CN105301132B - A kind of method that amino acid is combined in non-derived quick detection medicinal plant - Google Patents
A kind of method that amino acid is combined in non-derived quick detection medicinal plant Download PDFInfo
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Abstract
The invention discloses a kind of method that amino acid is combined in non-derived simultaneously quick detection medicinal plant, comprise the following steps：（1）The freezing of high-voltage pulse sour water solution combination amino acid and hydrolyzate and the analysis pre-treatment such as melt again；（2）Ultrahigh pressure liquid phase chromatogram Evaporative light scattering detector method is set up；（3）Contents of Amino Acids in medicinal plant.It is simple to operate that the present invention uses the sour hydrolysis amino acid of high-pressure pulse electric method to have, and sample hydrolysis is quick and complete, and reagent dosage is few, fast energy-saving, the advantages of environmental pollution is small.By analyzing pretreatment process addition freezing and redissolving link, it can be acted on by " ice is told ", the pollution of purification of target thing and reduction later stage to splitter extends the service life of chromatographic column；Analysis process uses water-fast silicagel column, and organic reagent consumption is few, environmental protection；More important point need not be derivative using EISD, easy to operate, and derivative reagent and side reaction are not introduced, significant for amino acid content in Accurate Determining medicinal plant.
Description
Technical field
The present invention relates to the analysis method that amino acid is combined in a kind of non-derived quick detection medicinal plant, belong to Chinese medicine
Constituent analysis field.
Background technology
Amino acid is the carboxylic acid containing amino, is the basic component units of biological function macro-molecular protein.In nature
In main with free amino acid and combine two kinds of forms of amino acid and exist.Two kinds of forms are all present in medicinal plant, except with battalion
Support value outer, indispensable effect is also played when playing drug effect.Secondly, the height of amino acid content, can plant as medicinal
The foundation that the thing property of medicine differentiates.Moreover, index acidic amino acid can be used for Qualitive test Chinese medicine.Therefore, amino acid in medicinal plant
Analysis differentiates that quality evaluation and its exploitation of Related product provide experimental basis for medicinal plant.Due to most of ammonia
The sour polarity of base is high, volatility is low, without strong chromophoric group, causes it to separate and detect extremely challenging.Amino-acid analyzer method
It is that amino acid analysis is most effective and most common method with chromatography, pretreatment is to ensure that analysis result accurately before analysis
One of key link.
There are traditional acid, alkali and enzyme hydrolysis method, and unconventional microwave acid, alkali and enzyme with reference to amino acid pre-treating method
Hydrolyze method.Its object is to disrupting tissue cell, make to discharge free amino acid with reference to the fracture of amino acid key.Traditional acid, alkali
Or enzyme hydrolysis method hydrolysis time is long, there is that hydrolytic process is cumbersome, high energy consumption is unsuitable for a large amount of samples of quick detection and amino
The shortcomings of sour percent hydrolysis is relatively low.In addition, the more conventional acid of microwave hydrolysis, alkali compare with enzyme hydrolysis method, it is time-consuming shorter, but it is micro-
The degree of accuracy of ripple method for hydrolysis awaits the discussion analysis and the accreditation gone together of Experimental comparison's data.
The percent hydrolysis of amino acids material is heavily dependent on the breaking-wall cell situation to specimen material, current broken wall
Method have the methods such as ultrasonic hydrolysis and microwave hydrolysis, but there are problems in them, such as time-consuming or clasmatosis not exclusively,
Cause to combine acid hydrolysis time consumption and energy consumption, hydrolysis efficiency is low.High-pressure pulse electric (High-voltage pulsed
Electric field, abbreviation HPEF) technology is a kind of new technology developed in recent years, mechanism is worn using cell membrane electricity
Hole principle makes breaking-wall cell in moment, causes cell membrane potential chaotic, and cell membrane and cell membrane generation are reversible or irreversible broken
It is bad, cellular component outflow.HPEF methods introduce the pre-treatment field of amino acid analysis, with processing time is short, energy consumption is low, be difficult to draw
Purpose product is played the advantages of be denatured, to more objective evaluation and resources of medicinal plant is utilized, preserves the ecological environment, saves the energy,
Reduction labour cost has great importance.
In addition, traditional amino acid hydrolyticsolution goes to the past extraction pillar for needing to use costliness to be further purified in upper prop,
To reduce pollution of the later stage to analysis chromatographic column.The method that this patent is then redissolved using amino acid hydrolyticsolution is freezed so that cold
Partial impurities after jelly insoluble in hydrolyzate solvent are got rid of by way of filtering and centrifuging, and can lower purifying cost significantly
Operated with simplifying.
The method of determined amino acid mainly has amino-acid analyzer method and chromatography.Amino-acid analyzer is due to expensive
It is high and lack general applicability with dedicated analysis post price.Chromatographic process mainly has liquid chromatography (LC), gas chromatography
(GC), capillary electrophoresis (CE), ion-exchange chromatography (IEC) and liquid chromatography-mass spectrography/mass spectrography (LC-MS/MS).From
Derivative angle can be summarized as two kinds of derivatization method and non-derivatization method.It is derivative first then to pass through liquid chromatogram-uv detection method, liquid phase
Chromatogram-fluorescence detection, gas chromatography (GC), capillary electrophoresis (CE) or liquid chromatography-mass spectrography/mass spectrography (LC-MS/
MS) method is detected.Secondly non-derivatization method is mainly HPLC (UPLC)-ELSD methods and HPLC (UPLC)-MS methods and ion is handed over
Colour changing spectrometry.
Pass through ultraviolet, fluorescence and mass spectrography detection amino acid, although sensitivity and separating degree obtain larger journey after derivatization
The raising of degree.But, there is more drawback, if any derivatization method can not detect imino acid and sulfur-containing amino acid, derivative is steady
, there is excessive derivative reagent and accessory substance interference, time-consuming and Individual amino acids derivatization is difficult and is not suitable for large quantities of in qualitative difference
The problems such as detection of secondary sample.In addition, current non-derivatization method, uses special method bag, chromatographic column specially and dedicated stream mostly
Dynamic phase, such as WatersAccQTagTM Ultra C18 (1.7 μm, 2.1 × 100mm), AccQTag Ultra Eluent A
And AccQTagUltra Eluent B etc. due to expensive without economical and practical.
The content of the invention
The present invention provides a kind of non-derived while combining the method for amino acid in quick detection medicinal plant.For early stage ammonia
Deficiency in terms of the pre-treatment of base acid analysis and analysis, proposes brand-new quick determination method.
The technical solution of the present invention is as follows：Histocyte rupture is carried out to sample using high-pressure pulse electric method first
And sour water solution, then hydrolyzate freezing is melted again, finally sample liquid entered using ultrahigh pressure liquid phase chromatogram-evaporative light-scattering method
Row Contents of Amino Acids.It is simple to operate that the present invention uses the sour hydrolysis amino acid of high-pressure pulse electric method to have, and sample hydrolysis is fast
Fast complete, reagent dosage is few, fast energy-saving, the advantages of environmental pollution is small.In addition, analysis pretreatment process is redissolved using freezing,
Purification of target thing is acted on by " ice is told ", the pollution to splitter is reduced, extends the service life of chromatographic column；Adopted during analysis
With water-fast C18 posts, organic reagent consumes few, environmental protection；Finally using EISD without derivative, operation letter
Just, derivative reagent and side reaction are not introduced, is suitable for the detection of big batch sample, there is preferable practical value and prospect.
A kind of non-derived of the present invention is while combine the analysis method of amino acid, mainly in quick detection medicinal plant
Comprise the following steps：
1. the freezing of high-voltage pulse sour water solution combination amino acid and hydrolyzate and melt analysis pre-treatment again；
Take medicinal plant 1.0g to be measured in hydrolysis pipe, add 6mol × L-1Hydrochloric acid solution, deoxygenation good seal sets electric field
Intensity 10kv × cm-1-30kv×cm-1, solid-liquid ratio 1:5-1:15 and burst length 30-90S carries out high voltage pulse method hydrolysis, treats
It is cooled to after room temperature and hydrolyzate is placed into subzero 20-60 degree freezing 2-4 hours, then takes out normal temperature and dissolve, then filtering, takes filter
Liquid 1mL nitrogen evaporators are dried up, and with flowing phased soln, cross 0.22 μm of membrane filtration standby；
2. ultrahigh pressure liquid phase chromatogram-Evaporative light scattering detector method is set up；
Accurately weigh each standard items 0.1mol × L-1Hydrochloric acid is configured to 0.5-10nmol × L-1Amino standard acid solution,
Using Hilic chromatographic columns (referring to Fig. 1), Amide chromatographic columns (referring to Fig. 2) and hydrophily C18 chromatographic columns (refer to figure
3) it is separated, is that mobile phase carries out gradient elution with acetonitrile-water (containing the fluorine butyric acid of 0.1-1% nine and trifluoroacetic acid),
Gradient elution (A is ion pair acetonitrile phase, and B is ion pair aqueous phase)：0-0.8min, 0%A；0.8-1.5min, 12%A；1.5-
4min, 15%A；4-5min, 61%A；5.1-8min, 61%A；8-8.1min, 0%A；8.1-10min, 0%A；Analysis time
10min.Flow velocity 0.3-0.5ml × min is set-1, 60-80 DEG C of ELSD drift pipes temperature, UPLC- on carrier gas flux 20-40PSI
ELSD is detected.Each concentration distinguishes the μ L of sample introduction 2 and surveys its peak area, and using the logarithm of sample introduction concentration as abscissa, the logarithm of peak area is
Ordinate draws standard curve.
3. Contents of Amino Acids in medicinal plant：
According to above-mentioned liquid phase chromatogram condition, the amino acid hydrolyzed to medicinal plant in prepare liquid sample carries out separation detection.
The μ L of sample introduction 2, determine peak area, and the content of amino acid in medicinal plant is calculated according to standard curve.
The freezing of step 1 high-voltage pulse sour water solution combination amino acid and hydrolyzate of the present invention and melt again before analysis
Processing, be preferably：
Take medicinal plant 1.0g to be measured in hydrolysis pipe, add 6mol × L-1Hydrochloric acid solution, deoxygenation good seal sets electric field
Intensity is 20kv × cm-1, solid-liquid ratio 1:10 and the burst length be that 90S carries out high voltage pulse method hydrolysis, being cooled to after room temperature will
Hydrolyzate is placed subzero 20-60 degree and freezed 2-4 hours, then takes out normal temperature and dissolves, has then filtered and taken filtrate 1mL nitrogen evaporators to blow
It is dry, after flowing phased soln, cross 0.22 μm of membrane filtration standby.
Step 2 ultrahigh pressure liquid phase chromatogram of the present invention-Evaporative light scattering detector method is set up：
Accurately weigh each standard items 0.1mol × L-1Hydrochloric acid is configured to 0.5-10nmol × L-1Amino standard acid solution,
Using hydrophily C18 chromatograms post separation (referring to accompanying drawing 3), with acetonitrile-water (containing 0.7% 9 fluorine butyric acid and 0.5% trifluoro second
Acid) carry out gradient elution, gradient elution for mobile phase (A is ion pair acetonitrile phase, and B is ion pair aqueous phase)：0-0.8min, 0%
A；0.8-1.5min, 12%A；1.5-4min, 15%A；4-5min, 61%A；5.1-8min, 61%A；8-8.1min, 0%A；
8.1-10min, 0%A；Analysis time 10min.Flow velocity 0.4ml × min is set-1, ELSD drift pipe temperature 70 Cs, carrier gas flux
The upper UPLC-ELSD detections of 30PSI.Each concentration distinguishes the μ L of sample introduction 2 and surveys its peak area, using the logarithm of sample introduction concentration as abscissa, peak
The logarithm of area is that ordinate draws standard curve.
According to above-mentioned liquid phase chromatogram condition, the amino acid hydrolyzed to medicinal plant in prepare liquid sample carries out separation detection.
The μ L of sample introduction 2, determine peak area, and the content of amino acid in medicinal plant is calculated according to standard curve.Each amino acid peak sequence is
1. glycine, 2. serines, 3. aspartic acids, 4. glutamic acid, 5. threonines, 6. alanine, 7. cystines, 8. proline, 9.
Valine, 10. methionine, 11. lysines, 12. histidines, 13. isoleucines, 14. arginine, 15. leucines, 16. phenylpropyl alcohols
Propylhomoserin, 17. tryptophans.
The positive effect of the present invention is：There is simple to operate, sample using the sour hydrolysis amino acid of high-pressure pulse electric method
Hydrolysis is quick and complete, and reagent dosage is few, fast energy-saving, the advantages of environmental pollution is small.In addition, analysis pretreatment process addition freezing
And link is redissolved, it can be acted on by " ice is told ", the pollution of purification of target thing and reduction later stage to splitter extends making for chromatographic column
Use the life-span；Furthermore, analysis process uses water-fast C18 posts, and organic reagent consumption is few, environmental protection；More important point is used
EISD is easy to operate without derivative, derivative reagent and side reaction is not introduced, for Accurate Determining medicinal plant
Middle amino acid content is significant.
Brief description of the drawings
Fig. 1 is Hilic chromatogram post separation figures；
Fig. 2 is Amide chromatogram post separation figures；
Fig. 3 is hydrophily C18 chromatogram post separation figures.
Embodiment
With reference to the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described.
Instrument and reagent
High-pressure pulse electric extraction system, the waveform of the pulse power is Exponential Decay Wave, frequency-adjustable.Separately contain high-voltage pulse
Power supply and extraction workpiece (oscillograph and process chamber), Ultra Performance Liquid Chromatography instrument Acquity UPLC (Waters companies),
EISD (Waters companies), work station (Empower-2)；AL204 electronic balances (plum Teller-support benefit instrument
Device Co., Ltd)；Ultrasonic cleaner (Kunshan Shu Mei ultrasonic cleaner KQ-500DE)；Centrifuge (the big-and-middle HC- of Anhui section
3513 supercentrifuges)；Acetonitrile is chromatographically pure (Fisher companies)；(Sigma is public for trifluoroacetic acid, hyptafluorobutyric acid and nine fluorine valeric acids
Department), water is ultra-pure water (Millopre), amino acid standard items (Nat'l Pharmaceutical ＆ Biological Products Control Institute), and other reagents are point
Analysis is pure.
Embodiment 1
A kind of non-derived is while combine the method for amino acid in quick detection ginseng
Ginseng picks up from Jilin Province's Ji'an City, and sample is the dry root of 5 years stranger's ginsengs, is collected in September, 2014.
1. samples of Ginseng high-voltage pulse sour water solution, the analysis pre-treatment such as freezing and redissolution：
Take ginseng to be measured sample 1.0g in hydrolysis pipe, add 6mol × L-1Hydrochloric acid solution, deoxygenation good seal sets electric field
Intensity is 20kv × cm-1, solid-liquid ratio 1:10 and the burst length be that 90S carries out high voltage pulse method hydrolysis, being cooled to after room temperature will
Hydrolyzate is placed subzero 20-60 degree and freezed 2-4 hours, then takes out normal temperature and dissolves, has then filtered and taken filtrate 1mL nitrogen evaporators to blow
It is dry, after flowing phased soln, cross 0.22 μm of membrane filtration standby；
2. ultrahigh pressure liquid phase chromatogram-Evaporative light scattering detector method is set up；
Accurately weigh each standard items 0.1mol × L-1Hydrochloric acid is configured to 0.5-10nmol × L-1Amino standard acid solution,
Using hydrophily C18 chromatogram post separations, entered with acetonitrile-water (containing 0.7% 9 fluorine butyric acid and 0.5% trifluoroacetic acid) for mobile phase
Row gradient elution, gradient elution (A is ion pair acetonitrile phase, and B is ion pair aqueous phase)：0-0.8min, 0%A；0.8-1.5min,
12%A；1.5-4min, 15%A；4-5min, 61%A；5.1-8min, 61%A；8-8.1min, 0%A；8.1-10min, 0%
A；Analysis time 10min.Flow velocity 0.4ml × min is set-1, ELSD drift pipe temperature 70 Cs, UPLC- on carrier gas flux 30PSI
ELSD is detected.Each concentration distinguishes the μ L of sample introduction 2 and surveys its peak area, and using the logarithm of sample introduction concentration as abscissa, the logarithm of peak area is
Ordinate draws standard curve.
3. the measure of amino acid is combined in ginseng：
Determined amino acid result in 3.1 ginsengs
According to above-mentioned liquid phase chromatogram condition, the amino acid hydrolyzed to ginseng in prepare liquid sample carries out separation detection.Sample introduction 2
μ L, determine peak area, and the content of amino acid in ginseng is calculated according to standard curve.In September, 2014 is adopted in Jilin Province's Ji'an City
Ginseng to be combined in Contents of Amino Acids, ginseng amino acid peak sequence be 1. glycine, 2. serines, 3. asparagus fern ammonia
Acid, 4. glutamic acid, 5. threonines, 6. alanine, 7. cystines, 8. proline, 9. valines, 10. methionine, 11. lysines,
12. histidine, 13. isoleucines, 14. arginine, 15. leucines, 16. phenylalanines, 17. tryptophans.6 times measure be averaged
Value is as shown in table 1.As can be seen from the table in 17 kinds of amino acid contained by ginseng, secondly arginine content highest is glutamic acid
And aspartic acid, methionine content is minimum.Total amino acid average content is 0.951mg × g-1。
3.2 repeated experiment
Same samples of Ginseng is taken to prepare 6 parts of sample solutions by identical experimental method, the μ L of sample introduction 2 are measured, as a result sent out
Now except the RSD of cystine is 3.06%, outside 3.00%, other are respectively less than 3.00%.Trace it to its cause, it may be possible to because
Cystine is in acid hydrolysis, because a small amount of oxygen is present and causes the cystine containing sulfydryl to have a little oxidation difference.
3.3 Precision Experiment
Same samples of Ginseng pretreatment liquid is taken, continuous sample introduction 6 times determines each amino acid content, and its RSD is 0.28-
0.59%.
3.4 stability experiment
Same samples of Ginseng pretreatment liquid is taken, respectively 0,8,16,24 hours sample introductions, even if 24 hours its peak areas of difference
Also very stable, its RSD is 0.28-0.86%.
3.5 degrees of accuracy are tested
The preparation of recovery span amino acid mother liquor：Precision weighs the mixed mark of amino acid of 105 DEG C of dryings 3 hours, is placed in 100mL appearances
In measuring bottle, it is dissolved in water and is diluted to scale, shake up and produce.
Reclaim the preparation of samples of Ginseng liquid：6 parts of samples of Ginseng pretreatment liquid 8mL is measured, 10mL volumetric flasks are respectively placed in
In, every part of accurate above-mentioned recovery span amino acid mother liquor 2mL of addition is finally diluted with water to scale, shaken up, produces recovery ginseng
Sample liquid.
Contents of Amino Acids is carried out by above-mentioned chromatographic condition, and the rate of recovery is calculated according to following calculation formula,
Calculation formula：The rate of recovery=(measured amount-sample size)/addition * 100%
As a result show, each the recovery of amino acid is in 98.7-103.4% scopes.RSD is 0.96%, therefore can draw this
The preferable conclusion of the assay method rate of recovery.
Embodiment 2
Non-derived is while combine the method for amino acid in the quick detection bark of eucommia
The bark of eucommia is purchased from enshi city, is purchased in December, 2014：
1. Eucommia Samples high-voltage pulse sour water solution, the analysis pre-treatment such as freezing and redissolution：
Take Eucommia Samples 1.0g to be measured in hydrolysis pipe, add 6mol × L-1Hydrochloric acid solution, deoxygenation good seal sets electric field
Intensity is 20kv × cm-1, solid-liquid ratio 1:10 and the burst length be that 90S carries out high voltage pulse method hydrolysis, being cooled to after room temperature will
Hydrolyzate is placed subzero 20-60 degree and freezed 2-4 hours, then takes out normal temperature and dissolves, has then filtered and taken filtrate 1mL nitrogen evaporators to blow
It is dry, after flowing phased soln, cross 0.22 μm of membrane filtration standby；
2. ultrahigh pressure liquid phase chromatogram-Evaporative light scattering detector method is set up；
Accurately weigh each standard items 0.1mol × L-1Hydrochloric acid is configured to 0.5-10nmol × L-1Amino standard acid solution,
Using hydrophily C18 chromatogram post separations, entered with acetonitrile-water (containing 0.7% 9 fluorine butyric acid and 0.5% trifluoroacetic acid) for mobile phase
Row gradient elution, gradient elution (A is ion pair acetonitrile phase, and B is ion pair aqueous phase)：0-0.8min, 0%A；0.8-1.5min,
12%A；1.5-4min, 15%A；4-5min, 61%A；5.1-8min, 61%A；8-8.1min, 0%A；8.1-10min, 0%
A；Analysis time 10min.Flow velocity 0.4ml × min is set-1, ELSD drift pipe temperature 70 Cs, UPLC- on carrier gas flux 30PSI
ELSD is detected.Each concentration distinguishes the μ L of sample introduction 2 and surveys its peak area, and using the logarithm of sample introduction concentration as abscissa, the logarithm of peak area is
Ordinate draws standard curve.
3. the measure of amino acid is combined in the bark of eucommia：
Determined amino acid result in 3.1 barks of eucommia
According to above-mentioned liquid phase chromatogram condition, the amino acid hydrolyzed to the bark of eucommia in prepare liquid sample carries out separation detection.Sample introduction 2
μ L, determine peak area, and the content of amino acid in the bark of eucommia is calculated according to standard curve.To in September, 2014 purchased from enshi city
It is 1. glycine, 2. serines, 3. asparagus fern ammonia that the bark of eucommia, which is combined amino acid peak sequence in Contents of Amino Acids, the bark of eucommia,
Acid, 4. glutamic acid, 5. threonines, 6. alanine, 7. cystines, 8. proline, 9. valines, 10. methionine, 11. lysines,
12. histidine, 13. isoleucines, 14. arginine, 15. leucines, 16. phenylalanines, 17. tryptophans.6 times measure be averaged
Value is as shown in table 2.As can be seen from the table in 17 kinds of amino acid contained by the bark of eucommia, secondly content of glutamic acid highest is arginine
And aspartic acid, cystine is minimum.Total amino acid average content is 1.518g × kg-1。
3.2 repeated experiment
Same Eucommia Samples are taken to prepare 6 parts of sample solutions by identical experimental method, the μ L of sample introduction 2 are measured, as a result removed
The RSD of cystine is 3.11%, and outside 3.00%, other are respectively less than 3.00%.Trace it to its cause, it may be possible to because Guang ammonia
Acid is in acid hydrolysis, because a small amount of oxygen is present and causes the cystine containing sulfydryl to have a little oxidation difference.
3.3 Precision Experiment
Same Eucommia Samples pretreatment liquid is taken, continuous sample introduction 6 times determines each amino acid content, and its RSD is 0.33-
0.51%.
3.4 stability experiment
Same Eucommia Samples pretreatment liquid is taken, respectively 0,8,16,24 hours sample introductions, even if 24 hours its peak areas of difference
Also very stable, its RSD is 0.65-1.31%.
3.5 degrees of accuracy are tested
The preparation of recovery span amino acid mother liquor：Precision weighs the mixed mark of amino acid of 105 DEG C of dryings 3 hours, is placed in 100mL appearances
In measuring bottle, it is dissolved in water and is diluted to scale, shake up and produce.
Reclaim the preparation of Eucommia Samples liquid：6 parts of Eucommia Samples pretreatment liquid 8mL is measured, 10mL volumetric flasks are respectively placed in
In, every part of accurate above-mentioned recovery span amino acid mother liquor 2mL of addition is finally diluted with water to scale, shaken up, produces the recovery bark of eucommia
Sample liquid.
Contents of Amino Acids is carried out by above-mentioned chromatographic condition, and the rate of recovery is calculated according to following calculation formula,
Calculation formula：The rate of recovery=(measured amount-sample size)/addition * 100%
As a result show, each the recovery of amino acid is in 98.1-102.8% scopes.RSD is 0.92%, therefore can draw this
The preferable conclusion of the assay method rate of recovery.
Embodiment 3
Non-derived is while combine the method for amino acid in the quick detection coptis
The coptis is purchased from Hubei Laifeng County, is purchased in July, 2014：
1. coptis sample high-voltage pulse sour water solution, the analysis pre-treatment such as freezing and redissolution：
Take coptis sample 1.0g to be measured in hydrolysis pipe, add 6mol × L-1Hydrochloric acid solution, deoxygenation good seal sets electric field
Intensity is 20kv × cm-1, solid-liquid ratio 1:10 and the burst length be that 90S carries out high voltage pulse method hydrolysis, being cooled to after room temperature will
Hydrolyzate is placed subzero 20-60 degree and freezed 2-4 hours, then takes out normal temperature and dissolves, has then filtered and taken filtrate 1mL nitrogen evaporators to blow
It is dry, after flowing phased soln, cross 0.22 μm of membrane filtration standby；
2. ultrahigh pressure liquid phase chromatogram-Evaporative light scattering detector method is set up；
Accurately weigh each standard items 0.1mol × L-1Hydrochloric acid is configured to 0.5-10nmol × L-1Amino standard acid solution,
Using hydrophily C18 chromatogram post separations, entered with acetonitrile-water (containing 0.7% 9 fluorine butyric acid and 0.5% trifluoroacetic acid) for mobile phase
Row gradient elution, gradient elution (A is ion pair acetonitrile phase, and B is ion pair aqueous phase)：0-0.8min, 0%A；0.8-1.5min,
12%A；1.5-4min, 15%A；4-5min, 61%A；5.1-8min, 61%A；8-8.1min, 0%A；8.1-10min, 0%
A；Analysis time 10min.Flow velocity 0.4ml × min is set-1, ELSD drift pipe temperature 70 Cs, UPLC- on carrier gas flux 30PSI
ELSD is detected.Each concentration distinguishes the μ L of sample introduction 2 and surveys its peak area, and using the logarithm of sample introduction concentration as abscissa, the logarithm of peak area is
Ordinate draws standard curve.
3. the measure of amino acid is combined in the coptis：
According to above-mentioned liquid phase chromatogram condition, the amino acid hydrolyzed to ginseng in prepare liquid sample carries out separation detection.Sample introduction 2
μ L, determine peak area, and the content of amino acid in ginseng is calculated according to standard curve.
Determined amino acid result in 3.1 coptiss
Contents of Amino Acids, 1. glycine, 2. ammonia are combined purchased from Hubei city of Laifeng County coptis in July, 2014
Acid, 3. aspartic acids, 4. glutamic acid, 5. threonines, 6. alanine, 7. cystines, 8. proline, 9. valines, 10. egg ammonia
Acid, 11. lysines, 12. histidines, 13. isoleucines, 14. arginine, 15. leucines, 16. phenylalanines, 17. tryptophans.
The average value of 6 measure is as shown in table 3.As can be seen from the table in 17 kinds of amino acid contained by the coptis, aspartate content is most
Height, is secondly threonine and glycine, and methionine content is minimum.Total amino acid average content is 0.550mg × g-1。
3.2 repeated experiment
Same coptis sample is taken to prepare 6 parts of sample solutions by identical experimental method, the μ L of sample introduction 2 are measured, as a result removed
The RSD of cystine is 3.12%, and outside 3.00%, other are respectively less than 3.00%.Trace it to its cause, it may be possible to because Guang ammonia
Acid is in acid hydrolysis, because a small amount of oxygen is present and causes the cystine containing sulfydryl to have a little oxidation difference.
3.3 Precision Experiment
Same coptis sample pre-treatments liquid is taken, continuous sample introduction 6 times determines each amino acid content, and its RSD is 0.88-
1.59%.
3.4 stability experiment
Same coptis sample pre-treatments liquid is taken, respectively 0,8,16,24 hours sample introductions, even if 24 hours its peak areas of difference
Also very stable, its RSD is 0.78-1.26%.
3.5 degrees of accuracy are tested
The preparation of recovery span amino acid mother liquor：Precision weighs the mixed mark of amino acid of 105 DEG C of dryings 3 hours, is placed in 100mL appearances
In measuring bottle, it is dissolved in water and is diluted to scale, shake up and produce.
Reclaim the preparation of coptis sample liquid：6 parts of coptis sample pre-treatments liquid 8mL is measured, 10mL volumetric flasks are respectively placed in
In, every part of accurate above-mentioned recovery span amino acid mother liquor 2mL of addition is finally diluted with water to scale, shaken up, produces recovery ginseng
Sample liquid.
Contents of Amino Acids is carried out by above-mentioned chromatographic condition, and the rate of recovery is calculated according to following calculation formula,
Calculation formula：The rate of recovery=(measured amount-sample size)/addition * 100%
As a result show, each the recovery of amino acid is in 98.8-103.1% scopes.RSD is 0.92%, therefore can draw this
The preferable conclusion of the assay method rate of recovery.
It is emphasized that：It the above is only presently preferred embodiments of the present invention, not make any formal to the present invention
Limitation, every technical spirit according to the present invention is to any simple modification made for any of the above embodiments, and equivalent variations are with modifying,
Belong to the category of technical solution of the present invention, should not be construed as limiting the invention.
Determined amino acid result in the ginseng of table 1.
Determined amino acid result in the bark of eucommia of table 2.
Determined amino acid result in the coptis of table 3.：
Claims (3)
1. a kind of non-derived combines the analysis method of amino acid in quick detection medicinal plant simultaneously, comprise the following steps：
1）The freezing of high-voltage pulse sour water solution combination amino acid and hydrolyzate and melt analysis pre-treatment again；
Take medicinal plant 1.0g to be measured in hydrolysis pipe, add 6mol × L-1Hydrochloric acid solution, deoxygenation sealing, sets electric-field intensity
10kv×cm-1-30 kv×cm-1, solid-liquid ratio 1:5-1:15 and burst length 30-90S carries out high voltage pulse method hydrolysis, to be cooled
Hydrolyzate is placed into subzero 20-60 degree after to room temperature to freeze 2-4 hours, then takes out normal temperature and dissolves, then filtering, takes filtrate
1mL nitrogen evaporators are dried up, and with flowing phased soln, cross 0.22 um membrane filtrations standby；
2）Ultrahigh pressure liquid phase chromatogram-Evaporative light scattering detector method is set up：
Accurately weigh each standard items 0.1mol × L-1Hydrochloric acid is configured to 0.5-10 nmol × L-1Amino standard acid solution, use
Hydrophilic acrylic silicon chromatographic column is separated to it, with the acetonitrile containing 0.7% 9 fluorine butyric acid and 0.5% trifluoroacetic acid ion pair-contain
0.7% 9 fluorine butyric acid and the water of 0.5% trifluoroacetic acid ion pair are that mobile phase carries out gradient elution, gradient elution：Wherein A be from
Son is to acetonitrile phase, and B is ion pair aqueous phase, 0-0.8min, 0%A；0.8-1.5min, 12%A；1.5-4min, 15%A；4-
5min, 61%A；5.1-8min, 61%A；8-8.1min, 0%A；8.1-10min, 0%A；Analysis time 10min；Flow velocity is set
0.3-0.5 ml×min-1, 60-80 DEG C of ELSD drift pipes temperature, UPLC-ELSD detections on carrier gas flux 20-40 PSI；It is each dense
Sample introduction 2uL surveys its peak area to degree respectively, using the logarithm of sample introduction concentration as abscissa, and the logarithm of peak area draws standard for ordinate
Curve；
3）Contents of Amino Acids in medicinal plant：
According to above-mentioned liquid phase chromatogram condition, the amino acid hydrolyzed to medicinal plant in prepare liquid sample carries out separation detection：Sample introduction
2uL, determines peak area, and the content of amino acid in medicinal plant is calculated according to standard curve.
2. a kind of non-derived described in claim 1 combines the analysis method of amino acid in quick detection medicinal plant simultaneously, its
It is characterised by：
Step 1）For；
Take medicinal plant 1.0g to be measured in hydrolysis pipe, add 6mol × L-1Hydrochloric acid solution, deoxygenation good seal sets electric-field intensity
For 20kv × cm-1, solid-liquid ratio 1:10 and the burst length be 90S carry out high voltage pulse method hydrolysis, be cooled to after room temperature will hydrolysis
Liquid is placed subzero 20-60 degree and freezed 2-4 hours, then takes out normal temperature and dissolves, and has then filtered and has taken filtrate 1mL nitrogen evaporators to dry up,
Flow after phased soln, cross 0.22 um membrane filtrations standby.
3. a kind of non-derived described in claim 1 combines the analysis method of amino acid in quick detection medicinal plant simultaneously, its
It is characterised by：
Step 2）For；
Accurately weigh each standard items 0.1mol × L-1Hydrochloric acid is configured to 0.5-10 nmol × L-1Amino standard acid solution, use
Hydrophilic acrylic silicon chromatogram post separation, with the acetonitrile containing 0.7% 9 fluorine butyric acid and 0.5% trifluoroacetic acid ion pair-contain 0.7% 9 fluorine
Butyric acid and the water of 0.5% trifluoroacetic acid ion pair are that mobile phase carries out gradient elution, gradient elution：A is ion pair acetonitrile phase, B
For ion pair aqueous phase：0-0.8min, 0%A；0.8-1.5min, 12%A；1.5-4min, 15%A；4-5min, 61%A；5.1-
8min, 61%A；8-8.1min, 0%A；8.1-10min, 0%A；The min of analysis time 10；0.4 ml of flow velocity × min is set-1,
UPLC-ELSD is detected on ELSD drift pipe temperature 70 Cs, the PSI of carrier gas flux 30；Each concentration distinguishes sample introduction 2uL and surveys its peak area,
Using the logarithm of sample introduction concentration as abscissa, the logarithm of peak area draws standard curve for ordinate.
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