CN102768246A - Flavonoid metabolic profiling analysis method based on nanofluidic channel liquid chromatogram/mass spectrum - Google Patents
Flavonoid metabolic profiling analysis method based on nanofluidic channel liquid chromatogram/mass spectrum Download PDFInfo
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Abstract
The invention discloses a nanofluidic channel liquid chromatogram/mass spectrum method of soybean flavonoid metabolic profiling analysis. By the adoption of a simple sample pretreatment method, under the conditions of high enrichment capacity of channels and optimized segregation analysis, profiling analysis of 73 flavonoids can be realized by minimal sample size. The analysis method provided by the invention is simple and rapid, has advantages of small sample amount and good repeatability, and is suitable for batch analysis of real samples.
Description
Technical field
The present invention relates to the analytical chemistry field, is a kind ofly to receive the method that the soybean flavonoids metabolic profile of flow liquid phase chromatogram/mass spectrometry (LC-Chip/MS) analyzes based on chip.
Background technology
An important directions of modern analysis chemical developer is exactly development traceization, integrated compartment analysis new method.For sensitivity and the flux that improves analysis, reduce analysis cost and sample consumption, development traceization, integrated analytical approach have become the field, forward position of current research.The flow liquid phase chromatographic technique of receiving is a special kind of skill that last century, the nineties grew up, because of its have sample consume little, analysis speed is fast and the advantage of easy of integrationization has attracted researcher's extensive concern, becomes the research focus in the microminiaturized analysis field.The chip that occurs is recently received flow liquid phase chromatogram/mass spectrometer system (LC-Chip/MS), and the complicacy used in sample concentration and separation function and the electrospray mass spectrometer of flow liquid phase system is connected and sprayer seamlessly combines receiving; Leakage and system's dead volume have been reduced significantly; Simplify workflow, improved sensitivity and the reliability analyzed.The LC-Chip/MS technology has been used to researchs such as protein group, oligosaccharides at present; The characteristics that it is easy to operate, highly sensitive and the sample consumption is few are also having great advantage aspect the metabolism group research, but up to now based on the technological rarely seen report of the metabolism group profile analysis of LC-Chip/MS.
Flavonoids is the one big type of derivative compound that is distributed widely in the plant, and known flavonoids has the 3000-4000 kind at present, and wherein the main flavonoids in the food roughly is divided into four types, is respectively flavanones, flavones, flavonols and isoflavones.What research was more at present is the isoflavones in the soybean, and it receives much concern because of having physiological functions such as prophylaxis of cancer, angiocardiopathy and osteoporosis as one type of secondary metabolite in the soybeans they grow process.Except that isoflavones, other flavone compounds in the soybean also have different physiological functions, as its content and distribution are studied, and are associated with metabolic pathway, then can obtain more comprehensive, complete information.Up to the present, the bibliographical information of Flavonoid substances analysis in the soybean mostly is confined to measure the general flavone content after certain several flavonoids metabolin or the hydrolysis, the report of only several pieces of profile analysis also needs second extraction and purge process.Not only can reduce drain on manpower and material resources and extract relatively fast and analyze, and can improve the flux of analysis greatly.
The LC-Chip/MS analytic system is as the microminiaturized representative of instrument; Except that reducing the sample size; Its key components-chip also has stronger dirigibility; We adopt customized high enrichment capacity chip not only can increase substantially detection sensitivity, simplify the sample pretreatment process, under the compartment analysis condition of optimizing, also can realize the compartment analysis of more flavone compounds.This method has simple to operate, highly sensitive advantage, can foundation be provided to the further research that the soybean sample of different planting environments and genetic background is analyzed to the soybean flavonoids.
Summary of the invention
The chip that the objective of the invention is to set up the analysis of a kind of soybean flavonoids metabolic profile is received flow liquid phase chromatogram/mass spectrometry method, makes preprocessing process more simple, and amount of samples still less and can obtain more flavonoids metabolin information.
For realizing above-mentioned purpose, the technical scheme that the present invention adopts is following:
The chip of a kind of soybean flavonoids metabolic profile analysis is received flow liquid phase chromatogram/mass spectrum (LC-Chip/MS) method:
(1) add the soya seeds sample of extract ultrasonic centrifugal after, supernatant is directly gone up an appearance analysis, the sample pretreatment process is simple, need not enrichment etc. preprocessing process; (2) supernatant is directly gone up the appearance analysis, adopts the compartment analysis condition of high enrichment capacity chip and optimization, can realize the profile analysis of more flavone compounds, and 1 μ L sample can obtain the metabolic profile of 73 kinds of flavone compounds in the soybean in 22 minutes;
High enrichment capacity chip is made up of 500nL high capacity enriching column, long 150mm analytical column and nozzle needle, and the packing material of enriching column and analytical column is Zorbax SB-C18; Enriching column links to each other through six-way valve with analytical column, and nozzle needle is fixed in an end of analytical column.
Concrete steps are following,
(1) takes by weighing a certain amount of soya seeds sample in glass tube; The extraction solvent (WS that contains methyl alcohol 10-30%) that in every arm, adds mark naringenin, isoquercitrin and each 0.5-2 μ g/mL of rutin in containing; Centrifugal after ultrasonic 10-20 minute, supernatant moved to be used for chip in the sample introduction bottle and receive flow liquid phase chromatogram/mass spectrometry analysis.
(2) the concrete parameter of LC-Chip/MS analytical approach is: what this analysis was finally selected for use is high enrichment capacity chip; This chip is by high capacity enriching column (500nL; ID 75 μ m), analytical column (form, and the packing material of enriching column and analytical column is Zorbax SB-C18 (5 μ m) by 75 μ m * 150mm) and nozzle needle (10 μ m ID).
Enriching column links to each other through six-way valve with analytical column; 4. 1. the two ends of enriching column link to each other with the interface of six-way valve respectively; 3. one end of analytical column links to each other with the interface of six-way valve, and nozzle needle is fixed in the other end of analytical column, the interface of six-way valve 2. as the moving phase end, it links to each other with B with mobile phase A through the nano pump; The interface of six-way valve 6. the waste liquid end, be the waste liquid outlet end, the interface of six-way valve 5. as the sample end, it links to each other with the sample storage container with eluent through the kapillary pump.
During enrichment, chip is connected the sample end through the kapillary pump and is introduced the sample in the sample storage container and on enriching column, realize enrichment with the waste liquid end, this moment six-way valve interface 5., 4., 1., 6. connection successively, sample end eluent is the acetonitrile water that contains volume 2%;
During sample analysis, connect moving phase end and analytical column through the nano pump, sample gets into analysis channel, this moment six-way valve interface 2., 1., 4., 3. be communicated with successively; Analyzing mobile phase A is the WS that contains volume 0.1% formic acid, and B is the acetonitrile that contains volume 0.1% formic acid; Condition of gradient elution: during 0min 90/10 (A/B, V/V), during 13min 70/30 (A/B, V/V), during 18min 5/95 (A/B, V/V) and keep 4min, during 22.1min 90/10 (A/B, V/V), start gradient 90/10 (A/B, V/V) balance 8min subsequently; Wherein 0-13min is that first section linear gradient elution, 13-18min are second section linear gradient elution, and 18-22.1min is the 3rd a section gradient elution;
The flow velocity of kapillary pump is 4-6 μ L/min, the flow velocity 0.3-0.6 μ L/min of nano pump; Sample size is 0.1-1 μ L, and the enriching column flush volume is 3-4 μ L behind the sample introduction; Effluent imports the mass spectrometer system detection without shunting behind the post of analytical column;
The mass spectrum condition is: electric spray ion source, adopt positive ion mode to detect; Use high-purity N 2 assistant spray ionization and desolventizings, 300 ℃ of dry gas temperature, the dry gas flow velocity is 4L/min; Capillary voltage is 1800-1900V; Centroid type collection mass spectrometric data; Mass scanning scope m/z 100～1000; Can obtain the metabolic profile spectrum of soybean flavonoids under these conditions, i.e. the TIC figure (Fig. 1) of soybean flavonoids LC-Chip/MS analysis.
The effect that the present invention has is: the autonomous standardized program of setting up is all adopted in the storage of sample, pre-service and analysis, avoids introducing personal error.
The preprocessing process that is adopted is simple, does not need secondary enrichment and purifying, can avoid the partial loss of purpose compound, has reduced the preprocessing process error simultaneously, has guaranteed the stability of analytical approach.Adopt microminiaturized analytical instrument and autonomous customized high power capacity enrichment chip, under the analysis condition of optimizing, saved analysis time and amount of samples, improved analysis throughput, combine with preprocessing process and can identify more target compound.In addition, adopt dissimilar interior marks and quality control sample to proofread and correct the error that the small drift of instrument response brings in preprocessing process and the experiment, further guaranteed the stability of this method.The method repeatability of 9 characteristic ions in the sample is investigated the result see table 1, its relative standard deviation is 1.50-7.66%, has further proved the stability of method that this patent is set forth.
Description of drawings
The TIC figure (A) that Fig. 1 soybean flavonoids LC-Chip/MS analyzes and the EIC figure (B) of typical ion.
The PLS-DA shot chart of the different plantation of Fig. 2 place soybean sample.Sample of each some expression, t representes that sample is at the major component projection value.◆: plantation Jinan, place, ▲: plantation Jilin, place.
Fig. 3 is high enrichment capacity chip structure synoptic diagram among the present invention.1 eluent among the figure, 2 waste liquids, 3 enriching columns, 4 moving phases, 5 analytical columns, 6 nozzle needles.
Embodiment
1. soya seeds sample collection
5 soybean varieties are respectively: Ji educates 73, long farming 16, nine farmings 30, lotus beans 12, middle yellow 13.Wherein luckyly educate 73, the plantation place of long farming 16 and nine farmings 30 is Jilin.Lotus beans 12 are Jinan with middle yellow 13 plantation place.Being placed on-80 ℃ of refrigerators after the soya seeds sample collection preserves.
2. analytical approach
2.1 soya seeds sample pre-service
The soya seeds sample takes out from-80 ℃ of refrigerators, waits for that in exsiccator its temperature returns to room temperature.Every kind of soya seeds sample is placed in the comminutor pulverizes, cross 60 mesh sieves, obtain the powder of uniform particle diameter.Soya seeds powder branch is filled in the plastic tube, subsequent use.
Take by weighing 5 kind soya seeds samples each 3 parts (every part of 0.1g), place the 5mL glass tube respectively.
Take by weighing every kind of soya seeds sample 0.5g, mix, as quality control (QC) sample.Take by weighing 6 parts in QC sample (every part of 0.1g) then, place the 5mL glass tube respectively.
Mark naringenin 0.4mg and isoquercitrin, each 1.6mg of rutin add the 1mL acetonitrile in taking by weighing in the 1.5mL centrifuge tube.Mediate, ultrasonic, make it to dissolve fully, obtain the mixed mark solution that concentration is respectively naringenin 0.4mg/mL, isoquercitrin 1.6mg/mL, rutin 1.6mg/mL.
Get 10mL water, 39.95mL methyl alcohol, ultrasonic mixing adds above-mentioned mixed mark solution 50 μ L as extracting solvent in extracting solvent, be total to 50mL behind the mixing and extract solvent.
In each glass tube, add 2mL and extract solvent, ultrasonic 20 minutes.Take out 1mL and move in the eppendorf pipe,, under the 14000rpm centrifugal 10 minutes, supernatant is moved to the sample introduction bottle in 4 ℃.
2.2 chip is received flow liquid phase chromatogram/mass spectrometry analysis
As shown in Figure 3; High enrichment capacity chip is by high capacity enriching column (500nL; ID 75 μ m), analytical column (form, and the packing material of enriching column and analytical column is Zorbax SB-C18 (
5 μ m) by 75 μ m * 150mm) and nozzle needle (10 μ m ID).
Enriching column links to each other through six-way valve with analytical column; 4. 1. the two ends of enriching column link to each other with the interface of six-way valve respectively; 3. one end of analytical column links to each other with the interface of six-way valve, and nozzle needle is fixed in the other end of analytical column, the interface of six-way valve 2. as the moving phase end, it links to each other with B with mobile phase A through the nano pump; The interface of six-way valve 6. the waste liquid end, be the waste liquid outlet end, the interface of six-way valve 5. as the sample end, it links to each other with the sample storage container with eluent through the kapillary pump;
Chip is connected the sample end through the kapillary pump and is introduced sample (sample storage container) and on enriching column, realize enrichment with the waste liquid end, this moment six-way valve interface 5., 4., 1., 6. connection successively, sample end eluent is the acetonitrile water that contains volume 2%;
During sample analysis, connect moving phase end and analytical column through the nano pump, sample gets into analysis channel, this moment six-way valve interface 2., 1., 4., 3. be communicated with successively.Analyzing mobile phase A is the WS that contains volume 0.1% formic acid, and B is the acetonitrile that contains volume 0.1% formic acid.Condition of gradient elution (wherein 0-13min is that first section linear gradient elution, 13-18min are second section linear gradient elution, and 18-22.1min is the 3rd a section gradient elution): during 0min 90/10 (A/B, V/V); During 13min 70/30 (A/B, V/V), 5/95 (A/B during 18min; V/V) and keep 4min, and during 22.1min 90/10 (A/B, V/V); Start gradient 90/10 (A/B, V/V) balance 8min subsequently.The flow velocity of kapillary pump is 4 μ L/min, the flow velocity 0.30 μ L/min of nano pump; Sample size is 1 μ L, and the enriching column flush volume is 4 μ L behind the sample introduction, analyzes and adopts the recoil pattern to reduce the bands of a spectrum broadening; Effluent imports the mass spectrometer system detection without shunting behind the post.The mass spectrum condition is: electric spray ion source (ES I), adopt positive ion mode to detect; Use high-purity N 2 assistant spray ionization and desolventizings, 300 ℃ of dry gas temperature, the dry gas flow velocity is 4L/min; Capillary voltage is 1800-1900V; Centroid type collection mass spectrometric data; Mass scanning scope m/z100～1000.Can obtain the TIC figure that soybean flavonoids LC-Chip/MS analyzes under these conditions.
The chip of 15 soya seeds samples is received the chromatogram/mass spectrophotometry of flow liquid phase in proper order for carrying out at random; In analytic process; Earlier once, after per minute is analysed 3 samples then, more once with QC appearance sample introduction with QC appearance sample introduction; Through guaranteeing the reappearance of QC sample retention time and response signal, guarantee that the experimentation chips receives flow liquid phase chromatogram/GC-MS and have stable preferably.Results suggest, the repeatability of QC sample is good in whole analysis time, tangible change of component do not occur, and drift does not appear in retention time, and table 1 and table 2 are the investigation result of method repeatability and instrument precision.
3. pattern-recognition and mark screening
In order to investigate the soybean flavonoids metabolic profile difference in different plantations place; We adopt qualitative analysis software (MassHunter Qualitative Analysis) that the flavone compound of all soya seeds samples is carried out data matrix of peak extraction formation; Promptly be matched to a peak table that constitutes by retention time, mass number and peak area to the measurement data of each sample, reduce systematic error through total peak area normalization simultaneously.(Umetrics, Umea Sweden) carry out PLS-DA and analyze to utilize SIMCA-P 11.5 then.SPSS 13.0 is adopted in non-parametric test, and (SPSS Inc., Chicago IL) carries out.
Fig. 2 is the PLS-DA shot chart of different plantations place soybean.Therefrom can know; The soybean sample that 9 plantation places are Jilin is that being projected on the first principal component of soybean sample in Jinan can well be separated with 6 plantation places, shows that there is significantly difference in the flavonoids metabolic profiles of the soybean sample in different plantations place.With the information extraction of VIP＞1.0 and carry out the independent sample non-parametric test; Think classification is had the compound of significant contribution through the variable (p＜0.05) of non-parametric test; They are respectively glycitein O-hexoside; Genistein O-hexoside, daidzeinO-hexoside malonylated, Flavonoid substances (table 3) such as daidzein O-hexoside malonylated.
Based on simple to operate, the highly sensitive advantage of this method, can the flavonoids in the soybean sample of different cultivars and genetic background be done further analysis, for its further investigation provides foundation.
The repeatability of table 1 soybean flavonoids LC-Chip/MS analytical approach is investigated (n=4)
In a few days (n=5) that table 2 soybean flavonoids LC-Chip/MS analyzes and (n=3) precision investigation in the daytime
There is the flavone compound of significant difference in the different plantation of table 3 place
For further specifying the characteristics of the said method of this patent, the applicant compares the chip of different samples preprocess method and different enrichment capacity.
1) adopt the SPE method that the soybean sample is carried out second extraction and purifying, to improve the response of low concentration compound.With the difference of embodiment one be behind the ultrasonic 20min of soybean sample, to carry out preenrichment and purifying by normal process with Oasis HLBSPE pillar; Normal process is: cross the SPE pillar behind 10 times of the soybean sample extraction liquid 1ml dilute with waters of ultrasonic 20min; Flow velocity is about 10ml/min; The back is with 1ml water and 0.05ml methanol wash, and the target extract uses 0.5ml methyl alcohol and 0.5ml ethanol/methylene (1: 1) to wash respectively at last, redissolves with 1ml water after the freeze-drying of collection liquid; Last appearance is analyzed, and all analysis conditions and parameter are all identical with embodiment one.The result finds; After adopting second extraction; The response of most compounds has 1-2 increase doubly; But the daidzein 7 of some compound such as m/z 751,4 '-O-di-(hexoside malonylated) detect less than, this explanation second extraction and purge process can cause the loss of part target compound.Though and a little less than the method for once extracting among the embodiment one response slightly, can detect more target compound, and can also shorten pretreatment time greatly and reduce solvent consumption.
2) adopt standard chips (40nL) that soybean sample extraction liquid is analyzed, all pretreatment conditions and analytical parameters are all identical with embodiment one.The result finds; Because the enrichment capacity of 40nL chip is less; Former thereby the loss that chemical compound lot is big because of polarity or abundance is lower; Like the glycitein O-hexoside malonylated of m/z 533, the glycitein-O-pentosylhexoside of m/z 579 and glycitein 7,4 '-O-d ihexoside of m/z 609 etc.And adopt our customized big enrichment capacity chip; In conjunction with simple sample pretreatment; Can realize the profile analysis of 73 kinds of Flavonoid substances; Further proved the superiority of the said method of this patent, this method may extend to the compartment analysis of Flavonoid substances in the soybean sample of more kinds and different genetic backgrounds.
The present invention adopts simple sample preprocess method, and under the compartment analysis condition of high enrichment capacity chip and optimization, small sample size can be realized the profile analysis of 73 kinds of flavone compounds.Analytical approach of the present invention is simple, quick, and the little and good reproducibility of sample consumption is suitable for the batch quantity analysis of actual sample.
Claims (4)
1. receive flow liquid phase chromatogram/mass spectral flavonoids metabolic profile analytical approach based on chip, it is characterized in that:
(1) add the soya seeds sample of extract ultrasonic centrifugal after, supernatant is directly gone up an appearance analysis; (2) supernatant is directly gone up the appearance analysis, and high enrichment capacity chip compartment analysis condition: high enrichment capacity chip is made up of 500nL high capacity enriching column, long 150mm analytical column and nozzle needle, and the packing material of enriching column and analytical column is Zorbax SB-C18; Enriching column links to each other through six-way valve with analytical column, and nozzle needle is fixed in an end of analytical column.
2. according to the said method of claim 1; It is characterized in that: take by weighing the soya seeds sample in glass tube in the step (1); The extraction solvent that in every arm, adds mark naringenin, isoquercitrin and each 0.5-2 μ g/mL of rutin in containing; Extracting solvent is the WS that contains methyl alcohol 10-30%, centrifugal after ultrasonic 10-20 minute, and supernatant is used for LC-Chip/MS and analyzes.
3. according to the said method of claim 1, it is characterized in that:
Enriching column links to each other through six-way valve with analytical column; 4. 1. the two ends of enriching column link to each other with the interface of six-way valve respectively; 3. one end of analytical column links to each other with the interface of six-way valve, and nozzle needle is fixed in the other end of analytical column, the interface of six-way valve 2. as the moving phase end, it links to each other with B with mobile phase A through the nano pump; The interface of six-way valve 6. the waste liquid end, be the waste liquid outlet end, the interface of six-way valve 5. as the sample end, it links to each other with the sample storage container with eluent through the kapillary pump.
4. according to the said method of claim 3, it is characterized in that:
What step (2) was selected for use is high enrichment capacity chip; This chip is by high capacity enriching column (500nL; ID 75 μ m), analytical column (form, and the packing material of enriching column and analytical column is Zorbax SB-C18 (
5 μ m) by 75 μ m * 150mm) and nozzle needle (10 μ m ID);
During enrichment, chip is connected the sample end through the kapillary pump and is introduced the sample in the sample storage container and on enriching column, realize enrichment with the waste liquid end, this moment six-way valve interface 5., 4., 1., 6. connection successively, sample end eluent is the acetonitrile water that contains volume 2%;
During sample analysis, connect moving phase end and analytical column through the nano pump, sample gets into analysis channel, this moment six-way valve interface 2., 1., 4., 3. be communicated with successively; Analyzing mobile phase A is the WS that contains volume 0.1% formic acid, and B is the acetonitrile that contains volume 0.1% formic acid; Condition of gradient elution: during 0min 90/10 (A/B, V/V), during 13min 70/30 (A/B, V/V), during 18min 5/95 (A/B, V/V) and keep 4min, during 22.1min 90/10 (A/B, V/V), subsequently start gradient 90/10 (A/B, V/V); Wherein 0-13min is that first section linear gradient elution, 13-18min are second section linear gradient elution, and 18-22.1min is the 3rd a section gradient elution;
The flow velocity of kapillary pump is 4-6 μ L/min, the flow velocity 0.3-0.6 μ L/min of nano pump; Sample size is 0.1-1 μ L, and the enriching column flush volume is 3-4 μ L behind the sample introduction; Effluent imports the mass spectrometer system detection without shunting behind the post of analytical column;
The mass spectrum condition is: electric spray ion source, adopt positive ion mode to detect; Use high-purity N 2 assistant spray ionization and desolventizings, 300 ℃ of dry gas temperature, the dry gas flow velocity is 4L/min; Capillary voltage is 1800-1900V; Mass scanning scope m/z 100～1000; Can obtain the metabolic profile spectrum of soybean flavonoids under these conditions.
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| WO2020213672A1 (en) * | 2019-04-16 | 2020-10-22 | 花王株式会社 | Method of predicting soybean yield |
| JP2020174668A (en) * | 2019-04-16 | 2020-10-29 | 花王株式会社 | Method of predicting soybean yield |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108333266A (en) * | 2017-12-28 | 2018-07-27 | 西北工业大学 | A kind of high pressure resistant minicore chip liquid chromatogram |
| CN108426970A (en) * | 2018-03-16 | 2018-08-21 | 绿城农科检测技术有限公司 | The method that enzymolysis stability by investigating candidate peptide fragment filters out the feature peptide fragment of milk powder allergen protein |
| WO2020213672A1 (en) * | 2019-04-16 | 2020-10-22 | 花王株式会社 | Method of predicting soybean yield |
| JP2020174668A (en) * | 2019-04-16 | 2020-10-29 | 花王株式会社 | Method of predicting soybean yield |
| CN113748337A (en) * | 2019-04-16 | 2021-12-03 | 花王株式会社 | Soybean yield prediction method |
| US20220198360A1 (en) * | 2019-04-16 | 2022-06-23 | Kao Corporation | Method of predicting soybean yield |
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