CN105301161B - Pre-column electrochemical derivatization one-time total quantity measuring method for malachite green and leucomalachite green in aquatic products - Google Patents
Pre-column electrochemical derivatization one-time total quantity measuring method for malachite green and leucomalachite green in aquatic products Download PDFInfo
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Abstract
The invention provides a pre-column electrochemical derivatization one-time total quantity measuring method for malachite green and leucomalachite green in aquatic products and belongs to one-time total quantity measuring methods for converting leucomalachite green into malachite green. The key of the method is that it is found that after a leucomalachite green solution is subjected to oxidation voltage +650 mv of an electrochemical Coulomb detector (ECD), averagely 98.86% of leucomalachite green is converted into malachite green, original malachite green is unchanged, and the total concentration of leucomalachite green and malachite green can be measured through a high performance liquid chromatograph in the one-time measurement process of leucomalachite green and malachite green in a sample. Sample pre-treatment time is greatly shortened, use of reagent consumables is reduced, and electrochemical precolumn derivatization is used for replacing post-column derivatization of lead dioxide in the national standard. The method has the advantages of being simple in sample pre-treatment, good in purification effect, high in derivatization rate and high in recovery rate.
Description
Technical field
The invention belongs to the assay method of malachite green and concealed malachite green, more particularly to exists concealed malachite green
Column front derivation turns to the single-time measurement total amount method of malachite green.
Background technology
Malachite green (Malachite Green, MG) is a kind of triphenylmethane chemicals.Industry is commonly used for silk, hair
The dyestuff of skin and paper.Because the oxidizing potential of malachite green and some amino acid of pathogen constitutive enzyme are close, divide in cell
Compete with it when splitting and hinder the formation of protein peptides, cause cell division to be suppressed, so as to play antibacterial, insecticidal action.By
It is obvious, cheap in its bactericidal effect, it is easy to use, once by many countries by it extensively with preventing and treating water mold in aquaculture
Disease, kills fish ectoparasites and the mould in fish-egg.The how precious fish food security incident of 2006 allows malachite green to cause society
The extensive concern of meeting.
Research in recent years finds, malachite green and its in animal body metabolite concealed malachite green (leucomalachite
Green, LMG) the triphenylmenthane chemical functional group in molecular structure confirmed with teratogenesis, carcinogenic, mutagenesis, Er Qie
It is long with the residence time in environment in fish body, it is not easy to be degraded, and can be by food chain transport to mammal and the mankind, to people
Body health and ecological environment produce very big harm.Simultaneously it can dissolve the zinc in adsorbed water body, cause the acute zinc of aquatic animal
Poisoning.Therefore, many countries such as America and Europe, Japan, Canada are all classified as aquaculture forbidden drugses.
China lists in malachite green from May, 2002《The veterinary drug and compound inventory of food animal disabling》.But, by
In malachite green antimycotic antiseptic effect substantially, it is cheap to be easy to get, it is easy to use, while lacking effective alternative medicine, also have
People during aquaculture and aquatic product transportation stealthily using and remain incessant after repeated prohibition. so far still often by aquatic products detect.
At present, national standard method is mainly high performance liquid chromatography and the tandem mass spectrometry of liquid chromatogram one, is to make after extraction
Chemical reagent brown lead oxide is used, concealed malachite green is changed into and determine after malachite green.But brown lead oxide consumption is uncertain
Or the titanium dioxide lead block of filling is uneven, oxidation efficiency is uncontrollable, and the quantity that concealed malachite green is changed into into malachite green is not
It is controllable, it is not high to the accuracy of concealed malachite green detection, and also with use time growth, weakening causes reappearance not
It is good.
The content of the invention
It is an object of the invention to provide high efficiency, it is simple to operate, at high proportion concealed malachite green changed into into malachite green
Afterwards, the disposable inspection of the ultraviolet-visible detector of high performance liquid chromatography one of concealed malachite green and malachite green merging amount is carried out
The method for surveying total amount.
Present disclosure is：
Electrochemical derivatization single-time measurement total amount method before the post of aquatic products Malachite Green and concealed malachite green, its step
It is rapid as follows：
First, reagent
Standard substance：Malachite green (content 98.0%)；With stability malachite green (content 98.0%)；
(chromatographically pure, content is more than 99.9%) for acetonitrile；
(chromatographically pure, content is more than 98%) for glacial acetic acid；
(chromatographically pure, content is more than 99.9%) for anhydrous acetic acid ammonium；
(chromatographically pure, content is more than 99%) for dichloromethane；
Acidic alumina solid absorbent (50-200 μm of particle diameter)；
(analysis is pure, and content is more than 99.8%) for sodium chloride.
Stability malachite green is colourless.
2nd, the solution to be determined of standard substance malachite green is prepared (referred to as：Standard liquid A to be determined)
Standard substance malachite green 10mg is accurately weighed, with acetonitrile malachite green is dissolved, be configured to concentration containing malachite green
For the standard substance malachite green solution to be determined of 1.0mg/L, A standard liquids to be determined, stand-by chromatograph detection had both been obtained；
3rd, the solution to be determined (standard liquid abbreviation B to be determined) of standard substance concealed malachite green is prepared
Standard substance concealed malachite green 10mg is accurately weighed, with acetonitrile concealed malachite green is dissolved, be configured to containing peacock
Malachite green concentration is the standard substance concealed malachite green solution to be determined of 1.0mg/L, has both obtained B standard liquids to be determined, treats
Detected with chromatograph.
4th, (sample abbreviation C to be determined is molten to prepare the solution to be determined of sample material malachite green and concealed malachite green
Liquid)
(1) solution is prepared：
Prepare buffer solution：Weigh 3.85g anhydrous acetic acid ammoniums to be dissolved in 1000mL distilled water, glacial acetic acid adjusts pH to 4.5；
Cross 0.45 μm of water system filter membrane；Because dissolution is fast under conditions of malachite green and concealed malachite green pH4.5, all there is respectively chemistry
Stability, the dissolution of malachite green and concealed malachite green is to increase the reason for anhydrous acetic acid ammonium, and glacial acetic acid has for small molecule
Machine acid, the molecule destruction to malachite green and concealed malachite green is little.
Prepare extract：By acetonitrile: dichloromethane is standby after mixing for 2: 1 by volume；Due to malachite green and recessiveness
Dissolubility is good under conditions of malachite green acetonitrile+dichloromethane, with standard substance malachite green and standard substance stability malachite
Green dissolution experiment, its solubility is both greater than respectively 90%；So with this mixing ratio of the two materials；
(2) sample C sample solutions to be determined are prepared
Take and be put in 50mL centrifuge tubes after 2.0g testing samples are smashed to pieces, add ammonium acetate buffer solution 3mL and extract
5mL, after being mixed with being vortexed, in being put into ultrasonic extractor, promotes malachite green and concealed malachite green to be dissolved in ultrasonic wave
In liquid, with ultrasonic wave dissolution 30min, acidic alumina solid absorbent 1g is added, add sodium chloride 1g, extraction of ocean eddies
2min, 8000r/min rotating speeds centrifugation 5min, takes supernatant in 45 DEG C of rotary evaporated to dryness, with the acetonitrile of 2.0mL on centrifuge
Dissolving, 0.45 μm of organic membrane filtration, the filtered fluid is the solution to be determined containing malachite green and concealed malachite green, both C
Sample solution to be determined；
The effect for adding acidic alumina solid absorbent is to remove oil substances, prevents removed oil substances
Interference is determined to malachite green and concealed malachite green.1g sodium chloride is added to increase the molten of malachite green and concealed malachite green
Go out.
5th, the chromatographic test strip part of high performance liquid chromatograph
High performance liquid chromatograph is set to：Use C18Chromatographic column (150mm × 4.6mm, 5 μm), mobile phase is acetonitrile；pH
Ammonium acetate buffer solution=78 of 4.5 states: 22 (volume ratios), flow velocity 1.0mL/min, the μ L of sample size 20,35 DEG C of column temperature.
6th, with standard liquid correction malachite green A to be determined is in the absorbing wavelength of high performance liquid chromatograph and determines mistake
Difference a% is (referred to as：The evaluated error of malachite green is a%)：
(6.1) field recognition is powered up：It is A standard liquids to be determined above-mentioned steps " five " the standard liquid of malachite green
Under the chromatographic test strip part of high performance liquid chromatograph, first A standard liquids to be determined are flow through electrochemistry coulometric detector (ECD)
Oxidation voltage+650mv, flow velocity 1.0mL/min, then through C18Chromatographic column, detects under UV-detector detection 618nm wavelength and contains
The concentration value of malachite green；Both the concentration value containing malachite green in A standard liquids to be determined had been measured；With dense containing malachite green
Angle value a1 compares with the standard density 1.0mg/L of step " two " Malachite Green, and the error for obtaining measure malachite green is
A1%；
(6.2) not powered field recognition：It is A standard liquid above-mentioned steps to be determined the standard liquid of malachite green
Under the chromatographic test strip part of " five " high performance liquid chromatograph, first A standard liquids to be determined are flow through electrochemistry coulometric detector
(ECD) oxidation voltage 0.0mv, flow velocity 1.0mL/min, then through C18Chromatographic column, examines under UV-detector detection 618nm wavelength
Survey the concentration value containing malachite green；Both the concentration value containing malachite green in A standard liquids to be determined had been measured；With containing malachite green
Concentration value a2 compare with the standard density 1.0mg/L of step " two " Malachite Green, obtain malachite green evaluated error
For a2%；
Work as a1%=a2%, or when the error of (a1%-a2%)/100 is less than one thousandth, it is average with a1% and a2%
Value is used as a%；After a% is less than one of percentage, next step measure is carried out.
7th, concealed malachite green has been measured by oxygen into after malachite green, used high-efficient liquid phase color with B standard liquids to be determined
Evaluated error b% of spectrometer is (referred to as：The evaluated error of concealed malachite green is b%)：
Power-up field recognition：It is B standard liquids to be determined above-mentioned steps " five " the standard liquid of concealed malachite green
Under the chromatographic test strip part of high performance liquid chromatograph, first B standard liquids to be determined are flow through electrochemistry coulometric detector (ECD)
Oxidation voltage+650mv, flow velocity 1.0mL/min, concealed malachite green malachite green is reduced into, then through C18After chromatographic column,
The concentration value that the malachite green for changing into is detected under 618nm wavelength is detected with UV-detector；With the malachite green for changing into
Concentration value compares with the standard density 1.0mg/L of concealed malachite green in step " two ", obtains the measure of concealed malachite green
Error is b%.
Solution containing concealed malachite green is firstly flowed through electrochemistry coulometric detector (ECD) oxidation voltage+650mv, is flowed
Fast 1.0mL/min, it is that the core in the present invention is sent out that just the oxidation of the concealed malachite green in this solution can be changed into malachite green
Bright technology, i.e. inventive point in the present invention.And this method for oxidation, can make concealed malachite green average 98.86% be changed into hole
Sparrow malachite green, the evaluated error of concealed malachite green is b% average 98.86%.So, the present invention is made the recessive hole in sample
In once determining, can measure total concentration c (mg/L) of concealed malachite green and malachite green has for sparrow malachite green and malachite green
Experimental basis.
It is the result of many experiments from+650mv in our oxidation experiment, many experiments are proved, containing recessive hole
When the solution of sparrow malachite green firstly flows through electrochemistry coulometric detector (ECD) oxidation voltage+650mv, concealed malachite green oxidation is changed into
The effect of malachite green is best.
8th, total concentration c (mg/L) of C sample solution Malachite Greens to be determined and concealed malachite green is determined；
Determine under the conditions of added electric field：The sample solution sample C to be determined color of step " five " high performance liquid chromatograph
Under spectrum testing conditions, sample solution C to be determined is through electrochemistry coulometric detector (ECD) oxidation voltage+650mv, flow velocity
1.0mL/min, malachite green content does not change, and concealed malachite green is oxidized to malachite green, then through C18After chromatographic column, use
UV-detector detects the detection under 618nm wavelength, determines C sample solution Malachite Greens to be determined and recessive peacock
Total concentration c (mg/L) of malachite green.
The experimental result of field energy bioassay standard solution malachite green, and also step are powered up according to step " six " (6.1)
" seven " power-up field energy determines standard liquid concealed malachite green by oxygen into the experimental result of malachite green, illustrates this step " eight "
Power-up field energy determines total concentration c (mg/L) of sample solution Malachite Green and concealed malachite green.
9th, concentration d (mg/L) of C sample solution Malachite Green sums to be determined is determined；
Determine in the case where current field condition is not added with：Sample solution sample C to be determined step " five " high performance liquid chromatograph
Under chromatographic test strip part, sample solution C to be determined is through electrochemistry coulometric detector (ECD) oxidation voltage 0.0mv, flow velocity
1.0mL/min, malachite green content does not change, and concealed malachite green content does not also change, then through C18Chromatographic column and other things
After matter is separated, detected under 618nm wavelength with UV-detector detection, determine C sample solution Malachite Greens to be determined
Content be d (mg/L), the amount of concealed malachite green is not contained in d (mg/L).
Due to being not added with current field condition, concealed malachite green can not be changed into malachite green.It is not powered then in this step " nine "
Field condition, can only measure concentration d (mg/L) of malachite green sum.
Tenth, the concealed malachite green concentration for calculating measure malachite green sum in sample is e (mg/L)：
The actual concentrations of sample Malachite Green are f=d × 100 ÷ a (mg/L)
The actual concentrations of concealed malachite green are g=(c-d) × 100 ÷ b (mg/L) in sample
The actual concentrations of sample Malachite Green and concealed malachite green are e=f+g (mg/L)；
Actual concentrations with sample Malachite Green and concealed malachite green are that e (mg/L) is converted in 2.0g testing samples
The actual amount of malachite green and concealed malachite green.
Advantages of the present invention：
This research high performance liquid chromatography, sample solution to be determined electrochemistry coulometric detector (ECD) oxidation is flow through
Voltage+650mv, flow velocity 1.0mL/min, just can be oxidized to malachite green, such that it is able to disposable concealed malachite green
Determine total concentration c (mg/L) of sample solution Malachite Green and concealed malachite green.It is set to determine this in aquatic products etc.
The total amount of two Toxics becomes very convenient.And present invention also offers amendment malachite green and concealed malachite green it is total
Concentration c (mg/L) error, obtains side of the actual actual concentrations of sample Malachite Green and concealed malachite green for e (mg/L)
Method.
The present invention is than prior art determination sample Malachite Green and the actual actual concentrations of concealed malachite green, the present invention
Greatly shorten time for sample pretreatment and reduce the use of reagent consumptive material, with electrochemistry column front derivation titanium dioxide in GB is replaced
The post-column derivation of lead.With sample pre-treatments are simple, good purification, the characteristics of derivative rate is high, the rate of recovery is good.
Most critical of the present invention be found that recessive malachite solution through electrochemistry coulometric detector (ECD) oxidation voltage+
650mv, after flow velocity 1.0mL/min, concealed malachite green average 98.86% is changed into malachite green, and original malachite green is not
Change, makes the present invention the concealed malachite green and malachite green in sample in once determining, and can measure recessive peacock
Total concentration c (mg/L) of malachite green and malachite green has foundation.
Specific embodiment
Electrochemical derivatization single-time measurement total amount before the post of embodiment 1, aquatic products Malachite Green and concealed malachite green
Method
Electrochemical derivatization single-time measurement total amount method before the post of aquatic products Malachite Green and concealed malachite green, its step
It is rapid as follows：
First, reagent
Standard substance：Malachite green (content 98.0%)；With stability malachite green (content 98.0%)；
(chromatographically pure, content is more than 99.9%) for acetonitrile；
(chromatographically pure, content is more than 98%) for glacial acetic acid；
(chromatographically pure, content is more than 99.9%) for anhydrous acetic acid ammonium；
(chromatographically pure, content is more than 99%) for dichloromethane；
Acidic alumina solid absorbent (50-200 μm of particle diameter)；
(analysis is pure, and content is more than 99.8%) for sodium chloride.
2nd, the solution to be determined of standard substance malachite green is prepared (referred to as：Standard liquid A to be determined)
Standard substance malachite green 10mg is accurately weighed, with acetonitrile malachite green is dissolved, be configured to concentration containing malachite green
For the standard substance malachite green solution to be determined of 1.0mg/L, A standard liquids to be determined, stand-by chromatograph detection had both been obtained.
3rd, the solution to be determined (standard liquid abbreviation B to be determined) of standard substance concealed malachite green is prepared
Standard substance concealed malachite green 10mg is accurately weighed, with acetonitrile concealed malachite green is dissolved, be configured to containing peacock
Malachite green concentration is the standard substance concealed malachite green solution to be determined of 1.0mg/L, has both obtained B standard liquids to be determined, treats
Detected with chromatograph.
4th, the solution to be determined (abbreviation of sample material malachite green and concealed malachite green is prepared：Sample C to be determined
Solution)
(1) solution is prepared：
Prepare buffer solution：Weigh 3.85g anhydrous acetic acid ammoniums to be dissolved in 1000mL distilled water, glacial acetic acid adjusts pH to 4.5；
Cross 0.45 μm of water system filter membrane；
Prepare extract：By acetonitrile: dichloromethane is standby after mixing for 2: 1 by volume.
(2) sample C sample solutions to be determined are prepared
Take and be put in 50mL centrifuge tubes after 2.0g testing samples are smashed to pieces, add ammonium acetate buffer solution 3mL and extract
5mL, after being mixed with being vortexed, in being put into ultrasonic extractor, promotes malachite green and concealed malachite green to be dissolved in ultrasonic wave
In liquid, with ultrasonic wave dissolution 30min, acidic alumina solid absorbent 1g is added, add sodium chloride 1g, extraction of ocean eddies
2min, 8000r/min rotating speeds centrifugation 5min, takes supernatant in 45 DEG C of rotary evaporated to dryness, with the acetonitrile of 2.0mL on centrifuge
Dissolving, 0.45 μm of organic membrane filtration, the filtered fluid is the solution to be determined containing malachite green and concealed malachite green, both C
Sample solution to be determined.
5th, the chromatographic test strip part of high performance liquid chromatograph
High performance liquid chromatograph is set to：Use C18Chromatographic column (150mm × 4.6mm, 5 μm), mobile phase is acetonitrile；pH
Ammonium acetate buffer solution=78 of 4.5 states: 22 (volume ratios), flow velocity 1.0mL/min, the μ L of sample size 20,35 DEG C of column temperature.
6th, with standard liquid correction malachite green A to be determined is in the absorbing wavelength of high performance liquid chromatograph and determines mistake
Difference a% is (referred to as：The evaluated error of malachite green is a%)：
(6.1) field recognition is powered up：It is A standard liquids to be determined above-mentioned steps " five " the standard liquid of malachite green
Under the chromatographic test strip part of high performance liquid chromatograph, first A standard liquids to be determined are flow through electrochemistry coulometric detector (ECD)
Oxidation voltage+650mv, flow velocity 1.0mL/min, then through C18Chromatographic column, detects under UV-detector detection 618nm wavelength and contains
The concentration value of malachite green；Both the concentration value containing malachite green in A standard liquids to be determined had been measured；With dense containing malachite green
Angle value a1 compares with the standard density 1.0mg/L of step " two " Malachite Green, and the error for obtaining measure malachite green is
A1%.
(6.2) not powered field recognition：It is A standard liquid above-mentioned steps to be determined the standard liquid of malachite green
Under the chromatographic test strip part of " five " high performance liquid chromatograph, first A standard liquids to be determined are flow through electrochemistry coulometric detector
(ECD) oxidation voltage 0.0mv, flow velocity 1.0mL/min, then through C18Chromatographic column, examines under UV-detector detection 618nm wavelength
Survey the concentration value containing malachite green；Both the concentration value containing malachite green in A standard liquids to be determined had been measured；With containing malachite green
Concentration value a2 compare with the standard density 1.0mg/L of step " two " Malachite Green, obtain malachite green evaluated error
For a2%；
Work as a1%=a2%, or when the error of (a1%-a2%)/100 is less than one thousandth, it is average with a1% and a2%
Value is used as a%；After a% is less than one of percentage, next step measure is carried out.
7th, concealed malachite green has been measured by oxygen into after malachite green, used high-efficient liquid phase color with B standard liquids to be determined
Evaluated error b% of spectrometer is (referred to as：The evaluated error of concealed malachite green is b%)：
It is B standard liquid to be determined above-mentioned steps " five " high performance liquid chromatography the standard liquid of concealed malachite green
Under the chromatographic test strip part of instrument, first B standard liquids to be determined flow through electrochemistry coulometric detector (ECD) oxidation voltage+
650mv, flow velocity 1.0mL/min, concealed malachite green malachite green is reduced into, then through C18After chromatographic column, ultraviolet inspection is used
Survey the concentration value that the malachite green for changing into is detected under device detection 618nm wavelength；With the concentration value of the malachite green for changing into
The standard density 1.0mg/L of concealed malachite green compares in step " two ", and the evaluated error for obtaining concealed malachite green is
B%.
8th, total concentration c (mg/L) of C sample solution Malachite Greens to be determined and concealed malachite green is determined；
Determine under the conditions of added electric field：The sample solution sample C to be determined color of step " five " high performance liquid chromatograph
Under spectrum testing conditions, sample solution C to be determined is through electrochemistry coulometric detector (ECD) oxidation voltage+650mv, flow velocity
1.0mL/min, malachite green content does not change, and concealed malachite green is oxidized to malachite green, then through C18After chromatographic column, use
UV-detector detects the detection under 618nm wavelength, determines C sample solution Malachite Greens to be determined and recessive malachite
Green total concentration c (mg/L).
9th, concentration d (mg/L) of C sample solution Malachite Green sums to be determined is determined；
Determine in the case where current field condition is not added with：Sample solution sample C to be determined step " five " high performance liquid chromatograph
Under chromatographic test strip part, sample solution C to be determined is through electrochemistry coulometric detector (ECD) oxidation voltage 0.0mv, flow velocity
1.0mL/min, malachite green content does not change, and concealed malachite green content does not also change, then through C18Chromatographic column and other things
After matter is separated, detected under 618nm wavelength with UV-detector detection, determine C sample solution Malachite Greens to be determined
Content be d (mg/L), the amount of concealed malachite green is not contained in d (mg/L).
Tenth, the concealed malachite green concentration for calculating measure malachite green sum in sample is e (mg/L)：
The actual concentrations of sample Malachite Green are f=d × 100 ÷ a (mg/L)
The actual concentrations of concealed malachite green are g=(c-d) × 100 ÷ b (mg/L) in sample
The actual concentrations of sample Malachite Green and concealed malachite green are e=f+g (mg/L)；
Actual concentrations with sample Malachite Green and concealed malachite green are that e (mg/L) is converted in 2.0g testing samples
The actual amount of malachite green and concealed malachite green.
Claims (1)
1. electrochemical derivatization single-time measurement total amount method, its step before the post of aquatic products Malachite Green and concealed malachite green
It is as follows：
First, reagent
Standard substance：Malachite green, content 98.0%；And concealed malachite green, content 98.0%；
Acetonitrile, chromatographically pure, content is more than 99.9%；
Glacial acetic acid, chromatographically pure, content is more than 98%；
Anhydrous acetic acid ammonium, chromatographically pure, content is more than 99.9%；
Dichloromethane, chromatographically pure, content is more than 99%；
Acidic alumina solid absorbent, 50-200 μm of particle diameter；
Sodium chloride, analyzes pure, and content is more than 99.8%；
2nd, the solution to be determined of standard substance malachite green is prepared
Standard substance malachite green 10mg is accurately weighed, with acetonitrile malachite green is dissolved, being configured to concentration containing malachite green is
The standard substance malachite green solution to be determined of 1.0mg/L, that is, obtain A standard liquids to be determined, stand-by chromatograph detection；
3rd, the solution to be determined of standard substance concealed malachite green is prepared
Standard substance concealed malachite green 10mg is accurately weighed, with acetonitrile concealed malachite green is dissolved, be configured to containing malachite green
Concentration is the standard substance concealed malachite green solution to be determined of 1.0mg/L, that is, obtain B standard liquids to be determined, stand-by color
Spectrometer is detected；
4th, the solution to be determined of sample material malachite green and concealed malachite green is prepared
(1) solution is prepared：
Prepare buffer solution：Weigh 3.85g anhydrous acetic acid ammoniums to be dissolved in 1000mL distilled water, glacial acetic acid adjusts pH to 4.5；Cross 0.45
μm water system filter membrane；
Prepare extract：By acetonitrile: dichloromethane is standby after mixing for 2: 1 by volume；
(2) sample C sample solutions to be determined are prepared
Take and be put in 50mL centrifuge tubes after 2.0g testing samples are smashed to pieces, add ammonium acetate buffer solution 3mL and extract 5mL, use
It is vortexed after mixing, it is molten with the intracellular malachite green of ultrasonic wave promotion organization and concealed malachite green in being put into ultrasonic extractor
Solution, with ultrasonic wave dissolution 30min, adds acidic alumina solid absorbent 1g in liquid, adds sodium chloride 1g, extraction of ocean eddies
2min, 8000r/min rotating speeds centrifugation 5min, takes supernatant in 45 DEG C of rotary evaporated to dryness, with the acetonitrile of 2.0mL on centrifuge
Dissolving, 0.45 μm of organic membrane filtration, the filtered fluid is the solution to be determined containing malachite green and concealed malachite green, i.e. C
Sample solution to be determined；
5th, the chromatographic test strip part of high performance liquid chromatograph
High performance liquid chromatograph is set to：Use C18 chromatographic columns, 150mm × 4.6mm, 5 μm, mobile phase is volume ratio 78: 22
Acetonitrile：The ammonium acetate buffer solution of pH=4.5, flow velocity 1.0mL/min, the μ L of sample size 20,35 DEG C of column temperature；
6th, absorbing wavelength and evaluated error of the malachite green in high performance liquid chromatograph is corrected with standard liquid A to be determined
A%：
(6.1) field recognition is powered up：It is A standard liquids to be determined above-mentioned steps " five " efficient the standard liquid of malachite green
Under the chromatographic test strip part of liquid chromatograph, first A standard liquids to be determined are flow through electrochemistry coulometric detector (ECD) oxidation
Voltage+650mv, flow velocity 1.0mL/min, then through C18 chromatographic columns, under UV-detector detection 618nm wavelength apertures are detected
The concentration value of sparrow malachite green；Measure the concentration value containing malachite green in A standard liquids to be determined；With the concentration containing malachite green
Value a1 compares with the standard density 1.0mg/L of step " two " Malachite Green, and the error for obtaining measure malachite green is a1%；
(6.2) not powered field recognition：It is A standard liquids to be determined above-mentioned steps " five " high the standard liquid of malachite green
Under the chromatographic test strip part of effect liquid phase chromatogram instrument, first A standard liquids to be determined are flow through electrochemistry coulometric detector (ECD) oxygen
Change voltage 0.0mv, flow velocity 1.0mL/min, then through C18 chromatographic columns, under UV-detector detection 618nm wavelength apertures are detected
The concentration value of sparrow malachite green；Measure the concentration value containing malachite green in A standard liquids to be determined；With the concentration containing malachite green
Value a2 compares with the standard density 1.0mg/L of step " two " Malachite Green, and the evaluated error for obtaining malachite green is a2%；
Work as a1%=a2%, or when the error of (a1%-a2%)/100 is less than one thousandth, made with the mean value of a1% and a2%
For a%；After a% is less than one of percentage, next step measure is carried out；
7th, concealed malachite green has been measured by oxygen into after malachite green, used high performance liquid chromatograph with B standard liquids to be determined
Evaluated error b%：
It is B standard liquid to be determined above-mentioned steps " five " high performance liquid chromatographs the standard liquid of concealed malachite green
Under chromatographic test strip part, first B standard liquids to be determined are flow through electrochemistry coulometric detector (ECD) oxidation voltage+650mv,
Flow velocity 1.0mL/min, concealed malachite green malachite green is reduced into, then after C18 chromatographic columns, is examined with UV-detector
Survey the concentration value that the malachite green for changing into is detected under 618nm wavelength；With the concentration value and step of the malachite green for changing into
The standard density 1.0mg/L of concealed malachite green compares in " two ", and the evaluated error for obtaining concealed malachite green is b%；
8th, total concentration c of C sample solution Malachite Greens to be determined and concealed malachite green, mg/L are determined；
Determine under the conditions of added electric field：Sample solution sample C to be determined is examined with the chromatogram of step " five " high performance liquid chromatograph
Under the conditions of survey, sample solution C to be determined is through electrochemistry coulometric detector (ECD) oxidation voltage+650mv, flow velocity 1.0mL/
Min, malachite green content does not change, and concealed malachite green is oxidized to malachite green, then after C18 chromatographic columns, with ultraviolet
Detector detects and detected under 618nm wavelength, determines C sample solution Malachite Greens to be determined and concealed malachite green
Total concentration c, mg/L；
9th, concentration d of C sample solution Malachite Greens to be determined, mg/L are determined；
Determine in the case where current field condition is not added with：The sample solution sample C to be determined chromatogram of step " five " high performance liquid chromatograph
Under testing conditions, sample solution C to be determined is through electrochemistry coulometric detector (ECD) oxidation voltage 0.0mv, flow velocity 1.0mL/
Min, malachite green content does not change, and concealed malachite green content does not also change, then separates with other materials through C18 chromatographic columns
Afterwards, detected under 618nm wavelength with UV-detector detection, determine the content of C sample solution Malachite Greens to be determined
For d, mg/L, the amount of concealed malachite green is not contained in d；
Tenth, the actual concentrations for calculating sample Malachite Green and concealed malachite green are e, mg/L：
The actual concentrations of sample Malachite Green are f=d × 100 ÷ a, mg/L；
The actual concentrations of concealed malachite green are g=(c-d) × 100 ÷ b, mg/L in sample；
The actual concentrations of sample Malachite Green and concealed malachite green are e=f+g, mg/L；
2.0g testing samples Malachite Green and hidden is converted into the actual concentrations e of sample Malachite Green and concealed malachite green
The actual amount of property malachite green.
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| CN110187040B (en) * | 2019-06-26 | 2021-08-17 | 渤海大学 | Sample pretreatment method for simultaneously detecting malachite green residue in freshwater aquaculture water body by liquid chromatography-visible light and fluorescence |
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| CN103399096B (en) * | 2013-08-01 | 2014-07-23 | 浙江省海洋水产研究所 | Method for detecting content of malachite green and metabolin thereof in sediment of aquaculture environment |
| CN103389349B (en) * | 2013-08-01 | 2014-07-23 | 浙江省海洋水产研究所 | Method for detecting content of malachite green and metabolin thereof in aquiculture environment water body |
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