CN110376307A - The method for detecting residue of aquatic products Malachite Green - Google Patents
The method for detecting residue of aquatic products Malachite Green Download PDFInfo
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Abstract
The present invention relates to the method for detecting residue of aquatic products Malachite Green, belong to the technical field of malachite green detection method comprising following steps: S1: the preparation of solution；S2: mixed-powder is prepared；S3:SPE column fills column；S4: Solid Phase Extraction；S5: High Performance Liquid Chromatography/Mass Spectrometry detection.The detection method rate of recovery provided by the invention is high, favorable reproducibility.
Description
Technical field
The present invention relates to the technical fields of malachite green detection method, more particularly, to the residual of aquatic products Malachite Green
Detection method.
Background technique
Peacock green belongs to a kind of industrial dye, and treatment saprolegniasis, cheek mildew and environment are often used in aquaculture
Disinfection etc..But malachite green and its metabolite leucogentian violet are eliminated slowly in animal body, and the residence time is long, and are had
There are carcinogenic, teratogenesis and mutagenesis.
Currently, effective control of government agencies at all levels has been obtained in aquaculture the phenomenon that Misuse malachite green, but
Still there are a small number of fishermen using malachite green, needs to be monitored aquaculture, the use of strict control malachite green.
Therefore, establish that a kind of rate of recovery is high, the malachite green detection technique of favorable reproducibility is necessary.
Summary of the invention
The purpose of the present invention one is to provide a kind of method for detecting residue of aquatic products Malachite Green, this method rate of recovery
High, favorable reproducibility.
Above-mentioned purpose of the invention has the technical scheme that
The method for detecting residue of aquatic products Malachite Green, comprising the following steps:
S1: mixing internal standard standard solution and sample solution the preparation of solution: are prepared；
S2: prepare mixed-powder: it is molten for chitosan is made in 1% acetum to dissolve the chitosan in 6-8mL mass fraction
Then diatomite and active carbon is added in liquid, the NaOH solution that mass fraction is 5% is added dropwise, and being adjusted to PH is 9, and solution starts to coagulate
After knot, then the HCl that mass fraction is 5%, which is added dropwise, is in neutrality mixed solution, and dry 1-2h, grinds after taking-up at 100-110 DEG C
It is broken into powder and mixed-powder is made；Wherein, chitosan, diatomite, active carbon weight fraction ratio be 1:1.2-1.7:7-8.5；
S3:SPE column fills column: mixed-powder and carbon nanotube being mixed to prepare filler, and filler is fitted into SPE column；Wherein, carbon
The weight fraction ratio of nanotube and mixed-powder is 1:1.5-2.8；
S4: it Solid Phase Extraction: takes the acetonitrile that 2-4mL mass fraction is 20% to activate SPE column, the sample solution in S1 is carried out
The mixing that then acetonitrile that sample is successively 80% with 2-4mL mass fraction, 2-4mL acetonitrile, ammonium acetate weight fraction ratio are 1:1
Solution is eluted, and is collected eluent and is concentrated into and closely does, and acetonitrile is added and is settled to 1mL, and 1mL5mol/L ammonium acetate, shake is added
It swings rear spare；
S5: Spectrometry High Performance Liquid Chromatography/Mass Spectrometry detection: is carried out to the sample solution after shaking in step S4；Chromatography
Condition are as follows:
Mobile phase A: the formic acid solution that mass fraction is 0.1%；
Mobile phase B: acetonitrile；Flow velocity is 0.25mL/min；2 μ L of sample volume.
By using above-mentioned technical proposal, pre-treatment, the filler of SPE column are carried out to sample solution using solid phase extraction techniques
Selecting is self-made fill, is first uniformly mixed chitosan, diatomite and active carbon and mixed-powder is made；Chitosan, diatom
Soil and active carbon all have stronger adsorptivity, after being configured to mixed-powder with certain proportion, then are uniformly mixed with carbon nanotube
Filler is made；Mixed-powder can be filled in the gap of carbon nanotube, when carrying out Solid Phase Extraction, can effectively adsorb malachite green and hidden
Color malachite green；Then malachite green and leucogentian violet are eluted with the mode of acetonihile gradient elution, with acetonitrile with
Ammonium acetate weight fraction ratio be 1:1 mixed solution, eluted again, improve malachite green and leucogentian violet elution
Completeness；Finally use high performance liquid chromatography detection, the rate of recovery height of detection method provided by the invention, favorable reproducibility.
The present invention is further arranged to: in the step S3, the weight fraction ratio of carbon nanotube and mixed-powder is 1:
2.0-2.4。
By using above-mentioned technical proposal, carbon nanotube is nanotube-shaped, and the granularity of mixed-powder compares carbon nanotube
Small, the compactness that filler is filled in SPE column can be improved in the proportion of strict control mixed-powder and carbon nanotube, reduces carbon nanometer
Adsorption effect and elution effect to malachite green and leucogentian violet are improved in gap between pipe.
The present invention is further arranged to: in the step S4, it is concentrated into such a way that nitrogen is blown after collection eluent and closely does,
The temperature that nitrogen is blown is 45 DEG C.
It by using above-mentioned technical proposal, is concentrated in such a way that nitrogen is blown, can quickly remove the moisture in eluent, accelerated
Speed is concentrated, reduces the concentration spent time, in addition, the mode that nitrogen is blown can also reduce malachite green and leucogentian violet damage
A possibility that mistake, improves the rate of recovery.
The present invention is further arranged to: in the step S4, after eluent constant volume and concussion, after 0.2 μm of membrane filtration
It is tested again.
By using above-mentioned technical proposal, the degree of purity of eluent after constant volume can be improved after membrane filtration, be conducive to
The fluency of subsequent high performance liquid chromatography sample introduction.
The present invention is further arranged to: it is described mixing internal standard standard solution preparation the following steps are included:
(a) malachite green, leucogentian violet, deuterated malachite green, deuterated leucogentian violet standard items are taken respectively, use acetonitrile
It is formulated as the standard reserving solution of 100 μ g/mL respectively；
(b) standard reserving solution for drawing 1mL malachite green and leucogentian violet respectively is with dilution in acetonitrile after mixing
The hybrid standard stock solution of 2ng/mL, 4ng/mL, 8ng/mL, 10ng/mL, 20ng/mL, 30ng/mL；
(c) standard reserving solution for drawing the deuterated malachite green of 1mL and deuterated leucogentian violet respectively, uses acetonitrile after mixing
It is diluted to the mixing internal standard standard reserving solution of 2ng/mL, 4ng/mL, 8ng/mL, 10ng/mL, 20ng/mL, 30ng/mL.
The present invention is further arranged to: the preparation method of the sample solution has been smashed to pieces the following steps are included: weighing 4-6g
Sample is added 200-205 μ L and mixes internal standard standard solution, and 10-15mL acetonitrile is added, and ultrasonic oscillation is mentioned with homogenate again after extracting
It takes, washs homogenate cutter head with acetonitrile, merge that with acetonitrile to be settled to 25mL after supernatant spare.
By using above-mentioned technical proposal, the loss of mixing internal standard standard solution can be reduced by washing homogenate cutter head with acetonitrile
Amount improves accuracy when detection.
The present invention is further arranged to: the high performance liquid chromatography takes gradient elution, gradient elution program are as follows: 0-
3.5min: Mobile phase B keeps 15%；3.5-4.5min: Mobile phase B keeps 50%；4.5-6min: Mobile phase B keeps 95%；6-
7min: Mobile phase B keeps 15%.
In conclusion advantageous effects of the invention are as follows:
1. filler is made in mixed-powder and carbon nanotube, mixed-powder be can be filled in the gap of carbon nanotube, carry out solid phase extraction
When taking, malachite green and leucogentian violet can be effectively adsorbed, improves the concentration effect to malachite green and leucogentian violet,
Elution effect is improved with the mode of gradient elution again, in conjunction with high-efficient liquid phase chromatogram technology, so that detection side provided by the invention
The rate of recovery height of method, favorable reproducibility；
2. the compactness that filler is filled in SPE column can be improved in the proportion of strict control mixed-powder and carbon nanotube, reduce carbon
Adsorption effect and elution effect to malachite green and leucogentian violet are improved in gap between nanotube.
Specific embodiment
Embodiment 1
One, the method for detecting residue of aquatic products Malachite Green, comprising the following steps:
S1: the preparation of solution:
Prepare mixing internal standard standard solution:
(a) malachite green, leucogentian violet, deuterated malachite green, deuterated leucogentian violet standard items are taken respectively, use acetonitrile
It is formulated as the standard reserving solution of 100 μ g/mL respectively；
(b) standard reserving solution for drawing 1mL malachite green and leucogentian violet respectively is with dilution in acetonitrile after mixing
The hybrid standard stock solution of 2ng/mL, 4ng/mL, 8ng/mL, 10ng/mL, 20ng/mL, 30ng/mL；
(c) standard reserving solution for drawing the deuterated malachite green of 1mL and deuterated leucogentian violet respectively, uses acetonitrile after mixing
It is diluted to the mixing internal standard standard reserving solution of 2ng/mL, 4ng/mL, 8ng/mL, 10ng/mL, 20ng/mL, 30ng/mL.
It prepares sample solution: weighing 4g and smashed sample to pieces, 200 μ L are added and mix internal standard standard solution, 10mL acetonitrile is added,
It is extracted again with homogenate after ultrasonic oscillation extraction, is washed with acetonitrile and be homogenized cutter head, be settled to 25mL with acetonitrile after merging supernatant
It is spare.
S2: it prepares mixed-powder: dissolving the chitosan in the acetum that 6mL mass fraction is 1% and chitosan is made
Then diatomite and active carbon is added in solution, the NaOH solution that mass fraction is 5% is added dropwise, and being adjusted to PH is 9, and solution starts
After condensation, then the HCl that mass fraction is 5%, which is added dropwise, is in neutrality mixed solution, the dry 1h at 100 DEG C, ground after taking-up at
Mixed-powder is made in powder；Wherein, chitosan, diatomite, active carbon weight fraction ratio be 1:1.2:7；
S3:SPE column fills column: mixed-powder and carbon nanotube being mixed to prepare filler, and filler is fitted into SPE column；Wherein, carbon
The weight fraction ratio of nanotube and mixed-powder is 1:1.5；
S4: Solid Phase Extraction: taking the acetonitrile that 2mL mass fraction is 20% to activate SPE column, and the sample solution in S1 is carried out loading,
Then with the acetonitrile of 2mL: ammonium acetate is that the mixed solution of 1:1 is eluted, and collects eluent and nitrogen blows and is concentrated at 45 DEG C
It is close dry, acetonitrile is added and is settled to 1mL, 1mL5mol/L ammonium acetate is added, it is spare through 0.2 μm of membrane filtration after concussion；
S5: Spectrometry High Performance Liquid Chromatography/Mass Spectrometry detection: is carried out to the sample solution after shaking in step S4；Chromatography
Condition are as follows:
Mobile phase A: the formic acid solution that mass fraction is 0.1%；
Mobile phase B: acetonitrile；Flow velocity is 0.25mL/min；2 μ L of sample volume；
Gradient elution program is shown in Table 1:
The gradient elution program of 1 embodiment 1 of table
| Time/min | Mobile phase A/% | Mobile phase B/% |
| 0-3.5 | 85 | 15 |
| 3.5-4.5 | 50 | 50 |
| 4.5-6 | 5 | 95 |
| 6-7 | 85 | 15 |
According to the chromatographic condition of the above high performance liquid chromatography, produced with internal mark method determination sample solution Malachite Green and its metabolism
The content of object leucogentian violet.
Two, linear relationship and detection limit
It is measured according to the above chromatographic condition, respectively using malachite green and the peak area Y of leucogentian violet as ordinate, with to be checked
The mass concentration X for surveying object is that abscissa draws standard curve and calculates equation of linear regression and detection limit, the results are shown in Table 2.
The linear relationship and detection limit of 2 embodiment 1 of table
| Equation of linear regression | Related coefficient | Detection limit (μ g/kg) | |
| Malachite green | Y=1.1435x+0.3241 | 0.9999 | 1.4 |
| Leucogentian violet | Y=0.8232x+1.6841 | 0.9998 | 1.6 |
As can be seen from Table 2, the related coefficient of malachite green and the equation of linear regression of leucogentian violet reach 0.9998 and with
On, detection limit is in 2 μ g/kg hereinafter, illustrating that the detection method of the present embodiment has preferable linear and higher sensitivity.
Three, measurement result
The measurement result of 3 embodiment 1 of table
| Residual quantity (μ g/kg) | |
| Malachite green | 14.23 |
| Leucogentian violet | 16.75 |
Four, the rate of recovery and Precision Experiment
It is detected using negative sample (without peacock green and leucomalachite green), with the method for adding standard solution in sample,
According to same detection method, recovery of standard addition experiment is carried out, each concentration does 3 Duplicate Samples, and each sample is in identical condition
Lower continuous survey 5 times, calculate relative standard deviation；It the results are shown in Table 4.
The recovery of standard addition and relative standard deviation of 4 embodiment 1 of table
| Average recovery of standard addition (%) | Relative standard deviation (%) | |
| Malachite green | 98.5 | 3.3 |
| Leucogentian violet | 97.6 | 4.1 |
By table 4 as it can be seen that average recovery of standard addition is up to 97.5% or more, relative standard deviation is respectively less than 5%, illustrates this implementation
The detection method accuracy of example is higher.
Embodiment 2
The method for detecting residue of aquatic products Malachite Green, comprising the following steps:
S1: the preparation of solution:
Prepare mixing internal standard standard solution:
(a) malachite green, leucogentian violet, deuterated malachite green, deuterated leucogentian violet standard items are taken respectively, use acetonitrile
It is formulated as the standard reserving solution of 100 μ g/mL respectively；
(b) standard reserving solution for drawing 1mL malachite green and leucogentian violet respectively is with dilution in acetonitrile after mixing
The hybrid standard stock solution of 2ng/mL, 4ng/mL, 8ng/mL, 10ng/mL, 20ng/mL, 30ng/mL；
(c) standard reserving solution for drawing the deuterated malachite green of 1mL and deuterated leucogentian violet respectively, uses acetonitrile after mixing
It is diluted to the mixing internal standard standard reserving solution of 2ng/mL, 4ng/mL, 8ng/mL, 10ng/mL, 20ng/mL, 30ng/mL.
It prepares sample solution: weighing 5g and smashed sample to pieces, 202 μ L are added and mix internal standard standard solution, 13mL acetonitrile is added,
It is extracted again with homogenate after ultrasonic oscillation extraction, is washed with acetonitrile and be homogenized cutter head, be settled to 25mL with acetonitrile after merging supernatant
It is spare.
S2: it prepares mixed-powder: dissolving the chitosan in the acetum that 7mL mass fraction is 1% and chitosan is made
Then diatomite and active carbon is added in solution, the NaOH solution that mass fraction is 5% is added dropwise, and being adjusted to PH is 9, and solution starts
After condensation, then the HCl that mass fraction is 5%, which is added dropwise, is in neutrality mixed solution, and dry 1.5h, grinds after taking-up at 105 DEG C
Mixed-powder is made at powder；Wherein, chitosan, diatomite, active carbon weight fraction ratio be 1:1.4:7.8；
S3:SPE column fills column: mixed-powder and carbon nanotube being mixed to prepare filler, and filler is fitted into SPE column；Wherein, carbon
The weight fraction ratio of nanotube and mixed-powder is 1:2.2；
S4: Solid Phase Extraction: taking the acetonitrile that 3mL mass fraction is 20% to activate SPE column, and the sample solution in S1 is carried out loading,
Then with the acetonitrile of 3mL: ammonium acetate is that the mixed solution of 1:1 is eluted, and collects eluent and nitrogen blows and is concentrated at 45 DEG C
It is close dry, acetonitrile is added and is settled to 1mL, 1mL5mol/L ammonium acetate is added, it is spare through 0.2 μm of membrane filtration after concussion；
S5: Spectrometry High Performance Liquid Chromatography/Mass Spectrometry detection: is carried out to the sample solution after shaking in step S4；Chromatography
Condition are as follows:
Mobile phase A: the formic acid solution that mass fraction is 0.1%；
Mobile phase B: acetonitrile；Flow velocity is 0.25mL/min；2 μ L of sample volume；
Gradient elution program is shown in Table 5:
The gradient elution program of 5 embodiment 2 of table
| Time/min | Mobile phase A/% | Mobile phase B/% |
| 0-3.5 | 85 | 15 |
| 3.5-4.5 | 50 | 50 |
| 4.5-6 | 5 | 95 |
| 6-7 | 85 | 15 |
Two, linear relationship and detection limit
It is measured according to the above chromatographic condition, respectively using malachite green and the peak area Y of leucogentian violet as ordinate, with to be checked
The mass concentration X for surveying object is that abscissa draws standard curve and calculates regression equation and detection limit, the results are shown in Table 6.
The linear relationship and detection limit of 6 embodiment 2 of table
| Equation of linear regression | Related coefficient | Detection limit (μ g/kg) | |
| Malachite green | Y=1.2721x+0.6864 | 0.9999 | 1.3 |
| Leucogentian violet | Y=0.7541x+1.2574 | 0.9999 | 1.5 |
By table 6 as it can be seen that the related coefficient of malachite green and the equation of linear regression of leucogentian violet reaches 0.9999, detection
It limits in 1.6 μ g/kg hereinafter, illustrating that the detection method of the present embodiment has preferable linear and higher sensitivity.
Three, measurement result
The measurement result of 7 embodiment 1 of table
| Residual quantity (μ g/kg) | |
| Malachite green | 12.55 |
| Leucogentian violet | 13.69 |
Four, the rate of recovery and Precision Experiment
It is detected using negative sample (without peacock green and leucomalachite green), with the method for adding standard solution in sample,
According to same detection method, recovery of standard addition experiment is carried out, each concentration does 3 Duplicate Samples, and each sample is in identical condition
Lower continuous survey 5 times, calculate relative standard deviation；It the results are shown in Table 8.
The recovery of standard addition and relative standard deviation of 8 embodiment 2 of table
| Average recovery of standard addition (%) | Relative standard deviation (%) | |
| Malachite green | 99.1 | 3.2 |
| Leucogentian violet | 98.2 | 3.7 |
By table 8 as it can be seen that average recovery of standard addition is up to 98.0% or more, relative standard deviation is respectively less than 4%, illustrates this implementation
The detection method accuracy of example is higher.
Embodiment 3
The method for detecting residue of aquatic products Malachite Green, comprising the following steps:
S1: the preparation of solution:
Prepare mixing internal standard standard solution:
(a) malachite green, leucogentian violet, deuterated malachite green, deuterated leucogentian violet standard items are taken respectively, use acetonitrile
It is formulated as the standard reserving solution of 100 μ g/mL respectively；
(b) standard reserving solution for drawing 1mL malachite green and leucogentian violet respectively is with dilution in acetonitrile after mixing
The hybrid standard stock solution of 2ng/mL, 4ng/mL, 8ng/mL, 10ng/mL, 20ng/mL, 30ng/mL；
(c) standard reserving solution for drawing the deuterated malachite green of 1mL and deuterated leucogentian violet respectively, uses acetonitrile after mixing
It is diluted to the mixing internal standard standard reserving solution of 2ng/mL, 4ng/mL, 8ng/mL, 10ng/mL, 20ng/mL, 30ng/mL.
It prepares sample solution: weighing 6g and smashed sample to pieces, 205 μ L are added and mix internal standard standard solution, 15mL acetonitrile is added,
It is extracted again with homogenate after ultrasonic oscillation extraction, is washed with acetonitrile and be homogenized cutter head, be settled to 25mL with acetonitrile after merging supernatant
It is spare.
S2: it prepares mixed-powder: dissolving the chitosan in the acetum that 8mL mass fraction is 1% and chitosan is made
Then diatomite and active carbon is added in solution, the NaOH solution that mass fraction is 5% is added dropwise, and being adjusted to PH is 9, and solution starts
After condensation, then the HCl that mass fraction is 5%, which is added dropwise, is in neutrality mixed solution, the dry 2h at 110 DEG C, ground after taking-up at
Mixed-powder is made in powder；Wherein, chitosan, diatomite, active carbon weight fraction ratio be 1:1.7:8.5；
S3:SPE column fills column: mixed-powder and carbon nanotube being mixed to prepare filler, and filler is fitted into SPE column；Wherein, carbon
The weight fraction ratio of nanotube and mixed-powder is 1:2.8；
S4: Solid Phase Extraction: taking the acetonitrile that 4mL mass fraction is 20% to activate SPE column, and the sample solution in S1 is carried out loading,
Then with the acetonitrile of 4mL: ammonium acetate is that the mixed solution of 1:1 is eluted, and collects eluent and nitrogen blows and is concentrated at 45 DEG C
It is close dry, acetonitrile is added and is settled to 1mL, 1mL5mol/L ammonium acetate is added, it is spare through 0.2 μm of membrane filtration after concussion；
S5: Spectrometry High Performance Liquid Chromatography/Mass Spectrometry detection: is carried out to the sample solution after shaking in step S4；Chromatography
Condition are as follows:
Mobile phase A: the formic acid solution that mass fraction is 0.1%；
Mobile phase B: acetonitrile；Flow velocity is 0.25mL/min；2 μ L of sample volume；
Gradient elution program is shown in Table 9:
The gradient elution program of 9 embodiment 3 of table
| Time/min | Mobile phase A/% | Mobile phase B/% |
| 0-3.5 | 85 | 15 |
| 3.5-4.5 | 50 | 50 |
| 4.5-6 | 5 | 95 |
| 6-7 | 85 | 15 |
Two, linear relationship and detection limit
It is measured according to the above chromatographic condition, respectively using malachite green and the peak area Y of leucogentian violet as ordinate, with to be checked
The mass concentration X for surveying object is that abscissa draws standard curve and calculates regression equation and detection limit, the results are shown in Table 10.
The linear relationship and detection limit of 10 embodiment 3 of table
| Equation of linear regression | Related coefficient | Detection limit (μ g/kg) | |
| Malachite green | Y=1.0763x+0.3561 | 0.9998 | 1.3 |
| Leucogentian violet | Y=0.8246x+0.6934 | 0.9999 | 1.5 |
By table 10 as it can be seen that the related coefficient of malachite green and the equation of linear regression of leucogentian violet reach 0.9998 and with
On, detection limit is in 1.6 μ g/kg hereinafter, illustrating that the detection method of the present embodiment has preferable linear and higher sensitivity.
Three, measurement result
The measurement result of 11 embodiment 1 of table
| Residual quantity (μ g/kg) | |
| Malachite green | 11.67 |
| Leucogentian violet | 13.35 |
Three, the rate of recovery and Precision Experiment
It is detected using negative sample (without peacock green and leucomalachite green), with the method for adding standard solution in sample,
According to same detection method, recovery of standard addition experiment is carried out, each concentration does 3 Duplicate Samples, and each sample is in identical condition
Lower continuous survey 5 times, calculate relative standard deviation；It the results are shown in Table 12.
The recovery of standard addition and relative standard deviation of 12 embodiment 3 of table
| Average recovery of standard addition (%) | Relative standard deviation (%) | |
| Malachite green | 98.7 | 3.5 |
| Leucogentian violet | 98.6 | 3.8 |
By table 12 as it can be seen that average recovery of standard addition is up to 98.5% or more, relative standard deviation is respectively less than 4%, illustrates this reality
The detection method accuracy for applying example is higher.
Embodiment 4
The method for detecting residue of aquatic products Malachite Green, with embodiment 2 the difference is that: in step S3, carbon nanotube
Weight fraction ratio with mixed-powder is 1:2.0.
Two, linear relationship and detection limit
It is measured according to the above chromatographic condition, respectively using malachite green and the peak area Y of leucogentian violet as ordinate, with to be checked
The mass concentration X for surveying object is that abscissa draws standard curve and calculates regression equation and detection limit, the results are shown in Table 13.
The linear relationship and detection limit of 13 embodiment 4 of table
| Equation of linear regression | Related coefficient | Detection limit (μ g/kg) | |
| Malachite green | Y=1.2251x+0.7569 | 0.9997 | 1.2 |
| Leucogentian violet | Y=0.8644x+1.3251 | 0.9999 | 1.4 |
By table 13 as it can be seen that the related coefficient of malachite green and the equation of linear regression of leucogentian violet reach 0.9997 and with
On, detection limit is in 1.5 μ g/kg hereinafter, illustrating that the detection method of the present embodiment has preferable linear and higher sensitivity.
Three, measurement result
The measurement result of 14 embodiment 4 of table
| Residual quantity (μ g/kg) | |
| Malachite green | 12.81 |
| Leucogentian violet | 15.33 |
Four, the rate of recovery and Precision Experiment
It is detected using negative sample (without peacock green and leucomalachite green), with the method for adding standard solution in sample,
According to same detection method, recovery of standard addition experiment is carried out, each concentration does 3 Duplicate Samples, and each sample is in identical condition
Lower continuous survey 5 times, calculate relative standard deviation；It the results are shown in Table 15.
The recovery of standard addition and relative standard deviation of 15 embodiment 4 of table
| Average recovery of standard addition (%) | Relative standard deviation (%) | |
| Malachite green | 99.0 | 3.7 |
| Leucogentian violet | 99.1 | 4.3 |
By table 15 as it can be seen that average recovery of standard addition is up to 99.0% or more, relative standard deviation is respectively less than 5%, illustrates this reality
The detection method accuracy for applying example is higher.
Embodiment 5
The method for detecting residue of aquatic products Malachite Green, with embodiment 2 the difference is that: in step S3, carbon nanotube
Weight fraction ratio with mixed-powder is 1:2.4.
Two, linear relationship and detection limit
It is measured according to the above chromatographic condition, respectively using malachite green and the peak area Y of leucogentian violet as ordinate, with to be checked
The mass concentration X for surveying object is that abscissa draws standard curve and calculates regression equation and detection limit, the results are shown in Table 16.
The linear relationship and detection limit of 16 embodiment 5 of table
| Equation of linear regression | Related coefficient | Detection limit (μ g/kg) | |
| Malachite green | Y=1.0155x+1.2243 | 0.9999 | 1.5 |
| Leucogentian violet | Y=0.6958x+1.8712 | 0.9999 | 1.7 |
By table 16 as it can be seen that the related coefficient of malachite green and the equation of linear regression of leucogentian violet reach 0.9999 and with
On, detection limit is in 2 μ g/kg hereinafter, illustrating that the detection method of the present embodiment has preferable linear and higher sensitivity.
Three, measurement result
The measurement result of 17 embodiment 1 of table
| Residual quantity (μ g/kg) | |
| Malachite green | 13.51 |
| Leucogentian violet | 16.24 |
Four, the rate of recovery and Precision Experiment
It is detected using negative sample (without peacock green and leucomalachite green), with the method for adding standard solution in sample,
According to same detection method, recovery of standard addition experiment is carried out, each concentration does 3 Duplicate Samples, and each sample is in identical condition
Lower continuous survey 5 times, calculate relative standard deviation；It the results are shown in Table 18.
The recovery of standard addition and relative standard deviation of 18 embodiment 5 of table
| Average recovery of standard addition (%) | Relative standard deviation (%) | |
| Malachite green | 98.5 | 3.3 |
| Leucogentian violet | 97.6 | 4.1 |
By table 18 as it can be seen that average recovery of standard addition is up to 97.5% or more, relative standard deviation is respectively less than 5%, illustrates this reality
The detection method accuracy for applying example is higher.
Comparative example 1
One, it is detected referring to the detection method in standard GB/T/T 19857-2005.
Two, linear relationship and detection limit
Respectively using malachite green and the peak area Y of leucogentian violet as ordinate, with the mass concentration X of object to be detected for horizontal seat
Plotting standard curve simultaneously calculates equation of linear regression and detection limit, the results are shown in Table 19.
The linear relationship and detection limit of 19 comparative example 1 of table
| Equation of linear regression | Related coefficient | Detection limit (μ g/kg) | |
| Malachite green | Y=1.2468x+0.9374 | 0.9996 | 2.0 |
| Leucogentian violet | Y=0.7782x+1.2367 | 0.9995 | 2.0 |
Three, measurement result
The measurement result of 20 comparative example 1 of table
| Residual quantity (μ g/kg) | |
| Malachite green | 10.56 |
| Leucogentian violet | 12.33 |
Four, the rate of recovery and Precision Experiment
It is detected using negative sample (without peacock green and leucomalachite green), with the method for adding standard solution in sample,
According to same detection method, recovery of standard addition experiment is carried out, each concentration does 3 Duplicate Samples, and each sample is in identical condition
Lower continuous survey 5 times, calculate relative standard deviation；It the results are shown in Table 21.
The recovery of standard addition and relative standard deviation of 21 comparative example 1 of table
| Average recovery of standard addition (%) | Relative standard deviation (%) | |
| Malachite green | 97.1 | 5.2 |
| Leucogentian violet | 97.5 | 5.6 |
It can be seen that by table 19,20,21, in comparative example 1, the phase relation of the equation of linear regression of malachite green and leucogentian violet
Several and recovery of standard addition is below the data in embodiment 1-5, and detection limit is higher than the data in embodiment 1-5, and comparative example 1
Relative standard deviation be greater than 5%, and embodiment 1-5 is below 5%, illustrates the present invention in fixed extractor technology with mixed powder
The mixture of end and carbon nanotube can effectively adsorb malachite green and leucogentian violet as filler, and with acetonitrile and acetic acid
The mixture of ammonium carries out liquid phase analysis as eluant, eluent by the way of gradient elution, to more sensitively detect aquatic products
The residual of Malachite Green.
In conclusion detection method rate of recovery height provided by the present invention and favorable reproducibility.
The embodiment of present embodiment is presently preferred embodiments of the present invention, not limits protection of the invention according to this
Range, therefore: the equivalence changes that all structures under this invention, shape, principle are done, should all be covered by protection scope of the present invention it
It is interior.
Claims (7)
1. the method for detecting residue of aquatic products Malachite Green, it is characterised in that: the following steps are included:
S1: mixing internal standard standard solution and sample solution the preparation of solution: are prepared；
S2: prepare mixed-powder: it is molten for chitosan is made in 1% acetum to dissolve the chitosan in 6-8mL mass fraction
Then diatomite and active carbon is added in liquid, the NaOH solution that mass fraction is 5% is added dropwise, and being adjusted to PH is 9, and solution starts to condense
Afterwards, then the HCl that mass fraction is 5%, which is added dropwise, is in neutrality mixed solution, the dry 1-2h at 100-110 DEG C, ground after taking-up at
Mixed-powder is made in powder；Wherein, chitosan, diatomite, active carbon weight fraction ratio be 1:1.2-1.7:7-8.5；
S3:SPE column fills column: mixed-powder and carbon nanotube being mixed to prepare filler, and filler is fitted into SPE column；Wherein, carbon
The weight fraction ratio of nanotube and mixed-powder is 1:1.5-2.8；
S4: Solid Phase Extraction: taking the acetonitrile that 2-4mL mass fraction is 20% to activate SPE column, and the sample solution in S1 is carried out loading,
Then with the acetonitrile of 2-4mL: ammonium acetate is that the mixed solution of 1:1 is eluted, and collects eluent and is concentrated into close dry, addition second
Nitrile is settled to 1mL, and 1mL5mol/L ammonium acetate is added, spare after concussion；
S5: Spectrometry High Performance Liquid Chromatography/Mass Spectrometry detection: is carried out to the sample solution after shaking in step S4；Chromatography
Condition are as follows:
Mobile phase A: the formic acid solution that mass fraction is 0.1%；
Mobile phase B: acetonitrile；Flow velocity is 0.25mL/min；2 μ L of sample volume.
2. the method for detecting residue of aquatic products Malachite Green according to claim 1, it is characterised in that: the step S3
In, the weight fraction ratio of carbon nanotube and mixed-powder is 1:2.0-2.4.
3. the method for detecting residue of aquatic products Malachite Green according to claim 1, it is characterised in that: the step S4
In, it is concentrated into such a way that nitrogen is blown after collection eluent and closely does, the temperature that nitrogen is blown is 45 DEG C.
4. the method for detecting residue of aquatic products Malachite Green according to claim 1, it is characterised in that: the step S4
In, after eluent constant volume and concussion, tested again after 0.2 μm of membrane filtration.
5. the method for detecting residue of aquatic products Malachite Green according to claim 1, it is characterised in that: in the mixing
Mark standard solution preparation the following steps are included:
(a) malachite green, leucogentian violet, deuterated malachite green, deuterated leucogentian violet standard items are taken respectively, use acetonitrile
It is formulated as the standard reserving solution of 100 μ g/mL respectively；
(b) standard reserving solution for drawing 1mL malachite green and leucogentian violet respectively is with dilution in acetonitrile after mixing
The hybrid standard stock solution of 2ng/mL, 4ng/mL, 8ng/mL, 10ng/mL, 20ng/mL, 30ng/mL；
(c) standard reserving solution for drawing the deuterated malachite green of 1mL and deuterated leucogentian violet respectively, uses acetonitrile after mixing
It is diluted to the mixing internal standard standard reserving solution of 2ng/mL, 4ng/mL, 8ng/mL, 10ng/mL, 20ng/mL, 30ng/mL.
6. the method for detecting residue of aquatic products Malachite Green according to claim 5, it is characterised in that: the sample is molten
The preparation method of liquid has smashed sample to pieces the following steps are included: weighing 4-6g, and 200-205 μ L is added and mixes internal standard standard solution, adds
Enter 10-15mL acetonitrile, ultrasonic oscillation is extracted with homogenate again after extracting, and is washed homogenate cutter head with acetonitrile, is used after merging supernatant
It is spare that acetonitrile is settled to 25mL.
7. the method for detecting residue of aquatic products Malachite Green according to claim 1, it is characterised in that: the efficient liquid
Phase chromatography takes gradient elution, gradient elution program are as follows: 0-3.5min: Mobile phase B keeps 15%；3.5-4.5min: Mobile phase B
Keep 50%；4.5-6min: Mobile phase B keeps 95%；6-7min: Mobile phase B keeps 15%.
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Application publication date: 20191025 |