CN109709232A - The remaining determination method of Bravo in aquatic animal tissue - Google Patents
The remaining determination method of Bravo in aquatic animal tissue Download PDFInfo
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- CN109709232A CN109709232A CN201910082702.1A CN201910082702A CN109709232A CN 109709232 A CN109709232 A CN 109709232A CN 201910082702 A CN201910082702 A CN 201910082702A CN 109709232 A CN109709232 A CN 109709232A
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Abstract
The invention discloses a kind of aquatic animal tissue in the remaining determination method of Bravo, belong to detection technique field, the determination method the following steps are included: 1) aquatic animal tissue in Bravo extraction；2) Bravo detection in aquatic animal tissue.This method is in completing aquatic animal tissue on the basis of Bravo extraction, sample pre-treatments, chromatographic condition and detection method etc. are optimized, so that the determination method treatment effeciency is high, quickly detection, high sensitivity, especially Bravo can be precisely quantified in aquatic animal is organized, provide reliable analysis means for the contents level and distribution characteristics of Bravo in general census aquatic animal tissue.
Description
Technical field
The present invention relates to detection technique field, the remaining detection point of Bravo in specifically a kind of aquatic animal tissue
Analysis method.
Background technique
Bravo (chlorothalonil) chemical name is daconil M, is a kind of non-internal-absorting, wide spectrum, efficiently
Protective fungicide, there is preventive and therapeutic effect to the fungal disease of various crop.Its sterilization mechanism is and the phosphorus in fungal cell
Acid glycerol aldehyde dehydrogenase is had an effect, and in conjunction with the protein containing cysteine in dehydrogenase body, makes dehydrogenase loss of activity, is broken
The metabolism of bad fungal cell, so as to cause the death of fungi.
Since production, the use in world's agricultural production so far alreadys exceed 50 years Bravo, due to extensive and long
Phase uses, and the residual of Bravo is all detected in crops and natural ecological environment, has to environment and food safety potential
It threatens.Bravo can cause the symptoms such as scytitis, eyes discomfort and gastrointestinal irritation, to fish, shellfish to people and mammal low toxicity
The aquatiles high poison such as class, Environmental protection general administration have been classified as one of the substance that the mankind may be made carcinogenic.I
Bravo has been included in monitoring range for the first time by " standards for drinking water quality " of state's revision in 2012.
201210338528.0 disclose it is a kind of quickly, sensitive detection fruit and vegetable surfaces pesticide (including Bravo) it is residual
The method stayed, using positive and negative ion mode, establishes ion mobility spectrometry inspection with Ion mobility spectrometry for basic detection technique
Survey fast qualitative, the semi-quantitative analysis method of pesticide residue.The sampling slice of different pesticide samples will be contained, dry out solvent is sent into
It carries out testing and analyzing in ion mobility spectrometry Thermal desorption sample injector and obtains detection signal, it is strong by sample peak transit time and signal
Degree carries out quantitative and semi-quantitative analysis.This method is quantitative and semi-quantitative analysis, cannot accurately analyze the concrete content of Bravo
It is horizontal.
201210137297.7 disclose a kind of method for detecting Fluazuron residual quantity in cattle tissue, and this method is by ox group
Tissue samples are after extracting, purifying, using acetonitrile-water as mobile phase, the detection of HPLC- UV detection method；Liquid chromatogram
Condition are as follows: chromatographic column, AgilentC18,5 μm, 150mm × 4.6mmi.d.；Mobile phase, the acetonitrile-water that volume ratio is 68/32；
Flow velocity: 1.2mLmin-1；Detection wavelength: 260nm；Column temperature: 25 DEG C；Sampling volume: 20mL.In cattle tissue measured by this method
The LOD of Fluazuron is 0.005mgkg-1；LOQ fat is 0.01mgkg-1, hetero-organization 0.02mgkg-1, Neng Gouman
The requirement that foot is analyzed about Fluazuron medicament residue in animal food.But its detection limit is still higher.
Therefore, it needs to develop the remaining determination method of Bravo suitable for aquatic animal tissue at present, is complete
It generally investigates the contents level of Bravo and distribution characteristics in aquatic animal tissue and reliable analysis means is provided in face.
Summary of the invention
The purpose of the present invention is to provide the remaining determination method of Bravo in a kind of aquatic animal tissue, operation letters
List, method is reliable, the rate of recovery is reasonable, and recovery of standard addition is 70~110%, RSD less than 10%, and the lower (inspection of detection limit
Rising limit can achieve 0.012 μ g/L), it has broad application prospects in aquatile analysis detection field.
The technical scheme adopted by the invention to solve the technical problem is that:
The remaining determination method of Bravo in aquatic animal tissue, comprising the following steps:
(1) aquatic animal tissue in Bravo extraction
1. aquatic animal tissue sample and protease is taken to be placed in centrifuge tube one, ultrasound is vortexed, and stands；
2. extraction: 1. being stood in the centrifuge tube one finished in step and extractant is added, be vortexed, CH is added3COONa and nothing
Water MgSO4, it is vortexed, vibrates, centrifugation is taken out extract liquor and freezed；
3. taking centrifuge tube two that N- propyl ethylenediamine, C is added18, anhydrous MgSO4, be then added step 2. in chilled extraction
Take liquid, be vortexed, then centrifugation takes supernatant, nitrogen blow it is close it is dry after, stabilizer is added, with n-hexane constant volume, excessively organic filter membrane is obtained
To sample to be tested；
For N- propyl ethylenediamine (PSA) with for the similar adsorbent of NH2, PSA is respectively 10.1 Hes there are two amino PKa value
10.9.There is ion-exchange capacity more stronger than nh 2 column.Retention mechanism: weak anionic exchanges (aqueous solution) or polarity effect
(non-polar organic solvent), complexing.The polarity ratio NH2 of PSA is weak, and PSA can generate chelating effect with metal ion, for mentioning
Take metal ion strong affinity and high capacity that can effectively remove fatty acid, organic acid and some polarity pigments and sugar.It uses extensively
In the processing of plant farming residual analysis sample.Predominantly forward extraction.
C18With hydrophobic effect, there is suction-operated to nonpolar component, retention mechanism: strong nonpolar action.It is main to use
In reversed phase extraction, it is suitable for nonpolarity to moderately polar compound, such as antibiotic, barbiturate, phthalazines, caffeine, medicine
Object, dyestuff, aromatic oil, liposoluble vitamin, fungicide, herbicide, environment pesticide, PAH and PCB residual, food additives,
Carbohydrate replaces rouge, phenol, phthalic acid ester, steroids, table activator, theophylline, water soluble vitamin to hydroxy-methylbenzene acid
Raw element etc..
(2) it detects
1. drawing standard curve, the method for drafting standard curve described herein can be led using existing detection and analysis technology
Common Specification Curve of Increasing method in domain, selected by titer concentration also without limitation.
2. the sample to be tested that step (1) 3. obtains is placed in sample injection bottle, detected with GC-ECD.
Wherein, the aquatic animal tissue is aquatic products meat, poultry meat and domestic animals meat aquatic animal tissue；
Preferably, aquatic animal tissue is the flesh of fish, shrimp, any one in crab meat；
Preferably, the aquatic animal tissue is the flesh of fish.
Further, step (1) 1. in pH be 7-8, such as 7.1,7.2,7.3 ... 8；Temperature is 40-50 DEG C, such as
Temperature in the above ranges such as 40 DEG C, 42 DEG C, 45 DEG C, 50 DEG C；Ultrasonic power is 200-400W, selectable within this range
Power；Further, ultrasonic time 2min-2h；Further, ultrasonic time 10min-40min；Further, surpass
The sound time is 30min；
Further, step (1) 1. in pH be 7.5, temperature be 45 DEG C, ultrasonic power 300W.
Further,
Step (1) is 2., (1) 3. middle centrifuge tube is polypropylene centrifuge tube；
Further, step (1) 2. in cooling time be 1-8h, be optionally 1h, 2h, 3h, 5h, 8h etc. within the scope of this
Any time.Further, step (1) 2. in cooling time be 3-6h, any time within the scope of this such as 3h, 5h, 6h；Into one
Step ground, 2. middle cooling time is 4h to step (1)；
Further, step (1) 2. described in extractant be 100% methanol or acid acetonitrile solution；
Further, the acid acetonitrile solution is formic acid/acetonitrile mixed solution or acetic acid/acetonitrile mixed solution；
Further, the formic acid/acetonitrile mixed solution is the acetonitrile solution that formic acid volume fraction is 0.5-2%；
Acetic acid/the acetonitrile mixed solution is the acetonitrile solution that acetic acid volume fraction is 0.5-2%；
Further, the formic acid/acetonitrile mixed solution is the acetonitrile solution that formic acid volume fraction is 1%；
Acetic acid/the acetonitrile mixed solution is the acetonitrile solution that acetic acid volume fraction is 1%；
Further, the acid acetonitrile solution is acetic acid/acetonitrile mixed solution.
Further, step (1) 2. in extraction conditions are as follows: 70 DEG C of extraction temperature, preheating time 5min, stablize extraction time
5min；
Further, this extraction step recycles 1-5 times, extraction step described herein are as follows: 1. stands and finishes in step
Extractant is added in centrifuge tube one, is vortexed, CH is added3COONa and anhydrous MgSO4, it being vortexed, vibrates, extract liquor is taken out in centrifugation,
The step of circulation are as follows: after taking out extract liquor, extractant is added in centrifuge tube one again again, is vortexed, vibrates, extraction is taken out in centrifugation
Take liquid；If need to recycle again, after taking out extract liquor, extractant is added in centrifuge tube one again again, is vortexed, vibrates, centrifugation,
Take out extract liquor；It recycles according to this.
Further, this extraction step recycles 3 times.The extract liquor that extraction step circulate operation obtains needs to merge.
Further, step (1) 2. in CH3COONa and anhydrous MgSO4Mass ratio be 1:(0.2-10)；
Further, step (1) 2. in CH3COONa and anhydrous MgSO4Mass ratio be 1:4；
Further, anhydrous MgSO4It need to be pre-processed, the pre-treatment step are as follows: the calcination 2- at 380-520 DEG C
8h is fitted into ground glass stoppered bottle after cooling, is placed in drier and saves.
Further, step (1) 3. described in stabilizer be formic acid.
Further, the method that step (2) 1. draws standard curve are as follows: preparation Bravo concentration be 20ppb, 50ppb,
The standard solution of 100ppb, 200ppb, 400ppb, 500ppb, are detected with GC-ECD, draw standard curve；
Further, in the step (2), when GC-ECD is detected,
Detecting instrument is Agilent 7890A type gas chromatograph,
Detector is μ-ECD,
Chromatographic column type is HP-5 quartz capillary column (30m × 0.32mm × 0.25 μm),
Nitrogen flow is 1mL/min,
Sample volume is 1 μ L,
Injector temperature is 250 DEG C.
Further, Bravo is after ultrasonic protein digestion in the aquatic animal tissue, recovery of standard addition 70
Less than 10%, detection is limited to 0.012 μ g/L by~110%, RSD.
Further technical solution is, the remaining determination method of Bravo in a kind of aquatic animal tissue, including with
Lower step:
(1) aquatic animal tissue in Bravo extraction
1. 5.0g flesh of fish sample and 10mL protease, are placed in polypropylene centrifuge tube one, adjusting pH is 7.5,300W ultrasound,
It is vortexed, stands；
2. extraction: 1. being stood in step and 10mL acetic acid volume fraction is added in the polypropylene centrifuge tube one finished as 1%
Acetonitrile solution is vortexed, and CH is added3COONa and anhydrous MgSO4, it is vortexed, vibrates, centrifugation takes out extract liquor and freezes 4h；
This step 2. in, 70 DEG C of extraction temperature, preheating time 5min, stablize extraction time 5min；This extraction step circulation 3
It is secondary, merge extract liquor three times；
That is: it extracts: 1. being stood in step and 10mL acetic acid volume fraction is added in the polypropylene centrifuge tube one finished as 1%
Acetonitrile solution, stablizes extraction time 5min by 70 DEG C of extraction temperature, preheating time 5min；Then it is vortexed, CH is added3COONa and nothing
Water MgSO4, it is vortexed, vibrates, centrifugation takes out extract liquor and freezes 4h；
3. taking 4mL centrifuge tube two that 50mg N- propyl ethylenediamine, 100mg C is added18, the anhydrous MgSO of 300mg4, then it is added
Step 2. in chilled extract liquor, be vortexed, then centrifugation takes supernatant, nitrogen blow it is close it is dry after, stabilizer is added, uses n-hexane
Constant volume, excessively organic filter membrane, obtains sample to be tested；
This step 3. in anhydrous MgSO4Using preceding first through following pretreatment: the calcination 4h at 450 DEG C is packed into after cooling
In ground glass stoppered bottle, it is placed in drier and saves.
(2) it detects
The sample to be tested that step (1) obtains is placed in sample injection bottle, is detected with GC-ECD, detecting instrument Agilent
7890A type gas chromatograph,
Detector is μ-ECD,
Chromatographic column type is HP-5 quartz capillary column (30m × 0.32mm × 0.25 μm),
Nitrogen flow is 1mL/min,
Sample volume is 1 μ L,
Injector temperature is 250 DEG C.
The beneficial effects of the present invention are:
1, the remaining determination method of Bravo in aquatic animal of the invention tissue, hundred be suitable in biological tissue
Bacterium is clear, can the tested Bravo of enrichment method, reduce detection limit, and the sensitivity of ameliorative way, and efficient and sensible, adaptability
High analysis instrument ensure that the accuracy of Bravo testing result in surface water and the flesh of fish.
2, aquatic animal tissue in the remaining determination method of Bravo reusing, stability and the rate of recovery all
It is significantly improved, Bravo in aquatic animal tissue can be extracted.
3, the remaining determination method of Bravo is easy to operate in aquatic animal tissue, and method is reliable, can batch processing；
Save solvent；Bravo is extracted and is enriched in achievable aquatic animal tissue, and extraction and enrichment are more abundant, to improve
Detection sensitivity and accuracy.
4, the remaining determination method of Bravo by optimization chromatography, Mass Spectrometry Conditions and reduces base in aquatic animal tissue
Mass effect etc. improves separating capacity and sensitivity.
Detailed description of the invention
Fig. 1 standards calibration curve and matrix matching calibration standard.
Specific embodiment
Below by specific embodiment, the present invention is further described, it is noted that for the ordinary skill of this field
For personnel, without departing from the principle of the present invention, several variations and modifications can also be made, these also should be regarded as belonging to
Protection scope of the present invention.
1, instrument and sample
1.1, instrument:
Detecting instrument is Agilent 7890A type gas chromatograph, Agilent Technologies (China) Co., Ltd
Detector is μ-ECD, Agilent Technologies (China) Co., Ltd
Chromatographic column type is HP-5 quartz capillary column (30m × 0.32mm × 0.25 μm),
Nitrogen evaporator: DC12H series nitrogen purges instrument, Town in Shanghai spectrum experiment Science and Technology Co., Ltd.
1.2, sample: the carp captured from following sampling area takes its structure of fish muscle.
2, test method and result
Draw standard curve: preparing Bravo concentration is 20ppb, 50ppb, 100ppb, 200ppb, 400ppb, 500ppb
Standard solution, detected with GC-ECD, with inner mark method ration, method detection limit (MDL) and method quantitative limit (MQL) are respectively adopted
3 times and 1 times of signal-to-noise ratio are calculated, and signal-to-noise ratio is using Masshunter software (Agilent) to addition low concentration Bravo
Sample calculated, using 20ppb, 50ppb, 100ppb, 200ppb, 400ppb, 500ppb as abscissa, corresponding peak face
Product is ordinate, establishes standards calibration curve and matrix matching calibration curve respectively, as shown in Fig. 1.
Embodiment 1: structure of fish muscle
The fish (choose in the present embodiment is carp) captured from sampling waters, takes its structure of fish muscle, weighs the fish of 5.0g
The protease of meat sample product and 10mL are added in polypropylene centrifuge tube one, and adjusting pH is 7.5, after 300W power ultrasound 30min, whirlpool
1min is revolved, 30min is stood.
It is 1% acetonitrile solution that 10mL acetic acid volume fraction is added into 50mL centrifuge tube, and preheating time 5min keeps extraction
Temperature 70 C stablizes extraction 5min；0.5g NaAc, the anhydrous MgSO of 2g is added in vortex 1min4, vortex 1min, shaking table oscillation
15min, 5000rpm are centrifuged 5min, and syringe takes out most extract liquor, after taking out extract liquor, again again in centrifuge tube one
Addition acetic acid volume fraction is 1% acetonitrile solution, and preheating time 5min is kept for 70 DEG C of extraction temperature, stablizes extraction 5min；It is vortexed
1min vibrates 15min, and 5000rpm is centrifuged 5min, takes out extract liquor；After then taking out extract liquor, it is added in centrifuge tube one again again
Extractant is vortexed, and vibrates, and extract liquor is taken out in centrifugation；It recycles 3 times according to this, merges extract liquor three times, be placed in -18 DEG C of refrigerators
Middle freezing 4h.
Extract liquor of the 1mL by freezing is taken out in 4mL polypropylene centrifuge tube two, 50mg is added in advance in centrifuge tube two
N- propyl ethylenediamine, 100mg C18, the anhydrous MgSO of 300mg4(anhydrous MgSO4Using preceding first through following pretreatment: at 450 DEG C
Calcination 4h is added in centrifuge tube two after cooling), vortex 1min, 8000rpm are centrifuged 5min.500 μ L supernatants are taken, nitrogen is blown closely
After dry, 10 μ L formic acid are added as stabilizer, is settled to 500 μ L with n-hexane, crosses 0.22 μm of organic filter membrane, obtain to test sample
Product.
The sample to be tested that above-mentioned steps are obtained is placed in sample injection bottle, is detected with GC-ECD, detecting instrument Agilent
7890A type gas chromatograph, testing conditions are as follows:
Detector is μ-ECD,
Chromatographic column type is HP-5 quartz capillary column (30m × 0.32mm × 0.25 μm),
Nitrogen flow is 1mL/min,
Sample volume is 1 μ L,
Injector temperature is 250 DEG C.
In the present embodiment, detection is limited to 0.012 μ g/L.
Embodiment 2-6
Compared with Example 1, difference place is only that, 10mL acetic acid volume fraction is that 1% acetonitrile solution successively replaces with
Acetic acid volume fraction is 0% acetonitrile solution, acetic acid volume fraction is 2% acetonitrile solution, acetic acid volume fraction is that 3% acetonitrile is molten
Liquid, acetic acid volume fraction are 4% acetonitrile solution, acetic acid volume fraction is 5% acetonitrile solution.
The detection of embodiment 2-6 is limited to 0.012 μ g/L.
Embodiment 7
Compared with Example 1, difference place is only that, by " the anhydrous MgSO of 0.5g NaAc, 2g4" replace with " 1g NaAc,
The anhydrous MgSO of 1.5g4”。
In the present embodiment, detection is limited to 0.012 μ g/L.
Embodiment 8 (extractant is methanol)
The fish (choose in the present embodiment is carp) captured from sampling waters, takes its structure of fish muscle, weighs the fish of 5.0g
The protease of meat sample product and 10mL are added in polypropylene centrifuge tube one, and adjusting pH is 7.5, after 300W power ultrasound 30min, whirlpool
1min is revolved, 30min is stood.
10mL methanol is added into 50mL centrifuge tube, preheating time 5min is kept for 70 DEG C of extraction temperature, stablizes extraction
5min；0.5g NaAc, the anhydrous MgSO of 2g is added in vortex 1min4, vortex 1min, shaking table oscillation 15min, 5000rpm centrifugation
5min, syringe take out most extract liquor, and after taking out extract liquor, methanol, preheating time is added in centrifuge tube one again again
5min is kept for 70 DEG C of extraction temperature, stablizes extraction 5min；Vortex 1min vibrates 15min, and 5000rpm is centrifuged 5min, takes out extraction
Take liquid；After then taking out extract liquor, extractant is added in centrifuge tube one again again, is vortexed, vibrates, extract liquor is taken out in centrifugation；According to
This circulation 3 times, merges extract liquor three times, is placed in -18 DEG C of refrigerators and freezes 4h.
Extract liquor of the 1mL by freezing is taken out in 4mL polypropylene centrifuge tube two, 50mg is added in advance in centrifuge tube two
N- propyl ethylenediamine, 100mg C18, the anhydrous MgSO of 300mg4(anhydrous MgSO4Using preceding first through following pretreatment: at 450 DEG C
Calcination 4h is added in centrifuge tube two after cooling), vortex 1min, 8000rpm are centrifuged 5min.500 μ L supernatants are taken, nitrogen is blown closely
After dry, 10 μ L formic acid are added as stabilizer, is settled to 500 μ L with n-hexane, crosses 0.22 μm of organic filter membrane, obtain to test sample
Product.
The sample to be tested that above-mentioned steps are obtained is placed in sample injection bottle, is detected with GC-ECD, detecting instrument Agilent
7890A type gas chromatograph, testing conditions are as follows:
Detector is μ-ECD,
Chromatographic column type is HP-5 quartz capillary column (30m × 0.32mm × 0.25 μm),
Nitrogen flow is 1mL/min,
Sample volume is 1 μ L,
Injector temperature is 250 DEG C.
In the present embodiment, detection is limited to 0.012 μ g/L.
It is detected using the flesh of fish that the method for embodiment 1 chooses following sampling position, hundred bacterium in the Fish Tissue of selection
Clear testing result such as the following table 1.
For the present invention during sample test, every 10 samples set batch processing blank, control sample mark-on in parallel and
Sample is parallel.Control sample is using clean matrix, and procedure blank investigates pollution whether is introduced in extraction process, controls sample
The parallel accuracy and reproducibility for investigating method parallel with sample of mark-on.
Measure its recovery of standard addition:
The fish (choose in the present embodiment is carp) captured from sampling waters, takes its structure of fish muscle, weighs the fish of 5.0g
The protease of meat sample product and 10mL are added in polypropylene centrifuge tube one, add the standard working solution of Bravo, make its concentration
0.2 μ g/kg, adjusting pH is 7.5, and after 300W power ultrasound 30min, vortex 1min stands 30min.
Other operations are the same as embodiment 1.Its recovery of standard addition is detected and calculated, as a result see the table below 1.
Bravo detectable concentration in 1 flesh of fish of table
The shrimp for taking the xiangtan, hunan province Yuhu District village Shang Lian fishing ground takes shrimp tissue (taking 3 samples, b-1, b-2, b-3), uses
The method of embodiment 1 detects taken shrimp tissue, testing result such as the following table 2 of Bravo in the shrimps tissue of selection.
Sampling spot and testing result such as the following table 2:
Measure its recovery of standard addition:
The shrimp (choose in the present embodiment is shrimp) captured from sampling waters, takes its shrimp tissue, weighs the shrimp of 5.0g
The protease of sample and 10mL are added in polypropylene centrifuge tube one, add the standard working solution of Bravo, make its concentration 0.2
μ g/kg, adjusting pH is 7.5, and after 300W power ultrasound 30min, vortex 1min stands 30min.
Other operations are the same as embodiment 1.Its recovery of standard addition is detected and calculated, as a result see the table below 2.
Bravo detectable concentration in 2 shrimp of table
The crab for taking the xiangtan, hunan province Yuhu District village Shang Lian fishing ground takes crab meat tissue (taking 3 samples, c-1, c-2, c-3), uses
The method of embodiment 1 detects taken crab meat tissue, testing result such as the following table 3 of Bravo in the crab class loading of selection.
Sampling spot and testing result such as the following table 3:
Measure its recovery of standard addition:
The crab (choose in the present embodiment is crab) captured from sampling waters, takes its crab meat tissue, weighs the crab meat of 5.0g
The protease of sample and 10mL are added in polypropylene centrifuge tube one, add the standard working solution of Bravo, make its concentration 0.2
μ g/kg, adjusting pH is 7.5, and after 300W power ultrasound 30min, vortex 1min stands 30min.
Other operations are the same as embodiment 1.Its recovery of standard addition is detected and calculated, as a result see the table below 3.
Bravo detectable concentration in 3 crab meat of table
The flesh of fish that the method that embodiment 2-8 is respectively adopted chooses following sampling position is detected, the Fish Tissue of selection
The testing result of middle Bravo such as the following table 4.
Measure its recovery of standard addition:
From sampling waters capture fish (chosen in the present embodiment be hengyang, hunan province city, the town great Pu virtue village, Hengdong County carp
Fish), its structure of fish muscle is taken, the protease of the flesh of fish sample and 10mL that weigh 5.0g is added in polypropylene centrifuge tube one, addition hundred
The clear standard working solution of bacterium makes 0.2 μ g/kg of its concentration, and adjusting pH is 7.5, after 300W power ultrasound 30min, is vortexed
1min stands 30min.
Other operations are the same as embodiment 2-8.Its recovery of standard addition is detected and calculated, as a result see the table below 4.
Bravo detectable concentration in 4 flesh of fish of table
Other aquatic animals are organized, such as other fish, detection method can be according to the sides in above-described embodiment
Method carries out analysis detection.
It above are only the preferred embodiment of the invention, be not restricted to the present invention.Those skilled in the art is come
It says, other various forms of variations or variation can also be made on the basis of the above description.There is no need and unable to all
Embodiment illustrate.And the obvious changes or variations that thus scheme is extended out are still in protection of the invention
Within the scope of.
Claims (10)
1. the remaining determination method of Bravo in aquatic animal tissue, which comprises the following steps:
(1) aquatic animal tissue in Bravo extraction
1. aquatic animal tissue sample and protease is taken to be placed in centrifuge tube one, ultrasound is vortexed, and stands；
2. extraction: 1. being stood in the centrifuge tube one finished in step and extractant is added, be vortexed, CH is added3COONa and anhydrous
MgSO4, it is vortexed, vibrates, centrifugation is taken out extract liquor and freezed；
3. taking centrifuge tube two that N- propyl ethylenediamine, C is added18, anhydrous MgSO4, be then added step 2. in chilled extract liquor,
Be vortexed, then centrifugation takes supernatant, nitrogen blow it is close it is dry after, stabilizer is added, with n-hexane constant volume, excessively organic filter membrane is obtained to be measured
Sample；
(2) it detects
1. drawing standard curve；
2. the sample to be tested that step (1) 3. obtains is placed in sample injection bottle, detected with GC-ECD.
2. the remaining determination method of Bravo in aquatic animal tissue according to claim 1, which is characterized in that
Aquatic animal tissue is the flesh of fish, shrimp, any one in crab meat；
Preferably, the aquatic animal tissue is the flesh of fish.
3. the remaining determination method of Bravo in aquatic animal tissue according to claim 1, which is characterized in that
Step (1) 1. in pH be 7-8, temperature be 40-50 DEG C, ultrasonic power 200-400W；Further, ultrasonic time is
2min-2h；Further, ultrasonic time 10min-40min；Further, ultrasonic time 30min；
Further, step (1) 1. in pH be 7.5, temperature be 45 DEG C, ultrasonic power 300W.
4. the remaining determination method of Bravo in aquatic animal tissue according to claim 1, which is characterized in that
Step (1) is 2., (1) 3. middle centrifuge tube is polypropylene centrifuge tube；
Further, 2. middle cooling time is 1-8h to step (1)；Further, 2. middle cooling time is 3-6h to step (1)；Into
One step, 2. middle cooling time is 4h to step (1)；
Further, step (1) 2. described in extractant be 100% methanol or acid acetonitrile solution；
Further, the acid acetonitrile solution is formic acid/acetonitrile mixed solution or acetic acid/acetonitrile mixed solution；
Further, the formic acid/acetonitrile mixed solution is the acetonitrile solution that formic acid volume fraction is 0.5-2%；
Acetic acid/the acetonitrile mixed solution is the acetonitrile solution that acetic acid volume fraction is 0.5-2%；
Further, the formic acid/acetonitrile mixed solution is the acetonitrile solution that formic acid volume fraction is 1%；
Acetic acid/the acetonitrile mixed solution is the acetonitrile solution that acetic acid volume fraction is 1%；
Further, the acid acetonitrile solution is acetic acid/acetonitrile mixed solution.
5. the remaining determination method of Bravo in aquatic animal tissue according to claim 1, which is characterized in that
Step (1) 2. in extraction conditions are as follows: 70 DEG C of extraction temperature, preheating time 5min, stablize extraction time 5min；
Further, extraction step recycles 1-5 times；Further, this extraction step recycles 3 times.
6. the remaining determination method of Bravo in aquatic animal tissue according to claim 1, which is characterized in that
Step (1) 2. in CH3COONa and anhydrous MgSO4Mass ratio be 1:(0.2-10)；
Further, step (1) 2. in CH3COONa and anhydrous MgSO4Mass ratio be 1:4；
Further, anhydrous MgSO4It need to be pre-processed, the pre-treatment step are as follows: the calcination 2-8h at 380-520 DEG C, it is cooling
It is fitted into ground glass stoppered bottle afterwards, is placed in drier and saves.
7. the remaining determination method of Bravo in aquatic animal tissue according to claim 1, which is characterized in that
Step (1) 3. described in stabilizer be formic acid.
8. the remaining determination method of Bravo in aquatic animal tissue according to claim 1, which is characterized in that
The method that step (2) 1. draws standard curve are as follows: preparation Bravo concentration be 20ppb, 50ppb, 100ppb, 200ppb,
The standard solution of 400ppb, 500ppb, are detected with GC-ECD, draw standard curve；
Further, the step (2) 2. in, GC-ECD detect when,
Detecting instrument is Agilent 7890A type gas chromatograph,
Detector is μ-ECD,
Chromatographic column type is HP-5 quartz capillary column (30m × 0.32mm × 0.25 μm),
Nitrogen flow is 1mL/min,
Sample volume is 1 μ L,
Injector temperature is 250 DEG C.
9. the remaining determination method of Bravo in aquatic animal tissue according to claim 1, which is characterized in that institute
Stating Bravo in aquatic animal tissue, after ultrasonic protein digestion, recovery of standard addition is 70~110%, RSD less than 10%,
Detection is limited to 0.012 μ g/L.
10. the remaining determination method of Bravo in -9 any aquatic animal tissues according to claim 1, feature
It is, comprising the following steps:
(1) aquatic animal tissue in Bravo extraction
1. 5.0g flesh of fish sample and 10mL protease, are placed in polypropylene centrifuge tube one, adjusting pH is 7.5,300W ultrasound, whirlpool
Rotation is stood；
2. extraction: 1. being stood in step and the acetonitrile that 10mL acetic acid volume fraction is 1% is added in the polypropylene centrifuge tube one finished
Solution is vortexed, and CH is added3COONa and anhydrous MgSO4, it is vortexed, vibrates, centrifugation takes out extract liquor and freezes 4h；
This step 2. in, 70 DEG C of extraction temperature, preheating time 5min, stablize extraction time 5min；This extraction step recycles 3 times,
Merge extract liquor three times；
3. taking 4mL centrifuge tube two that 50mg N- propyl ethylenediamine, 100mg C is added18, the anhydrous MgSO of 300mg4, step is then added
2. chilled extract liquor in is vortexed, then centrifugation takes supernatant, nitrogen blow it is close it is dry after, stabilizer is added, with n-hexane constant volume,
Organic filter membrane is crossed, sample to be tested is obtained；
This step 3. in anhydrous MgSO4Using preceding first through following pretreatment: the calcination 4h at 450 DEG C is packed into ground glass after cooling
In glass bottle, it is placed in drier and saves.
(2) it detects
1. drawing standard curve: preparing Bravo concentration is 20ppb, 50ppb, 100ppb, 200ppb, 400ppb, 500ppb
Standard solution is detected with GC-ECD, draws standard curve；
2. the sample to be tested that step (1) obtains is placed in sample injection bottle, detected with GC-ECD, detecting instrument Agilent
7890A type gas chromatograph,
Detector is μ-ECD,
Chromatographic column type is HP-5 quartz capillary column (30m × 0.32mm × 0.25 μm),
Nitrogen flow is 1mL/min,
Sample volume is 1 μ L,
Injector temperature is 250 DEG C.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110779995A (en) * | 2019-10-18 | 2020-02-11 | 石家庄君乐宝乳业有限公司 | Method for detecting content of residual pesticide in dairy product |
| CN112083115A (en) * | 2020-09-25 | 2020-12-15 | 上海市农产品质量安全中心 | Kit for detecting residual quantity of 7 barbiturates in raw fresh milk |
| CN114002348A (en) * | 2021-10-27 | 2022-02-01 | 江苏新河农用化工有限公司 | Method for detecting content of terephthalonitrile or isophthalonitrile and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1790008A (en) * | 2005-12-27 | 2006-06-21 | 云南农业大学 | Gas chromatography analysis method for 5-chloro-2,4,6-trifluoro-1,3-bezenedicarbonitrile |
| CN105334277A (en) * | 2014-06-04 | 2016-02-17 | 中粮营养健康研究院有限公司 | Rapid pre-treatment method and detection method used for wine pesticide residues analysis |
| CN105866272A (en) * | 2016-03-31 | 2016-08-17 | 青岛正方元信公共卫生检测有限公司 | A method of detecting pesticide residues in a healthcare product |
| CN106383180A (en) * | 2016-08-19 | 2017-02-08 | 中华人民共和国日照出入境检验检疫局 | A method of detecting a plurality of pesticide residues in silkworm pupae |
| CN106483232A (en) * | 2016-10-18 | 2017-03-08 | 山东拜尔检测有限公司 | The method for quick of Multiple Pesticides residual in a kind of soil |
| CN107132305A (en) * | 2017-07-18 | 2017-09-05 | 泉州出入境检验检疫局综合技术服务中心 | The detection method of organochlorine class compound in leather |
-
2019
- 2019-01-28 CN CN201910082702.1A patent/CN109709232B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1790008A (en) * | 2005-12-27 | 2006-06-21 | 云南农业大学 | Gas chromatography analysis method for 5-chloro-2,4,6-trifluoro-1,3-bezenedicarbonitrile |
| CN105334277A (en) * | 2014-06-04 | 2016-02-17 | 中粮营养健康研究院有限公司 | Rapid pre-treatment method and detection method used for wine pesticide residues analysis |
| CN105866272A (en) * | 2016-03-31 | 2016-08-17 | 青岛正方元信公共卫生检测有限公司 | A method of detecting pesticide residues in a healthcare product |
| CN106383180A (en) * | 2016-08-19 | 2017-02-08 | 中华人民共和国日照出入境检验检疫局 | A method of detecting a plurality of pesticide residues in silkworm pupae |
| CN106483232A (en) * | 2016-10-18 | 2017-03-08 | 山东拜尔检测有限公司 | The method for quick of Multiple Pesticides residual in a kind of soil |
| CN107132305A (en) * | 2017-07-18 | 2017-09-05 | 泉州出入境检验检疫局综合技术服务中心 | The detection method of organochlorine class compound in leather |
Non-Patent Citations (2)
| Title |
|---|
| 柴继业等: "基于QuEChERs - GC/ECD 快速检测贝类中有机氯农药及多氯联苯残留", 《水产养殖》 * |
| 王彬: "QuEChERS_气相色谱法鱼肉中多氯联苯残留分析方法的研究", 《粮食流通技术》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110779995A (en) * | 2019-10-18 | 2020-02-11 | 石家庄君乐宝乳业有限公司 | Method for detecting content of residual pesticide in dairy product |
| CN112083115A (en) * | 2020-09-25 | 2020-12-15 | 上海市农产品质量安全中心 | Kit for detecting residual quantity of 7 barbiturates in raw fresh milk |
| CN114002348A (en) * | 2021-10-27 | 2022-02-01 | 江苏新河农用化工有限公司 | Method for detecting content of terephthalonitrile or isophthalonitrile and application thereof |
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