CN105784453B - The pre-treating method of avermectin and ivermectin residue detection in the flesh of fish - Google Patents
The pre-treating method of avermectin and ivermectin residue detection in the flesh of fish Download PDFInfo
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- CN105784453B CN105784453B CN201610149310.9A CN201610149310A CN105784453B CN 105784453 B CN105784453 B CN 105784453B CN 201610149310 A CN201610149310 A CN 201610149310A CN 105784453 B CN105784453 B CN 105784453B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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Abstract
The invention belongs to the pre-treating method technical field of the quick selective mechanisms of fishing medicine of cultured freshwater fish parasitic diseases prevention, more particularly to the residual enzyme-linked pre-treating method that quick selective mechanisms are immunized of avermectin in the flesh of fish and ivermectin, the pre-treating method is based on dispersive solid-phase extraction and removes impurity principle, with NaCl-H2The acetonitrile extracting solution of O-AL N-acetonitrile saturation n-hexane d SPE purification systems and the flesh of fish mixes, purify grease removal at the same time, acetonitrile solution layer is taken to be used for follow-up Enzyme immunoassay, compared with prior art, the d SPE purification techniques that this method is completed using high power capacity preferably, low cost, one step of purification, realize the extraction and cleaning to remaining AVM and IVM in aquatic products, the pretreatment process is simple and quick, easy to operate, inexpensive, high power capacity feature, is adapted to the quick selective mechanisms of enzyme linked immunological.
Description
Technical field
The invention belongs to the pre-treating method of the quick selective mechanisms of fishing medicine of cultured freshwater fish parasitic diseases prevention
Technical field, is used for avermectin and the residual enzyme-linked immune quick sieve of ivermectin in the freshwater aquiculture flesh of fish more particularly to one kind
The pre-treating method that Selected Inspection is surveyed.
Background technology
Avermectin (AVM) and ivermectin (IVM) they are a kind of macrolide antibiotics insecticides of broad-spectrum high efficacy,
Application is wider in terms of freshwater aquiculture.Such medicine is fat-soluble good, in animal body residual effect time length, has nerve and hair to fish
Toxicity is educated, long term frequent uses, and potential wind can be brought safely to breeding environment safety, fish quality and human health
Danger, therefore, it is imperative to strengthen avermectin and ivermectin in the security risk monitoring that aquaculture is applied, in aquatic products
Such medicament residue detection technique is particularly quick selective mechanisms technical research, realizes that fish quality is effectively supervised
Important technology supports, to ensureing that consumer's health has and its significance.
At present, AVM the and IVM method for detecting residue applied to cultured freshwater fish mainly have liquid-matter method, liquid chromatogram-
Fluorescence method and enzyme-linked immunization, first two method expensive equipment, requirement are high, compared to first two method, enzyme-linked immunization
Specific good, high sensitivity, analysis capacity is big, is more suitable for quick selective mechanisms, application easy to spread.
Since sample-pretreating method is the important component of instrument analytical method, if sensitive to be applicable in, is simple and quick
Directly determine the precise and high efficiency of analysis testing result.Therefore except the traditionally pollution such as the more liquid liquid distribution of application, Solid Phase Extraction
Weight, consume outside high, step complexity pretreatment technology, answers weight of the new pretreatment technology of research application as analysis method research
Point, such as matrix solid phase dispersion extraction (MSPD), dispersive solid-phase extraction (d-SPE), the reverse Solid Phase Extraction of multi-functional impurity absorption
(MAS), molecular engram solid phase extraction etc..Wherein, dispersive solid-phase extraction (d-SPE) is the preceding place just to have grown up in recent years
Reason technology, is that solid phase extraction adsorbents particle is dispersed in the extract of sample, by acutely shaking, centrifuging, to remove extraction
The impurity such as lipid in liquid is taken, purification one step completion, is one with " quick, simple, cheap, effective, stable, safety " characteristic
Quickly, low cost, suitable for more residual sample pre-treating methods of a large amount of sample detections.
The sample-pretreating method of AVM and IVM multi-residue determinations is more traditional solid phase in the existing aquatic products in China
(SPE) method of extraction, complicated, of high cost, more demanding to experimental provision, grasp difficulty is big, therefore researches and develops sensitive, steady
It is particularly important that selective mechanisms pre-treating method quick suitable for enzyme linked immunological that is fixed, simple and quick, of low cost, being easy to grasp.
In conclusion there is an urgent need for using enzyme linked immunosorbent assay as detection means among the prior art, primary study will divide
Dissipate solid phase extraction techniques and be applied to the sample-pretreating method of avermectin and ivermectin residue detection in aquatic products.
The content of the invention
The technical problems to be solved by the invention：In view of the deficiencies of the prior art and defect, the present invention provide a kind of flesh of fish
The pre-treating method of middle avermectin and ivermectin residue detection, this method is using high power capacity preferably, low cost, purification one
The d-SPE purification techniques completed is walked, realizes the extraction and cleaning to remaining AVM and IVM in aquatic products, which has letter
The features such as list is quick, easy to operate, inexpensive, high power capacity, is adapted to the quick selective mechanisms of enzyme linked immunological.
The pre-treating method of avermectin and ivermectin residue detection in the flesh of fish, it is characterized in that：The pre-treating method bag
Include following steps：
It is placed in Step 1: weighing minced fillet sample 2g in 50mL centrifuge tubes；
Step 2: adding 6mL acetonitriles into the centrifuge tube, it is vortexed and is mixed with 2000r/min on whirlpool mixing shaker
Co oscillation 2min, centrifuges 5min with 4000r/min on centrifuge, takes supernatant stand-by；
Step 3: weighing 1g sodium chloride, 2mL water, 1.5g neutral aluminas respectively, it is placed in centrifuge tube, mixes, mixed
Close liquid；
Step 4: the supernatant that step 2 is obtained is added in the mixed liquor obtained through step 3, add 2mL acetonitriles and satisfy
And n-hexane, after vortex mixes, to centrifuge 5min more than or equal to 4000r/min or stand to separating liquid completely on centrifuge
Surface layer, wherein, intermediate layer is acetonitrile solution layer；
Step 5: by step 1 to step 4,400 μ L acetonitrile layer solution nitrogen are taken to be blown to dry, for enzyme-linked immunoassay；
Step 6: the pre-treating method of avermectin and ivermectin residue detection finishes in the flesh of fish.
Minced fillet sample 2g is weighed accurately to 0.01g in the step 1.
1.5g neutral aluminas in the step 3, using neutral alumina as dispersive solid-phase extraction adsorbent, using standard
Addition experiment enzymoimmunoassay, remaining avermectin and ivermectin in the flesh of fish are measured using enzyme-linked immunoassay instrument
Blank value and the rate of recovery.
By above-mentioned designing scheme, the present invention can bring following beneficial effect：Impurity is removed based on dispersive solid-phase extraction
Principle, with NaCl-H2The flesh of fish extracting solution of O-AL-N-acetonitrile saturation n-hexane d-SPE purification systems and acetonitrile mixes, together
When purify grease removal, take acetonitrile solution layer to be used for follow-up Enzyme immunoassay.
1st, d-SPE purification techniques is applied to the remaining enzyme linked immunosorbent detections of AVM and IVM in aquatic products, the preceding place first
Reason process has the characteristics that simple and quick, easy to operate, inexpensive, high power capacity, is adapted to the quick selective mechanisms of enzyme linked immunological.
2nd, propose first and optimize NaCl-H2The d-SPE of O-AL-N-acetonitrile saturation n-hexane-acetonitrile extracting solution is net
Change system.
3rd, pre-treating method provided by the invention, simplifies sample pretreatment process, realizes that purification, one step of degreasing process are complete
Into.
4th, use neutral alumina to remove impurity for dispersive solid-phase extraction adsorbent, realize that AVM's and IVM in aquatic products is more
Residue detection.
5th, in the d-SPE mixed solutions of this method, only acetonitrile solution layer is used for follow-up test, how to keep acetonitrile solution with
Other solution thoroughly separate, and are the key factors of the accuracy of influence method and sensitivity.This method Loading sequence is reasonable, first will
Supernatant is added in the mixed solution as made from sodium chloride, water, neutral alumina, adds 2mL acetonitrile saturation n-hexanes, vortex
It is Clear ＆ Transparent to centrifuge 5min more than or equal to 4000r/min or stand to layering completely on centrifuge after mixing, take second
Nitrile solution layer is used for subsequent operation, to ensure d-SPE operations accurately and reliably.
6th, the remaining Enzyme-Linked Immunospots of AVM and IVM and existing Enzyme-Linked Immunospot in the aquatic products based on d-SPE
Compare, easy to operate, cost is low, multi-residue determination, high sensitivity, and pre-treating method is different, and detection time shortens.
Brief description of the drawings
The invention will be further described with embodiment for explanation below in conjunction with the accompanying drawings：
Fig. 1 is the operational flowchart of the pre-treating method of avermectin and ivermectin residue detection in the flesh of fish of the present invention.
Embodiment
As shown in Figure 1, this method cardinal principle：Impurity principle is removed based on dispersive solid-phase extraction, with NaCl-H2O—
The flesh of fish extracting solution of AL-N-acetonitrile saturation n-hexane d-SPE purification systems and acetonitrile mixes, while purifies grease removal, takes acetonitrile molten
Liquid layer is used for follow-up Enzyme immunoassay.
The pre-treating method of avermectin and ivermectin residue detection in the flesh of fish, it is characterized in that：The pre-treating method bag
Include following steps：
It is placed in Step 1: weighing minced fillet sample 2g in 50mL centrifuge tubes；
Step 2: adding 6mL acetonitriles into the centrifuge tube, it is vortexed and is mixed with 2000r/min on whirlpool mixing shaker
Co oscillation 2min, centrifuges 5min with 4000r/min on centrifuge, takes supernatant stand-by；
Step 3: weighing 1g sodium chloride, 2mL water, 1.5g neutral aluminas respectively, it is placed in centrifuge tube, mixes, mixed
Close liquid；
Step 4: the supernatant that step 2 is obtained is added in the mixed liquor obtained through step 3, add 2mL acetonitriles and satisfy
And n-hexane, after vortex mixes, to centrifuge 5min more than or equal to 4000r/min or stand to separating liquid completely on centrifuge
Surface layer, wherein, intermediate layer is acetonitrile solution layer；
Step 5: by step 1 to step 4,400 μ L acetonitrile layer solution nitrogen are taken to be blown to dry, for enzyme-linked immunoassay；
Step 6: the pre-treating method of avermectin and ivermectin residue detection finishes in the flesh of fish.
Minced fillet sample 2g is weighed accurately to 0.01g in the step 1.
1.5g neutral aluminas in the step 3, using neutral alumina as dispersive solid-phase extraction adsorbent, using standard
Addition experiment enzymoimmunoassay, remaining avermectin and ivermectin in the flesh of fish are measured using enzyme-linked immunoassay instrument
Blank value and the rate of recovery.
Elaborate with reference to the prior art to the advance of pre-treating method provided by the invention：
First, existing national standard with document report in related aquatic products avermectin and ivermectin method for detecting residue and
Pretreatment technology contrast is shown in Table 1.
AVM, IVM method for detecting residue and pretreatment technology contrast in 1 existing aquatic products of table
Upper table finds out that either detecting instrument uses liquid chromatograph/mass spectrometer, liquid chromatogram or enzyme-linked immunoassay instrument, water
The sample pre-treatments of avermectin and ivermectin residue detection are extracted using acetonitrile in product, and Solid Phase Extraction (SPE) method is net
Change, impurity absorption pattern, collect whole effluxes, and acetonitrile is SPE sole operation solvents, wherein unique is not both SPE pillars
There are AL-N and AL-B.Its purification pattern and SPE modes of operation, the main function for illustrating SPE pillars are to remove impurity.Though this method
So it is widely used, but the SPE pillars of commercialization are of high cost, SPE operations will be through overactivation-loading-elution-elution, easily
Sample loss is caused, it is more demanding to operating personnel, it need to could be completed by special equipments such as solid-phase extracting instruments.
Based on above reason, enzyme-linked immunization specificity is good, quick selective mechanisms requirement to adapt to, and is reaching same removal
Under the premise of impurity purpose and ensuring method sensitivity, we are using high power capacity preferably, low cost, the d- for purifying step completion
SPE purification techniques, researchs and develops more residual sample pre-treating methods inexpensive, easy to operate, suitable for a large amount of sample detections.
D-SPE pretreatment technologies are applied to the remaining enzyme of AVM, IVM in aquatic products by pre-treating method provided by the invention
Connection be immunized quick selective mechanisms have not yet to see all reports, compared with existing pretreatment technology, difference have it is following some：
1st, d-SPE purification techniques is applied to the remaining enzyme linked immunosorbent detections of AVM and IVM in aquatic products first.
By it was found that, this method replaces SPE with d-SPE, realize in aquatic products remain AVM and IVM extraction it is net
Change, simple and quick, easy to operate, inexpensive, the high power capacity feature of prominent pretreatment process, are adapted to enzyme linked immunological quickly to screen inspection
Survey.
2nd, propose first and optimize NaCl-H2The d-SPE of O-AL-N-acetonitrile saturation n-hexane-acetonitrile extracting solution is net
Change system.
D-SPE is purified based on dispersive solid-phase extraction adsorbent adsorbing contaminant principle, in selection d-SPE purification systems
When, to ensure that the rate of recovery of pre-treating method should consider following factor at the same time：One be to try to reduce acetonitrile extracting solution in AVM and
Suction-operateds of the IVM in Al-N；Second, ensureing catharsis of the AL-N to acetonitrile extracting solution, particularly aquatic products matrix is complicated,
Protein, fat and other lipids in acetonitrile extracting solution etc. disturb the removal of enzyme-linked immunoassay material.This method is by H2O
Addition improve the polarity of d-SPE purification systems and destroy absorption of the Al-N to AVM and IVM in acetonitrile solution, the addition of NaCl has
Help the layering removal of impurities of scavenging solution, H2The O and NaCl synergistic effect common guarantee rate of recovery of pretreatment process.In addition, acetonitrile
The addition of saturation n-hexane greatly reduces in sample extracting solution fat to enzyme-linked immunoassay on the premise of the rate of recovery is ensured
Interference.
3rd, pre-treating method provided by the invention, simplifies sample pretreatment process, realizes that purification, one step of degreasing process are complete
Into.
This method is with mono- step of d-SPE instead of four activation of existing SPE purifications, loading, elution, elution process steps
Suddenly, n-hexane grease removal and in dispersive solid-phase extraction process is carried out at the same time, purification, the completion of one step of degreasing process is realized, is ensureing
On the premise of the rate of recovery, simplify operation, reduce cost, complement each other with the high selectivity of enzyme-linked immunization, realize aquatic products
Middle avermectin and the remaining high sensitivity detection of ivermectin.
4th, impurity is removed using Al-N dispersive solid-phase extractions adsorbent, realizes more residuals inspection of AVM and IVM in aquatic products
Survey.
It is demonstrated experimentally that AL-B is better than Al-N in d-SPE purification systems to AVM and IVM suction-operateds, pre-treatment can be reduced
The process rate of recovery, therefore, we different using the single residue detection of AL-B SPE columns from existing enzyme-linked immunosorbent assay method
Method is dispersed in the acetonitrile solution with certain polarity using Al-N dispersive solid-phase extraction agent and realizes that AVM's and IVM is how residual
Stay detection.
5th, key point control, ensuring method validity, stability are realized.
In the d-SPE mixed solutions of this method, only acetonitrile solution layer is used for follow-up test, how to keep acetonitrile solution and its
He thoroughly separates solution, is the key factor of the accuracy of influence method and sensitivity.This method sums up key control element
Ensure d-SPE operations accurately and reliably, i.e., rationally control Loading sequence, mixing time, layering is Clear ＆ Transparent, using acetonitrile saturation just
Hexane, takes acetonitrile layer to be used for subsequent operation.
6th, the remaining Enzyme-Linked Immunospots of AVM and IVM and existing Enzyme-Linked Immunospot in the aquatic products based on d-SPE
Compare, easy to operate, cost is low, multi-residue determination, high sensitivity, and pre-treating method is different, and detection time shortens, and difference is detailed
It is shown in Table 2.
The remaining enzymes of AVM and IVM exempt from detection method and exempt from detection method difference with existing enzyme in 2 d-SPE of table purification aquatic products
2nd, methods experiment of the invention analysis
1st, method and technology parameter determines
The pre-treating method of avermectin and ivermectin residue detection in the freshwater aquiculture flesh of fish provided by the invention, mainly
It is main to include extraction and purification suitable for the quick selective mechanisms of enzyme linked immunological.Extraction：Mainly use acetonitrile extraction of ocean eddies；Purification：
Completed using one step of dispersive solid-phase extraction method；Concentration：Blown using nitrogen or vacuum decompression distills；Dissolving：Using ELISA reagent
The redissolution working solution that box provides fully is vortexed dissolving, for enzyme-linked immunoassay.
Main operational steps are as follows：Uniform minced fillet sample 2g (accurately to 0.01g) is weighed in 50mL centrifuge tubes, is added
6mL acetonitriles, 2000r/min vortex mixeds concussion 2min, 4000r/min centrifugation 5min, supernatant add the 1g chlorinations mixed
Sodium, 2mL water, 1.5g neutral alumina mixed liquors centrifuge tube in, add 2mL acetonitrile saturation n-hexanes, after vortex mixes, with
5min is centrifuged more than or equal to 4000r/min or standing is Clear ＆ Transparent to being layered, and takes acetonitrile layer solution nitrogen among 400 μ L to be blown to
It is dry, for enzyme-linked immunoassay.
The present invention realizes avermectin and the how residual enzyme-linked of ivermectin in aquatic products and quick selective mechanisms, tool is immunized
There is the features such as simple, quick, sensitive, to stablize.
The present invention compares dispersive solid-phase extraction d-SPE, the reverse Solid Phase Extraction of multi-functional impurity absorption (MAS) and traditional
The sample purification methods such as liquid-liquid distribution extraction (LLE), flesh of fish mark-on test enzyme-linked immunoassay as a result, finding multi-functional impurity
Adsorb the reverse Solid phase extraction method rate of recovery and be below 60%, the liquid-liquid distribution extracting and purifying method rate of recovery is not detect, is said
The former bright operational losses are more, and the more interferases of the latter's impurity exempt from method measure, and clean-up effect is bad, and the dispersive solid-phase extraction method of purification
The rate of recovery meets the quick selective mechanisms requirement of enzyme linked immunological between 60%~120%, is optimal pre-treatment purification method.
The present invention also compares different dispersive solid-phase extraction agent acidic aluminas (AL-A), neutral alumina (AL-N) and alkali
Property aluminium oxide (AL-B) clean-up effect, standard addition experiment enzyme-linked immunoassay the result shows that, be scattered using neutral alumina
The blank determination of solid phase extraction adsorbents is worth the relatively low and rate of recovery closest to 100%, is optimum purification extraction adsorbent.Change
The different amounts (1.0g, 1.5g, 2.0g) for becoming neutral alumina carry out standard addition experiment enzyme-linked immunoassay, 1.5g neutral oxygens
Change aluminium is optimum amount, its clean-up effect and the rate of recovery are best.
The present invention optimizes Al-N- acetonitrile dispersive solid-phase extraction d-SPE systems to further improve the rate of recovery, foundation and
Experimental result is as follows.On the one hand, 2mL polar solvents H is introduced2O, neutral alumina is reduced by the polarity for increasing acetonitrile solution environment
Absorption of the aluminium in acetonitrile to avermectin and ivermectin, improves the rate of recovery to a certain extent.On the other hand, due to H2O
It is miscible with acetonitrile, the difficulty of concentration is added, by the layering for adding NaCl promotion acetonitrile phase and water phase, while salting-out effect is more
Be conducive to AVM and IVM and be easier to distribution in acetonitrile phase, can also improve the rate of recovery.3rd, for higher fatty acid sample, add 2ml
Acetonitrile saturation n-hexane is into above-mentioned scattered SPE systems, you can effectively remove sample fat, and can reduce n-hexane to AVM and
The solution loss of IVM, ensures the rate of recovery.Mark-on test Enzyme immunoassay the result shows that, n-hexane (acetonitrile saturation)-NaCl-
H2O-AL-N-acetonitrile dispersive solid-phase extraction purification systems are better than AL-N-acetonitrile dispersive solid-phase extraction purification systems, its blank
It is worth minimum, 2 μ g/kg of detection limit, and the rate of recovery of 2 μ g/kg, 4 μ g/kg standards addition and precision are in 60%~120% and
Within 30%, meet relevant criterion requirement.
2nd, method performance indicator
This pre-treating method performance indicator detection limit, accuracy and precision are carried out by commercialization enzyme linked immunological kit
Enzyme-linked immunoassay, it is as a result as follows.
2.1 detection limit：3 are shown in Table with 20 parts of parallel processing measure of blank flesh of fish sample and result of calculation, detection is limited to 1.183
μg/kg。
Quantitative limit：Carry out standard addition experiment to the blank flesh of fish with the AVM and IVM of 2 μ g/kg concentration respectively, carry out respectively
Six parallel laboratory tests, calculate the rate of recovery and the coefficient of variation.Experimental result, AVM and IVM are respectively in the μ g/kg of 1.258 μ g/kg~1.720
With the μ g/kg of 1.210 μ g/kg~1.672, the rate of recovery is 62.9%~86% and 60.5%~83.6%, and different coefficient is respectively less than
30%.Therefore either AVM or IVM, this method are 2 μ g/kg to the quantitative limit of standard addition sample.
3 blank sample of table measures and result of calculation statistical form (μ g/kg)
2.2 accuracy and precision
Carry out standard addition experiment to the blank flesh of fish with 2 μ g/kg, 4 μ g/kg concentration AVM and IVM respectively, and carry out respectively
Six is parallel, calculates the rate of recovery and the coefficient of variation is shown in Table 4.
4 accuracy of table, Precision Experiment data
In conclusion with remaining AVM and IVM in above-mentioned d-SPE pre-treating methods extraction and cleaning aquatic products, exempt from through enzyme-linked
The detection limit of epidemic disease measuring, AVM and IVM are 2 μ g/kg, and the rate of recovery on 2 μ g/kg, 4 μ g/kg addition concentration level is equal
For 60%~120%, variation within batch coefficient≤30%, interassay coefficient of variation≤40%.The result shows that the aquatic products based on d-SPE
AVM and IVM residual samples pre-treating method meets country on the quick selective mechanisms requirement of enzyme linked immunological in product.
Claims (3)
1. the pre-treating method of avermectin and ivermectin residue detection in the flesh of fish, it is characterized in that：The pre-treating method includes
Following steps：
It is placed in Step 1: weighing minced fillet sample 2g in 50mL centrifuge tubes；
Step 2: adding 6mL acetonitriles into the centrifuge tube, shaken on whirlpool mixing shaker with 2000r/min vortex mixeds
2min is swung, 5min is centrifuged with 4000r/min on centrifuge, takes supernatant stand-by；
Step 3: weighing 1g sodium chloride, 2mL water, 1.5g neutral aluminas respectively, it is placed in centrifuge tube, mixes, mixed
Liquid；
Step 4: the supernatant that step 2 is obtained is added in the mixed liquor obtained through step 3,2mL acetonitriles saturation is being added just
Hexane, after vortex mixes, to centrifuge 5min more than or equal to 4000r/min or stand to separating liquid level completely on centrifuge
Layer, wherein, intermediate layer is acetonitrile solution layer；
Step 5: by step 1 to step 4,400 μ L acetonitrile layer solution nitrogen are taken to be blown to dry, for enzyme-linked immunoassay；
Step 6: the pre-treating method of avermectin and ivermectin residue detection finishes in the flesh of fish.
2. the pre-treating method of avermectin and ivermectin residue detection in the flesh of fish according to claim 1, its feature
It is：Minced fillet sample 2g is weighed accurately to 0.01g in the step 1.
3. the pre-treating method of avermectin and ivermectin residue detection in the flesh of fish according to claim 1, its feature
It is：1.5g neutral aluminas in the step 3, using neutral alumina as dispersive solid-phase extraction adsorbent, are added real using standard
Enzymoimmunoassay is tested, the blank value of remaining avermectin and ivermectin in the flesh of fish is measured using enzyme-linked immunoassay instrument
And the rate of recovery.
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| FI3922241T3 (en) * | 2013-02-01 | 2023-11-28 | Celator Pharmaceuticals Inc | Remote loading of sparingly water-soluble drugs into liposomes |
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