CN114965743A - Metabolite two-dimensional center cutting gas chromatography-mass spectrometry separation method - Google Patents
Metabolite two-dimensional center cutting gas chromatography-mass spectrometry separation method Download PDFInfo
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- 238000000926 separation method Methods 0.000 title claims abstract description 34
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 title claims abstract description 26
- 239000002207 metabolite Substances 0.000 title claims description 19
- 238000004321 preservation Methods 0.000 claims abstract description 27
- 238000010438 heat treatment Methods 0.000 claims abstract description 12
- 239000007789 gas Substances 0.000 claims description 69
- 238000001819 mass spectrum Methods 0.000 claims description 15
- XYHKNCXZYYTLRG-UHFFFAOYSA-N 1h-imidazole-2-carbaldehyde Chemical compound O=CC1=NC=CN1 XYHKNCXZYYTLRG-UHFFFAOYSA-N 0.000 claims description 10
- WLAMNBDJUVNPJU-UHFFFAOYSA-N 2-methylbutyric acid Chemical compound CCC(C)C(O)=O WLAMNBDJUVNPJU-UHFFFAOYSA-N 0.000 claims description 10
- GWYFCOCPABKNJV-UHFFFAOYSA-M 3-Methylbutanoic acid Natural products CC(C)CC([O-])=O GWYFCOCPABKNJV-UHFFFAOYSA-M 0.000 claims description 10
- GWYFCOCPABKNJV-UHFFFAOYSA-N beta-methyl-butyric acid Natural products CC(C)CC(O)=O GWYFCOCPABKNJV-UHFFFAOYSA-N 0.000 claims description 10
- UAXOELSVPTZZQG-UHFFFAOYSA-N tiglic acid Natural products CC(C)=C(C)C(O)=O UAXOELSVPTZZQG-UHFFFAOYSA-N 0.000 claims description 10
- 230000001681 protective effect Effects 0.000 claims description 9
- 239000001307 helium Substances 0.000 claims description 7
- 229910052734 helium Inorganic materials 0.000 claims description 7
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 claims description 7
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- SYSQUGFVNFXIIT-UHFFFAOYSA-N n-[4-(1,3-benzoxazol-2-yl)phenyl]-4-nitrobenzenesulfonamide Chemical class C1=CC([N+](=O)[O-])=CC=C1S(=O)(=O)NC1=CC=C(C=2OC3=CC=CC=C3N=2)C=C1 SYSQUGFVNFXIIT-UHFFFAOYSA-N 0.000 description 1
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/52—Physical parameters
- G01N30/54—Temperature
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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Abstract
The invention belongs to the technical field of analytical chemistry, and discloses a two-dimensional center cutting gas chromatography-mass spectrometry separation system, which comprises a gas phase column incubator system and a valve body column incubator system; the gas column incubator system comprises a one-dimensional chromatographic column, a two-dimensional chromatographic column and a gas column incubator; the valve body column temperature box comprises a six-way valve A, a six-way valve B, a six-way valve bracket, a column temperature box body and a heating module; the heating module is positioned near the six-way valve A and the six-way valve B and is used for heating the six-way valve A and the six-way valve B; one end of the one-dimensional chromatographic column is communicated with the sample inlet through a pipeline, and the other end of the one-dimensional chromatographic column is communicated with A1 of the six-way valve A through a connecting two-way valve A, a heat preservation pipe 1 and a pipeline; one end of the two-dimensional chromatographic column is communicated with A2 of the six-way valve A through a connecting two-way valve B, a heat preservation pipe 2 and a pipeline, and the other end of the two-dimensional chromatographic column is communicated with B6 of the six-way valve B through a connecting two-way valve C, a heat preservation pipe 3 and a pipeline; the invention has good repeatability, high stability, rapidness and reliability.
Description
Technical Field
The invention belongs to the technical field of analytical chemistry.
Background
In recent years, modern chromatography has become one of the important tools for analytical separation of components of complex systems in the field of analytical chemistry. For separation of components in complex samples, one mode of separation often fails to provide sufficient peak capacity and chromatographic peak resolution. Therefore, combining different modes of separation methods to construct a multidimensional chromatographic separation system is an effective way to solve the problem. Since the concept of multidimensional separation was proposed in 1984, along with the continuous improvement of chromatographic methods and the development of technologies such as column switching control, multidimensional separation technology has been rapidly developed and applied in many scientific fields such as biology, chemistry and environment. In the field of gas chromatography, multidimensional separation technology mainly comprises two types, namely center cutting type two-dimensional chromatography and full two-dimensional gas chromatography. The center cutting type two-dimensional chromatography expands the separation capability of the one-dimensional chromatography, partial fractions after pre-separation from the first chromatographic column are re-injected into the second chromatographic column for further separation, other components in the sample are emptied or cut by the center, and the separation of the components of interest can be realized by increasing the times of center cutting. The two-dimensional gas chromatography combines two chromatographic columns which have different separation mechanisms and are independent with each other in a series connection mode to form the two-dimensional gas chromatography, the two chromatographic columns are connected by a modulator, and the modulator plays the roles of trapping, focusing and re-transmitting. And each chromatographic peak separated by the first chromatographic column is modulated by the modulator and then sent to the second chromatographic column in a pulse mode for further separation.
Compared with one-dimensional chromatography, the two multidimensional separation technologies have obvious advantages in the aspects of separation and research of samples such as petroleum, plant volatile oil, blood plasma and the like. However, due to the problems of high price, high maintenance cost, complex data processing and the like of the full two-dimensional gas chromatography/mass spectrum, for some samples which are not particularly complex and only contain a small amount of isomers or substances with similar boiling points, the two-dimensional (center-cut) gas chromatograph-mass spectrometer with low price, low maintenance cost and large analysis flux can sufficiently meet the actual scientific research requirements of people.
At present, MDGCGC-MS of Shimadzu and Dean switch GCMS of Agilent are mature commercial two-dimensional (center cutting) gas chromatograph-mass spectrometer, but the instruments usually need to install two detectors (hydrogen flame or mass spectrum) for one-dimensional and two-dimensional chromatographic columns to respectively detect components flowing out of the two chromatographic columns, and sometimes even need to install two gas phase column incubators. This greatly increases the cost of the instrument and the cost of maintenance. Therefore, it is urgently needed to develop a two-dimensional (center-cut) gas chromatograph-mass spectrometer with low price and simple maintenance to meet the increasing demands in the aspects of scientific research, application detection and the like.
Disclosure of Invention
In view of the above, the invention discloses a two-dimensional center-cut gas chromatography-mass spectrometry separation method for metabolites, which comprises the following steps: setting the six-way valve A and the six-way valve B to be in a state 1, and separating the substance to be detected collected by the sample inlet by using the one-dimensional chromatographic column; meanwhile, the two-dimensional chromatographic column is in an APC protective gas protection state; the state 1 is: the communication between A1 and A6, between A2 and A3, between A3 and A5, between A5 and A4, between B1 and B2, between B4 and B3, between B3 and B5 and between B5 and B6 of the six-way valve A; setting the states of the six-way valve A and the six-way valve B to be switched from the state 1 to the state 2 at the corresponding starting time of the target peak, and separating the object to be measured in a selected time period by the one-dimensional chromatographic column and the two-dimensional chromatographic column; the state 2 is: the communication between A1 and A2, between A4 and A3, between A3 and A5, between A5 and A6, between B1 and B6, between B2 and B3, between B3 and B5 and between B5 and B4 of the six-way valve A; setting the states of the six-way valve A and the six-way valve B to be switched from the state 2 to the state 3 at the corresponding end time of the target peak, connecting the two-dimensional chromatographic column with the mass spectrum in the helium protection, and continuously separating the object to be detected; the state 3 is: the six-way valve A is communicated with A1 and A6, A2 and A3, A3 and A5, A5 and A4, B1 and 6, B2 and B3, B3 and B5 and B5 and B4.
In a specific embodiment of the present invention, the start time is 19.5min and the end time is 20.5 min.
In a specific embodiment of the present invention, the gas temperature programming for separating the analyte collected by the sample inlet by the one-dimensional chromatographic column is as follows: the initial column temperature was 35 ℃ followed by 5 ℃ to 130 ℃ per minute, 30 ℃ to 230 ℃ per minute, held for 16 minutes.
In a specific embodiment of the present invention, the method for determining the corresponding start time and the corresponding end time of the target peak includes: setting the six-way valve A and the six-way valve B to be in a state 1, separating an object to be detected collected by a sample inlet through a one-dimensional chromatographic column, allowing the object to be detected to enter a mass spectrum system to obtain a mass spectrum, determining a characteristic peak of the object to be detected, and determining corresponding start time and corresponding end time of a target peak; meanwhile, the two-dimensional chromatographic column is in an APC protective gas protection state; the state 1 is: the six-way valve A is communicated with A1 and A6, A2 and A3, A3 and A5, A5 and A4, B1 and B2, B4 and B3, B3 and B5 and B5 and B6.
In a specific embodiment of the present invention, the gas temperature programming of the analyte separated by the one-dimensional chromatographic column and the two-dimensional chromatographic column for a selected period of time is: the initial column temperature was 35 deg.C, followed by 5 deg.C to 130 deg.C per minute, 30 deg.C to 160 deg.C per minute, 50 deg.C to 40 deg.C per minute, 1.5 deg.C to 120 deg.C per minute, 5 deg.C to 150 deg.C per minute, 30 deg.C to 230 deg.C per minute, and held for 5 minutes.
In a specific embodiment of the present invention, the analyte is a mixture containing 2-methylbutyric acid and isovaleric acid.
The invention also discloses a system of the metabolite two-dimensional center cutting gas chromatography-mass spectrometry separation method, which comprises a gas phase column incubator system and a valve body column incubator system; the gas column incubator system comprises a one-dimensional chromatographic column, a two-dimensional chromatographic column and a gas column incubator; the valve body column temperature box comprises a six-way valve A, a six-way valve B, a six-way valve bracket, a column temperature box body and a heating module; the heating module is positioned near the six-way valve A and the six-way valve B and is used for heating the six-way valve A and the six-way valve B; one end of the one-dimensional chromatographic column is communicated with the sample inlet through a pipeline, and the other end of the one-dimensional chromatographic column is communicated with A1 of the six-way valve A through a connecting two-way valve A, a heat preservation pipe 1 and a pipeline; one end of the two-dimensional chromatographic column is communicated with A2 of the six-way valve A through a connecting two-way valve B, a heat preservation pipe 2 and a pipeline, and the other end of the two-dimensional chromatographic column is communicated with B6 of the six-way valve B through a connecting two-way valve C, a heat preservation pipe 3 and a pipeline; the A1 of the six-way valve A is communicated with one end of the one-dimensional chromatographic column through a heat preservation pipe 1, a connecting two-way A and a pipeline, the A4 is communicated with an APC protective gas pipeline, the A3 is constantly communicated with the A5, and the A6 is communicated with the B2 of the six-way valve B through a pipeline; the B1 of the six-way valve B is communicated with a mass spectrometry system through a heat preservation pipe 4 and a pipeline, the B2 is connected with the A6 of the six-way valve A through a pipeline, and the B3 is constantly communicated with the B5.
In a specific embodiment of the present invention, the one-dimensional chromatographic column is a gas chromatographic column, and the gas chromatographic column is a HP-INNOWAX gas capillary chromatographic column.
In a specific embodiment of the present invention, the two-dimensional chromatography column is a gas chromatography column, and the gas chromatography column is a DB-624 gas chromatography column.
In a specific embodiment of the invention, the one-dimensional chromatography column and the two-dimensional chromatography column have orthogonality.
The invention adopts a two-dimensional gas chromatography-mass spectrometry (2DGC-MS) technology which is modified by two-dimensional (center cutting) on the basis of a one-dimensional Shimadzu gas chromatography-mass spectrometry (GC-MS) to carry out two-dimensional (center cutting) analysis on metabolites which are difficult to separate and identify in complex samples such as plant extracts, microbial fermentation strains and the like so as to identify substances with similar structures or boiling points which cannot be separated in the one-dimensional gas chromatography-mass spectrometry analysis.
The invention has good repeatability, high sensitivity, peak capacity and stability, is rapid and reliable, and is suitable for two-dimensional gas chromatography-mass spectrometry analysis of complex metabolites of various biological samples such as animal and insect species, plants, microbial fermentation samples and the like.
Drawings
FIG. 1 is a schematic view of a two-dimensional center-cut integrated gas mass separation system.
Wherein, 1 is a valve body column incubator, 2 is a gas column incubator, 3 is a one-dimensional chromatographic column, 4 is a two-dimensional chromatographic column, 6 is a six-way valve A, 7 is a six-way valve B, 8 is a sample inlet, 9 is a connecting two-way valve A, 10 is a heat preservation pipe 1, 11 is a connecting two-way valve B, 12 is a heat preservation pipe 2, 13 is a connecting two-way valve C, 14 is a heat preservation pipe 3, and 15 is a heat preservation pipe 4.
FIG. 2 is a TIC chart of one-dimensional gas chromatography of seven short-chain fatty acids.
FIG. 3 is a one-dimensional gas mass spectrum of a mixed standard of 2-methylbutyric acid and isovaleric acid.
FIG. 4 is a two-dimensional (center cut) gas mass analysis TIC chart of 2-methylbutyric acid and isovaleric acid.
FIG. 5 is a mass spectrum of 2-methylbutyric acid and isovaleric acid.
FIG. 6 is a graph of two-dimensional (center cut) gas mass analysis TIC of a sample of microbial inoculum.
Detailed Description
Example 1
The two-dimensional center cutting gas chromatography-mass spectrometry separation system comprises a gas phase column incubator system and a valve body column incubator system;
the gas column incubator system comprises a one-dimensional chromatographic column, a two-dimensional chromatographic column and a gas column incubator;
the valve body column temperature box comprises a six-way valve A, a six-way valve B, a six-way valve bracket, a column temperature box body and a heating module; the heating module is positioned near the six-way valve A and the six-way valve B and is used for heating the six-way valve A and the six-way valve B;
one end of the one-dimensional chromatographic column is communicated with the sample inlet through a pipeline, and the other end of the one-dimensional chromatographic column is communicated with A1 of the six-way valve A through a connecting two-way valve A, a heat preservation pipe 1 and a pipeline; the one-dimensional chromatographic column is a gas chromatographic column, and the gas chromatographic column is an HP-INNOWAX gas capillary chromatographic column. One end of the two-dimensional chromatographic column is communicated with A2 of the six-way valve A through a connecting two-way valve B, a heat preservation pipe 2 and a pipeline, and the other end of the two-dimensional chromatographic column is communicated with B6 of the six-way valve B through a connecting two-way valve C, a heat preservation pipe 3 and a pipeline; the two-dimensional chromatographic column is a gas chromatographic column, and the gas chromatographic column is a DB-624 gas chromatographic column.
The one-dimensional chromatographic column and the two-dimensional chromatographic column have orthogonality.
The A1 of the six-way valve A is communicated with one end of the one-dimensional chromatographic column through a heat preservation pipe 1, a connecting two-way A and a pipeline, the A4 is communicated with an APC protective gas (high-purity helium) pipeline, the A3 is constantly communicated with the A5, and the A6 is communicated with the B2 of the six-way valve B through a pipeline;
b1 of the six-way valve B is communicated with a mass spectrometry system through a heat preservation pipe 4 and a pipeline, B2 is connected with A6 of the six-way valve A through a pipeline, B3 is constantly communicated with B5, and B4 is communicated with an exhaust port pipeline;
the six-way valve A and the six-way valve B are filled with long-fiber glass wool in the space of the valve body column temperature box to improve the heat preservation effect of the valve body column temperature box.
Example 2
The two-dimensional center cutting gas-mass combination separation method for the metabolites comprises the following steps:
1. collecting a plurality of microbial zymogen liquid samples and short chain fatty acid mixed standard samples.
2. Pretreating a microbial fermentation bacteria liquid sample, injecting a sample of the bacteria liquid to be detected with ethyl acetate, extracting the sample with the same volume, and taking an ethyl acetate layer sample to be detected. And diluting the short-chain fatty acid mixed standard substance by using ethyl acetate to be detected.
3. The one-dimensional gas mass analysis determines the corresponding start time and end time of the target peak of the two-dimensional gas mass analysis.
Analyzing the short-chain fatty acid mixed standard substance by using a two-dimensional (center cutting) gas chromatography-mass spectrometer (based on the one-dimensional gas chromatography-mass spectrometry system modification of GC-MS QP 2010Ultra of Japan Shimadzu corporation), and performing the one-dimensional gas chromatography-mass spectrometry analysis by using the specific steps as follows:
setting the states of the six-way valve A and the six-way valve B as 1, wherein the specific setting of the state 1 is as follows: the a1 and a6 of six-way valve a, a2 and A3, A3 and a5, a5 and a4, B1 and B2, B4 and B3, B3 and B5, and B5 and B6.
The one-dimensional chromatographic column separates the substances to be detected collected by the sample inlet, the separated substances to be detected sequentially enter A1 and A2 of the six-way valve A through the two-way valve A and the heat preservation pipe 1, sequentially enter B2 and B1 of the six-way valve B through the pipeline, and enter the mass spectrum system through the heat preservation pipe 4, so that one-dimensional gas mass analysis is realized.
Meanwhile, the APC protective gas enters through A4 of the six-way valve A and sequentially passes through A5, A3 and A2, enters the two-dimensional chromatographic column through the heat preservation pipe 2, the connecting two-way valve B and the pipeline, enters B6 of the six-way valve B through the connecting two-way valve C, the heat preservation pipe 3 and the pipeline, sequentially passes through B6, B5, B3 and B4, and finally emptying and protecting the two-dimensional chromatographic column are achieved.
The specific conditions of the gas instrument for separating the substance to be detected collected by the sample inlet through the one-dimensional chromatographic column are as follows:
the gas chromatographic column is HP-INNOWAX gas capillary chromatographic column (30m X0.25 mu m X0.25.25 mu m), and the carrier gas of the mobile phase is high-purity helium with the purity of 99.999 percent. The gas phase temperature programming is as follows: the initial column temperature was 35 ℃ followed by 5 ℃ to 130 ℃ per minute, 30 ℃ to 230 ℃ per minute, held for 16 minutes. The flow rate is 1mL/min, and the APC gas path pressure is 45 kPa. No-shunt sampling mode. The ion source is an EI source. The temperatures of the sample inlet, the ion source and the ion source interface are all 230 ℃. The temperature of the valve body column incubator is 250 ℃. The data acquisition time is 8min to 38min, and the molecular weight range is 29-500 m/z.
As can be seen from FIG. 2, under one-dimensional conditions, the two isomers of 2-methylbutyric acid and isovaleric acid are not chromatographically separated. Under the conditions of bombardment by a gaseous EI ion source, the mass spectral data typically obtained is not the actual molecular weight information of the species, and in most cases is its fragmentation information. As can be seen from FIG. 3, the mass spectrum fragment peaks of 2-methylbutyric acid and isovaleric acid are overlapped, and thus accurate qualitative and quantitative analysis thereof cannot be performed.
4. Selecting a chromatographic peak of 19.7min as the corresponding starting time of a target peak of two-dimensional (center cutting) gas quality analysis through one-dimensional gas quality analysis, and analyzing the short-chain fatty acid mixed standard substance by secondary sample injection, wherein the method specifically comprises the following steps:
State 3 of six-way valve a and six-way valve B: the six-way valve A is communicated with A1 and A6, A2 and A3, A3 and A5, A5 and A4, B1 and 6, B2 and B3, B3 and B5 and B5 and B4.
(1) Setting the six-way valve A and the six-way valve B to be in a state 1, separating the to-be-detected object collected by the sample inlet by using the one-dimensional chromatographic column, and emptying the two-dimensional chromatographic column in an APC (automatic Power control) protective gas state;
the specific conditions of the gas instrument for separating the substance to be detected collected by the sample inlet by the one-dimensional chromatographic column are as follows:
the gas chromatographic column was a HP-INNOWAX gas capillary chromatographic column (30m X0.25.25 μm X0.25.25 μm) with a mobile phase carrier gas of 99.999% high purity helium. The gas phase temperature programming is as follows: the initial column temperature was 35 ℃ followed by 5 ℃ to 130 ℃ per minute, 30 ℃ to 230 ℃ per minute, held for 16 minutes. The flow rate is 1mL/min, and the APC gas path pressure is 45 kPa. No-shunt sampling mode. The ion source is an EI source. The temperatures of the sample inlet, the ion source and the ion source interface are all 230 ℃. The temperature of the valve body column incubator is 250 ℃. The data acquisition time is 8min to 38min, and the molecular weight range is 29-500 m/z.
(2) Setting the states of a six-way valve A and a six-way valve B to be switched from a state 1 to a state 2 according to the corresponding starting time of a target peak of two-dimensional gas quality analysis selected by the one-dimensional gas quality analysis, namely when the time is 19.5min, and connecting a one-dimensional chromatographic column and a two-dimensional chromatographic column in series to ensure that the target peak is switched into the two-dimensional chromatographic column; the object to be detected is further separated in the two-dimensional chromatographic column, and the object to be detected in a selected time period is separated by the one-dimensional chromatographic column and the two-dimensional chromatographic column. The state 2 is: the six-way valve A is communicated with A1 and A2, A4 and A3, A3 and A5, A5 and A6, B1 and B6, B2 and B3, B3 and B5 and B5 and B4.
The specific conditions of the gas instrument for separating the substance to be detected in the selected time interval by the one-dimensional chromatographic column and the two-dimensional chromatographic column are as follows:
the one-dimensional gas chromatography column is HP-INNOWAX gas capillary chromatography column (30m X0.25.25 μm X0.25.25 μm), and the two-dimensional gas chromatography column is DB-624 gas chromatography column (30m X0.25.25 μm X1.40.40 μm). The mobile phase carrier gas was 99.999% high purity helium.
The gas phase temperature programming of the two chromatographic columns is as follows: the initial column temperature was 35 deg.C, followed by 5 deg.C to 130 deg.C per minute, 30 deg.C to 160 deg.C per minute, to 160 deg.C immediately at 50 deg.C to 40 deg.C per minute, 1.5 deg.C to 120 deg.C per minute, 5 deg.C to 150 deg.C per minute, 30 deg.C to 230 deg.C per minute, and held for 5 minutes. The flow rate is 1mL/min, and the APC gas path pressure is 45 kPa. No-shunt sampling mode. The ion source is an EI source. The temperatures of the sample inlet, the ion source and the ion source interface are all 230 ℃. The temperature of the valve body column incubator is 250 ℃. The data acquisition time is 8min to 65min, and the molecular weight range is 29-500 m/z.
(3) And (3) according to the corresponding end time of a target peak of two-dimensional gas mass analysis selected by the one-dimensional gas mass analysis, namely when the time is 20.5min, setting the states of the six-way valve A and the six-way valve B to be switched from the state 2 to the state 3, emptying the one-dimensional chromatographic column in helium protection, connecting the two-dimensional chromatographic column with a mass spectrum to continuously separate a target object to be detected in a selected time period, finally separating the target peak in the second-dimensional chromatographic column, and finally entering the mass spectrum for detection.
As can be seen from FIG. 4, the two isomers of 2-methylbutyric acid and isovaleric acid are well separated on the second dimension chromatogram. Also, as can be seen from fig. 5, the mass spectrum fragment peaks of 2-methylbutyric acid and isovaleric acid are very different and, when they are chromatographically separated, they can be accurately qualitatively and quantitatively analyzed.
5. Subsequently, the microbial inoculum sample was subjected to two-dimensional (center cut) gas mass analysis by the method described above (FIG. 6). As can be seen from FIG. 6, 2-methylbutyric acid and isovaleric acid were also well separated from the microbial fermentation broth sample by this method.
The invention relates to a novel metabolite two-dimensional (center cutting) gas chromatography-mass spectrometry system established on a two-dimensional (center cutting) gas chromatography-mass spectrometry technology (2DGC-MS) after two-dimensional (center cutting) transformation is carried out on the basis of a one-dimensional Shimadzu gas chromatography-mass spectrometry instrument (GC-MS). The method is suitable for performing two-dimensional (center cutting) gas chromatography-mass spectrometry analysis on complex metabolites of various biological samples such as animal and insect samples, plant samples, microbial fermentation samples and the like. The method has the advantages of good repeatability, high sensitivity, high peak capacity and high stability, and is rapid and reliable.
Claims (10)
1. The two-dimensional center cutting gas-mass combined separation method for the metabolites is characterized by comprising the following steps of:
setting the six-way valve A and the six-way valve B to be in a state 1, and separating the to-be-detected object collected by the sample inlet by the one-dimensional chromatographic column; meanwhile, the two-dimensional chromatographic column is in an APC protective gas protection state; the state 1 is: the communication between A1 and A6, between A2 and A3, between A3 and A5, between A5 and A4, between B1 and B2, between B4 and B3, between B3 and B5 and between B5 and B6 of the six-way valve A;
setting the states of the six-way valve A and the six-way valve B to be switched from the state 1 to the state 2 at the corresponding starting time of the target peak, and separating the object to be measured in a selected time period by the one-dimensional chromatographic column and the two-dimensional chromatographic column; the state 2 is: the communication between A1 and A2, between A4 and A3, between A3 and A5, between A5 and A6, between B1 and B6, between B2 and B3, between B3 and B5 and between B5 and B4 of the six-way valve A;
setting the states of the six-way valve A and the six-way valve B to be switched from the state 2 to the state 3 at the corresponding end time of the target peak, connecting the two-dimensional chromatographic column with the mass spectrum in the helium protection, and continuously separating the object to be detected; the state 3 is: the six-way valve A is communicated with A1 and A6, A2 and A3, A3 and A5, A5 and A4, B1 and 6, B2 and B3, B3 and B5 and B5 and B4.
2. The two-dimensional center-cut GC-MS separation method of a metabolite according to claim 1, wherein said start time is 19.5min and said end time is 20.5 min.
3. The two-dimensional center-cut gas-mass spectrometry separation method for the metabolites according to claim 1, wherein the gas-phase temperature programming for separating the analytes collected by the sample inlet by the one-dimensional chromatographic column is as follows: the initial column temperature was 35 ℃ followed by 5 ℃ to 130 ℃ per minute, 30 ℃ to 230 ℃ per minute, held for 16 minutes.
4. The two-dimensional center-cut GC-MS separation method of metabolites according to claim 1, wherein the corresponding start time and the corresponding end time of said target peak are determined by:
setting the six-way valve A and the six-way valve B to be in a state 1, separating an object to be detected collected by a sample inlet through a one-dimensional chromatographic column, allowing the object to be detected to enter a mass spectrum system to obtain a mass spectrum, determining a characteristic peak of the object to be detected, and determining corresponding start time and corresponding end time of a target peak; meanwhile, the two-dimensional chromatographic column is in an APC protective gas protection state; the state 1 is: the six-way valve A is communicated with A1 and A6, A2 and A3, A3 and A5, A5 and A4, B1 and B2, B4 and B3, B3 and B5 and B5 and B6.
5. The two-dimensional center-cut gas-mass spectrometry separation method for the metabolites according to claim 1, wherein the gas-phase temperature programming of the analyte for the selected time period of the separation of the one-dimensional chromatographic column and the two-dimensional chromatographic column is as follows: the initial column temperature was 35 deg.C, followed by 5 deg.C to 130 deg.C per minute, 30 deg.C to 160 deg.C per minute, 50 deg.C to 40 deg.C per minute, 1.5 deg.C to 120 deg.C per minute, 5 deg.C to 150 deg.C per minute, 30 deg.C to 230 deg.C per minute, and held for 5 minutes.
6. The two-dimensional center-cut GC-MS separation method of a metabolite according to claim 1, wherein said analyte is a mixture containing 2-methylbutyric acid and isovaleric acid.
7. The two-dimensional center-cut GC-MS separation method system for metabolites according to claim 1, comprising a gas phase column oven system and a valve body column oven system;
the gas column incubator system comprises a one-dimensional chromatographic column, a two-dimensional chromatographic column and a gas column incubator;
the valve body column temperature box comprises a six-way valve A, a six-way valve B, a six-way valve bracket, a column temperature box body and a heating module; the heating module is positioned near the six-way valve A and the six-way valve B and is used for heating the six-way valve A and the six-way valve B;
one end of the one-dimensional chromatographic column is communicated with the sample inlet through a pipeline, and the other end of the one-dimensional chromatographic column is communicated with A1 of the six-way valve A through a connecting two-way valve A, a heat preservation pipe 1 and a pipeline;
one end of the two-dimensional chromatographic column is communicated with A2 of the six-way valve A through a connecting two-way valve B, a heat preservation pipe 2 and a pipeline, and the other end of the two-dimensional chromatographic column is communicated with B6 of the six-way valve B through a connecting two-way valve C, a heat preservation pipe 3 and a pipeline;
the A1 of the six-way valve A is communicated with one end of the one-dimensional chromatographic column through a heat preservation pipe 1, a connecting two-way A and a pipeline, the A4 is communicated with an APC protective gas pipeline, the A3 is constantly communicated with the A5, and the A6 is communicated with the B2 of the six-way valve B through a pipeline;
the B1 of the six-way valve B is communicated with a mass spectrometry system through a heat preservation pipe 4 and a pipeline, the B2 is connected with the A6 of the six-way valve A through a pipeline, and the B3 is constantly communicated with the B5.
8. The system for two-dimensional center-cut GC-MS separation of a metabolite according to claim 7, wherein said one-dimensional chromatographic column is a gas chromatographic column, and said gas chromatographic column is a HP-INNOWAX gas capillary chromatographic column.
9. The system for the two-dimensional center-cut GC-MS separation method of a metabolite according to claim 7, wherein said two-dimensional chromatographic column is a gas chromatographic column, and said gas chromatographic column is a DB-624 gas chromatographic column.
10. The system for two-dimensional center-cut GC-MS separation of a metabolite of claim 7, wherein said one-dimensional chromatographic column and said two-dimensional chromatographic column have orthogonality.
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