CN110514766B - Method for determining trichloromethylpyridine in food - Google Patents

Method for determining trichloromethylpyridine in food Download PDF

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CN110514766B
CN110514766B CN201910856422.1A CN201910856422A CN110514766B CN 110514766 B CN110514766 B CN 110514766B CN 201910856422 A CN201910856422 A CN 201910856422A CN 110514766 B CN110514766 B CN 110514766B
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trichloropicoline
chloropyridine
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CN110514766A (en
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李立
李荷丽
杨洋
韩世鹤
陈捷
尹昱
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Chinese Academy of Inspection and Quarantine CAIQ
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G01—MEASURING; TESTING
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract

The invention discloses a method for determining trichloromethylpyridine in food, which comprises the steps of extracting a sample by acetonitrile, dispersing and purifying by ethylenediamine-N-propyl silanized silica gel (PSA), octadecylsilane chemically bonded silica (C18) and Graphitized Carbon Black (GCB) matrixes, deriving a purified solution by methyl esterification, carrying out back extraction by ethyl acetate, determining and confirming by a gas chromatography-mass spectrometry/mass spectrometer, and quantifying by an internal standard method. The recovery rate, detection limit, precision and other technical indexes of the determination method meet requirements, the determination method is particularly applied to detection of sweet corn, wheat, sorghum, corn and popcorn, the reproducibility is good, the determination method is simple and convenient to operate, the result is accurate, and the determination method has guiding significance in the field of entry-exit inspection and quarantine.

Description

Method for determining trichloromethylpyridine in food
Technical Field
The invention relates to the technical field of analytical chemistry, in particular to a method for determining trichloromethylpyridine in food.
Background
Since nitrapyrin has selective activity to azotobacter, it can be used as nitrogen oxide inhibitor and soil nitrogen fertilizer synergist, commonly called azopirine. When the compound is applied together with urea and nitrogen fertilizer, the oxidation of ammonium ions in soil can be delayed, and the compound is an important pesticide and medical intermediate. It is rapidly degraded in plants, animals and soil to 6-chloropyridine-2-carboxylic acid, the only significant chemical residue produced after application of azapirine.
At present, the detection of trichloropicoline and 6-chloropyridine-2-carboxylic acid, a metabolite thereof, in plant-derived foods in China is still a blank, so that a detection method suitable for detecting the residual quantity of trichloropicoline in import and export plant-derived foods is very necessary to be established, the quality of agricultural products is ensured, and the enterprise management and domestic detection institution level is integrally improved.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide the method for measuring the trichloromethyl pyridine in the plant-derived food, the method is simple and convenient to operate, accurate in result, high in repeatability, and all technical indexes such as the recovery rate, the detection limit and the precision meet the requirements.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a method for measuring trichloropicoline in food comprises the following steps:
1) preparing a blank sample extracting solution: extracting a sample without containing trichloropicoline and 6-chloropyridine-2-carboxylic acid residues by acetonitrile, dispersing and purifying by using an ethylenediamine-N-propyl silanized silica gel (PSA), octadecylsilane chemically bonded silica (C18) and Graphitized Carbon Black (GCB) matrix, and performing methyl esterification derivatization on a purified solution to prepare a blank sample extracting solution; the actual sample is also processed according to the steps;
2) preparation of a standard solution:
the standard solution comprises a standard stock solution, an intermediate standard stock solution and a derivative mixed standard use solution;
respectively weighing 10mg of trichloropicoline and 6-chloropyridine-2-carboxylic acid standard substances, and preparing 1000mg/L of the standard stock solution by using methanol;
respectively sucking a proper amount of standard stock solutions, and diluting with methanol to prepare 40mg/L of the intermediate standard stock solution;
respectively sucking 0.50mL of trichloropicoline and 6-chloropyridine-2-carboxylic acid standard stock solutions into a colorimetric tube, performing derivatization in the step 1), using a blank sample extracting solution to fix the volume to 1.0mL, and preparing the derivatized mixed standard use solution of 20 mg/L;
3) preparing an internal standard solution:
weighing 10mg of 2-picolinic acid methyl ester, dissolving with ethyl acetate, transferring into a 10mL volumetric flask, fixing the volume, uniformly mixing to obtain an internal standard stock solution, and diluting the internal standard stock solution with ethyl acetate to prepare an internal standard solution of 10 mg/L;
4) matrix mix standard working solution: absorbing a certain volume of derivative mixed standard use solution, diluting the derivative mixed standard use solution into matrix standard working solution with applicable concentration by using a blank sample extracting solution according to needs, and preparing the matrix standard working solution on site;
5) determination of the standard working curve: absorbing a certain amount of derivative mixed standard use solution, adding 40 mu L of internal standard solution, diluting the derivative mixed standard use solution into 0.0mg/L, 0.025mg/L, 0.050mg/L, 0.10mg/L, 0.20mg/L and 0.40mg/L standard working solutions by blank sample extracting solution step by step, preparing a series of matrix mixed standard working solutions after filtering, determining by a gas chromatography-mass spectrometer, and drawing a standard curve by taking the ratio of the pesticide quantitative ion peak area to the internal standard substance quantitative ion peak area as a vertical coordinate and the ratio of the pesticide standard solution mass concentration to the internal standard substance mass concentration as a horizontal coordinate;
6) gas chromatography-mass spectrometry/mass spectrometry: wherein the chromatographic column is a THERMOTR-35MS quartz capillary column; the chromatographic column temperature was: maintaining at 70 deg.C for 1.5min, heating to 180 deg.C at 20 deg.C/min, heating to 210 deg.C at 5 deg.C/min, heating to 280 deg.C at 25 deg.C/min, and maintaining for 5 min; carrier gas: helium with purity more than or equal to 99.999% and flow rate of 1.2 mL/min; sample inlet temperature: 250 ℃; sample introduction amount: 1 mu L of the solution;
7) according to the detection result of the step 6), quantifying by adopting an internal standard method, and calculating the contents of trichloropicoline and 6-chloropyridine-2-carboxylic acid in the sample according to the formulas (1) and (2):
calibration of a standard curve: by
Figure GDA0003477160640000021
Find a and b, then
Figure GDA0003477160640000022
Figure GDA0003477160640000023
In the formula:
As-peak area of trichloropicoline or 6-chloropyridine-2-carboxylic acid in the standard solution;
A′is-peak area of internal standard methyl 2-picolinate in standard solution;
cs-the concentration of trichloropicoline or 6-chloropyridine-2-carboxylic acid in mg/L of the standard solution;
c′is-the concentration of the internal standard methyl 2-picolinate in mg/L in the standard solution;
c-the concentration of trichloropicoline or 6-chloropyridine-2-carboxylic acid in mg/L in the sample solution obtained from the standard working curve;
cis-the concentration of the internal standard methyl 2-picolinate in the sample solution is in mg/L;
a is the peak area of trichloropicoline or 6-chloropicolinic acid in the sample;
Ais-peak area of internal standard 2-picolinic acid methyl ester in the sample;
x is the content of trichloropicoline or 6-chloropyridine-2-carboxylic acid in the sample, and the unit is mg/kg;
v is the constant volume of the final sample liquid, and the unit is mL;
m is the sample amount represented by the final sample solution, and the unit is g; the food is selected from wheat, sorghum, corn and popcorn.
Further, the step 1) comprises the extraction of sweet corn and any one of wheat, sorghum, corn except the sweet corn and popcorn; wherein the extraction steps of the sweet corn are as follows: weighing 10g of sample into a 50mL centrifuge tube, adding 10mL of acetonitrile and 0.1mL of formic acid, vortexing for 1min, extracting for 15min by shaking in an oscillator, adding 3g of sodium chloride, shaking for 5min, centrifuging for 5min at 4500r/min, taking 4mL of supernatant, and purifying; the extraction steps of any one of wheat, sorghum, corn except sweet corn and popcorn are as follows: weighing a sample in a centrifuge tube of 5g to 50mL, adding 15g of sea sand, adding 5mL of water, shaking for dispersion, adding 10mL of acetonitrile and 0.1mL of formic acid, swirling for 1min, shaking in an oscillator for extraction for 15min, adding 3g of sodium chloride, shaking for 5min, centrifuging for 5min at 4500r/min, taking 4mL of supernatant, and purifying.
Further, the purification step in the step 1) is as follows: 4mL of a sample liquid to be purified was added to a solution containing 50mg of ethylenediamine-N-propylsilanized silica gel (PSA), 100mg of octadecylsilane chemically bonded silica (C18), 100mg of Graphitized Carbon Black (GCB), 200mg of MgSO 24And (3) in a 10mL plastic centrifuge tube, uniformly mixing for 1min in a vortex manner, centrifuging for 5min at 4500r/min, accurately sucking 2mL of purified solution into a 10mL test tube, drying by nitrogen in a water bath at 40 ℃, rapidly adding 1mL of methanol, shaking and dissolving residues, and waiting for derivatization.
Further, the derivation step in the step 1) is: placing 1mL of purified methanol solution to be derivatized in an ice-water bath, slowly adding 200 μ l of concentrated sulfuric acid, derivatizing for 30min at 55 ℃, blowing the purified methanol solution to about 400ul in a 50 ℃ water bath, adding 5mL of saturated sodium chloride solution, shaking and mixing uniformly, transferring to a 50mL centrifuge tube A, washing the derivatized test tube with 5mL of ethyl acetate, transferring to a centrifuge tube A, shaking and extracting, transferring an ethyl acetate layer to a clean 50mL centrifuge tube B, extracting the water phase with 3mL of ethyl acetate and 3mL of ethyl acetate twice, combining the organic phases in a centrifuge tube B, adding 2mL of 2% sodium sulfate solution to a centrifuge tube B, washing the organic phase, transferring the ethyl acetate layer, passing through anhydrous sodium sulfate to a nitrogen blowing colorimetric tube, leaching the anhydrous sodium sulfate with a small amount of ethyl acetate, collecting the whole filtrate, blowing nitrogen and concentrating at 40 ℃, adding 40ul of internal standard (4.13), diluting to 2mL with ethyl acetate, adding anhydrous sodium sulfate, passing through a 0.2 mu m microporous filter membrane, and carrying out gas chromatography-mass spectrometry/mass spectrometry determination analysis.
Further, the condition in the step 6) is to determine that the sample and the matrix mixed standard working solution are mixed, if the deviation between the retention time of the chromatographic peak of the substance to be detected and the retention time of the matrix standard working solution is within +/-2.5%; and the relative abundance of the qualitative ion pair is consistent with that of the matrix standard working solution with similar concentration, and if the deviation does not exceed the maximum allowable deviation range, the sample can be judged to have the corresponding object to be detected.
Further, in the step 7), the reference retention time of trichloropicoline and the reference retention time of 6-chloropyridine-2-carboxylic acid are about 7.32min and 7.97min, respectively.
Further, in the step 7), the quantitative limit of the trichloropicoline and the 6-chloro-2-pyridinecarboxylic acid is 0.025mg/kg for sweet corn, and 0.05mg/kg for wheat, sorghum, corn except for the sweet corn and popcorn.
According to the method for determining trichloromethylpyridine in food, a sample is extracted by acetonitrile, is dispersed and purified by an ethylenediamine-N-propylsilanized silica gel (PSA), an octadecylsilane chemically bonded silica gel (C18) and a Graphitized Carbon Black (GCB) matrix, a purified solution is subjected to methyl esterification derivatization, then is subjected to ethyl acetate back extraction, and is determined and confirmed by a gas chromatography-mass spectrometer, and the amount of the trichloromethylpyridine in the food is determined by an internal standard method. The recovery rate, the detection limit, the precision and other technical indexes of the determination method meet the requirements, the determination method is particularly applied to the detection of sweet corn, wheat, sorghum, corn except the sweet corn and popcorn, the reproducibility is good, the determination method is simple and convenient to operate, the result is accurate, and the determination method has guiding significance in the field of entry-exit inspection and quarantine.
Drawings
FIG. 1 is a full-scan mass spectrum of a trichloromethylpyridine standard substance of the invention;
FIG. 2 is a full-scan mass spectrum of a 6-chloro-2-pyridinecarboxylic acid methyl ester standard substance according to the present invention;
FIG. 3 is an MRM chromatogram of a standard of trichloropicoline, methyl 6-chloro-2-pyridinecarboxylate and methyl 2-pyridinecarboxylate (internal standard) according to the present invention;
FIG. 4.1 is a working curve of the sweet corn substrate 6-chloro-2-pyridinecarboxylic acid of the present invention;
FIG. 4.2 is a working curve of 6-chloro-2-pyridinecarboxylic acid, a wheat substrate, according to the present invention;
FIG. 4.3 is a working curve of sorghum substrate 6-chloro-2-pyridinecarboxylic acid of the present invention;
FIG. 4.4 is a working curve for corn substrate 6-chloro-2-pyridinecarboxylic acid in addition to sweet corn according to the present invention;
FIG. 4.5 is a working curve of popcorn substrate 6-chloro-2-pyridinecarboxylic acid of the present invention;
FIG. 5.1 is a working curve of trichloropicoline as a sweet corn substrate in accordance with the present invention;
FIG. 5.2 is a working curve of the wheat substrate trichloropicoline of the present invention;
FIG. 5.3 is a working curve of trichloropicoline as sorghum substrate according to the invention;
FIG. 5.4 is a working curve of corn substrate trichloropicoline in addition to sweet corn according to the invention;
FIG. 5.5 is a working curve of the popcorn substrate trichloropicoline of the present invention;
FIG. 6.1 is a blank (trichloromethylpyridine) MRM chromatogram of sweet corn according to the invention;
FIG. 6.2 is a chromatogram of a labeled (0.025 mg/kg trichloromethylpyridine) MRM of sweet corn of the present invention;
FIG. 6.3 is a sweet corn blank (6-chloro-2-pyridinecarboxylic acid) MRM chromatogram of the present invention;
FIG. 6.4 is a chromatogram of a labeled (0.025 mg/kg 6-chloro-2-pyridinecarboxylic acid) MRM of sweet corn according to the present invention;
FIG. 7.1 is a wheat blank (trichloromethylpyridine) MRM chromatogram of the present invention;
FIG. 7.2 is a chromatogram of labeled (0.05 mg/kg of trichloromethylpyridine) MRM of wheat according to the present invention;
FIG. 7.3 is a MRM chromatogram of wheat blank (6-chloro-2-pyridinecarboxylic acid) according to the present invention;
FIG. 7.4 is a chromatogram of labeled (0.05 mg/kg of 6-chloro-2-pyridinecarboxylic acid) MRM of wheat of the present invention;
FIG. 8.1 is a MRM chromatogram of sorghum blank (trichloromethylpyridine) according to the invention;
FIG. 8.2 is a chromatogram of labeled (0.05 mg/kg of trichloromethylpyridine) MRM of sorghum in accordance with the present invention;
FIG. 8.3 is a MRM chromatogram of sorghum blank (6-chloro-2-pyridinecarboxylic acid) according to the present invention;
FIG. 8.4 is a chromatogram of labeled (0.05 mg/kg 6-chloro-2-pyridinecarboxylic acid) MRM of sorghum in accordance with the present invention;
FIG. 9.1 is a maize blank (trichloropicoline) MRM chromatogram of the present invention, excluding sweet maize;
FIG. 9.2 is a chromatogram of a labeled (0.05 mg/kg of nitrapyrin) MRM of a corn of the present invention, excluding sweet corn;
FIG. 9.3 is a MRM chromatogram of a blank (6-chloro-2-pyridinecarboxylic acid) from maize of the present invention, excluding sweet maize;
FIG. 9.4 is a chromatogram of a labeled (0.05 mg/kg 6-chloro-2-pyridinecarboxylic acid) MRM of a corn of the present invention other than sweet corn;
FIG. 10.1 is a popcorn blank (trichloromethylpyridine) MRM chromatogram of the present invention;
FIG. 10.2 is a chromatogram of a popcorn spiked (0.05 mg/kg nitrapyrin) MRM of the present invention;
FIG. 10.3 is a popcorn blank (6-chloro-2-pyridinecarboxylic acid) MRM chromatogram of the present invention;
FIG. 10.4 is a chromatogram of the popcorn addition marker (0.05 mg/kg 6-chloro-2-pyridinecarboxylic acid) MRM of the present invention.
Detailed Description
In the description of the present embodiment, the terms "upper", "lower", "front", "rear", "left", "right", and the like indicate orientations or positional relationships based on those shown in the drawings, and are only for convenience of describing the present invention and simplifying the description, and do not indicate or imply that the referred device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present invention. Furthermore, the terms "first," "second," and the like are used for descriptive purposes only and are not to be construed as specifically indicating or implying relative importance.
In order to more clearly illustrate the technical solution of the present invention, the following description is made in the form of specific embodiments.
Examples
This example is a measurement of the content of trichloropicoline and 6-chloro-2-pyridinecarboxylic acid in food. The principle of the determination method is that a sample is extracted by acetonitrile, is dispersed and purified by ethylenediamine-N-propyl silanized silica gel (PSA), octadecylsilane chemically bonded silica (C18) and Graphitized Carbon Black (GCB) matrixes, purified liquid is derived by methyl esterification, and then is back extracted by ethyl acetate, and the internal standard method is used for quantification. The method is suitable for qualitative and quantitative determination of trichloromethylpyridine and 6-chloropyridine-2-carboxylic acid metabolite in wheat, sorghum, corn except sweet corn, sweet corn and popcorn.
The reagents in this example are analytically pure except for special indications, and the water is primary water specified in GB/T6682-.
The required materials include:
acetonitrile: carrying out chromatographic purification; ethyl acetate: carrying out chromatographic purification; methanol: carrying out chromatographic purification; sodium chloride;
anhydrous magnesium sulfate: burning at 650 ℃ for 4h, cooling to room temperature in a drier, and storing in a sealed container for later use;
anhydrous sodium sulfate: burning at 650 ℃ for 4h, cooling to room temperature in a drier, and storing in a sealed container for later use;
concentrated sulfuric acid; formic acid.
Solution preparation: saturated sodium chloride solution: 36g of sodium chloride dissolved in 100mL of water; 2% sodium sulfate solution: 2g sodium sulfate, dissolved in 100mL water;
standard substance: nitrapyrin (Nitrapyrin), CAS No: 1929-82-4, formula: c6H3Cl4N, the purity is more than or equal to 99.4%;
6-chloropyridine-2-carboxylic acid (6-Chloro-2-pyridinecarboxylic acid), CAS No: 4684-94-0, molecular formula: c6H4ClNO2The purity is more than or equal to 99.0 percent;
methyl 2-picolinate (Methyl picolinate), CAS No: 2459-07-6, formula: c7H7NO2Purity is more than or equal to 98.0 percent, and an internal standard substance.
Solution preparation:
preparing a blank sample extracting solution: extracting, purifying and deriving a sample without trichloropicoline and 6-chloropyridine-2-carboxylic acid residues to prepare a blank sample extracting solution;
preparation of a standard solution:
the standard solution comprises a standard stock solution, an intermediate standard stock solution and a derivative mixed standard use solution;
respectively weighing 10mg of trichloropicoline and 6-chloropyridine-2-carboxylic acid standard substances, and preparing 1000mg/L of the standard stock solution by using methanol;
respectively sucking a proper amount of standard stock solutions, and diluting with methanol to prepare 40mg/L of the intermediate standard stock solution;
respectively sucking 0.50mL of trichloropicoline and 6-chloropyridine-2-carboxylic acid standard stock solutions into a colorimetric tube, diluting the solution to a constant volume of 1.0mL by using a blank sample extracting solution after derivatization, and preparing the derivative mixed standard use solution of 20 mg/L;
preparing an internal standard solution:
weighing 10mg of 2-picolinic acid methyl ester, dissolving with ethyl acetate, transferring into a 10mL volumetric flask, fixing the volume, uniformly mixing to obtain an internal standard stock solution, and diluting the internal standard stock solution with ethyl acetate to prepare an internal standard solution of 10 mg/L;
matrix mix standard working solution: and (3) absorbing a certain volume of derivative mixed standard use solution, diluting the derivative mixed standard use solution into matrix standard working solution with an appropriate concentration by using a blank sample extracting solution according to needs, and preparing the matrix standard working solution on site.
The material comprises: ethylenediamine-N-propylsilanized silica gel (PSA): 40-60 μm; octadecylsilane bonded silica (C18): 40-60 μm; graphitized Carbon Black (GCB): 40-120 μm; microporous filter membrane: 13 mm. times.0.22. mu.m, organic phase.
The apparatus and device comprises: gas chromatography-triple quadrupole mass spectrometer: is provided with an electron bombardment source (EI); analytical balance: the dose is 0.0001g and 0.01g respectively; a centrifuge: the rotating speed is not lower than 4500 r/min; a tissue triturator; a horizontal oscillator; a vortex mixer; heating the water bath shaking table; nitrogen blowing instrument: the temperature can be controlled.
Sample preparation and storage: replacing about 500g of surface sample with sweet corn and popcorn, mashing in a tissue mashing machine, filling into a polyethylene bottle or bag, sealing and marking; wheat, sorghum, corn except sweet corn, replacing about 500g of a surface sample, crushing the sample to ensure that the whole sample can pass through a standard mesh screen with the diameter of 425 mu m, putting the crushed sample into a polyethylene bottle or bag, sealing and marking; and (3) sample preservation: the samples are stored respectively according to the test and the standby, and are stored at the temperature of 18 ℃ below zero, and the samples are prevented from being polluted or the content of residues is prevented from changing in the sampling and sample preparation operation processes.
The measuring steps in the invention comprise:
1) extracting samples, namely extracting sweet corn and any one of wheat, sorghum, corn except the sweet corn and popcorn;
extraction of sweet corn: weighing 10g (accurate to 0.01g) of sample into a 50mL centrifuge tube, adding 10mL acetonitrile and 0.1mL formic acid, vortexing for 1min, extracting for 15min by shaking in an oscillator, adding 3g sodium chloride, shaking for 5min, centrifuging for 5min at 4500r/min, taking 4mL of supernatant, and purifying.
Extracting wheat, sorghum, corn except sweet corn and popcorn: weighing 5g (accurate to 0.01g) of sample into a 50mL centrifuge tube, adding 15g of sea sand, adding 5mL of water, shaking for dispersion, adding 10mL of acetonitrile and 0.1mL of formic acid, swirling for 1min, shaking and extracting for 15min in an oscillator, adding 3g of sodium chloride, shaking for 5min, centrifuging for 5min at 4500r/min, taking 4mL of supernatant, and purifying.
The determination method of the invention selects acetonitrile for extraction, and 0.1mL of formic acid is added, aiming at extracting 6-chloro-2-pyridine carboxylic acid into an acetonitrile layer well in an acidic acetonitrile system, and experiments prove that the recovery rate of 6-chloro-2-pyridine carboxylic acid is very low without adding formic acid during extraction.
2) Purification
4mL of a sample liquid to be purified was added to a solution containing 50mg of ethylenediamine-N-propylsilanized silica gel (PSA), 100mg of octadecylsilane chemically bonded silica (C18), 100mg of Graphitized Carbon Black (GCB), 200mg of MgSO 24In a 10mL plastic centrifuge tube, vortex and mix for 1 min. Centrifuging at 4500r/min for 5min, accurately sucking 2mL of purified solution into a 10mL test tube, drying with nitrogen in water bath at 40 ℃, rapidly adding 1mL of methanol, shaking to dissolve residue, and allowing to derivatize.
In the measuring method of the invention, after the sample is extracted by acetonitrile, some polar and medium-polar impurities are also extracted into the acetonitrile, such as vitamins, pigments and the like, and the ethylene diamine-N-propyl silanized silica gel (PSA), octadecylsilane chemically bonded silica (C18) and Graphitized Carbon Black (GCB) are selected as adsorbents according to the properties of the impurities. Their amounts were then optimized by taking 1.0ml of 100. mu.g/L standard solution and purifying with 50mg, 75mg, 100mg, 200mg adsorbents, respectively, in parallel 6 times, taking the average recovery, comparing the purification results for different amounts and finally determining the amounts of adsorbents as 50mg PSA, 100mg C18 and 100mg GCB.
3) Derivatisation
1mL of the purified methanol solution to be derivatized is placed in an ice-water bath, 200. mu.l of concentrated sulfuric acid is slowly added, derivatization is carried out for 30min at 55 ℃, and the solution is blown to about 400ul in a water bath at 50 ℃.
Adding 5ml of saturated sodium chloride solution, shaking and mixing uniformly, transferring to a 50ml centrifuge tube A, washing a derivative test tube by using 5ml of ethyl acetate, transferring to a centrifuge tube A, shaking and extracting, transferring an ethyl acetate layer to a clean 50ml centrifuge tube B, adding 3ml of ethyl acetate and 3ml of ethyl acetate into a water phase respectively, extracting twice, combining an organic phase in a centrifuge tube B, adding 2ml of 2% sodium sulfate solution into the centrifuge tube B to wash the organic phase, transferring the ethyl acetate layer, passing through anhydrous sodium sulfate in a nitrogen blowing colorimetric tube, leaching the anhydrous sodium sulfate by using a small amount of ethyl acetate, collecting all filtrates, blowing nitrogen at 40 ℃ for concentration, adding 40ul of internal standard solution, fixing the volume to 2ml by using ethyl acetate, adding anhydrous sodium sulfate for dewatering, passing through a 0.2 mu m microporous filter membrane, and waiting for GC-MS/MS analysis.
The conditions for the derivation were optimized in this example,
determination of the derivation time: taking 1mL of 6-chloropyridine-2-carboxylic acid solution prepared from methanol at a concentration of 200ng/mL, adding 200 microliters of concentrated sulfuric acid, and selecting derivatization time for 10min, 20min, 30min and 60min respectively, wherein the derivatization time is 10min, the response is lowest, and the derivatization time is 20min, slightly increases and then becomes stable. Therefore, the derivatization time was 30 min.
Determination of the derivatization temperature: taking 1mL of 6-chloropyridine-2-carboxylic acid solution prepared from methanol at a concentration of 200ng/mL, adding 100 microliters of concentrated sulfuric acid, and performing derivatization for 30min at 30 ℃ and 55 ℃ respectively, wherein the determination result shows that the temperature tends to be stable. Therefore, the derivatization time was 55 ℃.
Derivatization reaction, wherein the dosage of concentrated sulfuric acid is selected: in the derivation process, the dosage of 50 mul, 100 mul, 200 mul and 300 mul concentrated sulfuric acid is selected, and experimental results show that if the standard solution is derived and 100 mul concentrated sulfuric acid is added, the derivation can be completed and the conversion rate is stable, but after the sample matrix is added, the conversion rate of the derivation is reduced unstably because the impurities consume sulfuric acid, so that 200 mul concentrated sulfuric acid is selected when in derivation.
The extraction conditions of the derivative 6-chloro-2-pyridinecarboxylic acid methyl ester are optimized:
after derivatization, the methanol needs to be dried by blowing, and then the ethyl acetate is used for back extraction, otherwise, the methanol content in the saturated sodium chloride solution is high, so that the solubility of the derivative 6-chloro-2-pyridinecarboxylic acid methyl ester in the water phase is increased, and the extraction efficiency of the 6-chloro-2-pyridinecarboxylic acid methyl ester is obviously reduced, and the experimental result is shown in table 1.
TABLE 1 comparison of extraction efficiencies under different conditions
Figure GDA0003477160640000091
Experimental results show that methanol needs to be blown dry as much as possible during back extraction after derivatization, three times of extraction are needed, and the recovery of the 6-chloro-2-pyridinecarboxylic acid methyl ester can meet the requirements.
Selection of extraction reagent of derivative 6-chloro-2-pyridinecarboxylic acid methyl ester:
n-hexane and ethyl acetate are respectively selected to carry out back extraction on the derivative 6-chloro-2-picolinic acid methyl ester, and experimental results show that the recovery rate of the 6-chloro-2-picolinic acid methyl ester is only about 60% by using n-hexane for extraction, and the recovery rate of the 6-chloro-2-picolinic acid methyl ester is more than 90% by using ethyl acetate for extraction. Therefore, ethyl acetate is selected as the extraction reagent of 6-chloro-2-pyridinecarboxylic acid methyl ester after the derivatization reaction.
4) Gas chromatography-mass spectrometry/mass spectrometry
Instrument reference conditions:
a) a chromatographic column: THERMOTR-35MS quartz capillary column; 30m × 0.25mm × 0.25 μm, or equivalent;
b) temperature of the column: maintaining at 70 deg.C for 1.5min, heating to 180 deg.C at 20 deg.C/min, heating to 210 deg.C at 5 deg.C/min, heating to 280 deg.C at 25 deg.C/min, and maintaining for 5 min;
c) carrier gas: helium with purity more than or equal to 99.999% and flow rate of 1.2 mL/min;
d) sample inlet temperature: 250 ℃;
e) sample introduction amount: 1 mu L of the solution;
f) and (3) sample introduction mode: no shunt sampling;
g) electron bombardment source: 70 eV;
h) ion source temperature: 230 ℃;
i) transmission line temperature: 280 ℃;
j) solvent retardation: 3 min;
the selection of chromatographic conditions mainly comprises the selection of chromatographic columns and chromatographic column temperature conditions, the interaction of pyridine rings in molecular structures of trichloropicoline and 6-chloropyridine-2-carboxylic acid and silicon hydroxyl in chromatographic column packing can cause the tailing of chromatographic peaks, the 6-chloropyridine-2-carboxylic acid has strong polarity, is sensitive to light, heat and oxygen, is easy to oxidize, is not beneficial to being directly analyzed by a gas chromatography-mass spectrometer, and is subjected to methyl esterification reaction to derive the 6-chloropyridine-2-carboxylic acid to generate the 6-chloro-2-pyridinecarboxylic acid methyl ester, so that the polarity is reduced, the stability is improved, and the chromatographic column has good retention behavior. In the experiment, four chromatographic columns of DB-1701, DB-5MS, DB-INOVWAX and TR-35MS were selected for the experiment. The THERMOTR-35MS quartz capillary column is selected for separation through experiments, the coating layer of the chromatographic column is (35% -phenyl) -polysiloxane stationary liquid, the peak types of trichloropicoline and 6-chloro-2-pyridinecarboxylic acid methyl ester are sharp, no tailing exists, and the sensitivity of the 6-chloro-2-pyridinecarboxylic acid methyl ester is remarkably improved compared with the first three chromatographic columns. Can make the object to be measured obtain good separation, and the chromatographic column cost is moderate.
By utilizing EI source, under a positive ion mode, performing primary mass spectrometry (Q1 scanning) on trichloropicoline and 6-chloro-2-pyridinecarboxylic acid methyl ester respectively to obtain a parent ion peak, and performing secondary mass spectrometry (daughter ion scanning) on the parent ion peak to obtain fragment ion information, thereby determining quantitative and qualitative ion pairs.
The method adopts the time selection ion mode, and the ion scanning is only carried out in a narrow area of the corresponding retention time, so that more pairs of ions can obtain longer residence time in the same whole scanning process, the sensitivity is improved, and the mutual interference between peaks is reduced as much as possible.
Data were collected in the MRM mode, and the best response was achieved for each ion pair by optimizing the collision energy, see table 2.
TABLE 2 Retention time and multiple reactions monitoring ion and collision energy
Figure GDA0003477160640000101
The obtained standard substance full-scan mass spectrogram of trichloropicoline and 6-chloro-2-picolinic acid methyl ester and the MRM chromatogram of the trichloropicoline, 6-chloro-2-picolinic acid methyl ester and 2-picolinic acid methyl ester (internal standard) standard substance are shown in FIGS. 1-3.
Determination of the Standard working Curve
Absorbing a certain amount of derivative mixed standard use solution, adding 40 mu L of internal standard solution, diluting the standard use solution into 0.0mg/L, 0.025mg/L, 0.050mg/L, 0.10mg/L, 0.20mg/L and 0.40mg/L standard working solutions by blank sample extracting solution step by step, preparing a series of matrix mixed standard working solutions after filtering, measuring by a gas chromatography-mass spectrometer, taking the ratio of the pesticide quantitative ion peak area to the internal standard substance quantitative ion peak area as a vertical coordinate and the ratio of the pesticide standard solution mass concentration to the internal standard substance mass concentration as a horizontal coordinate, measuring under the GC-MS/MS experimental conditions determined by the measuring method, and drawing a standard curve, wherein the standard curve is shown in reference figures 4.1-5.5. The results show that the concentrations of the trichloropicoline and the 6-chloro-2-pyridinecarboxylic acid are significant linearly within the range of 0.025-0.40 mg/L.
And (3) measuring the lower limit: the method has the following quantitative limits of trichloropicoline and 6-chloro-2-pyridinecarboxylic acid: the sweet corn is 0.025mg/kg, and the wheat, the sorghum, the corn except the sweet corn and the popcorn are all 0.05 mg/kg.
For qualitative and quantitative measurements:
and (3) qualitative determination: determining the sample and matrix mixed standard working solution according to the conditions, if the deviation of the chromatographic peak retention time of the substance to be determined and the retention time of the matrix standard working solution is within +/-2.5%; and the relative abundance of the qualitative ion pair is consistent with that of the matrix standard working solution with similar concentration, and the deviation does not exceed the range specified in the table 3, so that the corresponding object to be detected exists in the sample.
TABLE 3 maximum permissible deviation of relative ion abundance in qualitative terms
Relative ion abundance ＞50％ More than 20% to 50% More than 10% to 20% ≤10％
Allowable relative deviation ±20％ ±25％ ±30％ ±50％
Quantitative determination: in the method, an internal standard calibration curve method is adopted for quantitative determination, in order to reduce the influence of the matrix on the quantitative determination, a standard curve drawn by a matrix standard working solution is adopted for the quantitative determination, and the response value of the object to be measured in the measured sample is ensured to be within a linear range. The reference retention times of trichloropicoline and 6-chloropyridine-2-carboxylic acid under the chromatographic conditions were about 7.32min and 7.97min, respectively. The MRM chromatograms of the corresponding trichloropicoline, methyl 6-chloro-2-pyridinecarboxylate and methyl 2-picolinate (internal standard) standards are shown in figure 3.
The matrix effect means that the co-efflux component in the sample analysis solution other than the analyte changes the response value of the analyte, thereby affecting the accuracy and reproducibility of the quantitative analysis. Experiments show that the trichloropicoline and the 6-chloro-2-pyridinecarboxylic acid methyl ester have a matrix enhancement effect, the trichloropicoline matrix effect is about 28%, and the 6-chloro-2-pyridinecarboxylic acid methyl ester matrix effect is about 11%.
After the sample is measured, according to the detection result, the content of trichloropicoline and 6-chloropyridine-2-carboxylic acid in the sample is calculated according to the formulas (1) and (2) by adopting an internal standard method:
calibration of a standard curve: by
Figure GDA0003477160640000121
Find a and b, then
Figure GDA0003477160640000122
Figure GDA0003477160640000123
In the formula:
As-peak area of trichloropicoline or 6-chloropyridine-2-carboxylic acid in the standard solution;
A′is-peak area of internal standard methyl 2-picolinate in standard solution;
cs-the concentration of trichloropicoline or 6-chloropyridine-2-carboxylic acid in mg/L of the standard solution;
c′is-the concentration of the internal standard methyl 2-picolinate in mg/L in the standard solution;
c-the concentration of trichloropicoline or 6-chloropyridine-2-carboxylic acid in mg/L in the sample solution obtained from the standard working curve;
cis-the concentration of the internal standard methyl 2-picolinate in the sample solution is in mg/L;
a is the peak area of trichloropicoline or 6-chloropicolinic acid in the sample;
Ais-peak area of internal standard 2-picolinic acid methyl ester in the sample;
x is the content of trichloropicoline or 6-chloropyridine-2-carboxylic acid in the sample, and the unit is mg/kg;
v is the constant volume of the final sample liquid, and the unit is mL;
m represents the amount of sample in g.
FIGS. 6.1-10.4 are MRM chromatograms of samples of trichloropicoline and 6-chloro-2-pyridinecarboxylic acid blank and samples added and recovered, respectively.
The precision of actual samples such as sweet corn, wheat, sorghum, corn except the sweet corn, popcorn and the like are respectively measured, and the precision and the recovery rate data are shown in table 4 after the addition and recovery tests of trichloropicoline and 6-chloro-2-pyridinecarboxylic acid with different concentrations are carried out.
Meanwhile, the method is verified by five units, and three levels of addition recovery experiments are carried out on the sweet corn, the wheat, the sorghum, the corn except the sweet corn and the popcorn, and the verification results are shown in table 5.
The results show that the technical indexes of the detection method such as recovery rate, detection limit and precision meet the requirements, the method is applied to detection of sweet corn, wheat, sorghum, corn except the sweet corn and popcorn, the reproducibility is good, the detection method has the advantages of simplicity and convenience in operation and accurate results, and can be widely applied to food detection.
Finally, it is to be understood that the above embodiments are merely exemplary embodiments taken to illustrate the principles of the present invention, which is not intended to be limiting. It will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention, and these changes and modifications are to be considered as within the scope of the invention.
Table 4 results of recovery and precision of trichloropicoline and 6-chloro-2-pyridinecarboxylic acid addition in room (n ═ 6)
Figure GDA0003477160640000131
Figure GDA0003477160640000141
Figure GDA0003477160640000151
TABLE 5 summary of the results of the recovery and precision of the addition of trichloropicoline and 6-chloro-2-pyridinecarboxylic acid between the chambers
Figure GDA0003477160640000152
Figure GDA0003477160640000161
Figure GDA0003477160640000171

Claims (7)

1. The method for determining trichloropicoline in food is characterized by comprising the following steps:
1) preparing a blank sample extracting solution: extracting a sample without containing trichloropicoline and 6-chloropyridine-2-carboxylic acid residues by acetonitrile, dispersing and purifying by using an ethylenediamine-N-propyl silanized silica gel (PSA), octadecylsilane chemically bonded silica (C18) and Graphitized Carbon Black (GCB) matrix, and performing methyl esterification derivatization on a purified solution to prepare a blank sample extracting solution; the actual sample is also processed according to the steps;
2) preparation of a standard solution:
the standard solution comprises a standard stock solution, an intermediate standard stock solution and a derivative mixed standard use solution;
respectively weighing 10mg of trichloropicoline and 6-chloropyridine-2-carboxylic acid standard substances, and preparing 1000mg/L of the standard stock solution by using methanol;
respectively sucking a proper amount of standard stock solutions, and diluting with methanol to prepare 40mg/L of the intermediate standard stock solution;
respectively sucking 0.50mL of trichloropicoline and 6-chloropyridine-2-carboxylic acid standard stock solutions into a colorimetric tube, performing derivatization in the step 1), using the blank sample extracting solution to fix the volume to 1.0mL, and preparing the derivatized mixed standard use solution of 20 mg/L;
3) preparing an internal standard solution:
weighing 10mg of 2-picolinic acid methyl ester, dissolving with ethyl acetate, transferring into a 10mL volumetric flask, fixing the volume, uniformly mixing to obtain an internal standard stock solution, and diluting the internal standard stock solution with ethyl acetate to prepare an internal standard solution of 10 mg/L;
4) matrix mix standard working solution: absorbing a certain volume of derivative mixed standard use solution, diluting the derivative mixed standard use solution into matrix standard working solution with applicable concentration by using a blank sample extracting solution according to needs, and preparing the matrix standard working solution on site;
5) determination of the standard working curve: absorbing a certain amount of derivative mixed standard use solution, adding 40 mu L of internal standard solution, diluting the derivative mixed standard use solution into 0.0mg/L, 0.025mg/L, 0.050mg/L, 0.10mg/L, 0.20mg/L and 0.40mg/L standard working solutions by blank sample extracting solution step by step, preparing a series of matrix mixed standard working solutions after filtering, determining by a gas chromatography-mass spectrometer, and drawing a standard curve by taking the ratio of the pesticide quantitative ion peak area to the internal standard substance quantitative ion peak area as a vertical coordinate and the ratio of the pesticide standard solution mass concentration to the internal standard substance mass concentration as a horizontal coordinate;
6) gas chromatography-mass spectrometry/mass spectrometry: wherein the chromatographic column is a THERMOTR-35MS quartz capillary column; the chromatographic column temperature was: maintaining at 70 deg.C for 1.5min, heating to 180 deg.C at 20 deg.C/min, heating to 210 deg.C at 5 deg.C/min, heating to 280 deg.C at 25 deg.C/min, and maintaining for 5 min; carrier gas: helium with purity more than or equal to 99.999% and flow rate of 1.2 mL/min; sample inlet temperature: 250 ℃; sample introduction amount: 1 mu L of the solution;
7) according to the detection result of the step 6), quantifying by adopting an internal standard method, and calculating the contents of trichloropicoline and 6-chloropyridine-2-carboxylic acid in the sample according to the formulas (1) and (2):
calibration of a standard curve: by
Figure 95486DEST_PATH_IMAGE001
To obtainaAndbthen, then
Figure 12626DEST_PATH_IMAGE002
（1）
Figure 323522DEST_PATH_IMAGE003
（2）
In the formula:
A s ——peak area of trichloropicoline or 6-chloropyridine-2-carboxylic acid in the standard solution;
A ' is ——peak area of internal standard 2-picolinic acid methyl ester in the standard solution;
c s ——the concentration of trichloropicoline or 6-chloropyridine-2-carboxylic acid in the standard solution is mg/L;
c ' is ——the concentration of the internal standard 2-picolinic acid methyl ester in the standard solution is mg/L;
c——test from standard working curveThe concentration of trichloropicoline or 6-chloropyridine-2-carboxylic acid in the sample solution is mg/L;
c is ——the concentration of the internal standard 2-picolinic acid methyl ester in the sample solution is mg/L;
A——peak area of trichloropicoline or 6-chloropicolinic acid in the sample;
A is ——peak area of internal standard 2-picolinic acid methyl ester in the sample;
X——the content of trichloropicoline or 6-chloropyridine-2-carboxylic acid in the sample is mg/kg;
V——the volume of the final sample solution is determined by mL;
m——the sample amount represented by the final sample solution is g;
the food is selected from wheat, sorghum, corn and popcorn.
2. The determination method according to claim 1, wherein the step 1) comprises extraction of sweet corn and extraction of any one of wheat, sorghum, corn other than sweet corn and popcorn; wherein the extraction steps of the sweet corn are as follows: weighing 10g of sample into a 50mL centrifuge tube, adding 10mL of acetonitrile and 0.1mL of formic acid, vortexing for 1min, extracting for 15min by shaking in an oscillator, adding 3g of sodium chloride, shaking for 5min, centrifuging for 5min at 4500r/min, taking 4mL of supernatant, and purifying; the extraction steps of any one of wheat, sorghum, corn except sweet corn and popcorn are as follows: weighing a sample in a centrifuge tube of 5g to 50mL, adding 15g of sea sand, adding 5mL of water, shaking for dispersion, adding 10mL of acetonitrile and 0.1mL of formic acid, swirling for 1min, shaking in an oscillator for extraction for 15min, adding 3g of sodium chloride, shaking for 5min, centrifuging for 5min at 4500r/min, taking 4mL of supernatant, and purifying.
3. The assay method according to claim 1, wherein the purification step in step 1) is: adding 4mL of sample liquid to be purified into a 10mL plastic centrifuge tube containing 50mg of ethylenediamine-N-propylsilanized silica gel (PSA), 100mg of octadecylsilane chemically bonded silica (C18), 100mg of Graphitized Carbon Black (GCB) and 200mg of MgSO4, uniformly mixing by vortex for 1min, centrifuging at 4500r/min for 5min, accurately sucking 2mL of purified liquid into a 10mL test tube, drying by nitrogen in water bath at 40 ℃ and rapidly adding 1mL of methanol to shake and dissolve residues to be derived.
4. The assay method according to claim 1, wherein the deriving step in step 1) is: placing 1mL of purified methanol solution to be derivatized in an ice-water bath, slowly adding 200 μ l of concentrated sulfuric acid, derivatizing for 30min at 55 ℃, blowing the purified methanol solution to about 400ul in a 50 ℃ water bath, adding 5mL of saturated sodium chloride solution, shaking and mixing uniformly, transferring to a 50mL centrifuge tube A, washing the derivatized test tube with 5mL of ethyl acetate, transferring to a centrifuge tube A, shaking and extracting, transferring an ethyl acetate layer to a clean 50mL centrifuge tube B, extracting the water phase with 3mL of ethyl acetate twice, combining the organic phases in a centrifuge tube B, adding 2mL of 2% sodium sulfate solution to a centrifuge tube B, washing the organic phase, transferring the ethyl acetate layer, passing through anhydrous sodium sulfate to a nitrogen blowing colorimetric tube, leaching the anhydrous sodium sulfate with a small amount of ethyl acetate, collecting all filtrates, blowing nitrogen at 40 ℃ for concentration, adding 40% of internal standard, diluting to 2mL with ethyl acetate, adding anhydrous sodium sulfate for removing water, passing through a 0.2 μm microporous filter membrane, and (5) carrying out gas chromatography-mass spectrometry/mass spectrometry determination analysis.
5. The assay method according to claim 1, wherein the conditions in step 6) are such that the sample and matrix mixed standard working solution is assayed if the retention time of the chromatographic peak of the substance to be assayed is within ± 2.5% of the retention time of the matrix standard working solution; and the relative abundance of the qualitative ion pair is consistent with that of the matrix standard working solution with similar concentration, and if the deviation does not exceed the maximum allowable deviation range, the sample can be judged to have the corresponding object to be detected.
6. The method according to claim 1, wherein the reference retention times of the nitrapyrin and the 6-chloropyridine-2-carboxylic acid in step 7) are about 7.32min and 7.97min, respectively.
7. The assay of claim 1, wherein in step 7), the limits of quantitation of the trichloropicoline and the 6-chloro-2-pyridinecarboxylic acid are 0.025mg/kg for sweet corn and 0.05mg/kg for wheat, sorghum, corn other than sweet corn, and popcorn.
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