CN107153101B - Detection method of plant-derived feed polyoxin - Google Patents

Abstract

The invention relates to a method for detecting polyoxin in plant-derived feed, which comprises the following steps: pretreating the material; extracting and purifying; qualitative analysis and determination; quantitative determination; and calculating and verifying the result. The method has the advantages of high identification accuracy and accurate positioning, is representative in the detection of polyoxin in China, and has good application prospect.

Description

Detection method of plant-derived feed polyoxin

Technical Field

The invention belongs to the field of pesticide residue detection, and particularly relates to a method for detecting polyoxin in plant-derived feed.

Background

In recent years, with the disablement of high-toxicity and high-residue chemical pesticides, green, environment-friendly, low-toxicity and low-residue biological pesticides are more and more favored by farmers. The polyoxin has the effects of inhibiting germ sporulation and disease spot enlargement, and is widely applied to preventing and treating various diseases such as cucumber downy mildew, wheat powdery mildew, corn smut, ginseng black spot, apple and pear gray spot, rice sheath blight and the like. However, the toxicity research of the polyoxin on human and livestock is relatively lagged at present, and the toxicological action condition is not clear. And the crops such as rice, corn and wheat which are polluted by polyoxin are taken as feed, are eaten by pigs, cattle, sheep and the like, can be accumulated in the human body, enter the human body through a food chain and cause certain harm. At present, the polyoxin determination method comprises a microbiological method, a capillary electrophoresis-electrochemiluminescence method and a high performance liquid chromatography, and the capillary electrophoresis-electrochemiluminescence method and the high performance liquid chromatography are adopted at home mostly. The measuring methods are used for measuring a series of polyoxin metabolites produced by fermentation, and have the advantages of low sensitivity, long time consumption and poor precision.

Disclosure of Invention

The invention aims to solve the technical problem of providing a method for detecting the polyoxin in the plant-derived feed, which detects whether the polyoxin is contained in the plant-derived feed by adopting the steps of pretreatment of materials, extraction and purification, qualitative analysis and determination, quantitative determination, result calculation and verification and the like, has the advantages of high identification accuracy and accurate positioning, is representative in the detection of the polyoxin in China, and has good application prospect.

The invention relates to a method for detecting polyoxin in plant-derived feed, which comprises the following steps:

(1) taking a plant-derived feed sample, grinding and sieving; then adding a formic acid acetonitrile solution (according to the mass-volume ratio of 2.5g:20ml), uniformly mixing, performing ultrasonic extraction, centrifuging, blow-drying, and dissolving by using an ammonia water solution to obtain a sample to be purified;

(2) pre-washing an extraction column with acetonitrile and water in sequence, transferring the sample to be purified into the extraction column, removing a sample loading solution, eluting with a glacial acetic acid acetonitrile water solution, collecting an eluent, drying, and fixing the volume;

(3) and performing qualitative analysis and quantitative determination by adopting a liquid chromatography-tandem mass spectrum, and finally determining the content of the polyoxin in the sample by calculation.

The concentration of the formic acid acetonitrile solution in the step (1) is 3% (formic acid: acetonitrile: 3:97) -8% (formic acid: acetonitrile: 8: 92); the concentration of the aqueous ammonia solution is 1% (aqueous ammonia: water: 1:99) to 3% (aqueous ammonia: water: 3: 97).

The centrifugation speed in the step (1) is 5000r/min, and the centrifugation time is 3-7 min.

The concentration of the glacial acetic acid acetonitrile solution in the step (2) is 5% (glacial acetic acid: acetonitrile: 5:95) -9% (glacial acetic acid: acetonitrile: 9: 91).

The extraction column in the step (2) is a MAX solid phase extraction column.

And (3) the deviation between the retention time of the substance to be detected in the sample and the retention time of the standard solution in the qualitative analysis in the step (3) is not more than +/-3% of the retention time of the standard solution.

And (3) in the step (3), the matrix standard working solution is adopted for quantitative determination to draw a standard curve, and the response value of the polyoxin in the test sample is ensured to be in the linear range of the instrument.

The calculation formula in the step (3) is as follows:

in the formula: c: the concentration of the corresponding polyoxin in the sample is mu g/mL; v: the volume is determined by the unit of mL; f: dilution times; m: sample mass in g.

The absolute difference between two independent measurements obtained under repeated conditions must not exceed 15% of the arithmetic mean.

Advantageous effects

The method detects whether the plant source feed contains the polyoxin or not by adopting the steps of pretreatment, extraction and purification of materials, qualitative analysis and determination, quantitative determination, result calculation and verification and the like, has the advantages of high identification accuracy and accurate positioning, is representative in the polyoxin detection in China, and has good application prospect.

Drawings

FIG. 1 is a 0.1mg/L polyoxin chromatogram.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

Example 1

Taking plant-derived feeds of Hebei, Shandong, Fujian and the like:

(1) pretreating the material; taking 500g of a representative feed sample, reducing by at least 100g by a quartering method, grinding, sieving with a sample sieve with the aperture of 0.9mm, uniformly mixing, putting into a closed container, and storing in a dark place at a low temperature for later use;

(2) extraction and purification

Extraction: weighing 2.5g of sample, placing the sample into a 50.0mL centrifuge tube, adding 20mL of 4% formic acid acetonitrile solution, uniformly mixing by vortex for 1min, placing the mixture in an ultrasonic extraction device for 30min, then centrifuging the mixture for 5min at 5000r/min, taking 8mL of supernatant, blowing the supernatant to be nearly dry by a nitrogen instrument at 50 ℃, dissolving the extracted sample by 1mL of 2.5% ammonia water solution, and vortex for 30s to be purified;

purifying: pre-washing the mixed cation solid-phase extraction column with 3.0mL of acetonitrile and water in sequence, transferring the sample to be purified into the column, discarding the sample solution, eluting with 10mL of 6% glacial acetic acid acetonitrile water solution, collecting the eluent in a 20mL glass test tube, drying by using a nitrogen instrument, and performing constant volume measurement with 1mL of acetonitrile water solution to obtain the sample to be detected.

(3) And (3) qualitative analysis and determination: liquid chromatography-tandem mass spectrometry is adopted for determination:

chromatographic and mass spectral conditions:

a chromatographic column: hypersil GOLD Amino column, 30mm × 3.0mm (i.d.), 1.9 μm or equivalent size chromatographic column;

the mobile phase and gradient elution conditions are shown in table 1:

TABLE 1 mobile phase and gradient elution conditions

Time/min	Flow rate/(mL/min)	Mobile phase A (1% formic acid)/%)	Mobile phase B (methanol)/%)
				0	0.25	15	85
0.5	0.25	15	85
				1.0	0.25	50	50
4.0	0.25	50	50
				5.0	0.25	15	85
6.0	0.25	15	85

Column temperature: 40 ℃;

sample introduction volume: 5 mu L of the solution;

an ion source: ESI;

the scanning mode is as follows: scanning negative ions;

the detection mode is as follows: monitoring multiple reactions;

electrospray voltage: 4500V;

flow rate of atomizing gas: 15.0L/min;

flow rate of drying gas: 3.0L/min;

heating module temperature: 400 ℃;

temperature of DL tube: at 250 ℃ to obtain a mixture.

The ionization mode, qualitative ion pair, quantitative ion pair, retention time and collision energy reference values are shown in table 2:

TABLE 2 ionization mode, qualitative ion pair, quantitative ion pair, retention time and reference value of collision energy

Under the same experiment conditions, the deviation between the retention time of the substance to be detected in the sample and the retention time of the standard solution is not more than +/-3% of the retention time of the standard solution, the relative abundance of the qualitative ions of each component in the sample is compared with the relative abundance of the corresponding qualitative ions in the standard solution with the mass concentration close to the mass concentration, and the deviation is not more than the range specified in the table 3, so that the corresponding substance to be detected exists in the sample can be judged.

Table 3 maximum allowable deviation (%) of relative ion abundance in qualitative confirmation

Relative ion abundance	＞50	20～50	10～20	<10
					Allowable relative deviation	±20	±25	±30	±50

(4) Quantitative determination

The liquid chromatogram-tandem mass spectrum in the standard is quantitatively measured by an external standard-standard curve method. In order to reduce the influence of the matrix on the quantification, the standard solution for quantification is used for drawing a standard curve by adopting a matrix standard working solution, and the response value of the polyoxin in the test sample is ensured to be in the linear range of the instrument. The multi-reaction monitoring pattern of the standard solution of polyoxin under the chromatographic and mass spectrometric conditions is shown in figure 1.

(5) Result calculation verification

The polyoxin content (X) in the sample is expressed in mass fraction (mg/kg) and is calculated by the following formula:

in the formula: c: the concentration of the corresponding polyoxin B in the sample solution is in micrograms per milliliter (μ g/mL); v: volume fixed in milliliters (mL); f: dilution times; m: sample mass in grams (g).

The absolute difference between two independent measurements obtained under repeated conditions must not exceed 15% of the arithmetic mean.

And (5) determining the content of the polyoxin in the detected plant source feed through the detection of the steps (1) to (5).

The results are as follows:

Claims (8)

1. a method for detecting a plant-derived feed polyoxin comprises the following steps:

(1) taking a plant-derived feed sample, grinding and sieving; then adding a formic acid acetonitrile solution, uniformly mixing, performing ultrasonic extraction, centrifuging, blow-drying, and dissolving by using an ammonia water solution to obtain a sample to be purified;

(2) pre-washing an extraction column with acetonitrile and water in sequence, transferring the sample to be purified into the extraction column, removing a sample loading solution, eluting with a glacial acetic acid acetonitrile water solution, collecting an eluent, drying, and fixing the volume; wherein the extraction column is an MAX solid phase extraction column;

(3) performing qualitative analysis and quantitative determination by adopting liquid chromatography-tandem mass spectrometry, and finally determining the content of the polyoxin in the sample by calculation; the chromatographic and mass spectrometric conditions were:

a chromatographic column: HypersilGOLD Amino column;

column temperature: 40 ℃;

sample introduction volume: 5 mu L of the solution;

an ion source: ESI;

the scanning mode is as follows: scanning negative ions;

the detection mode is as follows: monitoring multiple reactions;

electrospray voltage: 4500V;

flow rate of atomizing gas: 15.0L/min;

flow rate of drying gas: 3.0L/min;

heating module temperature: 400 ℃;

temperature of DL tube: 250 ℃;

the mobile phase and gradient elution conditions were as follows:

time/min Flow rate/(mL/min) Mobile phase A (1% formic acid)/%) Mobile phase B (methanol)/%) 0 0.25 15 85 0.5 0.25 15 85 1.0 0.25 50 50 4.0 0.25 50 50 5.0 0.25 15 85 6.0 0.25 15 85

。

2. The method for detecting polyoxin in plant-derived feed according to claim 1, wherein the method comprises the following steps: the concentration of the formic acid acetonitrile solution in the step (1) is 3% -8%; the concentration of the ammonia solution is 1-3%.

3. The method for detecting polyoxin in plant-derived feed according to claim 1, wherein the method comprises the following steps: the centrifugation speed in the step (1) is 5000r/min, and the centrifugation time is 5-6 min.

4. The method for detecting polyoxin in plant-derived feed according to claim 1, wherein the method comprises the following steps: the concentration of the glacial acetic acid acetonitrile solution in the step (2) is 5% -9%.

5. The method for detecting polyoxin in plant-derived feed according to claim 1, wherein the method comprises the following steps: and (3) the deviation between the retention time of the substance to be detected in the sample and the retention time of the standard solution in the qualitative analysis in the step (3) is not more than +/-3% of the retention time of the standard solution.

6. The method for detecting polyoxin in plant-derived feed according to claim 1, wherein the method comprises the following steps: and (3) in the step (3), the matrix standard working solution is adopted for quantitative determination to draw a standard curve, and the response value of the polyoxin in the test sample is ensured to be in the linear range of the instrument.

7. The method for detecting polyoxin in plant-derived feed according to claim 1, wherein the method comprises the following steps: the calculation formula in the step (3) is as follows:

in the formula: c: the concentration of the corresponding polyoxin in the sample is mu g/mL; v: the volume is determined by the unit of mL; f: dilution times; m: sample mass in g.

8. The method for detecting polyoxin in plant-derived feed according to claim 1, wherein the method comprises the following steps: the absolute difference between two independent measurements obtained under repeated conditions must not exceed 15% of the arithmetic mean.

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