CN106770742A - A kind of ceftiofur metabolite detection method and kit - Google Patents
A kind of ceftiofur metabolite detection method and kit Download PDFInfo
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- CN106770742A CN106770742A CN201611112591.7A CN201611112591A CN106770742A CN 106770742 A CN106770742 A CN 106770742A CN 201611112591 A CN201611112591 A CN 201611112591A CN 106770742 A CN106770742 A CN 106770742A
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- 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
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Abstract
The invention discloses a kind of ceftiofur metabolite detection method and kit, including chromatograph, PDAD, chromatographic column, eddy mixer, electric-heated thermostatic water bath, centrifuge, assay balance, refiner, nitrogen drying instrument and dual water distillation apparatus, Ceftiofur standard items, acetonitrile, methyl alcohol are chromatographically pure, dithioerythritol（Purity 99%）, iodoacetamide（Purity 99%）, trifluoroacetic acid and sodium tetraborate etc..The present invention also establishes the method that Ceftiofur residual in metabolin is determined with high-efficient liquid phase chromatogram technology, and metabolin sample is extracted through dithioerythritol, and iodoacetamide derives, C18 solid phase column extracting and purifyings, methanol solution is eluted, and is as a result shown, the method is easy to operate, quick, sensitivity is high.
Description
Technical field
The present invention relates to technical field of biological, more particularly to a kind of ceftiofur metabolite detection method and reagent
Box.
Background technology
This product is off-white color to pale yellow powder.Insoluble in water, slightly soluble, almost insoluble in ethanol in acetone.For
Antimicrobial.Cephalosporins is semi-synthetic third generation animal specific cynnematin.Sodium salt and hydrochloride are made for injection
With.
Absorb rapid after this product intramuscular and hypodermic injection, high with tissue drug concentration in blood, effective blood drug concentration is maintained
Time is long, eliminates slow, long half time.It is rapid in 15MIN to be absorbed in generation in blood plasma after pig intramuscular injection this product to ox
The de- furoyl Ceftiofur of one-level metabolin（Desfuroyl ceftiofur,DFC）Because lactam nucleus are not damaged, its
Antibacterial activity is essentially identical with Ceftiofur.DFC can further form the inactive fluidisation of DFC NACs two in tissue
Thing.Apparent volume of distribution≤the 1L/KG of this product.Pig, sheep, after ox multiple dose intramuscular injection in kidney concentration highest, be secondly
Lung, liver, fat and muscle, can typically be maintained above the concentration of MIC.Such as pig presses 3mg/kg intramuscular injections, 1 times a day, is used in conjunction 3
My god, its tissue concentration is respectively：Blood plasma 3.25, respiratory tract 0.60, skin 0.57, lung 0.49, tonsillotome 0.30, joint fluid
0.29.Ox presses 1mg/kg intramuscular injections, 1 times a day, is used in conjunction 5 days, and its tissue concentration is respectively blood plasma 1.0（7.5H after injection）Close
Section liquid 0.65（2.5H after injection）, lung 0.36, jejunum 0.34.It is main with inactive free metabolin-DFC cysteamines in milk
The formation of sour disulphide occurs.The excretion of cephalo plug furan is relatively slow, and there is obvious species variation the half-life period of animal（Horse, ox is continuous
Sheep, pig, dog, chicken, the T1/2 of turkey）Respectively 3.15,7.12,2.83,14.5,4.12,6.77 and 7.45H.But major part can
By urine and discharge in excrement in 24H after intramuscular injection.DFC degrades rapidly to inactive compound in ox in the excrement of pig and chicken,
So needing a kind of ceftiofur metabolite detection method and kit.
The content of the invention
Based on the technical problem that background technology is present, the present invention proposes a kind of ceftiofur metabolite detection method and examination
Agent box, with various aggregation of data monitoring characteristics, solves the problems, such as existing animal metabolism analyte detection Ceftiofur.
The present invention provides following technical scheme：A kind of ceftiofur metabolite detection method and kit, including chromatograph,
PDAD, chromatographic column, eddy mixer, electric-heated thermostatic water bath, centrifuge, assay balance, refiner, nitrogen
Drying instrument and dual water distillation apparatus, Ceftiofur standard items, acetonitrile, methyl alcohol are chromatographically pure, dithioerythritol（Purity 99%）, iodine
Acetamide（Purity 99%）, trifluoroacetic acid and sodium tetraborate etc., it is characterised in that：The ceftiofur metabolite detection method and examination
Agent box detecting step is：
S1, prepare main solution, main solution include standard reserving solution, standard working solution, borate buffer, phosphate buffer,
Dithioerythritol extract solution, iodoacetamide solution and mobile phase；
Prepare before the detection of S2, thing to be detected and equipment, including：（1）Feed of the full price without antibacterials, complete feed is continuously fed
The animal of ten days is supported, collection animal metabolism thing is standby afterwards,（2）Chromatographic column：AgilentZorbaxXDB-C18（4.6m×
250mm × 5 μm, I.D）,（3）Detector：UV-detector, Detection wavelength 266nm；（4）Mobile phase is water：Trifluoroacetic acid: second
Nitrile=800: 1: 200（Volume ratio）；（5）Column temperature：30℃；Flow velocity：1mL/min；Sample size：100μL；
The extraction and purification of S3, sample, are extracted and derive, and animal metabolism thing is stirred, and are transferred to the clean small beakers of 100mL
In, it is stirred for uniformly, weighing with glass bar（5.0±0.1）G metabolin samples, put in 50mL tool plug centrifuge tubes, add 0.4%
Dithioerythritol extract solution 10mL, the 1~2min that is vortexed is mixed, and 50 DEG C of water-bath 30min, every 3~5min vortex oscillations 1 time, shake
It is immediately placed in water-bath after swinging, room temperature is placed in after 30min, 4% iodoacetamide solution 3mL is added after cooling, the 1~2min that is vortexed is mixed
Even, room temperature lucifuge derives 30min, every 10min, is vortexed and mixes 1 time, and derivative adds 0.025mol/L phosphoric acid buffers after finishing
Liquid 5mL, regulation pH value to 2.5~3.0, in 4 DEG C of 10000r/min centrifugation 20min, takes supernatant and is placed in another centrifuge tube, standby
With purification, C18 solid-phase extraction columns are activated in advance with 3mL methyl alcohol, 3mL phosphate buffers, then by supernatant addition post, used
The ultrapure water wash of 10mL, vacuum solid-phase extraction device is drained, and is finally eluted with 5mL methanol solutions, collects eluent, 45 DEG C of nitrogen
Drying, with 0.5mL flow phased soln, vortex oscillation 5min, ultrasonic 5min, 0.22 μm of filtering with microporous membrane, taking 100 μ L is carried out
HPLC is detected；
S4, the rate of recovery and precision determines, claims metabolin 5g, is placed in 50mL polypropylene centrifuge tubes, be separately added into 0.1,1.0,
Each 1mL of standard working solution of 10.0 μ g/mL, after extracting and purify through the above method, cephalo in egg sample is tried to achieve in HPLC detections
The rate of recovery of the thiophene furan in 0.02,0.20,2.00 μ g/g levels, during measure, the treatment of each concentration determines 5 within same working day
It is secondary（In a few days）；5 batches are determined in different operating day（In the daytime）, per 5 repetitions of batch, chromatographic peak in a few days and in the daytime is calculated respectively
Area average and standard error, seek in a few days and in the daytime relative standard deviation（RS）；
S5, sensitivity determination weigh 5 parts of blank eggs and are homogenized each 5g, are placed in 50mL polypropylene centrifuge tubes, Jia 0.05 respectively,
0.10th, each 1mL of standard working solution of 0.25,0.50,1.00 μ g/mL, make the homogenate of every 1g eggs containing Ceftiofur 0.01,0.02,
0.05th, 0.10,0.20 μ g, extract, purify as stated above.
Preferably, the standard reserving solution in step S1 weighs 10.0mg Ceftiofur standard items including precision, is placed in
In 100mL volumetric flasks, dissolved with phosphate buffer and diluted and be settled to scale, concentration be 100 μ g/mL Ceftiofur storage
Standby liquid, is made into series standard concentration with phosphate buffer before use, and standard working solution includes taking 1mL Ceftiofur storing solutions, according to
Secondary dilution with phosphate buffer is settled to 10.00,5.00,1.00,0.50,0.25,0.10,0.05 μ g/mL, borate buffer
Including 0.05mol/L borate buffers（PH value 9）, sodium tetraborate 19.05g, potassium chloride 3.70g are accurately weighed, it is settled to
1000mL, phosphate buffer includes 0.025mol/L phosphate buffers（PH value 7）：3.4g potassium dihydrogen phosphates accurately are weighed, is added water
PH value is adjusted to 7.0 with potassium hydroxide after about 700mL, 1000mL is settled to, and dithioerythritol extract solution includes that 0.4% 2 sulphur is red
Moss alcohol extract：4.0g dithioerythritols are weighed, is dissolved in 1000mL0.05mol/L borate buffers（It is now with the current）, iodine second
Amide solution includes 4% iodoacetamide solution：4.0g iodoacetamides are weighed, is dissolved in 100mL0.025mol/L phosphate buffers
（It is now with the current）, mobile phase measures 800mL water respectively, and then 1mL trifluoroacetic acids, 200mL acetonitriles mix.
Preferably, prepare before thing to be detected and equipment detection in step S2, including：（1）Feeding of the full price without antibacterials
Material,（2）Chromatographic column：AgilentZorbaxXDB-C18（4.6m × 250mm × 5 μm, I.D）,（3）Detector：Ultraviolet detection
Device, Detection wavelength 266nm；（4）Mobile phase is water：Trifluoroacetic acid: acetonitrile=800: 1: 200（Volume ratio）；（5）Column temperature：30℃；
Flow velocity：1mL/min；Sample size：100μL.
Preferably, in step S3 sample extraction and purification, extract derive, take supernatant and be placed in another centrifuge tube, it
Standby purification afterwards, is in advance activated C18 solid-phase extraction columns with 3mL methyl alcohol, 3mL phosphate buffers, and supernatant then is added into post
In, with the ultrapure water wash of 10mL, vacuum solid-phase extraction device is drained, and is finally eluted with 5mL methanol solutions, collects eluent, 45
The drying of DEG C nitrogen, phased soln is flowed with 0.5mL, and vortex oscillation 5min, ultrasonic 5min, 0.22 μm of filtering with microporous membrane take 100 μ L
Carry out HPLC detections.
Beneficial effects of the present invention：Establish the side that Ceftiofur residual in metabolin is determined with high-efficient liquid phase chromatogram technology
Method, metabolin sample is extracted through dithioerythritol, and iodoacetamide derives, C18 solid phase column extracting and purifyings, methanol solution wash-out, knot
Fruit shows that the method is easy to operate, quick, sensitivity is high.
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：A kind of ceftiofur metabolite detection method and kit, including chromatograph,
PDAD, chromatographic column, eddy mixer, electric-heated thermostatic water bath, centrifuge, assay balance, refiner, nitrogen
Drying instrument and dual water distillation apparatus, Ceftiofur standard items, acetonitrile, methyl alcohol are chromatographically pure, dithioerythritol（Purity 99%）, iodine
Acetamide（Purity 99%）, trifluoroacetic acid and sodium tetraborate etc., it is characterised in that：The ceftiofur metabolite detection method and examination
Agent box detecting step is：
S1, prepare main solution, main solution include standard reserving solution, standard working solution, borate buffer, phosphate buffer,
Dithioerythritol extract solution, iodoacetamide solution and mobile phase, including standard reserving solution weigh 10.0mg cephalo thiophenes including precision
Furan standard items, are placed in 100mL volumetric flasks, are dissolved with phosphate buffer and diluted and are settled to scale, concentration be 100 μ g/
The Ceftiofur storing solution of mL, is made into series standard concentration with phosphate buffer before use, and standard working solution includes taking 1mL cephalos
Thiophene furan storing solution, is diluted with phosphate buffer be settled to 10.00,5.00,1.00,0.50,0.25,0.10,0.05 μ g/ successively
ML, borate buffer includes 0.05mol/L borate buffers（PH value 9）, accurately weigh sodium tetraborate 19.05g, potassium chloride
3.70g, is settled to 1000mL, and phosphate buffer includes 0.025mol/L phosphate buffers（PH value 7）：Accurately weigh 3.4g phosphoric acid
Potassium dihydrogen, pH value is adjusted to 7.0 after the about 700mL that adds water with potassium hydroxide, is settled to 1000mL, and dithioerythritol extract solution includes
0.4% dithioerythritol extract solution：4.0g dithioerythritols are weighed, is dissolved in 1000mL0.05mol/L borate buffers（Now match somebody with somebody
Now use）, iodoacetamide solution include 4% iodoacetamide solution：4.0g iodoacetamides are weighed, 100mL0.025mol/L phosphoric acid is dissolved in
In buffer solution（It is now with the current）, mobile phase measures 800mL water respectively, and then 1mL trifluoroacetic acids, 200mL acetonitriles mix；
Prepare before the detection of S2, thing to be detected and equipment, including：（1）Feed of the full price without antibacterials, complete feed is continuously fed
The animal of ten days is supported, collection animal metabolism thing is standby afterwards,（2）Chromatographic column：AgilentZorbaxXDB-C18（4.6m×
250mm × 5 μm, I.D）,（3）Detector：UV-detector, Detection wavelength 266nm；（4）Mobile phase is water：Trifluoroacetic acid: second
Nitrile=800: 1: 200（Volume ratio）；（5）Column temperature：30℃；Flow velocity：1mL/min；Sample size：100μL；
The extraction and purification of S3, sample, are extracted and derive, and animal metabolism thing is stirred, and are transferred to the clean small beakers of 100mL
In, it is stirred for uniformly, weighing with glass bar（5.0±0.1）G metabolin samples, put in 50mL tool plug centrifuge tubes, add 0.4%
Dithioerythritol extract solution 10mL, the 1~2min that is vortexed is mixed, and 50 DEG C of water-bath 30min, every 3~5min vortex oscillations 1 time, shake
It is immediately placed in water-bath after swinging, room temperature is placed in after 30min, 4% iodoacetamide solution 3mL is added after cooling, the 1~2min that is vortexed is mixed
Even, room temperature lucifuge derives 30min, every 10min, is vortexed and mixes 1 time, and derivative adds 0.025mol/L phosphoric acid buffers after finishing
Liquid 5mL, regulation pH value to 2.5~3.0, in 4 DEG C of 10000r/min centrifugation 20min, takes supernatant and is placed in another centrifuge tube, standby
With purification, C18 solid-phase extraction columns are activated in advance with 3mL methyl alcohol, 3mL phosphate buffers, then by supernatant addition post, used
The ultrapure water wash of 10mL, vacuum solid-phase extraction device is drained, and is finally eluted with 5mL methanol solutions, collects eluent, 45 DEG C of nitrogen
Drying, with 0.5mL flow phased soln, vortex oscillation 5min, ultrasonic 5min, 0.22 μm of filtering with microporous membrane, taking 100 μ L is carried out
HPLC is detected；
S4, the rate of recovery and precision determines, claims metabolin 5g, is placed in 50mL polypropylene centrifuge tubes, be separately added into 0.1,1.0,
Each 1mL of standard working solution of 10.0 μ g/mL, after extracting and purify through the above method, cephalo in egg sample is tried to achieve in HPLC detections
The rate of recovery of the thiophene furan in 0.02,0.20,2.00 μ g/g levels, during measure, the treatment of each concentration determines 5 within same working day
It is secondary（In a few days）；5 batches are determined in different operating day（In the daytime）, per 5 repetitions of batch, chromatographic peak in a few days and in the daytime is calculated respectively
Area average and standard error, seek in a few days and in the daytime relative standard deviation（RS）；
S5, sensitivity determination weigh 5 parts of blank eggs and are homogenized each 5g, are placed in 50mL polypropylene centrifuge tubes, Jia 0.05 respectively,
0.10th, each 1mL of standard working solution of 0.25,0.50,1.00 μ g/mL, make the homogenate of every 1g eggs containing Ceftiofur 0.01,0.02,
0.05th, 0.10,0.20 μ g, extract, purify as stated above.
Embodiment one
A kind of ceftiofur metabolite detection method and kit, including chromatograph, PDAD, chromatographic column, whirlpool
Whirlpool blender, electric-heated thermostatic water bath, centrifuge, assay balance, refiner, nitrogen drying instrument and dual water distillation apparatus, cephalo
Thiophene furan standard items, acetonitrile, methyl alcohol are chromatographically pure, dithioerythritol（Purity 99%）, iodoacetamide（Purity 99%）, trifluoroacetic acid and
Sodium tetraborate etc., it is characterised in that：The ceftiofur metabolite detection method and kit detecting step are：
S1, prepare main solution, main solution include standard reserving solution, standard working solution, borate buffer, phosphate buffer,
Dithioerythritol extract solution, iodoacetamide solution and mobile phase, including standard reserving solution weigh 10.0mg cephalo thiophenes including precision
Furan standard items, are placed in 100mL volumetric flasks, are dissolved with phosphate buffer and diluted and are settled to scale, concentration be 100 μ g/
The Ceftiofur storing solution of mL, is made into series standard concentration with phosphate buffer before use, and standard working solution includes taking 1mL cephalos
Thiophene furan storing solution, is diluted with phosphate buffer be settled to 10.00,5.00,1.00,0.50,0.25,0.10,0.05 μ g/ successively
ML, borate buffer includes 0.05mol/L borate buffers（PH value 9）, accurately weigh sodium tetraborate 19.05g, potassium chloride
3.70g, is settled to 1000mL, and phosphate buffer includes 0.025mol/L phosphate buffers（PH value 7）：Accurately weigh 3.4g phosphoric acid
Potassium dihydrogen, pH value is adjusted to 7.0 after the about 700mL that adds water with potassium hydroxide, is settled to 1000mL, and dithioerythritol extract solution includes
0.4% dithioerythritol extract solution：4.0g dithioerythritols are weighed, is dissolved in 1000mL0.05mol/L borate buffers（Now match somebody with somebody
Now use）, iodoacetamide solution include 4% iodoacetamide solution：4.0g iodoacetamides are weighed, 100mL0.025mol/L phosphoric acid is dissolved in
In buffer solution（It is now with the current）, mobile phase measures 800mL water respectively, and then 1mL trifluoroacetic acids, 200mL acetonitriles mix；
Prepare before the detection of S2, thing to be detected and equipment, including：（1）Feed of the full price without antibacterials, complete feed is continuously fed
The animal of ten days is supported, collection animal metabolism thing is standby afterwards,（2）Chromatographic column：AgilentZorbaxXDB-C18（4.6m×
250mm × 5 μm, I.D）,（3）Detector：UV-detector, Detection wavelength 266nm；（4）Mobile phase is water：Trifluoroacetic acid: second
Nitrile=800: 1: 200（Volume ratio）；（5）Column temperature：30℃；Flow velocity：1mL/min；Sample size：100μL；
The extraction and purification of S3, sample, are extracted and derive, and animal metabolism thing is stirred, and are transferred to the clean small beakers of 100mL
In, it is stirred for uniformly, weighing 4.9g metabolin samples with glass bar, put in 50mL tool plug centrifuge tubes, add 0.4% 2 sulphur red
Moss alcohol extract 10mL, the 1~2min that is vortexed is mixed, 50 DEG C of water-bath 30min, every 3~5min vortex oscillations 1 time, is stood after vibration
It is put into water-bath, room temperature is placed in after 30min, add 4% iodoacetamide solution 3mL, vortex 1min to mix after cooling, room temperature is kept away
Light derives 30min, every 10min, is vortexed and mixes 1 time, and derivative adds 0.025mol/L phosphate buffer 5mL, regulation after finishing
PH value, in 4 DEG C of 10000r/min centrifugation 20min, takes supernatant and is placed in another centrifuge tube to 2.8, and 3mL first is used in standby purification
Alcohol, 3mL phosphate buffers by the activation of C18 solid-phase extraction columns, then by supernatant addition post, are drenched with 10mL ultra-pure waters in advance
Wash, vacuum solid-phase extraction device is drained, finally eluted with 5mL methanol solutions, collect eluent, 0.5mL is used in 45 DEG C of nitrogen dryings
Flowing phased soln, vortex oscillation 5min, ultrasonic 6min, 0.22 μm of filtering with microporous membrane, taking 100 μ L carries out HPLC detections；
S4, the rate of recovery and precision determines, claims metabolin 5g, is placed in 50mL polypropylene centrifuge tubes, be separately added into 0.1,1.0,
Each 1mL of standard working solution of 10.0 μ g/mL, after extracting and purify through the above method, cephalo in egg sample is tried to achieve in HPLC detections
The rate of recovery of the thiophene furan in 0.02,0.20,2.00 μ g/g levels, during measure, the treatment of each concentration determines 5 within same working day
It is secondary（In a few days）；5 batches are determined in different operating day（In the daytime）, per 5 repetitions of batch, chromatographic peak in a few days and in the daytime is calculated respectively
Area average and standard error, seek in a few days and in the daytime relative standard deviation（RS）；
S5, sensitivity determination weigh 5 parts of blank eggs and are homogenized each 5g, are placed in 50mL polypropylene centrifuge tubes, Jia 0.05 respectively,
0.10th, each 1mL of standard working solution of 0.25,0.50,1.00 μ g/mL, make the homogenate of every 1g eggs containing Ceftiofur 0.01,0.02,
0.05th, 0.10,0.20 μ g, extract, purify as stated above.
Embodiment two
A kind of ceftiofur metabolite detection method and kit, including chromatograph, PDAD, chromatographic column, whirlpool
Whirlpool blender, electric-heated thermostatic water bath, centrifuge, assay balance, refiner, nitrogen drying instrument and dual water distillation apparatus, cephalo
Thiophene furan standard items, acetonitrile, methyl alcohol are chromatographically pure, dithioerythritol（Purity 99%）, iodoacetamide（Purity 99%）, trifluoroacetic acid and
Sodium tetraborate etc., it is characterised in that：The ceftiofur metabolite detection method and kit detecting step are：
S1, prepare main solution, main solution include standard reserving solution, standard working solution, borate buffer, phosphate buffer,
Dithioerythritol extract solution, iodoacetamide solution and mobile phase, including standard reserving solution weigh 10.0mg cephalo thiophenes including precision
Furan standard items, are placed in 100mL volumetric flasks, are dissolved with phosphate buffer and diluted and are settled to scale, concentration be 100 μ g/
The Ceftiofur storing solution of mL, is made into series standard concentration with phosphate buffer before use, and standard working solution includes taking 1mL cephalos
Thiophene furan storing solution, is diluted with phosphate buffer be settled to 10.00,5.00,1.00,0.50,0.25,0.10,0.05 μ g/ successively
ML, borate buffer includes 0.05mol/L borate buffers（PH value 9）, accurately weigh sodium tetraborate 19.05g, potassium chloride
3.70g, is settled to 1000mL, and phosphate buffer includes 0.025mol/L phosphate buffers（PH value 7）：Accurately weigh 3.4g phosphoric acid
Potassium dihydrogen, pH value is adjusted to 7.0 after the about 700mL that adds water with potassium hydroxide, is settled to 1000mL, and dithioerythritol extract solution includes
0.4% dithioerythritol extract solution：4.0g dithioerythritols are weighed, is dissolved in 1000mL0.05mol/L borate buffers（Now match somebody with somebody
Now use）, iodoacetamide solution include 4% iodoacetamide solution：4.0g iodoacetamides are weighed, 100mL0.025mol/L phosphoric acid is dissolved in
In buffer solution（It is now with the current）, mobile phase measures 800mL water respectively, and then 1mL trifluoroacetic acids, 200mL acetonitriles mix；
Prepare before the detection of S2, thing to be detected and equipment, including：（1）Feed of the full price without antibacterials, complete feed is continuously fed
The animal of ten days is supported, collection animal metabolism thing is standby afterwards,（2）Chromatographic column,（3）Detector,（4）Mobile phase is water,（5）Post
Temperature；
The extraction and purification of S3, sample, are extracted and derive, and animal metabolism thing is stirred, and are transferred to the clean small beakers of 100mL
In, it is stirred for uniformly, weighing 5.1g metabolin samples with glass bar, put in 50mL tool plug centrifuge tubes, add 0.4% 2 sulphur red
Moss alcohol extract 10mL, vortex 1.5min are mixed, 50 DEG C of water-bath 30min, every 4min vortex oscillations 1 time, are put immediately after vibration
Enter in water-bath, room temperature is placed in after 30min, 4% iodoacetamide solution 3mL is added after cooling, be vortexed 1.5 mixings, room temperature lucifuge derives
30min, every 10min, is vortexed and mixes 1 time, and derivative adds 0.025mol/L phosphate buffer 5mL after finishing, regulation pH value to
2.8, in 4 DEG C of 10000r/min centrifugation 25min, take supernatant and be placed in another centrifuge tube, standby purification, with 3mL methyl alcohol, 3mL
Phosphate buffer in advance by C18 solid-phase extraction columns activation, then by supernatant add post in, with the ultrapure water wash of 10mL, vacuum
Solid-phase extraction device is drained, and is finally eluted with 5mL methanol solutions, collects eluent, and 0.5mL mobile phases are used in 45 DEG C of nitrogen dryings
Dissolving, vortex oscillation 5min, ultrasonic 8min, 0.22 μm of filtering with microporous membrane, taking 100 μ L carries out HPLC detections；
S4, the rate of recovery and precision determines, claims metabolin 5g, is placed in 50mL polypropylene centrifuge tubes, be separately added into 0.1,1.0,
Each 1mL of standard working solution of 10.0 μ g/mL, after extracting and purify through the above method, cephalo in egg sample is tried to achieve in HPLC detections
The rate of recovery of the thiophene furan in 0.02,0.20,2.00 μ g/g levels；
S5, sensitivity determination weigh 5 parts of blank eggs and are homogenized each 5g, are placed in 50mL polypropylene centrifuge tubes, Jia 0.05 respectively,
0.10th, each 1mL of standard working solution of 0.25,0.50,1.00 μ g/mL, make the homogenate of every 1g eggs containing Ceftiofur 0.01,0.02,
0.05th, 0.10,0.20 μ g, extract, purify as stated above.
Embodiment three
A kind of ceftiofur metabolite detection method and kit, including chromatograph, PDAD, chromatographic column, whirlpool
Whirlpool blender, electric-heated thermostatic water bath, centrifuge, assay balance, refiner, nitrogen drying instrument and dual water distillation apparatus, cephalo
Thiophene furan standard items, acetonitrile, methyl alcohol are chromatographically pure, dithioerythritol（Purity 99%）, iodoacetamide（Purity 99%）, trifluoroacetic acid and
Sodium tetraborate etc., it is characterised in that：The ceftiofur metabolite detection method and kit detecting step are：
S1, prepare main solution, main solution include standard reserving solution, standard working solution, borate buffer, phosphate buffer,
Dithioerythritol extract solution, iodoacetamide solution and mobile phase, including standard reserving solution weigh 10.0mg cephalo thiophenes including precision
Furan standard items, are placed in 100mL volumetric flasks, are dissolved with phosphate buffer and diluted and are settled to scale, concentration be 100 μ g/
The Ceftiofur storing solution of mL, is made into series standard concentration with phosphate buffer before use, and standard working solution includes taking 1mL cephalos
Thiophene furan storing solution, is diluted with phosphate buffer be settled to 10.00,5.00,1.00,0.50,0.25,0.10,0.05 μ g/ successively
ML, borate buffer includes 0.05mol/L borate buffers（PH value 9）, accurately weigh sodium tetraborate 19.05g, potassium chloride
3.70g, is settled to 1000mL, and phosphate buffer includes 0.025mol/L phosphate buffers（PH value 7）：Accurately weigh 3.4g phosphoric acid
Potassium dihydrogen, pH value is adjusted to 7.0 after the about 700mL that adds water with potassium hydroxide, is settled to 1000mL, and dithioerythritol extract solution includes
0.4% dithioerythritol extract solution：4.0g dithioerythritols are weighed, is dissolved in 1000mL0.05mol/L borate buffers（Now match somebody with somebody
Now use）, iodoacetamide solution include 4% iodoacetamide solution：4.0g iodoacetamides are weighed, 100mL0.025mol/L phosphoric acid is dissolved in
In buffer solution（It is now with the current）, mobile phase measures 800mL water respectively, and then 1mL trifluoroacetic acids, 200mL acetonitriles mix；
Prepare before the detection of S2, thing to be detected and equipment, including：（1）Feed of the full price without antibacterials,（2）Chromatographic column,（3）
Detector,（4）Mobile phase is water,（5）Column temperature.
The extraction and purification of S3, sample, are extracted and derive, and animal metabolism thing is stirred, and are transferred to clean small of 100mL
In beaker, it is stirred for uniformly, weighing 5.1g metabolin samples with glass bar, is put in 50mL tool plug centrifuge tubes, adds 0.4% 2
Sulphur erythrite extract solution 10mL, vortex 2min are mixed, 50 DEG C of water-bath 30min, every 5min vortex oscillations 1 time, after vibration immediately
It is put into water-bath, room temperature is placed in after 30min, 4% iodoacetamide solution 3mL is added after cooling, be vortexed and mix, room temperature lucifuge is spread out
Life, every 10min, is vortexed and mixes 1 time, and derivative adds 0.025mol/L phosphate buffer 5mL after finishing, and adjusts pH value to 3.0,
In 4 DEG C of 10000r/min centrifugation 20min, take supernatant and be placed in another centrifuge tube, standby purification, with methyl alcohol, phosphate buffer
In advance by the activation of C18 solid-phase extraction columns, then by supernatant addition post, ultrapure water wash, vacuum solid-phase extraction device is used to take out
It is dry, finally eluted with methanol solution, collect eluent, 45 DEG C of nitrogen dryings flow phased soln with 0.5mL, vortex oscillation 5min,
Ultrasonic 7min, 0.22 μm of filtering with microporous membrane, taking 100 μ L carries out HPLC detections；
S4, the rate of recovery and precision determines, claims metabolin 5g, is placed in 50mL polypropylene centrifuge tubes, be separately added into 0.1,1.0,
Each 1mL of standard working solution of 10.0 μ g/mL, after extracting and purify through the above method, HPLC detections；
S5, sensitivity determination weigh 5 parts of blank eggs and are homogenized each 5g, are placed in 50mL polypropylene centrifuge tubes, Jia 0.05 respectively,
0.10th, each 1mL of standard working solution of 0.25,0.50,1.00 μ g/mL, make the homogenate of every 1g eggs containing Ceftiofur 0.01,0.02,
0.05th, 0.10,0.20 μ g, extract, purify as stated above.
The above, the only present invention preferably specific embodiment, but protection scope of the present invention is not limited thereto,
Any one skilled in the art the invention discloses technical scope in, technology according to the present invention scheme and its
Inventive concept is subject to equivalent or change, should all be included within the scope of the present invention.
Claims (4)
1. a kind of ceftiofur metabolite detection method and kit, including chromatograph, PDAD, chromatographic column,
Eddy mixer, electric-heated thermostatic water bath, centrifuge, assay balance, refiner, nitrogen drying instrument and dual water distillation apparatus, head
Spore thiophene furan standard items, acetonitrile, methyl alcohol are chromatographically pure, dithioerythritol（Purity 99%）, iodoacetamide（Purity 99%）, trifluoroacetic acid
With sodium tetraborate etc., it is characterised in that：The ceftiofur metabolite detection method and kit detecting step are：
S1, prepare main solution, main solution include standard reserving solution, standard working solution, borate buffer, phosphate buffer,
Dithioerythritol extract solution, iodoacetamide solution and mobile phase；
Prepare before the detection of S2, thing to be detected and equipment, including：（1）Feed of the full price without antibacterials,（2）Chromatographic column；（3）
Detector；（4）Mobile phase is water；
The extraction and purification of S3, sample, are extracted and derive, and animal metabolism thing is stirred, and are transferred to the clean small beakers of 100mL
In, it is stirred for uniformly, weighing with glass bar（5.0±0.1）G metabolin samples, put in 50mL tool plug centrifuge tubes, add 0.4%
Dithioerythritol extract solution 10mL, the 1~2min that is vortexed is mixed, and 50 DEG C of water-bath 30min, every 3~5min vortex oscillations 1 time, shake
It is immediately placed in water-bath after swinging, room temperature is placed in after 30min, 4% iodoacetamide solution 3mL is added after cooling, the 1~2min that is vortexed is mixed
Even, room temperature lucifuge derives 30min, every 10min, is vortexed and mixes 1 time, and derivative adds 0.025mol/L phosphoric acid buffers after finishing
Liquid 5mL, regulation pH value to 2.5~3.0, in 4 DEG C of 10000r/min centrifugation 20min, takes supernatant and is placed in another centrifuge tube；
S4, the rate of recovery and precision determines, claims metabolin 5g, is placed in 50mL polypropylene centrifuge tubes, be separately added into 0.1,1.0,
Each 1mL of standard working solution of 10.0 μ g/mL, after extracting and purify through the above method, cephalo in egg sample is tried to achieve in HPLC detections
The rate of recovery of the thiophene furan in 0.02,0.20,2.00 μ g/g levels, during measure, the treatment of each concentration determines 5 within same working day
It is secondary（In a few days）；5 batches are determined in different operating day（In the daytime）, per 5 repetitions of batch, chromatographic peak in a few days and in the daytime is calculated respectively
Area average and standard error, seek in a few days and in the daytime relative standard deviation（RS）；
S5, sensitivity determination weigh 5 parts of blank eggs and are homogenized each 5g, are placed in 50mL polypropylene centrifuge tubes, Jia 0.05 respectively,
0.10th, each 1mL of standard working solution of 0.25,0.50,1.00 μ g/mL, make the homogenate of every 1g eggs containing Ceftiofur 0.01,0.02,
0.05th, 0.10,0.20 μ g, extract, purify as stated above.
2. a kind of ceftiofur metabolite detection method according to claim 1 and kit, it is characterised in that：Step S1
In standard reserving solution weigh 10.0mg Ceftiofur standard items including precision, be placed in 100mL volumetric flasks, use phosphate buffer
Dissolve and dilute and be settled to scale, concentration be 100 μ g/mL Ceftiofur storing solution, matched somebody with somebody with phosphate buffer before use
Into series standard concentration, standard working solution includes taking 1mL Ceftiofur storing solutions, is diluted with phosphate buffer be settled to successively
10.00th, 5.00,1.00,0.50,0.25,0.10,0.05 μ g/mL, borate buffer includes 0.05mol/L borate buffers
（PH value 9）, sodium tetraborate 19.05g, potassium chloride 3.70g are accurately weighed, 1000mL is settled to, phosphate buffer includes
0.025mol/L phosphate buffers（PH value 7）：3.4g potassium dihydrogen phosphates accurately are weighed, is adjusted with potassium hydroxide after the about 700mL that adds water
Section pH value is settled to 1000mL to 7.0, and dithioerythritol extract solution includes 0.4% dithioerythritol extract solution：Weigh 4.0g bis-
Sulphur erythrite, is dissolved in 1000mL0.05mol/L borate buffers（It is now with the current）, iodoacetamide solution include 4% iodoacetamide
Solution：4.0g iodoacetamides are weighed, is dissolved in 100mL0.025mol/L phosphate buffers（It is now with the current）, mobile phase measures respectively
800mL water is taken, then 1mL trifluoroacetic acids, 200mL acetonitriles mix.
3. a kind of ceftiofur metabolite detection method according to claim 1 and kit, it is characterised in that：Step S2
In thing to be detected and equipment detection before prepare, including：（1）Feed of the full price without antibacterials,（2）Chromatographic column：
AgilentZorbaxXDB-C18（4.6m × 250mm × 5 μm, I.D）,（3）Detector：UV-detector, Detection wavelength
266nm；（4）Mobile phase is water：Trifluoroacetic acid: acetonitrile=800: 1: 200（Volume ratio）；（5）Column temperature：30℃；Flow velocity：1mL/
min；Sample size：100μL.
4. a kind of ceftiofur metabolite detection method according to claim 1 and kit, it is characterised in that：Step S3
The extraction and purification of middle sample, extract derive, take supernatant and be placed in another centrifuge tube, afterwards standby purification, with 3mL methyl alcohol,
3mL phosphate buffers in advance by the activation of C18 solid-phase extraction columns, during supernatant then added into post, with the ultrapure water wash of 10mL, very
Empty solid-phase extraction device is drained, and is finally eluted with 5mL methanol solutions, collects eluent, and 45 DEG C of nitrogen dryings are flowed with 0.5mL
Phased soln, vortex oscillation 5min, ultrasonic 5min, 0.22 μm of filtering with microporous membrane, taking 100 μ L carries out HPLC detections.
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