CN108226351A - The method for detecting Avermectins medicine - Google Patents
The method for detecting Avermectins medicine Download PDFInfo
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- CN108226351A CN108226351A CN201611129560.2A CN201611129560A CN108226351A CN 108226351 A CN108226351 A CN 108226351A CN 201611129560 A CN201611129560 A CN 201611129560A CN 108226351 A CN108226351 A CN 108226351A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
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Abstract
The method that the present invention proposes detection Avermectins medicine, including：(1) sample, n-hexane, water and acetonitrile are mixed, and obtained mixed liquor is centrifuged, obtain the mixed liquor with upper and lower two layers of first separating liquids, collect the first separating liquid of lower floor；(2) water, acetonitrile and the first separating liquid of upper strata are mixed, and obtained mixed liquor is centrifuged, obtains the mixed liquor with upper and lower two layers of second separating liquids, collect the second separating liquid of lower floor；(3) merge the second separating liquid of the first separating liquid of lower floor and lower floor, add in water and triethylamine, obtain prepare liquid；(3) with chromatography column purification prepare liquid, it is purified product；(4) purified product is performed the derivatization, obtains derivatization product；(5) derivatization product is detected using liquid chromatography, and Avermectins medicine content in sample is determined based on testing result.The method of the present invention can accurately detect Avermectins medicine.
Description
Technical field
The present invention relates to field of food.In particular it relates to detect the method for Avermectins medicine.
Background technology
Avermectins medicine includes ivermectin, avermectin, doractin and eprinomectin.Avermectin is
Efficiently, the antibiotic insecticide of wide spectrum has very strong osmosis on blade, can kill subepidermal pest, and residual effect
Phase is long；Ivermectin is novel wide spectrum, efficient, less toxic antibiotics antiparasitic agent, and ivermectin is avermectin
Derivative, ivermectin are widely used in ox, sheep, horse, the gastrointestinal nematode parasites of pig, thread lungworm and parasitic arthropod, the enteron aisle of dog
Nematode, ear mite, itch mite, heartworm and microfilaria and poultry gastrointestinal nematode and vermin, are substantially carried out being subcutaneously injected；
Doractin is Novel macrocyclic lactone anti-parasite medicine, and nematode, insect and mite are respectively provided with and efficiently kill effect, mainly into
Row is subcutaneously injected；The amount of per injection is 0.2mg/kg weight；Eprinomectin insecticidal properties and other Avermectins medicines
Object is the same, is substantially carried out being subcutaneously injected.
However, the method for detection Avermectins medicine still has much room for improvement at present.
Invention content
The present invention is directed to solve at least one technical problem in the prior art at least to a certain extent.For this purpose,
The method that the present invention proposes detection Avermectins medicine, this method can accurately detect Avermectins medicine.
It should be noted that the present invention is the following discovery based on inventor and completes：
GB 29696-2013 discloses the how remaining high performance liquid chromatography detection of Avermectins medicine in a kind of milk
Method (hereinafter referred to as National Standard Method).But the detection object of National Standard Method is milk, and it is higher to be not particularly suited for detection fat content
Butter and cream detect Avermectins medicine in butter and cream using this method and remain more, and accuracy is poor, the rate of recovery compared with
It is low.
In view of this, inventor has found by many experiments, uses n-hexane and water to isolate in butter or cream
Grease, so as to prevent it from being interfered to Avermectins medicine extraction, purification, derivatization and chromatography detection.As a result, according to this
The method of the detection Avermectins medicine of inventive embodiments can accurately detect Avermectins medicine.
For this purpose, the present invention proposes a kind of method for detecting Avermectins medicine.The method includes：(1) by described in
Sample, n-hexane, water and acetonitrile are mixed, and obtained mixed liquor is centrifuged, and obtain having upper and lower two layers first
The mixed liquor of separating liquid collects the first separating liquid of lower floor；(2) water, acetonitrile and the first separating liquid of the upper strata are mixed, and
Obtained mixed liquor is centrifuged, obtains the mixed liquor with upper and lower two layers of second separating liquids, lower floor second is collected and detaches
Liquid；(3) merge first separating liquid of lower floor and the second separating liquid of lower floor, add in water and triethylamine, obtain prepare liquid；(3) it uses
Prepare liquid described in chromatography column purification, is purified product；(4) purified product is performed the derivatization, obtains derivatization product；
(5) the derivatization product is detected, and determine Avermectin in the sample based on testing result using liquid chromatography
Plain class medicament contg, wherein, the sample is butter or cream；The Avermectins medicine is selected from ivermectin, Avermectin
At least one of element, doractin and eprinomectin.
Inventor obtains above-mentioned optimum detection methodology by many experiments.Specifically, it is big due to existing in butter or cream
Grease is measured, and Avermectins medicine to be detected is covered by grease, influences to detect.N-hexane, water and second are first used as a result,
Nitrile is mixed with sample, so as to effectively Avermectins medicine be made to be detached with grease, is obtained mainly dissolved with Avermectins
The acetonitrile-water layer (lower floor) of drug and mainly dissolved with the n-hexane layer (upper strata) of grease.Thereby, it is possible to be effectively prevented grease
Interference to testing result.Then, it is mixed with water, acetonitrile with n-hexane layer, a small amount of AVM hereinafter being mixed with is extracted with further
Bacteriums drug.
The offer of n-hexane can fully extract grease；The presence of water accelerates the dissolving of butter or cream, so as to carry
High detection efficiency, Avermectins medicine can be dissolved in acetonitrile, in order to subsequent operation.N-hexane, water and acetonitrile association
Same-action is that inventor obtains by many experiments to detach the processing mode of Avermectins medicine and grease, using at this
The separating effect of reason mode is preferable, and compared with detach in batches (for example, first with the grease that n-hexane will be extracted in sample,
Non- n-hexane layer is collected, then the Avermectins medicine in n-hexane layer is further extracted with water and acetonitrile), when shortening detection
Between.
In addition, above-mentioned steps (1) and (2) carry out in same centrifuge tube, i.e., in step (1), after centrifugation, under absorption
The first separating liquid of layer, the first separating liquid of upper strata are retained in centrifuge tube, then the operation of step (3) is carried out in centrifuge tube.As a result,
So that easy to operate, the used time is short, and avoids causing damages in liquid transfer process, so as to influence the accuracy of testing result.
The method of detection Avermectins medicine according to embodiments of the present invention can accurately detect avermectin as a result,
Class drug.
According to an embodiment of the invention, the method for above-mentioned detection Avermectins medicine can also have following supplementary technology
Feature：
According to an embodiment of the invention, the method further includes：Separately by the ivermectin standard items,
Avermectin standard items, doractin standard items and eprinomectin standard items carry out gradient dilution, obtain various concentration
Ivermectin standard solution, avermectin standard solution, doractin standard solution and eprinomectin standard items
Solution；Separately by the ivermectin standard solution of the various concentration, avermectin standard solution, doractin
Standard solution and eprinomectin standard solution are dried, and by obtained product simultaneously with the purified product
Step (4) and (5) is carried out successively.
Inventor has found, using will contain ivermectin standard items, avermectin standard items, doractin mark in National Standard Method
The mixed liquor of quasi- product and eprinomectin standard items is purified and derivatization operation, still, obtained peak area-dense
The related coefficient of scale directrix curve is relatively low, so as to ensure the accuracy of testing result.Further, inventor has found, no
It is operated in the form of mixed liquor, each Avermectins medicine is independently operated, the phase of obtained standard curve
Relationship number ensure that the accuracy of testing result close to 1.Detection Avermectins medicine according to embodiments of the present invention as a result,
Method can accurately detect Avermectins medicine.
According to an embodiment of the invention, the purification includes：It is extracted successively with acetonitrile 5mL and cleaning solution 5mL activated solids
Column takes the prepare liquid to cross column, drains off naturally, drains 5min；Isooctane 3mL is added to wash the solid-phase extraction column, drains 5min,
Acetonitrile 5mL is eluted, and is collected eluent in 10mL test tubes, is dried up in 60 DEG C of water-bath nitrogen, then added in again into the test tube
1mL acetonitriles, vortex oscillation 1min dry up in 60 DEG C of water-bath nitrogen, obtain the purified product.
Directly eluent is performed the derivatization after 60 DEG C of water-bath nitrogen dry up in National Standard Method.But inventor has found, receives
During collecting eluent, easily there is a small amount of eluent to remain in inboard wall of test tube position on the upper side, can not be contacted with bottom liquid, dry up
Afterwards, a small amount of Avermectins medicine is sticked to inner wall position on the upper side, can not carry out subsequent derivation, cause testing result relatively low.Into
And inventor adds in acetonitrile into the test tube after drying, and carry out vortex oscillation, since acetonitrile is not easy to hang by many experiments
Wall, so as to wash Avermectins medicine remaining on wall, so as to ensure that the accuracy of testing result.As a result,
The method of detection Avermectins medicine according to embodiments of the present invention can accurately detect Avermectins medicine.
According to embodiments of the present invention, in step (1), the temperature of the water is 40~60 DEG C.Inventor passes through many experiments
It was found that after sample is mixed with 40~60 DEG C of water, butter or creamer dissolved are can speed up, improves detection efficiency.If temperature
It is too low, butter or cream can not be fully dissolved, extends detection time；If temperature is excessively high, shadow can be caused to Avermectins medicine
It rings.According to a preferred embodiment of the invention, the temperature of the water is 50 DEG C.Detection AVM hereinafter according to embodiments of the present invention as a result,
The method of bacteriums drug can accurately detect Avermectins medicine.
According to embodiments of the present invention, based on sample described in 3~6g, in step (1), the use of the n-hexane, acetonitrile and water
Amount is respectively 10mL, 10mL and 5mL.Inventor obtains aforementioned proportion relationship by many experiments, on this condition can be abundant
Cream or butter are dissolved, and is sufficiently separated grease and Avermectins medicine, i.e., grease is present in n-hexane, avermectin
Class drug is present in acetonitrile and water, so as to efficiently extract out Avermectins medicine, prevents grease from interfering.If n-hexane is used
It measures less, will lead to not fully extract grease；If the dosage of acetonitrile and water is excessive, dilution Avermectins medicine is equivalent to,
By causing, the peak area that following liquid-phase chromatography detects is too small, is easy to cause error.As a result, according to embodiments of the present invention detection Ah
The method of dimension bacteriums drug can accurately detect Avermectins medicine.
According to embodiments of the present invention, based on sample described in 3~6g, in step (2), the dosage of the water and acetonitrile is respectively
5mL and 10mL.Inventor has found, can further extract the Avermectins medicine in n-hexane layer on this condition, so as to
Ensure the accuracy of testing result.If water and acetonitrile content are less, will lead to not fully extract grease；If the use of acetonitrile and water
Amount is excessive, is equivalent to dilution Avermectins medicine, and by causing, the peak area that following liquid-phase chromatography detects is too small, be easy to cause mistake
Difference.The method of detection Avermectins medicine according to embodiments of the present invention can accurately detect Avermectins medicine as a result,
Object.
According to embodiments of the present invention, the derivatization includes：Derivatization reagent A liquid is added in into the purified product successively
100 μ L and derivatization reagent B liquid 150 μ L, closed, whirling motion 10s, sequentially add 50 μ L of 50 μ L of glacial acetic acid and triethylamine, whirling motion
10s in room temperature confined reaction 30min, is eventually adding 650 μ L of methanol and mixing, to obtain the derivatization product.As a result,
The method of detection Avermectins medicine according to embodiments of the present invention can accurately detect Avermectins medicine.
According to embodiments of the present invention, the liquid chromatography testing conditions are as follows：Chromatographic column：Symmetry C18 chromatographic columns
(250 × 4.6mm, 5 μm of grain size)；Mobile phase：Acetonitrile-aqueous solution, wherein acetonitrile concentration are 90 volume %；Column oven：30℃；Swash
Send out wavelength：365nm；Launch wavelength：475nm；Sample size：20μL；Flow velocity：1.0mL/min.As a result, according to embodiments of the present invention
The method of detection Avermectins medicine can accurately detect Avermectins medicine.
In another aspect of this invention, the present invention proposes a kind of method for detecting Avermectins medicine.According to this hair
Bright embodiment, the method includes：
1) butter or cream 5.0g are weighed in 50mL centrifuge tubes, add in 10mL n-hexanes, 50 DEG C of hot water of 5.0mL and
10m1 acetonitriles, whirling motion oscillation 1min, 8000r/min centrifugation 10min, obtain the mixed liquor with upper and lower two layers of first separating liquids,
Collect the first separating liquid of lower floor；
2) 5.0mL water and acetonitrile 10mL, whirling motion oscillation 1min, 8000r/min will be added in first separating liquid of upper strata
Centrifuge 10min, obtain the mixed liquor with upper and lower two layers of second separating liquids, collect the second separating liquid of lower floor and with the lower floor
First separating liquid mixes, and adds in 10mL water into obtained mixed liquor and 50 μ L triethylamines, mixing obtain prepare liquid；
3) prepare liquid is taken to cross column, is drained off naturally, is taken out with acetonitrile 5mL and cleaning solution 5mL activation C18 solid-phase extraction columns successively
Dry 5min；Isooctane 3mL is added to wash the C18 solid-phase extraction columns, drains 5min, acetonitrile 5mL elutions collect eluent in 10mL
It in test tube, is dried up in 60 DEG C of water-bath nitrogen, 1mL acetonitriles, vortex oscillation 1min, in 60 DEG C of water-baths is then added in into the test tube
Nitrogen dries up, and is purified product；
4) eprinomectin, avermectin, doractin and ivermectin standard items are separately subjected to gradient
Dilution, obtains respective various concentration standard working solution；
The standard working solution 1mL of various concentration is respectively taken in different 5mL test tubes, is dried up in 60 DEG C of nitrogen, obtains difference
Eprinomectin, avermectin, doractin and the ivermectin product of concentration；
5) respectively to the eprinomectin containing purified product, various concentration, avermectin, doractin and Yi Wei bacterium
The test tube of plain product sequentially adds 100 μ L derivatization reagent A liquid and 150 μ L derivatization reagent B liquid, closed, whirling motion 10s, successively
50 μ L of acetic acid and triethylamine 50 μ L, whirling motion 10s on the rocks in room temperature confined reaction 30min, add in 650 μ L mixings of methanol, filter,
Respectively obtain respective derivatization product；
6) the respective derivatization product is subjected to liquid chromatographic detection, by the Ai Pulinuo of obtained various concentration
Rhzomorph, avermectin, doractin and ivermectin derivatization product peak area and its corresponding concentration draw standard curve；
7) the corresponding peak area of the derivatization product of the purified product is substituted into the standard curve, be calculated described
Eprinomectin, avermectin, doractin and ivermectin content in butter or cream,
The liquid chromatographic detection condition is as follows：
Chromatographic column：Symmetry C18 chromatographic columns (250 × 4.6mm, 5 μm of grain size)；
Mobile phase：Acetonitrile-aqueous solution, wherein acetonitrile concentration are 90 volume %；
Column oven：30℃；
Excitation wavelength：365nm；
Launch wavelength：475nm；
Sample size：20μL；
Flow velocity：1.0mL/min.
The method of detection Avermectins medicine according to embodiments of the present invention can accurately detect avermectin as a result,
Class drug.
The additional aspect and advantage of the present invention will be set forth in part in the description, and will partly become from the following description
It obtains significantly or is recognized by the practice of the present invention.
Specific embodiment
The embodiment of the present invention is described below in detail.The embodiments described below is exemplary, and is only used for explaining this hair
It is bright, and be not considered as limiting the invention.
It should be noted that term " first ", " second " are only used for description purpose, and it is not intended that instruction or hint phase
To importance or the implicit quantity for indicating indicated technical characteristic.Define " first " as a result, the feature of " second " can be with
Express or implicitly include one or more this feature.Further, in the description of the present invention, unless otherwise saying
Bright, " multiple " are meant that two or more
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that following
Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Particular technique or item are not specified in embodiment
Part, it is carried out according to the described technology of document in the art or condition or according to product description.Agents useful for same or instrument
Production firm person is not specified in device, and being can be with conventional products that are commercially available.
Embodiment 1
In this embodiment, Avermectins medicine is detected in following manner：
1st, reagent and material
Eprinomectin, avermectin, ivermectin standard items：Content >=98%；Doractin standard items：Content >=
94.3%；
Methanol：Chromatographically pure；
Acetonitrile：Chromatographically pure；
Trifluoroacetic anhydride；
Triethylamine：It analyzes pure；
Isooctane；
Water；
Solid phase extraction column：C18,500mg/6mL；
N- methylimidazoles.
2nd, the preparation of solution
(1) standard solution
200 μ g/mL Avermectins medicine standard reserving solutions：Precision weighs eprinomectin, avermectin, Doramectin
Element and each 10mg of ivermectin standard items are dissolved with acetonitrile respectively in different 50mL measuring bottles and are diluted to scale, be configured to
The Avermectins medicine standard reserving solution of a concentration of 200 μ g/mL.
(2) typical medicine is configured
Cleaning solution：Take acetonitrile 30mL, water 70mL and 20 μ L of triethylamine, mixing.
Derivatization reagent A liquid：N- methylimidazoles 1mL, acetonitrile 1mL are taken, mixing is now with the current.
Derivatization reagent B liquid：Trifluoroacetic anhydride 1mL, acetonitrile 2mL are taken, mixing is now with the current.
2nd, step
(1) sample to be tested (butter or cream) 5.0g (being accurate to 0.01g) samples are weighed in 50mL centrifuge tubes, are added in
The n-hexane of 10mL adds the hot water of 50 DEG C of 5.0mL, adds acetonitrile 10mL, whirling motion oscillation 1min, 8000r/min centrifugation
10min takes subnatant.5.0mL water and acetonitrile 10mL are added in upper liquid, repeats to extract primary, merging subnatant, and adding twice
Enter 10mL water, 50 μ L triethylamines, mixing obtains prepare liquid；
(2) prepare liquid is taken to cross column, is drained off naturally, is taken out with acetonitrile 5mL and cleaning solution 5mL activation C18 solid-phase extraction columns successively
Dry 5min；Isooctane 3mL is added to wash C18 solid-phase extraction columns, drains 5min, acetonitrile 5mL elutions collect eluent in 10mL test tubes
In, it is dried up in 60 DEG C of water-bath nitrogen, then adds 1mL acetonitriles, vortex oscillation 1min is dried up in 60 DEG C of water-bath nitrogen, obtained
Purified product；
(3) it is respectively that eprinomectin, avermectin, doractin and ivermectin standard reserving solution progress gradient is dilute
It releases, obtains various concentration standard working solution；
The standard working solution 1mL of various concentration is respectively taken in different 5mL test tubes, is dried up in 60 DEG C of nitrogen, obtains difference
Eprinomectin, avermectin, doractin and the ivermectin product of concentration；
(4) respectively to the eprinomectin containing purified product, various concentration, avermectin, doractin and Yi Wei
The test tube of rhzomorph product adds 100 μ L of derivatization reagent A liquid, derivatization reagent B liquid 150 μ L, closed, whirling motion 10s successively, adds successively
50 μ L of glacial acetic acid and triethylamine 50 μ L, whirling motion 10s in room temperature confined reaction 30min, add 650 μ L mixings of methanol, filter, respectively
Obtain respective derivatization product；
(5) respective derivatization product is subjected to liquid chromatographic detection, by the Ai Pulinuo bacterium of obtained various concentration
Element, avermectin, doractin and ivermectin peak area and its corresponding concentration draw standard curve, as shown in table 1.
The corresponding peak area of obtained sample to be tested is substituted into standard curve, Ai Pulinuo in sample to be tested is calculated
Rhzomorph, avermectin, doractin and ivermectin content.
Liquid chromatographic detection condition is as follows：
Chromatographic column：Symmetry C18 chromatographic columns (250 × 4.6mm, 5 μm of grain size)；
Mobile phase：Acetonitrile-aqueous solution, wherein acetonitrile concentration are 90 volume %；
Column oven：30℃；
Excitation wavelength：365nm；
Launch wavelength：475nm；
Sample size：20μL；
Flow velocity：1.0mL/min.
1 standard curve of table
| Component | Equation of linear regression | Related coefficient (R2) |
| Ivermectin | Y=455.0655x-205.4248 | 0.9987 |
| Avermectin | Y=682.3748x+423.5340 | 0.9965 |
| Doractin | Y=394.2759x+415.7921 | 0.9959 |
| Eprinomectin | Y=616.9756x-837.4960 | 0.9987 |
Embodiment 2
In this embodiment, the rate of recovery is measured in following manner：
(1) anhydrous butter oil without ivermectin, avermectin, doractin and eprinomectin or butter are distinguished
It is mixed with ivermectin, avermectin, doractin and eprinomectin standard items storing solution, obtains corresponding mark-on
Sample；
(2) ivermectin, avermectin, doractin and Ai Puli in mark-on sample are measured using the method for embodiment 1
Promise rhzomorph content；
(3) rate of recovery is calculated based on following equation：
Ivermectin, avermectin, doractin or eprinomectin content in the rate of recovery (%)=mark-on sample ×
100/ theoretical value additive amount.
The results are shown in Table 2.As can be seen that the rate of recovery between 90~110%, illustrates the accuracy of the method for the present invention
It is higher.
2 rate of recovery of table
Embodiment 3
The rate of recovery is measured according to the method for embodiment 2, wherein, eprinomectin, avermectin, doractin and Yi Wei
The pitch-based sphere of each component of rhzomorph, parallel determination seven times；
Testing result is as shown in table 3, it can be seen that relative standard deviation is relatively low, illustrate the precision of the method for the present invention compared with
It is high.
3 repeatability of table
Comparative example 1
Avermectins medicine is detected according to the method for embodiment 2, difference lies in step (3), by Ai Pulinuo bacterium
Element, avermectin, doractin and ivermectin standard reserving solution are mixed, and obtained mixed liquor progress gradient is dilute
It releases, obtains various concentration standard working solution.
As a result as shown in table 4 and 5.If as can be seen that various Avermectins medicines are mixed, operated, obtained
The related coefficient of the standard curve arrived is relatively low, and the rate of recovery is relatively low, so as to ensure the accuracy of testing result.According to implementation
The method of example 1 independently operates various Avermectins medicines, and the rate of recovery is higher, and the accuracy of testing result is higher.
4 standard curve of table
| Component | Equation of linear regression | Related coefficient (R2) |
| Ivermectin | Y=384.5639x-1,376.8649 | 0.9469 |
| Avermectin | Y=553.8149x+4,880.2492 | 0.8972 |
| Doractin | Y=433.0299x+1,981.8924 | 0.9441 |
| Eprinomectin | Y=671.9270x+2,269.3002 | 0.9577 |
5 rate of recovery of table
Comparative example 2
According to embodiment 2 method detect Avermectins medicine, difference lies in, in step (2), collect eluent in
In 10mL test tubes, dried up in 60 DEG C of water-bath nitrogen, be purified product.
The results are shown in Table 6, during collecting eluent, easily has a small amount of eluent to remain in inboard wall of test tube portion on the upper side
Position, can not contact with bottom liquid, and after drying, a small amount of Avermectins medicine is sticked to inner wall position on the upper side, can not carry out follow-up
Derivatization causes the rate of recovery relatively low, and relative standard deviation is higher, and testing result is inaccurate.In embodiment 1, after drying
Acetonitrile is added in test tube, and carries out vortex oscillation, since acetonitrile is not easy wall built-up, so as to by Avermectins remaining on wall
Drug is washed, so as to ensure that the accuracy of testing result.
The result of 6 comparative example 2 of table
Comparative example 3
Avermectins medicine is detected according to the method for embodiment 2, difference lies in the step of, embodiment 1, the temperature of hot water
Spend is 30 DEG C.
The results are shown in Table 7, and hot water temperature is too low, extends the thawing time of butter, anhydrous butter oil, leads to detection efficiency
It is relatively low.But the temperature of hot water it is low when influence to testing result it is smaller.
7 rate of recovery of table
Comparative example 4
Avermectins medicine is detected according to the method for embodiment 2, difference lies in (1) the step of, embodiment 1, hot water
Temperature be 100 DEG C.
The results are shown in Table 8, and hot water temperature is excessively high, and eprinomectin, avermectin, doractin, ivermectin are not
Stablize, easily decompose, the rate of recovery is than relatively low.
8 rate of recovery of table
Comparative example 5
Avermectins medicine is detected according to the method for embodiment 2, difference lies in (1) the step of, embodiment 1, processing
When butter and anhydrous butter oil, n-hexane, acetonitrile content are respectively：1-20ml、10ml.
The results are shown in Table 9, when the additive amount of acetonitrile is 10ml, increases with the addition of n-hexane, Ai Pulinuo bacterium
Element, avermectin, doractin, ivermectin the rate of recovery gradually increase；After the amount of n-hexane increases to 10ml, recycling
The result of rate is stablized, and illustrating the amount of n-hexane increases to after 10ml, it is no longer necessary to increase.
9 rate of recovery of table
Comparative example 5
Avermectins medicine is detected according to the method for embodiment 2, difference lies in (1) the step of, embodiment 1, sample
It is dried up in 60 DEG C of water-bath nitrogen, the volume for adding in acetonitrile is respectively 5ml, 3ml, 2ml, 1ml, 0.5ml.
The results are shown in Table 10, and when the addition of acetonitrile is 1.0ml, the rate of recovery of sample detection meets the requirements, reason
Amount to add in acetonitrile can redissolve the sample on test tube wall in acetonitrile completely, and after drying up again, when analyte derivative treats
Surveying component can be combined with derivating agent completely, improve the rate of recovery.Since the additive amount of derivatization reagent during derivatization is less
Residual sample if the addition of acetonitrile is very few, fully can not be washed, cause the rate of recovery relatively low by (totally 250 μ L)；If acetonitrile
Addition is excessive, and remaining sample is still had after drying and is stayed on the test tube wall of bottom, can not be connect with less amount of derivatization reagent
It touches, it is relatively low so as to also lead to the rate of recovery.
10 rate of recovery of table
In the description of this specification, reference term " one embodiment ", " example ", " is specifically shown " some embodiments "
The description of example " or " some examples " etc. means specific features, structure, material or the spy for combining the embodiment or example description
Point is contained at least one embodiment of the present invention or example.In the present specification, schematic expression of the above terms are not
It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be in office
It is combined in an appropriate manner in one or more embodiments or example.In addition, without conflicting with each other, the skill of this field
Art personnel can tie the different embodiments or examples described in this specification and the feature of different embodiments or examples
It closes and combines.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is impossible to limitation of the present invention is interpreted as, those of ordinary skill in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, changes, replacing and modification.
Claims (9)
- A kind of 1. method for detecting Avermectins medicine, which is characterized in that including：1) butter or cream 5.0g are weighed in 50mL centrifuge tubes, adds in 10mL n-hexanes, 50 DEG C of hot water of 5.0mL and 10ml second Nitrile, whirling motion oscillation 1min, 8000r/min centrifugation 10min, obtains the mixed liquor with upper and lower two layers of first separating liquids, under collection The first separating liquid of layer；2) 5.0mL water and acetonitrile 10mL, whirling motion oscillation 1min, 8000r/min centrifugation will be added in first separating liquid of upper strata 10min, obtains the mixed liquor with upper and lower two layers of second separating liquids, collect the second separating liquid of lower floor and with the lower floor first Separating liquid mixes, and adds in 10mL water into obtained mixed liquor and 50 μ L triethylamines, mixing obtain prepare liquid；3) prepare liquid is taken to cross column, is drained off naturally, is taken out with acetonitrile 5mL and cleaning solution 5mL activation C18 solid-phase extraction columns successively Dry 5min；Isooctane 3mL is added to wash the C18 solid-phase extraction columns, drains 5min, acetonitrile 5mL elutions collect eluent in 10mL It in test tube, is dried up in 60 DEG C of water-bath nitrogen, 1mL acetonitriles, vortex oscillation 1min, in 60 DEG C of water-baths is then added in into the test tube Nitrogen dries up, and is purified product；4) eprinomectin, avermectin, doractin and ivermectin standard items are separately subjected to gradient dilution, Obtain respective various concentration standard working solution；The standard working solution 1mL of the various concentration is respectively taken in different 5mL test tubes, is dried up in 60 DEG C of nitrogen, obtains difference Eprinomectin, avermectin, doractin and the ivermectin product of concentration；5) it is produced respectively to the eprinomectin containing purified product, various concentration, avermectin, doractin and ivermectin The test tube of object sequentially adds 100 μ L derivatization reagent A liquid and 150 μ L derivatization reagent B liquid, and closed, whirling motion 10s is sequentially added 50 μ L of glacial acetic acid and triethylamine 50 μ L, whirling motion 10s in room temperature confined reaction 30min, add in 650 μ L mixings of methanol, filter, point Respective derivatization product is not obtained；6) the respective derivatization product is subjected to liquid chromatographic detection, by the Ai Pulinuo bacterium of obtained various concentration Element, avermectin, doractin and ivermectin derivatization product peak area and its corresponding concentration draw standard curve；7) the corresponding peak area of the derivatization product of the purified product is substituted into the standard curve, the butter is calculated Or eprinomectin, avermectin, doractin and ivermectin content in cream,The liquid chromatographic detection condition is as follows：Chromatographic column：Symmetry C18 chromatographic columns (250 × 4.6mm, 5 μm of grain size)；Mobile phase：Acetonitrile-aqueous solution, wherein acetonitrile concentration are 90 volume %；Column oven：30℃；Excitation wavelength：365nm；Launch wavelength：475nm；Sample size：20μL；Flow velocity：1.0mL/min.
- A kind of 2. method for detecting Avermectins medicine, which is characterized in that including：(1) sample, n-hexane, water and acetonitrile are mixed, and obtained mixed liquor is centrifuged, had The mixed liquor of upper and lower two layers of first separating liquids collects the first separating liquid of lower floor；(2) water, acetonitrile and the first separating liquid of the upper strata are mixed, and obtained mixed liquor is centrifuged, obtained Mixed liquor with upper and lower two layers of second separating liquids collects the second separating liquid of lower floor；(3) merge first separating liquid of lower floor and the second separating liquid of lower floor, add in water and triethylamine, obtain prepare liquid；(3) prepare liquid described in chromatography column purification, is purified product；(4) purified product is performed the derivatization, obtains derivatization product；(5) the derivatization product is detected using liquid chromatography, and based on testing result determine in the sample Ah Bacteriums medicament contg is tieed up,Wherein,The sample is butter or cream；The Avermectins medicine is selected from at least one of ivermectin, avermectin, doractin and eprinomectin.
- 3. method according to claim 2, which is characterized in that further comprise：Separately by the ivermectin standard items, avermectin standard items, doractin standard items and Ai Pulinuo bacterium Plain standard items carry out gradient dilution, obtain the ivermectin standard solution, avermectin standard solution, Duola of various concentration Rhzomorph standard solution and eprinomectin standard solution；Separately by the ivermectin standard solution of the various concentration, avermectin standard solution, doractin Standard solution and eprinomectin standard solution are dried, and by obtained product simultaneously with the purified product Step (4) and (5) is carried out successively.
- 4. method according to claim 2, which is characterized in that the purification includes：Successively with acetonitrile 5mL and cleaning solution 5mL activated solid extraction columns, the prepare liquid is taken to cross column, is drained off naturally, is drained 5min；Isooctane 3mL is added to wash the solid-phase extraction column, drains 5min, acetonitrile 5mL elutions collect eluent in 10mL test tubes In, it is dried up in 60 DEG C of water-bath nitrogen, then adds in 1mL acetonitriles, vortex oscillation 1min, in 60 DEG C of water-bath nitrogen into the test tube again Air-blowing is done, and obtains the purified product.
- 5. method according to claim 2, which is characterized in that in step (1), the temperature of the water is 40~60 DEG C, preferably 50℃。
- 6. method according to claim 2, which is characterized in that based on sample described in 3~6g, in step (1), it is described just oneself The dosage of alkane, acetonitrile and water is respectively 10mL, 10mL and 5mL.
- 7. method according to claim 2, which is characterized in that based on sample described in 3~6g, in step (2), the water and second The dosage of nitrile is respectively 5mL and 10mL.
- 8. method according to claim 2, which is characterized in that the derivatization includes：100 μ L and 150 μ L of derivatization reagent B liquid of derivatization reagent A liquid, closed, whirling motion are added in into the purified product successively 10s sequentially adds 50 μ L of glacial acetic acid and triethylamine 50 μ L, whirling motion 10s, in room temperature confined reaction 30min, is eventually adding methanol 650 μ L and mixing, to obtain the derivatization product.
- 9. method according to claim 2, which is characterized in that the liquid chromatography testing conditions are as follows：Chromatographic column：Symmetry C18 chromatographic columns (250 × 4.6mm, 5 μm of grain size)；Mobile phase：Acetonitrile-aqueous solution, wherein acetonitrile concentration are 90 volume %；Column oven：30℃；Excitation wavelength：365nm；Launch wavelength：475nm；Sample size：20μL；Flow velocity：1.0mL/min.
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