CN108303473A - Biological sample preparation method and applications and metabolin qualitative and quantitative analysis method - Google Patents
Biological sample preparation method and applications and metabolin qualitative and quantitative analysis method Download PDFInfo
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- CN108303473A CN108303473A CN201711021691.3A CN201711021691A CN108303473A CN 108303473 A CN108303473 A CN 108303473A CN 201711021691 A CN201711021691 A CN 201711021691A CN 108303473 A CN108303473 A CN 108303473A
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Abstract
The present invention provides for before carrying out gas chromatograph and mass spectrograph combination detection and analysis to the metabolin in the organism sample from organism, biological sample is handled to obtain the biological sample preparation method and applications and metabolin qualitative and quantitative analysis method of pre-treatment sample, biological sample preparation method therein is used for the processing before being carried out to the biological sample from organism and obtains that the preceding processing sample that metabolin carries out gas chromatograph and mass spectrograph combination detection and analysis can be used for, it is characterized by comprising the following steps：Step 1, extraction process；Step 2, derivatization；Step 3, standardization and impurity elimination, wherein in step 1, extraction biological sample is obtained by freeze-drying.
Description
Technical field
The invention belongs to chemical fields, and in particular to a kind of for the generation in the organism sample from organism
It before thanking object progress gas-chromatography and mass spectrometer detection and analysis, prepared by biological sample, obtains the life of processed sample
Object sample preparation methods and its application in metabolin qualitative and quantitative analysis and corresponding metabolin qualitative and quantitative analysis side
Method.
Background technology
There is the metabolin that a large amount of various approach generate, some are to be metabolized generation from organism itself in organism
, some are the micropopulation common metabolic generations in organism and organism, are more also the microorganisms in organism
Group's own metabolism generates.The metabolin that each approach generates performs its own function, and mutually acts synergistically on the whole of organism
Body function, so how to detect that more metabolins further to analyze, become the important research of current metabolite analysis
Target.
And the metabolite concentration difference that various approach generate is huge, also multifarious in physicochemical property, especially human body is (raw
Object) the metabolin that generates in the organism (host) of enterobacteriaceae and enterobacteriaceae produced with organism (host) common metabolic
Raw Co metabolism object, including short chain fatty acids, amino acid, benzoyl and phenyl derivatives, indole derivatives, lipid, bile acid,
Choline, vitamin and polyamines class etc., they have completely different physics and chemical property, therefore how to be put down with as few as possible
Platform measures the great challenge of metabolin as much as possible simultaneously.
Spread out currently, above-mentioned short chain fatty acids, amino acid, benzoyl and the phenyl of being mentioned to can be detected simultaneously there has been no method
A variety of small molecule metabolites such as biology, indole derivatives, lipid, bile acid, choline, vitamin and polyamines class.
Invention content
The present invention provides a kind of for carrying out gas-chromatography to the metabolin in the organism sample from organism
Before being tested and analyzed with mass spectrometer, the biological sample that pre-treatment sample is prepared is carried out to biological sample, and its be metabolized
Application in object qualitative and quantitative analysis and corresponding metabolin qualitative and quantitative analysis method.
To achieve the goals above, present invention employs following technical solutions：
The present invention provides a kind of biological sample preparation methods, for the place before being carried out to the biological sample from organism
Reason obtains that the pre-treatment sample that metabolin carries out gas-chromatography and mass spectrometer detection and analysis can be used for, which is characterized in that packet
Include following steps：Step 1, extraction process extracts the metabolin in biological sample to obtain extraction biological sample；Step 2,
Derivatization under predetermined derivatization conditions, performs the derivatization extraction biological sample to obtain derivative biological sample after completing step 1
This；Step 3, the reservation being mixed to get by a variety of linear paraffin standard items is added into derivative biological sample in standardization and impurity elimination
It after standard of index mixture of substances, carries out impurity elimination and obtains pre-treatment sample, wherein in step 1, carried by freeze-drying
Take biological sample.
Biological sample preparation method provided by the invention, also has the feature that：Wherein, the condition of freeze-drying be
Temperature is to be dried in vacuo at -80 DEG C to -30 DEG C.
Biological sample preparation method provided by the invention, also has the feature that：Wherein, the vacuum degree of vacuum is
0.1mbar-0.002mbar。
Biological sample preparation method provided by the invention, also has the feature that：Wherein, step 1 is specially：Step
1.1, take the biological sample of the first predetermined volume in centrifuge tube；Step 1.2, the volume with the biological sample in step 1.1 is pressed
Than cold methanol is added for the first predetermined volume ratio；Step 1.3, after cold methanol being added, the condition for being -20 DEG C to -30 DEG C in temperature
Lower standing 20min-30min, under this condition, albumen precipitation is abundant.Then in the item that temperature is 4 DEG C, centrifugal force is 16000g
10min is centrifuged under part, the supernatant that centrifugation obtains, which is fitted into freeze-drying in sample introduction bottle, obtains extraction biological sample.
Biological sample preparation method provided by the invention, also has the feature that：Wherein, the first predetermined volume is 100-
500μL。
Biological sample preparation method provided by the invention, also has the feature that：Wherein, the first predetermined volume ratio is：
The biological sample of first predetermined volume：Cold methanol volume=1 of addition:3.
Biological sample preparation method provided by the invention, also has the feature that：Wherein, biological sample is organism
Serum.
Biological sample preparation method provided by the invention, also has the feature that：Wherein, step 1 is specially：Take first
The biological sample of predetermined volume is fitted into freeze-drying in sample introduction bottle and obtains extraction biological sample.
Biological sample preparation method provided by the invention, also has the feature that：Wherein, the first predetermined volume is 100-
1000μL。
Biological sample preparation method provided by the invention, also has the feature that：Wherein, the first predetermined volume is 100-
500μL。
Biological sample preparation method provided by the invention, also has the feature that：Wherein, the first predetermined volume is 250-
1000μL。
Biological sample preparation method provided by the invention, also has the feature that：Wherein, organism sample is organism
Urine or saliva in it is one or more.
Biological sample preparation method provided by the invention, also has the feature that：Biological sample is micro- life of organism
Object cell, step 1 are specially：Step 1.1, prepare biological sample, the cell from organism is collected in centrifuge tube, in temperature
Degree is 4 DEG C, centrifugal force centrifuges 10min under being 200g-1000g, and under this condition, cell will not rupture, and metabolin is caused to lose,
It can be detached again with the culture solution on upper layer simultaneously, then after being cleaned with PBS solution, the PBS solution that 1mL is added is resuspended to obtain cell liquid and makees
For biological sample；Step 1.2, homogenate is counted, number of cells first is carried out to cell suspension with digital automatic cell counter
Accurate metering, when count results meet predetermined value, centrifugation again obtains cell, discards upper layer PBS solution, and water and Leng Jia is added
Alcohol (volume ratio is the second predetermined volume ratio) is homogenized to obtain cell homogenates liquid；Step 1.3, it centrifuges, is 4 in temperature
DEG C, centrifugal force 10min is centrifuged to cell homogenates liquid under conditions of being 16000g, the obtained supernatant of centrifugation is transferred to sample introduction bottle
In be freeze-dried to obtain extraction biological sample.
Biological sample preparation method provided by the invention, also has the feature that：Wherein, the predetermined amount of cell sample is
5×106-5×107It is a.
Biological sample preparation method provided by the invention, also has the feature that：Wherein, the second predetermined volume ratio is：
The volume of water：Volume=1 of cold methanol:4.
Biological sample preparation method provided by the invention, also has the feature that：Predetermined derivatization conditions are：Temperature is
Under the alkaline condition of 20-25 degree (pH value range 10-12).
Biological sample preparation method provided by the invention, also has the feature that：Wherein, step 2 is specially：By hydrogen-oxygen
Change sodium solution, methanol solution, pyridine solution and derivative reagent, by the ratio that the volume ratio between them is third predetermined volume ratio
Example sequentially adds, and NaOH solution is added and adds methanol solution and pyridine solution after the dissolving of pre-treatment sample, mixes at this time
Derivative reagent is added after even, and 30s is finally shaken under the vibration condition of 2,500-3,000rpm and obtains derivative biological sample, hydrogen
A concentration of 1M of sodium hydroxide solution.
Biological sample preparation method provided by the invention, also has the feature that：Derivative reagent tries for methylchloroformate
Agent.
Biological sample preparation method provided by the invention, also has the feature that：Third predetermined volume ratio is：Hydroxide
Sodium solution：Pyridine solution：Methanol solution：The volume ratio of derivative reagent is 10：8：1.7:1.
Biological sample preparation method provided by the invention, also has the feature that：Step 3 is specially：Step 3.1, will
Retention index standard substance mixture, which is added in chloroformic solution, obtains the chloroformic solution of standard items containing retention index；Step 3.2, will
The chloroformic solution of standard items containing retention index is added in the ratio that the volume ratio with derivative biological sample is the 4th predetermined volume ratio
In derivative biological sample, under the vibration condition of 2,500-3,000rpm, 10s is shaken, obtains mark sample mixing sheet；Step 3.3, press with
The NaHCO3 solution that a concentration of 50mM is added for the ratio of the 5th predetermined volume ratio for the volume ratio of mark sample mixing sheet is added to mark sample mixing
In this, 10s is shaken under the vibration condition of 2,500-3,000rpm, under conditions of temperature is 4 DEG C, centrifugal force is 2000g from
Heart 10min is to be layered to obtain layering solution；Step 3.4, the chloroform layer for being located at layering bottom in solution will be layered to be transferred to containing 100mg
Pre-treatment biological sample is used as in the gas phase sample bottle of anhydrous sodium sulfate.
Biological sample preparation method provided by the invention, also has the feature that：Wherein, the 4th predetermined volume ratio is 1:
1。
Biological sample preparation method provided by the invention, also has the feature that：Wherein, the 5th predetermined volume ratio is 1:
2。
Biological sample preparation method provided by the invention, also has the feature that：Retention index standard substance mixture
Include the linear paraffin standard items from C8-C30.
Biological sample preparation method provided by the invention, also has the feature that：Metabolin is micro- life in host
The Co metabolism object that object group is metabolized the metabolin of generation in host or micropopulation and host's common metabolic generate either more than
Two kinds of summation.
Biological sample preparation method provided by the invention, also has the feature that：Host is human body, and micropopulation is behaved
Intestinal flora in body.
Biological sample preparation method provided by the invention, also has the feature that：Metabolin be aliphatic acid, phenyl class,
It is one or more in indoles, lipid, bile acid, choline, vitamin or polyamines class.
The present invention also provides a kind of application of biological sample preparation method in metabolin qualitative and quantitative analysis, feature exists
In：Wherein, biological sample preparation method is above-mentioned biological sample preparation method.
The present invention also provides a kind of metabolin qualitative and quantitative analysis methods, by gas-chromatography and mass spectrometer to coming from
The biological sample of organism carries out qualitative and quantitative analysis and obtains information to be analyzed, to analyze the metabolin in organism,
It is characterized by comprising the following steps：Step 1, biological sample pre-treatment carry out pre-treatment to biological sample and obtain pre-treatment
Biological sample；Step 2, detection acquisition information, is detected pre-treatment biological sample by gas-chromatography and mass spectrometer
And collect information to be analyzed corresponding with metabolin；Step 3, qualitative and quantitative analysis, based on including and various criterion substance
Derivative distinguish the standard database of corresponding different Information in Mass Spectra, qualitative and quantitative analysis is carried out to information to be analyzed,
In, step 1 is carried out using above-mentioned biological sample preparation method.
Metabolin qualitative and quantitative analysis method provided by the invention, also has the feature that：In step 2, gas phase color
The temperature control of spectrometer uses temperature programming method：Initial temperature is 35-45 DEG C, 1min-2min is kept, with 10 DEG C/min-25
DEG C/rate of min rises to 260 DEG C -300 DEG C, 290 DEG C -320 then are risen to the rate of 40 DEG C/min-, keeps 2min-
5min, total time 15.25min-28.25.
Metabolin qualitative and quantitative analysis method provided by the invention, also has the feature that：The condition of gas chromatograph
For：Chromatographic column is capillary chromatographic column, and length 30m, internal diameter 0.25mm, packing material is the crosslinking poly- silicon of diphenyldimethyl
One kind in oxygen alkane, middle polarity cross-linked phase or low-to-moderate polarity phase, filling thickness are 1.4 μm of -0.25mm, the range of temperature in use
It is -60 DEG C -350 DEG C；Injector temperature is 270 DEG C；Transfer tube temperature is 270 DEG C.
Metabolin qualitative and quantitative analysis method provided by the invention, also has the feature that：Mass spectrometric use condition
For：Ion source temperature is 220 DEG C；Sampling volume is 0.5-1 μ L；Splitless injecting samples；Ion source voltage is 70eV；Scanning of the mass spectrum model
It encloses for 38-650m/z；Acquisition rate is 20-25spectra/s.
Metabolin qualitative and quantitative analysis method provided by the invention, also has the feature that：Wherein, standard database is also
Include distinguishing corresponding different retention indexs from the corresponding derivative matter of various criterion substance.
Invention effect
Biological sample preparation method provided by the invention and its application and metabolin qualitative and quantitative analysis method, due to
In biological sample preparation method, when extracting processing to the metabolin in biological sample, extracted by freeze-drying
Biological sample, in this way, the moisture in extraction biological sample greatly reduces so that the content concn of wherein metabolin is concentrated,
Avoid metabolin due to low content due in being analyzed in subsequent quantitation concentration it is too low, it is difficult to the problem of detected, significantly
Improve the recall rate of metabolin.Therefore, as soon as by using a detection technique platform, subsequent quick separating energy can be passed through
More kinds of metabolins is detected, to can finally realize simultaneously to short chain fatty acids, amino acid, benzoyl and phenyl derivative
A variety of metabolin qualitative and quantitative analysis such as object, indole derivatives, lipid, bile acid, choline, vitamin and polyamines class；Also,
Concentration of each metabolin in subsequent quantitation analysis is improved on the whole, so that the result accuracy that detection obtains is more
It is good, improve the accuracy and reliability of the entire metabolin quantitative analysis of detection technique platform.
Description of the drawings
Fig. 1 detects the corresponding principal component scores figure obtained after the different kind organism sample in embodiment for embodiment.
Fig. 2 is that each biological sample obtained after being detected in embodiment and retention index standard substance mixture are corresponding
Total ion chromatogram；
Fig. 3 is each biological sample detected in embodiment and the corresponding chloro-carbonic acid of retention index standard substance mixture
The total ion chromatogram of methyl ester derivation.
Specific implementation mode
Below with to human serum, urine metabolin and Escherichia coli in metabolin be detected, to people
For body and Co metabolism object, the individual metabolin of enterobacteriaceae of the enterobacteriaceae in human body are analyzed, illustrate this in conjunction with attached drawing
The specific implementation mode of invention.For specific method or material used in embodiment, those skilled in the art can be at this
On the basis of inventive technique thinking, conventional replacement is carried out according to existing technology and is selected, the embodiment of the present invention is not limited solely to
Specific record.
Experimental method used in embodiment is conventional method unless otherwise specified；Used material, reagent
Deng being commercially available unless otherwise specified.
Embodiment 1
Prepare biological sample：
76 that no identity identification information is selected from sample database (NMMS), and above-mentioned 76 corresponding serum is taken to distinguish
As serum biological sample, corresponding urine respectively as urine biology sample, namely there are 76 parts of serum biological samples and 76 parts
Urine biology sample；
Prepare cell biological with E.coli BL 21 (being purchased from Sigma-Aldrich, St.Louis, MO) Bacillus coli cells
Sample, for being verified to the metabolin detected in 76 above-mentioned biological samples.
Quality Control sample：The sample bought from Sigma-Aldrich (St.Louis, MO, USA) is as serum Quality Control sample；
The mixing of each urine biology sample is as urine Quality Control sample；The mixing of each cell biological sample is as cell Quality Control sample
This.
Step 1, biological sample pre-treatment, specifically includes：
Step 1, extraction process,
Extraction process extracts to obtain extraction biological sample to the metabolin in biological sample：
1. the extraction process of serum biological sample and serum Quality Control sample
(1) extraction process of serum biological sample
Since the metabolin in biological sample has water-soluble, also have fat-soluble, some are easy denaturation or degradation, and have
Content it is very low, it is difficult to detect, so the design of this process, mainly allows the metabolin in biological sample that can obtain quickly
And adequately extraction, specially：
Step 1.1, take the serum biological sample of the first predetermined volume in centrifuge tube, the first predetermined volume is 100-500 μ
L, dosage is too many, and the matrix of biological sample can pollute instrument detector when being detected, and causes the sensitivity of instrument to decline, dosage
Very little, then the content being detected is inadequate, it is difficult to detect the low metabolite of content, most preferably 100 μ L, both can guarantee at this time
All metabolins can be detected accurately, and be avoided that instrument is polluted by biological sample.
Step 1.2, it is that cold methanol, this reality is added in the first predetermined volume ratio to press with the volume ratio of the biological sample in step 1
It applies in example, the first predetermined volume ratio is：The biological sample of first predetermined volume：Cold methanol volume=1 of addition:3.When serum is given birth to
When object sample is 100-500 μ L, then the cold methanol of 300-1500 μ L is added, the best cold methanol that 300 μ L are added.Methanol, which has, to be promoted
Into the function of reaction, and if room temperature methanol is added, exothermic reaction can be occurred by meeting water, and this can make certain metabolins drop
Solution, to cause these metabolins to lose, so, it can be occurred to avoid such case using cold methanol solvate, also, cold first
The low temperature of alcohol can also allow albumen precipitation more complete, because albumen is easier to be precipitated at low temperature, be avoided that albumen pair in this way
The negative effect of derivatization reaction.And the time too many, sample can be caused to be freeze-dried, very little then egg of addition is added in cold methanol
Precipitate in vain it is insufficient, to influence derivative efficiency, so it is 1 to select the first predetermined volume ratio:3 ratio is added.
Step 1.3, after cold methanol being added, 20min-30min is stood under conditions of temperature is -20 DEG C to -30 DEG C, at this
Under the conditions of, albumen precipitation is abundant.Then temperature be in 4 DEG C, centrifugal force be 16000g under conditions of centrifuge 10min, will centrifuge
Obtained supernatant is fitted into freeze-drying in sample introduction bottle and obtains the extraction biological sample of serum.
After being freeze-dried, due to drying out, biological sample is concentrated significantly so that the concentration of metabolin therein
It is sufficiently high, so substantially increasing testing result, especially greatly improve the recall rate of the metabolin of low content.The present embodiment
In, the condition of freeze-drying is：- 40 DEG C to -30 DEG C of temperature, vacuum degree are 0.1mbar to 0.002mbar.Under this condition, both
It can guarantee that the metabolin in biological sample is non-volatile or degrades, also can guarantee to obtain enough dryings.
(2) extraction process of serum Quality Control sample
Operating process same as serum biological sample extraction process is carried out to serum Quality Control sample and obtains serum Quality Control sample
Biological sample is extracted in this Quality Control.
2. the extraction process of urine biology sample and urine Quality Control sheet
(1) extraction process of urine biology sample
It takes the biological sample of the first predetermined volume to be fitted into sample introduction bottle to be urinated under the freeze-drying of predetermined lyophilisation condition
The extraction biological sample of liquid biological sample.In the present embodiment, operating process is identical as in serum biological sample, and the first of urine
Predetermined volume is with the also identical of serum biological sample.
(2) extraction process of urine Quality Control sample
The same operating process with the processing of urine biology sample extraction is carried out to urine Quality Control sample and obtains urine Quality Control sample
This extraction biological sample.
3. the extraction process of cell biological sample and cell Quality Control biological sample
(1) extraction process of cell biological sample
Here the cell biological sample namely the large intestine in the present embodiment that cell biological sample generally refers to microorganism
The cell biological sample of bacillus.
Specially：
Step 1.1, prepare biological sample, the cell from organism is collected in 50mL centrifuge tubes, be 4 in temperature
DEG C, centrifugal force be 200g-1000g under centrifuge 10min, under this condition, cell will not rupture, and metabolin is caused to lose, simultaneously
It can be detached again with the culture solution on upper layer.After being cleaned with PBS solution, the PBS solution that 1mL is added is resuspended to obtain cell suspension conduct
Cell biological sample；
Step 1.2, homogenate is counted, number of cells counting is carried out to cell suspension with TC20 automatic cell counters, when
When count results meet predetermined amount, water and cold methanol volume ratio are added for the second predetermined volume ratio into cell suspension and carried out
Homogenate obtains cell homogenates liquid.
In the present embodiment, predetermined amount is 5 × 106-5×107A, the too many cell of cell quantity can lead to matrix effect, pollution
Detector, instrumental sensitivity decline, and cell quantity is very little, and the low metabolin of content is then difficult to detect.
In the present embodiment, the second predetermined volume ratio is：
The volume of water：Volume=1 of cold methanol:4.
The amount that water and cold methanol are added will be in rational range, and cold methanol is very little, and it is insufficient to not only result in albumen precipitation,
Contaminated ion source, lowering apparatus detection sensitivity, while fat-soluble metabolin can be caused to be difficult to fully extract again.Methanol volume mistake
It is big then drying time can be caused longer.
Step 1.3, it centrifuges, cell homogenates liquid is centrifuged under conditions of temperature is 4 DEG C, centrifugal force is 16000g
The supernatant that centrifugation obtains is transferred to the extraction biological sample sheet for being freeze-dried to obtain cell in sample introduction bottle by 10min.
Step 2, derivatization
After completing step 1, under predetermined derivatization conditions, each corresponding extraction biological sample that step 1 obtains is divided
It does not perform the derivatization to obtain each corresponding derivative biological sample.Predetermined derivatization conditions are：Temperature is the alkaline condition of 20-25 degree
Under, pH value range is preferably 10-12.It is more stable that derivative efficiency is carried out under this condition.
The design of this process, primarily to accelerating derivatization reaction efficiency, specially：
The biological sample that step 1 obtains is put into 4 DEG C of low temperature sample injection disc, by sodium hydroxide solution, methanol solution, pyrrole
Pyridine solution and derivative reagent are added sequentially to step 1 in the ratio that the volume ratio between them is third predetermined volume ratio and obtain
To extraction biological sample in perform the derivatization reaction.In the present embodiment, derivative reagent is methylchloroformate reagent (MCF).
Wherein, a concentration of 1M of sodium hydroxide solution, the concentration it is selected by make decision：In organism, especially
The pH value for being the different biological samples in human body can be different, can also change with morbid state, and the reason of this variation
Buffer salt balance mainly in organism changes, so the extraction sample obtained after being extracted to biological sample spreads out
When biochemical, this buffer salt system needs relatively high strong acid or highly basic to break, and what is selected in the present embodiment is highly basic
System.
Third predetermined volume ratio is in the present embodiment：Sodium hydroxide solution：Pyridine solution：Methanol solution：Methylchloroformate
Volume ratio be 10：8：1.7:1, specifically, in the present embodiment, sodium hydroxide solution, methanol solution, pyridine solution and chloromethane
The volume of sour methyl ester agent is respectively：200 μ L, 167 μ L, 34 μ L and 20 μ L.Such volume ratio cooperation, derivatization reaction efficiency
Highest.
And the process of above-mentioned addition is：NaOH solution is added after being dissolved at the pre-treatment sample of lyophilised state, then adds
Enter methanol solution and pyridine solution, methanol solution is added and pyridine solution carries out mixing, then adds MCF reagents, finally exists
30s is shaken under the vibration condition of 2,500-3,000rpm obtains derivative biological sample.Under the vibration condition, each solvent is anti-
It can be sufficiently mixed during answering uniformly, accelerate derivatization reaction.
The reaction of MCF derivative reagents is active, and under above-mentioned vibration condition, a fiercer shaking can be kept, Jin Erneng
So that the solution being added after MCF reagents obtains quick and sufficient dispersion, solvent reaction process is sufficiently mixed uniformly, in this way can be with
Ensure the rate of derivative reaction.
By above-mentioned derivatization process, serum biological sample, serum Quality Control sample, urine biology sample, urine are respectively obtained
Quality Control sample, cell biological sample and the respective derivative biological sample of cell Quality Control sample.
Step 3, standardization and impurity elimination
To the derivative biological sample of each serum, urine and cell and corresponding Quality Control sample to being obtained in step 2
In be separately added into the retention index standard substance mixture being mixed to get by a variety of linear paraffin standard items after, carry out impurity elimination obtain
Pre-treatment sample.
The design of this process, primarily to the standard items as data analysis standard reference after examination with computer are added, and
By the impurity removal in derivative biological sample, specially：
Step 3.1, retention index standard substance mixture is added in chloroformic solution and obtains standard items containing retention index
Chloroformic solution, be added retention index standard substance mixture effect be in order to when qualitative analysis as retention index
The reference of (Retention Indices), in the present embodiment retention index standard substance mixture by C8-C30 linear paraffin
Standard items mix namely the retention index standard substance mixture includes linear paraffin standard items from C8-C30.
Step 3.2, the chloroformic solution of standard items containing retention index is made a reservation for by the volume ratio with derivative biological sample for the 4th
Volume ratio is added in derivative biological sample, under the vibration condition of 2,500-3,000rpm, shakes 10s.
In the present embodiment, the volume ratio of the volume of the chloroformic solution of standard items containing retention index of addition and derivative biological sample
It is 1:1 namely the 4th predetermined volume ratio be 1:1.The chloroform of standard items containing retention index that 400 μ L are about added in the present embodiment is molten
Liquid.
The shaking of the process, can be by solution mixing, while can further carry out the extraction of metabolin.
Step 3.3, it is added a concentration of 50mM's in the ratio that the volume ratio with mark sample mixing sheet is the 5th predetermined volume ratio
NaHCO3Solution be added to mark sample mixing sheet in, shake 10s under the vibration condition of 2,500-3,000rpm, temperature be 4 DEG C, from
Centrifugation 10min is to be layered to obtain layering solution under conditions of mental and physical efforts are 2000g.
In the present embodiment, the NaHCO of addition3The volume ratio of the volume of solution and mark sample mixing sheet is 1:2 namely the 5th predetermined
Volume ratio is 1:2.In the present embodiment, the NaHCO of about 400 μ L is added3Solution.Here NaHCO is added3Aqueous solution is for neutralizing
The excessive acid ion generated, while so that organic solvents, chloroform is more prone to and aqueous phase separation.
The shaking of the process, can be by solution mixing, while can further carry out the extraction of metabolin, and can will invest support
Substance on disk cleans in solution.
Step 3.4, it will be layered the gas for being located at the chloroform layer for being layered bottom in solution and being transferred to the anhydrous sodium sulfate containing 100mg later
Pre-treatment biological sample is used as in phase sample bottle, for the ease of distinguishing and illustrating, for the sample as Quality Control, with pre-treatment matter
Biological sample is controlled to indicate.
Step 2, detection acquisition information, is detected pre-treatment biological sample by gas-chromatography and mass spectrometer
And collect information to be analyzed corresponding with metabolin.
During being somebody's turn to do, the temperature control of gas chromatograph uses temperature programming method, and temperature programming control setting is good
It is bad, it directly affects finally to the speed of the substance separating rate in preceding biological sample, to influence the quality of separating resulting, in turn
Last qualitative and quantitative analysis is served conclusive.
In order to reach above-mentioned separating effect, inventor is had found by long-term practice, can guarantee the time short enough, separation
Enough substances, and reach baseline between each substance and clearly detach, it is desirable that the use of gas chromatograph meets following item
Part：
1, the use condition of gas chromatograph is：Chromatographic column is capillary chromatographic column, length 30m, internal diameter 0.25mm,
And the packing material in chromatographic column is one be crosslinked in diphenyldimethyl polysiloxanes, middle polarity cross-linked phase or low-to-moderate polarity phase
Kind, and filling thickness be 1.4 μm of -0.25mm, ranging from 60 DEG C -350 DEG C of temperature in use；The injection port temperature of gas chromatograph
Degree is 270 DEG C, and transfer tube temperature is 270 DEG C.
2 also, total detection time is controlled in a relatively short period of time using temperature programming method as far as possible：
Use Restek Rxi-5ms chromatographic columns in the present embodiment, the conceptual design of temperature programming method can there are four types of,
It is as follows respectively：
Scheme one, initial temperature are 45 DEG C, keep 2min, rise to 300 DEG C with the rate of 12 DEG C/min, keep 5min,
Total detection time is 28.25min；
Scheme two, initial temperature are 45 DEG C, keep 1min, 260 DEG C are risen to the rate of 10 DEG C/min, with 40 DEG C/min
Rate rise to 320 DEG C, it is 26min to keep 2min, total detection time；
Scheme three, initial temperature are 45 DEG C, keep 1min, 260 DEG C are risen to the rate of 15 DEG C/min, with 40 DEG C/min
Rate rise to 320 DEG C, it is 18.83min to keep 2min, total detection time；
Scheme four, initial temperature are 45 DEG C, keep 1min, 260 DEG C are risen to the rate of 20 DEG C/min, with 40 DEG C/min
Rate rise to 320 DEG C, it is 15.25min to keep 2min, total detection time.
Detection is done using above-mentioned four kinds of schemes to the serum of this implementation and urine biology sample respectively to compare, as a result such as
Shown in table 1.
The GC separation parameter optimization process of 1 serum of table and urine specimen
From table 1 it follows that total time most short can control can detect more generations at 15.25min, the program
Thank to product (peak number), stronger peak height, half-peak breadth (PWHH) is narrower, and peak purity (PP) is more preferable.
Further, it is suggested that adoptable chromatographic column also has：Restek Rxi-624Sil chromatographic columns, the length of 30m, internal diameter
For 0.25mm, packing material is middle polarity cross-linked phase, and 1.4 μm of thickness, maximum temperature is 300-320 DEG C；Restek Rtx-624
Chromatographic column, the length of 30m, internal diameter 0.25mm, packing material are low-to-moderate polarity phase, 1.4 μm of thickness, use temperature range
For:- 20 DEG C of 240 DEG C of to, it is as follows using temperature programming method accordingly：
Initial temperature is 35 DEG C, keeps 2min, 260 DEG C is risen to the rate of 25 DEG C/min, with the rate of 40 DEG C/min
290 DEG C are risen to, it is 17.75min to keep 5min, total detection time.
In addition, in order to can guarantee enough Mass Spectrometer Method effects, rapidly carried out with the substance that can be detached to gas-chromatography
Mass Spectrometer Method obtains corresponding information to be analyzed, so as to accurately identify these information to be analyzed, and then realizes to these
Information to be analyzed more precisely qualitative, quantitative, in the present embodiment, mass spectrometric use condition is：Ion source temperature is 220 DEG C；
Sampling volume is 0.5-1 μ L；Splitless injecting samples；Ion source voltage is 70eV；Scanning of the mass spectrum ranging from 38-650m/z；Acquisition speed
Rate is 20-25spectra/s.Specifically, LECO Pegasus HT TOF mass spectrographs may be used to be detected.In the condition
Under, each metabolin can have good signal, ensure the accuracy of quantitative analysis.
Restek Rxi-5ms chromatographic columns are finally used in the present embodiment, and are detected using scheme four.
According to above-mentioned, shared serum, urine and cell three classes biological sample, to the upper machine sequence of every class biological sample
It is：Every 14 corresponding pre-treatment biological samples, into the corresponding pre-treatment Quality Control sample (QC) of a needle.Per class biological sample equal six
Batch is into complete.
Step 3, qualitative and quantitative analysis, the database of the derivative foundation of the standard items substance based on business procurement, we
The retention index and ms fragment information for removing the different substances to be analyzed of comparison, it is accurate qualitative fixed to being carried out to information to be analyzed to realize
Amount analysis, specially：
Data processing：The initial data that chromatography and mass spectrograph combination collect is exported as into NetCDF formats, is adopted
It is (including data prediction, baseline correction, flat that data analysis is carried out with ChromaTOF (v4.50, Leco Co., CA, USA) software
Cunning, noise reduction, deconvolution, metabolin retrieval and calculated by peak area etc.), obtain different letter to be analyzed corresponding from different metabolic object
Breath, which includes mass spectrometry parameters and retention index (RI).
Qualitative analysis：Based on standard database, it is analysed to information and carries out mass spectrum with the standard substance in standard database
The matching of parameter and retention index (RI) obtains matching degree, when matching degree is more than 70%, then can be corresponded to qualitative information to be analyzed
Metabolin be corresponding standard substance.
Quantitative analysis：Combined standard curve provides；Final data will export as CSV formats, and information includes sample name, generation
Thank object, Kovats-RI, quota ion, peak area and concentration；Using the method for establishing standard curve, final quantitative knot is obtained
Fruit.
In the present embodiment, standard database is carried out by the derivative obtained after deriving to various criterion substance
Machine qualitative and quantitative analysis obtains, and includes the criterion numeral that corresponding different Information in Mass Spectra are distinguished from the derivative of various criterion substance
According to library.According to the metabolin ingredient contained in prediction biological sample, it is commercially available the chemicals of standard substance.
Fig. 1 detects the corresponding principal component scores figure obtained after the different kind organism sample in embodiment for embodiment.
As shown in Figure 1, in figure, (A) light color circle indicates that the principal component that serum detection of biological samples goes out, dark circle indicate
The principal component that serum Quality Control detection of biological samples goes out, the R2X=0.504 of two kinds of principal components；In figure, (B) dark color circle indicates urine
The principal component of liquid biological sample, light circle indicate the principal component principal component of urine Quality Control biological sample, the R2X of two kinds of principal components
=0.479；Also, QC sample standard deviations are gathered at one in Fig. 1.
So it can be seen from figure 1 that this method has good reproducibility.
Fig. 2 is that each biological sample obtained after being detected in embodiment and retention index standard substance mixture are corresponding
Total ion chromatogram；
Fig. 3 is each biological sample detected in embodiment and the corresponding chloro-carbonic acid of retention index standard substance mixture
The total ion chromatogram of methyl ester derivation.
Retention index standard substance mixture is indicated with retention index in Fig. 2 and Fig. 3.
The temperature programming control using the present embodiment is can be seen that from such as Fig. 2, display can detect more under this condition
Metabolite, stronger peak height, half-peak breadth is narrower, and peak purity is more preferable, and especially Fig. 3 is shown：Due to rational temperature programming
Control just separates 12 kinds of short chain fatty acids in 3min.
In the present embodiment, generation that the biological sample from human body and the cell biological pattern detection from Escherichia coli are arrived
Thank to object, obtain the content of the corresponding metabolin of each biological sample as shown in Table 2 after quantitative analysis, in table 2 blank be less than
Detection limit：
The quantitative result of serum of the table 2 from human body, urine and the metabolin detected in Escherichia coli
From Table 2, it can be seen that using this method, the metabolite content that can be detected can quantify and arrive denier, explanation
By this method, the extremely low metabolin of content can be detected.
Verify example 1
This verification example is for verifying the method reproducibility of embodiment.
Using the step one in the method for embodiment, 6 hybrid standard product and biological sample are independently prepared, and use
Step two in the method for embodiment and step 3 carry out continuously upper machine analysis.Testing result is shown in Table 3.
Table 3 verifies the methodology parameter of example 1
Related coefficient is calculated by concentration and respective area in a linear regression model (LRM)s in table 3；B detection limits (LOD) take S/
The concentration of standard substance when N values are 6；C reproducibility is by retention index standard substance mixture and the serum mixed respectively, urine
Metabolite concentration is calculated in biological sample；* indicate that RSD values are more than 20%.
Table 3 is shown, removes metabolin of the concentration near quantitative limit, and the RSD values of 90% or more metabolin are less than 15%,
Have good reproducibility, illustrates the method favorable reproducibility of embodiment.
Verify example 2
This verification example is used to verify the stability of the method for embodiment.
Each biological sample is handled using step 1 in the method for embodiment to obtain different pre-treatment biology samples
This, upper machine testing is carried out after each pre-treatment biological sample is carried out following storages respectively later again：
The 2nd day, the 4th day and the 6th day of storage, and storage temperature is at -80 DEG C.
Every time detection be all made of RI standard items rectify an instrument interval long-time after sample introduction response variance.As a result such as 4 institute of table
Show.
Table 4 is that verification example 2 carries out the testing result after different storages
From table 4, it can be seen that the content that different number of days detects is close, and in controlled range, about 80% derivatization
Metabolite is stored at -80 DEG C and can be detected in 4 days, and error is less than 20%, since sample volatilizees in six days, can lead to sample
This metabolin detected value is higher, so it is proposed that sample detects completion in 4 days, in short, using the step one in embodiment
The pre-treatment biological sample obtained after pre-treatment is carried out, stability is preferable.
Embodiment effect
The biological sample preparation method of embodiment offer and its application and metabolin qualitative and quantitative analysis method, due to
In biological sample preparation method, when extracting processing to the metabolin in biological sample, extracted by freeze-drying
Biological sample, in this way, the moisture in extraction biological sample greatly reduces so that the wherein content concentration of metabolin thus is avoided that
Due to low content metabolin due in being analyzed in subsequent quantitation concentration it is too low, it is difficult to detected, substantially increase metabolism
The recall rate of object.Therefore, by using a detection technique platform, more kinds of metabolism of subsequent quick separating can be detected
Object, so as to it is final realize simultaneously to short chain fatty acids, amino acid, benzoyl and phenyl derivatives, indole derivatives, lipid,
A variety of metabolin qualitative and quantitative analysis such as bile acid, choline, vitamin and polyamines class；Also, each generation is improved on the whole
Thank to concentration of the object in subsequent quantitation analysis, so that the result correctness that detection obtains is more preferable, and then it is flat to improve detection
The accuracy of the entire metabolin quantitative analysis of platform.
Further, the pre-treatment biological sample that the biological preparation of the step one in embodiment is handled has
Good stability, notable advantage include：On the one hand, due to the metabolism analyte detection in the biological sample of organism, often
The biological sample material needed is more, and the metabolin of detection is also more, as a result also more accurate, so big data analysis is needed, this
Sample can spend very long detection time, generally can not complete within one day, so when biological sample quantity is more, can carry out in batches
Pre-treatment obtains the pre-treatment biological sample of all biological samples, is then stored at -80 DEG C, then completes detection in batches, this
Sample can eliminate the preparation error between criticizing, and the storage of stable sample can also ensure testing result within error range；Another party
Face, if the data analysis discovery of testing result is problematic, it is also possible to upper machine testing verify, at this moment can carry out again
When step 1, the pre-treatment biological sample of the backup handled can be directly used in again upper machine, when both saving when needed
Between, and due to being obtained with a pre-treatment, moreover it is possible to keep the consistency of pre-treatment, raising to detect the accurate of verification again
Property；
It further, can be by entirety since the control of the temperature of the gas chromatograph in embodiment uses temperature programming method
Time control is in 15.25min-28.25min, so that the detection technique platform of the method using the present embodiment, it can be shorter
Time realize efficiently separating to a variety of metabolins for extracting, so as to realize simultaneously to short chain fatty acids, amino acid, benzene
Numerous kinds and the concentration such as formyl and phenyl derivatives, indole derivatives, lipid, bile acid, choline, vitamin and polyamines class
Metabolin qualitative and quantitative analysis.
In addition, in the present embodiment, the biological sample used for serum, urine, may be used also by the biological sample as the present invention
To use saliva, at this time in step 1, the first predetermined volume of saliva is 250-1000 μ L, 500 μ L of optimal selection.
In addition, as the present embodiment, host is human body, and the metabolin of detection is：Intestinal flora in human body is in human body
It is metabolized in the Co metabolism object that the metabolin generated or intestinal flora are generated with human body common metabolic, the preparation method as the present invention
And its application and metabolism determination method, host can be all living things body, metabolin can be independent microorganism generation
The metabolin thanked, can be from the microorganism in host and the host symbiosis metabolin or microorganism in host
Metabolin.
In addition, in embodiment, derivative reagent is methylchloroformate reagent, as the present invention, derivative reagent can select
With all similar derivative reagent is acted on methylchloroformate reagent.
In addition, the biological sample preparation method as the present invention and the metabolism object detecting method that is related to, can use life
Object sample preparation methods choose the data results that representative biological sample carries out pre-treatment and upper machine, selection and standard
The retention index of the substance metabolin consistent with Information in Mass Spectra matching degree, or can be but not each with matched standard substance
The high metabolin of matching degree between biological sample is also their shared metabolins, is put into standard database, as unknown
Shared metabolin library, when carrying out pre-treatment and upper machine testing and data processing to all biological samples, using above-mentioned selection
The metabolin of retention index and Information in Mass Spectra out is matched, and qualitative point of the metabolin in biological sample may be implemented
Analysis, substantially increases the accuracy and recall rate of metabolin qualitative analysis in biological sample in this way.
Claims (32)
1. a kind of biological sample preparation method for handling before carrying out the biological sample from organism obtains that generation can be used for
Thank to the pre-treatment sample that object carries out gas-chromatography and mass spectrometer detection and analysis, which is characterized in that include the following steps：
Step 1, extraction process extracts the metabolin in the biological sample to obtain extraction biological sample；
Step 2, derivatization under predetermined derivatization conditions, performs the derivatization the extraction biological sample after completing step 1
Obtain derivative biological sample；
Step 3, standardization and impurity elimination are added into the derivative biological sample and are mixed to get by a variety of linear paraffin standard items
After retention index standard substance mixture, carries out impurity elimination and obtains the pre-treatment sample,
Wherein, in step 1, the extraction biological sample is obtained by freeze-drying.
2. biological sample preparation method according to claim 1, it is characterised in that：
Wherein, the condition of freeze-drying be -80 DEG C to -30 DEG C in temperature at be dried in vacuo.
3. biological sample preparation method according to claim 2, it is characterised in that：
Wherein, the vacuum degree of vacuum is 0.1mbar-0.002mbar.
4. biological sample preparation method according to claim 1, it is characterised in that：
Wherein, the step 1 is specially：
Step 1.1, take the biological sample of the first predetermined volume in centrifuge tube；
Step 1.2, it is that cold methanol is added in the first predetermined volume ratio to press with the volume ratio of the biological sample in step 1.1；
Step 1.3, after the cold methanol is added, 20min-30min is stood under conditions of temperature is -20 DEG C to -30 DEG C, at this
Under the conditions of, albumen precipitation is abundant.Then 10min is centrifuged under conditions of temperature is 4 DEG C, centrifugal force is 16000g, will centrifuged
To supernatant be fitted into sample introduction bottle freeze-drying and obtain the extraction biological sample.
5. biological sample preparation method according to claim 4, it is characterised in that：
Wherein, first predetermined volume is 100-500 μ L.
6. biological sample preparation method according to claim 4, it is characterised in that：
Wherein, first predetermined volume ratio is：
The biological sample of first predetermined volume：Cold methanol volume=1 of addition:3.
7. the biological sample preparation method according to claim 1 to 6 any one, it is characterised in that：
Wherein, the biological sample is the serum of the organism.
8. biological sample preparation method according to claim 1, it is characterised in that：
Wherein, the step 1 is specially：
It takes the biological sample of the first predetermined volume to be fitted into freeze-drying in sample introduction bottle and obtains the extraction biological sample.
9. biological sample preparation method according to claim 8, it is characterised in that：
Wherein, first predetermined volume is 100-1000 μ L.
10. biological sample preparation method according to claim 9, it is characterised in that：
Wherein, first predetermined volume is 100-500 μ L.
11. biological sample preparation method according to claim 9, it is characterised in that：
Wherein, first predetermined volume is 250-1000 μ L.
12. the biological sample pre-treating method according to any one of claims 1 to 3,8 to 11, it is characterised in that：
Wherein, the organism sample is the urine or one or more in saliva of the organism.
13. biological sample preparation method according to claim 1, it is characterised in that：
The biological sample is the microbial cell of the organism, and the step 1 is specially：
Step 1.1, prepare biological sample, the cell from the organism is collected in centrifuge tube, temperature be 4 DEG C, from
Mental and physical efforts are to centrifuge 10min under 200g-1000g, then after being cleaned with PBS solution, the PBS solution that 1mL is added is resuspended to obtain institute
Cell liquid is stated as the biological sample；
Step 1.2, homogenate is counted, it is accurate that number of cells first is carried out to the cell suspension with digital automatic cell counter
It counts, when count results meet predetermined value, centrifugation again obtains cell, discards upper layer PBS solution, and water and cold methanol is added
(volume ratio is the second predetermined volume ratio) is homogenized to obtain cell homogenates liquid；
Step 1.3, it centrifuges, the cell homogenates liquid is centrifuged under conditions of temperature is 4 DEG C, centrifugal force is 16000g
The supernatant that centrifugation obtains is transferred in sample introduction bottle and is freeze-dried to obtain the extraction biological sample by 10min.
14. biological sample preparation method according to claim 13, it is characterised in that：
Wherein, the predetermined amount of the cell sample is 5 × 106-5×107It is a.
15. biological sample preparation method according to claim 13, it is characterised in that：
Wherein, second predetermined volume ratio is：The volume of water：Volume=1 of cold methanol:4.
16. biological sample preparation method according to claim 1, it is characterised in that：
The predetermined derivatization conditions are：Temperature is under the alkaline condition of 20-25 degree.
17. biological sample preparation method according to claim 1, it is characterised in that：
Wherein, the step 2 is specially：
It is predetermined for third by the volume ratio between them by sodium hydroxide solution, methanol solution, pyridine solution and derivative reagent
The ratio of volume ratio sequentially adds, and the NaOH solution is added and adds the methanol after pre-treatment sample dissolving
Solution and the pyridine solution, add the derivative reagent at this time after mixing, finally in the vibrator bar of 2,500-3,000rpm
30s is shaken under part obtains the derivative biological sample, a concentration of 1M of the sodium hydroxide solution.
18. biological sample preparation method according to claim 17, it is characterised in that：
Wherein, the derivative reagent is methylchloroformate reagent.
19. biological sample preparation method according to claim 17, it is characterised in that：
The third predetermined volume ratio is：
The sodium hydroxide solution：The pyridine solution：The methanol solution：The volume ratio of the derivative reagent is 10：8：
1.7:1。
20. biological sample preparation method according to claim 1, it is characterised in that：
Step 3 is specially：
Step 3.1, the retention index standard substance mixture is added in chloroformic solution and obtains standard items containing retention index
Chloroformic solution；
Step 3.2, it is the by described press the chloroformic solution of standard items containing retention index with the volume ratio of the derivative biological sample
The ratio of four predetermined volume ratios is added in the derivative biological sample, under the vibration condition of 2,500-3,000rpm, shaking
10s obtains mark sample mixing sheet；
Step 3.3, it is added a concentration of 50mM's in the ratio that the volume ratio with the mark sample mixing sheet is the 5th predetermined volume ratio
NaHCO3Solution is added in the mark sample mixing sheet, and 10s is shaken under the vibration condition of 2,500-3,000rpm, is 4 in temperature
DEG C, centrifugal force be 2000g under conditions of centrifugation 10min be layered to obtain layering solution；
Step 3.4, the chloroform layer that layering bottom is located in the layering solution is transferred to the gas phase sample of the anhydrous sodium sulfate containing 100mg
The pre-treatment biological sample is used as in product bottle.
21. biological sample preparation method according to claim 20, it is characterised in that：
Wherein, the 4th predetermined volume ratio is 1:1.
22. biological sample preparation method according to claim 20, it is characterised in that：
Wherein, the 5th predetermined volume ratio is 1:2.
23. biological sample preparation method according to claim 1, it is characterised in that：
The retention index standard substance mixture includes the linear paraffin standard items from C8-C30.
24. biological sample preparation method according to claim 1, it is characterised in that：
The metabolin is that the micropopulation in host is metabolized the metabolin of generation or the micropopulation in the host
The summation of the Co metabolism object generated with host's common metabolic either both the above.
25. biological sample preparation method according to claim 24, it is characterised in that：
The host is human body, and the micropopulation is the intestinal flora in human body.
26. biological sample preparation method according to claim 1, it is characterised in that：
The metabolin is one kind in aliphatic acid, phenyl class, indoles, lipid, bile acid, choline, vitamin or polyamines class
Or it is a variety of.
27. a kind of application of biological sample preparation method in metabolin qualitative and quantitative analysis, it is characterised in that：
Wherein, the biological sample preparation method is the biological sample preparation method described in any one of claim 1-26.
28. a kind of metabolin qualitative and quantitative analysis method, by gas-chromatography and mass spectrometer to the biology from organism
Sample is detected analysis and obtains information to be analyzed, to analyze the metabolin in the organism, which is characterized in that packet
Include following steps：
Step 1, biological sample pre-treatment carry out pre-treatment to the biological sample and obtain pre-treatment biological sample；
Step 2, detection acquisition information, is detected the pre-treatment biological sample by gas-chromatography and mass spectrometer
And collect information to be analyzed corresponding with the metabolin；
Step 3, qualitative and quantitative analysis, based on including and the corresponding different mass spectrums letter of the derivative of various criterion substance difference
The standard database of breath carries out qualitative and quantitative analysis to the information to be analyzed,
Wherein, step 1 is carried out using the biological sample preparation method described in any one of claim 1 to 26.
29. metabolin qualitative and quantitative analysis method according to claim 28, it is characterised in that：
In step 2,
The temperature control of the gas chromatograph uses temperature programming method：Initial temperature is 35-45 DEG C, keeps 1min-2min,
Rise to 260 DEG C -300 DEG C with the rate of 10 DEG C/min-25 DEG C/min, then with the rate of 40 DEG C/min rise to 290 DEG C -
320, kept for 2min-5min, total time 15.25min-28.25min.
30. metabolin qualitative and quantitative analysis method according to claim 28, it is characterised in that：
The condition of the gas chromatograph is：
Chromatographic column is capillary chromatographic column, and length 30m, internal diameter 0.25mm, packing material is that crosslinking diphenyldimethyl is poly-
One kind in siloxanes, middle polarity cross-linked phase or low-to-moderate polarity phase, filling thickness are 1.4 μm of -0.25mm, the range of temperature in use
It is 60 DEG C -350 DEG C；Injector temperature is 270 DEG C；Transfer tube temperature is 270 DEG C.
31. metabolin qualitative and quantitative analysis method according to claim 28, it is characterised in that：
The mass spectrometric use condition is：Ion source temperature is 220 DEG C；Sampling volume is 0.5-1 μ L；Splitless injecting samples；From
Component voltage is 70eV；Scanning of the mass spectrum ranging from 38-650m/z；Acquisition rate is 20-25spectra/s.
32. metabolin qualitative and quantitative analysis method according to claim 28, it is characterised in that：
Wherein, the standard database further includes distinguishing corresponding difference from the corresponding derivative matter of the different standard substances
Retention index.
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