阅读说明：本技术 一种测定植物性食品中唑嘧菌胺残留量的液相色谱串联质谱法 (Liquid chromatography-tandem mass spectrometry method for determining residual quantity of ametoctradin in plant food ) 是由 熊敏 于 2019-08-30 设计创作，主要内容包括：本发明属于食品安全技术领域,涉及一种测定植物性食品中唑嘧菌胺残留量的液相色谱串联质谱法。称取均匀样品,以甲醇均质提取,无水硫酸镁和PSA净化,高速冷冻离心后,上清液旋蒸至近干,80％乙腈溶解残渣,过Oasis PRiME HLB柱净化,经0.22μm滤膜过滤,经液相色谱串联质谱检测,外标法定量。本发明所述检测方法前处理步骤简单新颖,除杂效果好,方法的灵敏度、回收率高,测定结果的精密度好,可以有效的检测植物性食品中唑嘧菌胺残留量。(The invention belongs to the technical field of food safety, and relates to a liquid chromatography tandem mass spectrometry method for determining ametoctradin residue in plant food. Weighing a uniform sample, carrying out homogeneous extraction by methanol, purifying by anhydrous magnesium sulfate and PSA, carrying out high-speed freezing and centrifugation, carrying out rotary evaporation on a supernatant until the supernatant is nearly dry, dissolving residues by 80% acetonitrile, purifying by an Oasis PRIME HLB column, filtering by a 0.22 mu m filter membrane, detecting by liquid chromatography-tandem mass spectrometry, and quantifying by an external standard method. The detection method disclosed by the invention has the advantages of simple and novel pretreatment steps, good impurity removal effect, high sensitivity and recovery rate of the method, and good precision of the measurement result, and can be used for effectively detecting the residual quantity of ametoctradin in plant food.)

1. A liquid chromatography tandem mass spectrometry method for determining ametoctradin residual quantity in plant food is characterized by comprising the following steps:

(1) extraction:

weighing 10g of a uniform sample in a 50ml centrifuge tube, adding 30ml of methanol, carrying out homogeneous extraction, carrying out refrigerated centrifugation at 8000r/min, taking supernatant, repeatedly extracting residues once with 20ml of methanol, combining the supernatant, adding 6g of anhydrous magnesium sulfate and 150mg of N-propyl ethylenediamine solid-phase adsorbent, shaking, transferring the supernatant to a rotary evaporation bottle, carrying out rotary evaporation at 40 ℃ till the mixture is nearly dry, redissolving an 80% acetonitrile solution, and fixing the volume to 5ml to obtain a solution to be purified;

(2) purifying:

purifying the liquid to be purified by an Oasis PRIME HLB column; keeping the sample loading process at 1 drop/second, collecting effluent, filtering with 0.22 μm filter membrane to obtain sample solution, and performing liquid chromatography tandem mass spectrometry;

(3) and (3) determination:

the standard solution and the sample solution were measured under the following conditions of liquid chromatography tandem mass spectrometry:

a. chromatographic conditions are as follows:

a chromatographic column: c18, 1.7 μm, 2.1mm × 50 mm;

mobile phase: the phase A is 0.1 percent formic acid solution and the phase B is 0.1 percent formic acid acetonitrile solution, and the gradient elution is carried out by the procedure of initial mobile phase proportion and 0.1 percent formic acid water solution; 0.1% formic acid acetonitrile solution 95: 5; 2min, 0.1% aqueous formic acid: 0.1% formic acid acetonitrile solution 95: 5; 5.5min, 0.1% aqueous formic acid: 0.1% formic acid acetonitrile solution 0: 100; 6min, 0.1% aqueous formic acid: 0.1% formic acid acetonitrile solution 0: 100; 7min, 0.1% aqueous formic acid: 0.1% formic acid acetonitrile solution 95: 5; 7.5min, 0.1% aqueous formic acid: 0.1% formic acid acetonitrile solution 95: 5;

flow rate: 0.5 mL/min;

sample introduction amount: 5.0 mu L;

column temperature: 40 ℃;

b. mass spectrum conditions:

an ion source: an ESI electrospray ion source;

the scanning mode is as follows: scanning positive ions;

the detection mode is as follows: monitoring MRM multiple reactions;

ion source temperature: 120 ℃;

the temperature of the desolvation: at 450 ℃;

the parent ion was 276.4, the quantitative ion was 149.4, and the qualitative ion was 176.3;

the cone voltage is 50V, the quantitative ion collision energy is 35eV, and the qualitative ion pair collision energy is 40 eV.

Technical Field

The invention belongs to the technical field of food safety, and particularly relates to a method for determining ametoctradin residual quantity in plant food by liquid chromatography-tandem mass spectrometry.

Technical Field

The ametoctradin is a triazolopyrimidine bactericide, belongs to a mitochondrial respiration inhibitor, is a high-selectivity bactericide, has a control effect on downy mildew and phytophthora oomycetes fungi, has extremely strong residual activity and rain resistance, can be redistributed in leaves, protects the healthy growth of crops, and fully exerts the growth potential. At the beginning of 2010, the ametoctradin is registered in Romania and is mainly used for preventing late blight and downy mildew on grapes; 7 months 2010, approved registration in the uk; in the same year, registration was made in the netherlands; in 2011, the ametoctradin product Initium comes on the market; 2012, registered in australia, canada, italy and the united states, for fruit trees, vegetables, hops, grapes, potatoes and ornamental plants; on 1/8.2013, ametoctradin was listed on the registered active ingredient list of the european union pesticide registration regulation for grapes, vegetables and potatoes; 8, 6 months and 2013, 98 percent of ametoctradin raw pesticide is temporarily registered in China; to date, ametoctradin has been registered in more than 50 countries worldwide. Including grapes, potatoes, tomatoes, lettuce and other vegetables. Ametoctradin has a unique mechanism of action, which makes it the only existing fungicide in this category, and therefore has no cross-resistance with other commercial fungicides, which makes ametoctradin an ideal tool for fungal resistance management on specialty crops.

The temporary limit of the ametoctradin is formulated in GB 2763-2016 maximum limit of pesticide residues in food in China, and the temporary limit of the ametoctradin in grapes, cucumbers and potatoes is respectively 2mg/kg, 1mg/kg and 0.05 mg/kg. However, no corresponding detection method is formulated at present.

The method for detecting the residual quantity of the ametoctradin serving as a vegetable food is developed, can meet the urgent requirements of production, sale and supervision and spot inspection of agricultural products on the detection of the ametoctradin, and has a wide market prospect of 49 new pesticides which are globally registered or marketed in 2016. The boscalid-simethiprole compound pesticide is applied to vegetables.

At present, the detection research on the ametoctradin focuses on the analysis of pesticide components and the content of the ametoctradin in environmental soil, and the content is high, so that the detection analysis is mostly carried out by adopting a liquid chromatography. For example, the study on the dimethomorph-ametoctradin suspension agent high performance liquid chromatography analysis method published by 2010 of Jiangyifei et al in pesticide science and administration, the study on the detection method on the ametoctradin residue in soil ultra performance liquid chromatography published by 2012 of Guozjing et al in pesticide science and administration, and the like.

The current research on the detection of the residual quantity of ametoctradin in plant food is less. Liu Lei et al 2017, "food safety quality inspection academy" published "ultra-high performance liquid chromatography-tandem mass spectrometry for measuring dimethomorph and ametoctradin in pepper and cultivation soil thereof, samples of the pepper and the soil are extracted by acetonitrile and water, and after salting out, supernatant is taken for testing, and the detection limit in the pepper and the soil can reach 10 mu g/kg. However, the method is simple, liquid quality is tested after extraction and salting-out, and the method is not suitable for various vegetable foods with complex matrixes and has a small application range.

"determination of amplitude in residual in front of and in front of tables by modified liquid, easy, chemical, effective, regular, and safe method using excellent chromatography/chromatography in" published by Mingfeng Hu et al 2015 in Food Chemistry ". The ametoctradin is mainly detected aiming at vegetable and fruit substrates. According to different fruits and vegetables such as apples and cucumbers, different QuChers fillers are tried to treat samples to remove interference substances such as pigments and sugar in the samples. However, the cleaning effect is not ideal for other samples, such as grain rape samples with high oil content and tea samples with substrate charge.

A.S. Komarova et al, 2017, published in Journal of Analytical Chemistry, "Determination of acetone in plant reagents and environmental samples by HPLC with an UV detector", soil and some plant foods are extracted with acetone aqueous solution, purified by solid phase extraction, detected by a liquid chromatography ultraviolet detector, and the detection limit can reach 5-10 mug/kg. The method adopts solid phase extraction and purification, and has wide application range and low detection limit. However, false positive results are likely to occur when the detection is performed by a liquid chromatography ultraviolet detector. The traditional solid phase extraction column has complicated extraction steps and long time consumption.

Therefore, the detection method for determining the residual quantity of ametoctradin in the plant food, which is disclosed by the invention, has the advantages of simple and novel pretreatment steps, good purification effect and high recovery rate, is necessary.

Disclosure of Invention

The invention aims to solve the technical problem of being suitable for a method for detecting the residual quantity of the ametoctradin in the plant food, effectively extracting the residual ametoctradin in the plant food and well removing interferents. The detection limit can meet the requirement of national standard on the maximum residue limit of ametoctradin in brown rice, paddy and apples.

The technical scheme of the invention is realized by the following steps:

(1) extraction:

weighing 10g of a uniform sample in a 50ml centrifuge tube, adding 30ml of methanol, carrying out homogeneous extraction, carrying out refrigerated centrifugation at 8000r/min, taking supernatant, repeatedly extracting residues once with 20ml of methanol, combining the supernatant, adding 6g of anhydrous magnesium sulfate and 150mg of N-propyl ethylenediamine solid-phase adsorbent, shaking, transferring the supernatant to a rotary evaporation bottle, carrying out rotary evaporation at 40 ℃ till the mixture is nearly dry, redissolving an 80% acetonitrile solution, and fixing the volume to 5ml to obtain a solution to be purified;

(2) purifying:

purifying the liquid to be purified by an Oasis PRIME HLB column; keeping the sample loading process at 1 drop/second, collecting effluent, filtering with 0.22 μm filter membrane to obtain sample solution, and performing liquid chromatography tandem mass spectrometry;

(3) and (3) determination:

the standard solution and the sample treatment solution were measured under the following conditions of liquid chromatography tandem mass spectrometry:

a. chromatographic conditions are as follows:

a chromatographic column: c18, 1.7 μm, 2.1 mm. times.50 mm

Mobile phase: 0.1% formic acid solution (A) + 0.1% formic acid acetonitrile solution (B), gradient elution, procedure starting mobile phase ratio, 0.1% formic acid aqueous solution; 0.1% formic acid acetonitrile solution 95: 5; 2min, 0.1 percent formic acid water solution to 0.1 percent formic acid acetonitrile solution 95 to 5; 5.5min, 0.1% formic acid water solution to 0.1% formic acid acetonitrile solution 0: 100; 6min, 0.1 percent formic acid water solution to 0.1 percent formic acid acetonitrile solution 0: 100; 7min, 0.1 percent formic acid water solution to 0.1 percent formic acid acetonitrile solution 95: 5; 7.5min, 0.1% formic acid water solution to 0.1% formic acid acetonitrile solution 95: 5;

flow rate: 0.5 mL/min;

sample introduction amount: 5.0 mu L;

column temperature: 40 ℃;

b. mass spectrum conditions:

an ion source: an ESI electrospray ion source;

the scanning mode is as follows: scanning positive ions;

the detection mode is as follows: monitoring MRM multiple reactions;

ion source temperature: 120 ℃;

the temperature of the desolvation: 450 deg.C

The parent ion was 276.4, the quantitative ion was 149.4, and the qualitative ion was 176.3;

the cone voltage is 50V, the quantitative ion collision energy is 35eV, and the qualitative ion pair collision energy is 40 eV.

Drawings

FIG. 1 is a 10 mu g/L ametoctradin standard solution chromatogram

Detailed description of the invention

The invention will be further illustrated by the following examples. The examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, and the preferred methods and materials described herein are exemplary only.

Example 1

1. Instruments and reagents

High performance liquid chromatography-tandem mass spectrometer: waters, UPLC-Xevo TQ, USA;

standard substance: ametoctradin, 100 mg/L;

methanol: carrying out chromatographic purification;

acetonitrile: carrying out chromatographic purification;

formic acid: analytical purity

Anhydrous magnesium sulfate: analyzing and purifying;

Oasis PRiME HLB：6mL，200mg；

the water used in the method is first-grade water.

2. Conditions of instrumental analysis

a. Liquid chromatography conditions:

a chromatographic column: waters, BEH C18, 1.7 μm, 2.1 mm. times.50 mm

Mobile phase: 0.1% formic acid in water (A) + 0.1% formic acid in acetonitrile (B), gradient elution, procedure starting with mobile phase ratio, 0.1% formic acid in water; 0.1% formic acid acetonitrile solution 95: 5; 2min, 0.1 percent formic acid water solution to 0.1 percent formic acid acetonitrile solution 95 to 5; 5.5min, 0.1% formic acid water solution to 0.1% formic acid acetonitrile solution 0: 100; 6min, 0.1 percent formic acid water solution to 0.1 percent formic acid acetonitrile solution 0: 100; 7min, 0.1 percent formic acid water solution to 0.1 percent formic acid acetonitrile solution 95: 5; 7.5min, 0.1% formic acid water solution to 0.1% formic acid acetonitrile solution 95: 5;

flow rate: 0.3 mL/min;

sample introduction amount: 5.0 mu L;

column temperature: 40 ℃;

b. mass spectrum conditions:

an ion source: an ESI electrospray ion source;

the scanning mode is as follows: scanning positive ions;

the detection mode is as follows: monitoring MRM multiple reactions;

ion source temperature: 120 ℃;

the temperature of the desolvation: 450 deg.C

The parent ion was 276.4, the quantitative ion was 149.4, and the qualitative ion was 176.3;

the cone voltage is 50V, the quantitative ion collision energy is 35eV, and the qualitative ion pair collision energy is 40 eV.

3. Linear equation of equations

Preparing a standard stock solution: weighing 10mg of ametoctradin standard substance, dissolving with methanol, and metering to 10mL, wherein the concentration of the standard stock solution is 1000 mg/L.

Preparing a standard working solution by using the standard solution: the concentrations were 0.005mg/L, 0.010mg/L, 0.020mg/L, 0.100 mg/L and 0.200 mg/L. The measurement was carried out under the above-mentioned instrumental analysis conditions, and the quantification was carried out by external standard method. The results of linear regression using the peak area y as ordinate and the concentration x as abscissa are shown in Table 1

TABLE 1 Carbamil retention time, regression equation and linear correlation coefficient

4. Sample pretreatment method

(1) Extraction:

weighing 10g of a uniform sample in a 50ml centrifuge tube, adding 30ml of methanol, carrying out homogeneous extraction, carrying out refrigerated centrifugation at 8000r/min, taking supernatant, repeatedly extracting residues once with 20ml of methanol, combining the supernatant, adding 6g of anhydrous magnesium sulfate and 150mg of N-propyl ethylenediamine solid-phase adsorbent, shaking, transferring the supernatant to a rotary evaporation bottle, carrying out rotary evaporation at 40 ℃ till the mixture is nearly dry, redissolving an 80% acetonitrile solution, and fixing the volume to 5ml to obtain a solution to be purified;

(2) purifying:

purifying the liquid to be purified by an Oasis PRIME HLB column; keeping the sample loading process at 1 drop/second, collecting effluent, filtering with 0.22 μm filter membrane to obtain sample solution, and performing liquid chromatography tandem mass spectrometry;

5. sample assay

Weighing 6 parts of sample, processing the sample according to the pretreatment method described in 4, detecting according to the analysis conditions of the instrument described in 2, and performing qualitative determination, external standard method quantification on retention time and ion abundance ratio. The results of the experiment are shown in Table 2

TABLE 2 determination of ametoctradin in apple samples

6. Precision and recovery

The results of the precision and recovery test of the present method are shown in Table 3.

TABLE 3 recovery rate test results

7. Detection limit

The detection limit of the ametoctradin is 0.3 mu g/kg, and the quantification limit is 1 mu g/kg.