CN110501450A - The detection method of brilliant green in soil - Google Patents
The detection method of brilliant green in soil Download PDFInfo
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- CN110501450A CN110501450A CN201910909255.2A CN201910909255A CN110501450A CN 110501450 A CN110501450 A CN 110501450A CN 201910909255 A CN201910909255 A CN 201910909255A CN 110501450 A CN110501450 A CN 110501450A
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
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Abstract
The invention discloses the detection methods of brilliant green in soil.The detection method includes the following steps: brilliant green and deuterated malachite green standard solution is respectively prepared, and obtains brilliant green standard solution and deuterated malachite green standard solution；The deuterated malachite green standard solution is added in the brilliant green standard solution, standard working solution is prepared into；The deuterated malachite green standard solution dissolution is added in soil, obtains sample liquid；The sample liquid is used into Solid Phase Extraction column extracting, forms extracting solution；The standard working solution and the extracting solution are detected using liquid chromatography mass combined instrument respectively.The detection method of brilliant green in soil of the present invention can reduce object loss, improve dosing accuracy.
Description
Technical field
The present invention relates to test and analyze technical field, and in particular to the detection method of brilliant green in soil.
Background technique
Brilliant green belongs to alkaline triphenylmethane dye, and fungicide and antiparasitic agent are commonly used in aquaculture, by
There is mutagenesis, teratogenesis and carcinogenesis in triphenylmethane, American-European and China has forbidden to use triphen in aquaculture
Methane class dyestuff is contaminated pond waters and soil due to having used brilliant green in aquaculture process, subsequent
It is easy to form residual in fish body in breeding process, therefore fish culture need to carry out effective monitoring to pond waters and soil.
The detection method of the existing brilliant green of foreign countries, and there are no the correlation techniques of brilliant green detection in soil and water for the country at present.
External correlation technique uses external standard method substantially at present, and object loss is bigger in extraction process, quantitative accurate
Property is not high.
Summary of the invention
Based on this, the present invention it is necessary to provide one kind can reduce object loss, improve dosing accuracy soil in it is bright
Rotten green detection method.
In order to achieve the object of the present invention, the invention adopts the following technical scheme:
The present invention also provides a kind of detection methods of brilliant green in soil, include the following steps:
Standard solution is respectively prepared in brilliant green and deuterated malachite green, obtains brilliant green standard solution and deuterated malachite
Green standard solution；
The deuterated malachite green standard solution is added in the brilliant green standard solution, it is bent to be prepared into standard work
Line；
The deuterated malachite green standard solution and extracting solution is added in soil, obtains sample liquid after extracted and purification；
The sample liquid is used into Solid Phase Extraction column purification, forms extracting solution；
The standard working solution and the extracting solution are detected using liquid chromatography mass combined instrument respectively；
If the chromatographic peak retention time of mass chromatography peak retention time and the standard working solution in the extracting solution exists
It is consistent within the scope of tolerance, and the relative abundance of two ion pairs of brilliant green and the standard work in the extracting solution
The relative abundance for making liquid is consistent within the scope of tolerance, then contains object brilliant green in the soil；Otherwise it is free of
There is brilliant green.
The detection method of brilliant green in above-mentioned soil can promote the extraction effect of brilliant green using solid phase extraction method
Rate guarantees the accuracy of detection；Use deuterated malachite green for internal standard, can offset in extraction process the loss of object and
Impurity interferes the interference of bring instrument matrix effect, guarantees the object rate of recovery close to 100%, and it is quantitative accurate to greatly strengthen
Property.
In some of embodiments, when needing the content of brilliant green in soil described in quantitative measurment, the detection method
Include the following steps:
Prepare the standard working solution of various concentration, and make the deuterated malachite green in the standard working solution with
The concentration of the deuterated malachite green in the extracting solution is consistent；
The standard working solution of various concentration is detected, using concentration as abscissa, the area of chromatographic peak is made of ordinate
Standard curve；
Corresponding extracting solution will be obtained after sample extraction, purification, detects the extracting solution；
The content of the brilliant green is calculated according to following formula；
Wherein, X is the content of brilliant green in the soil, μ g/kg；C is the extracting solution read from standard curve
The content of middle object brilliant green, ng/mL；V is the volume of the extracting solution, mL；M is the sample weighting amount of the soil, g；N is dilute
Release multiple.
In some of embodiments, after the completion of the extracting solution detection, before the content for calculating the brilliant green, or in institute
Before stating extracting solution detection, also there are following steps: the deuterated malachite green standard solution is added in blank sample, using solid
Mutually extraction column extracting forms blank and detects liquid；The blank is detected using liquid chromatography mass combined instrument and detects liquid, the extraction
The chromatographic peak area of liquid and the chromatographic peak area of blank detection liquid subtract each other, and substitute into the standard curve and calculate the brilliant green
Content.
In some of embodiments, described the step of standard solution is made in deuterated malachite green specifically: by the deuterium
Simultaneously constant volume is dissolved for malachite green acetonitrile solution；Described the step of standard solution is made in brilliant green specifically: will be described bright
Rotten green acetonitrile solution dissolution and constant volume.
It is described that the deuterated malachite green standard solution dissolution is added in soil in some of embodiments, obtain sample liquid
The step of specifically: the soil is ground, be separately added into the deuterated malachite green standard solution, ammonium acetate buffer solution,
P-methyl benzenesulfonic acid solution and acetonitrile solution are uniformly dispersed, centrifugation, take supernatant that water and methylene chloride is added after standing, take after standing
The concentration of lower layer's solution, obtains sample liquid.
In some of embodiments, it is described by the sample liquid use Solid Phase Extraction column extracting, formed extracting solution the step of
Specifically: neutral alumina column is activated with acetonitrile solution, the sample liquid is added, elutes the neutral alumina with acetonitrile solution
Aluminium column collects eluent, the eluent is evaporated to dryness, and adds acetonitrile solution dissolution, and filtering takes filtrate, obtains extracting solution.
Liquid phase chromatogram condition in some of embodiments, in the liquid chromatography mass combined instrument are as follows: chromatographic column is
WATERS UPLC BEH C18 chromatographic column, mobile phase are acetonitrile+5mmol/L ammonium acetate gradient elution containing 0.1% formic acid.
Mass Spectrometry Conditions in some of embodiments, in the liquid chromatography mass combined instrument are as follows: electric spray ion source, just
It is ion scan, electron spray voltage 4000V, capillary voltage 100V, assistor pressure 35psi, 11L/min flow blowback air, bright
Rotten green two ion pair 358.3/297.2 and 358.3/341.2 and deuterated malachite green ion pair 334.2/318.2.
Liquid phase chromatogram condition in some of embodiments, in the liquid chromatography mass combined instrument are as follows: flow velocity 0.3mL/
Min, 5 μ L of sample volume.
In some of embodiments, the deviation range of the relative abundance permission are as follows: relative abundance > 50%, allow ±
20% deviation；Relative abundance 20%-50% allows ± 25% deviation；Relative abundance 10%-20% allows ± 30%
Deviation；Relative abundance < 10% allows ± 50% deviation.
Detailed description of the invention
Fig. 1 is the brilliance green using the standard working solution of the detection method detection of brilliant green in soil of the present invention
Spectrogram；
Fig. 2 is the deuterated peacock using the standard working solution of the detection method detection of brilliant green in soil of the present invention
Malachite green chromatogram；
Fig. 3 is brilliant green chromatogram in the soil detected using the detection method of brilliant green in soil of the present invention；
Fig. 4 is using deuterated malachite is green in the soil of the detection method detection of brilliant green in soil of the present invention
Spectrogram；
Fig. 5 is the brilliant green chromatography using the soil mark-on of the detection method detection of brilliant green in soil of the present invention
Figure；
Fig. 6 is to detect the deuterated of liquid using the soil mark-on of the detection method detection of brilliant green in soil of the present invention
Malachite green chromatogram.
Specific embodiment
It to facilitate the understanding of the present invention, below will be to invention is more fully described.But the present invention can be to be permitted
Mostly different form is realized, however it is not limited to embodiment described herein.On the contrary, purpose of providing these embodiments is makes
It is more thorough and comprehensive to the understanding of the disclosure.
Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention
The normally understood meaning of technical staff is identical.Term as used herein in the specification of the present invention is intended merely to description tool
The purpose of the embodiment of body, it is not intended that in the limitation present invention.
The present invention provides a kind of detection method of brilliant green in soil, includes the following steps:
Standard solution is respectively prepared in brilliant green and deuterated malachite green, obtains brilliant green standard solution and deuterated malachite
Green standard solution；
Deuterated malachite green standard solution is added in brilliant green standard solution, standard working curve is prepared into；
The deuterated malachite green standard solution and extracting solution is added in soil, obtains sample liquid after extracted and purification；
The sample liquid is used into Solid Phase Extraction column purification, forms extracting solution；
Examination criteria working solution and extracting solution are distinguished using liquid chromatography mass combined instrument；
If the chromatographic peak retention time of mass chromatography peak retention time and standard working solution in extracting solution is in tolerance
It is consistent in range, and the relative abundance of two ion pairs and the relative abundance of standard working solution of brilliant green exist in extracting solution
It is consistent within the scope of tolerance, then contains object brilliant green in soil；Otherwise brilliant green is not contained.
Soil matrix is complicated, and the impurity contained is more, and brilliant green content is lower, detects the brilliant green in soil, magnificent
It is green that procrypsis brilliant green is easily converted in the case where impurity is more, therefore in the detection process, need strict control extraction side
Method and qualitative-and-quantitative method, otherwise Detection accuracy is difficult to ensure.
This method extracts the brilliant green in soil using solid phase extraction method, can remove except most highly polar and weak
Polar impurity has reached preferable clean-up effect, can promote the extraction efficiency of brilliant green, guarantees the accuracy of detection；Using
Deuterated malachite green is internal standard, can offset the loss of object and impurity interference bring instrument matrix effect in extraction process
It should interfere, guarantee that the object rate of recovery close to 100%, greatly strengthens quantitative accuracy.
Above-mentioned method can be with qualitative detection brilliant green.But certain occasions may require quantitatively being detected, to hold soil
The concentration of brilliant green in earth.
When needing the content of brilliant green in quantitative measurment soil, which includes the following steps:
The standard working solution of various concentration is prepared, and makes the deuterium in the deuterated malachite green and extracting solution in standard working solution
It is consistent for the concentration of malachite green；
The standard working solution for detecting various concentration, using concentration as abscissa, the area of chromatographic peak is that standard is made in ordinate
Curve；
Corresponding extracting solution will be obtained after sample extraction, purification, detects the extracting solution；
The content of brilliant green is calculated according to following formula；
Wherein, X is the content of brilliant green in the soil, μ g/kg；C is the extracting solution read from standard curve
The content of middle object brilliant green, ng/mL；V is the volume of the extracting solution, mL；M is the sample weighting amount of the soil, g；N is dilute
Release multiple.
Adopt can use the content that internal standard detection method accurately detects brilliant green in soil with the aforedescribed process, significantly
Improve quantitative accuracy.
In one embodiment, in order to keep quantitative detection result more acurrate, influence of the solution being added to chromatographic peak area is prevented,
After the completion of extracting solution detection, before the content for calculating brilliant green, or before extracting solution detection, also progress blank test, blank
Test includes the following steps: that deuterated malachite green standard solution for blank sample is added, using Solid Phase Extraction column extracting, is formed empty
White detection liquid；Liquid is detected using liquid chromatography mass combined instrument detection blank, the chromatographic peak area and blank of extracting solution detect liquid
Chromatographic peak area subtract each other, substitute into standard curve calculate brilliant green content.Thus can to avoid addition solution for inspection
The influence for surveying result, so that the content of the brilliant green detected is more accurate.I.e. in addition to sample is not added, the method for blank test with
The detection method of sample is consistent.
In one embodiment, the step of standard solution is made in deuterated malachite green specifically: by deuterated malachite green second
The dissolution of nitrile solution and constant volume；The step of standard solution is made in brilliant green specifically: brilliant green is dissolved and determined with acetonitrile solution
Hold.Standard solution is made as internal standard using deuterated malachite green, the loss of object and miscellaneous can be offset in extraction process
Matter interferes the interference of bring instrument matrix effect.
It is multiple that a small amount of acetonitrile solution dissolution constant volume wherein can be used, guarantee that deuterated malachite green dissolution is complete.And it is every
Its concentration is different after dissolution is primary, and condition of storage is also different, therefore the time for dissolving obtained lysate preservation every time is different, can
It is taken out and is used in different time sections using the holding time.
The organic matter that acetonitrile solution therein can also can dissolve deuterated malachite green using others replaces, such as second
The solution such as acid, methanol.
In one embodiment, the step of standard solution dissolution is added, obtains sample liquid by soil specifically: soil is ground, point
Deuterated malachite green standard solution, ammonium acetate buffer solution, p-methyl benzenesulfonic acid solution and acetonitrile solution are not added to be uniformly dispersed, from
The heart takes after standing supernatant that water and methylene chloride is added, and stands the concentration of Hou Qu lower layer solution, obtains sample liquid.
Brilliant green is easily converted to procrypsis brilliant green in the case where impurity is more, and ammonium acetate buffer solution provides stabilization
Faintly acid system, be added p-methyl benzenesulfonic acid facilitate brilliant green formed ion pair, ensure that brilliant green in the steady of extraction process
It is qualitative, while aqueous phase solution has high osmosis, can greatly enhance interpenetrating for Extraction solvent and sample substrate, enhances
Extraction efficiency.Therefore it ensure that the stability of object in extraction process, and substantially increase the extraction efficiency of object.
Methylene chloride and water liquid-liquid extraction are added in extracting solution, when three kinds of acetonitrile, methylene chloride, water solvents mix,
Acetonitrile is mutually dissolved with methylene chloride, and water phase and other two kinds of demixing of solvents, a large amount of water-solubility impurity are dissolved in water, pass through
Experiment shows that the basic fully dissolved of object in the mixed solvent of acetonitrile and methylene chloride, is eliminated greatly by liquid-liquid extraction mode
The water-solubility impurity of amount.The weaker impurity of depolarization can be removed after neutral alumina SPE column, liquid-liquid extraction and Solid Phase Extraction combine
Mode can remove most highly polar and low pole impurity, reached preferable clean-up effect.
Soil before grinding, preferably first carries out air-drying processing, removes biggish stone and other impurity.Then it crosses
2.0mm aperture sieve is smashed to pieces completely with homogenizer or dismembyator, is uniformly mixed and is used as sample.
In one embodiment, the step of using Solid Phase Extraction column extracting by sample liquid, form extracting solution specifically: by neutral oxygen
Change aluminium column to be activated with acetonitrile solution, sample liquid is added, elutes neutral alumina column with acetonitrile solution, collect eluent, will elute
Liquid is evaporated to dryness, and adds acetonitrile solution dissolution, and filtering takes filtrate, obtains extracting solution.Neutral alumina column is living using acetonitrile solution
Change, neutral alumina column is allowed to elute the highly polar and low pole impurity in acetonitrile solution；Again through evaporation, dissolution and mistake
Step is filtered, the brilliant green efficiently extracted in solution can be.
Liquid phase chromatogram condition in one embodiment, in liquid chromatography mass combined instrument are as follows: chromatographic column is WATERS UPLC
BEH C18 chromatographic column, mobile phase are acetonitrile+5mmol/L ammonium acetate gradient elution containing 0.1% formic acid.Flow velocity 0.3mL/min,
5 μ L of sample volume.Test of many times proves, is detected under this condition, can preferably detect the brilliant green in sample, reduces
Interference in detection process.
Mass Spectrometry Conditions in one embodiment, in liquid chromatography mass combined instrument are as follows: electric spray ion source, cation scanning,
Electron spray voltage 4000V, capillary voltage 100V, assistor pressure 35psi, 11L/min flow blowback air, brilliant green two from
Son is to 358.3/297.2 and 358.3/341.2 and deuterated malachite green ion pair 334.2/318.2.Test of many times proves, at this
Under the conditions of detected, can preferably detect the brilliant green in sample, reduce the interference in detection process.Certainly, liquid phase
Chromatographic condition and Mass Spectrometry Conditions can also be replaced with the other conditions of similar equal performance.
In one embodiment, when whether containing brilliant green in judging soil, the deviation range of relative abundance permission are as follows: opposite
Abundance > 50% allows ± 20% deviation；Relative abundance 20%-50% allows ± 25% deviation；Relative abundance 10%-
20%, allow ± 30% deviation；Relative abundance < 10% allows ± 50% deviation.
The deviation range that chromatographic peak retention time allows is ± 5%.
Hereinafter embodiments of the present invention will be further illustrated by embodiment.
Embodiment
The detection method of brilliant green in soil provided in this embodiment, comprising the following steps:
S1. standard reserving solution is prepared
S1a. it prepares standard reserving solution: deuterated malachite green standard reserving solution: accurately weighing deuterated malachite green standard items
10.0mg is in 50mL beaker, and with the dissolution of a small amount of acetonitrile and constant volume is to 50mL brown volumetric flask, mixes, concentration is 200 μ g/mL.
The solution can save 6 months under the conditions of -18 DEG C.
Brilliant green standard reserving solution: accurately weighing brilliant green standard items 10.0mg in 50mL beaker, molten with a small amount of acetonitrile
It solves and constant volume is to 50mL brown volumetric flask, mix, concentration is 200 μ g/mL.The solution can save 6 months under the conditions of -18 DEG C.
S2. standard solution is prepared
S2a. from drawing 0.5mL in brilliant green standard reserving solution respectively into 100mL brown volumetric flask, and it is fixed with acetonitrile
Hold, mix, concentration is 1.0 μ g/mL, which can save 1 month under the conditions of 4 DEG C；0.5mL is drawn again to 50mL brown capacity
Bottle in, and use acetonitrile constant volume, mix, concentration be 0.01 μ g/mL (the solution prepared before use), obtain brilliant green standard solution.
0.5mL is drawn from deuterated malachite green standard reserving solution into 100mL brown volumetric flask, and with acetonitrile constant volume,
It mixes, obtains standard solution, concentration is 1.0 μ g/mL.The solution can in refrigerator 4 DEG C save 1 month.Obtain deuterated malachite green
Standard solution.
S3. standard solution is prepared
S3a. it makes standard curve: diluting deuterated malachite green standard solution and brilliant green standard with 50% acetonitrile water respectively
Solution, will deuterated malachite green standard solution be added brilliant green standard solution in, be respectively configured 0.5ng/mL, 1.0ng/mL,
The standard working solution of 2.0ng/mL, 5.0ng/mL and 10.0ng/mL respectively contain deuterated malachite green 5.0ng/mL.The solution faces use
Now match.
S4. prepared by sample
It takes pedotheque 2.0kg in ventilation natural air drying, removes biggish stone and other impurity, then cross 2.0mm
Aperture sieve is smashed to pieces completely with homogenizer or dismembyator, is uniformly mixed, as laboratory testing sample.
S5. brilliant green extracts
10g sample (being accurate to 0.01g) is accurately weighed in 80mL centrifuge tube, the deuterated malachite green mark of 1.0 μ g/mL is added
4mL0.1mol/L ammonium acetate buffer solution, 4mL1.0mol/L p-methyl benzenesulfonic acid solution and 50mL acetonitrile is added in quasi- 50 μ L of solution,
5min is centrifuged with speed the homogeneous 30s, 4000r/min of 10000r/min with homogenizer, supernatant is poured into equipped with 20mL water, 40mL
In the separatory funnel of methylene chloride, 5min is shaken up and down, and stratification sops up upper strata aqueous phase, and it is above-mentioned to add the repetition of 20mL water
Operation, takes lower layer's solution in chicken heart bottle, 40 DEG C are concentrated to dryness, and obtain sample liquid.
Sample liquid is added 10mL acetonitrile and elutes chicken heart bottle, takes the neutral alumina column of 2mL overactivation (on neutral alumina column
1cm high anhydrous sodium sulfate is filled out at end, is activated using preceding with 5mL acetonitrile), it is collected into 150mL brown chicken heart bottle, the elution of 20mL acetonitrile
Neutral alumina column, eluent are all collected into same chicken heart bottle, 40 DEG C of decompression rotary evaporated to dryness, use 2mL acetonitrile+water afterwards
(1+1) solution, which is vortexed, to be eluted, and is taken 0.2 μm of organic membrane filtration of clear liquid, is obtained extracting solution.
S6. examination with computer: taking the injection LC-MS instrument detection of 5 μ L extracting solutions, and specific test condition is as follows:
Liquid phase chromatogram condition:
A, chromatographic column: 1.7 μm of C18 of WATERS UPLC BEH, 100*2.1mm or the suitable person of performance
B, mobile phase: acetonitrile+5mmol/L ammonium acetate (containing 0.1% formic acid) gradient elution
C, flow velocity: 0.3mL/min
D, sample volume: 5 μ L
Mass Spectrometry Conditions:
A, ion source: electric spray ion source
B, scanning mode: cation scanning
C, detection mode: multiple-reaction monitoring
D, electron spray voltage: 4000V
E, capillary voltage: 100V
F, assistor pressure: 35psi
G, blowback air: 11L/min
H brilliant green ion pair: 358.3/297.2 (quantitative) 358.3/341.2
Deuterated malachite green ion pair: 334.2/318.2
S7. result judgement: Fig. 1 and Fig. 2 is the chromatogram of standard working solution；Fig. 3 and Fig. 4 is the chromatography of sample extracting solution
Figure.If with the chromatographic peak retention time and the chromatographic peak retention time one of the standard working solution with concentration of the extracting solution of concentration
It causes.And in sample two daughter ions of target compound relative abundance it is consistent with the relative abundance of standard solution, relative abundance
Deviation is no more than the relative deviation allowed, then can determine whether that there are brilliant greens in sample.
S8. quantitative measurment
Deuterated malachite green standard solution and brilliant green standard solution are diluted with 50% acetonitrile water respectively, by deuterated malachite
Green standard solution is added in brilliant green standard solution, configuration 0.5ng/mL, 1.0ng/mL, 2.0ng/mL, 5.0ng/mL,
The standard working solution of 10.0ng/mL respectively contains deuterated malachite green 5.0ng/mL, with standard working solution sample introduction, with standard working solution
Concentration be abscissa, chromatographic peak area is ordinate, draws standard working curve, extracting solution isometric sample introduction measurement, internal standard
Standard measure；Brilliant green content in extracting solution should be within standard curve concentration range.
S9. mark-on reclaims
Mark-on is carried out using blank sample, adds the deuterated malachite green standard solution of 0.01 μ g/mL with 1.0mL liquid-transfering gun
500 μ L are in blank sample, that is, addition concentration is 0.5 μ g/kg pitch-based sphere.
S10. blank test
In addition to not weighing sample, step S5-S6 step is repeated, obtains blank detection liquid, detection blank detects liquid.Fig. 5 and Fig. 6
The chromatogram of liquid is detected for blank.
S11. result calculates
The residual quantity of brilliant green in sample is calculated according to following formula:
In formula: X is the content of brilliant green in soil, μ g/kg；C is the object from the extracting solution read on standard curve
The content of brilliant green, ng/mL；V is the volume of extracting solution, mL；M is the sample weighting amount of soil, g；N is extension rate.
By Fig. 1 to Fig. 6 it is found that accurately quantitative detection the content of brilliant green in soil can be gone out using this method.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (10)
1. the detection method of brilliant green in soil, which comprises the steps of:
Standard solution is respectively prepared in brilliant green and deuterated malachite green, obtains brilliant green standard solution and deuterated malachite green mark
Quasi- solution；
The deuterated malachite green standard solution is added in the brilliant green standard solution, standard working curve is prepared into；
The deuterated malachite green standard solution and extracting solution is added in soil, obtains sample liquid after extracted and purification；
The sample liquid is used into Solid Phase Extraction column purification, forms extracting solution；
The standard working solution and the extracting solution are detected using liquid chromatography mass combined instrument respectively；
If the chromatographic peak retention time of mass chromatography peak retention time and the standard working solution in the extracting solution is allowing
It is consistent in deviation range, and the relative abundance of two ion pairs of brilliant green and the standard working solution in the extracting solution
Relative abundance be consistent within the scope of tolerance, then in the soil contain object brilliant green；Otherwise without containing bright
It is rotten green.
2. the detection method of brilliant green in soil according to claim 1, which is characterized in that when needing described in quantitative measurment
In soil when the content of brilliant green, the detection method includes the following steps:
Prepare the standard working solution of various concentration, and make the deuterated malachite green in the standard working solution with it is described
The concentration of the deuterated malachite green in extracting solution is consistent；
The standard working solution for detecting various concentration, using concentration as abscissa, the area of chromatographic peak is that standard is made in ordinate
Curve；
Corresponding extracting solution will be obtained after sample extraction, purification, detects the extracting solution；
The content of the brilliant green is calculated according to following formula；
Wherein, X is the content of brilliant green in the soil, μ g/kg；C is the mesh from the extracting solution read on standard curve
Mark the content of object brilliant green, ng/mL；V is the volume of the extracting solution, mL；M is the sample weighting amount of the soil, g；N is dilution times
Number.
3. the detection method of brilliant green in soil according to claim 2, which is characterized in that the extracting solution detection is completed
Afterwards, before the content for calculating the brilliant green, or before extracting solution detection, also there are following steps: by blank sample
The deuterated malachite green standard solution is added, using Solid Phase Extraction column extracting, forms blank and detects liquid；Using liquid chromatogram matter
It composes combined instrument and detects the blank detection liquid, the chromatographic peak area of the chromatographic peak area of the extracting solution and blank detection liquid
Subtract each other, substitutes into the content that the standard curve calculates the brilliant green.
4. the detection method of brilliant green in soil according to claim 1, which is characterized in that described by deuterated malachite green
The step of standard solution is made specifically: deuterated malachite green acetonitrile solution is dissolved into simultaneously constant volume；It is described by brilliant green
The step of standard solution is made specifically: brilliant green acetonitrile solution is dissolved into simultaneously constant volume.
5. the detection method of brilliant green in soil according to claim 1, which is characterized in that it is described soil is added described in
The step of deuterated malachite green standard solution dissolves, and obtains sample liquid specifically: the soil is ground, is separately added into described deuterated
Malachite green standard solution, ammonium acetate buffer solution, p-methyl benzenesulfonic acid solution and acetonitrile solution are uniformly dispersed, centrifugation, after standing
It takes supernatant that water and methylene chloride is added, stands the concentration of Hou Qu lower layer solution, obtain sample liquid.
6. the detection method of brilliant green in soil according to claim 1, which is characterized in that described to adopt the sample liquid
With Solid Phase Extraction column extracting, the step of forming extracting solution specifically: activate neutral alumina column with acetonitrile solution, described in addition
Sample liquid elutes the neutral alumina column with acetonitrile solution, collects eluent, the eluent is evaporated to dryness, is added
Acetonitrile solution dissolution, filtering take filtrate, obtain extracting solution.
7. the detection method of brilliant green in soil according to claim 1, which is characterized in that the liquid chromatography mass connection
With the liquid phase chromatogram condition in instrument are as follows: chromatographic column be WATERS UPLC BEH C18 chromatographic column, mobile phase be acetonitrile+contain 0.1%
The 5mmol/L ammonium acetate gradient elution of formic acid.
8. the detection method of brilliant green in soil according to claim 1 or claim 7, which is characterized in that the liquid chromatogram matter
Compose the Mass Spectrometry Conditions in combined instrument are as follows: electric spray ion source, cation scanning, electron spray voltage 4000V, capillary voltage
100V, assistor pressure 35psi, 11L/min flow blowback air, brilliant green two ion pair 358.3/297.2 and 358.3/
341.2 and deuterated malachite green ion pair 334.2/318.2.
9. the detection method of brilliant green in soil according to claim 7, which is characterized in that the liquid chromatography mass connection
With the liquid phase chromatogram condition in instrument are as follows: flow velocity 0.3mL/min, 5 μ L of sample volume.
10. the detection method of brilliant green in soil according to claim 1, which is characterized in that the relative abundance allows
Deviation range are as follows: relative abundance > 50%, allow ± 20% deviation；Relative abundance 20%-50%, allow ± 25% it is inclined
Difference；Relative abundance 10%-20% allows ± 30% deviation；Relative abundance < 10% allows ± 50% deviation.
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