CN109374773B - Analysis method of panda fecal metabolome - Google Patents
Analysis method of panda fecal metabolome Download PDFInfo
- Publication number
- CN109374773B CN109374773B CN201811403389.9A CN201811403389A CN109374773B CN 109374773 B CN109374773 B CN 109374773B CN 201811403389 A CN201811403389 A CN 201811403389A CN 109374773 B CN109374773 B CN 109374773B
- Authority
- CN
- China
- Prior art keywords
- sample
- panda
- mixing
- methanol
- derivatization
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
-
- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- 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
- G01N30/7206—Mass spectrometers interfaced to gas chromatograph
-
- 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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/025—Gas chromatography
-
- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
Abstract
The invention provides an analysis method of panda fecal metabolome, belonging to the technical field of biotechnology and metabolome. The analysis method of the panda fecal metabolome provided by the invention comprises the steps of mixing and dissolving a sample and an internal standard solution, then drying in vacuum, performing derivatization, and performing derivatization treatment; through gas chromatography-mass spectrometry, the panda feces can be accurately analyzed, and the metabolism condition of the pandas is tracked; has better research and utilization values.
Description
Technical Field
The invention relates to the technical field of biotechnology and metabolome, in particular to an analysis method of panda fecal metabolome.
Background
Metabonomics is a complete set of research on low molecular weight compounds, and is an ideal omics method for researching intestinal metabolic activity. Many metabolites, hormones and vitamins secreted by microorganisms can be reabsorbed by the host and affect circulating metabolites, such as branched chain amino acids and trefoil factor 3, which have been shown to be associated with insulin resistance and intestinal syndrome, respectively. The fecal metabolome provides relevant information about the metabolic interactions between the intestinal flora, the diet and the host. In combination with 16S progeny sequencing, the metabolome is able to provide a comprehensive phenotype of host-microbiome interaction.
Pandas are one of the most representative bears in China and are considered as signs of national wealth. Even with this flagship position, pandas remain a fragile species that relies on protection, with hundreds of them being protected by zoos and breeding centers around the world. Because female pandas do not have enough breast milk to feed both litters, a mix of breast milk and artificial formula is often used. Meanwhile, adult pandas mainly eat bamboo, so the intestinal flora of adult pandas is adapted to the diet eating bamboo, and the adaptation is most capable of digesting cellulose. However, no research report on the panda fecal metabolome exists at present.
Disclosure of Invention
The invention aims to provide an analysis method of panda fecal metabolome, which can better follow the metabolism condition of pandas by analyzing the panda fecal metabolome.
In order to achieve the above purpose of the invention, the following technical scheme is adopted:
a method for analyzing panda fecal metabolome comprises the following steps:
mixing a panda excrement sample with the extraction solvent-internal standard mixed solution to obtain a mixed sample;
grinding the mixed sample, centrifuging for the first time, taking a supernatant sample, concentrating and drying to obtain a powder sample;
mixing and dissolving a powder sample, sodium hydroxide, methanol and a pyridine solution to obtain a derivatization initial sample;
adding methyl chloroformate into the derivatization initial sample twice, mixing by vortex for 25-35s, adding chloroform and sodium bicarbonate solution, and mixing by vortex for 8-15s to obtain a derivatization sample;
and centrifuging the derivatization sample, and carrying out gas chromatography-mass spectrometry on a lower chloroform layer sample.
Compared with the prior art, the invention has the beneficial effects that: the analysis method of the panda fecal metabolome provided by the invention comprises the steps of mixing and dissolving a sample and an internal standard solution, then drying in vacuum, performing derivatization, and performing derivatization treatment; through gas chromatography-mass spectrometry, the panda feces can be accurately analyzed, and the metabolism condition of the pandas is tracked; has better research and utilization values.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
In the specification, um represents amu, Atomic mass unit, and the scanning speed μ/s represents elementary Atomic mass unit dalton (symbol: u) per second.
The method for analyzing the panda fecal metabolome according to the embodiment of the present invention will be specifically described below.
A method for analyzing panda fecal metabolome comprises the following steps:
mixing a panda excrement sample with the extraction solvent-internal standard mixed solution to obtain a mixed sample;
grinding the mixed sample, centrifuging for the first time, taking a supernatant sample, concentrating and drying to obtain a powder sample;
mixing and dissolving a powder sample, sodium hydroxide, methanol and a pyridine solution to obtain a derivatization initial sample;
adding methyl chloroformate into the derivatization initial sample twice, mixing by vortex for 25-35s, adding chloroform and sodium bicarbonate solution, and mixing by vortex for 8-15s to obtain a derivatization sample;
and centrifuging the derivatization sample, and carrying out gas chromatography-mass spectrometry on a lower chloroform layer sample.
The accurate metabolism condition of the pandas can be obtained by performing derivatization treatment on the excrement of the pandas and performing gas chromatography-mass spectrometry. Because the metabolic condition can be influenced by the change of the dietary structure of the pandas, different dietary habits of the pandas can be analyzed by analyzing the pandas of different ages and the pandas of different stages; and the dietary structure of the young pandas can be detected, and the digestion condition of the young pandas on cellulose can be analyzed.
In some embodiments of the invention, the capillary column for gas chromatography-mass spectrometry is ZB-1701(30 m.times.250 μm.times.0.15 μm, with a 5 meter guard column); the electron impact ionization energy in the analysis was 65-75 eV.
Through efficient analysis, more accurate experimental data can be obtained; the metabolic data can be analyzed relatively comprehensively through electron impact ionization energy of 65-75 eV.
In some embodiments of the invention, the flow rate of the helium stream is 0.8-1.2mL/min at 290 deg.C; the temperatures of the auxiliary, mass quadrupole and mass source are 240-255 ℃, 225-235 ℃ and 145-155 ℃; the scanning speed is 1.562 mu/s; the solvent delay time is 5-6.5 min.
Under the condition that the flow rate of the helium flow is 0.8-1.2mL/min, single molecules are ionized through high-temperature gasification and ionization, and the types and the molecular contents of the molecules in the sample can be conveniently obtained; the specificity is strong, the sensitivity is high and the detection speed is high; facilitating high throughput analysis.
In some embodiments of the invention, the frequency of milling the mixed sample is 30Hz for 150-200s, and the milling beads are added before milling, the speed of primary centrifugation is 15000g-18000g, and the time is 12-18 min.
Grinding beads are added, so that a sample can be ground at a certain frequency, and the sample can be ground into suspension with a proper particle size; the sample can be uniformly ground through the frequency of 30Hz, and the subsequent ionization and analysis are facilitated.
Through high-speed centrifugation for 12-18min, metabolites and residues can be well separated, and the influence of the residues on an analysis result is avoided.
In some embodiments of the present invention, the concentration and drying process is performed in vacuum for 200min and 150-.
Through vacuum concentration and drying, the metabolites are prevented from being oxidized by additional chemical reaction and the like, and the actual condition of the sample can be better and truer ensured. The actually measured data can truly reflect the actual metabolic condition.
In some embodiments of the invention, sodium hydroxide, methanol and pyridine are added to the powder sample in a 6:5:1 volume ratio.
The metabolites in the sample can be well ensured to be stably preserved by adding the sodium hydroxide, the methanol and the pyridine.
In some embodiments of the invention, the speed of centrifugation of the derivatized sample is 1400rpm to 1700rpm for 250 seconds and the temperature is 4 ℃.
The derivatized sample can be dissolved in a solvent by centrifugation, and the sample dissolved in chloroform can be separated by centrifugation.
In some embodiments of the invention, the chloroform and sodium bicarbonate solution are added in a 1:1 volume ratio.
The metabolites can be effectively dissolved through chloroform and sodium bicarbonate to form a sample to be detected, and detection and analysis are convenient to carry out.
In some embodiments of the invention, the extraction solvent-internal standard mixture is one of methanol 80%, isopropanol acetonitrile: water (3:2:2), methanol (100%), methanol: chloroform (3:1), acetonitrile (100%) and acetonitrile: chloroform (3:1) in that order.
Various molecules in the metabolites can be extracted through different extraction solvent-internal standard mixed liquids, so that the metabolites are extracted to the maximum extent, and the problem of missed detection is avoided.
In some embodiments of the invention, the concentration of the sodium bicarbonate solution is 40-55 mmol/L.
The sodium bicarbonate with proper concentration can play a certain buffering role and also can protect the sample from being damaged.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The embodiment provides an analysis method of panda fecal metabolome, which can analyze the metabolic condition of pandas, continuously track the metabolic condition of pandas and analyze the development trend of the metabolic condition through longitudinal comparison; risks can be predicted by transversely comparing the metabolic conditions of different pandas; the method comprises the following steps:
1.1, drying the feces of the pandas for about 5 hours to obtain a feces dried sample;
1.2 grinding and uniformly mixing the dried excrement sample, weighing 10mg of the sample, and adding the sample into a spiral cap tube;
1.3 adding 600 mu L of methanol as an extraction-internal standard solution into a spiral cap tube, and mixing to obtain a mixed sample;
1.4 adding metal or ceramic grinding beads into the screw cap tube of each mixed sample, grinding and mixing the samples by a grinder at the frequency of 30Hz for 150 s; centrifuging at 15000g for 12min, and collecting supernatant;
1.5, carrying out vacuum freeze concentration and drying on the supernatant for 150min to obtain a powder sample;
1.6 mixing and dissolving 200 mu L of sodium hydroxide, 167 mu L of methanol and 34 mu L of pyridine solution with the powder sample to obtain a derivatization initial sample;
1.7 mixing the derivatization initial sample with 20 mu L of methyl chloroformate, vortexing for 25s, adding 20 mu L of methyl chloroformate again, mixing, vortexing for 25s, and then, adding 400 mu L of chloroform and 400 mu L of 40mmol/L sodium bicarbonate solution, and vortexing for 10s to obtain a derivatization sample;
1.8 centrifuging the derivatized sample at 1400rpm and 4 ℃ for 250s, and taking the lower chloroform layer sample;
1.9 carrying out gas chromatography-mass spectrometry on the chloroform layer sample; the capillary column for gas chromatography-mass spectrometry was ZB-1701(30 m.times.250. mu.m.times.0.15 μm, containing 5m guard column); the electron impact ionization energy in the analysis was 65 eV; the flow rate of the helium flow is 0.8mL/min at 290 ℃; the temperatures of the assist, mass quadrupole and mass source were 240 ℃, 225 ℃ and 145 ℃; the detection quality ranges from 30um to 550um (i.e., 30amu-550 amu); the scanning speed is 1.562 mu/s; the solvent delay time was 5 min.
Example 2
The embodiment provides an analysis method of panda fecal metabolome, which can analyze the metabolic condition of pandas, continuously track the metabolic condition of pandas and analyze the development trend of the metabolic condition through longitudinal comparison; risks can be predicted by transversely comparing the metabolic conditions of different pandas; the method comprises the following steps:
1.1, drying the feces of the pandas for about 5 hours to obtain a feces dried sample;
1.2 grinding and uniformly mixing the dried excrement sample, weighing 10mg of the sample, and adding the sample into a spiral cap tube;
1.3 adding 600 mu L of mixed solution of isopropanol, acetonitrile and water (3:2:2) into a spiral cap tube to serve as extraction solvent-internal standard mixed solution, and mixing to obtain a mixed sample;
1.4 adding metal or ceramic grinding beads into the screw cap tube of each mixed sample, grinding and mixing the samples by a grinder at the frequency of 30Hz for 200 s; then 18000g is centrifuged for 18min, and the supernatant is taken;
1.5, carrying out vacuum freeze concentration and drying on the supernatant for 200min to obtain a powder sample;
1.6 mixing and dissolving 200 mu L of sodium hydroxide, 167 mu L of methanol and 34 mu L of pyridine solution with the powder sample to obtain a derivatization initial sample;
1.7 mixing the derivatization initial sample with 20 mu L of methyl chloroformate, vortexing for 35s, adding 20 mu L of methyl chloroformate again, mixing, vortexing for 35s, and then, adding 400 mu L of chloroform and 400 mu L of 55mmol/L sodium bicarbonate solution, and vortexing for 10s to obtain a derivatization sample;
1.8 centrifuging the derivatization sample for 320s at 1700rpm and 4 ℃, and taking a lower chloroform layer sample;
1.9 carrying out gas chromatography-mass spectrometry on the chloroform layer sample; the capillary column for gas chromatography-mass spectrometry was ZB-1701(30 m.times.250. mu.m.times.0.15 μm, containing 5m guard column); the electron impact ionization energy in the analysis is 75 eV; the flow rate of the helium flow is 1.2mL/min at 290 ℃; the temperatures of the assist, mass quadrupole and mass source were 255 ℃, 235 ℃ and 155 ℃; the detection quality ranges from 30um to 550 um; the scanning speed is 1.562 mu/s; the solvent delay time was 6.5 min.
Example 3
The embodiment provides an analysis method of panda fecal metabolome, which can analyze the metabolic condition of pandas, continuously track the metabolic condition of pandas and analyze the development trend of the metabolic condition through longitudinal comparison; risks can be predicted by transversely comparing the metabolic conditions of different pandas; the method comprises the following steps:
1.1, drying the feces of the pandas for about 5 hours to obtain a feces dried sample;
1.2 grinding and uniformly mixing the dried excrement sample, weighing 10mg of the sample, and adding the sample into a spiral cap tube;
1.3 adding 600 mu L of mixed solution of methanol and chloroform (3:1) into a spiral cap tube to serve as extraction solvent-internal standard mixed solution, and mixing to obtain a mixed sample;
1.4 adding metal or ceramic grinding beads into the screw cap tube of each mixed sample, grinding and mixing the samples by a grinder at the frequency of 30Hz for 180 s; then, 17000g of the supernatant is centrifuged for 15min, and the supernatant is taken;
1.5, carrying out vacuum freeze concentration and drying on the supernatant for 180min to obtain a powder sample;
1.6 mixing and dissolving 200 mu L of sodium hydroxide, 167 mu L of methanol and 34 mu L of pyridine solution with the powder sample to obtain a derivatization initial sample;
1.7 mixing the derivatization initial sample with 20 mu L of methyl chloroformate, vortexing for 30s, adding 20 mu L of methyl chloroformate again, mixing, vortexing for 30s, and then adding 400 mu L of chloroform and 400 mu L of 40mmol/L sodium bicarbonate solution, and vortexing for 10s to obtain a derivatization sample;
1.8 centrifuging the derivatization sample for 300s at 1500rpm and 4 ℃, and taking a lower chloroform layer sample;
1.9 carrying out gas chromatography-mass spectrometry on the chloroform layer sample; the capillary column for gas chromatography-mass spectrometry was ZB-1701(30 m.times.250. mu.m.times.0.15 μm, containing 5m guard column); the electron impact ionization energy in the analysis was 65 eV; the flow rate of the helium flow is 0.8mL/min at 290 ℃; the temperatures of the assist, mass quadrupole and mass source were 240 ℃, 225 ℃ and 145 ℃; the detection quality ranges from 30um to 550 um; the scanning speed is 1.562 mu/s; the solvent delay time was 5 min.
Example 4
The embodiment provides an analysis method of panda fecal metabolome, which can analyze the metabolic condition of pandas, continuously track the metabolic condition of pandas and analyze the development trend of the metabolic condition through longitudinal comparison; risks can be predicted by transversely comparing the metabolic conditions of different pandas; the method comprises the following steps:
1.1, drying the feces of the pandas for about 5 hours to obtain a feces dried sample;
1.2 grinding and uniformly mixing the dried excrement sample, weighing 10mg of the sample, and adding the sample into a spiral cap tube;
1.3 adding 600 mu L of acetonitrile (100%) serving as an extraction solvent-internal standard mixed solution into a spiral cap tube, and mixing to obtain a mixed sample;
1.4 adding metal or ceramic grinding beads into the screw cap tube of each mixed sample, grinding and mixing the samples by a grinder at the frequency of 30Hz for 180 s; then, 17000g of the supernatant is centrifuged for 15min, and the supernatant is taken;
1.5, carrying out vacuum freeze concentration and drying on the supernatant for 180min to obtain a powder sample;
1.6 mixing and dissolving 200 mu L of sodium hydroxide, 167 mu L of methanol and 34 mu L of pyridine solution with the powder sample to obtain a derivatization initial sample;
1.7 mixing the derivatization initial sample with 20 mu L of methyl chloroformate, vortexing for 30s, adding 20 mu L of methyl chloroformate again, mixing, vortexing for 30s, and then adding 400 mu L of chloroform and 400 mu L of 40mmol/L sodium bicarbonate solution, and vortexing for 10s to obtain a derivatization sample;
1.8 centrifuging the derivatization sample for 300s at 1500rpm and 4 ℃, and taking a lower chloroform layer sample;
1.9 carrying out gas chromatography-mass spectrometry on the chloroform layer sample; the capillary column for gas chromatography-mass spectrometry was ZB-1701(30 m.times.250. mu.m.times.0.15 μm, containing 5m guard column); the electron impact ionization energy in the analysis was 65 eV; the flow rate of the helium flow is 0.8mL/min at 290 ℃; the temperatures of the assist, mass quadrupole and mass source were 240 ℃, 225 ℃ and 145 ℃; the detection quality ranges from 30um to 550 um; the scanning speed is 1.562 mu/s; the solvent delay time was 5 min.
Example 5
The embodiment provides an analysis method of panda fecal metabolome, which can analyze the metabolic condition of pandas, continuously track the metabolic condition of pandas and analyze the development trend of the metabolic condition through longitudinal comparison; risks can be predicted by transversely comparing the metabolic conditions of different pandas; the method comprises the following steps:
1.1, drying the feces of the pandas for about 5 hours to obtain a feces dried sample;
1.2 grinding and uniformly mixing the dried excrement sample, weighing 10mg of the sample, and adding the sample into a spiral cap tube;
1.3 adding 600 mu L of acetonitrile-chloroform (3:1) as an extraction solvent-internal standard mixed solution into a spiral cap tube, and mixing to obtain a mixed sample;
1.4 adding metal or ceramic grinding beads into the screw cap tube of each mixed sample, grinding and mixing the samples by a grinder at the frequency of 30Hz for 180 s; then, 17000g of the supernatant is centrifuged for 15min, and the supernatant is taken;
1.5, carrying out vacuum freeze concentration and drying on the supernatant for 180min to obtain a powder sample;
1.6 mixing and dissolving 200 mu L of sodium hydroxide, 167 mu L of methanol and 34 mu L of pyridine solution with the powder sample to obtain a derivatization initial sample;
1.7 mixing the derivatization initial sample with 20 mu L of methyl chloroformate, vortexing for 30s, adding 20 mu L of methyl chloroformate again, mixing, vortexing for 30s, and then adding 400 mu L of chloroform and 400 mu L of 40mmol/L sodium bicarbonate solution, and vortexing for 10s to obtain a derivatization sample;
1.8 centrifuging the derivatization sample for 300s at 1500rpm and 4 ℃, and taking a lower chloroform layer sample;
1.9 carrying out gas chromatography-mass spectrometry on the chloroform layer sample; the capillary column for gas chromatography-mass spectrometry was ZB-1701(30 m.times.250. mu.m.times.0.15 μm, containing 5m guard column); the electron impact ionization energy in the analysis was 65 eV; the flow rate of the helium flow is 0.8mL/min at 290 ℃; the temperatures of the assist, mass quadrupole and mass source were 240 ℃, 225 ℃ and 145 ℃; the detection quality ranges from 30um to 550 um; the scanning speed is 1.562 mu/s; the solvent delay time was 5 min.
Examples of the experiments
In this experimental example, a sample was processed and the result was analyzed by the analysis method of the panda fecal metabolome of example 3.
And (4) analyzing results: the metabolites were deconvoluted and characterized using AMDIS software and MCF mass spectral libraries.
And (3) identification standard: more than 85% of the spectra fit the library spectra and are within a one minute window of the respective meteorological mass spectrum retention time. The chromatographic heights (relative concentrations) of the reference ions for each metabolite were extracted using MassOmics R-based software and corrected for relative metabolite concentrations with the internal reference d 4-alanine concentration and stool sample dry weight. Statistical analysis was performed using SPSS software version 24.0, R-based software, and Microsoft Excel. The repeatability of the extraction method was examined using Principal Component Analysis (PCA) and Coefficient of Variation (CV). The R software was used to render two-dimensional projections of Principal Component Analysis (PCA) and point maps. The coverage of metabolites from the six extraction protocols was compared and shown in the Venturi chart by Adobe Illustrator.
Experiment six extraction solvents for extracting panda feces metabonomics analysis and evaluation
Repeatability: 80% methanol and isopropanol: acetonitrile: the water (isopropanol: acetonitrile: water, 3:2:2) has the best extraction repeatability for adult pandas and juvenile pandas respectively. Methanol (80%) and isopropanol acetonitrile water (3:2:2) showed the most reproducible coefficients of variance (coefficient of variance: 9.8%, 8.7%) across the biochemical classes identified in adult panda and juvenile panda feces, respectively. Importantly, the coefficient of variance of acetonitrile (100%) showed minimal reproducibility for both adult pandas (coefficient of variance: 27.5%) and young pandas (coefficient of variance: 25.4%). The extraction reproducibility of isopropanol acetonitrile water (3:2:2) in the feces of panda pups was very excellent for unsaturated long chain fatty acids (CV: 5.13%) and saturated long chain fatty acids (CV: 5.23%), while the coefficient of variance for amino acids was inferior to other extraction solvents but better than acetonitrile (100%). Therefore, isopropanol, acetonitrile, water (3:2:2) is an ideal fatty acid extraction solvent, not an ideal solvent for amino acid extraction. Potential reason for the superior reproducibility of isopropanol acetonitrile to water (3:2:2) in extracting fatty acids may be that isopropanol is an inhibitor of phospholipase D and thus protects the native form of lipids.
Metabolite coverage: among metabolites extracted from adult panda feces, the order of extraction solvents was methanol (80%; metabolites (110 metabolites), isopropanol acetonitrile: water (3:2: 2; 108 metabolites), methanol (100%; 104 metabolites), methanol: chloroform (3: 1; 100 metabolites), acetonitrile (100%; 84 metabolites) and acetonitrile: chloroform (3: 1; 73 metabolites.) the extraction solvent metabolites were ranked to cover panda baby feces as isopropanol: acetonitrile: water (3:2: 2; 120 metabolites), methanol (80%; 107 metabolites), methanol (100%; 106 metabolites), methanol: chloroform (3: 1; 102 metabolites), acetonitrile (100%; 85 metabolites), acetonitrile: chloroform (3: 1; 79 metabolites.) furthermore, the hexagonal wien diagram illustrates that methanol synthetic solvent (methanol (80%) and methanol: chloroform (3:1)) cover metabolites in the feces The highest overlap rate was found in adult pandas, whereas in young pandas the overlap rate was highest for coverage metabolites of isopropanol, acetonitrile, water (3:2:2) and methanol (100%). Overall, methanol (80%) and isopropanol acetonitrile water (3:2:2) cover the largest range of all identified metabolites in adult pandas and juvenile pandas, respectively.
Extraction efficiency: the extraction efficiency is determined by the sum of all peak intensities of the extracted metabolites of similar biochemical classes. In adult panda feces metabolome, the peak intensity of amino acid, benzoic acid derivative, long-chain unsaturated fatty acid and methanol (80%) in TCA cycle intermediate is the highest; the isopropanol, acetonitrile and water (3:2:2) have the highest extraction rate on the amino acid derivatives and secondary metabolites; methanol (100%) gives the best extraction of nicotinamide/vitamins and long chain unsaturated fatty acids; and acetonitrile (100%) is an alkane. In contrast, for infant panda fecal metabolisms, isopropanol acetonitrile water (3:2:2) not only showed the highest peak intensity in all biochemical classes including central carbon metabolism, but also the highest extraction rates for long-chain saturated fatty acids and medium-chain saturated fatty acids, with the only exceptions being medium-chain fatty acids (highest extraction rate of 100% methanol) and alkanes. It is noted that acetonitrile (100%) and chloroform (3:1) extract paraffins have the highest peak intensities, while amino acids, amino acid derivatives, benzoic acids, TCA cycle intermediates and secondary metabolites have the lowest peak intensities, both in adult panda feces and in young pandas. Since these two extraction solvents are nonpolar solvents, they are highly efficient extraction solvents for nonpolar compounds such as alkanes, but have low extraction efficiency for polar compounds such as amino acids and TCA cycle intermediates.
In summary, the analysis method for panda fecal metabolome provided by the embodiment of the invention can extract the component to be detected in panda feces in the maximum range and the maximum efficiency through the optimized extraction agent, so that the detection result is more accurate and reliable.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (6)
1. An analysis method of a panda fecal metabolome is characterized by comprising the following steps: mixing a panda excrement sample with the extraction solvent-internal standard mixed solution to obtain a mixed sample; grinding the mixed sample, centrifuging for the first time to obtain a supernatant sample, and concentrating and drying to obtain a powder sample; mixing and dissolving the powder sample, sodium hydroxide, methanol and pyridine solution to obtain a derivatization initial sample; adding methyl chloroformate into the derivatization initial sample twice, mixing by vortex for 25-35s, adding chloroform and sodium bicarbonate solution, and mixing by vortex for 8-15s to obtain a derivatization sample; centrifuging the derivatization sample and carrying out gas chromatography-mass spectrometry on a lower chloroform layer sample;
the capillary column for gas chromatography-mass spectrometry was ZB-1701; the electron impact ionization energy in the analysis is 65-75 eV;
at 290 ℃, the flow rate of the helium flow is 0.8-1.2 mL/min; the temperatures of the auxiliary, mass quadrupole and mass source are 240-255 ℃, 225-235 ℃ and 145-155 ℃; the scanning speed is 1.562 mu/s; the solvent delay time is 5-6.5 min;
mixing and dissolving 200 mu L of sodium hydroxide, 167 mu L of methanol and 34 mu L of pyridine solution with the powder sample;
the powder sample is obtained by concentrating and drying a 10mg panda excrement sample;
the extraction solvent-internal standard mixed solution is one of 80% of methanol, 3:2:2 of isopropanol to acetonitrile to water, 100% of methanol, 3:1 of methanol to chloroform, 100% of acetonitrile or 3:1 of acetonitrile to chloroform, and the internal reference in the extraction solvent-internal standard mixed solution is d 4-alanine.
2. The method for analyzing the fecal metabolome of pandas according to claim 1, wherein the frequency of grinding the mixed sample is 30Hz for 150-200s, and grinding beads are added before the grinding, and the speed of the primary centrifugation is 15000g-18000g for 12-18 min.
3. The method for analyzing the panda fecal metabolome according to claim 1, wherein the concentration and drying process to obtain the powder sample is vacuum concentration and drying process, wherein the concentration and drying time is 150-.
4. The method for analyzing the fecal metabolome of panda according to claim 1, wherein the speed of centrifuging the derivatized sample is 1400rpm-1700rpm, the time is 250 seconds and 320 seconds, and the temperature is 4 ℃.
5. The method for analyzing panda fecal metabolome according to claim 1, wherein the chloroform and the sodium bicarbonate solution are added in a volume ratio of 1: 1.
6. The method for analyzing panda fecal metabolome according to claim 1, wherein the concentration of the sodium bicarbonate solution is 40-55 mmol/L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811403389.9A CN109374773B (en) | 2018-11-22 | 2018-11-22 | Analysis method of panda fecal metabolome |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811403389.9A CN109374773B (en) | 2018-11-22 | 2018-11-22 | Analysis method of panda fecal metabolome |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109374773A CN109374773A (en) | 2019-02-22 |
| CN109374773B true CN109374773B (en) | 2020-05-22 |
Family
ID=65382732
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811403389.9A Active CN109374773B (en) | 2018-11-22 | 2018-11-22 | Analysis method of panda fecal metabolome |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109374773B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008083027A (en) * | 2006-09-27 | 2008-04-10 | Korea Inst Of Science & Technology | Analytical method for difference of metabolic substance between both biological sample groups, using gas chromatograph-mass spectrometer |
| CN105651908A (en) * | 2016-03-02 | 2016-06-08 | 中国药科大学 | GC-MS (gas chromatography-mass spectrometer)-based method for quantifying eleven types of short-chain fatty acids in intestinal contents and fecal samples |
| CN107121517A (en) * | 2017-07-05 | 2017-09-01 | 赛思莱（厦门）生物科技有限公司 | A kind of sample preparation processing method studied for excrement and soil metabolism group |
| CN108287207A (en) * | 2017-04-24 | 2018-07-17 | 麦特绘谱生物科技（上海）有限公司 | Biological sample preparation method and applications and metabolin qualitative and quantitative analysis method |
-
2018
- 2018-11-22 CN CN201811403389.9A patent/CN109374773B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008083027A (en) * | 2006-09-27 | 2008-04-10 | Korea Inst Of Science & Technology | Analytical method for difference of metabolic substance between both biological sample groups, using gas chromatograph-mass spectrometer |
| US7678579B2 (en) * | 2006-09-27 | 2010-03-16 | Korea Institute Of Science And Technology | Method for analysis metabolite difference between biological samples |
| CN105651908A (en) * | 2016-03-02 | 2016-06-08 | 中国药科大学 | GC-MS (gas chromatography-mass spectrometer)-based method for quantifying eleven types of short-chain fatty acids in intestinal contents and fecal samples |
| CN108287207A (en) * | 2017-04-24 | 2018-07-17 | 麦特绘谱生物科技（上海）有限公司 | Biological sample preparation method and applications and metabolin qualitative and quantitative analysis method |
| CN107121517A (en) * | 2017-07-05 | 2017-09-01 | 赛思莱（厦门）生物科技有限公司 | A kind of sample preparation processing method studied for excrement and soil metabolism group |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109374773A (en) | 2019-02-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Patel et al. | Plants metabolome study: Emerging tools and techniques | |
| CN112689755A (en) | Mass spectrometry for detecting and quantifying microflora-related metabolites | |
| CN110573478B (en) | Mass spectrometry method for detecting and quantifying organic acid metabolites | |
| CN114324619B (en) | Method for measuring prochloraz and metabolite residue in cereal grains and application thereof | |
| Gupta et al. | Seaweed metabolomics: a new facet of functional genomics | |
| CN109374773B (en) | Analysis method of panda fecal metabolome | |
| CN109459506B (en) | Rapid sample pretreatment method for detecting polychlorinated biphenyl in tea | |
| Sedaghat et al. | Extraction and preconcentration of hemin from human blood serum and breast cancer supernatant | |
| Zheng et al. | Qualitative‐quantitative analysis of multi‐mycotoxin in milk using the high‐performance liquid chromatography‐tandem mass spectrometry coupled with the quick, easy, cheap, effective, rugged and safe method | |
| CN112666287B (en) | GCMSMS-based high-throughput analysis method and application of animal intestinal flora metabolites | |
| Huang et al. | Determination of six eugenol residues in aquatic products by gas chromatography-orbitrap mass spectrometry | |
| Böttcher et al. | Analysis of phenolic choline esters from seeds of Arabidopsis thaliana and Brassica napus by capillary liquid chromatography/electrospray‐tandem mass spectrometry | |
| CN111057055A (en) | Multichannel mass spectrum derivatization reagent for detecting hydroxyl polycyclic aromatic hydrocarbon, and preparation method and application thereof | |
| CN110003035B (en) | Stable isotope labeled 3-ethyl aminobenzoate-D5And method for preparing the same | |
| Jones et al. | Bridging the gap: basic metabolomics methods for natural product chemistry | |
| CN112362796A (en) | Method for detecting biotoxin in bee pollen and application thereof | |
| CN103926368B (en) | Method for extracting biotin from corn steep liquor and thin layer chromatography (TLC) scanning detection method of biotin | |
| Li et al. | Determination of three oryzanols in rice by mixed-mode solid-phase extraction coupled with high performance liquid chromatography-tandem mass spectrometry | |
| Llorente et al. | Rice grain quality benchmarking through profiling of volatiles and metabolites in grains using gas chromatography mass spectrometry | |
| CN113720945B (en) | Quick detection method for organic phosphate flame retardant and metabolite thereof in urine based on QuEChRS method | |
| Zhang et al. | Validation and quantification of genomic 5-carboxylcytosine (5caC) in mouse brain tissue by liquid chromatography-tandem mass spectrometry | |
| Shi et al. | Salting-out assisted liquid/liquid extraction coupled with low-temperature purification for analysis of endocrine-disrupting chemicals in milk and infant formula by ultra high performance liquid chromatography-tandem mass spectrometry | |
| CN112129856A (en) | Method for identifying ellagic acid from gallnut | |
| CN111257449A (en) | Method for determining stable hydrogen isotope ratio of acetic acid methyl sites in vinegar beverage | |
| CN111257439A (en) | Method for detecting hydroxyl polybrominated diphenyl ethers in aquatic products by solid-phase extraction-ultra-high performance liquid chromatography tandem mass spectrometry |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |