CN107389848A - The extraction of D types amino acid and assay method in a kind of poisons in freshwater - Google Patents

The extraction of D types amino acid and assay method in a kind of poisons in freshwater Download PDF

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CN107389848A
CN107389848A CN201710614278.1A CN201710614278A CN107389848A CN 107389848 A CN107389848 A CN 107389848A CN 201710614278 A CN201710614278 A CN 201710614278A CN 107389848 A CN107389848 A CN 107389848A
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amino acid
opa
extraction
solution
poisons
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孙福红
吴丰昌
郭飞
朱元荣
穆云松
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Chinese Research Academy of Environmental Sciences
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Chinese Research Academy of Environmental Sciences
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/89—Inverse chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/14—Preparation by elimination of some components
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/067—Preparation by reaction, e.g. derivatising the sample
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/14—Preparation by elimination of some components
    • G01N2030/146—Preparation by elimination of some components using membranes

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Abstract

The present invention relates to a kind of extraction of amino acid and assay method, specifically discloses the extraction of D types amino acid and assay method in a kind of poisons in freshwater.This method comprises the following steps：S1. sample；S2. handled using the acid-hydrolyzed method of microwave radiation technology gas phase；S3. N isobutyryl D cysteines and OPA are dissolved into methanol solution, dilute to obtain OPA derivating agent using cushioning liquid；S4. testing sample is taken, sequentially adds OPA derivating agent and cushioning liquid, vortice mixes, and places carry out derivatization reaction at room temperature；S5. it is measured using high performance liquid chromatography fluorescence detector.The present invention uses the acid-hydrolyzed pre-treating method of microwave radiation technology gas phase, realizes to the extraction of D types amino acid and measure in poisons in freshwater, has the advantages that easy to operate, the rate of recovery is high, method precision is high.

Description

The extraction of D types amino acid and assay method in a kind of poisons in freshwater
Technical field
The present invention relates to a kind of extraction of amino acid and assay method, and in particular to D types amino acid in a kind of poisons in freshwater Extraction and assay method.
Background technology
Amino acid is a kind of important organic compounds containing amino and carboxyl, and it is the weight of water body and Organic Matter In Sediments Part is wanted, is played in the environmental geochemistry cyclic process of aquatic ecosystem nutriment (carbon, nitrogen, sulphur, phosphorus etc.) Important function.Amino acid is the basic composition unit of protein, and the a-amino acid of constitutive protein matter shares kind more than 20 in nature, In addition to glycine, the alpha-carbon atom in all a-amino acids is chiral carbon, therefore amino acid has D types and two kinds of space structures of L-type Type.Protein in organism is nearly all made up of L-type amino acid, and D type amino acid contents are few.D types amino acid comes Its is special for source electrode, exists only in the cell membrane of some bacteriums and plant.Bacterium is growing and D type amino is produced in metabolic process Sour (including D types aspartic acid, D types alanine, D types serine and D types glutamic acid), D type amino acid is not susceptible to degrade, can Preserve and accumulate for a long time in the environment, available for identification Source Organic Matter and Transport And Transformation process.
In recent decades, China rivers and lakes body eutrophication problem is on the rise, organic matter in Freshwater ecosystems Content is high.Poisons in freshwater organic matter is significantly different in physicochemical characteristics and structure composition, source and Transport And Transformation process etc. In seawater.Source Organic Matter is stablized single relatively in briny environment, mainly biology vital movement produce, the content of organic matter compared with It is low.And poisons in freshwater Source Organic Matter is extremely complex, have it is endogenous with two kinds of external source, endogenous processes mostly come from water plant and Zoobenthos decomposition, microbial life activity, bacterium catabolism, deposit release etc., outer source procedure then mostlys come from stream The organic substance of input, companion in domain production and living process (such as discharge of industrial wastewater discharge, mine wastewater, the use of agriculture chemical) With the input of the nutriments such as nitrogen phosphorus.Water body occur eutrophication after, biological activity is active, the content of organic matter acutely increase and Aggregation, the change of increase and water body physical and chemical condition plus nitrogen and phosphorus load, change following for organic matter in aquatic ecosystem Ring gauge is restrained, and causes composition and structure, source, degraded, sedimentation rate and environmental effect of organic matter etc. to occur significantly Change.
The separation and Extraction of D types amino acid and determination techniques can be organic matter in Freshwater ecosystems in freshwater lake water body Complicated source provides technical support with process identification, helps to illustrate body eutrophication process and the origin cause of formation, is controlled for eutrophication Reason provides science and technology support.At present for the extraction of D types amino acid in poisons in freshwater and assay method there is not yet relevant report.
The content of the invention
It is an object of the invention to provide the extraction of D type amino acid and assay method in a kind of poisons in freshwater.This method is realized To D types amino acid (including D types aspartic acid, D types alanine, D types serine and D types glutamic acid) separation and Extraction in fresh water And measure, have the advantages that finding speed is fast, the rate of recovery is high, method precision is high.
The technical scheme is that：The extraction of D types amino acid and assay method in a kind of poisons in freshwater, including following step Suddenly：
S1. sample
Water body example crosses 0.45 μm of filter membrane and goes the removal of impurity, and under the conditions of -4 DEG C, dark is sealed；
S2. water sample pre-processes
Carried out using the acid-hydrolyzed method of microwave radiation technology gas phase：Water sample to be measured is taken, adds ascorbic acid solution to prevent from treating Survey amino acid in solution to aoxidize, add hydrochloric acid solution, be put into microwave dissolver after mixing, carry out clearing up to obtain hydrolyzate；
After the hydrolyzate is cooled into room temperature, neutrality is neutralized to alkali lye, obtains testing sample, sealing is to be measured；
S3. prepared by derivating agent
N- isobutyryls-D-Cys and OPA (OPA) are dissolved into methanol solution, it is dilute using cushioning liquid Release so that the concentration of N- isobutyryls-D-Cys is 5-6g/L and the concentration of OPA is 2-2.5g/L, resulting solution For OPA derivating agent (OPA derivating agents), 4 DEG C are sealed；
S4. derivatization reaction
Testing sample is taken, sequentially adds OPA derivating agent and cushioning liquid, vortice is mixed, placed at room temperature, Ensure that with OPA derivatization reaction fully occurs for amino acid；
S5. Instrument measuring
It is measured using high performance liquid chromatography-fluorescence device；Using anti-phase C18 performance liquid chromatographic columns to D types and L Type amino acid is separated；Wherein, chromatographic column is：LiChrospher 100RP-18 chromatographic columns；The composition of mobile phase is：Flowing Phase A uses 35-45mmol L-1Potassium dihydrogen phosphate buffer solution, pH=12；Mobile phase B uses methanol：Acetonitrile=13:1 (volume Than)；Using gradient elution.
Further, what the water sample pre-processed concretely comprises the following steps：1ml water samples to be measured are taken, add 100-120 μ l 12mmol L-1Ascorbic acid solution adds 0.5-0.6ml 6mol L to prevent that amino acid aoxidizes in solution to be measured-1Salt Acid solution, it is transferred to after mixing in counteracting tank, counteracting tank is put into micro-wave digestion reaction vessel, to micro-wave digestion reaction vessel Add 25ml 6mol L-1Seal, vacuumize after hydrochloric acid solution, be filled with nitrogen, cleared up.
Further, the microwave dissolver power output is 600W, and automatic heating program is：It is warming up in 2-4min 150 DEG C, 150 DEG C of holding 30-35min, after sample is cooled to 80 DEG C, EP (end of program).
Further, the microwave dissolver power output is 600W, and automatic heating program is：It is warming up in 3min 150 DEG C, 150 DEG C of holding 32.5min, after sample is cooled to 80 DEG C, EP (end of program).
Further, the alkali lye is the mixing of one or both of NaOH solution and KOH solution, more preferably NaOH solution.
Further, the cushioning liquid of the step S3 and step S4 are that pH value is 9.5, and concentration is 0.5mol L-1Boric acid Cushioning liquid.
Further, the detailed process of the derivating agent preparation is：By 0.05-0.06g N- isobutyryls-D-Cys It is dissolved into 0.02-0.03g OPAs in 1ml methanol solutions, the use of pH value is 9.5, concentration is 0.5mol L-1Boric acid delays Rush solution and be diluted to 10ml, resulting solution is OPA derivating agent.
D type amino acid contents are determined as column front derivation agent using OPA.In the N- isobutyryls-D- containing sulfydryl In the presence of cysteine, derivatization reaction can occur rapidly with amino acid for OPA derivating agent, and derivative products are through high phase High sensitivity fluoroscopic examination can be carried out after liquid chromatogram separation, and this method has that easy to operate, reaction rate is fast, reagent is not done The advantages that disturbing measure.
Further, the derivatization reaction is：Testing sample is taken, OPA derivating agent is sequentially added and pH value is 9.5, concentration is 0.5mol L-1Borate buffer solution, vortice mix, and place 20-30min at room temperature, ensure amino acid and neighbour Derivatization reaction fully occurs for phthalaldehyde；Wherein, testing sample, OPA derivating agent and pH value are 9.5, and concentration is 0.5mol L-1The volume ratio of borate buffer solution is：1：(1-1.2)：(1-1.2).
Further, the derivatization reaction concretely comprises the following steps：Accurate 100 μ l testing samples of drawing are placed in sample bottle, Sequentially add 100 μ l OPAs derivating agents, 100 μ l pH value be 9.5, concentration is 0.5mol L-1Borate buffer solution, whirlpool Spigot mixes, and places 20-30min at room temperature, ensures that with OPA derivatization reaction fully occurs for amino acid.After derivative, adopt It is measured with high performance liquid chromatography-fluorescence device.
Further, the separation detection condition of the high performance liquid chromatography-fluorescence device is：Mobile phase A uses 40mmol L-1Potassium dihydrogen phosphate buffer solution, pH=12, cross 0.22 μm of filter membrane；Mobile phase B uses methanol：Acetonitrile=13:1 (body Product ratio), cross 0.22 μm of filter membrane.
Further, the gradient elution program of the separation detection of the high performance liquid chromatography-fluorescence device is as follows：
Flow velocity is 0.8ml min-1, 30 DEG C of column temperature, automatic sampler is equipped with, sample size is 10-30 μ l, and detector is fluorescence Detector, excitation wavelength 350nm, launch wavelength 420nm.
Further, the extraction of D type amino acid and assay method also include method precision and can in the poisons in freshwater Investigated by property, i.e. step S6. method precisions and recovery of standard addition：Add four kinds of D types of 3 concentration levels respectively in the sample Amino acid standard liquid, each 3 repetitions of concentration level, step S1-S5 is carried out, to investigate method precision and mark-on reclaims Rate.
The present invention is hydrolyzed using vapor phase acid, and water body example only dilutes 10 times after resolution, fully ensures that amino acid can Detected by instrument.Further, since the content of D type amino acid and the complexity of interference component in fresh water, acid amount increasing can make D type amino acid in water body is fully hydrolyzed, and hydrolysis is more thorough, and measurement result is accurate；Meanwhile derivatization reaction by water body example with spreading out The proportioning of raw agent and derivative agent concentration have a great influence, and the amount and concentration of the invention by controlling derivating agent, ensure that D type amino acid Derivatization reaction fully occurs with derivating agent, is further advantageous to the Accurate Determining of result.
Elution time length during Instrument measuring of the present invention, elution program is fine, the 4 kinds of D type amino acid isolated through instrument Peak type is symmetrical, tailing peak and bifurcated peak does not occur, and is not disturbed mutually between each material peak, and separating effect is preferable.
The present invention uses the acid-hydrolyzed pre-treating method of microwave radiation technology gas phase, realizes to D types amino acid in poisons in freshwater Extraction and measure, have the advantages that easy to operate, the rate of recovery is high, method precision is high.The acid-hydrolyzed side of microwave radiation technology gas phase Method substantially reduces analysis time, improves detection efficiency.
Embodiment
Embodiment 1
The extraction of D types amino acid and assay method, comprise the following steps in a kind of poisons in freshwater：
S1. sample
Water body example crosses 0.45 μm of filter membrane and goes the removal of impurity, and under the conditions of -4 DEG C, dark is sealed；
S2. water sample pre-processes
Carried out using the acid-hydrolyzed method of microwave radiation technology gas phase：Water sample to be measured is taken, adds ascorbic acid solution to prevent from treating Survey amino acid in solution to aoxidize, add hydrochloric acid solution, be put into microwave dissolver after mixing, carry out clearing up to obtain hydrolyzate；
After the hydrolyzate is cooled into room temperature, neutrality is neutralized to alkali lye, obtains testing sample, sealing is to be measured；
S3. prepared by derivating agent
N- isobutyryls-D-Cys and OPA are dissolved into methanol solution, is diluted, made using cushioning liquid The concentration for obtaining N- isobutyryls-D-Cys is 5-6g/L and the concentration of OPA is 2-2.5g/L, and resulting solution is neighbour Phthalaldehyde derivating agent, 4 DEG C are sealed；
S4. derivatization reaction
Testing sample is taken, sequentially adds OPA derivating agent and cushioning liquid, vortice is mixed, placed at room temperature, Ensure that with OPA derivatization reaction fully occurs for amino acid；
S5. Instrument measuring
It is measured using high performance liquid chromatography-fluorescence device；Using anti-phase C18 performance liquid chromatographic columns to D types and L Type amino acid is separated；Wherein, chromatographic column is：LiChrospher 100RP-18 chromatographic columns；The composition of mobile phase is：Flowing Phase A uses 35-45mmol L-1Potassium dihydrogen phosphate buffer solution, pH=12；Mobile phase B uses methanol：Acetonitrile=13:1 (volume Than)；Using gradient elution.
Embodiment 2
The present embodiment describes D type amino in poisons in freshwater in detail by taking China typical case eutrophic lake-Taihu Lake water body as an example The extraction of acid and assay method, comprise the following steps：
S1. sample
Take Taihu Lake middle of a lake area water body example to cross 0.45 μm of filter membrane and go the removal of impurity and algae, under the conditions of -4 DEG C, dark sealing is protected Deposit and take back laboratory.
S2. water sample pre-processes
Carried out using the acid-hydrolyzed method of microwave radiation technology gas phase：1ml water samples to be measured are taken, add 100 μ l12mmol L-1It is anti-bad Hematic acid solution and 0.5ml 6mol L-1Hydrochloric acid solution, it is transferred to after mixing in counteracting tank, counteracting tank is put into micro-wave digestion reaction In container, 25ml 6mol L are added to micro-wave digestion reaction vessel-1Seal, vacuumize after hydrochloric acid solution, be filled with nitrogen, carry out Clear up to obtain hydrolyzate；Wherein, microwave dissolver power output is 600W, and automatic heating program is：150 DEG C are warming up in 3min, 150 DEG C of holding 32.5min, after sample is cooled to 80 DEG C, EP (end of program).
After hydrolyzate is cooled into room temperature, 10ml is settled to after being neutralized to neutrality with NaOH solution, obtains testing sample, is sealed It is to be measured；
S3. prepared by derivating agent
0.05g N- isobutyryls-D-Cys and 0.02g OPAs are dissolved into 1ml methanol solutions, used PH value is 9.5, and concentration is 0.5mol L-1Borate buffer solution is diluted to 10ml, and resulting solution is OPA derivating agent, and 4 DEG C it is sealed.
S4. derivatization reaction
Accurate 100 μ l testing samples of drawing are placed in sample bottle, sequentially add 100 μ l OPAs derivating agents, 100 μ l PH value is 9.5, and concentration is 0.5mol L-1Borate buffer solution, vortice mix, and place 20-30min at room temperature.
S5. Instrument measuring
Prepare D types amino acid (D types aspartic acid, D types alanine, D types serine and D types glutamic acid) Standard Stock solutions 1mmol L-1, it is 0.001-0.1mmol L to be diluted to series concentration-1Machine does standard curve on standard working solution.
It is measured using high performance liquid chromatography-fluorescence device；Using anti-phase C18 performance liquid chromatographic columns to D types and L Type amino acid is separated, wherein, chromatographic column is：LiChrospher 100RP-18 chromatographic columns.
Testing conditions are：Mobile phase A uses 40mmol L-1Potassium dihydrogen phosphate buffer solution, pH=12, cross 0.22 μm of filter Film；Mobile phase B uses methanol：Acetonitrile=13:1 (volume ratio), cross 0.22 μm of filter membrane；Gradient elution program is as follows：
Flow velocity is 0.8ml min-1, 30 DEG C of column temperature, automatic sampler is equipped with, sample size is 20 μ l, and detector is examined for fluorescence Survey device, excitation wavelength 350nm, launch wavelength 420nm.
Using said extracted and assay method, D types aspartic acid, D types alanine, D types in Taihu Lake middle of a lake area water body are measured The content of glutamic acid and D type serines is respectively 0.19,0.13,0.09,0.15 μm of ol L-1, it is shown in Table 1.
S6. method precision and recovery of standard addition
Add four kinds of D type amino acid standard liquids of 3 concentration levels, each 3 weights of concentration level respectively in the sample It is multiple, 1 is specifically shown in Table, step S1-S5 is carried out, to investigate method precision and recovery of standard addition.
The rate of recovery of table 1D type amino acid and the relative standard deviation (n=3) of measure
As a result show, D types aspartic acid, D types alanine, the recovery of standard addition of D types glutamic acid and D type serines are respectively 89.5-104.6%, 93.2-105.6%, 90.7-103.3%, 95.1-97.4%, relative standard deviation are respectively 3.79- 4.69%th, 1.38-3.78%, 2.58-4.02%, 2.13-3.97%.For the rate of recovery more than 90%, relative standard deviation is low In 5%.Therefore, the method rate of recovery of the invention is higher, and method precision is high, meets requirement of experiment.
Embodiment 3
The extraction of D types amino acid and assay method, comprise the following steps in a kind of poisons in freshwater：
S1. sample
Take Taihu Lake middle of a lake area water body example to cross 0.45 μm of filter membrane and go the removal of impurity and algae, under the conditions of -4 DEG C, dark sealing is protected Deposit and take back laboratory.
S2. water sample pre-processes
Carried out using the acid-hydrolyzed method of microwave radiation technology gas phase：1ml water samples to be measured are taken, add 110 μ l 12mmol L-1It is anti- Bad hematic acid solution and 0.55ml 6mol L-1Hydrochloric acid solution, is transferred in counteracting tank after mixing, and counteracting tank is put into micro-wave digestion In reaction vessel, 25ml 6mol L are added to micro-wave digestion reaction vessel-1Seal, vacuumize after hydrochloric acid solution, be filled with nitrogen, Carry out clearing up to obtain hydrolyzate；Wherein, microwave dissolver power output is 600W, and automatic heating program is：150 are warming up in 2min DEG C, 150 DEG C of holding 30min, after sample is cooled to 80 DEG C, EP (end of program).
After hydrolyzate is cooled into room temperature, 10ml is settled to after being neutralized to neutrality with NaOH solution, obtains testing sample, is sealed It is to be measured；
S3. prepared by derivating agent
0.06g N- isobutyryls-D-Cys and 0.02g OPAs are dissolved into 1ml methanol solutions, used PH value is 9.5, and concentration is 0.5mol L-1Borate buffer solution is diluted to 10ml, and resulting solution is OPA derivating agent, and 4 DEG C it is sealed.
S4. derivatization reaction
Accurate 100 μ l testing samples of drawing are placed in sample bottle, sequentially add 110 μ l OPAs derivating agents, 110 μ l PH value is 9.5, and concentration is 0.5mol L-1Borate buffer solution, vortice mix, and place 20-30min at room temperature.
S5. Instrument measuring
Prepare D types amino acid (D types aspartic acid, D types alanine, D types serine and D types glutamic acid) Standard Stock solutions 1mmol L-1, it is 0.001-0.1mmol L to be diluted to series concentration-1Machine does standard curve on standard working solution.
It is measured using high performance liquid chromatography-fluorescence device；Using anti-phase C18 performance liquid chromatographic columns to D types and L Type amino acid is separated, wherein, chromatographic column is：LiChrospher 100RP-18 chromatographic columns.
Testing conditions are：Mobile phase A uses 40mmol L-1Potassium dihydrogen phosphate buffer solution, pH=12, cross 0.22 μm of filter Film；Mobile phase B uses methanol：Acetonitrile=13:1 (volume ratio), cross 0.22 μm of filter membrane；Gradient elution program is as follows：
Flow velocity is 0.8ml min-1, 30 DEG C of column temperature, automatic sampler is equipped with, sample size is 10 μ l, and detector is examined for fluorescence Survey device, excitation wavelength 350nm, launch wavelength 420nm.
Embodiment 4
The extraction of D types amino acid and assay method, comprise the following steps in a kind of poisons in freshwater：
S1. sample
Take Taihu Lake middle of a lake area water body example to cross 0.45 μm of filter membrane and go the removal of impurity and algae, under the conditions of -4 DEG C, dark sealing is protected Deposit and take back laboratory.
S2. water sample pre-processes
Carried out using the acid-hydrolyzed method of microwave radiation technology gas phase：1ml water samples to be measured are taken, add 120 μ l 12mmol L-1It is anti- Bad hematic acid solution and 0.6ml 6mol L-1Hydrochloric acid solution, it is transferred to after mixing in counteracting tank, it is anti-that counteracting tank is put into micro-wave digestion Answer in container, 25ml 6mol L are added to micro-wave digestion reaction vessel-1Seal, vacuumize after hydrochloric acid solution, be filled with nitrogen, enter Row clears up to obtain hydrolyzate；Wherein, microwave dissolver power output is 600W, and automatic heating program is：150 are warming up in 4min DEG C, 150 DEG C of holding 35min, after sample is cooled to 80 DEG C, EP (end of program).
After hydrolyzate is cooled into room temperature, 10ml is settled to after being neutralized to neutrality with KOH solution, obtains testing sample, is sealed It is to be measured；
S3. prepared by derivating agent
0.06g N- isobutyryls-D-Cys and 0.03g OPAs are dissolved into 1ml methanol solutions, used PH value is 9.5, and concentration is 0.5mol L-1Borate buffer solution is diluted to 10ml, and resulting solution is OPA derivating agent, and 4 DEG C it is sealed.
S4. derivatization reaction
Accurate 100 μ l testing samples of drawing are placed in sample bottle, sequentially add 120 μ l OPAs derivating agents, 120 μ l PH value is 9.5, and concentration is 0.5mol L-1Borate buffer solution, vortice mix, and place 20-30min at room temperature.
S5. Instrument measuring
Prepare D types amino acid (D types aspartic acid, D types alanine, D types serine and D types glutamic acid) Standard Stock solutions 1mmol L-1, it is 0.001-0.1mmol L to be diluted to series concentration-1Machine does standard curve on standard working solution.
It is measured using high performance liquid chromatography-fluorescence device；Using anti-phase C18 performance liquid chromatographic columns to D types and L Type amino acid is separated, wherein, chromatographic column is：LiChrospher 100RP-18 chromatographic columns.
Testing conditions are：Mobile phase A uses 40mmol L-1Potassium dihydrogen phosphate buffer solution, pH=12, cross 0.22 μm of filter Film；Mobile phase B uses methanol：Acetonitrile=13:1 (volume ratio), cross 0.22 μm of filter membrane；Gradient elution program is as follows：
Flow velocity is 0.8ml min-1, 30 DEG C of column temperature, automatic sampler is equipped with, sample size is 30 μ l, and detector is examined for fluorescence Survey device, excitation wavelength 350nm, launch wavelength 420nm.
Above-mentioned detailed description is illustrating for one of them possible embodiments of the present invention, and the embodiment is simultaneously not used to The scope of the claims of the present invention is limited, all equivalence enforcements or change without departing from carried out by the present invention, is intended to be limited solely by the technology of the present invention In the range of scheme.

Claims (7)

1. the extraction of D types amino acid and assay method in a kind of poisons in freshwater, it is characterised in that comprise the following steps：
S1. sample
Water body example crosses 0.45 μm of filter membrane and goes the removal of impurity, and under the conditions of -4 DEG C, dark is sealed；
S2. water sample pre-processes
Carried out using the acid-hydrolyzed method of microwave radiation technology gas phase：Water sample to be measured is taken, it is to be measured molten to prevent to add ascorbic acid solution Amino acid aoxidizes in liquid, adds hydrochloric acid solution, is put into microwave dissolver after mixing, carries out clearing up to obtain hydrolyzate；
After the hydrolyzate is cooled into room temperature, neutrality is neutralized to alkali lye, obtains testing sample, sealing is to be measured；
S3. prepared by derivating agent
N- isobutyryls-D-Cys and OPA are dissolved into methanol solution, diluted using cushioning liquid so that N- The concentration of isobutyryl-D-Cys is 5-6g/L and the concentration of OPA is 2-2.5g/L, and resulting solution is adjacent benzene two Formaldehyde-derived agent, 4 DEG C are sealed；
S4. derivatization reaction
Testing sample is taken, sequentially adds OPA derivating agent and cushioning liquid, vortice is mixed, placed at room temperature, is ensured With OPA derivatization reaction fully occurs for amino acid；
S5. Instrument measuring
It is measured using high performance liquid chromatography-fluorescence device；Using anti-phase C18 performance liquid chromatographic columns to D types and L-type ammonia Base acid is separated；Wherein, chromatographic column is：LiChrospher 100RP-18 chromatographic columns；The composition of mobile phase is：Mobile phase A Using 35-45mmol L-1Potassium dihydrogen phosphate buffer solution, pH=12；Mobile phase B uses methanol：Acetonitrile=13:1 (volume ratio)； Using gradient elution.
2. the extraction of D type amino acid and assay method in a kind of poisons in freshwater according to claim 1, it is characterised in that The water sample pretreatment concretely comprises the following steps：1ml water samples to be measured are taken, add 100-120 μ l 12mmol L-1Ascorbic acid solution To prevent that amino acid aoxidizes in solution to be measured, 0.5-0.6ml 6mol L are added-1Hydrochloric acid solution, resolution is transferred to after mixing In tank, counteracting tank is put into micro-wave digestion reaction vessel, 25ml 6mol L are added to micro-wave digestion reaction vessel-1Hydrochloric acid is molten Seal, vacuumize after liquid, be filled with nitrogen, cleared up.
3. the extraction of D type amino acid and assay method in a kind of poisons in freshwater according to claim 2, it is characterised in that The microwave dissolver power output is 600W, and automatic heating program is：150 DEG C are warming up in 2-4min, 150 DEG C of holding 30- 35min, after sample is cooled to 80 DEG C, EP (end of program).
4. the extraction of D type amino acid and assay method in a kind of poisons in freshwater according to claim 1, it is characterised in that The alkali lye is the mixing of one or both of NaOH solution and KOH solution.
5. the extraction of D type amino acid and assay method in a kind of poisons in freshwater according to claim 1, it is characterised in that Detailed process prepared by the derivating agent is：By 0.05-0.06g N- isobutyryls-D-Cys and 0.02-0.03g neighbour's benzene two Formaldehyde is dissolved into 1ml methanol solutions, the use of pH value is 9.5, concentration is 0.5mol L-1Borate buffer solution is diluted to 10ml, Resulting solution is OPA derivating agent.
6. the extraction of D type amino acid and assay method in a kind of poisons in freshwater according to claim 1, it is characterised in that The derivatization reaction is：Testing sample is taken, it is 9.5 to sequentially add OPA derivating agent and pH value, and concentration is 0.5mol L-1 Borate buffer solution, vortice mix, and place 20-30min at room temperature, ensure that amino acid fully derives with OPA Reaction；Wherein, testing sample, OPA derivating agent and pH value are 9.5, and concentration is 0.5mol L-1Borate buffer solution Volume ratio is：1：(1-1.2)：(1-1.2).
7. the extraction of D type amino acid and assay method in a kind of poisons in freshwater according to claim any one of 1-6, it is special Sign is that the separation detection condition of the high performance liquid chromatography-fluorescence device is：Mobile phase A uses 40mmol L-1Di(2-ethylhexyl)phosphate Hydrogen potassium cushioning liquid, pH=12, cross 0.22 μm of filter membrane；Mobile phase B uses methanol：Acetonitrile=13:1 (volume ratio), cross 0.22 μm Filter membrane；Gradient elution program is as follows：
Flow velocity is 0.8ml min-1, 30 DEG C of column temperature, sample size is 10-30 μ l, and detector is fluorescence detector, and excitation wavelength is 350nm, launch wavelength 420nm.
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Application publication date: 20171124