CN109917040A - A method of a variety of intestinal flora organic acids are quantified based on LC-MS - Google Patents
A method of a variety of intestinal flora organic acids are quantified based on LC-MS Download PDFInfo
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Abstract
The application, which discloses, a kind of carries out quantitative method to a variety of intestinal flora organic acids based on LC-MS, which comprises the sample is pre-processed, then performed the derivatization with derivatization reagent by the preparation of sample；The preparation of blank sample；LC-MS detects the sample and the blank sample；The quantitative analysis of sample.The application also provides "dead" 6 carbon, 13 stable isotope labeling 3- nitrophenyl hydrazine or 2- nitrophenyl hydrazine is marked on the purposes detected in intestinal flora organic acid as in.The method of the application is based on highly sensitive triple level four bars mass spectrographs；6 "dead" 13 stable isotope labelings of carbon13C6The introducing of -3NPH derivatization reagent guarantees quantitative accuracy.
Description
Technical field
This application involves analytical chemistry fields, a kind of based on effect liquid phase chromatogram/triple level four bars especially but not limited to being related to
The method that mass spectrum quantifies intestinal flora organic acid.
Background technique
Organic acid refers to some organic compounds with acidity.The most common organic acid is carboxylic acid, it is a kind of participation
The important substance of organism vital movement such as participates in tricarboxylic acid cycle, maintains body energy supply etc.；The occurrence and development of many diseases are all
It is related with metabolism of organic acids imbalance, such as cardiovascular disease, Alzheimer disease, diabetes；And short chain fatty acids are enterobacteriaceaes
There is the primary product that group's fermentation polysaccharides generate adjusting colony balance and the expression of antitumor, anti-inflammatory and controlling gene etc. to make
With；Middle chain, long chain fatty acids are other than as film rouge, moreover it is possible to adjust energetic supersession.In recent years, intestinal flora and host's phase interaction
With being the new research hotspot of life science, intestinal flora is unclear to the direct or indirect physiological effect of host.Therefore, in order to
The interaction between enteric microorganism and host is preferably studied, establish a kind of can detect the highly sensitive of a variety of organic acids simultaneously
The analysis method of degree is extremely urgent.It is done in addition, some organic acids such as acetic acid, palmitinic acid etc. usually will appear strong background
It disturbs, leads to the inaccuracy of quantitative result.These background contaminations are evaluated, realize that the work of accurate quantitative analysis is essential.
There are many contents that method is used to measure organic acid, including gas-chromatography, Capillary Electrophoresis, efficient liquid phase at present
Chromatography etc., for non-volatile organic acids and middle long chain fatty acids, gas chromatography-mass spectrum (gas chromatography-mass
Spectrometry, GC-MS) analysis usually requires the derivatizations such as esterification increasing its volatility and is detected, but GC-MS
Reproducibility and complex matrices analytic ability make the technology receive certain limitation.With the fast development of analytical technology, liquid
Matter joint technology becomes mainstream technology because of the advantages such as its selectivity is strong, high sensitivity, sample pre-treatments are simple.
Summary of the invention
It is the general introduction to the theme being described in detail herein below.This general introduction is not the protection model in order to limit claim
It encloses.
The application is based on the highly sensitive mass spectrometric Selective reaction monitoring of triple level four bars (selected reaction
Monitoring, SRM) mode carries out Data Detection, in addition to common high abundance organic acid, more low contents and important can be covered
Organic acid.But fatty acid metabolin keeps the method limited due to a lack of fragments characteristic ion.To solve fatty acid metabolin
The problem of lacking fragments characteristic ion, the organic acid to volatilize in the sample of underivatized can't detect in LC-MS, fat
The organic acid of acids does not have fragments characteristic, it is also difficult to be detected.
Based on problem above, the application introduces 3- nitrobenzophenone hydrazine (3-nitrophenylhydrazine, 3-NPH) or 2-
The derivative reaction of nitrophenyl hydrazine, performs the derivatization carboxyl, to improve its second order ms behavior and improve the spirit of instrument detection
Sensitivity.This application provides a kind of derivative reaction based on 3-NPH or 2-NPH, cover up to 42 kinds of volatility or non-volatile
The organic acid of property quantifies；The quantitative approach of the application is based on highly sensitive triple level four bars mass spectrographs；"dead" 6
A 13 stable isotope labeling of carbon13C6The introducing of -3NPH derivatization reagent makes each standard items have corresponding internal standard, in this way
It ensure that quantitative accuracy, and save experimental cost；Be added blank reaction, background correction interference so that data can
Guaranteed by property.
Specifically, quantitative side is carried out to a variety of intestinal flora organic acids based on LC-MS this application provides a kind of
Method, which comprises
The sample is pre-processed, is then performed the derivatization with derivatization reagent by the preparation of sample；
The preparation of blank sample；
LC-MS detects the sample and the blank sample；
The quantitative analysis of sample.
In this application, the sample can be fresh intestinal contents, excrement, tissue or blood.
In this application, the extraction of sample may include: to extract sample with acetonitrile.
In this application, the derivatization may include:
The mixture of the sample and acetonitrile is centrifuged, takes supernatant that 3- nitrophenyl hydrazine, 1- (3- dimethylamino third is added
Base) -3- ethyl-carbodiimide hydrochloride (EDCHCl) and pyridine solution, react 15min-30min at 25 DEG C -60 DEG C, nitrogen
It is dried up under gas shielded spare.
In this application, when the sample is fresh intestinal contents, excrement or tissue, 10mg-100mg is taken, or work as
When the sample is blood, 10 μ L-100 μ L are taken, it is homogeneous dilute that the progress of 0.1-1mL 50%-70% acetonitrile (ACN) aqueous solution is added
It releases, is vortexed and mixes 1min-3min.By mixture in 3000rpm-12000rpm room temperature 10min-20min, take on 40 μ L-100 μ L
Clearly, the 50%-70% acetonitrile solution of the 3-NPH of 10 μ L-50 μ L 200mM, 25 μ L 120mM 1- (3- dimethylaminos third are added
Base) -3- ethyl-carbodiimide hydrochloride (EDC) solution, wherein solvent is the 50%-70% acetonitrile solution comprising 6% pyridine.
At 25 DEG C -60 DEG C, 15min-30min is reacted, is dried up under nitrogen protection spare.
In this application, the preparation of the blank sample may include:
By acetonitrile, 3- nitrophenyl hydrazine, 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride and pyridine solution,
15min-30min is reacted at 25 DEG C -60 DEG C, is dried up under nitrogen protection spare.
In this application, the preparation of the blank sample may include: to take 40 μ L-100 μ L 50%-70%ACN, be added
The 50%-70%ACN solution of the 3-NPH of 10 μ L-50 μ L 200mM, 25 μ L 120mM are mixed with 1- (the 3- dimethylamino of 6% pyridine
Propyl) -3- ethyl-carbodiimide hydrochloride (EDC) 50%-70% acetonitrile solution.At 25 DEG C -60 DEG C, 15min- is reacted
30min is dried up spare under nitrogen protection.
In this application, in LC-MS detection, liquid phase chromatogram condition: chromatographic column: ACQUITY UPLC BEH
C18；Chromatographic column temperature: 35 DEG C -55 DEG C；1 μ L-10 μ L of sample volume；Flow velocity: 0.2mL/min-0.3mL/min；Flow phase composition: A phase
For water, B phase is acetonitrile；Gradient: 0-1min, 2%B, 1-34min, 2-98%B, 34-40min, 98%B, 40-45min,
2%B, the percentage are percent by volume.
Mass Spectrometry Conditions: being detected, electrospray ionisation using the mass spectrometric Selective reaction monitoring mode of triple level four bars, is born
Ion mode scanning, spray voltage: 3000V；Ion transfer tube temperature: 320 DEG C；Auxiliary temperature degree: 300 DEG C；Sheath gas:
35arb；Auxiliary gas flow speed: 10arb；The resolution ratio of mass analyzer Q1 and Q2: 0.7FWHM；Collision gas pressure is
1.5mTorr。
In this application, the method can be with simultaneous quantitative 20-60 kind intestinal flora organic acid.
In this application, the method can be with 42 kinds of intestinal flora organic acids of simultaneous quantitative, and the organic acid includes following
In any work it is more kinds of: formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, caproic acid, enanthic acid, n-nonanoic acid, the last of the ten Heavenly stems
Acid, hendecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, 16 carbon monoenoic acids, heptadecane
Acid, octadecanoid acid, 18 carbon monoenoic acids, octadecadienoic acid, octatecatrienoic acid, nonadecylic acid, eicosatetraenoic acid, 20
Carbon 5 alkene acid, docosahexaenoic acid, lignoceric acid, tetracosa carbon monoenoic acid, acetoacetate, beta-hydroxybutyric acid, citric acid, crow
Head acid, isocitric acid, α-ketoglutaric acid, fumaric acid, succinic acid, malic acid, oxaloacetic acid, pyruvic acid, lactic acid.
In this application, the quantitative calculating of sample includes:
With the ratio of the peak area at the characteristic ion peak of the standard items after the derivatization and corresponding internal standard peak area
For ordinate, concentration is abscissa, draws the standard curve of internal standard method,
The peak area ratio of each compound in the sample is first deducted into blank reaction, then brings the standard song into
Line obtains the content of organic acid in the sample.
In certain embodiments of the application, qualitative analysis can be carried out first: be extracted by Tracefinder software
The chromatographic peak of sample and the standard items appearance time comparison after retention time, with derivatization carry out qualitative analysis, and to being detected
Compound carry out chromatographic peak integral.Then, then quantitative analysis is carried out: with the characteristic ion peak of the standard items after derivatization
Peak area is ordinate with the ratio of corresponding internal standard peak area, and concentration is abscissa, draws the standard curve of internal standard method.It will
The peak area ratio of each compound in sample first deducts blank reaction, then brings standard curve into, obtains having in tested sample
The absolute content of machine acid.
In this application, the quantitative analysis further includes standard items mother liquor: the standard items of above-mentioned organic acid are taken respectively,
Initial concentration is 50mM, and to prepare the mixed standard solution of organic acid, mixing mother liquor final concentration is respectively 200 μM, uses 50%-
70%ACN successively dilutes following concentration point: 100 μM, 50 μM, 25 μM, 10 μM, 5 μM, 2.5 μM, 1 μM, 500nM, 250nM,
100nM, 50nM, 25nM, 10nM, 5nM, 2.5nM, 1nM, 0.5nM, 0.25nM, 0.1nM take 40 μ L-100 μ L titers spare.
In certain embodiments of the application, reaction is performed the derivatization to sample and standard items: taking the acetonitrile of sample molten
The acetonitrile solution of liquid and standard items is separately added into the 50%-70%ACN solution of the 3-NPH of 10 μ L-50 μ L 200mM, 25 μ L
120mM 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC) solution, wherein solvent is to include 6% pyridine
50%-70% acetonitrile solution.At 25 DEG C -60 DEG C, 15min-30min is reacted, is dried up under nitrogen protection spare.
In this application, internal standard derivative reaction: 10 μ L-50 μ L are added in the standard solution for taking organic acid to mix
200mM's13C6The 50%-70%ACN solution of -3NPH adds 1- (the 3- dimethylamino that 25 μ L 120mM are mixed with 6% pyridine
Propyl) -3- ethyl carbodiimide solution (EDC).At 25 DEG C -60 DEG C, 15min-30min is reacted, dilutes 10 times with 70%ACN
It is spare.
Liquid chromatogram separation: ultra performance liquid chromatography (Ultrahigh Performance Liquid is utilized
Chromatography, UPLC) system and reverse chromatograms analytical column carry out chromatographic isolation, and liquid phase chromatogram condition includes: chromatographic column:
ACQUITY UPLC BEH C18, chromatographic column temperature: 35 DEG C -55 DEG C；1 μ L-10 μ L of sample volume；Flow velocity: 0.2mL/min-0.3mL/
min；Flowing phase composition: A Xiang Weishui, B phase is acetonitrile, Mass Spectrometer Method: is selected in triple level four bars mass spectrographs using high sensitivity
It selects reaction monitoring mode to be detected, electrospray ionisation, negative ion mode scanning, spray voltage: 3000V；Ion transfer tube temperature
Degree: 320 DEG C；Auxiliary temperature degree: 300 DEG C；Sheath gas: 35arb；Auxiliary gas flow speed: 10arb；Mass analyzer Q1 and Q2
Resolution ratio: 0.7FWHM；Collision gas pressure is 1.5mTorr.
Data analysis: mark product, internal standard, blank reaction and sample are extracted using Tracefinder qualitative and quantitative analysis software
Chromatographic retention, parent ion and its feature daughter ion information of middle collected organic acid, and to obtained chromatographic peak face
It is integrated.It is qualitative to metabolin progress by the retention time of standard items, it substitutes into standard curve and metabolin is quantified
Analysis.
Due to the adoption of the above technical solution, compared with prior art, present invention has the advantage that
1. the quantitative approach of the application covers the relevant organic acid of more intestinal floras, enteron aisle can be further studied
Interaction between microorganism and host.
2. the quantitative approach of the application can be met multiple to the greatest extent based on highly sensitive triple level four bars mass spectrographs
The detection of organic acid in miscellaneous sample.
3. 13 stable isotope labeling of carbon13C6The introducing of -3NPH derivatization reagent can make each standard items have phase
The internal standard answered guarantees quantitative accuracy, saves experimental cost.
4. blank reaction is added, carry out the interference of organic acid in background correction, so that the reliability of data is guaranteed.
Other features and advantage will illustrate in the following description, also, partly become from specification
It obtains it is clear that being understood and implementing the application.The purpose of the application and other advantages can be by specifications, right
Specifically noted structure is achieved and obtained in claim and attached drawing.
Detailed description of the invention
Attached drawing is used to provide to further understand technical scheme, and constitutes part of specification, with this
The embodiment of application is used to explain the technical solution of the application together, does not constitute the limitation to technical scheme.
Fig. 1 is 42 kinds of organic acids and interior target total ion chromatogram in stool in mice sample.
Fig. 2 is the organic acid content detected in stool in mice sample.
Fig. 3 figure is the organic acid content detected in mouse liver sample.
Specific embodiment
For the purposes, technical schemes and advantages of the application are more clearly understood, below in conjunction with attached drawing to the application
Embodiment be described in detail.It should be noted that in the absence of conflict, in the embodiment and embodiment in the application
Feature can mutual any combination.
Embodiment 1: the content of organic acid in measurement stool in mice sample
1) Sample pretreatment: collecting mouse fresh excreta sample, weighs 86mg excrement, 860 μ L 50%ACN is added, by sample
After this homogenate, 30 μ L supernatants are taken after 12000rpm is centrifuged 15min, the 3-NPH, 25 μ L 120mM of 25uL 200mM is added
Acetonitrile solution reacted 30min, isometric internal standard was added, blows under nitrogen protection at 40 DEG C EDC (containing 6% pyridine)
It is dry, it is to be analyzed；LC-MS analysis:
2) liquid-phase condition: ACQUITY UPLC BEH C18 chromatographic column (2.1*100mm, 1.7 μM)；Column temperature: 40 DEG C；Stream
Speed: 0.25mL/min；Mobile phase: A is 100% water, and B is 100% acetonitrile.Gradient: 0-1min, 2%B, 1-34min, 2-
98%B, 34-40min, 98%B, 40-45min, 2%B.1 μ L of sample volume.
Mass Spectrometry Conditions: heating electric spray ion source (Heating electrospray ion source, HESI), ion
320 DEG C of transfer tube temperature；300 DEG C of temperature degree of auxiliary；Sheath gas: 35arb；Assist gas: 10arb；The spraying electricity of anionic textiles mode
Pressure: 3.5kV；Scan pattern: Selective reaction monitoring mode；The resolution ratio of mass analyzer Q1 and Q2 are 0.7FWHM；Collision gas is
Argon gas, collision atmospheric pressure are 1.5mTorr.The information of mass spectroscopy parent ion, daughter ion and collision energy is shown in Table 1.
The ion pair information that table 1 monitors
3) accuracy and precision of method: derivative reaction and upper machine testing with step 1) and 2) prepare three height
The mixed liquor of 42 standard items of middle low concentration is as Quality Control sample (n=3), the accuracy and precision of comparative approach, specifically
It the results are shown in Table 2.
The accuracy and precision of 2 the present processes of table
4) qualitative and quantitative analysis: firstly, when extracting chromatographic peak and the reservation in sample by Tracefinder software
Between, it is compared with the standard items appearance time after 42 kinds of derivatizations to carry out qualitative analysis, and color is carried out to compound detected
The integral of spectral peak.Secondly, with the peak area at the characteristic ion peak of the standard items after derivatization and corresponding interior target peak area
Ratio is ordinate, and concentration is abscissa, draws standard curve.Finally, by the peak area ratio of each compound in sample
Blank reaction is deducted, standard curve is brought into, obtains the absolute content of organic acid in stool in mice sample.Fig. 1 is quantitative mouse excrement
Just in sample organic acid total ion current figure.Fig. 2 is the organic acid content detected in stool in mice sample.
Embodiment 2: the content of organic acid in measurement mouse liver sample
Weigh mouse fresh liver sample 50mg, 500 μ L 70%ACN be added, after sample is homogenized, through 12000rpm from
It takes 30 μ L supernatants after heart 15min, is added the 3-NPH of 25 μ L 200mM, 25 μ L 120mM EDC (containing 6% pyridine) solution,
At 40 DEG C, 30min is reacted, isometric internal standard is added, dries up under nitrogen protection, it is to be analyzed；Upper machine testing condition is the same as example 1
In 2), sampling volume be 5 μ L.Data analysis process is with 4 in example 1).Fig. 3 has for what is detected in mouse liver sample
Machine acid content.
Although embodiment disclosed by the application is as above, the content only for ease of understanding the application and use
Embodiment is not limited to the application.Technical staff in any the application fields, is taken off not departing from the application
Under the premise of the spirit and scope of dew, any modification and variation, but the application can be carried out in the form and details of implementation
Scope of patent protection, still should be subject to the scope of the claims as defined in the appended claims.
Claims (12)
1. a kind of method for quantifying a variety of intestinal flora organic acids based on LC-MS, which comprises
The sample is pre-processed, is then performed the derivatization with derivatization reagent by the preparation of sample；
The preparation of blank sample；
LC-MS detects the sample and the blank sample；
The quantitative analysis of sample.
2. according to the method described in claim 1, wherein, the derivatization reagent is 3- nitrophenyl hydrazine or 2- nitrophenyl hydrazine.
3. according to the method described in claim 1, wherein, the sample is fresh intestinal contents, excrement, tissue or blood.
4. according to the method described in claim 1, wherein, the extraction of sample includes: to extract sample with acetonitrile, methanol or ethyl alcohol.
5. according to the method described in claim 1, wherein, the derivatization includes:
The mixture of the sample and acetonitrile is centrifuged, takes supernatant that derivatization reagent, 1- (3- dimethylamino-propyl) -3- is added
Ethyl-carbodiimide hydrochloride and pyridine solution react 15min-30min at 25 DEG C -60 DEG C, dry up under nitrogen protection spare.
6. according to the method described in claim 1, wherein, the preparation of the blank sample includes:
By acetonitrile, derivatization reagent, 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride and pyridine solution, 25
15min-30min is reacted at DEG C -60 DEG C, is dried up under nitrogen protection spare.
7. according to the method described in claim 1, wherein, the quantitative analysis includes addition internal standard compound, the internal standard compound nothing
6 carbon of radioactivity, 13 stable isotope labeling nitrophenyl hydrazine, then performs the derivatization again.
8. according to the method described in claim 1, wherein, in LC-MS detection, liquid phase chromatogram condition: chromatographic column: reverse phase
Chromatographic column；Chromatographic column temperature: 35 DEG C -55 DEG C；1 μ L-10 μ L of sample volume；Flow velocity: 0.2mL/min-0.3mL/min；Flow phase composition:
A Xiang Weishui, B phase is acetonitrile；Gradient: 0-1min, volume ratio 2%B, 1-34min, volume ratio 2%-98%B, 34-
40min, volume ratio 98%B, 40-45min, volume ratio 2%B,
Mass Spectrometry Conditions: it is detected using the mass spectrometric Selective reaction monitoring mode of triple level four bars, electrospray ionisation, anion
Mode scans, spray voltage: 3000V；Ion transfer tube temperature: 320 DEG C；Auxiliary temperature degree: 300 DEG C；Sheath gas: 35arb；
Auxiliary gas flow speed: 10arb；The resolution ratio of mass analyzer Q1 and mass analyzer Q2: 0.7FWHM；The pressure of collision gas
For 1.5mTorr.
9. according to the method described in claim 1, wherein, the method simultaneous quantitative 20-60 kind intestinal flora organic acid.
10. according to the method described in claim 9, wherein, 42 kinds of intestinal flora organic acids of the method simultaneous quantitative are described to have
Machine acid include: formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, caproic acid, enanthic acid, n-nonanoic acid, capric acid, hendecanoic acid,
Dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, 16 carbon monoenoic acids, Heptadecanoic acide, octadecanoid acid,
18 carbon monoenoic acids, octadecadienoic acid, octatecatrienoic acid, nonadecylic acid, eicosatetraenoic acid, eicosapentaenoic acid, two
Dodecahexaene acid, lignoceric acid, tetracosa carbon monoenoic acid, acetoacetate, beta-hydroxybutyric acid, citric acid, aconitic acid, different lemon
Any one of acid, α-ketoglutaric acid, fumaric acid, succinic acid, malic acid, oxaloacetic acid, pyruvic acid, lactic acid or more.
11. according to the method described in claim 1, wherein, the quantitative analysis of sample includes:
It extracts the chromatographic peak in sample and the standard items appearance time after retention time, with derivatization compares to carry out qualitative point
Analysis, and the integral of chromatographic peak is carried out to compound detected；
It with the ratio of corresponding internal standard peak area is vertical with the peak area at the characteristic ion peak of the standard items after the derivatization
Coordinate, concentration are abscissa, draw the standard curve of internal standard method；
The peak area ratio of each compound in the sample is first deducted into blank reaction, then brings the standard curve into, is obtained
The content of organic acid into the sample.
12. "dead" 6 carbon, 13 stable isotope labeling 3- nitrophenyl hydrazine or 2- nitrophenyl hydrazine are marked on analytical chemistry in being used as,
Purposes preferably in detection intestinal flora organic acid.
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