阅读说明：本技术 一种蔬菜中灭蝇胺残留量的快速检测方法 (Method for rapidly detecting residual quantity of cyromazine in vegetables ) 是由 成艳 彭桂芝 刘祥燕 张廷翠 曹慧艳 郭晓华 郑萌萌 秦瑶 张永勤 于 2019-11-27 设计创作，主要内容包括：本发明公开了一种蔬菜中灭蝇胺的快速检测方法,属于农药检测技术领域。检测方法包括如下步骤：将蔬菜样品粉碎,充分混匀,准确称取蔬菜样品置于离心管中,加入2%乙酸乙腈,使用均质器进行均质且边均质边加入无水Na<Sub>2</Sub>SO<Sub>4</Sub>,NaCL,之后进行离心,离心后取上清液1mL用于净化。净化结束后将处理好的样品于高效液相色谱-质谱仪进行定性和定量分析,得到蔬菜中灭蝇胺的含量。本发明的有益效果在于：本发明检测方法中灭蝇胺的色谱峰响应良好,2.37分钟就能够出峰,仪器响应较高,从而有效的节省了进样的时间,提高了灭蝇胺的检测效率。本发明检测方法中,灭蝇胺的检测的准确率明显提高,样品的加标回收率在90.01%～104.81%之间,相对标准偏差在0.50%～3.49%之间。(The invention discloses a rapid detection method of cyromazine in vegetables, and belongs to the technical field of pesticide detection. The detection method comprises the following steps: crushing a vegetable sample, fully and uniformly mixing, accurately weighing the vegetable sample, placing the vegetable sample in a centrifuge tube, adding 2% acetic acid acetonitrile, homogenizing by using a homogenizer, and adding anhydrous Na while homogenizing 2 SO 4 NaCL, followed by centrifugation, after which 1mL of supernatant was taken for clarification. And after purification, qualitatively and quantitatively analyzing the treated sample in a high performance liquid chromatography-mass spectrometer to obtain the content of cyromazine in the vegetables. The invention has the beneficial effects that: the chromatographic peak response of the cyromazine in the detection method is good, the cyromazine can generate peaks in 2.37 minutes, and the response of an instrument is higher, so that the sample introduction time is effectively saved, and the detection efficiency of the cyromazine is improved. In the detection method, the detection accuracy of cyromazine is obviously improved, and the standard recovery rate of the sample is 90.01-104.81 percent and the relative standard deviation is between 0.50 percent and 3.49 percent.)

1. A method for rapidly detecting residual quantity of cyromazine in vegetables is characterized by comprising the following steps:

step 1: preparation of Standard solutions

(1) Preparation of a cyromazine standard stock solution (100. mu.g/mL): accurately weighing a proper amount of cyromazine standard substance in a 10mL volumetric flask, dissolving the cyromazine standard substance with acetonitrile, fixing the volume to a scale, and storing the solution at 4 ℃ in a dark place;

(2) preparation of a standard intermediate solution of cyromazine (10. mu.g/mL): accurately sucking 1mL of cyromazine standard stock solution (100 mu g/mL) into a 10mL volumetric flask, dissolving the cyromazine standard stock solution with acetonitrile, and fixing the volume to a scale;

(3) preparing a standard working solution of cyromazine: preparing standard intermediate solution (10 mug/mL) of cyromazine with blank matrix extracting solution into standard solutions with the concentrations of 2ng/mL, 4ng/mL, 10ng/mL, 20ng/mL and 50ng/mL respectively for later use, and drawing a calibration curve;

step 2: sample pretreatment:

(1) sample extraction

Crushing a vegetable sample, fully and uniformly mixing, accurately weighing the vegetable sample, placing the vegetable sample in a centrifuge tube, adding 2% acetic acid acetonitrile, homogenizing by using a homogenizer, and adding anhydrous Na while homogenizing₂SO₄And NaCL, then centrifuging, and taking 1mL of supernatant for purification after centrifuging;

(2) sample purification

A. Placing the strong cation exchange column in a solid phase extraction device, pre-washing and activating by using 5mL of methanol, balancing by using 5mL of water, and immediately transferring 1mL of supernatant into an SCX column when a solvent reaches the surface of an adsorption layer of the column;

B. washing the strong cation exchange column with 6mL each of 2% formic acid water and methanol in turn, discarding effluent liquid and draining the small column;

C. eluting strong cation exchange column with 6mL ammonia water methanol (5+95) for 3 times, collecting in round bottom flask, concentrating to 0.5mL below 40 deg.C on rotary evaporator, blowing dry with nitrogen, diluting to 1mL with acetonitrile, passing through 0.22 μm organic filter membrane, and determining with high performance liquid chromatography-mass spectrometer;

(3) sample detection

Performing qualitative and quantitative analysis on the treated sample by using a high performance liquid chromatography-mass spectrometer to calculate the content of cyromazine in the vegetables;

sampling 5.0 mu L of sample solution to be detected, and analyzing and detecting by using a high performance liquid chromatography-tandem mass spectrometer to obtain a total ion flow graph, a quantitative ion chromatogram and a qualitative ion pair relative abundance chromatogram of the sample solution to be detected;

analyzing and measuring the cyromazine standard working solution prepared in the step 1 by using a high performance liquid chromatography-tandem mass spectrometer to obtain a total ion flow diagram, a quantitative ion chromatogram and a qualitative ion pair relative abundance chromatogram of the standard solution;

analyzing the retention time of the cyromazine, a qualitative ion pair (m/z)167/108 and a quantitative ion pair (m/z)167/85, and preparing a cyromazine standard curve by chromatographic peaks of the qualitative ion pair and the quantitative ion pair;

according to chromatographic peaks of qualitative ion pairs and quantitative ion pairs of cyromazine in a sample solution to be detected, combining a standard curve, calculating to obtain the concentration Ci of pesticide residue in the sample solution to be detected, and calculating to obtain the content Xi of cyromazine in vegetables according to the following formula, wherein the content calculation formula is as follows:

in the formula:

xi- -residual amount of cyromazine in milligrams per kilogram (mg/kg) in the sample;

ci- - -solution concentration of the component to be measured in nanograms per milliliter (ng/mL) from the standard curve;

v- -volume of sample solution in milliliters (mL);

m- -the mass of the sample represented by the sample solution in grams (g);

the liquid chromatogram conditions of the high performance liquid chromatogram-mass spectrometer are as follows: waters BEH hilc (1.7um, 2.1X 100 mm); sample introduction volume is 5 uL; mobile phase A: 0.2% formic acid acetonitrile, mobile phase B: 0.2% formic acid water; flow rate: 0.3 mL/min; gradient: 0min: a: B =95:5,2 min: a: B =75:25, 4-5 min: a: b =50:50, 5.1 min: a: B =95: 5;

the MS/MS mass spectrum conditions of the high performance liquid chromatography-mass spectrum/mass spectrometer are as follows: an ion source: an electrospray ion source; ion source temperature: 400 ℃;

an ionization mode: ESI +; drying gas: the temperature is 330 ℃, and the flow rate is 9L/min; sheath gas: the temperature is 350 ℃, and the flow rate is 12L/min; atomizing gas pressure: 36 psi; capillary voltage: 4500V, scanning mode: multiple Reaction Monitoring (MRM).

2. The assay of claim 1, wherein the vegetable sample comprises: spinach, cabbage, green sword bean, okra and taro seed.

3. The detection method according to claim 1, wherein the weight of the vegetable sample/the amount of acetonitrile acetate is 5g/25 ml; the vegetable sample comprises the following components in percentage by mass: anhydrous Na₂SO₄: the NaCL is 5:5: 2.

4. The detection method according to claim 1, wherein the rotation speed of the homogenizer in the step 2 is 8000 r/min.

5. The detection method according to claim 1, wherein the centrifugation in step 2 is carried out under 4000r/min for 5 min.

6. The detection method according to claim 1, wherein the detection limit of the detection method is 0.010 mg/kg.

Technical Field

The invention relates to the technical field of pesticide detection, in particular to a rapid detection method of cyromazine in vegetables.

Background

Cyromazine, also known as Cyromazine, is a triazine compound with a chemical name of N-cyclopropyl-1, 3, 5-triazine-2, 4, 6-triamine, is a triazine compound, has a regulating effect on insect growth, belongs to a novel high-efficiency, low-toxicity and nitrogen-containing heterocyclic pesticide, has special physiological activity on controlling dipteran insect larvae and pupae, has a systemic conduction effect, can interfere ecdysis and pupation, and causes the morphological distortion of the dipteran insect larvae and the pupae to inhibit or incomplete adult eclosion, thereby achieving the purposes of controlling excessive dipteran insect breeding and reducing the number of insects, and being an ecological pesticide with better effect on controlling dipteran insect pests at present.

At present, the cyromazine residues in vegetables are very serious in China, and the detection standard of the cyromazine in the existing vegetables is NY/T1725-one 2009 high performance liquid chromatography. The standard is that cyromazine in a sample is extracted by ammonium acetate-acetonitrile mixed solution, pi electrons on a six-membered ring at 215nm are excited, and the pi electrons are detected by an ultraviolet detector. And (4) according to the retention time of the standard substance peak, performing qualitative determination and performing quantitative determination by an external standard method. The instrument used in the method has low detection sensitivity, can only quantify according to the peak-out time, and has long sample introduction time, so the detection effect of the cyromazine residue is not ideal, the recovery rate is low, and the detection speed is slow. Therefore, a new method for detecting cyromazine in vegetables with accurate detection effect and high detection speed is urgently needed at present.

Disclosure of Invention

The invention aims to provide a detection method for cyromazine in vegetables, which has good detection effect and high detection speed.

In order to achieve the purpose, the invention provides the following technical scheme:

a method for rapidly detecting residual quantity of cyromazine in vegetables comprises the following steps:

step 1: preparation of Standard solutions

(1) Preparation of a cyromazine standard stock solution (100. mu.g/mL): accurately weighing a proper amount of cyromazine standard substance in a 10mL volumetric flask, dissolving the cyromazine standard substance with acetonitrile, fixing the volume to a scale, and storing the cyromazine standard substance at 4 ℃ in a dark place.

(2) Preparation of a standard intermediate solution of cyromazine (10. mu.g/mL): accurately sucking 1mL of cyromazine standard stock solution (100 mu g/mL) into a 10mL volumetric flask, dissolving the cyromazine standard stock solution with acetonitrile, and fixing the volume to a scale.

(3) Preparing a standard working solution of cyromazine: the cyromazine standard intermediate solution (10 mug/mL) is prepared into standard solutions with the concentrations of 2ng/mL, 4ng/mL, 10ng/mL, 20ng/mL and 50ng/mL respectively by using a blank matrix extracting solution for later use, and is used for drawing a calibration curve.

Step 2: sample pretreatment:

(1) sample extraction

Crushing a vegetable sample, fully and uniformly mixing, accurately weighing the vegetable sample, placing the vegetable sample in a centrifuge tube, adding 2% acetic acid acetonitrile, homogenizing by using a homogenizer, and adding anhydrous Na while homogenizing₂SO₄NaCL, followed by centrifugation, after which 1mL of supernatant was taken for clarification.

(2) Sample purification

A. The strong cation exchange column is placed in a solid phase extraction device, pre-washing and activation are carried out by using 5mL of methanol, then 5mL of water is used for balancing, and when the solvent reaches the surface of the adsorption layer of the column, 1mL of supernatant is immediately transferred to the SCX column.

B. The strong cation exchange column was washed with 6mL each of 2% formic acid water and methanol in sequence, the effluent was discarded and the column was drained.

C. The strong cation exchange column was eluted 3 times with 6mL of aqueous ammonia methanol (5+95) and collected in a round bottom flask. Concentrating to 0.5mL below 40 deg.C on a rotary evaporator, blowing dry with nitrogen, diluting to 1mL with acetonitrile, filtering with 0.22 μm organic filter membrane, and measuring with HPLC-MS/MS.

(3) Sample detection

And (3) carrying out qualitative and quantitative analysis on the treated sample by using a high performance liquid chromatography-mass spectrometer to calculate the content of the cyromazine in the vegetables.

A. Sampling 5.0 mu L of sample solution to be detected, and analyzing and detecting by using a high performance liquid chromatography-tandem mass spectrometer to obtain a total ion flow graph, a quantitative ion chromatogram and a qualitative ion pair relative abundance chromatogram of the sample solution to be detected;

B. analyzing and measuring the cyromazine standard working solution prepared in the step 1 by using a high performance liquid chromatography-tandem mass spectrometer to obtain a total ion flow diagram, a quantitative ion chromatogram and a qualitative ion pair relative abundance chromatogram of the standard solution;

C. and analyzing the retention time of the cyromazine, a qualitative ion pair (m/z)167/108 and a quantitative ion pair (m/z)167/85, and preparing chromatographic peaks of the qualitative ion pair and the quantitative ion pair to obtain a cyromazine standard curve.

D. According to chromatographic peaks of qualitative ion pairs and quantitative ion pairs of cyromazine in a sample solution to be detected, combining a standard curve, calculating to obtain the concentration Ci of pesticide residue in the sample solution to be detected, and calculating to obtain the content Xi of cyromazine in vegetables according to the following formula, wherein the content calculation formula is as follows:

in the formula:

xi- -residual amount of cyromazine in milligrams per kilogram (mg/kg) in the sample;

ci- - -solution concentration of the component to be measured in nanograms per milliliter (ng/mL) from the standard curve;

v- -volume of sample solution in milliliters (mL);

m- -the mass of the sample represented by the sample solution in grams (g).

The liquid chromatogram conditions of the high performance liquid chromatogram-mass spectrometer are as follows: waters BEH hilc (1.7um, 2.1X 100 mm); sample introduction volume is 5 uL; mobile phase A: 0.2% formic acid acetonitrile, mobile phase B: 0.2% formic acid water; flow rate: 0.3 mL/min; gradient: 0min, a: B: 95:5,2 min: a, B is 75:25, 4-5 min: a: b50: 50,5.1 min: and A and B are 95: 5.

The MS/MS mass spectrum conditions of the high performance liquid chromatography-mass spectrum/mass spectrometer are as follows: an ion source: an electrospray ion source; ion source temperature: at 400 ℃. An ionization mode: ESI +; drying gas: the temperature is 330 ℃, and the flow rate is 9L/min; sheath gas: the temperature is 350 ℃, and the flow rate is 12L/min; atomizing gas pressure: 36 psi; capillary voltage: 4500V, scanning mode: multiple Reaction Monitoring (MRM).

Preferably, the vegetable sample comprises: spinach, cabbage, green sword bean, okra and taro seed.

Preferably, the weight of the vegetable sample/the amount of acetonitrile acetate used is 5g/25 ml; the vegetable sample comprises: anhydrous Na2SO 4: the NaCL was 5g:5g:2 g.

Preferably, the rotating speed of the homogenizer in the step 2 is 8000 r/min.

Preferably, the centrifugation condition in the step 2 is 4000r/min for 5 min.

Preferably, the detection limit of the detection method is 0.010 mg/kg.

The invention has the beneficial effects that:

1. compared with the prior art, the detection method provided by the invention effectively saves the sample injection time, realizes the rapid detection of the cyromazine in the vegetables, and improves the detection efficiency of the cyromazine.

2. In the detection method, the detection accuracy of the cyromazine is obviously improved, the detection limit of the cyromazine is 0.010mg/kg, the standard addition recovery rate is 90.01-104.81%, and the relative standard deviation is 0.50-3.49%.

3. The detection method disclosed by the invention dilutes the sample through pretreatment, so that the pollution of the sample to an instrument is reduced.

4. The detection method disclosed by the invention is simple to operate and good in stability, and has excellent standard-adding recovery rates in spinach, cabbage, green sword bean, okra and taro seeds, and the standard-adding recovery rate result is stable.

5. The invention adopts blank matrix to prepare the standard working solution, and solves the problem that the recovery rate is influenced by over-low on-machine data of the sample due to large inhibition effect of the matrix of the sample.

6. The invention adopts a strong cation exchange column (SCX) to purify a sample, and solves the problems that the prior purifying column is commonly used: c18 solid phase extraction column, amino solid phase extraction column (NH)₂) And graphitized carbon black pillars (Carb) and the like have serious interception and adsorption problems on cyromazine.

Drawings

FIG. 1 is a graph of the calibration curve for cyromazine

FIG. 2 is a total ion flow diagram of a cyromazine standard substance

FIG. 3 is a quantitative ion chromatogram of cyromazine

FIG. 4 is chromatogram of relative abundance of cyromazine qualitative ion

Detailed Description

In order to clearly illustrate the technical features of the present solution, the present solution is explained below by way of specific embodiments.

The reagents and materials used in the invention are:

acetonitrile (chromatographically pure); methanol (chromatographically pure); ammonia, acetic acid, formic acid (guaranteed purity); 2% acetonitrile acetate: measuring 20mL of acetic acid into a 1L volumetric flask, and metering the volume to the scale with acetonitrile; 2% formic acid water: measuring 2mL of formic acid in a 100mL volumetric flask, and metering the volume to a scale with water; ammonia methanol (5+ 95): sucking 5mL of ammonia water into a 100mL volumetric flask, and metering the volume to the scale with methanol; cyromazine standard (CAS number: 66215-27-8): the purity is more than or equal to 98 percent; strong cation exchange column (SCX): 500mg/3mL, SEPUKE.

The instrument used in the invention is as follows:

agilent1290/6460 high performance liquid chromatography-tandem mass spectrometer with ESI source; waters BEH hilc chromatography column (1.7um, 2.1X 100 mm); a food processor; analytical balance: 0.01g, 0,1 mg; a homogenizer: 6000-36000 r/min; a rotary evaporator; a solid phase extraction device.