CN110470780B - Identification method for protein feed raw material mixed with terramycin dregs - Google Patents
Identification method for protein feed raw material mixed with terramycin dregs Download PDFInfo
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Abstract
An identification method of protein feed raw material mixed with terramycin dregs relates to the field of feed quality safety detection. The identification method comprises the following steps: extracting the protein feed raw material by using a first solvent, diluting by using a second solvent, and filtering to obtain a solution to be detected. Taking oxytetracycline, tetracycline and anhydrotetracycline as markers of oxytetracycline dregs, separating the liquid to be detected by liquid chromatography, detecting by mass spectrometry, and identifying whether the markers exist in the protein feed raw material, wherein the first solvent is an organic solvent. The identification method has the advantages of small sample dosage, simple pretreatment process, short detection time, high sensitivity and accurate result, and can effectively identify whether the protein feed raw material is doped with the terramycin dregs.
Description
Technical Field
The application relates to the field of feed quality safety detection, in particular to an identification method of protein feed raw materials doped with terramycin dregs.
Background
Protein feed raw materials (bean pulp, cottonseed meal and rapeseed meal) are important feed protein sources and are widely applied to livestock and poultry breeding. The terramycin dregs are dregs produced by filtering the residual fermentation culture medium after producing terramycin by biological fermentation, and have certain crude protein. There has been a medium report that the illegal culturist can be used by adding terramycin dregs into feed protein raw material instead. As the feed raw materials containing the terramycin dregs are used in the breeding process, the feed has potential risk hazards of harming the health of bred animals, influencing food safety, inducing the generation of bacterial drug resistance and the like, the No. 176 bulletin issued by the ministry of agriculture, the ministry of health and the national drug administration in China clearly stipulates that the terramycin dregs are forbidden to be added into the feed for use.
At present, although the existing detection technology at home and abroad can accurately measure the content of the terramycin in the feed protein raw material, the existing detection technology cannot identify whether the content is caused by adding a terramycin prototype drug or adding terramycin dregs.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The application provides an identification method for protein feed raw materials doped with terramycin dregs, which aims to improve the problem that the existing identification method cannot distinguish whether the terramycin in the feed protein raw materials is caused by adding terramycin prototype drugs or the terramycin dregs is doped.
The identification method for the protein feed raw material mixed with the oxytetracycline pharmaceutical dregs provided by the embodiment of the application comprises the following steps:
extracting the protein feed raw material by using a first solvent, diluting by using a second solvent, and filtering to obtain a solution to be detected.
Taking oxytetracycline, tetracycline and anhydrotetracycline as markers of oxytetracycline dregs, separating the liquid to be detected by liquid chromatography, detecting by mass spectrometry, and identifying whether the markers exist in the protein feed raw material.
Wherein the first solvent is an organic solvent.
According to the identification method for the terramycin residue doped in the protein feed raw material, terramycin, tetracycline and anhydrotetracycline are used as markers of the terramycin residue, and a liquid chromatography-mass spectrometry combined method is used to effectively obtain whether the protein feed raw material contains the markers, if the markers are contained, the protein feed raw material is doped with the terramycin residue, and if the markers are not contained, or only one or two of the markers are contained, the protein feed raw material is not doped with the terramycin residue.
By adopting the identification method, whether the protein feed raw material contains the terramycin can be effectively identified, and whether the terramycin is caused by adding the terramycin prototype drug or adding the terramycin dregs can be effectively distinguished, namely, whether the protein feed raw material is added with the terramycin dregs can be effectively identified.
In addition, the identification method of the protein feed raw material mixed with the terramycin dregs according to the embodiment of the application also has the following additional technical characteristics:
in some embodiments shown in the present application, in mass spectrometry, the mass-to-charge ratio is 461.1555m/z as the characteristic fragment of oxytetracycline, the mass-to-charge ratio is 445.1606m/z as the characteristic fragment of tetracycline, and the mass-to-charge ratio is 443.1450m/z as the characteristic fragment of anhydrotetracycline.
In other words, in the step of mass spectrometric detection, the presence or absence of the characteristic fragments is confirmed in the obtained mass spectrogram to identify the presence or absence of the marker in the protein feed raw material.
In some embodiments illustrated herein, the step of subjecting the liquid to be tested to liquid chromatography comprises:
gradient elution was performed with mobile phase a and mobile phase B.
Wherein the mobile phase A is 1.5 mM-2.5 mM acetic ammonia solution, and the acetic ammonia solution contains 0.15% -0.25% formic acid by volume concentration.
Mobile phase B was acetonitrile.
In some embodiments illustrated herein, the step of gradient elution is performed as follows, in volume percent:
when the time is 0-0.5 min, the mobile phase A is kept at 95%, and the mobile phase B is kept at 5%.
When the time is 0.5-3.0 min, the mobile phase A is reduced to 85% from 95%, and the mobile phase B is increased to 15% from 5%.
And when the time is 3.0-10.0 min, the mobile phase A is reduced from 85% to 60%, and the mobile phase B is increased from 15% to 40%.
When 10.0-18.0 min, the mobile phase A is reduced from 60% to 0, and the mobile phase B is increased from 40% to 100%.
When the time is 18.0-23.0 min, the mobile phase A is kept at 0, and the mobile phase B is kept at 100%.
And when the time is 23.0-25.0 min, the mobile phase A is kept at 95%, and the mobile phase B is kept at 5%.
Optionally, the flow rate of the gradient elution is 0.3-0.5 mL/min.
Alternatively, in the step of liquid chromatography separation, the stationary phase of the chromatographic column used is an octadecyl functional group.
Alternatively, the chromatography column is a Zorbax Eclipse Plus C18 chromatography column.
Optionally, the column temperature of the chromatography column is 38 ℃ to 42 ℃.
In some embodiments illustrated herein, mass spectrometry detection comprises: and detecting the product after liquid chromatography separation by adopting a quadrupole time-of-flight mass spectrum.
Optionally, the mass spectrometric detection conditions are:
an electrospray ionization source; scanning positive ions within the scanning range of 400-500 m/z; temperature of the drying gas: 250 ℃; flow rate of drying gas: 7L/min; atomizing gas pressure: 7 psi; temperature of sheath gas: 325 ℃; the flow rate of the sheath gas: 11L/min; nozzle voltage 200V; capillary voltage: 3 kV; capillary exit voltage: 120V; skimmer voltage 750V.
In some embodiments shown herein, the second solvent comprises an aqueous ammonia acetate solution having a concentration of 1.5mM to 2.5mM, wherein the aqueous ammonia acetate solution contains formic acid at a concentration of 0.15% to 0.25% by volume.
The matrix effect in mass spectrometry is reduced by dilution with the second solvent as described above.
In some embodiments shown herein, the protein feed material has a particle size of greater than or equal to 18 mesh.
Optionally, the particle size of the protein feed raw material is 18-40 meshes.
Under the conditions, the contact surface of the protein feed raw material and the first solvent is effectively increased, and the extraction effect is good.
In some embodiments illustrated herein, the first solvent comprises methanol.
The methanol can effectively permeate into protein feed raw materials, and the extraction effect is good.
In some embodiments illustrated herein, the step of subjecting the protein feed material to a first solvent comprises:
mixing a protein feed raw material and a first solvent according to the weight ratio of 1 g: (8 ml-12 ml) and shaking for extraction to obtain the extract.
Centrifuging the extractive solution, and collecting supernatant.
Optionally, centrifuging the extract at a rate of 5000r/min to 7000r/min for 4min to 6 min.
Optionally, the protein feed material comprises at least one of rapeseed meal, soybean meal, and cottonseed meal.
The identification method for the protein feed raw material mixed with the oxytetracycline pharmaceutical dregs provided by the embodiment of the application has the beneficial effects that: the method has the advantages of less sample consumption, simple pretreatment process, short detection time, higher sensitivity and accurate result. Meanwhile, terramycin, tetracycline and anhydrotetracycline are used as markers of terramycin dregs, and a liquid chromatography-mass spectrometry combined method is adopted, so that whether protein feed raw materials contain terramycin can be effectively identified, whether the terramycin is caused by adding a terramycin prototype drug or adding terramycin dregs can be effectively distinguished, and whether the protein feed raw materials are added with the terramycin dregs can be effectively identified.
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In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained from the drawings without inventive effort.
FIG. 1 is a chromatogram for extracting the characteristic fragment of oxytetracycline in example 1 of the present application;
FIG. 2 is a tetracycline feature fragment extraction chromatogram in example 1 of the present application;
FIG. 3 is a chromatogram for extracting a characteristic fragment of anhydrotetracycline in example 1 of the present application;
FIG. 4 is an ion diagram of the characteristic fragment of oxytetracycline in example 1 of the present application;
FIG. 5 is a tetracycline-characteristic fragment ion diagram of example 1 of the present application;
FIG. 6 is a characteristic fragment ion diagram of anhydrotetracycline in example 1 of this application.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions of the embodiments of the present application will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
In this application, the terms "first," "second," and the like are used merely to distinguish one description from another, and are not to be construed as indicating or implying relative importance.
The identification method of the protein feed raw material mixed with the oxytetracycline pharmaceutical dregs in the examples of the present application is specifically described below.
The protein feed raw material provided by the application comprises but is not limited to at least one of rapeseed meal, soybean meal and cottonseed meal. For example, the protein feed raw material is rapeseed meal, which is a byproduct of rapeseed which is used as a raw material after oil extraction, the protein content of the rapeseed is between 34% and 38%, the ratio of methionine to lysine in amino acid composition is high, the protein feed raw material belongs to one of three major protein raw materials (bean pulp, cottonseed meal and rapeseed meal) in China, and the protein feed raw material is an important feed protein source, wherein the protein feed raw material, such as the rapeseed meal, can be directly squeezed material residues or fermented material residues.
The terramycin dregs are dregs produced by filtering the residual fermentation culture medium after producing terramycin by biological fermentation, and cannot be effectively distinguished after being mixed with protein feed raw materials.
Because the terramycin can be added into the protein feed raw material and the terramycin dregs are forbidden to be added, the identification of the terramycin in the feed protein raw material caused by adding the terramycin prototype drug or adding the terramycin dregs has obvious significance for monitoring the quality of the feed protein raw material.
The terramycin dregs are dregs generated after filtering the residual fermentation culture medium after producing the terramycin by biological fermentation, the terramycin dregs contain not only the final product terramycin, but also medicine residues which are not completely extracted and secondary metabolites which are not evaluated in safety, and the inventor confirms that the terramycin, tetracycline and anhydrotetracycline are used as the markers of the terramycin dregs together from the secondary metabolites and the medicine residues which are not completely extracted through a large amount of screening and combination, so that the operation is simple, and whether the terramycin dregs are added in the feed protein raw materials or not can be effectively identified. When the marker only contains the terramycin and the tetracycline, or the marker only contains the terramycin and the anhydrotetracycline, verification proves that whether the terramycin dregs are added in the feed protein raw material or not can not be reasonably identified.
It should be noted that, the oxytetracycline, tetracycline and anhydrotetracycline are used as the markers, the identification method is the simplest, and other substances are added on the basis of oxytetracycline, tetracycline and anhydrotetracycline and are used as the markers together, which is also within the protection scope provided by the present application.
The identification method for the protein feed raw material mixed with the oxytetracycline pharmaceutical dregs comprises the following steps:
s1, extracting a protein feed raw material by using a first solvent, diluting the protein feed raw material by using a second solvent, and filtering to obtain a solution to be detected.
Optionally, the particle size of the protein feed raw material is larger than or equal to 18 meshes, the particle size of the protein feed raw material within the particle size range is small, the protein feed raw material can be fully contacted with the first solvent, related substances in the protein feed raw material can be fully extracted, and the extraction efficiency is improved.
In the actual identification operation process, the applicant finds that, because the protein feed raw material contains a certain humidity, during subsequent filtration, the particle size of the protein feed raw material is too small, so that the protein feed raw material is easy to adhere and block a sieve pore, and subsequent filtration cannot be effectively performed, so that the particle size of the protein feed raw material is further selected from 18 to 40 meshes, for example, the particle size of the protein feed raw material is any one value or a range value between any two values of 18 meshes, 20 meshes, 24 meshes, 28 meshes, 30 meshes, 32 meshes, 35 meshes and 40 meshes.
If the raw material particles of the protein feed are large, the raw material particles can be crushed and sieved before being extracted by the first solvent so as to meet the requirement of the particle size range.
Wherein the first solvent is an organic solvent which can effectively extract effective components in the protein feed raw material. Optionally, the first solvent comprises methanol, wherein the methanol has good permeability and can effectively extract effective components in the protein feed raw material.
Optionally, in one example shown herein, the step of subjecting the protein feed material to a first solvent comprises:
mixing a protein feed raw material and a first solvent according to the weight ratio of 1 g: (8 ml-12 ml) are mixed, for example, protein feed material is mixed with a first solvent according to a ratio of 1 g: 8ml, 1 g: 9ml, 1 g: 9.5ml, 1 g: 10ml, 1 g: 11ml, 1 g: mixing any solid-liquid ratio or any range value between two solid-liquid ratios in 12ml, and then oscillating and extracting to obtain an extracting solution; centrifuging the extractive solution, and collecting supernatant.
Wherein, except for the oscillation extraction mode, still standing extraction or stirring extraction can be adopted, wherein, the efficiency of the oscillation extraction and the stirring extraction is higher than that of the still standing extraction, and the extraction of related substances is more sufficient.
Optionally, centrifuging the extractive solution at 5000-7000 r/min for 4-6 min; optionally, the extract is centrifuged at 6000r/min for 5 min. Through the operation, impurities are effectively removed, and the low content of the impurities in the supernatant is ensured.
Wherein the second solvent includes, but is not limited to, water.
In some embodiments shown herein, the second solvent may be an aqueous solution of acetic acid with a concentration of 1.5mM to 2.5mM, for example, the second solvent is an aqueous solution of acetic acid with a concentration of 1.5mM, 1.7mM, 2mM, 2.3mM, 2.4mM, 2.5mM, or a range between any two of the above concentration values, wherein the aqueous solution of acetic acid contains 0.15 v/v% to 0.25 v/v% of formic acid, that is, the aqueous solution of acetic acid contains 1.5mM to 2.5mM of acetic acid, and the aqueous solution of acetic acid contains 0.15% to 0.25% of formic acid by volume. For example, the aqueous ammonia acetate solution contains formic acid at any one of 0.15 v/v%, 0.18 v/v%, 0.2 v/v%, 0.22 v/v%, 0.25 v/v% by volume or in any two values defined by the volume concentration. The matrix effect in mass spectrometry is reduced by dilution with the second solvent as described above.
The filtration can adopt an organic filter membrane and the like, wherein the organic filter membrane is a polytetrafluoroethylene filter membrane and the like, so that the diluted solution is effectively purified, the impurities in the solution to be detected are reduced, and the blockage of the mass spectrometry system pipeline by the particles is prevented.
S2, taking oxytetracycline, tetracycline and anhydrotetracycline as markers of oxytetracycline drug residues, and carrying out liquid chromatography separation and mass spectrometry detection on a liquid to be detected to identify whether the markers exist in the protein feed raw materials.
In other words, the method uses oxytetracycline, tetracycline and anhydrotetracycline as markers of oxytetracycline drug residues, and obtains whether protein feed raw materials contain the markers through a liquid chromatography-mass spectrometry combined method, if the markers are contained, the protein feed raw materials are doped with the oxytetracycline drug residues, if the markers are not contained, or only one or two of the markers are contained, the protein feed raw materials are not doped with the oxytetracycline drug residues.
Wherein, the liquid to be detected is separated by liquid chromatography, detected by mass spectrometry and detected by a liquid chromatography-mass spectrometer.
In some embodiments illustrated herein, the step of subjecting the liquid to be tested to liquid chromatography comprises: gradient elution was performed with mobile phase a and mobile phase B.
Wherein the mobile phase A is 1.5 mM-2.5 mM acetic ammonia solution, and the acetic ammonia solution contains 0.15 v/v% -0.25 v/v% formic acid; mobile phase B was acetonitrile. That is to say, the mobile phase A is acetic acid ammonia water solution, the concentration of the acetic acid ammonia is 1.5 mM-2.5 mM, meanwhile, the acetic acid ammonia water solution contains formic acid, and the volume concentration of the formic acid in the acetic acid ammonia water solution is 0.15% -0.25%.
In other words, mobile phase A is an aqueous ammonia acetate solution having a concentration of any one or a range between any two concentration values of 1.5mM, 1.7mM, 2mM, 2.3mM, 2.4mM, and 2.5mM, wherein the aqueous ammonia acetate solution contains formic acid at any one or a range defined by any two volume concentration values of 0.15 v/v%, 0.18 v/v%, 0.2 v/v%, 0.22 v/v%, and 0.25 v/v%.
In view of the above, in order to better perform the liquid chromatography separation on the solution to be detected diluted by the second solvent, optionally, the second solvent is the mobile phase a, that is, the second solvent is the same as the mobile phase a.
Note that during the gradient elution, the volume percentage of the mobile phase a + the mobile phase B is 100%.
Specifically, in some embodiments illustrated herein, the step of gradient elution is performed as follows, in volume percent:
when the time is 0-0.5 min, the mobile phase A is kept at 95%, and the mobile phase B is kept at 5%.
When the time is 0.5-3.0 min, the mobile phase A is reduced to 85% from 95%, and the mobile phase B is increased to 15% from 5%.
And when the time is 3.0-10.0 min, the mobile phase A is reduced from 85% to 60%, and the mobile phase B is increased from 15% to 40%.
When 10.0-18.0 min, the mobile phase A is reduced from 60% to 0, and the mobile phase B is increased from 40% to 100%.
When the time is 18.0-23.0 min, the mobile phase A is kept at 0, and the mobile phase B is kept at 100%.
And when the time is 23.0-25.0 min, the mobile phase A is kept at 95%, and the mobile phase B is kept at 5%.
Optionally, the flow rate of the gradient elution is 0.3-0.5 mL/min, for example, the flow rate of the gradient elution is any one of 0.3mL/min, 0.4mL/min, 0.5mL/min or between any two flow rate values. It is noted that references to intervening elements in this application include both endpoints.
Alternatively, in the step of liquid chromatography separation, the stationary phase of the chromatographic column used is an octadecyl functional group.
Alternatively, the chromatography column is a Zorbax Eclipse Plus C18 chromatography column.
Optionally, the column temperature of the chromatography column is 38 ℃ to 42 ℃, for example, the column temperature is any one of 38 ℃, 39 ℃, 40 ℃, 41 ℃, 42 ℃ or between any two temperature values.
In the embodiments provided herein, optionally, the mass spectrometry detection includes: and detecting the product after liquid chromatography separation by adopting a quadrupole time-of-flight mass spectrum.
The quadrupole time-of-flight mass spectrum is, for example, an Agilent6465LC-Q TOF quadrupole time-of-flight mass spectrum.
Optionally, the mass spectrometric detection conditions are:
an electrospray ionization source; scanning positive ions within the scanning range of 400-500 m/z; temperature of the drying gas: 250 ℃; flow rate of drying gas: 7L/min; atomizing gas pressure: 7 psi; temperature of sheath gas: 325 ℃; the flow rate of the sheath gas: 11L/min; nozzle voltage 200V; capillary voltage: 3 kV; capillary exit voltage: 120V; skimmer voltage 750V.
Optionally, in mass spectrometry, the mass-to-charge ratio is 461.1555m/z as a characteristic fragment of oxytetracycline, the mass-to-charge ratio is 445.1606m/z as a characteristic fragment of tetracycline, and the mass-to-charge ratio is 443.1450m/z as a characteristic fragment of anhydrotetracycline.
The features and properties of the present application are described in further detail below with reference to examples.
Example 1
An identification method for protein feed raw material mixed with terramycin dregs comprises the following steps:
s1, crushing the rapeseed dregs doped with the oxytetracycline dregs (10 percent, m/m) by a cyclone mill, wherein the crushed granularity reaches 18 meshes.
S2, weighing 0.5g of the sample crushed in the step S1 into a clean centrifuge tube, adding 5mL of methanol, carrying out vortex for 2 minutes, and placing on a shaker for shaking for 30 minutes for extraction. Centrifuging at 6000r/min for 5min, and collecting supernatant as extractive solution.
S3, sucking 0.5mL of the extraction solution obtained in the step S2, adding an equal volume of mobile phase A for dilution, and filtering by adopting a polytetrafluoroethylene filter membrane to obtain a purified solution to be detected.
S4, sucking 0.5mL of the solution to be detected obtained in the step S3, and performing liquid chromatography separation. The conditions for the liquid chromatographic separation were as follows:
the chromatographic column is a Zorbax Eclipse Plus C18 chromatographic column, the column temperature: 42 ℃.
The mobile phase A is: 2.0mM aqueous ammonia acetate (containing 0.2 v/v% formic acid), mobile phase B: and (3) acetonitrile. The flow rate was 0.5 mL/min.
③ adopting a gradient elution mode: and (3) keeping the mobile phase A at 95% and the mobile phase B at 5% in volume percentage within 0-0.5 min.
When the time is 0.5-3.0 min, the mobile phase A is reduced to 85% from 95%, and the mobile phase B is increased to 15% from 5%.
And when the time is 3.0-10.0 min, the mobile phase A is reduced from 85% to 60%, and the mobile phase B is increased from 15% to 40%.
When 10.0-18.0 min, the mobile phase A is reduced from 60% to 0, and the mobile phase B is increased from 40% to 100%.
When the time is 18.0-23.0 min, the mobile phase A is kept at 0, and the mobile phase B is kept at 100%.
And when the time is 23.0-25.0 min, the mobile phase A is kept at 95%, and the mobile phase B is kept at 5%.
Sample feeding amount: 20 ul.
S5, detecting the components separated by the liquid chromatography in the step S4 by a quadrupole time-of-flight mass spectrometry (Agilent6465LC-Q TOF) under the following mass spectrum conditions:
an electrospray ionization source; scanning positive ions within the scanning range of 400-500 m/z; temperature of the drying gas: 250 ℃; flow rate of drying gas: 7L/min; atomizing gas pressure: 7 psi; temperature of sheath gas: 325 ℃; the flow rate of the sheath gas: 11L/min; nozzle voltage 200V; capillary voltage: 3 kV; capillary exit voltage: 120V; skimmer voltage 750V.
S6, judging a result:
in the liquid chromatogram-mass spectrum, characteristic fragment extraction chromatogram of oxytetracycline extracted at 5.976min is shown in FIG. 1. The tetracycline feature fragment extraction chromatogram at 5.701min is shown in FIG. 2. The extraction chromatogram of the dehydrated tetracycline feature fragment at 7.994min is shown in FIG. 3. Through fragment ion analysis, the characteristic fragment ion 445.1606m/z of oxytetracycline is detected as shown in FIG. 4, the characteristic fragment ion 461.1555m/z of tetracycline is detected as shown in FIG. 5, and the characteristic fragment ion 443.1450m/z of anhydrotetracycline is detected as shown in FIG. 6. The result shows that the terramycin residue marker is detected from the rapeseed dregs, which indicates that the terramycin residue is contained, and the identification result is the same as the actual condition.
Example 2
An identification method for protein feed raw material mixed with terramycin dregs comprises the following steps:
s1, crushing the rapeseed dregs doped with the oxytetracycline dregs (10 percent, m/m) by a cyclone mill until the granularity reaches 20 meshes.
S2, weighing 0.5g of the sample crushed in the step S1 into a clean centrifuge tube, adding 4mL of methanol, vortexing for 2 minutes, and placing on a shaker for shaking for 30 minutes for extraction. Centrifuging at centrifugal force of 5000r/min for 6min after extraction, and collecting supernatant as extraction solution.
S3, sucking 0.5mL of the extraction solution obtained in the step S2, adding an equal volume of mobile phase A for dilution, and filtering by adopting a polytetrafluoroethylene filter membrane to obtain a purified solution to be detected.
S4, sucking 0.5mL of the solution to be detected obtained in the step S3, and performing liquid chromatography separation. The conditions for the liquid chromatographic separation were as follows:
the chromatographic column is a Zorbax Eclipse Plus C18 chromatographic column, the column temperature: at 38 ℃.
The mobile phase A is: 1.5mM aqueous ammonia acetate solution (containing 0.15 v/v% formic acid), mobile phase B: and (3) acetonitrile. The flow rate was 0.5 mL/min.
③ adopting a gradient elution mode: and (3) keeping the mobile phase A at 95% and the mobile phase B at 5% in volume percentage within 0-0.5 min.
When the time is 0.5-3.0 min, the mobile phase A is reduced to 85% from 95%, and the mobile phase B is increased to 15% from 5%.
And when the time is 3.0-10.0 min, the mobile phase A is reduced from 85% to 60%, and the mobile phase B is increased from 15% to 40%.
When 10.0-18.0 min, the mobile phase A is reduced from 60% to 0, and the mobile phase B is increased from 40% to 100%.
When the time is 18.0-23.0 min, the mobile phase A is kept at 0, and the mobile phase B is kept at 100%.
And when the time is 23.0-25.0 min, the mobile phase A is kept at 95%, and the mobile phase B is kept at 5%.
Sample feeding amount: 20 ul.
S5, detecting the components separated by the liquid chromatography in the step S4 by a quadrupole time-of-flight mass spectrometry (Agilent6465LC-Q TOF) under the following mass spectrum conditions:
an electrospray ionization source; scanning positive ions within the scanning range of 400-500 m/z; temperature of the drying gas: 250 ℃; flow rate of drying gas: 7L/min; atomizing gas pressure: 7 psi; temperature of sheath gas: 325 ℃; the flow rate of the sheath gas: 11L/min; nozzle voltage 200V; capillary voltage: 3 kV; capillary exit voltage: 120V; skimmer voltage 750V.
S6, judging a result:
in the liquid chromatogram-mass spectrum, the characteristic fragment of oxytetracycline is extracted at 5.976min by characteristic fragment extraction. The tetracycline feature fragment was extracted at 5.701 min. The anhydrotetracycline feature fragments were extracted at 7.994 min. The result shows that the terramycin residue marker is detected from the rapeseed dregs, which indicates that the terramycin residue is contained, and the identification result is the same as the actual condition.
Example 3
An identification method for protein feed raw material mixed with terramycin dregs comprises the following steps:
s1, crushing the rapeseed dregs doped with the oxytetracycline dregs (10 percent, m/m) by a cyclone mill until the granularity reaches 40 meshes.
S2, weighing 0.5g of the sample crushed in the step S1 into a clean centrifuge tube, adding 6mL of an extraction solvent (the extraction solvent is methanol), carrying out vortex for 2 minutes, and placing on a shaker for shaking for 30 minutes for extraction. Centrifuging at centrifugal force of 5000r/min for 6min after extraction, and collecting supernatant as extraction solution.
S3, sucking 0.5mL of the extraction solution obtained in the step S2, adding an isovolumetric mobile phase A for dilution, and filtering by adopting a polytetrafluoroethylene filter membrane to obtain a purified solution to be detected.
S4, sucking 0.5mL of the solution to be detected obtained in the step S3, and performing liquid chromatography separation. The conditions for the liquid chromatographic separation were as follows:
the chromatographic column is a Zorbax Eclipse Plus C18 chromatographic column, the column temperature: at 40 ℃.
The mobile phase A is: 2.0mM aqueous ammonia acetate (containing 0.2 v/v% formic acid), mobile phase B: acetonitrile at a flow rate of 0.5 mL/min.
③ adopting a gradient elution mode: and (3) keeping the mobile phase A at 95% and the mobile phase B at 5% in volume percentage within 0-0.5 min.
When the time is 0.5-3.0 min, the mobile phase A is reduced to 85% from 95%, and the mobile phase B is increased to 15% from 5%.
And when the time is 3.0-10.0 min, the mobile phase A is reduced from 85% to 60%, and the mobile phase B is increased from 15% to 40%.
When 10.0-18.0 min, the mobile phase A is reduced from 60% to 0, and the mobile phase B is increased from 40% to 100%.
When the time is 18.0-23.0 min, the mobile phase A is kept at 0, and the mobile phase B is kept at 100%.
And when the time is 23.0-25.0 min, the mobile phase A is kept at 95%, and the mobile phase B is kept at 5%.
Sample feeding amount: 20 ul.
S5, detecting the components separated by the liquid chromatography in the step S4 by a quadrupole time of flight mass spectrometry (Agilent6465LC-Q TOF), wherein the mass spectrometry conditions are as follows:
an electrospray ionization source; scanning positive ions within the scanning range of 400-500 m/z; temperature of the drying gas: 250 ℃; flow rate of drying gas: 7L/min; atomizing gas pressure: 7 psi; temperature of sheath gas: 325 ℃; the flow rate of the sheath gas: 11L/min; nozzle voltage 200V; capillary voltage: 3 kV; capillary exit voltage: 120V; skimmer voltage 750V.
S6, judging a result:
in the liquid chromatogram-mass spectrum, the characteristic fragment of oxytetracycline is extracted at 5.976min by characteristic fragment extraction. The tetracycline feature fragment was extracted at 5.701 min. The anhydrotetracycline feature fragments were extracted at 7.994 min. The result shows that the terramycin residue marker is detected from the rapeseed dregs, which indicates that the terramycin residue is contained, and the identification result is the same as the actual condition.
In conclusion, the identification method for the protein feed raw material mixed with the terramycin dregs has the advantages of less sample dosage, simple pretreatment process, short detection time, higher sensitivity and accurate result, and can effectively identify whether the protein feed raw material is mixed with the terramycin dregs.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (17)
1. An identification method for protein feed raw material mixed with terramycin dregs is used for identifying whether the source of terramycin contained in the protein feed is terramycin dregs or terramycin prototype drug, and is characterized by comprising the following steps:
extracting a protein feed raw material by using a first solvent, diluting by using a second solvent, and filtering to obtain a solution to be detected;
taking oxytetracycline, tetracycline and anhydrotetracycline as markers of oxytetracycline dregs, separating a liquid to be detected by liquid chromatography, detecting by mass spectrometry, and identifying whether the protein feed raw material has the markers;
wherein the first solvent is an organic solvent.
2. The identification method according to claim 1, wherein the characteristic fragment of oxytetracycline is represented by a mass-to-charge ratio of 461.1555m/z, the characteristic fragment of tetracycline is represented by a mass-to-charge ratio of 445.1606m/z, and the characteristic fragment of anhydrotetracycline is represented by a mass-to-charge ratio of 443.1450m/z in the mass spectrometric detection.
3. An authentication method according to claim 1, wherein the step of subjecting the liquid to be tested to liquid chromatography comprises:
performing gradient elution with mobile phase A and mobile phase B;
wherein the mobile phase A is 1.5 mM-2.5 mM ammonium acetate aqueous solution, and the ammonium acetate aqueous solution contains 0.15 v/v% -0.25 v/v% of formic acid;
the mobile phase B is acetonitrile.
4. The method of claim 3, wherein the step of gradient elution is performed in the following manner, in percentage by volume: when 0-0.5 min is reached, the mobile phase A is kept at 95%, and the mobile phase B is kept at 5%; at 0.5-3.0 min, the mobile phase A is reduced from 95% to 85%, and the mobile phase B is increased from 5% to 15%; at 3.0-10.0 min, the mobile phase A is reduced from 85% to 60%, and the mobile phase B is increased from 15% to 40%; at 10.0-18.0 min, the mobile phase A is reduced from 60% to 0, and the mobile phase B is increased from 40% to 100%; when the time is 18.0-23.0 min, the mobile phase A is kept at 0, and the mobile phase B is kept at 100%; and when the time is 23.0-25.0 min, the mobile phase A is kept at 95%, and the mobile phase B is kept at 5%.
5. The method according to claim 3, wherein the flow rate of the gradient elution is 0.3 to 0.5 mL/min.
6. The method of claim 1, wherein in the step of liquid chromatography, the stationary phase of the column is octadecyl functional group.
7. The method of claim 6, wherein the chromatography column is a Zorbax Eclipse PlusC18 chromatography column.
8. The identification method according to claim 6, wherein the column temperature of the chromatography column is 38 ℃ to 42 ℃.
9. The method of identification according to claim 1, wherein the mass spectrometric detection comprises: and detecting the product separated by the liquid chromatography by adopting a quadrupole time-of-flight mass spectrum.
10. The identification method according to claim 9, wherein the mass spectrometric detection conditions are:
an electrospray ionization source; scanning positive ions within the scanning range of 400-500 m/z; temperature of the drying gas: 250 ℃; flow rate of drying gas: 7L/min; atomizing gas pressure: 7 psi; temperature of sheath gas: 325 ℃; the flow rate of the sheath gas: 11L/min; nozzle voltage 200V; capillary voltage: 3 kV; capillary exit voltage: 120V; skimmer voltage 750V.
11. The identification method according to claim 1, wherein the second solvent comprises ammonium acetate with a concentration of 1.5 mM-2.5 mM, and the ammonium acetate contains 0.15 v/v% to 0.25 v/v% formic acid.
12. The identification method according to claim 1, wherein the particle size of the protein feed material is 18 mesh or more.
13. The identification method according to claim 1, wherein the particle size of the protein feed material is 18 to 40 mesh.
14. The method of identifying according to claim 1, wherein the first solvent comprises methanol.
15. The identification method according to claim 1, wherein the step of extracting the protein feed raw material with the first solvent comprises:
mixing the protein feed raw material with the first solvent according to a ratio of 1 g: (8 ml-12 ml) and carrying out shaking extraction to obtain an extracting solution;
and centrifuging the extracting solution and taking supernatant.
16. The method of claim 15, wherein the extract is centrifuged at 5000 to 7000r/min for 4 to 6 min.
17. The identification method according to claim 1, wherein the protein feed material comprises at least one of rapeseed meal, soybean meal, and cottonseed meal.
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