CN102768246B - 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 based on chip, to receive the method for soybean flavonoids metabolic profiling analysis of flow liquid phase Chromatography/Mass Spectrometry coupling (LC-Chip/MS).
Background technology
An important directions of Development of Modern Analytical Chemistry 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 Disciplinary Frontiers 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 study hotspot in microminiaturized analysis field.The chip occurred is recently received flow liquid phase Chromatography/Mass Spectrometry system (LC-Chip/MS) and is connected with the complexity of using in electrospray mass spectrometer with separation function with sprayer and seamlessly combines receiving the sample concentration of flow liquid phase system, decrease leakage and system dead volume, simplify workflow, improved sensitivity and the reliability analyzed.The LC-Chip/MS technology has been used to the 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 metabolism group research, but the rarely seen report of metabolism group profile analysis technology based on LC-Chip/MS up to now.
Flavonoids is to be distributed widely in the large class derivative compound of one in plant, and known flavonoids has the 3000-4000 kind at present, and wherein the main flavonoids in food roughly is divided into four classes, is respectively flavanones, flavones, flavonols and isoflavones.What research was more at present is the isoflavones in soybean, and it receives much concern because having the physiological functions such as pre-anti-cancer, angiocardiopathy and osteoporosis as the class secondary metabolite in the soybeans they grow process.Except isoflavones, other flavone compounds in soybean also have different physiological functions, as its content and distribution are studied, and are associated with metabolic pathway, can obtain more comprehensive, complete information.Up to the present, the bibliographical information of Flavonoid substances analysis in soybean mostly is confined to measure the general flavone content after certain several flavonoids metabolin or 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 greatly improve the flux of analysis.
The LC-Chip/MS analytic system is as the representative of equipment miniaturization, except reducing 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.It is simple to operate, highly sensitive that the method has advantages of, the further research that can be analyzed as the soybean flavonoids the soybean sample of different planting environments and genetic background provides foundation.
Summary of the invention
The chip that the object of the invention is to set up a kind of soybean flavonoids metabolic profiling analysis is received flow liquid phase Chromatography/Mass Spectrometry method, makes preprocessing process more simple, and amount of samples still less, and can obtain more flavonoids metabolin information.
For achieving the above object, the technical solution used in the present invention is as follows:
A kind of chip of soybean flavonoids metabolic profiling analysis is received flow liquid phase Chromatography/Mass Spectrometry (LC-Chip/MS) method:
(1) add the soya seeds sample of extract ultrasonic centrifugal after, the direct loading analysis of supernatant, the sample pretreatment process is simple, need not enrichment etc. preprocessing process; (2) the direct loading analysis of supernatant, adopt the compartment analysis condition of high enrichment capacity chip and optimization, can realize the profile analysis of more flavone compounds, 1 μ L sample can obtain soybean in 22 minutes in the metabolic profile of 73 kinds of flavone compounds;
High enrichment capacity chip is comprised of the large capacity enriching column of 500nL, long 150mm analytical column and nozzle needle, and the packing material of enriching column and analytical column is Zorbax SB-C18; Enriching column is connected by six-way valve with analytical column, and nozzle needle is fixed in an end of analytical column.
Concrete steps are as follows,
(1) take a certain amount of soya seeds sample in glass tube, add the extraction solvent (containing the aqueous solution of methyl alcohol 10-30%) containing interior mark naringenin, isoquercitrin and each 0.5-2 μ g/mL of rutin in every arm, centrifugal after ultrasonic 10-20 minute, supernatant is moved in sample injection bottle for chip and receives flow liquid phase Chromatography/Mass Spectrometry combination analysis.
(2) design parameter of LC-Chip/MS analytical approach is: this analysis final choice be high enrichment capacity chip, this chip is by large capacity enriching column (500nL, ID 75 μ m), analytical column (75 μ m * 150mm) and nozzle needle (10 μ m ID) form, the packing material of enriching column and analytical column is Zorbax SB-C18 (5 μ m).
Enriching column is connected by six-way valve with analytical column, 4. 1. the two ends of enriching column be connected with the interface of six-way valve respectively, 3. one end of analytical column is connected with the interface of six-way valve, nozzle needle is fixed in the other end of analytical column, the interface of six-way valve 2. as the mobile phase end, it is connected with B with mobile phase A by 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 is connected with the sample storage container with eluent by the kapillary pump.
During enrichment, chip connects the sample end by the kapillary pump and the waste liquid end is introduced the sample in the sample storage container and realize enrichment on enriching column, and now 5., 4., 1., 6. be communicated with successively, sample end eluent is the acetonitrile water containing volume 2% to the interface of six-way valve;
During sample analysis, by the nano pump, connect mobile phase end and analytical column, sample enters analysis channel, and now the interface of six-way valve 2., 1., 4., 3. be communicated with successively; Analyze mobile phase A for the aqueous solution containing volume 0.1% formic acid, B is the acetonitrile containing volume 0.1% formic acid; Condition of gradient elution: 90/10 (A/B, V/V) during 0min, 70/30 (A/B, V/V) during 13min, 5/95 (A/B, V/V) keep 4min, 90/10 (A/B during 22.1min during 18min, V/V), start gradient 90/10 (A/B, V/V) balance 8min subsequently; Wherein 0-13min is that first paragraph linear gradient elution, 13-18min are the second segment linear gradient elution, and 18-22.1min is the 3rd 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 after sample introduction, the enriching column flush volume is 3-4 μ L; After the post of analytical column, efflux imports the mass spectrometer system detection without shunting;
The mass spectrum condition is: electric spray ion source adopts 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 under these conditions the metabolic profile spectrum of soybean flavonoids, the TIC figure (Fig. 1) that soybean flavonoids LC-Chip/MS analyzes.
The effect that the present invention has is: the storage of sample, pre-service and analysis all adopt the autonomous standardized program of setting up, and avoid introducing personal error.
The preprocessing process 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, with preprocessing process, combine and can identify more target compound.In addition, adopt dissimilar interior mark and quality control sample to proofread and correct preprocessing process and test Instrumental to respond the error that small drift brings, further guaranteed the stability of the method.The method repeatability of 9 characteristic ions in sample is investigated and be the results are shown in Table 1, and its relative standard deviation is 1.50-7.66%, has further proved the stability of method that this patent is set forth.
The accompanying drawing explanation
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.Each point means a sample, and t means that sample is in the principal component projection value.◆: plantation Jinan, place, ▲: plantation Jilin, place.
Fig. 3 is high enrichment capacity chip structure schematic diagram in the present invention.1 eluent in figure, 2 waste liquids, 3 enriching columns, 4 mobile phases, 5 analytical columns, 6 nozzle needles.
Embodiment
1. soya seeds sample collection
5 soybean varieties are respectively: Ji is educated 73, is grown agriculture 16, nine agricultures 30, lotus beans 12, middle yellow 13.Wherein luckyly educate 73, the plantation place of long agriculture 16 and nine agricultures 30 is Jilin.Lotus beans 12 and middle yellow 13 plantation place are Jinan.Be placed on-80 ℃ of Refrigerator stores after the soya seeds sample collection.
2. analytical approach
2.1 soya seeds sample preprocessing
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 comminutor and pulverizes, cross 60 mesh sieves, obtain the powder of uniform particle diameter.The soya seeds powder is divided and is filled in plastic tube, standby.
Take 5 kind soya seeds samples each 3 parts (every part of 0.1g), be placed in respectively the 5mL glass tube.
Take every kind of soya seeds sample 0.5g, mix, as quality control (QC) sample.Then take 6 parts, QC sample (every part of 0.1g), be placed in respectively the 5mL glass tube.
Take interior mark naringenin 0.4mg and isoquercitrin, each 1.6mg of rutin in the 1.5mL centrifuge tube, add the 1mL acetonitrile.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 as extracting solvent, add above-mentioned mixed mark solution 50 μ L to extracting in solvent, mixes rear 50mL altogether and extract solvent.
In each glass tube, add 2mL to extract solvent, ultrasonic 20 minutes.Take out 1mL and move in the eppendorf pipe, in 4 ℃, under 14000rpm centrifugal 10 minutes, supernatant is moved to sample injection bottle.
2.2 chip is received flow liquid phase Chromatography/Mass Spectrometry combination analysis
As shown in Figure 3, high enrichment capacity chip is comprised of large capacity enriching column (500nL, ID 75 μ m), analytical column (75 μ m * 150mm) and nozzle needle (10 μ m ID), the packing material of enriching column and analytical column be Zorbax SB-C18 (
5 μ m).
Enriching column is connected by six-way valve with analytical column, 4. 1. the two ends of enriching column be connected with the interface of six-way valve respectively, 3. one end of analytical column is connected with the interface of six-way valve, nozzle needle is fixed in the other end of analytical column, the interface of six-way valve 2. as the mobile phase end, it is connected with B with mobile phase A by 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 is connected with the sample storage container with eluent by the kapillary pump;
Chip connects the sample end by the kapillary pump and the waste liquid end is introduced sample (sample storage container) and realize enrichment on enriching column, and now 5., 4., 1., 6. be communicated with successively, sample end eluent is the acetonitrile water containing volume 2% to the interface of six-way valve;
During sample analysis, by the nano pump, connect mobile phase end and analytical column, sample enters analysis channel, and now the interface of six-way valve 2., 1., 4., 3. be communicated with successively.Analyze mobile phase A for the aqueous solution containing volume 0.1% formic acid, B is the acetonitrile containing volume 0.1% formic acid.(wherein 0-13min is that first paragraph linear gradient elution, 13-18min are the second segment linear gradient elution to condition of gradient elution, 18-22.1min be the 3rd section gradient elution): 90/10 (A/B during 0min, V/V), 70/30 (A/B, V/V) during 13min, 5/95 (A/B during 18min, V/V) and keep 4min, 90/10 (A/B, V/V) during 22.1min, 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 after sample introduction, the enriching column flush volume is 4 μ L, analyzes and adopts the recoil pattern to reduce widening; After post, efflux imports the mass spectrometer system detection without shunting.The mass spectrum condition is: electric spray ion source (ES I) adopts 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 under these conditions the TIC figure that soybean flavonoids LC-Chip/MS analyzes.
The chip of 15 soya seeds samples is received flow liquid phase Gas Chromatography/Mass Spectrometry Analysis sequentially for carrying out at random, in analytic process, first by QC sample sample introduction once, then after 3 samples of every analysis, again by QC sample sample introduction once, by guaranteeing the reappearance of QC sample retention time and response signal, guarantee that the experimentation chips receives flow liquid phase Chromatography/Mass Spectrometry combined instrument stability is preferably arranged.Results suggest, in whole analysis time, the repeatability of QC sample is good, obvious change of component do not occur, and drift does not appear in retention time, the investigation result that table 1 and table 2 are 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) to carry out a data matrix of peak extraction formation to the flavone compound of all soya seeds samples, the measurement data of each sample is matched to a peak table consisted of retention time, mass number and peak area, reduces systematic error by total peak area normalization simultaneously.Then utilize SIMCA-P 11.5 (Umetrics, Umea, Sweden) to carry out the PLS-DA analysis.Non-parametric test adopts SPSS 13.0 (SPSS Inc., Chicago, IL) to carry out.
Fig. 2 is the PLS-DA shot chart of different plantations place soybean.Therefrom known, being projected on first principal component of soybean sample that 9 plantation places soybean sample that is Jilin is Jinan with 6 plantation places can well be separated, and shows that there is significantly difference in the flavonoids metabolic profiles of the soybean sample in different plantations place.By 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 by the variable (p<0.05) of non-parametric test, they are respectively glycitein O-hexoside, genistein O-hexoside, daidzeinO-hexoside malonylated, the Flavonoid substances (table 3) such as daidzein O-hexoside malonylated.
Simple to operate, the highly sensitive advantage based on this method, can be further analyzed the flavonoids in the soybean sample of different cultivars and genetic background, 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 illustrating the characteristics of the described method of this patent, the applicant compares the chip of different sample pretreating methods and different enrichment capacity.
1) adopt the SPE method to carry out second extraction and purifying to the soybean sample, to improve the response of low concentration compound.With the difference of embodiment mono-be to carry out preenrichment and purifying with Oasis HLBSPE pillar by normal process after the ultrasonic 20min of soybean sample, normal process is: 10 times of rear SPE pillars of crossing of the soybean sample extraction liquid 1ml dilute with water of ultrasonic 20min, flow velocity is about 10ml/min, rear by 1ml water and 0.05ml methanol wash, the target extract finally uses 0.5ml methyl alcohol and 0.5ml ethanol/methylene (1: 1) to rinse respectively, after collecting the liquid freeze-drying, with 1ml water, redissolve, loading is analyzed, and all analysis conditions and parameter are all identical with embodiment mono-.Found that, after adopting second extraction, the response of most compounds has 1-2 increase doubly, but some compound is as the daidzein 7 of m/z 751,4 '-O-di-(hexoside malonylated) can't detect, and this explanation second extraction and purge process can cause the loss of part target compound.Although and, a little less than the method for once extracting in embodiment mono-response slightly, more target compound can be detected, and can also greatly shorten pretreatment time and reduce solvent consumption.
2) adopt standard chips (40nL) to be analyzed soybean sample extraction liquid, all pretreatment conditions and analytical parameters are all identical with embodiment mono-.Found that, because the enrichment capacity of 40nL chip is less, former thereby the loss that chemical compound lot is large because of polarity or abundance is lower, as the glycitein-O-pentosylhexoside of glycitein O-hexoside malonylated, the m/z 579 of m/z 533 and the glycitein 7,4 ' of m/z 609-O-d ihexoside etc.And adopt our customized large 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 described method of this patent, the 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 preprocessing 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 sample consumption is little and reproducible, is suitable for the batch quantity analysis of actual sample.
Claims (4)
1. the chip of a soybean flavonoids metabolic profiling analysis is received flow liquid phase Chromatography/Mass Spectrometry method, it is characterized in that:
(1) add the soya seeds sample of extract ultrasonic centrifugal after, the direct loading analysis of supernatant; (2) the direct loading analysis of supernatant, high enrichment capacity chip compartment analysis condition: high enrichment capacity chip is comprised of the large capacity enriching column of 500nL, long 150mm analytical column and nozzle needle, and the packing material of enriching column and analytical column is Zorbax SB-C18; Enriching column is connected by six-way valve with analytical column, and nozzle needle is fixed in an end of analytical column; During sample analysis, by the nano pump, connect mobile phase end and analytical column, sample enters analysis channel, and now the second interface of six-way valve, first interface, the 4th interface and the 3rd interface are communicated with successively; Analyze mobile phase A for the aqueous solution containing volume 0.1% formic acid, B is the acetonitrile containing volume 0.1% formic acid; Condition of gradient elution: 10%B during 0min, 30%B during 13min, 95%B keep 4min during 18min, become start gradient 10%B balance 8min during 22.1min; Wherein 0-13min is that first paragraph linear gradient elution, 13-18min are the second segment linear gradient elution, and 18-22.1min is the 3rd 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 after sample introduction, the enriching column flush volume is 3-4 μ L; After the post of analytical column, efflux imports the mass spectrometer system detection without shunting;
The mass spectrum condition is: electric spray ion source adopts positive ion mode to detect; Use high-purity N
2Assistant spray ionization and desolventizing, 300 ℃ of dry gas temperature, the dry gas flow velocity is 4L/min; Capillary voltage is 1800-1900V; Mass scanning scope m/z100～1000; Can obtain under these conditions the metabolic profile spectrum of soybean flavonoids.
2. method according to claim 1, it is characterized in that: in step (1), take the soya seeds sample in glass tube, add the extraction solvent containing interior mark naringenin, isoquercitrin and each 0.5-2 μ g/mL of rutin in every arm, extract solvent for the aqueous solution containing methyl alcohol 80%, centrifugal after ultrasonic 10-20 minute, supernatant is analyzed for LC-Chip/MS.
3. method according to claim 1 is characterized in that:
Enriching column is connected by six-way valve with analytical column, the two ends of enriching column are connected with the 4th interface with the first interface of six-way valve respectively, one end of analytical column is connected with the 3rd interface of six-way valve, nozzle needle is fixed in the other end of analytical column, the second interface of six-way valve as the mobile phase end, it is connected with B with mobile phase A by the nano pump, the 6th interface of six-way valve is as the waste liquid outlet end, the 5th interface of six-way valve as the sample end, it is connected with the sample storage container with eluent by the kapillary pump.
4. method according to claim 3 is characterized in that:
What step (2) was selected is high enrichment capacity chip, and this chip is by 500nL, and the nozzle needle of the large capacity enriching column of ID75 μ m, the analytical column of 75 μ m * 150mm and 10 μ m ID forms, and the packing material of enriching column and analytical column is
The Zorbax SB-C18 of 5 μ m;
During enrichment, chip connects the sample end by the kapillary pump and the waste liquid end is introduced the sample in the sample storage container and realize enrichment on enriching column, now the 5th interface of six-way valve, the 4th interface, first interface and the 6th interface are communicated with successively, and sample end eluent is the acetonitrile water containing volume 2%.
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