阅读说明：本技术 一种超高效液相色谱-串联质谱测定水产品中三嗪类除草剂残留的方法 (Method for determining triazine herbicide residue in aquatic product by ultra-high performance liquid chromatography-tandem mass spectrometry ) 是由 彭婕 何力 居小倩 甘金华 陈建武 于 2020-07-15 设计创作，主要内容包括：本发明公开了一种超高效液相色谱-串联质谱测定水产品中三嗪类除草剂残留的方法,包括如下步骤：1、配制混合标准溶液；2、UPLC分析检测；3、HESI-MS/MS分析检测；4、建立标准曲线；5、水产样品的前处理；6、待检测样品的检测。该方法简单,能快速准确检测水产品中三嗪类除草剂的残留量,基质干扰少,测定结果准确。(The invention discloses a method for determining triazine herbicide residue in an aquatic product by ultra-high performance liquid chromatography-tandem mass spectrometry, which comprises the following steps: 1. preparing a mixed standard solution; 2. UPLC analysis and detection; 3. HESI-MS/MS analysis detection; 4. establishing a standard curve; 5. pretreatment of aquatic product samples; 6. and (5) detecting the sample to be detected. The method is simple, can quickly and accurately detect the residual quantity of the triazine herbicide in the aquatic product, has less matrix interference and accurate determination result.)

1. A method for determining triazine herbicide residue in aquatic products by ultra-high performance liquid chromatography-tandem mass spectrometry is characterized by comprising the following steps

1.1, preparing a mixed standard solution:

preparing N parts of mixed standard solutions with gradient concentration, wherein the triazine herbicides in each part of mixed standard solution are the same in type, the triazine herbicides in each part of mixed standard solution are the same in concentration, and the internal standard atrazine-D is contained in each part of mixed solution₅Mixing the internal standard substance atrazine-D in the standard solution₅The concentrations of (A) were the same, and the internal standard substance atrazine-D₅In the gradient concentration range of each triazine herbicide;

1.2, UPLC analysis and detection:

and (3) respectively carrying out UPLC analysis and detection on each mixed standard solution, wherein the UPLC conditions are as follows:

the chromatographic column adopts C₁₈A chromatographic column, wherein a mobile phase adopts a mixed solution of an organic phase and a water phase, the organic phase is acetonitrile, the water phase is 0.1 wt% formic acid solution, the flow rate of the mobile phase is 0.3-0.4mL/min, and the mobile phase is eluted by adopting a gradient elution mode;

1.3, HESI-MS/MS analysis and detection:

the detection conditions of the HESI-MS/MS analysis are as follows:

the ion source is a heating atmospheric pressure electric spray source, positive ion mode scanning is carried out, a reaction monitoring mode is selected for determination, the spray voltage is 3000-: the purity of argon is more than or equal to 99.999, 2.0mTorr, the half-peak width of Q1 is 0.7, and the half-peak width of Q3 is 0.7;

After HESI-MS/MS analysis and detection, the peak area of the sub-ion of each triazine herbicide and the internal standard atrazine-D under each concentration can be obtained₅The ratio of the peak areas of the daughter ions;

1.4, establishing a standard curve:

taking the concentration of each triazine herbicide as an abscissa, and taking the peak area of the daughter ion of each triazine herbicide and an internal standard atrazine-D under each concentration₅The ratio of the peak areas of the daughter ions is a vertical coordinate, drawing is carried out according to the data detected by each triazine herbicide, and a standard curve of each triazine herbicide is obtained after fitting, so that a function relation formula of the standard curve of each triazine herbicide is obtained;

1.5, pretreatment of aquatic product samples:

taking homogeneous muscle sample of aquatic animal, adding atrazine-D as internal standard substance₅Adding acetonitrile, sodium chloride and anhydrous magnesium sulfate in sequence, vortex oscillating, centrifuging, collecting supernatant, repeatedly extracting and mixing the supernatants, evaporating the supernatant to dryness, and collecting residueDissolving by using a solvent, adding a purifying agent, wherein the purifying agent is a mixture of ethylenediamine-N-propyl silane, octadecylsilane bonded silica gel and EMR-Lipid powder, performing vortex oscillation and centrifugation, and filtering the obtained supernatant by using a microporous filter membrane to obtain a sample to be detected;

1.6, detection of a sample to be detected:

analyzing and detecting the sample to be detected according to the method of the step 1.2-1.3 to obtain the peak area of the sub-ion of each triazine herbicide and the internal standard atrazine-D₅The ratio of the peak area of the daughter ion is obtained by mixing the peak area of the daughter ion of each triazine herbicide with the peak area of the internal standard substance atrazine-D₅Substituting the ratio of the ionic peak areas into the functional relation of the standard curve of the corresponding triazine herbicide to obtain the concentration of the corresponding triazine herbicide.

2. The method for determining triazine herbicide residue in aquatic products by ultra-high performance liquid chromatography-tandem mass spectrometry as claimed in claim 1, wherein the method comprises the following steps: the chromatographic column has a column length of 100mm, an inner diameter of 2.1mm, a packed particle size of 1.7 μm and a column temperature of 30 ℃.

3. The method for determining triazine herbicide residue in aquatic products by ultra high performance liquid chromatography-tandem mass spectrometry as claimed in claim 1, wherein the gradient elution conditions are as follows:

the first stage is as follows: the elution time is 0-2min, wherein the volume percentage of the organic phase is 15-25%, and the volume percentage of the water phase is 75-85%;

and a second stage: the elution time is 3-5min, wherein the volume percentage of the organic phase is gradually increased from 15-25% to 90-95%, and the volume percentage of the aqueous phase is gradually decreased from 75-85% to 5-10%;

And a third stage: the elution time is 2-3min, wherein the volume percentage of the organic phase is 90-95%, and the volume percentage of the water phase is 5-10%;

a fourth stage: the elution time is 0.1-0.5min, wherein the volume percentage of the organic phase is gradually reduced from 90-95% to 15-25%, and the volume percentage of the aqueous phase is gradually increased from 5-10% to 75-85%;

the fifth stage: the elution time is 1.5-4min, wherein the volume percentage of the organic phase is 15-25%, and the volume percentage of the water phase is 75-85%.

4. The method for determining triazine herbicide residue in aquatic products by ultra-high performance liquid chromatography-tandem mass spectrometry as claimed in claim 1, wherein the method comprises the following steps: in step 1.5, the solvent is methanol or acetonitrile.

5. The method for determining triazine herbicide residue in aquatic products by ultra-high performance liquid chromatography-tandem mass spectrometry as claimed in claim 1, wherein the method comprises the following steps: the microporous filter membrane is an organic microporous filter membrane, and the pore diameter of the microporous filter membrane is not more than 0.22 mu m.

Technical Field

The invention relates to the technical field of physicochemical inspection of pesticide residues in aquatic products, in particular to a method for determining triazine herbicide residues in aquatic products by using ultra-high performance liquid chromatography-tandem mass spectrometry.

Background

Triazine herbicides (Triazine herbicides) are highly selective herbicides that act by inhibiting plant photosynthesis and have been used as early as 50 s in the 20 th century. The herbicide is widely applied to the control of various weeds, has large dosage, stable property and long lasting period, and has more and more attention to the harm to human health and environment. It has been reported that such compounds may cause human cancers and congenital defects, and at the same time interfere with the normal function of hormones, which have been included in the list of endocrine disrupter compounds in many countries of the world. At present, the detection of triazine herbicide residues is mainly focused on grains, vegetables and environmental samples, and relatively few methods are used for detecting the triazine herbicide residues in aquatic products.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a method for determining triazine herbicide residue in an aquatic product by using ultra-high performance liquid chromatography-tandem mass spectrometry, the method is simple, the residual amount of triazine herbicide in the aquatic product can be rapidly and accurately detected, the matrix interference is less, and the determination result is accurate.

The technical scheme adopted for realizing the above purpose of the invention is as follows:

a method for determining triazine herbicide residue in aquatic products by ultra-high performance liquid chromatography-tandem mass spectrometry comprises the following steps

1. Preparing a mixed standard solution:

preparing N parts of mixed standard solutions with gradient concentration, wherein the triazine herbicides in each part of mixed standard solution are the same in type, the triazine herbicides in each part of mixed standard solution are the same in concentration, and the internal standard atrazine-D is contained in each part of mixed solution ₅Mixing the internal standard substance atrazine-D in the standard solution₅The concentrations of (A) were the same, and the internal standard substance atrazine-D₅In the gradient concentration range of each triazine herbicide;

2. UPLC analysis and detection:

and (3) respectively carrying out UPLC analysis and detection on each mixed standard solution, wherein the UPLC conditions are as follows:

the chromatographic column adopts C₁₈A chromatographic column, wherein a mobile phase adopts a mixed solution of an organic phase and a water phase, the organic phase is acetonitrile, the water phase is 0.1 wt% formic acid solution, the flow rate of the mobile phase is 0.3-0.4mL/min, and the mobile phase is eluted by adopting a gradient elution mode;

3. HESI-MS/MS analysis and detection:

the detection conditions of the HESI-MS/MS analysis are as follows:

the ion source is a heating atmospheric pressure electric spray source, positive ion mode scanning is carried out, a reaction monitoring mode is selected for determination, the spray voltage is 3000-: the purity of argon is more than or equal to 99.999, 2.0mTorr, the half-peak width of Q1 is 0.7, and the half-peak width of Q3 is 0.7;

after HESI-MS/MS analysis and detection, the peak area of the sub-ion of each triazine herbicide and the internal standard atrazine-D under each concentration can be obtained₅The ratio of the peak areas of the daughter ions;

4. Establishing a standard curve:

taking the concentration of each triazine herbicide as an abscissa, and taking the peak area of the daughter ion of each triazine herbicide and an internal standard atrazine-D under each concentration₅The ratio of the peak areas of the daughter ions is a vertical coordinate, drawing is carried out according to the data detected by each triazine herbicide, and a standard curve of each triazine herbicide is obtained after fitting, so that a function relation formula of the standard curve of each triazine herbicide is obtained;

5. pretreatment of aquatic product samples:

taking homogeneous muscle sample of aquatic animal, adding atrazine-D as internal standard substance₅Sequentially adding acetonitrile, sodium chloride and anhydrous magnesium sulfate, performing vortex oscillation, centrifuging, collecting supernatant, repeatedly extracting, mixing the supernatants, evaporating the supernatant, dissolving the residue with solvent, and adding purifying agent such as ethylenediamine-N-Propylsilane (PSA) and octadecylsilane chemically bonded silica (C)₁₈) And EMR-Lipid powder, centrifuging after vortex oscillation, and filtering the obtained supernatant with a microporous filter membrane to obtainTo a sample to be detected;

6. detecting a sample to be detected:

analyzing and detecting the sample to be detected according to the method in the step 2-3 to obtain the peak area of the sub-ion of each triazine herbicide and the internal standard atrazine-D ₅The ratio of the peak area of the daughter ion is obtained by mixing the peak area of the daughter ion of each triazine herbicide with the peak area of the internal standard substance atrazine-D₅Substituting the ratio of the ionic peak areas into the functional relation of the standard curve of the corresponding triazine herbicide to obtain the concentration of the corresponding triazine herbicide.

Further, the chromatographic column has a column length of 100mm, an inner diameter of 2.1mm, a packed particle size of 1.7 μm and a column temperature of 30 ℃.

Further, the conditions of the gradient elution are as follows:

the first stage is as follows: the elution time is 0-2min, wherein the volume percentage of the organic phase is 15-25%, and the volume percentage of the water phase is 75-85%;

and a second stage: the elution time is 3-5min, wherein the volume percentage of the organic phase is gradually increased from 15-25% to 90-95%, and the volume percentage of the aqueous phase is gradually decreased from 75-85% to 5-10%;

and a third stage: the elution time is 2-3min, wherein the volume percentage of the organic phase is 90-95%, and the volume percentage of the water phase is 5-10%;

a fourth stage: the elution time is 0.1-0.5min, wherein the volume percentage of the organic phase is gradually reduced from 90-95% to 15-25%, and the volume percentage of the aqueous phase is gradually increased from 5-10% to 75-85%;

the fifth stage: the elution time is 1.5-4min, wherein the volume percentage of the organic phase is 15-25%, and the volume percentage of the water phase is 75-85%.

Further, in step 5, the solvent is methanol or acetonitrile.

Furthermore, the microporous filter membrane is an organic microporous filter membrane, and the pore diameter of the microporous filter membrane is not more than 0.22 μm.

Compared with the prior art, the invention has the advantages and beneficial effects that:

1. according to the invention, the QuEChERS method is adopted to purify the samples, so that the time for purifying each aquatic product sample is greatly shortened, the conventional purifying agent is abandoned, the special purifying agent is adopted, the interference of the matrix is obviously reduced, the sensitivity and the accuracy are improved, and the residual condition of the triazine herbicide in the aquatic products can be accurately reflected.

2. The invention adopts an internal standard method and a special internal standard substance atrazine-D₅Compared with the common external standard method, the method can eliminate the influence on the analysis result caused by the fluctuation of instruments and operation conditions, thereby improving the precision of measurement and the accuracy and reliability of the result.

Drawings

FIG. 1 is a graph comparing the results of recovering muscle tissue of grass carp blank by using example 1 and comparative example 1 and adding 11 triazine herbicides at a concentration of 20. mu.g/kg.

Detailed Description

The present invention will be described in detail with reference to specific examples.