CN109557216B

CN109557216B - Identification method of tylosin filter residue in rapeseed meal

Info

Publication number: CN109557216B
Application number: CN201910059702.XA
Authority: CN
Inventors: 李阳; 樊霞; 索德成; 肖志明; 贾铮; 田静; 王石
Original assignee: Institute of Quality Standards and Testing Technology for Agro Products of Henan Academy of Agricultural Science
Current assignee: Institute of Quality Standards and Testing Technology for Agro Products of Henan Academy of Agricultural Science
Priority date: 2019-01-22
Filing date: 2019-01-22
Publication date: 2020-02-18
Anticipated expiration: 2039-01-22
Also published as: CN109557216A

Abstract

The invention relates to the field of feed quality and safety detection, in particular to a method for identifying tylosin filter residues mixed with rapeseed meal. The method comprises: extracting and removing impurities from a rapeseed meal sample to obtain a purified solution, and performing liquid chromatography separation and mass spectrometry detection on the purified solution; in the detection result, tylosin and demethylmacricin are used as tylosin Markers for ethoxycin filter residues. The method provided by the invention has few sample dosage, simple pretreatment process, short detection time, high sensitivity and accurate result, and can meet the detection purpose of identifying the filter residue mixed with tylosin in rapeseed meal.

Description

Identification method of filter residue mixed with tylosin in rapeseed dregs

Technical Field

The invention relates to the field of feed quality safety detection, in particular to an identification method of filter residue mixed with tylosin in rapeseed meal.

Background

The rapeseed meal is a byproduct obtained by taking rapeseed as a raw material and extracting oil, the protein content of the rapeseed is 34-38%, the ratio of methionine to lysine in amino acid composition is high, the rapeseed meal belongs to one of three protein raw materials (bean meal, cotton meal and rapeseed meal) in China, and the rapeseed meal is an important feed protein source and is widely applied to livestock and poultry breeding. The tylosin filter residue is waste residue generated after filtering the residual fermentation culture medium after producing the tylosin by biological fermentation, and has certain crude protein. The medium reports that tylosin filter residue is mixed into feed protein raw materials for replacement use by illegal culturists. Because the tylosin filter residue contains medicine residues which are not completely extracted and secondary metabolites which are not evaluated in safety, the feed raw materials containing the tylosin filter residue are used in the breeding process, and potential risk hazards of harming the health of bred animals, influencing food safety, inducing the generation of bacterial drug resistance and the like are caused. No. 176 bulletin issued by the ministry of agriculture, the ministry of health and the national drug administration in China clearly stipulates that tylosin filter residue is prohibited to be added into feed for use.

At present, the existing detection technology at home and abroad mainly detects tylosin prototype drugs, and although the content of tylosin in feed protein raw materials can be accurately measured, the reason of exceeding the standard of the drugs cannot be identified, namely, the reason of exceeding the standard caused by adding the tylosin prototype drug or adding tylosin filter residue cannot be distinguished, so that the situation that the quality of the drugs is difficult to determine in the supervision and law enforcement process is caused. Therefore, the agricultural administration department has the identification technical requirement in the supervision work of tylosin filter residue in feed protein raw materials.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide an identification method for tylosin filter residue doped in rapeseed meal, which takes tylosin and demethylated colicin as markers of the tylosin filter residue, extracts and purifies a sample by liquid-liquid extraction and solid-phase extraction, and separates and detects 2 markers by liquid chromatography-time-of-flight mass spectrometry, thereby realizing the identification of the tylosin filter residue doped in the feed rapeseed meal.

The invention relates to an identification method of tylosin-doped filter residue in rapeseed meal, which comprises the following steps:

extracting and removing impurities from a rapeseed meal sample to obtain a purified solution, and performing liquid chromatography separation and mass spectrometry detection on the purified solution;

and taking tylosin and demethylated colicin as markers of tylosin filter residue in the detection result for analysis.

The method provided by the invention can meet the detection aim of identifying the filter residue doped with tylosin in the rapeseed dregs.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a total ion flux extraction chromatogram of tylosin in an embodiment of the present invention;

FIG. 2 is a chromatogram of total ion flux extraction of demethylated colicin in an embodiment of the present invention;

FIG. 3 is a characteristic fragment ion diagram of tylosin in one embodiment of the present invention;

FIG. 4 is a characteristic fragment ion diagram of demethylated colicin in accordance with an embodiment of the present invention.

Detailed Description

In some embodiments, the liquid chromatography is gradient eluted with mobile phase a and mobile phase B;

the mobile phase A is 0.05 v/v% -0.15 v/v% formic acid aqueous solution; 0.07 v/v%, 0.10 v/v%, 0.13 v/v% aqueous formic acid solution can also be selected;

the mobile phase B is 0.05 v/v% -0.15 v/v% formic acid acetonitrile solution. 0.07 v/v%, 0.10 v/v%, 0.13 v/v% acetonitrile solution of formic acid can be selected;

in some embodiments, the gradient elution procedure is:

when the time is 0-1 min, 95 v/v% of a mobile phase A and 5 v/v% of a mobile phase B;

when the time is 1-5 min, 95 v/v% -70 v/v% of a mobile phase A and 5 v/v% -30 v/v% of a mobile phase B;

when the time is 5-11 min, the mobile phase A is 70 v/v% -5 v/v%, and the mobile phase B is 30 v/v% -95 v/v%;

when the time is 11-13 min, the mobile phase A is 5 v/v%, and the mobile phase B is 95 v/v%;

when 13.01-15 min, the mobile phase A is 95 v/v%, and the mobile phase B is 5 v/v%;

wherein, the floating range of the numerical parameter of the concentration of the mobile phase is +/-10%, or +/-8%, +/-5%, +/-3% of the numerical parameter.

In some embodiments, the gradient elution has a flow rate of 0.3 to 0.5 mL/min; 0.4mL/min can also be selected.

In some embodiments, the active functional group of the stationary phase of the chromatography column used for liquid chromatography is a pentafluorophenyl functional group;

in some embodiments, the chromatography column is a Kinetex F5 chromatography column;

in some embodiments, the chromatographic column has a column temperature of 38 ℃ to 42 ℃, optionally 40 ℃.

In some embodiments, the mass spectrometry employs a quadrupole-time-of-flight mass spectrometry;

in some embodiments, the conditions of the mass spectrum are:

an electrospray ionization source; scanning positive ions within the scanning range of 500-1000 m/z; flow rate of atomizing gas: 50 psi; auxiliary heating air flow: 50 psi; air flow of the air curtain: 50 psi; capillary temperature: 400 ℃; ionization voltage: 5500V; gas cluster voltage: 80V; collision energy: 35V; CES voltage: 15V;

wherein the above conditions relate to a floating range of the respective numerical parameter of ± 10%, or ± 8%, ± 5%, ± 3% of the numerical parameter.

In some embodiments, the extraction of the rapeseed meal sample is extraction using an extraction solvent;

the extraction solvent is formic acid solution containing 0.15 v/v% -0.25 v/v%, wherein the solvent is a mixture of acetonitrile and water, and the volume ratio of the acetonitrile to the water is (0.8-1.2) to (0.8-1.2);

preferably, the extraction solvent is a 0.2 v/v% formic acid solution, wherein the solvent is a mixture of acetonitrile and water, and the volume ratio of acetonitrile to water is 1: 1.

In some embodiments, the method for extracting a rapeseed meal sample comprises:

mixing the raw materials in a ratio of 1 g: mixing the rapeseed meal sample and the extraction solvent in a solid-liquid ratio (8 ml-12 ml) (or 1 g: 10 ml) and extracting by oscillation; centrifuging the extracting solution and taking supernatant;

in some embodiments, the centrifugation is 5000r/min to 7000r/min centrifugation for 4min to 6 min;

in some embodiments, the centrifugation is 6000r/min centrifugation for 5 min.

In some embodiments, the rapeseed meal sample has a particle size of greater than or equal to 18 mesh; preferably 18 to 40 mesh.

In some embodiments, the removing comprises removing impurities using an Oasis PRiME HLB column.

In some embodiments, the mass to charge ratio data at and around mass to charge ratios of 916.5264m/z and 772.4478m/z are characteristic fragments of tylosin, and the mass to charge ratio data at and around mass to charge ratios of 888.4951m/z and 596.3429m/z are characteristic fragments of desmethyl colicin.

The method provided by the invention has the advantages of less sample consumption, simple pretreatment process, short detection time, higher sensitivity and accurate result, and can meet the detection aim of identifying the filter residue doped with tylosin in the rapeseed dregs.

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. 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 commercially available.

Example 1

(1) Sample pretreatment

And (3) crushing the rapeseed dregs doped with the tylosin filter residue (10 percent, m/m) by adopting a cyclone mill, wherein the crushed granularity reaches 25 meshes.

(2) Liquid-liquid extraction

Weighing 0.5g of crushed sample into a clean centrifuge tube, adding 4mL of extraction solvent (the extraction solvent is acetonitrile/water mixed solution containing 0.15 v/v% formic acid, the volume ratio of acetonitrile to water is 0.8:1.2, swirling for 2 minutes, placing on a shaker, shaking for 30 minutes for extraction, centrifuging for 6 minutes under the centrifugal force of 5000r/min after extraction is finished, and taking supernatant as extraction solution.

(3) Solid phase extraction purification

0.5mL of the extract solution was aspirated through the Oasis PRIME HLB column, and if necessary, a vacuum was applied to increase the flow rate, and the filtrate was collected to obtain a purified solution.

(4) Liquid chromatography separation

0.5mL of the purified solution was aspirated, and the solution was diluted 4-fold with 10mM ammonium formate buffer solution (pH4.5) and subjected to liquid chromatography.

① the chromatographic column is a Kinetex F5 chromatographic column with a column temperature of 38 ℃;

② mobile phase A is 0.07 v/v% formic acid water solution, mobile phase B is 0.13 v/v% formic acid acetonitrile solution, and the flow rate is 0.3 mL/min;

③ adopts gradient elution mode, wherein the volume percentage of A is 0.00-1.00min, A is 95%, B is 5%, 1.00-5.00min, A is 95% -70%, B is 5% -30%, 5.00-11.00min, A is 70% -5%, B is 30% -95%, 11.00-13.00min, A is 5%, B is 95%, 13.01-15.00min, A is 95%, and B is 5%;

④ sample size is 20 ul;

(5) mass spectrometric detection

The components after liquid chromatography separation are detected by a four-stage rod-time-of-flight mass spectrum (Triple TOF5600+), and the mass spectrum conditions are as follows:

① electrospray ionization source;

② positive ion scan, scan range 500-;

③ atomizing gas (N)₂) Flow rate: 50 psi;

④ auxiliary heating gas (N)₂) Flow rate: 50 psi;

⑤ curtain gas flow (N)₂)：50psi；

⑥ capillary temperature 400 deg.C;

⑦ ionization voltage is 5500V;

⑧ gas cluster voltage is 80V;

⑨ collision energy 35V;

⑩ CES voltage: 15V.

Example 2

(1) Sample pretreatment

(2) Liquid-liquid extraction

Weighing 0.5g of crushed sample into a clean centrifuge tube, adding 6mL of extraction solvent (the extraction solvent is acetonitrile/water mixed solution containing 0.25 v/v% formic acid, the volume ratio of acetonitrile to water is 1.2:0.8, swirling for 2 minutes, placing on a shaker, shaking for 30 minutes for extraction, centrifuging for 4 minutes under the centrifugal force of 7000r/min after extraction is finished, and taking supernatant as extraction solution.

(3) Solid phase extraction purification

(4) Liquid chromatography separation

① chromatographic column is Kinetex F5 chromatographic column, and the column temperature is 42 ℃;

② mobile phase A is 0.12 v/v% formic acid water solution, mobile phase B is 0.08 v/v% formic acid acetonitrile solution, and the flow rate is 0.5 mL/min;

④ sample size is 20 ul;

(5) mass spectrometric detection

① electrospray ionization source;

② positive ion scan, scan range 500-;

③ atomizing gas (N)₂) Flow rate: 50 psi;

④ auxiliary heating gas (N)₂) Flow rate: 50 psi;

⑤ curtain gas flow (N)₂)：50psi；

⑥ capillary temperature 400 deg.C;

⑦ ionization voltage is 5500V;

⑧ gas cluster voltage is 80V;

⑨ collision energy 35V;

⑩ CES voltage: 15V.

Example 3

(1) Sample pretreatment

And (3) crushing the rapeseed dregs doped with the tylosin filter residue (10 percent, m/m) by adopting a cyclone mill, wherein the crushed granularity reaches 18 meshes.

(2) Liquid-liquid extraction

Weighing 0.5g of the crushed sample into a clean centrifuge tube, adding 5mL of an extraction solvent (the extraction solvent is an acetonitrile/water mixed solution containing 0.2% formic acid, and the volume ratio of acetonitrile to water is 1:1), vortexing for 2 minutes, and placing on a shaking table to shake for 30 minutes for extraction. Centrifuging at 6000r/min for 5min, and collecting supernatant as extractive solution.

(3) Solid phase extraction purification

(4) Liquid chromatography separation

① chromatographic column is Kinetex F5 chromatographic column, and the column temperature is 40 ℃;

② mobile phase A is 0.1% formic acid water solution, mobile phase B is 0.1% formic acid acetonitrile solution, and flow rate is 0.4 mL/min;

④ sample size is 20 ul;

(5) mass spectrometric detection

① electrospray ionization source;

② positive ion scan, scan range 500-;

③ atomizing gas (N)₂) Flow rate: 50 psi;

④ auxiliary heating gas (N)₂) Flow rate: 50 psi;

⑤ curtain gas flow (N)₂)：50psi；

⑥ capillary temperature 400 deg.C;

⑦ ionization voltage is 5500V;

⑧ gas cluster voltage is 80V;

⑨ collision energy 35V;

⑩ CES voltage: 15V.

(6) And (4) judging a result:

extracting total ion flow in liquid chromatogram-mass spectrogram, and extracting tylosin chromatogram peak at 7.465min as shown in FIG. 1; the chromatographic peak of demethylated colicin extracted at 5.620min is shown in FIG. 2; by fragment ion analysis, characteristic fragment ions 916.5264m/z and 772.4478m/z of tylosin were detected as shown in FIG. 3, and characteristic fragment ions 888.4951m/z and 596.3429m/z of desmethyl colicin were detected as shown in FIG. 4. The result shows that the marker of tylosin filter residue is detected in the rapeseed dregs, which indicates that the tylosin filter residue is contained.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. the identification method of adding tylosin filter residue in a rapeseed meal, is characterized in that, comprises:

After extracting and removing impurities from the rapeseed meal sample, a purified solution is obtained, and the purified solution is subjected to liquid chromatography separation and mass spectrometry detection;

In the test results, tylosin and Demethylmacrocin were used as markers of tylosin filter residues;

The liquid chromatography carries out gradient elution with mobile phase A and mobile phase B;

The mobile phase A is an aqueous formic acid solution of 0.05v/v% to 0.15v/v%;

The mobile phase B is 0.05v/v%～0.15v/v% formic acid in acetonitrile;

The procedure of the gradient elution is:

0～1min, mobile phase A 95v/v%, mobile phase B 5v/v%;

1～5min, mobile phase A 95v/v%～70v/v%, mobile phase B 5v/v%～30v/v%;

5～11min, mobile phase A 70v/v%～5v/v%, mobile phase B 30v/v%～95v/v%;

From 11 to 13 minutes, mobile phase A 5v/v%, mobile phase B 95v/v%;

13.01～15min, mobile phase A 95v/v%, mobile phase B 5v/v%;

Wherein, the floating range of the numerical parameter of the mobile phase concentration is ±10% of the numerical parameter;

The flow rate of the gradient elution is 0.3-0.5 mL/min;

The active functional group of the stationary phase of the chromatographic column used in the liquid chromatography is a pentafluorophenyl functional group;

The chromatographic column is a Kinetex F5 chromatographic column.

2 . The method according to claim 1 , wherein the column temperature of the chromatographic column is 38° C.˜42° C. 3 .

3. The method according to claim 1, wherein the mass spectrometry adopts quadrupole-time-of-flight mass spectrometry.

4. method according to claim 3, is characterized in that, the condition of described mass spectrometry is:

Electrospray ionization source; positive ion scanning, scanning range 500-1000m/z; atomizing gas flow: 50psi; auxiliary heating gas flow: 50psi; air curtain gas flow: 50psi; capillary temperature: 400℃; Cluster voltage: 80V; Collision energy: 35V; CES voltage: 15V;

The floating range of each numerical parameter involved in the above conditions is ±10% of the numerical parameter.

5. The method according to claim 1, wherein the extraction of the rapeseed meal sample is extraction using an extraction solvent;

The extraction solvent is a solution containing 0.15v/v%～0.25v/v% formic acid, wherein the solvent is a mixture of acetonitrile and water, and the volume ratio of acetonitrile to water is (0.8～1.2):(0.8～1.2).

6. The method according to claim 5, wherein the extraction method of the rapeseed meal sample comprises:

Mix the rapeseed meal sample with the extraction solvent and extract by shaking at a solid-to-liquid ratio of 1g:(8ml-12ml); after centrifuging the extract, take the supernatant.

7. The method according to claim 6, wherein the centrifugation is 5000r/min～7000r/min centrifugation for 4min～6min.

8. The method according to claim 6, wherein the diameter of the rapeseed meal sample is greater than or equal to 18 meshes.

9 . The method according to claim 6 , wherein the diameter of the rapeseed meal sample is 18-40 meshes. 10 .

10. The method according to claim 1, wherein the impurity removal is specifically performed by using an Oasis PRiME HLB cartridge.

11. The method according to claim 1, wherein the mass-to-charge ratio is 916.5264m/z and 772.4478m/z as the characteristic fragments of tylosin, and the mass-to-charge ratio is 888.4951m/z and 596.3429m /z as the characteristic fragment for demethylamcin.