; the second object of the present invention is to provide a kit for detecting metabolites.

The first object of the present invention can be achieved by the following technical solutions: a method for detecting metabolites in a human body, comprising the steps of:

s01: the booster is ingested by the subject and then the body fluid or exhaled breath of the subject is collected;

s02: converting the exhaled gas or body fluid into an object to be tested;

s03: and detecting the content of the target detection object in the body fluid or the exhaled breath.

Preferably, the enhancer is a substance containing saccharides having a glycemic index of greater than 20.

Preferably, in step S01, the intake of saccharide is 0.1g or more.

Preferably, in step S02, the exhaled gas is converted into the analyte by using a selective adsorption device; the selective adsorption device is an adsorption sampling tube filled with a stationary phase, and the stationary phase is coated with a coating liquid containing a derivative reagent prepared by using an organic solution; the derivative reagent is selected from one of 2, 4-dinitrophenylhydrazine, pentafluorophenylhydrazine and O- (2,3,4,5, 6-pentafluorobenzyl) hydroxylamine hydrochloride.

Preferably, the content of the derivatization reagent in the coating liquid is more than or equal to 0.01 percent, and the coating liquid is acidic. The volume content of 36-38% concentrated hydrochloric acid in the coating liquid is 0.1%.

Preferably, in step S02, the air bag is heated at a constant temperature, the exhaled air from the human body is introduced into the adsorption sampling tube at a constant flow rate, the adsorption sampling tube is eluted with an eluent, and the filtered solution is diluted with a constant volume agent to obtain a solution to be tested.

Preferably, the eluent and the constant volume agent are one or a mixture of two of n-hexane, ethyl acetate and acetonitrile.

Preferably, in step S01, the exhaled breath is collected by using the pre-treated air bag, and the treatment step is to introduce nitrogen or helium into the air bag at a temperature of 60-80 ℃, to make the gas stay in the air bag for at least 30 minutes, to extract the gas and to introduce new nitrogen or helium, and to repeat the steps for at least 4 times.

The first object of the present invention can be achieved by the following technical solutions: a metabolic capability detection kit is characterized by comprising an enhancer, a collecting device, a selective adsorption device, an eluent and a constant volume liquid.

Preferably, the selective adsorption device is an adsorption column filled with a stationary phase, and the stationary phase is coated with a coating liquid containing a derivatization reagent prepared from an organic solution; the derivative reagent is selected from one of 2, 4-dinitrophenylhydrazine, pentafluorophenylhydrazine and O- (2,3,4,5, 6-pentafluorobenzyl) hydroxylamine hydrochloride.

The working principle of the invention is as follows: the metabolic capacity of different people is reflected by the intake of the reinforcer, collecting exhaled air or body fluid of the human body and analyzing the concentration change curve of target products in the exhaled air.

Compared with the prior art, the invention has the following advantages:

1. the method has high sensitivity, and can quantify the acetoin in 62ng/L (calculated as the acetoin, 3L of exhaled gas) gas at the lowest. Good stability, and the RSD% is controlled within 5%. The precision is high, and the relative standard deviation of the quantitative result measured at different times is controlled within 15 percent.

2. On the basis of a reliable value, the method obtains a variation curve of the acetoin value of the person, so that the carbohydrate metabolism conditions of different persons can be accurately judged, and the variation curve is used for representing the microbial activity of the digestive tract.

3. The air bag needs to be pretreated, so that the influence of materials on the method is reduced, and the reliability is improved.

4. On the basis of a reliable value, the method obtains a variation curve of the acetoin value of the person, so that the carbohydrate metabolism conditions of different persons can be accurately judged, and the variation curve is used for representing the microbial activity of the digestive tract.

5. The derivatization reagent can be adsorbed on the stationary phase, acetoin can perform chemical reaction with the derivatization reagent coated on the stationary phase and has continuity, and the generated product can be eluted from the stationary phase.

6. The method has high reaction selectivity, and the adsorption sampling tube can only selectively absorb aldehyde and ketone substances but not other substances in exhaled air of a human body, so that the possible influence of other substrates in the exhaled air on acetoin is reduced, and the reliability of the method is improved.

7. After the air bag collects the exhaled air of the human body, the temperature of the air bag is between 60 and 80 ℃, namely, the exhaled air of the human body is heated and treated at constant temperature, so that the stability of a sample is improved, and the subsequent detection result is more accurate.

8. According to the invention, by means of ingestion of the enhancer, the content of acetoin in exhaled breath is amplified under the condition of consistent control of the ingestion amount, the sensitivity is enhanced, the error is reduced, and random interference on the result under a low numerical value is avoided.

Drawings

FIG. 1 is a schematic view showing the carbohydrate metabolism in three persons according to example 2 of the present invention;

FIG. 2 is a schematic representation of carbohydrate metabolism in a first person according to example 3 of the present invention;

FIG. 3 is a schematic representation of sugar metabolism in a second individual according to example 3 of the present invention;

FIG. 4 is a schematic representation of sugar metabolism in a third individual according to example 3 of the present invention.

Detailed Description

The following is a detailed embodiment of the present invention and the accompanying drawings are incorporated to illustrate the technical solution of the present invention

Further, the present invention is not limited to these examples.

(1) Preparation of Selective adsorption apparatus

Preparing an adsorption sampling tube, namely preparing a coating liquid containing a derivative reagent and with the content of 0.01-1% by using organic solvents such as n-hexane, ethyl acetate, acetonitrile and the like; the volume content of 36-38% concentrated hydrochloric acid in the coating liquid is 0.1%. Then filling 200mg of stationary phase filler (silica gel, C18, tenax-GR and the like can be selected) on the blank adsorption column; and finally, coating the coating liquid on an adsorption column filled with a stationary phase, wherein the coating method is carried out by adopting a direct coating method.

As the derivatizing reagent, 2, 4-Dinitrophenylhydrazine (DNPH), pentafluorophenylhydrazine (PFPH), O- (2,3,4,5, 6-pentafluorobenzyl) hydroxylamine hydrochloride (PFBHA) can be used.

Before coating, a blank adsorption column is firstly rinsed by corresponding solvents such as normal hexane, ethyl acetate, acetonitrile and the like, a negative pressure solid phase extraction system is used for removing rinsed organic reagents, then coating liquid is directly coated on the adsorption column, after the coating is saturated, a negative pressure solid phase extraction system is used for removing redundant coating liquid, a selective adsorption sampling tube capable of selectively adsorbing the exhaled breath aldehyde ketone substances is prepared, the adsorption column is dried by nitrogen, and the selective adsorption sampling tube is sealed and stored at 4 ℃ for standby.

And (3) carrying out constant temperature treatment on the gas in the gas bag, wherein the constant temperature treatment temperature can be selected from 40-70 ℃, then introducing the human body exhaled gas into the adsorption sampling tube at a constant flow rate (the flow rate is selected from 5-1000ml/min), then respectively eluting the adsorption sampling tube by using an eluent, and filtering to obtain the liquid to be detected.

Pretreatment of gas bags

The air bag is pretreated, and can be a Taidela air bag or a nalophen or PFV or air bag made of other materials.

And (3) putting the air bag into a constant temperature control box, setting the temperature at 60 ℃, filling nitrogen into the air bag, keeping the nitrogen in the air bag for 3-5 minutes, extracting the nitrogen, introducing the nitrogen again, repeating the steps for 4 times, and then carrying out cleaning verification on the air bag. The temperature, number of purges, gas introduced can be varied, and the other steps are as above.

Cleaning and verifying of the air bag: and after the air bag is cleaned, 3L of nitrogen is filled, the air bag is heated at the constant temperature of 50 ℃, the air bag is absorbed by an absorption sampling tube, all the gas in the bag is absorbed, the absorption sampling tube is eluted by a solvent and is subjected to constant volume to 2ml, and a filtration test background is obtained, and the bag can be used only after the background test meets the requirement (the quantitative result of the marker measured in the background of the sampling bag is not more than 10ug counted by formaldehyde).

Experiments prove that: when the temperature is 60-80 ℃, introducing nitrogen or helium into the air bag, staying in the air bag for 30 minutes or more, extracting and introducing, and repeating for 4-8 times, wherein the background test can meet the requirement.

Example 1

The reinforcing agent is configured: according to the weight ratio of glucose and water 1: 4-50, and preparing into aqueous glucose solution. The subject ingests an aqueous glucose solution orally, and total glucose is ingested in an amount of at least 0.1 g. After ingestion, the gas exhaled by the human body was collected by the pretreated air bags and collected by the air bags at 10 minutes, 25 minutes, and 55 minutes, respectively. (the time points for the air bag to collect exhaled human body gases include, but are not limited to, 10 minutes, 25 minutes, 55 minutes.)

Preparation of adsorption sampling tube

Preparing 2, 4-Dinitrophenylhydrazine (DNPH) into a coating liquid with DNPH content of 0.1% by using n-hexane; preparing an adsorption sampling tube according to the step (1), and rinsing the blank adsorption column by using n-hexane before coating.

Collection and treatment of exhaled air from human body

After the glucose aqueous solution is drunk, 3L of exhaled air of the human body is collected by the pretreated air bag; after this time, collection was carried out with air bags at intervals of 10 minutes, 25 minutes and 55 minutes. The human body exhaled air flows through the adsorption sampling tube at the flow rate of 50ml/min, and the temperature of the adsorption sampling tube and the air bag in the process is controlled at 40 ℃. And eluting the adsorption sampling tube by using eluant n-hexane, and fixing the volume to 2ml by using the n-hexane to be detected.

Detection of liquid to be detected by gas chromatography-tandem mass spectrometer

Selecting an acetoin-DNPH standard solution as the standard solution of the invention, and quantifying the acetoin in the exhaled breath according to an external standard method. And detecting the standard solution by using a gas chromatography-tandem mass spectrometer, and drawing a linear regression working curve by using the concentration content in the standard solution as a horizontal coordinate and the corresponding measured peak area as a vertical coordinate. The standard curve concentrations are shown in table 1.

TABLE 1 Standard Curve concentrations

The detection conditions of the gas chromatograph-mass spectrometer are as follows:

sample inlet temperature: 250 deg.C

A chromatographic column: rxi-5Sil MS,30 m.times.0.25 mm.times.0.25 μm

Column temperature procedure: 40 ℃ (1min) _15 ℃/min _300 ℃ (2min)

And (3) sample introduction mode: without diversion

The collection mode is as follows: MRM

Detector voltage: relative to tuning +0.8 KV;

the parameter settings are shown in table 2.

TABLE 2 parameter settings

The detection sensitivity of the method takes the signal-to-noise ratio 9 as a limit of quantification, and 2272ng/L can be detected in the gas components at the lowest. The instrumental detection sensitivity of acetoin is shown in table 3.

TABLE 3 instrumental detection sensitivity of acetoin

	(ppt)	(ppt)
			Concentration of reagent	1136.36	3409.09
Concentration in breath (3L)	757.58	2272.72

Example 2

The reinforcing agent is configured: according to the weight ratio of starch to water at 100 ℃ of 1: 2-100, and preparing into edible starch solution. The total intake of starch was 75g, and the subjects ingested orally. The gas exhaled by human body is collected by the air bag after pretreatment before intake, and after intake, the gas is collected by the air bag at 0,5,30,60,90 and 120 minutes. (the point in time at which the air bag collects exhaled breath from the body includes, but is not limited to, the above time.)

Preparation of adsorption sampling tube

Preparing 2, 4-Dinitrophenylhydrazine (DNPH) into a coating solution with the content of a derivatization reagent of 0.1 percent by using acetonitrile; preparing an adsorption sampling tube according to the step (1), and rinsing the blank adsorption column by using acetonitrile before coating.

Collection and treatment of exhaled air from human body

The human body exhaled air flows through the adsorption sampling tube at the flow rate of 100ml/min, and the temperature of the adsorption sampling tube and the air bag in the process is controlled at 70 ℃. And then eluting the adsorption sampling tube by using an eluent acetonitrile, and fixing the volume to 2ml by using the acetonitrile to be detected.

Detection of liquid chromatogram-tandem mass spectrum combined instrument on solution to be detected

Selecting an acetoin-DNPH standard solution as the standard solution of the invention, and quantifying the acetoin in the exhaled breath according to an external standard method. And detecting the standard solution by using a liquid chromatography-tandem mass spectrometer, and drawing a linear regression working curve by taking the concentration content in the standard solution as a horizontal coordinate and the corresponding measured peak area as a vertical coordinate. The standard curve concentrations are shown in table 4.

TABLE 4 Standard Curve concentrations

The set parameters of the liquid phase chromatography apparatus are shown in Table 5, and the mobile phase is selected from A0.5 mmol/L ammonium acetate buffer solution + B acetonitrile:

TABLE 5 liquid chromatograph parameters

Time (min)	Mobile phase A%	Mobile phase B%	Remarks for note
				3	50	50	Is free of
5	40	60	Is free of
				7.5	40	60	Is free of
8	30	90	Is free of
				9.5	30	90	Is free of
10	50	50	Is free of
				Program termination

The set mass spectrometer instrument parameters are shown in table 6:

TABLE 6 Instrument parameters

Name of Compound	m/z	Retention time	Event number	Reference ion
					Acetoin	267.00>152.00	4.632	2:MRM(-)	267.00>122.00

The detection sensitivity of the method takes a signal-to-noise ratio 9 as a quantitative limit, and the lowest energy in gas components reaches 62 ng/L. The sensitivity is shown in Table 7.

TABLE 7 instrumental detection sensitivity of acetoin

The stability is good, and the RSD% is controlled within 2%. The precision is high, and the relative standard deviation of all the markers is controlled within 8 percent (standard quality control of all the markers) according to the quantitative result of different time tests. Table 8 shows the stability measurements of the quantitative results of the markers during the control of the quality of the markers during the day and during the day. 50ug/L of acetoin-2, 4-dinitrophenylhydrazone is used as a quality control product, namely the content of the acetoin-DNPH in the quality control product is measured in different time by using the detection method, and the amount of the acetoin is obtained by calculation, so that the stability of the detection method is detected.

TABLE 8 stability measurements of daily and intraday quantitative results for marker substances

By means of ingestion of the enhancer, the content of acetoin in exhaled breath is amplified under the condition of consistent control of the ingestion amount, the sensitivity is enhanced, errors are reduced, and random interference on results under low numerical values is avoided. FIG. 1 is a graph showing the results of tests performed on 3 persons at different times after ingestion of starch, and it is seen that the characteristics of the curves of the persons are different, and the curves are demarcated by dotted lines, and the curves with high peaks or slow recovery peaks at the upper parts of the dotted lines represent that the microorganisms in the digestive tract are active in sugar metabolism, but not active in the other.

Example 3

The reinforcing agent is configured: according to lactose and 100 ℃ water 1: 2-100, and preparing into edible lactose solution. Lactose is ingested in a total amount of at least 10g and orally by the subject. After ingestion, the gas exhaled by the human body was collected by the air bag after pretreatment, and the air bag was used for collection at 5 minutes, 30 minutes, 60 minutes, and 90 minutes, respectively, while collecting saliva at that time point.

Preparation of adsorption sampling tube

Collection and treatment of exhaled air from human body

After the lactose solution is drunk, 3L of exhaled air of the human body is collected by the air bag after pretreatment; thereafter, collection was performed with an air bag at 5 minutes, 30 minutes, 60 minutes, and 90 minutes, respectively. The human body exhaled air flows through the adsorption sampling tube at the flow rate of 80ml/min, and the temperature of the adsorption sampling tube and the air bag in the process is controlled at 70 ℃. And then eluting the adsorption sampling tube by using an eluent acetonitrile, and fixing the volume to 2ml by using the acetonitrile to be detected.

Human saliva Collection and processing

After the lactose solution is drunk, 3L of exhaled air of the human body is collected by the air bag after pretreatment; thereafter, about 1ml of saliva was collected with a disposable sputum cup at 5 minutes, 30 minutes, 60 minutes, and 90 minutes, respectively. Saliva is sucked up by 50ul, added into acetonitrile eluent, and then the adsorption sampling tube is eluted by the mixed liquid and is made to 2ml by acetonitrile. And sealing the solution, and then placing the solution in a 50 ℃ oven for reaction for 1h to be tested.

The same as in example 2.

After the lactose is ingested, the acetoin components in the exhaled breath and the saliva are tested simultaneously, so that the aims of mutual verification and error reduction can be achieved. (see FIGS. 2-4, change in values before and after ingestion by 3 subjects). FIG. 3 is a carbohydrate metabolism profile of a first person of the invention; FIG. 3 is a carbohydrate metabolism profile of a second individual of the invention; FIG. 4 is a carbohydrate metabolism profile of a third individual of the invention.

Composition of the kit

The kit comprises a kit A adopting a liquid chromatogram-tandem mass spectrometer for detection and a kit B adopting a gas chromatogram-tandem mass spectrometer for detection

Items included in kit a are shown in table 9, items included in kit B are shown in table 10, and the contents should be verified according to the above-described method. The kit is stored at 4-8 ℃.

TABLE 9 kit Contents

TABLE 10 kit Contents

The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.

Claims

1. A method for detecting a metabolite, comprising the steps of:

s02: converting the exhaled gas or body fluid into an object to be tested;

2. The method according to claim 1, wherein the booster is a substance containing a saccharide having a glycemic index of greater than 20.

3. The method for detecting metabolites, according to claim 1, wherein the carbohydrate is ingested in an amount of 0.1g or more in step S01.

4. The method according to claim 1, wherein in step S02, the exhaled air is converted into the analyte by using a selective adsorption device; the selective adsorption device is an adsorption sampling tube filled with a stationary phase, and the stationary phase is coated with a coating liquid containing a derivative reagent prepared by using an organic solution; the derivative reagent is selected from one of 2, 4-dinitrophenylhydrazine, pentafluorophenylhydrazine and O- (2,3,4,5, 6-pentafluorobenzyl) hydroxylamine hydrochloride.

5. The method for detecting the metabolites according to claim 1, wherein the content of the derivatizing reagent in the coating solution is not less than 0.01%, and the coating solution is acidic.

6. The method according to claim 1, wherein in step S02, the air bag is heated at a constant temperature, the exhaled air from the human body is introduced into the adsorption sampling tube at a constant flow rate, the adsorption sampling tube is eluted with an eluent, and the filtrate is filtered and then diluted with a constant volume agent to obtain the solution to be tested.

7. The method for detecting the metabolites according to claim 1, wherein the eluent and the constant volume agent are one or a mixture of two of n-hexane, ethyl acetate and acetonitrile.

8. The method of claim 1, wherein the exhaled breath is collected by a pre-treated air bag in step S01, and the treatment step comprises introducing nitrogen or helium into the air bag at a temperature of 60-80 ℃ to allow the air to stay in the air bag for at least 30 minutes, withdrawing the air bag and introducing new nitrogen or helium, and repeating the steps at least 4 times.

9. A metabolic capability detection kit is characterized by comprising an enhancer, a collecting device, a selective adsorption device, an eluent and a constant volume liquid.

10. The kit for detecting metabolic capability according to claim 9, wherein the selective adsorption device is an adsorption column filled with a stationary phase, and the stationary phase is coated with a coating solution containing a derivatizing reagent prepared from an organic solution; the derivative reagent is selected from one of 2, 4-dinitrophenylhydrazine, pentafluorophenylhydrazine and O- (2,3,4,5, 6-pentafluorobenzyl) hydroxylamine hydrochloride.