CN105651874B - The special high-accuracy efficient liquid-phase chromatography method that gamma aminobutyric acid and 16 kinds of protein hydrolytic amino acids are detected simultaneously in milk-contained drink - Google Patents
The special high-accuracy efficient liquid-phase chromatography method that gamma aminobutyric acid and 16 kinds of protein hydrolytic amino acids are detected simultaneously in milk-contained drink Download PDFInfo
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Abstract
The invention discloses the special high-accuracy efficient liquid-phase chromatography method that gamma aminobutyric acid in a kind of milk-contained drink and 16 kinds of protein hydrolytic amino acids are detected simultaneously, set sample hydrolysis, derivation process, three operating procedures of upper post detection, then for reagent treatment and reagent dosage and processing parameter used in above-mentioned three operating procedure of property settings of milk-contained drink, so that the technological parameter of above-mentioned three operating procedure mutually tightly coordinates, realize to gamma aminobutyric acid in milk-contained drink and the single step high precision test of 16 kinds of protein hydrolytic amino acids.The present invention can realize the single step detection to the gamma aminobutyric acid in milk-contained drink and 16 kinds of protein hydrolytic amino acids, and with high sensitivity and superior stability.
Description
Technical field
The invention belongs to amino acid detection techniques field, γ-aminobutyric acid and 16 hatching egg plain boiled waters specially in milk-contained drink
Solve the efficient liquid-phase chromatography method that amino acid is detected simultaneously.
Background technology
γ-aminobutyric acid（GABA）It is a kind of naturally occurring nonprotein amino acid, is mammalian central nervous system
The inhibitory transmitter substance of system, is one of most important neurotransmitter in brain tissue.It can combine brain acceptor antianxity simultaneously
It is allowed to activate, is then acted synergistically with other material, prevents the information related to anxiety from arriving at mesencephalic centre, god can be reduced
Through first activity, the situation such as the uneasiness of the people that releive, pressure, tired, worried.
Ministry of Public Health approval GABA is new resource food within 2009, ferments and makes through Lactobacillus hilgardii by raw material of Pidolidone
Into can be applied in beverage and candy, dilated food etc..One of consumer goods that dairy products are generally eaten as people, GABA and ox
The combination of milk, can make people alleviate pressure while needed for supplementing daily nutrition well, reduce uneasiness.
For γ-aminobutyric acid, existing light industry standard QB/T 4587-2013《γ-aminobutyric acid》Normalization
Alpha-aminobutyric acid content detection method is provided in annex；Open sunshine etc.（2003）A meter embryo is determined using automatic derivatization before OPA posts
γ-aminobutyric acid in bud；Tu Yunfei etc.（2012）Using the gamma-amino in pre column Derivatization tealeaves
Butyric acid；Hu Xuelian etc.（2015）γ-aminobutyric acid using high effective liquid chromatography for measuring in blackberry fruit juice and blackberry beer.
For amino acid detection, existing national standards GB/T 5009.124-2003《The measure of amino acid in food》, for determining food
16 kinds of amino acid in product, but use full-automatic amino-acid analyzer.
These methods or the γ-aminobutyric acid in rice plumule, tealeaves, blackberry fruit juice and beer is detected, or
Detection to 16 kinds of amino acid in food, is detected simultaneously to γ-aminobutyric acid in milk-contained drink and 16 kinds of protein hydrolytic amino acids
Fast and accurately analyzing detecting method be also rarely reported.Therefore, γ-aminobutyric acid and 16 hatching egg plain boiled waters in milk-contained drink are carried out
The research work of amino acid Simultaneous Detection is solved, is had great importance to improving product quality, supervision.
The content of the invention
The technical problem to be solved in the present invention is to provide γ-aminobutyric acid in a kind of milk-contained drink and 16 kinds of proteolysis ammonia
The special high-accuracy efficient liquid-phase chromatography method of base acid detection simultaneously, with high sensitivity and superior stability.
In order to solve the above technical problems, the technical solution used in the present invention is as follows.
The special high-accuracy efficient liquid phase that γ-aminobutyric acid and 16 kinds of protein hydrolytic amino acids are detected simultaneously in milk-contained drink
Chromatographic process, sets sample hydrolysis, derivation process, three operating procedures of upper post detection, and then the characteristic for milk-contained drink is set
Reagent treatment and reagent dosage and processing parameter used in fixed above-mentioned three operating procedure so that the technique of above-mentioned three operating procedure
Parameter mutually tightly coordinates, and realizes high-precision to the single step of γ-aminobutyric acid in milk-contained drink and 16 kinds of protein hydrolytic amino acids
Degree detection.
As a preferred technical solution of the present invention, the detection method comprises the following steps：
A, prepare milk-contained drink hydrolyzation sample：Take milk-contained drink to be placed in hydrolysis pipe, add cold immediately after hydrochloric acid and phenol
Jelly processing, is then charged with inert gas and tightens hydrolysis pipe blind nut, then hydrolysis pipe is put into thermostatic drying chamber and is fully hydrolyzed,
Then hydrolyzate is taken out and filtered, taken filtrate to dry and be evaporated, again with the sample introduction of dissolving with hydrochloric acid and refined filtration to high performance liquid chromatography
In bottle；
B, hydrolyzation sample automatic column front derivation processing：At ambient temperature, sample obtained by absorption step A and with and boric acid
Buffer solution, OPA are mixed, then draw the mixing of 9- Fluorenylmethyl chloroformates, prepare loading；
C, high performance liquid chromatography detection γ-aminobutyric acid and 16 kinds of amino acid content：The lightning strip of high performance liquid chromatography
Part is, chromatographic column：General C18 liquid-phase chromatographic columns；Column temperature：40℃；Mobile phase A：Crystallization sodium acetate is added in pure water to mix, and is adjusted
PH adds tetrahydrofuran after adjusting；Mobile phase B：Crystallization sodium acetate is added in pure water to mix, and adjusts addition acetonitrile and methanol after pH to mix；
Condition of gradient elution setting is as follows：
| Sequence number | Time (min) | Mobile phase A % | Mobile phase B % | Flow velocity（mL/min） |
| 1 | 0.00 | 100-90 | 0-10 | 0.8-1.1 |
| 2 | 27.5 | 35-45 | 55-65 | 0.8-1.1 |
| 3 | 31.5 | 0 | 100 | 1.3-1.6 |
| 4 | 32 | 0 | 100 | 1.3-1.6 |
| 5 | 34 | 0 | 100 | 0.8-1.1 |
| 6 | 35.5 | 100-90 | 0-10 | 0.8-1.1 |
Testing conditions setting is as follows：Detector uses diode array or UV-detector；0-25min, setting detection ripple
Long 338nm；25-32min, setting Detection wavelength 262nm；32-35.5min, setting Detection wavelength 338nm.
As a preferred technical solution of the present invention, the detection method comprises the following steps, is related in following step
Parts by weight are calculated in grams, and the parts by volume being related to is calculated in units of milliliter：
A, prepare milk-contained drink hydrolyzation sample：Take the parts by weight of milk-contained drink 1 to be placed in hydrolysis pipe, add concentrated hydrochloric acid 1-3 bodies
Product part adds 6mol/L hydrochloric acid 10-15 parts by volume, adds the phenol 3-4 drops newly distilled, and hydrolysis pipe is put into refrigerant,
3min-5imn is freezed, high pure nitrogen is filled with and tightens blind nut under nitrogen charging gaseity, hydrolysis pipe is put into 109-111 DEG C of perseverance
In warm drying box, hydrolysis takes out cooling after 20-30 hours, opens hydrolysis pipe, and by hydrolyzate, all filtering is transferred in volumetric flask,
Take filtrate 1-2 parts by volume vacuum desiccator in 40 DEG C of -50 DEG C of dryings, be evaporated, the hydrochloric acid with 0.5-2 parts by volume 0.1mol/L is molten
Solution, then using 0.2 μm -0.8 μm of membrane filtration into high performance liquid chromatography sample injection bottle；
B, hydrolyzation sample automatic column front derivation processing：At ambient temperature, sample 0.5-2 volumes obtained by absorption step A
Part, borate buffer 3-7 parts by volume is drawn, OPA 0.5-2 parts by volume is drawn, mixed, then draw 9- fluorenyl methyl chlorine
Formic acid esters 0.5-2 parts by volume, is mixed, and prepares loading；
C, high performance liquid chromatography detection γ-aminobutyric acid and 16 kinds of amino acid content：The lightning strip of high performance liquid chromatography
Part is, chromatographic column：General C18 liquid-phase chromatographic columns；Column temperature：40℃；Mobile phase A：4-8 weight is added in 1000 parts by volume pure water
Part crystallization sodium acetate, is mixed, pH is adjusted to 7.15-7.25, adds 3-5 parts by volume tetrahydrofurans；Mobile phase B：400 parts by volume are pure
6-12 parts by weight Crystalline sodium acetates are added in water, are mixed, pH is adjusted to 7.15-7.25, add 600-1000 parts by volume of acetonitrile and
600-1000 parts by volume methanol, is mixed；Condition of gradient elution setting is as follows：
| Sequence number | Time (min) | Mobile phase A % | Mobile phase B % | Flow velocity（mL/min） |
| 1 | 0.00 | 100-90 | 0-10 | 0.8-1.1 |
| 2 | 27.5 | 35-45 | 55-65 | 0.8-1.1 |
| 3 | 31.5 | 0 | 100 | 1.3-1.6 |
| 4 | 32 | 0 | 100 | 1.3-1.6 |
| 5 | 34 | 0 | 100 | 0.8-1.1 |
| 6 | 35.5 | 100-90 | 0-10 | 0.8-1.1 |
Testing conditions setting is as follows：Detector uses diode array or UV-detector；0-25min, setting detection ripple
Long 338nm；25-32min, setting Detection wavelength 262nm；32-35.5min, setting Detection wavelength 338nm.
As a preferred technical solution of the present invention, the detection method comprises the following steps, is related in following step
Parts by weight are calculated in grams, and the parts by volume being related to is calculated in units of milliliter：
A, prepare milk-contained drink hydrolyzation sample：Take the parts by weight of milk-contained drink 1 to be placed in hydrolysis pipe, add the volume of concentrated hydrochloric acid 2
Part adds the parts by volume of 6mol/L hydrochloric acid 12, adds the phenol 3 newly distilled and drips, and hydrolysis pipe is put into refrigerant, freezed
4imn, is filled with high pure nitrogen and tightens blind nut under nitrogen charging gaseity, hydrolysis pipe is put into 110 DEG C of thermostatic drying chamber, water
Solution takes out cooling after 24 hours, open hydrolysis pipe, and by hydrolyzate, all filtering is transferred in 25mL volumetric flasks, takes the body of filtrate 1.5
Product part, in 45 DEG C of dryings, is evaporated, with 1 parts by volume 0.1mol/L dissolving with hydrochloric acid, then using 0.45 μm with vacuum desiccator
Membrane filtration is into high performance liquid chromatography sample injection bottle；
B, hydrolyzation sample automatic column front derivation processing：At ambient temperature, the parts by volume of sample 1 obtained by absorption step A, inhales
The parts by volume of borate buffer 5 is taken, the parts by volume of OPA 1 is drawn, mixed, then draw the volume of 9- Fluorenylmethyl chloroformates 1
Part, mix, prepare loading；
C, high performance liquid chromatography detection γ-aminobutyric acid and 16 kinds of amino acid content：The lightning strip of high performance liquid chromatography
Part is, chromatographic column：General C18 liquid-phase chromatographic columns；Column temperature：40℃；Mobile phase A：6 parts by weight are added in 1000 parts by volume pure water
Sodium acetate is crystallized, is mixed, pH is adjusted to 7.15-7.25, adds 4 parts by volume tetrahydrofurans；Mobile phase B：In 400 parts by volume pure water
9 parts by weight Crystalline sodium acetates are added, are mixed, pH is adjusted to 7.15-7.25, adds 800 parts by volume of acetonitrile and 800 parts by volume first
Alcohol, is mixed；Condition of gradient elution setting is as follows：
| Sequence number | Time (min) | Mobile phase A % | Mobile phase B % | Flow velocity（mL/min） |
| 1 | 0.00 | 100-90 | 0-10 | 0.8-1.1 |
| 2 | 27.5 | 35-45 | 55-65 | 0.8-1.1 |
| 3 | 31.5 | 0 | 100 | 1.3-1.6 |
| 4 | 32 | 0 | 100 | 1.3-1.6 |
| 5 | 34 | 0 | 100 | 0.8-1.1 |
| 6 | 35.5 | 100-90 | 0-10 | 0.8-1.1 |
Testing conditions setting is as follows：Detector uses diode array or UV-detector；0-25min, setting detection ripple
Long 338nm；25-32min, setting Detection wavelength 262nm；32-35.5min, setting Detection wavelength 338nm.
As a preferred technical solution of the present invention, the C18 liquid-phase chromatographic columns described in step C select X Bridge
C18 chromatographic columns, Hypersil ODS C18 chromatographic columns.
γ-aminobutyric acid and 16 kinds of standard amino acid derivative chromatograms that present invention detection is obtained, peak sequence is successively
For：（1）Asp, asparatate；（2）Glu, glutamic acid；（3）Ser, serine；（4）His, histidine；（5）Gly, glycine；
（6）Thr, threonine；（7）Arg, arginine；（8）Ala, alanine；（9）GABA, γ-aminobutyric acid；（10）Tyr, tyrosine；
（11）Val, valine；（12）Met, methionine；（13）Phe, phenylalanine；（14）Ile, isoleucine；（15）Leu, bright ammonia
Acid；（16）Lys, lysine；（17）Pro, proline.
It is using the beneficial effect produced by above-mentioned technical proposal：The present invention have developed specially for the characteristic of milk-contained drink
Reagent treatment and reagent dosage and processing parameter so that the technological parameter of detecting step mutually tightly coordinates, are realized pair
γ-aminobutyric acid and the single step high sensitivity of 16 kinds of protein hydrolytic amino acids, the detection of high stability in milk-contained drink, to carrying
High dairy products product quality and supervision have important practical significance.
Applicant has carried out campaign for the detection method of the present invention.Referring to accompanying drawing 1,2（Fig. 1 is γ-aminobutyric acid
With the separation spectrogram of 16 kinds of standard amino acids；Fig. 2 is γ-aminobutyric acid and 16 kinds of proteolysis in milk-contained drink in embodiment 1
The separation spectrogram of amino acid）, it is seen that detection method of the invention can be realized to γ-aminobutyric acid in milk-contained drink and 16 hatching eggs
The single step of white hydrolysis amino acid detects that separating spectrum is very clear and reliable simultaneously.
Meanwhile, the campaign of applicant shows that this detection method stability is high.Using its detect milk-contained drink in γ-
Aminobutyric acid and 16 kinds of proteolysis amino acid contents, detection are limited to 1.10pmol/ μ L -3.86pmol/ μ L, RSD and are respectively
3.62%-8.51% and 2.56%-11.63%, preci-sion and accuracy is high.Following table is γ-aminobutyric acid and 16 kinds of amino acid
Test result.
| Sequence number | Object | Test limit/（pmol/µL） | Precision/（g/100g） | The degree of accuracy/（%） |
| 1 | Asparatate | 1.10 | 0.138±0.008 | 88.1-100.2 |
| 2 | Glutamic acid | 1.30 | 0.341±0.017 | 87.4-104.9 |
| 3 | Serine | 2.27 | 0.081±0.004 | 97.4-108.6 |
| 4 | Histidine | 2.97 | 0.039±0.003 | 99.1-110.5 |
| 5 | Glycine | 2.21 | 0.031±0.002 | 86.7-109.1 |
| 6 | Threonine | 2.19 | 0.073±0.003 | 93.0-99.4 |
| 7 | Arginine | 2.18 | 0.053±0.003 | 88.8-100.1 |
| 8 | Alanine | 2.22 | 0.062±0.004 | 96.0-112.9 |
| 9 | γ-aminobutyric acid | 2.91 | 0.065±0.005 | 93.5-105.2 |
| 10 | Tyrosine | 2.47 | 0.062±0.003 | 87.4-98.7 |
| 11 | Valine | 2.61 | 0.079±0.005 | 103.6-109.8 |
| 12 | Methionine | 2.56 | 0.048±0.004 | 86.7-97.9 |
| 13 | Phenylalanine | 2.76 | 0.056±0.004 | 90.0-104.5 |
| 14 | Isoleucine | 2.74 | 0.95±0.003 | 101.5-111.9 |
| 15 | Leucine | 2.76 | 0.159±0.006 | 90.7-105.8 |
| 16 | Lysine | 1.53 | 0.126±0.005 | 95.7-106.9 |
| 17 | Proline | 3.86 | 0.136±0.008 | 101.6-108.1 |
Brief description of the drawings
Fig. 1 is the separation spectrogram of γ-aminobutyric acid and 16 kinds of standard amino acids.
Fig. 2 is the separation spectrogram of γ-aminobutyric acid and 16 kinds of protein hydrolytic amino acids in milk-contained drink（Embodiment 1）.
Embodiment
The present invention is described in detail in following examples.Various raw materials used in the present invention and items of equipment are conventional city
Product is sold, can be directly obtained by market purchase.
Embodiment 1
A, prepare milk-contained drink hydrolyzation sample
Take milk-contained drink 1.0g to be placed in hydrolysis pipe, add concentrated hydrochloric acid 2mL, add the phenol 3 newly distilled and drip, will hydrolyze
Pipe is put into refrigerant, is freezed 3imn, is filled with high pure nitrogen and tightens blind nut under nitrogen charging gaseity, and hydrolysis pipe is put into 110
DEG C thermostatic drying chamber in, hydrolysis 24 hours after take out cooling.Hydrolysis pipe is opened, hydrolyzate whole filtering is transferred into 25mL holds
In measuring bottle, take filtrate 1mL vacuum desiccators in 50 DEG C of dryings, be evaporated, with 1mL 0.1mol/L dissolving with hydrochloric acid, 0.45 μm
Membrane filtration comes in high performance liquid chromatography sample injection bottle；
The automatic column front derivation processing of B, hydrolysate, obtains derivative products
5 parts of borate buffer is drawn, 1 part of OPA is drawn, 1 part of sample is absorbed, mixed, then draw 9- fluorenyl methyls
1 part of chloro-formate, is mixed, and prepares loading；
C, high performance liquid chromatography are while γ-aminobutyric acid and 16 kinds of amino acid
The separation condition of high performance liquid chromatography is, chromatographic column：Hypersil ODS C18 liquid-phase chromatographic columns；Mobile phase A：
In 1000mL pure water, 8g crystallization sodium acetates are added, are mixed, pH is adjusted to 7.20；Add 5mL tetrahydrofurans；Mobile phase B：400mL is pure
In water, 12g crystallization sodium acetates are added, are mixed, pH is adjusted to 7.20；800mL acetonitriles and 800mL methanol are added, is mixed；Gradient elution
Condition see the table below：
| Sequence number | Time (min) | Mobile phase A % | Mobile phase B % | Flow velocity（mL/min） |
| 1 | 0.00 | 92 | 8 | 1.0 |
| 2 | 27.5 | 40 | 60 | 1.0 |
| 3 | 31.5 | 0 | 100 | 1.5 |
| 4 | 32 | 0 | 100 | 1.5 |
| 5 | 34 | 0 | 100 | 1.0 |
| 6 | 35.5 | 92 | 8 | 1.0 |
It is γ-aminobutyric acid and 16 kinds of protein hydrolytic amino acids in milk-contained drink under the conditions of embodiment 1 referring to accompanying drawing 2
Separate spectrogram；The peak sequence of each detectable substance is followed successively by：（1）Asp, asparatate；（2）Glu, glutamic acid；（3）Ser, silk ammonia
Acid；（4）His, histidine；（5）Gly, glycine；（6）Thr, threonine；（7）Arg, arginine；（8）Ala, alanine；（9）
GABA, γ-aminobutyric acid；（10）Tyr, tyrosine；（11）Val, valine；（12）Met, methionine；（13）Phe, phenylpropyl alcohol ammonia
Acid；（14）Ile, isoleucine；（15）Leu, leucine；（16）Lys, lysine；（17）Pro, proline.
Embodiment 2
A, prepare milk-contained drink hydrolyzation sample
Take milk-contained drink 1.0g to be placed in hydrolysis pipe, add concentrated hydrochloric acid 3mL, add the phenol 4 newly distilled and drip, will hydrolyze
Pipe is put into refrigerant, is freezed 5imn, is filled with high pure nitrogen and tightens blind nut under nitrogen charging gaseity, and hydrolysis pipe is put into 109
DEG C thermostatic drying chamber in, hydrolysis 24 hours after take out cooling.Hydrolysis pipe is opened, hydrolyzate whole filtering is transferred into 25mL holds
In measuring bottle, take filtrate 2mL vacuum desiccators in 40 DEG C of dryings, be evaporated, with 1mL 0.1mol/L dissolving with hydrochloric acid, 0.45 μm
Membrane filtration comes in high performance liquid chromatography sample injection bottle；
The automatic column front derivation processing of B, hydrolysate, obtains derivative products
5 parts of borate buffer is drawn, 1 part of OPA is drawn, 1 part of sample is absorbed, mixed, then draw 9- fluorenyl methyls
1 part of chloro-formate, is mixed, and prepares loading；
C, high performance liquid chromatography are while γ-aminobutyric acid and 16 kinds of amino acid
The separation condition of high performance liquid chromatography is, chromatographic column：X Bridge C18 liquid-phase chromatographic columns；Mobile phase A：
In 1000mL pure water, 6g crystallization sodium acetates are added, are mixed, pH is adjusted to 7.18；Add 4mL tetrahydrofurans；Mobile phase B：400mL is pure
In water, 10g crystallization sodium acetates are added, are mixed, pH is adjusted to 7.18；800mL acetonitriles and 800mL methanol are added, is mixed；Gradient elution
Condition see the table below：
| Sequence number | Time (min) | Mobile phase A % | Mobile phase B % | Flow velocity（mL/min） |
| 1 | 0.00 | 90 | 10 | 0.8 |
| 2 | 27.5 | 45 | 55 | 0.8 |
| 3 | 31.5 | 0 | 100 | 1.3 |
| 4 | 32 | 0 | 100 | 1.3 |
| 5 | 34 | 0 | 100 | 0.8 |
| 6 | 35.5 | 90 | 10 | 0.8 |
Detection obtains γ-aminobutyric acid and 16 kinds of standard amino acid derivative chromatograms, the peak sequence of each detectable substance according to
It is secondary to be：（1）Asp, asparatate；（2）Glu, glutamic acid；（3）Ser, serine；（4）His, histidine；（5）Gly, sweet ammonia
Acid；（6）Thr, threonine；（7）Arg, arginine；（8）Ala, alanine；（9）GABA, γ-aminobutyric acid；（10）Tyr, junket ammonia
Acid；（11）Val, valine；（12）Met, methionine；（13）Phe, phenylalanine；（14）Ile, isoleucine；（15）Leu, it is bright
Propylhomoserin；（16）Lys, lysine；（17）Pro, proline.
Embodiment 3
A, prepare milk-contained drink hydrolyzation sample
Take milk-contained drink 1.0g to be placed in hydrolysis pipe, add 6mol/L hydrochloric acid 10mL, add the phenol 3 newly distilled and drip,
Hydrolysis pipe is put into refrigerant, 3imn is freezed, is filled with high pure nitrogen and tightens blind nut under nitrogen charging gaseity, hydrolysis pipe is put
In the thermostatic drying chamber for entering 111 DEG C, hydrolysis takes out cooling after 24 hours.Hydrolysis pipe is opened, all filtering is transferred to by hydrolyzate
In 25mL volumetric flasks, take filtrate 2mL vacuum desiccators in 45 DEG C of dryings, be evaporated, with 1mL 0.1mol/L dissolving with hydrochloric acid,
0.45 μm of membrane filtration comes in high performance liquid chromatography sample injection bottle；
The automatic column front derivation processing of B, hydrolysate, obtains derivative products
5 parts of borate buffer is drawn, 1 part of OPA is drawn, 1 part of sample is absorbed, mixed, then draw 9- fluorenyl methyls
1 part of chloro-formate, is mixed, and prepares loading；
C, high performance liquid chromatography are while γ-aminobutyric acid and 16 kinds of amino acid
The separation condition of high performance liquid chromatography is, chromatographic column：Hypersil ODS C18 liquid-phase chromatographic columns；Mobile phase A：
In 1000mL pure water, 5g crystallization sodium acetates are added, are mixed, pH is adjusted to 7.25；Add 3mL tetrahydrofurans；Mobile phase B：400mL is pure
In water, 8g crystallization sodium acetates are added, are mixed, pH is adjusted to 7.25；800mL acetonitriles and 800mL methanol are added by volume, are mixed；
Condition of gradient elution see the table below：
| Sequence number | Time (min) | Mobile phase A % | Mobile phase B % | Flow velocity（mL/min） |
| 1 | 0.00 | 100 | 0 | 1.1 |
| 2 | 27.5 | 35 | 65 | 1.1 |
| 3 | 31.5 | 0 | 100 | 1.6 |
| 4 | 32 | 0 | 100 | 1.6 |
| 5 | 34 | 0 | 100 | 1.1 |
| 6 | 35.5 | 100 | 0 | 1.1 |
Detection obtains γ-aminobutyric acid and 16 kinds of standard amino acid derivative chromatograms, the peak sequence of each detectable substance according to
It is secondary to be：（1）Asp, asparatate；（2）Glu, glutamic acid；（3）Ser, serine；（4）His, histidine；（5）Gly, sweet ammonia
Acid；（6）Thr, threonine；（7）Arg, arginine；（8）Ala, alanine；（9）GABA, γ-aminobutyric acid；（10）Tyr, junket ammonia
Acid；（11）Val, valine；（12）Met, methionine；（13）Phe, phenylalanine；（14）Ile, isoleucine；（15）Leu, it is bright
Propylhomoserin；（16）Lys, lysine；（17）Pro, proline.
Embodiment 4
A, prepare milk-contained drink hydrolyzation sample
Take milk-contained drink 1.0g to be placed in hydrolysis pipe, add 6mol/L hydrochloric acid 15mL, add the phenol 4 newly distilled and drip,
Hydrolysis pipe is put into refrigerant, 5imn is freezed, is filled with high pure nitrogen and tightens blind nut under nitrogen charging gaseity, hydrolysis pipe is put
In the thermostatic drying chamber for entering 110 DEG C, hydrolysis takes out cooling after 24 hours.Hydrolysis pipe is opened, all filtering is transferred to by hydrolyzate
In 25mL volumetric flasks, take filtrate 1mL vacuum desiccators in 40 DEG C of dryings, be evaporated, with 1mL 0.1mol/L dissolving with hydrochloric acid,
0.45 μm of membrane filtration comes in high performance liquid chromatography sample injection bottle；
The automatic column front derivation processing of B, hydrolysate, obtains derivative products
5 parts of borate buffer is drawn, 1 part of OPA is drawn, 1 part of sample is absorbed, mixed, then draw 9- fluorenyl methyls
1 part of chloro-formate, is mixed, and prepares loading；
C, high performance liquid chromatography are while γ-aminobutyric acid and 16 kinds of amino acid
The separation condition of high performance liquid chromatography is, chromatographic column：X Bridge C18 liquid-phase chromatographic columns；Mobile phase A：
In 1000mL pure water, 4g parts of crystallization sodium acetates are added, are mixed, pH is adjusted to 7.15；Add 3.5mL tetrahydrofurans；Mobile phase B：
In 400mL pure water, 6g crystallization sodium acetates are added, are mixed, pH is adjusted to 7.15；By 800mL acetonitriles and 800mL methanol is added, mix；
Condition of gradient elution see the table below：
| Sequence number | Time (min) | Mobile phase A % | Mobile phase B % | Flow velocity（mL/min） |
| 1 | 0.00 | 95 | 5 | 0.9 |
| 2 | 27.5 | 42 | 58 | 0.9 |
| 3 | 31.5 | 0 | 100 | 1.4 |
| 4 | 32 | 0 | 100 | 1.4 |
| 5 | 34 | 0 | 100 | 0.9 |
| 6 | 35.5 | 100-90 | 0-10 | 0.9 |
Detection obtains γ-aminobutyric acid and 16 kinds of standard amino acid derivative chromatograms, the peak sequence of each detectable substance according to
It is secondary to be：（1）Asp, asparatate；（2）Glu, glutamic acid；（3）Ser, serine；（4）His, histidine；（5）Gly, sweet ammonia
Acid；（6）Thr, threonine；（7）Arg, arginine；（8）Ala, alanine；（9）GABA, γ-aminobutyric acid；（10）Tyr, junket ammonia
Acid；（11）Val, valine；（12）Met, methionine；（13）Phe, phenylalanine；（14）Ile, isoleucine；（15）Leu, it is bright
Propylhomoserin；（16）Lys, lysine；（17）Pro, proline.
Foregoing description only proposes as enforceable technical scheme of the invention, not as to its technical scheme single in itself
Restrictive condition.
Claims (3)
1. the efficient liquid-phase chromatography method that γ-aminobutyric acid and 16 kinds of protein hydrolytic amino acids are detected simultaneously in milk-contained drink, its
It is characterised by：Sample hydrolysis, derivation process, three operating procedures of upper post detection are set, then the characteristic for milk-contained drink is set
Determine reagent treatment and reagent dosage and processing parameter used in above three operating procedure so that above three operating procedure
Technological parameter mutually tightly coordinates, and realizes to γ-aminobutyric acid in milk-contained drink and the single step of 16 kinds of protein hydrolytic amino acids
High precision test；The detection method comprises the following steps that the parts by weight being related in following step are calculated in grams, are related to
Parts by volume calculated in units of milliliter：
A, prepare milk-contained drink hydrolyzation sample：Take the parts by weight of milk-contained drink 1 to be placed in hydrolysis pipe, add concentrated hydrochloric acid 1-3 parts by volume
Or 6mol/L hydrochloric acid 10-15 parts by volume is added, the phenol 3-4 drops newly distilled are added, hydrolysis pipe is put into refrigerant, freezed
3min-5min, is filled with high pure nitrogen and tightens blind nut under nitrogen charging gaseity, and the constant temperature that hydrolysis pipe is put into 109-111 DEG C is done
In dry case, hydrolysis takes out cooling after 20-30 hours, opens hydrolysis pipe, and by hydrolyzate, all filtering is transferred in volumetric flask, takes filter
Liquid 1-2 parts by volume vacuum desiccator is evaporated in 40 DEG C of -50 DEG C of dryings, with 0.5-2 parts by volume 0.1mol/L dissolving with hydrochloric acid,
Then using 0.2 μm -0.8 μm of membrane filtration into high performance liquid chromatography sample injection bottle；
B, hydrolyzation sample automatic column front derivation processing：At ambient temperature, sample 0.5-2 parts by volume obtained by absorption step A, inhales
Borate buffer 3-7 parts by volume is taken, OPA 0.5-2 parts by volume is drawn, mixed, then draw 9- Fluorenylmethyl chloroformates
0.5-2 parts by volume, is mixed, and prepares loading；
C, high performance liquid chromatography detection γ-aminobutyric acid and 16 kinds of amino acid content：The separation condition of high performance liquid chromatography is,
Chromatographic column：General C18 liquid-phase chromatographic columns；Column temperature：40℃；Mobile phase A：4-8 parts by weight knots are added in 1000 parts by volume pure water
Brilliant sodium acetate, is mixed, and pH is adjusted to 7.15-7.25, adds 3-5 parts by volume tetrahydrofurans；Mobile phase B：In 400 parts by volume pure water
6-12 parts by weight Crystalline sodium acetates are added, are mixed, pH is adjusted to 7.15-7.25, adds 600-1000 parts by volume of acetonitrile and 600-
1000 parts by volume methanol, are mixed；Condition of gradient elution setting is as follows：
Testing conditions setting is as follows：Detector uses diode array or UV-detector；0-25min, sets Detection wavelength
338nm；25-32min, setting Detection wavelength 262nm；32-35.5min, setting Detection wavelength 338nm.
2. γ-aminobutyric acid and 16 kinds of protein hydrolytic amino acids are detected simultaneously in milk-contained drink according to claim 1
Efficient liquid-phase chromatography method, it is characterised in that：The detection method comprises the following steps, the parts by weight being related in following step with
Gram calculated for unit, the parts by volume being related to calculates in units of milliliter：
A, prepare milk-contained drink hydrolyzation sample：Take the parts by weight of milk-contained drink 1 be placed in hydrolysis pipe in, add the parts by volume of concentrated hydrochloric acid 2 or
The parts by volume of 6mol/L hydrochloric acid 12 is added, the phenol 3 newly distilled is added and drips, hydrolysis pipe is put into refrigerant, 4min is freezed, fills
Enter high pure nitrogen and tighten blind nut under nitrogen charging gaseity, hydrolysis pipe is put into 110 DEG C of thermostatic drying chamber, hydrolyzed 24 hours
Cooling is taken out afterwards, opens and hydrolyzes pipe, all filtering is transferred in 25mL volumetric flasks by hydrolyzate, takes the parts by volume of filtrate 1.5 with very
Empty drier is evaporated in 45 DEG C of dryings, with 1 parts by volume 0.1mol/L dissolving with hydrochloric acid, then using 0.45 μm of membrane filtration
Into high performance liquid chromatography sample injection bottle；
B, hydrolyzation sample automatic column front derivation processing：At ambient temperature, the parts by volume of sample 1 obtained by absorption step A, draws boron
The parts by volume of acid buffer 5, draws the parts by volume of OPA 1, mixes, then draws the parts by volume of 9- Fluorenylmethyl chloroformates 1, mixes
It is even, prepare loading；
C, high performance liquid chromatography detection γ-aminobutyric acid and 16 kinds of amino acid content：The separation condition of high performance liquid chromatography is,
Chromatographic column：General C18 liquid-phase chromatographic columns；Column temperature：40℃；Mobile phase A：6 parts by weight Crystalline are added in 1000 parts by volume pure water
Sodium acetate, is mixed, and pH is adjusted to 7.15-7.25, adds 4 parts by volume tetrahydrofurans；Mobile phase B：Added in 400 parts by volume pure water
9 parts by weight Crystalline sodium acetates, are mixed, and pH is adjusted to 7.15-7.25, adds 800 parts by volume of acetonitrile and 800 parts by volume methanol, are mixed
It is even；Condition of gradient elution setting is as follows：
Testing conditions setting is as follows：Detector uses diode array or UV-detector；0-25min, sets Detection wavelength
338nm；25-32min, setting Detection wavelength 262nm；32-35.5min, setting Detection wavelength 338nm.
3. γ-aminobutyric acid and 16 kinds of protein hydrolytic amino acids are detected simultaneously in milk-contained drink according to claim 1 or 2
Efficient liquid-phase chromatography method, it is characterised in that：C18 liquid-phase chromatographic columns described in step C select X Bridge C18 chromatograms
Post, Hypersil ODS C18 chromatographic columns.
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| CN107843673A (en) * | 2017-12-11 | 2018-03-27 | 许昌学院 | A kind of method that gamma aminobutyric acid in white wine is quickly determined using LC MS |
| CN107894478A (en) * | 2017-12-18 | 2018-04-10 | 光明乳业股份有限公司 | A kind of method of gamma aminobutyric acid in semi-solid/solid dairy products using liquid chromatographic detection |
| CN108828106A (en) * | 2018-08-30 | 2018-11-16 | 上海市农业科学院 | A method of amino acid content in detection straw mushroom |
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| CN109180512B (en) * | 2018-10-22 | 2021-03-26 | 南昌大学 | Method for removing glutamic acid from gamma-aminobutyric acid fermentation liquor |
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