CN110470780A - The discrimination method of protein feed raw material admixture Terramycin-type waste water - Google Patents
The discrimination method of protein feed raw material admixture Terramycin-type waste water Download PDFInfo
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- CN110470780A CN110470780A CN201910862794.5A CN201910862794A CN110470780A CN 110470780 A CN110470780 A CN 110470780A CN 201910862794 A CN201910862794 A CN 201910862794A CN 110470780 A CN110470780 A CN 110470780A
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- terramycin
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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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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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
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
- G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
- G01N30/724—Nebulising, aerosol formation or ionisation
- G01N30/7266—Nebulising, aerosol formation or ionisation by electric field, e.g. electrospray
Abstract
A kind of protein feed raw material adds the discrimination method of Terramycin-type waste water, is related to feeding quality safety testing field.The discrimination method includes: to dilute protein feed raw material with the second solvent through the first solvent extraction, and filtering obtains prepare liquid.Using terramycin, tetracycline and dehydration tetracycline as the marker of Terramycin-type waste water, prepare liquid is separated through liquid chromatogram, Mass Spectrometer Method, identifying protein feed raw material whether there is marker, wherein the first solvent is organic solvent.The discrimination method amount of samples is few, and pretreatment process is simple, and detection time is short, sensitivity is higher, as a result accurately, can effectively identify whether protein feed raw material adds Terramycin-type waste water.
Description
Technical field
This application involves feeding quality safety testing fields, and it is mould to add soil in particular to a kind of protein feed raw material
The discrimination method of the plain dregs of a decoction.
Background technique
Protein feed raw material (dregs of beans, cotton dregs, rapeseed dregs) is important forage protein source, is widely used in livestock and poultry and supports
It grows.Terramycin-type waste water is that remaining fermentation medium generates waste residue after filtering after biofermentation produces terramycin, is had certain
Crude protein.Once there is media report, illegal culturist adds Terramycin-type waste water and substitutes use into forage protein raw material.Due to cultivation
Process is generated carefully using the feedstuff containing Terramycin-type waste water with cultivated animals health, influence food safety and induction is endangered
The potential risks hidden danger such as bacterium drug resistance, the Ministry of Agriculture, China, the Ministry of Public Health and National Drug Administration combine No. 176 of publication
Bulletin clear stipulaties, which are forbidden for Terramycin-type waste water being spiked into feed, to be used.
Although currently, both at home and abroad existing detection technique can terramycin content in Accurate Determining forage protein raw material,
It can not identify due to addition terramycin prototype medicine or add caused by Terramycin-type waste water.
In view of this, the present invention is specifically proposed.
Summary of the invention
The application provides a kind of discrimination method of protein feed raw material admixture Terramycin-type waste water, to improve existing identification side
Method, cannot be distinguished from terramycin in forage protein raw material is due to addition terramycin prototype medicine or to add caused by Terramycin-type waste water
The problem of.
The discrimination method of Terramycin-type waste water is added according to protein feed raw material provided by the embodiments of the present application comprising:
It by protein feed raw material through the first solvent extraction, is diluted with the second solvent, filtering obtains prepare liquid.
Using terramycin, tetracycline and dehydration tetracycline as the marker of Terramycin-type waste water, by prepare liquid through liquid chromatogram
Separation, Mass Spectrometer Method, identifying protein feed raw material whether there is marker.
Wherein, the first solvent is organic solvent.
The discrimination method of Terramycin-type waste water is added, according to the protein feed raw material of the embodiment of the present application with terramycin, Fourth Ring
Element and marker of the dehydration tetracycline as Terramycin-type waste water pass through Liquid Chromatography-Mass Spectrometry, effectively acquisition protein feed
Whether raw material contains marker, if containing marker, representing protein feed raw material admixture has Terramycin-type waste water, if without containing mark
Will object, or the marker contained only have one of which or two kinds, then representing has Terramycin-type waste water without adding.
It can effectively identify whether protein feed raw material contains terramycin using above-mentioned discrimination method, and can effectively distinguish
The terramycin is that is to say due to addition terramycin prototype medicine or caused by adding Terramycin-type waste water, effectively identification albumen
Whether feedstuff adds Terramycin-type waste water.
In addition, also being had according to the discrimination method that the protein feed raw material of the embodiment of the present application adds Terramycin-type waste water as follows
Additional technical characteristic:
It is 461.1555m/z as terramycin using mass-to-charge ratio in Mass Spectrometer Method in some embodiments shown in the application
Fragments characteristic, using mass-to-charge ratio be 445.1606m/z be used as tetracycline fragments characteristic, using mass-to-charge ratio for 443.1450m/z as
The fragments characteristic of dehydration tetracycline.
In other words, in the step of Mass Spectrometer Method, in obtained mass spectrogram, confirmation features described above fragment whether there is, with
Identifying protein feed raw material whether there is marker.
In some embodiments shown in the application, the step of prepare liquid is separated through liquid chromatogram, includes:
Gradient elution is carried out with mobile phase A and Mobile phase B.
Wherein, mobile phase A is 1.5mM~2.5mM acetic acid ammonia spirit, is containing volumetric concentration in acetic acid ammonia spirit
0.15%~0.25% formic acid.
Mobile phase B is acetonitrile.
In some embodiments shown in the application, the step of gradient elution in, with volume percentage, in the following way
It carries out:
When 0~0.5min, mobile phase A remains 95%, and Mobile phase B remains 5%.
When 0.5~3.0min, mobile phase A is decreased to 85% by 95%, and Mobile phase B increases to 15% by 5%.
When 3.0~10.0min, mobile phase A is decreased to 60% by 85%, and Mobile phase B increases to 40% by 15%.
When 10.0~18.0min, mobile phase A is decreased to 0 by 60%, and Mobile phase B increases to 100% by 40%.
When 18.0~23.0min, mobile phase A remains 0, and Mobile phase B remains 100%.
When 23.0~25.0min, mobile phase A remains 95%, and Mobile phase B remains 5%.
Optionally, the flow velocity of gradient elution is 0.3~0.5mL/min.
Optionally, in the step of liquid chromatogram separates, the stationary phase of chromatographic column used is octadecyl functional group.
Optionally, chromatographic column is Zorbax Eclipse Plus C18 chromatographic column.
Optionally, the column temperature of chromatographic column is 38 DEG C~42 DEG C.
In some embodiments shown in the application, Mass Spectrometer Method includes: using level four bars flight time mass spectrum to through liquid phase
Product after chromatographic isolation is detected.
Optionally, the condition of Mass Spectrometer Method are as follows:
Electrospray ionisation source；Cation scanning, scanning range 400-500m/z；Dry temperature degree: 250 DEG C；Dry gas stream
Amount: 7L/min；Atomization gas pressure: 7psi；Sheath temperature degree: 325 DEG C；Sheath throughput: 11L/min；Spray nozzle voltage 200V；Capillary
Voltage: 3kV；Capillary outlet voltage: 120V；Skimmer voltage 750V.
In some embodiments shown in the application, the second solvent includes that the Ammonium Acetate that concentration is 1.5mM~2.5mM is water-soluble
Liquid, the formic acid for being 0.15%~0.25% containing volumetric concentration in acetic acid ammonia spirit.
By the dilution of above-mentioned second solvent, the matrix effect in mass spectral analysis is reduced.
In some embodiments shown in the application, partial size >=18 mesh of protein feed raw material.
Optionally, the partial size of protein feed raw material is 18~40 mesh.
Under above-mentioned condition, effectively increase the contact surface of protein feed raw material and the first solvent, extraction effect is good.
In some embodiments shown in the application, the first solvent includes methanol.
Methanol can effectively penetrate into protein feed raw material, and extraction effect is good.
In some embodiments shown in the application, include: through the step of the first solvent extraction by protein feed raw material
By protein feed raw material with the first solvent according to 1g:(8ml~12ml) it mixes, mechanical shaking extraction, obtain extracting solution.
Supernatant is taken after extracting solution is centrifuged.
Optionally, extracting solution is centrifuged 4min~6min with the rate of 5000r/min~7000r/min.
Optionally, protein feed raw material includes at least one of rapeseed dregs, dregs of beans and cotton dregs.
The beneficial effect that protein feed raw material provided by the embodiments of the present application adds the discrimination method of Terramycin-type waste water is: sample
Product dosage is few, and pretreatment process is simple, and detection time is short, sensitivity is higher, as a result accurately.Simultaneously with terramycin, tetracycline and
Marker of the dehydration tetracycline as Terramycin-type waste water can effectively identify protein feed by Liquid Chromatography-Mass Spectrometry
Whether raw material contains terramycin, and can effectively distinguish the terramycin is due to addition terramycin prototype medicine or to add terramycin
It caused by the dregs of a decoction, that is to say, effectively identify whether protein feed raw material adds Terramycin-type waste water.
Detailed description of the invention
Technical solution in ord to more clearly illustrate embodiments of the present application, below will be to needed in the embodiment attached
Figure is briefly described, it should be understood that the following drawings illustrates only some embodiments of the application, therefore is not construed as pair
The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this
A little attached drawings obtain other relevant attached drawings.
Fig. 1 is terramycin fragments characteristic extraction chromatography figure in the embodiment of the present application 1；
Fig. 2 is tetracycline fragments characteristic extraction chromatography figure in the embodiment of the present application 1；
Fig. 3 is dehydration tetracycline fragments characteristic extraction chromatography figure in the embodiment of the present application 1；
Fig. 4 is terramycin fragments characteristic ion figure in the embodiment of the present application 1；
Fig. 5 is tetracycline fragments characteristic ion figure in the embodiment of the present application 1；
Fig. 6 is dehydration tetracycline fragments characteristic ion figure in the embodiment of the present application 1.
Specific embodiment
It, below will be in the embodiment of the present application to keep the purposes, technical schemes and advantages of the embodiment of the present application clearer
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
In this application, term " first ", " second " etc. are only used for distinguishing description, are not understood to indicate or imply phase
To importance.
The discrimination method for adding Terramycin-type waste water to the protein feed raw material of the embodiment of the present application below is specifically described.
Wherein, protein feed raw material provided by the present application include but is not limited in rapeseed dregs, dregs of beans and cotton dregs at least
It is a kind of.For example, protein feed raw material is rapeseed dregs, it is the by-product for being raw material after taking oil using rapeseed, egg for it
White matter content is between 34%~38%, and methionine and lysine ratio are higher in amino acid composition, belongs to three large protein of China
One of raw material (dregs of beans, cotton dregs, rapeseed dregs), is important forage protein source, wherein it should be noted that protein feed raw material,
Such as rapeseed dregs can be the material residue after directly squeezing, or the material residue after fermentation.
Terramycin-type waste water is that remaining fermentation medium generates waste residue after filtering after biofermentation produces terramycin, with egg
After white feedstuff mixing, can not effectively it be differentiated.
Due to terramycin can make an addition to protein feed raw material and Terramycin-type waste water is forbidden adding, identify forage protein
Terramycin is due to addition terramycin prototype medicine or caused by adding Terramycin-type waste water, to supervision forage protein original in raw material
The quality of material has significant meaning.
Terramycin-type waste water is that remaining fermentation medium generates waste residue after filtering after biofermentation produces terramycin, and soil is mould
The plain dregs of a decoction are other than containing final product terramycin, also containing the medicament residue not being fully extracted and without safety evaluatio
Secondary metabolite, inventor is from above-mentioned secondary metabolite, the medicament residue not being fully extracted, by largely sieving
Choosing, combination, when confirming the marker to use terramycin, tetracycline and dehydration tetracycline collectively as Terramycin-type waste water, operation
Simply, it can effectively identify whether forage protein raw material has added Terramycin-type waste water.When marker only contains terramycin and Fourth Ring
It is verified when plain or when marker only contains terramycin and dehydration tetracycline, whether can not reasonably identify forage protein raw material
Admixture has Terramycin-type waste water.
It should be noted that identified using terramycin, tetracycline and dehydration tetracycline three collectively as marker herein
Mode is the simplest, other substances is added on the basis of terramycin, tetracycline and dehydration tetracycline, collectively as label
Object, also in protection scope provided by the present application.
Protein feed raw material provided by the present application adds the discrimination method of Terramycin-type waste water comprising:
S1. protein feed raw material is diluted through the first solvent extraction with the second solvent, filtering obtains prepare liquid.
Optionally, the partial size of the partial size of protein feed raw material >=18 mesh, the protein feed raw material in the particle size range is small, can
It is sufficiently contacted with the first solvent, sufficiently extracts the related substances in protein feed raw material, improve extraction efficiency.
In actual identification operating process, it has been found that since protein feed raw material contains certain humidity, subsequent
It when filtering, since protein feed raw material partial size is too small, is easy to cohere, blocks sieve pore, it is subsequent to be effectively filtered, because
This still optionally further, the partial size of protein feed raw material is 18~40 mesh, such as the partial size of protein feed raw material is 18 mesh, 20
Mesh, 24 mesh, 28 mesh, 30 mesh, 32 mesh, 35 mesh, any value in 40 mesh or the value range between any two point value.
It holds above-mentioned, if protein feed raw material particle is larger, can be crushed, be sieved before through the first solvent extraction, with full
The requirement of the above-mentioned particle size range of foot.
Wherein, the first solvent is organic solvent, can effectively extract the effective component in protein feed raw material.It is optional
Ground, the first solvent include methanol, wherein methanol permeability is good, can effectively extract the effective component in protein feed raw material.
Optionally, in a kind of example shown in the application, include: through the step of the first solvent extraction by protein feed raw material
By protein feed raw material with the first solvent according to 1g:(8ml~12ml) it mixes, such as by protein feed raw material and
One solvent is according to any solid-liquid ratio in 1g:8ml, 1g:9ml, 1g:9.5ml, 1g:10ml, 1g:11ml, 1g:12ml or appoints
The value range anticipated between two solid-liquid ratios is mixed, then mechanical shaking extraction, obtains extracting solution；On being taken after extracting solution is centrifuged
Clear liquid.
Wherein, it other than the mode of mechanical shaking extraction, can also be extracted using standing to extract or stir, wherein oscillation mentions
It takes and stirs the efficiency extracted and be above standing extraction, and the extraction of related substances is more abundant.
Optionally, extracting solution is centrifuged 4min~6min with the rate of 5000r/min~7000r/min；Optionally, it will mention
Liquid is taken to be centrifuged 5min with the rate of 6000r/min.By aforesaid operations, impurity is effectively removed, guarantees impurity content in supernatant
It is low.
Wherein, the second solvent includes but is not limited to water.
In some embodiments shown in the application, the second solvent can be that the Ammonium Acetate that concentration is 1.5mM~2.5mM is water-soluble
Liquid, such as it is any concentration value in 1.5mM, 1.7mM, 2mM, 2.3mM, 2.4mM, 2.5mM or any that the second solvent, which is concentration,
The acetic acid ammonia spirit of value range between two concentration values, wherein contain 0.15v/v%~0.25v/ in the acetic acid ammonia spirit
The formic acid of v%, in other words in acetic acid ammonia spirit, the concentration of Ammonium Acetate is 1.5mM~2.5mM, while acetic acid ammonia spirit
In contain formic acid, volumetric concentration of the formic acid in acetic acid ammonia spirit is 0.15%~0.25%.Such as in acetic acid ammonia spirit
Formic acid containing any volumetric concentration in 0.15v/v%, 0.18v/v%, 0.2v/v%, 0.22v/v%, 0.25v/v% is appointed
The formic acid for two volume concentration value limited range values of anticipating.By the dilution of above-mentioned second solvent, reduce in mass spectral analysis
Matrix effect.
Wherein, the filtering such as organic filter membrane can be used in filtering, wherein organic filter membrane is, for example, teflon membrane filter etc., effectively
Solution after purification dilution, reduces the impurity in prepare liquid, prevents particulate matter from blocking mass spectrometry system pipeline.
S2. using terramycin, tetracycline and dehydration tetracycline as the marker of Terramycin-type waste water, by prepare liquid through liquid phase color
Spectrum separation, Mass Spectrometer Method, identifying protein feed raw material whether there is marker.
In other words, liquid phase color is passed through as the marker of Terramycin-type waste water using terramycin, tetracycline and dehydration tetracycline
Spectrum-Mass Spectrometry, obtains whether protein feed raw material contains marker, if containing marker, represents protein feed raw material
Admixture has Terramycin-type waste water, if not containing marker, or the marker contained only has one of which or two kinds, then represents and do not mix
Added with Terramycin-type waste water.
Wherein, prepare liquid is separated, Mass Spectrometer Method through liquid chromatogram, can be detected by liquid chromatography mass combined instrument.
In some embodiments shown in the application, the step of prepare liquid is separated through liquid chromatogram include: with mobile phase A and
Mobile phase B carries out gradient elution.
Wherein, mobile phase A is 1.5mM~2.5mM acetic acid ammonia spirit, in acetic acid ammonia spirit containing 0.15v/v%~
The formic acid of 0.25v/v%；Mobile phase B is acetonitrile.In other words mobile phase A is in acetic acid ammonia spirit, and the concentration of Ammonium Acetate is
1.5mM~2.5mM, while containing formic acid in acetic acid ammonia spirit, volumetric concentration of the formic acid in acetic acid ammonia spirit is
0.15%~0.25%.
In other words, it is any concentration value in 1.5mM, 1.7mM, 2mM, 2.3mM, 2.4mM, 2.5mM that mobile phase A, which is concentration,
Or the acetic acid ammonia spirit of the value range between any two concentration value, wherein in the acetic acid ammonia spirit containing 0.15v/v%,
The formic acid or any two volume of any volumetric concentration in 0.18v/v%, 0.2v/v%, 0.22v/v%, 0.25v/v% are dense
The formic acid of angle value limited range value.
Hold it is above-mentioned, in order to make using the second solvent dilution after prepare liquid, carry out liquid chromatogram separation better effect,
Optionally, the second solvent is mobile phase A, and in other words, the second solvent is identical as mobile phase A.
It is noted that during gradient elution, with volume percentage, mobile phase A+Mobile phase B=100%.
Specifically, in some embodiments shown in the application, the step of gradient elution in, with volume percentage, according to
Following manner carries out:
When 0~0.5min, mobile phase A remains 95%, and Mobile phase B remains 5%.
When 0.5~3.0min, mobile phase A is decreased to 85% by 95%, and Mobile phase B increases to 15% by 5%.
When 3.0~10.0min, mobile phase A is decreased to 60% by 85%, and Mobile phase B increases to 40% by 15%.
When 10.0~18.0min, mobile phase A is decreased to 0 by 60%, and Mobile phase B increases to 100% by 40%.
When 18.0~23.0min, mobile phase A remains 0, and Mobile phase B remains 100%.
When 23.0~25.0min, mobile phase A remains 95%, and Mobile phase B remains 5%.
Optionally, the flow velocity of gradient elution be 0.3~0.5mL/min, such as gradient elution flow velocity be 0.3mL/min,
Any flow speed value in 0.4mL/min, 0.5mL/min or between any two flow speed value.It is noted that the application institute
Be related between including two endpoint values.
Optionally, in the step of liquid chromatogram separates, the stationary phase of chromatographic column used is octadecyl functional group.
Optionally, chromatographic column is Zorbax Eclipse Plus C18 chromatographic column.
Optionally, the column temperature of chromatographic column is 38 DEG C~42 DEG C, such as column temperature is in 38 DEG C, 39 DEG C, 40 DEG C, 41 DEG C, 42 DEG C
Any temperature value or between any two temperature value.
In embodiment provided by the present application, optionally, Mass Spectrometer Method includes: using level four bars flight time mass spectrum to through liquid
Product after phase chromatographic isolation is detected.
Level four bars flight time mass spectrum is, for example, 6465 LC-Q TOF level four bars flight time mass spectrum of Agilent.
Optionally, the condition of Mass Spectrometer Method are as follows:
Electrospray ionisation source；Cation scanning, scanning range 400-500m/z；Dry temperature degree: 250 DEG C；Dry gas stream
Amount: 7L/min；Atomization gas pressure: 7psi；Sheath temperature degree: 325 DEG C；Sheath throughput: 11L/min；Spray nozzle voltage 200V；Capillary
Voltage: 3kV；Capillary outlet voltage: 120V；Skimmer voltage 750V.
It optionally, is fragments characteristic of the 461.1555m/z as terramycin using mass-to-charge ratio, with mass-to-charge ratio in Mass Spectrometer Method
Fragments characteristic for 445.1606m/z as tetracycline, it is broken for the feature of 443.1450m/z as dehydration tetracycline using mass-to-charge ratio
Piece.
The feature of the application and performance are described in further detail with reference to embodiments.
Embodiment 1
A kind of protein feed raw material adds the discrimination method of Terramycin-type waste water comprising:
S1. the rapeseed dregs that Terramycin-type waste water (10%, m/m) will be added is crushed using Cyclone mill, smashed granularity
Reach 18 mesh.
S2. the smashed sample of 0.5g step S1 is weighed into clean centrifuge tube, 5mL methanol is added, and is vortexed 2 minutes,
It is placed in shake 30 minutes on shaking table and extract.It is centrifuged 5min under the centrifugal force of 6000r/min after the completion of extraction, takes supernatant
As extraction solution.
S3. the extraction solution in 0.5mL step S2 is drawn, isometric mobile phase A is added to dilute, then uses polytetrafluoroethyl-ne
Alkene membrane filtration, the prepare liquid after being purified.
S4. the prepare liquid that 0.5mL step S3 is obtained is drawn, liquid chromatogram separation is carried out.The condition of liquid chromatogram separation is such as
Under:
1. chromatographic column is Zorbax Eclipse Plus C18 chromatographic column, column temperature: 42 DEG C.
2. mobile phase A are as follows: 2.0mM acetic acid ammonia spirit (formic acid containing 0.2v/v%), Mobile phase B are as follows: acetonitrile.Flow velocity is
0.5mL/min。
3. using gradient elution mode: with volume percentage, when 0~0.5min, mobile phase A remains 95%, flowing
Phase B remains 5%.
When 0.5~3.0min, mobile phase A is decreased to 85% by 95%, and Mobile phase B increases to 15% by 5%.
When 3.0~10.0min, mobile phase A is decreased to 60% by 85%, and Mobile phase B increases to 40% by 15%.
When 10.0~18.0min, mobile phase A is decreased to 0 by 60%, and Mobile phase B increases to 100% by 40%.
When 18.0~23.0min, mobile phase A remains 0, and Mobile phase B remains 100%.
When 23.0~25.0min, mobile phase A remains 95%, and Mobile phase B remains 5%.
4. sample volume: 20ul.
S5. the ingredient after the separation of step S4 liquid chromatogram is passed through into level four bars flight time mass spectrum (Agilent
6465LC-Q TOF) it detects, Mass Spectrometry Conditions are as follows:
Electrospray ionisation source；Cation scanning, scanning range 400-500m/z；Dry temperature degree: 250 DEG C；Dry gas stream
Amount: 7L/min；Atomization gas pressure: 7psi；Sheath temperature degree: 325 DEG C；Sheath throughput: 11L/min；Spray nozzle voltage 200V；Capillary
Voltage: 3kV；Capillary outlet voltage: 120V；Skimmer voltage 750V.
S6. result judgement:
It in liquid chromatography-mass spectrography figure, is extracted by fragments characteristic, terramycin fragments characteristic is extracted at 5.976min
Extraction chromatography figure is as shown in Figure 1.It is as shown in Figure 2 that tetracycline fragments characteristic extraction chromatography figure is extracted at 5.701min.In
It is as shown in Figure 3 that dehydration tetracycline fragments characteristic extraction chromatography figure is extracted at 7.994min.It is analyzed, is detected by fragment ion
Terramycin fragments characteristic ion 445.1606m/z is as shown in figure 4, detection tetracycline fragments characteristic ion 461.1555m/z such as Fig. 5
Shown, detection dehydration tetracycline fragments characteristic ion 443.1450m/z is as shown in Figure 6.The result shows that it is mould to detect soil in rapeseed dregs
Plain dregs of a decoction marker illustrates containing Terramycin-type waste water, which is the same as the actual situation.
Embodiment 2
A kind of protein feed raw material adds the discrimination method of Terramycin-type waste water comprising:
S1. the rapeseed dregs that Terramycin-type waste water (10%, m/m) will be added is crushed using Cyclone mill, smashed granularity
Reach 20 mesh.
S2. the smashed sample of 0.5g step S1 is weighed into clean centrifuge tube, 4mL methanol is added, and is vortexed 2 minutes,
It is placed in shake 30 minutes on shaking table and extract.It is centrifuged 6min under 5000r/min centrifugal force after the completion of extraction, supernatant is taken to make
To extract solution.
S3. the extraction solution of the 0.5mL in aspiration step S2, adds isometric mobile phase A to dilute, using polytetrafluoroethylene (PTFE)
Membrane filtration, the prepare liquid after being purified.
S4. the prepare liquid that 0.5mL step S3 is obtained is drawn, liquid chromatogram separation is carried out.The condition of liquid chromatogram separation is such as
Under:
1. chromatographic column is Zorbax Eclipse Plus C18 chromatographic column, column temperature: 38 DEG C.
2. mobile phase A are as follows: 1.5mM acetic acid ammonia spirit (formic acid containing 0.15v/v%), Mobile phase B are as follows: acetonitrile.Flow velocity is
0.5mL/min。
3. using gradient elution mode: with volume percentage, when 0~0.5min, mobile phase A remains 95%, flowing
Phase B remains 5%.
When 0.5~3.0min, mobile phase A is decreased to 85% by 95%, and Mobile phase B increases to 15% by 5%.
When 3.0~10.0min, mobile phase A is decreased to 60% by 85%, and Mobile phase B increases to 40% by 15%.
When 10.0~18.0min, mobile phase A is decreased to 0 by 60%, and Mobile phase B increases to 100% by 40%.
When 18.0~23.0min, mobile phase A remains 0, and Mobile phase B remains 100%.
When 23.0~25.0min, mobile phase A remains 95%, and Mobile phase B remains 5%.
4. sample volume: 20ul.
S5. the ingredient after the separation of step S4 liquid chromatogram is passed through into level four bars flight time mass spectrum (Agilent
6465LC-Q TOF) it detects, Mass Spectrometry Conditions are as follows:
Electrospray ionisation source；Cation scanning, scanning range 400-500m/z；Dry temperature degree: 250 DEG C；Dry gas stream
Amount: 7L/min；Atomization gas pressure: 7psi；Sheath temperature degree: 325 DEG C；Sheath throughput: 11L/min；Spray nozzle voltage 200V；Capillary
Voltage: 3kV；Capillary outlet voltage: 120V；Skimmer voltage 750V.
S6. result judgement:
It in liquid chromatography-mass spectrography figure, is extracted by fragments characteristic, it is broken that terramycin feature is extracted at 5.976min
Piece.Tetracycline fragments characteristic is extracted at 5.701min.Dehydration tetracycline fragments characteristic is extracted at 7.994min.As a result
Show to detect Terramycin-type waste water marker in rapeseed dregs, illustrate containing Terramycin-type waste water, the identification result and actual conditions phase
Together.
Embodiment 3
A kind of protein feed raw material adds the discrimination method of Terramycin-type waste water comprising:
S1. the rapeseed dregs that Terramycin-type waste water (10%, m/m) will be added is crushed using Cyclone mill, smashed granularity
Reach 40 mesh.
S2. the smashed sample of 0.5g step S1 is weighed into clean centrifuge tube, and 6mL Extraction solvent is added and (extracts molten
Agent is methanol), it is vortexed 2 minutes, is placed in shake 30 minutes on shaking table and extracts.In 5000r/min centrifugal force after the completion of extraction
Lower centrifugation 6min takes supernatant as extraction solution.
S3. the extraction solution in 0.5mL step S2 is drawn, adds isometric mobile phase A to dilute, is filtered using polytetrafluoroethylene (PTFE)
Film filtering, the prepare liquid after being purified.
S4. the prepare liquid that 0.5mL step S3 is obtained is drawn, liquid chromatogram separation is carried out.The condition of liquid chromatogram separation is such as
Under:
1. chromatographic column is Zorbax Eclipse Plus C18 chromatographic column, column temperature: 40 DEG C.
2. mobile phase A are as follows: 2.0mM acetic acid ammonia spirit (formic acid containing 0.2v/v%), Mobile phase B are as follows: acetonitrile, flow velocity are
0.5mL/min。
3. using gradient elution mode: with volume percentage, when 0~0.5min, mobile phase A remains 95%, flowing
Phase B remains 5%.
When 0.5~3.0min, mobile phase A is decreased to 85% by 95%, and Mobile phase B increases to 15% by 5%.
When 3.0~10.0min, mobile phase A is decreased to 60% by 85%, and Mobile phase B increases to 40% by 15%.
When 10.0~18.0min, mobile phase A is decreased to 0 by 60%, and Mobile phase B increases to 100% by 40%.
When 18.0~23.0min, mobile phase A remains 0, and Mobile phase B remains 100%.
When 23.0~25.0min, mobile phase A remains 95%, and Mobile phase B remains 5%.
4. sample volume: 20ul.
S5. it will pass through level four bars flight time mass spectrum (Agilent 6465LC- by ingredient after the separation of step S4 liquid chromatogram
Q TOF) it detects, Mass Spectrometry Conditions are as follows:
Electrospray ionisation source；Cation scanning, scanning range 400-500m/z；Dry temperature degree: 250 DEG C；Dry gas stream
Amount: 7L/min；Atomization gas pressure: 7psi；Sheath temperature degree: 325 DEG C；Sheath throughput: 11L/min；Spray nozzle voltage 200V；Capillary
Voltage: 3kV；Capillary outlet voltage: 120V；Skimmer voltage 750V.
S6. result judgement:
It in liquid chromatography-mass spectrography figure, is extracted by fragments characteristic, it is broken that terramycin feature is extracted at 5.976min
Piece.Tetracycline fragments characteristic is extracted at 5.701min.Dehydration tetracycline fragments characteristic is extracted at 7.994min.As a result
Show to detect Terramycin-type waste water marker in rapeseed dregs, illustrate containing Terramycin-type waste water, the identification result and actual conditions phase
Together.
To sum up, protein feed raw material provided by the present application adds the discrimination method of Terramycin-type waste water, and amount of samples is few, preceding place
Reason process is simple, and detection time is short, sensitivity is higher, as a result accurately, can effectively identify whether protein feed raw material adds soil
The mycin dregs of a decoction.
The above is only preferred embodiment of the present application, are not intended to limit this application, for those skilled in the art
For member, various changes and changes are possible in this application.Within the spirit and principles of this application, it is made it is any modification,
Equivalent replacement, improvement etc., should be included within the scope of protection of this application.
Claims (10)
1. the discrimination method that a kind of protein feed raw material adds Terramycin-type waste water, characterized in that it comprises:
It by protein feed raw material through the first solvent extraction, is diluted with the second solvent, filtering obtains prepare liquid；
Using terramycin, tetracycline and dehydration tetracycline as the marker of Terramycin-type waste water, prepare liquid is separated through liquid chromatogram,
Mass Spectrometer Method identifies the protein feed raw material with the presence or absence of the marker；
Wherein, first solvent is organic solvent.
2. discrimination method according to claim 1, which is characterized in that in the Mass Spectrometer Method, be with mass-to-charge ratio
Fragments characteristic of the 461.1555m/z as the terramycin is feature of the 445.1606m/z as the tetracycline using mass-to-charge ratio
Fragment is fragments characteristic of the 443.1450m/z as the dehydration tetracycline using mass-to-charge ratio.
3. discrimination method according to claim 1, which is characterized in that the step of separating the prepare liquid through liquid chromatogram
Include:
Gradient elution is carried out with mobile phase A and Mobile phase B；
Wherein, the mobile phase A is 1.5mM~2.5mM acetic acid ammonia spirit, contains 0.15v/ in the acetic acid ammonia spirit
The formic acid of v%~0.25v/v%；
The Mobile phase B is acetonitrile.
4. discrimination method according to claim 3, which is characterized in that in the step of the gradient elution, with volume basis
Than meter, carry out in the following way:
When 0~0.5min, the mobile phase A remains 95%, and the Mobile phase B remains 5%；
When 0.5~3.0min, the mobile phase A is decreased to 85% by 95%, and the Mobile phase B increases to 15% by 5%；
When 3.0~10.0min, the mobile phase A is decreased to 60% by 85%, and the Mobile phase B increases to 40% by 15%；
When 10.0~18.0min, the mobile phase A is decreased to 0 by 60%, and the Mobile phase B increases to 100% by 40%；
When 18.0~23.0min, the mobile phase A remains 0, and the Mobile phase B remains 100%；
When 23.0~25.0min, the mobile phase A remains 95%, and the Mobile phase B remains 5%；
Optionally, the flow velocity of the gradient elution is 0.3~0.5mL/min.
5. discrimination method according to claim 1, which is characterized in that used in the step of liquid chromatogram separates
The stationary phase of chromatographic column is octadecyl functional group；
Optionally, the chromatographic column is Zorbax Eclipse Plus C18 chromatographic column；
Optionally, the column temperature of the chromatographic column is 38 DEG C~42 DEG C.
6. discrimination method according to claim 1, which is characterized in that the Mass Spectrometer Method includes: to be flown using level four bars
Time mass spectrum detects the product after liquid chromatogram separation；
Optionally, the condition of the Mass Spectrometer Method are as follows:
Electrospray ionisation source；Cation scanning, scanning range 400-500m/z；Dry temperature degree: 250 DEG C；Dry gas stream amount:
7L/min；Atomization gas pressure: 7psi；Sheath temperature degree: 325 DEG C；Sheath throughput: 11L/min；Spray nozzle voltage 200V；Capillary electricity
Pressure: 3kV；Capillary outlet voltage: 120V；Skimmer voltage 750V.
7. discrimination method according to claim 1, which is characterized in that second solvent include concentration be 1.5mM~
2.5mM acetic acid ammonia spirit, the formic acid containing 0.15v/v%~0.25v/v% in the acetic acid ammonia spirit.
8. discrimination method according to claim 1, which is characterized in that the partial size of the protein feed raw material >=18 mesh；
Optionally, the partial size of the protein feed raw material is 18~40 mesh.
9. discrimination method according to claim 1, which is characterized in that first solvent includes methanol.
10. discrimination method according to claim 1, which is characterized in that mention the protein feed raw material through the first solvent
The step of taking include:
By the protein feed raw material with first solvent according to 1g:(8ml~12ml) it mixes, mechanical shaking extraction is extracted
Liquid；
Supernatant will be taken after extracting solution centrifugation；
Optionally, the extracting solution is centrifuged 4min~6min with the rate of 5000r/min~7000r/min；
Optionally, the protein feed raw material includes at least one of rapeseed dregs, dregs of beans and cotton dregs.
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