CN106770741A - Tylosin detection method and kit in a kind of poultry - Google Patents
Tylosin detection method and kit in a kind of poultry Download PDFInfo
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- CN106770741A CN106770741A CN201611112547.6A CN201611112547A CN106770741A CN 106770741 A CN106770741 A CN 106770741A CN 201611112547 A CN201611112547 A CN 201611112547A CN 106770741 A CN106770741 A CN 106770741A
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Abstract
The invention discloses a kind of kit of tylosin in poultry, the inside of the kit is placed with ammonium dihydrogen phosphate, anhydrous phosphoric acid ammonium dihydrogen, phosphoric acid, two hypophosphite monohydrate sodium dihydrogens, potassium dihydrogen phosphate, three hypophosphite monohydrate hydrogen dipotassiums, n-hexane, methyl alcohol, petroleum ether, tri-distilled water and dichloromethane, a kind of tylosin detection method in poultry, detection method comprises the following steps：S1, the ammonium dihydrogen phosphate of 0.1 0.5mol/L is taken out with pipettor, and the anhydrous phosphoric acid ammonium dihydrogen of 0.1 0.5g is weighed with electronic balance, is put into dissolving in beaker.Tylosin detection method and kit in the poultry, Practical Performance are good, and detection more facilitates, time saving and energy saving, detection accuracy is higher, and the residual tylosin in poultry accurately can be detected, harm of the tylosin to health of people effectively is avoided, cost is lower, and detection efficiency is high.
Description
Technical field
The present invention relates to tylosin detection technique field, tylosin detection method and reagent in specially a kind of poultry
Box.
Background technology
Tylosin (tylosin) is a kind of macrolide antibiotics that streptomyces fradiae is produced, since the eighties,
Tylosin is widely used as the special antibacterial growth-promoting agent of poultry in the world, and the mycoplasmal diseases and leather to treating poultry are blue
The infection of family name's positive bacteria, promote poultry growth, reduce the death rate and improve food conversion ratio and have obvious effect, tylosin is dynamic
Residual in thing tissue, can threaten food security, and the health to the mankind causes harm, No. 235 bulletins of the Ministry of Agriculture of China《Animal
Property food herbal medicine MRL》Tylosin is remained in animal food and has formulated MRL, wherein,
MRL of the tylosin in chicken gizzard tissue is defined for 0.13mg/kg, tylosin in Pork Tissue most
High residue limitation is 0.2mg/kg.
In recent years, the hot issue that food-safety problem is discussed by people always, the tylosin warp remained in poultry
Often it is raised, the tylosin serious harm health of people remained in poultry, the detection method of existing tylosin is most
It is microbial method and instrumental method, but the accuracy of microbial method is low, the high cost of instrumental method, excessively cumbersome, inspection
Efficiency is surveyed low, therefore, it is proposed that tylosin detection method and kit in a kind of poultry of efficient quick.
The content of the invention
（One）The technical problem of solution
In view of the shortcomings of the prior art, the invention provides tylosin detection method and kit in a kind of poultry, solve
The low problem of detection efficiency.
（Two）Technical scheme
To achieve the above object, the present invention provides following technical scheme：The kit of tylosin in a kind of poultry, the examination
The inside of agent box is placed with ammonium dihydrogen phosphate, anhydrous phosphoric acid ammonium dihydrogen, phosphoric acid, two hypophosphite monohydrate sodium dihydrogens, biphosphate
Potassium, three hypophosphite monohydrate hydrogen dipotassiums, n-hexane, methyl alcohol, petroleum ether, tri-distilled water and dichloromethane.
A kind of tylosin detection method in poultry, detection method comprises the following steps：
S1, the ammonium dihydrogen phosphate of 0.1-0.5mol/L is taken out with pipettor, and the anhydrous of 0.1-0.5g is weighed with electronic balance
Ammonium dihydrogen phosphate, is put into dissolving in beaker, in the environment of 4-10 DEG C, reacts 5-10min；
S2, measures the phosphoric acid of 12-14mL, adds water and is settled to 800-1000mL, and labeled as A liquid, weighs two hydrations of 30-32g
Sodium dihydrogen phosphate, adds water and is settled to 800-1000mL, and labeled as B liquid, take A liquid 450-455mL, 451.6mL, 453.5mL,
454.1mL, B liquid 545-550mL, 548.4mL, 546.5mL, 545.9mL, are configured to the phosphate buffer that PH is 2.1-2.5
I, and with the frequent real-time monitoring of pH value analyzer；
S3, the potassium dihydrogen phosphate of 4-5g and the three hypophosphite monohydrate hydrogen dipotassiums of 0.5-0.8g are weighed with electronic balance, are put into beaker
Dissolving, adds water and is settled to 450-500mL, is configured to the phosphate buffer II that PH is 5.5-5.8, and passed through with pH value analyzer
Normal real-time monitoring；
S4, weighs the poultry sample of 10-20g, is wrapped with filter paper after crushing and is put into apparatus,Soxhlet's, add 0.5-1g just oneself
Alkane, with 60-75 DEG C of water-bath degreasing to colourless, and take out dry it is standby；
S5, weighs sample after the degreasing of 1-2g, and is put into the centrifuge tube of refrigerated centrifuge, adds the methyl alcohol of 10-15mL, ultrasound
25-30min is extracted, after extraction is finished, in the environment of 5-10 DEG C, 8000-10000r/min centrifugation 10-15min take supernatant
Liquid, residue is extracted 2 times again, merges No. 3 extract solutions, in the environment of 4-10 DEG C, 4500-5000r/min centrifugation 5-10min,
And extract solution is labeled as solution A；
S6, after the completion of S5, takes out the solution A of 30-50mL and is put into the separatory funnel of 125mL, takes the petroleum ether of 30-40mL
And add in separatory funnel, 1-3min is vibrated, after stratification, layer solution is removed in the separatory funnel of 300mL, and add
The tri-distilled water of 10-20mL and the dichloromethane of 20-30mL, vibrate 3-5min, after stratification, lower floor's solution are collected in flask
In, then to 300mL separatory funnel add 20-30mL dichloromethane, vibrate 3-5 minutes, after stratification, by lower floor's solution
Collect in flask again；
S7, the dichloromethane layer that will be collected twice is evaporated in the environment of 50-60 DEG C, and labeled as A residues, takes the A of 0.5-1g
Residue, the acetonitrile of the phosphate buffer I and 0.5-1mL of 3-5mL are put into beaker, react 3-5min, and the A for taking 0.5-1g is residual
Slag, the acetonitrile of the phosphate buffer II and 0.5-1mL of 3-5mL are put into another beaker, 3-5min are reacted, for efficient liquid phase
Chromatogram is detected.
Preferably, the capacity of the centrifuge tube is 50mL.
Preferably, the quantity of the beaker, separatory funnel and centrifuge tube is at least five.
Preferably, the model AL204 of the electronic balance.
Preferably, the model SXT-06 of the apparatus,Soxhlet's.
Preferably, the model BPH-200A of the pH value analyzer.
Preferably, the model X-15R of the refrigerated centrifuge.
（Three）Beneficial effect
The invention provides tylosin detection method and kit in a kind of poultry, possesses following beneficial effect：
（1）Tylosin detection method and kit in the poultry, the detection method that the application is proposed, Practical Performance are good, lead to
Perphosphate buffer solution I and phosphate buffer II, coordinates the effect of high performance liquid chromatography, can be to the safe happy bacterium of sample interior
Element is effectively detected that detection more facilitates, time saving and energy saving, and detection accuracy is higher.
（2）Tylosin detection method and kit in the poultry, by being provided with kit, coordinate the application to be proposed
Tylosin detection method, the residual tylosin in poultry can accurately be detected, effectively avoid safe pleasure
Harm of the rhzomorph to health of people, cost is lower, and detection efficiency is high.
（3）Tylosin detection method and kit in the poultry, by supporting multiple beakers, separatory funnel and centrifugation
Pipe, can carry out repeated detection and contrast, it is ensured that the relative accuracy of experiment, the equipment that the application is previously mentioned, its model is equal
It is preferred model, more preferably, experiment effect more preferably, effectively reduces the deviation of detection to accuracy.
Specific embodiment
Based on the embodiment in the present invention, those of ordinary skill in the art are obtained under the premise of creative work is not made
The every other embodiment for obtaining, belongs to the scope of protection of the invention.
The present invention provides a kind of technical scheme：Place a kind of kit of tylosin in poultry, the inside of kit
There are ammonium dihydrogen phosphate, anhydrous phosphoric acid ammonium dihydrogen, phosphoric acid, two hypophosphite monohydrate sodium dihydrogens, potassium dihydrogen phosphate, three hypophosphite monohydrate hydrogen
Dipotassium, n-hexane, methyl alcohol, petroleum ether, tri-distilled water and dichloromethane.
Embodiment 1
A kind of tylosin detection method in poultry, detection method comprises the following steps：
S1, the ammonium dihydrogen phosphate of 0.1mol/L is taken out with pipettor, and the anhydrous phosphoric acid dihydro of 0.1g is weighed with electronic balance
Ammonium, is put into dissolving in beaker, in the environment of 4 DEG C, reacts 5min；
S2, measures the phosphoric acid of 12mL, adds water and is settled to 800mL, and labeled as A liquid, weighs the two hypophosphite monohydrate sodium dihydrogens of 30g,
Add water and be settled to 800mL, and labeled as B liquid, take A liquid 451.6mL, B liquid 548.4mL, be configured to the phosphate-buffered that PH is 2.1
Liquid I, and with the frequent real-time monitoring of pH value analyzer；
S3, the potassium dihydrogen phosphate of 4g and the three hypophosphite monohydrate hydrogen dipotassiums of 0.5g are weighed with electronic balance, are put into dissolving in beaker, plus
Water is settled to 450mL, is configured to the phosphate buffer II that PH is 5.5, and with the frequent real-time monitoring of pH value analyzer；
S4, weighs the poultry sample of 10g, is wrapped with filter paper after crushing and is put into apparatus,Soxhlet's, adds the n-hexane of 0.5g, uses
60 DEG C of water-bath degreasings to colourless, and take out dry it is standby；
S5, weighs sample after the degreasing of 1g, and is put into the centrifuge tube of refrigerated centrifuge, adds the methyl alcohol of 10mL, ultrasonic extraction
25min, after extraction is finished, in the environment of 5 DEG C, 8000r/min centrifugation 10min take supernatant, and residue is extracted 2 times again, is closed
And No. 3 extract solutions, in the environment of 4 DEG C, 4500r/min centrifugation 5min, and extract solution is labeled as solution A；
S6, after the completion of S5, takes out the solution A of 30mL and is put into the separatory funnel of 125mL, takes the petroleum ether of 30mL and adds
In separatory funnel, 1min is vibrated, after stratification, remove layer solution in the separatory funnel of 300mL, and add three steamings of 10mL
The dichloromethane of water and 20mL, vibrates 3min, after stratification, lower floor's solution is collected in flask, then to point liquid of 300mL
Funnel adds the dichloromethane of 20mL, vibrates 3 minutes, after stratification, lower floor's solution is collected in flask again；
S7, the dichloromethane layer that will be collected twice is evaporated in the environment of 50 DEG C, and labeled as A residues, take 0.5g A residues,
The acetonitrile of the phosphate buffer I and 0.5mL of 3mL is put into beaker, reacts 3min, takes A residues, the phosphate of 3mL of 0.5g
The acetonitrile of buffer solution II and 0.5mL is put into another beaker, 3min is reacted, for high performance liquid chromatography detection.
There are following characteristics compared with embodiment 2 and embodiment 3 in the detection method of the embodiment 1：
The detection used time is short, and detection speed is fast, and detection efficiency is high；
Detection accuracy and testing cost are below embodiment 2 and embodiment 3；
Detection accuracy >=94%.
Embodiment 2
A kind of tylosin detection method in poultry, detection method comprises the following steps：
S1, the ammonium dihydrogen phosphate of 0.3mol/L is taken out with pipettor, and the anhydrous phosphoric acid dihydro of 0.3g is weighed with electronic balance
Ammonium, is put into dissolving in beaker, in the environment of 6 DEG C, reacts 6min；
S2, measures the phosphoric acid of 13mL, adds water and is settled to 900mL, and labeled as A liquid, weighs the two hypophosphite monohydrate sodium dihydrogens of 31g,
Add water and be settled to 900mL, and labeled as B liquid, take A liquid 453.5mL, B liquid 546.5mL, be configured to the phosphate-buffered that PH is 2.3
Liquid I, and with the frequent real-time monitoring of pH value analyzer；
S3, the potassium dihydrogen phosphate of 4.5g and the three hypophosphite monohydrate hydrogen dipotassiums of 0.7g are weighed with electronic balance, are put into dissolving in beaker,
Add water and be settled to 480mL, be configured to the phosphate buffer II that PH is 5.6, and with the frequent real-time monitoring of pH value analyzer；
S4, weighs the poultry sample of 15g, is wrapped with filter paper after crushing and is put into apparatus,Soxhlet's, adds the n-hexane of 0.8g, uses
70 DEG C of water-bath degreasings to colourless, and take out dry it is standby；
S5, weighs sample after the degreasing of 1.5g, and is put into the centrifuge tube of refrigerated centrifuge, adds the methyl alcohol of 12mL, and ultrasound is carried
28min is taken, after extraction is finished, in the environment of 8 DEG C, 900r/min centrifugation 12min take supernatant, and residue is extracted 2 times again,
Merge No. 3 extract solutions, in the environment of 8 DEG C, 4800r/min centrifugation 8min, and extract solution is labeled as solution A；
S6, after the completion of S5, takes out the solution A of 40mL and is put into the separatory funnel of 125mL, takes the petroleum ether of 35mL and adds
In separatory funnel, 2min is vibrated, after stratification, remove layer solution in the separatory funnel of 300mL, and add three steamings of 15mL
The dichloromethane of water and 25mL, vibrates 4min, after stratification, lower floor's solution is collected in flask, then to point liquid of 300mL
Funnel adds the dichloromethane of 25mL, vibrates 4 minutes, after stratification, lower floor's solution is collected in flask again；
S7, the dichloromethane layer that will be collected twice is evaporated in the environment of 55 DEG C, and labeled as A residues, take 0.8g A residues,
The acetonitrile of the phosphate buffer I and 0.8mL of 4mL is put into beaker, reacts 4min, takes A residues, the phosphate of 4mL of 0.8g
The acetonitrile of buffer solution II and 0.8mL is put into another beaker, 4min is reacted, for high performance liquid chromatography detection.
There are following characteristics in the detection method of the embodiment 2：
The detection accuracy and testing cost of the embodiment 2 are above embodiment 1, but the detection speed of embodiment 2 is imitated with detection
Rate is below embodiment 1, and the detection time of embodiment 2 is longer than the detection time of embodiment 1；
The detection accuracy and testing cost of the embodiment 2 are below embodiment 3, but the detection speed of embodiment 2 is imitated with detection
Rate is above embodiment 3, and the detection time of embodiment 2 is shorter than the detection time of embodiment 3；
Detection accuracy >=96%.
Embodiment 3
A kind of tylosin detection method in poultry, detection method comprises the following steps：
S1, the ammonium dihydrogen phosphate of 0.5mol/L is taken out with pipettor, and the anhydrous phosphoric acid dihydro of 0.5g is weighed with electronic balance
Ammonium, is put into dissolving in beaker, in the environment of 10 DEG C, reacts 10min；
S2, measures the phosphoric acid of 14mL, adds water and is settled to 1000mL, and labeled as A liquid, weighs the two hypophosphite monohydrate sodium dihydrogens of 32g,
Add water and be settled to 1000mL, and labeled as B liquid, take A liquid 454.1mL, B liquid 545.9mL, be configured to the phosphate that PH is 2.5 and delay
Fliud flushing I, and with the frequent real-time monitoring of pH value analyzer；
S3, the potassium dihydrogen phosphate of 5g and the three hypophosphite monohydrate hydrogen dipotassiums of 0.8g are weighed with electronic balance, are put into dissolving in beaker, plus
Water is settled to 500mL, is configured to the phosphate buffer II that PH is 5.8, and with the frequent real-time monitoring of pH value analyzer；
S4, weighs the poultry sample of 20g, is wrapped with filter paper after crushing and is put into apparatus,Soxhlet's, the n-hexane of 1g is added, with 75
DEG C water-bath degreasing to colourless, and take out dry it is standby；
S5, weighs sample after the degreasing of 2g, and is put into the centrifuge tube of refrigerated centrifuge, adds the methyl alcohol of 15mL, ultrasonic extraction
30min, after extraction is finished, in the environment of 10 DEG C, 10000r/min centrifugation 15min take supernatant, and residue is extracted 2 times again,
Merge No. 3 extract solutions, in the environment of 10 DEG C, 5000r/min centrifugation 10min, and extract solution is labeled as solution A；
S6, after the completion of S5, takes out the solution A of 50mL and is put into the separatory funnel of 125mL, takes the petroleum ether of 40mL and adds
In separatory funnel, 3min is vibrated, after stratification, remove layer solution in the separatory funnel of 300mL, and add three steamings of 20mL
The dichloromethane of water and 30mL, vibrates 5min, after stratification, lower floor's solution is collected in flask, then to point liquid of 300mL
Funnel adds the dichloromethane of 30mL, vibrates 5 minutes, after stratification, lower floor's solution is collected in flask again；
S7, the dichloromethane layer that will be collected twice is evaporated in the environment of 60 DEG C, and labeled as A residues, takes A residues, the 5mL of 1g
The acetonitrile of phosphate buffer I and 1mL be put into beaker, react 5min, take A residues, the phosphate buffer II of 5mL of 1g
It is put into another beaker with the acetonitrile of 1mL, 5min is reacted, for high performance liquid chromatography detection.
There are following characteristics compared with embodiment 1 and embodiment 2 in the detection method of the embodiment 3：
Detection accuracy and testing cost are above embodiment 1 and embodiment 2；
Detection time is long, and detection speed is slow, and detection efficiency is low；
Detection accuracy >=98%.
In sum, tylosin detection method and kit in the poultry, the detection method that the application is proposed are practical
Performance is good, by phosphate buffer I and phosphate buffer II, coordinates the effect of high performance liquid chromatography, can be in sample
The tylosin in portion is effectively detected that detection more facilitates, time saving and energy saving, and detection accuracy is higher, by being provided with examination
Agent box, the tylosin detection method for coordinating the application to be proposed, can be carried out accurately to the residual tylosin in poultry
Detection, effectively avoids harm of the tylosin to health of people, and cost is relatively low, and detection efficiency is high, and detection accuracy >=
98%。
It should be noted that herein, such as first and second or the like relational terms are used merely to a reality
Body or operation make a distinction with another entity or operation, and not necessarily require or imply these entities or deposited between operating
In any this actual relation or order.And, term " including ", "comprising" or its any other variant be intended to
Nonexcludability is included, so that process, method, article or equipment including a series of key elements not only will including those
Element, but also other key elements including being not expressly set out, or also include being this process, method, article or equipment
Intrinsic key element.In the absence of more restrictions.By sentence " including one ... the key element for limiting, it is not excluded that
Also there is other identical element in the process including the key element, method, article or equipment ".
Although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
Understanding can carry out various changes, modification, replacement to these embodiments without departing from the principles and spirit of the present invention
And modification, the scope of the present invention be defined by the appended.
Claims (8)
1. in a kind of poultry tylosin kit, it is characterised in that：The inside of the kit is placed with biphosphate
Ammonium salt solution, anhydrous phosphoric acid ammonium dihydrogen, phosphoric acid, two hypophosphite monohydrate sodium dihydrogens, potassium dihydrogen phosphate, three hypophosphite monohydrate hydrogen dipotassiums, just oneself
Alkane, methyl alcohol, petroleum ether, tri-distilled water and dichloromethane.
2. tylosin detection method in a kind of poultry, it is characterised in that：Detection method comprises the following steps：
S1, the ammonium dihydrogen phosphate of 0.1-0.5mol/L is taken out with pipettor, and the anhydrous of 0.1-0.5g is weighed with electronic balance
Ammonium dihydrogen phosphate, is put into dissolving in beaker, in the environment of 4-10 DEG C, reacts 5-10min；
S2, measures the phosphoric acid of 12-14mL, adds water and is settled to 800-1000mL, and labeled as A liquid, weighs two hydrations of 30-32g
Sodium dihydrogen phosphate, adds water and is settled to 800-1000mL, and labeled as B liquid, take A liquid 450-455mL, 451.6mL, 453.5mL,
454.1mL, B liquid 545-550mL, 548.4mL, 546.5mL, 545.9mL, are configured to the phosphate buffer that PH is 2.1-2.5
I, and with the frequent real-time monitoring of pH value analyzer；
S3, the potassium dihydrogen phosphate of 4-5g and the three hypophosphite monohydrate hydrogen dipotassiums of 0.5-0.8g are weighed with electronic balance, are put into beaker
Dissolving, adds water and is settled to 450-500mL, is configured to the phosphate buffer II that PH is 5.5-5.8, and passed through with pH value analyzer
Normal real-time monitoring；
S4, weighs the poultry sample of 10-20g, is wrapped with filter paper after crushing and is put into apparatus,Soxhlet's, add 0.5-1g just oneself
Alkane, with 60-75 DEG C of water-bath degreasing to colourless, and take out dry it is standby；
S5, weighs sample after the degreasing of 1-2g, and is put into the centrifuge tube of refrigerated centrifuge, adds the methyl alcohol of 10-15mL, ultrasound
25-30min is extracted, after extraction is finished, in the environment of 5-10 DEG C, 8000-10000r/min centrifugation 10-15min take supernatant
Liquid, residue is extracted 2 times again, merges No. 3 extract solutions, in the environment of 4-10 DEG C, 4500-5000r/min centrifugation 5-10min,
And extract solution is labeled as solution A；
S6, after the completion of S5, takes out the solution A of 30-50mL and is put into the separatory funnel of 125mL, takes the petroleum ether of 30-40mL
And add in separatory funnel, 1-3min is vibrated, after stratification, layer solution is removed in the separatory funnel of 300mL, and add
The tri-distilled water of 10-20mL and the dichloromethane of 20-30mL, vibrate 3-5min, after stratification, lower floor's solution are collected in flask
In, then to 300mL separatory funnel add 20-30mL dichloromethane, vibrate 3-5 minutes, after stratification, by lower floor's solution
Collect in flask again；
S7, the dichloromethane layer that will be collected twice is evaporated in the environment of 50-60 DEG C, and labeled as A residues, takes the A of 0.5-1g
Residue, the acetonitrile of the phosphate buffer I and 0.5-1mL of 3-5mL are put into beaker, react 3-5min, and the A for taking 0.5-1g is residual
Slag, the acetonitrile of the phosphate buffer II and 0.5-1mL of 3-5mL are put into another beaker, 3-5min are reacted, for efficient liquid phase
Chromatogram is detected.
3. in a kind of poultry according to claim 2 tylosin detection method, it is characterised in that：The centrifuge tube
Capacity is 50mL.
4. in a kind of poultry according to claim 2 tylosin detection method, it is characterised in that：The beaker, point
The quantity of liquid funnel and centrifuge tube is at least five.
5. in a kind of poultry according to claim 2 tylosin detection method, it is characterised in that：The electronic balance
Model AL204.
6. in a kind of poultry according to claim 2 tylosin detection method, it is characterised in that：The surname extraction
The model SXT-06 of device.
7. in a kind of poultry according to claim 2 tylosin detection method, it is characterised in that：The pH value is determined
The model BPH-200A of instrument.
8. in a kind of poultry according to claim 2 tylosin detection method, it is characterised in that：The refrigerated centrifuge
The model X-15R of machine.
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