CN105158382A - Method for detecting fluopicolide in fruits and vegetables - Google Patents

Abstract

The invention relates to a method for detecting fluopicolide in fruits and vegetables. The method comprises sample extraction and purification, sample solution and standard operating fluid preparation, and liquid chromatograph-based separation and mass spectrum detection in order. Through use of the treatment means and appropriate technological parameters, the method can realize simple, fast and accurate determination of fluopicolide content of vegetables and fruits, has the advantages of high detection sensitivity, good stability and accurate result, and provides a reliable and fast detection method for qualitation and quantification of fluopicolide residue in fruits and vegetables and food safety supervision.

Description

The detection method of fluopicolide in a kind of fruits and vegetables

Technical field

The invention provides a kind of detection method of biological poisonous and harmful substance, the assay method of fluopicolide in a kind of vegetable and fruit is provided especially, belong to poisonous and harmful substance in agricultural byproducts and remain safety testing field.

Background technology

Fluopicolide (fluopicolide) chemical name is 2; the chloro-N-of 6-bis-[(the chloro-5-trifluoromethyl of 3--2-pyridine radicals) methyl] benzamide; the benzamide series bactericidal agent developed by Bayer Bitterfeld GmbH crop science company, has protection and broad-spectrum curing effect.

Fluopicolide molecular formula

Fluopicolide is mainly used on all kinds of vegetables and grape, to common oomycetes diseases such as downy mildew, epidemic disease, late blight, samping off, there is outstanding preventive effect, it mainly acts on cell membrane and intercellular specific proteins, because it has excellent system conductive and stronger thin layer penetration power, all there is good inhibiting effect to each Main Morphology of pathogen; To crop and Environmental security, be specially adapted to high-quality, green vegetables are produced; Can provide comprehensive for young leaves, stem, stem tuber, young fruit and protect lastingly, because medicament can absorb fast through blade face, so resistance of rainwater washing against, for vegetables diseases prevention in rainy season provides reliable guarantee.

The health of fluopicolide meeting harmful to human residual too much in right crops, therefore relevant regulations has all been made, as Britain MRLs to the fluopicolide maximum residue limit in crops by a lot of country: potato 0.02ppm, grape 2ppm; European Union MRLs: cucumber 0.5ppm, tomato 1ppm; China MRLs: tomato 0.1ppm, cucumber 0.5ppm, Chinese cabbage 0.5ppm, watermelon 0.1ppm, potato 0.05ppm, capsicum 0.1ppm.

Along with the raising day by day of food security standard, there is active demand for detecting the detection method that in fruits and vegetables, fluopicolide as much as possible is residual quick, sensitive, exactly, is also one of Focal point and difficult point problem furtherd investigate in current crops field.So up to now, carry out detecting fast there is no the reliable method of complete set for fluopicolide residual in fruits and vegetables, thus the present invention arises at the historic moment.The method can determine fluopicolide residual in fruits and vegetables simple and efficient, sensitive, exactly, greatly reduces testing cost and shortens detection time.

Summary of the invention

The object of the invention is to solve the problems of the prior art, a kind of easy, quick, accurate, highly sensitive, reproducible fluopicolide detection method is provided.

Described detection method of the present invention, comprises the steps:

(1) sample preparation: carry out extracting and purifying again after with an organic solvent being extracted by the sample of containing fluopicolide;

(2) standard solution preparation: by the same kind matrix blank sample of not containing fluopicolide by step (1) process, obtain the residue after sample extraction purification, add appropriate fluopicolide standard solution, vortex mixes, and obtains the fluopicolide hybrid standard working fluid of variable concentrations;

(3) solution detects, and sample solution is carried out liquid chromatograph with standard solution respectively in turn by following condition and is separated and Mass Spectrometer Method:

(3.1) liquid chromatograph is separated: AgilentProshell120EC-C18 (3.0mm × 100mm, 2.7 μm) chromatographic column; 0.1% aqueous formic acid is mobile phase A, methyl alcohol is that Mobile phase B carries out linear gradient elution; Flow velocity is 0.4mL/min; Column temperature is 30 DEG C; Sample size is 2 μ L, and quantivative approach is external standard method (peak area);

(3.2) Mass Spectrometer Method: ESI source holotype ionizes; Multiple-reaction monitoring (MRM); Sheath temperature degree is 350 DEG C; Sheath airshed is 12L/min; Atomization gas pressure is 40psi; Capillary voltage: 3000V (ESI+), 3500V (ESI-); Dry gas temperature is 300 DEG C; Dry gas flow is 7L/min-1;

(4) fluopicolide content in test liquid is calculated as follows:

$\mathrm{Ci}=\frac{\mathrm{Ai}}{\mathrm{As}}\×\mathrm{Cs}$

In formula: the concentration of fluopicolide in Ci-sample;

Ai-sample chromatographic peak area;

As-fluopicolide hybrid standard working fluid chromatographic peak area;

Cs-fluopicolide hybrid standard working fluid concentration.

In the above-mentioned steps (1) of detection method of the present invention, sample extraction method as an embodiment is: accurately take 20g and be accurate to the sample of 0.1g in 250mL triangular flask, add 40mL acetonitrile, homogenate 1min in high-speed homogenization machine, collect filtrate in the 200mL tool plug graduated cylinder that 5-7gNaCl is housed, after cover lid, thermal agitation 1min, left at room temperature 1h, get supernatant 10mL, evaporating solvent is near dry, and residue 2mL methylene chloride-methanol dissolves.

In the above-mentioned steps (1) of detection method of the present invention, sample extraction method as an embodiment is: be transferred to by sample in florisil silica solid-phase extraction column, use methylene chloride-methanol washing sample bottle again, each 4mL, wash 2 times, and cleansing solution is all proceeded in solid-phase extraction column, collect whole efflux, after evaporating solvent near doing, redissolve with 5mL methyl alcohol, and cross 0.22 μm of organic phase filter membrane, obtain sample solution to be detected.

Wherein, described methylene chloride-methanol volume ratio is 95: 5.

Wherein, described evaporating solvent is not particularly limited to the nearly dry embodiment adopted, and does near as blown by high-purity Nitrogen evaporator gentleness under naturally can volatilize, heat volatilization, water-bath, for obtaining sample fast, preferably adopts bath temperature to can be 40-55 DEG C.

Wherein, filter the organic film used and be not particularly limited, as can be teflon, its aperture can be 0.22-0.45 μm, its objective is to filter and removes impurity, in order to avoid affect subsequent detection.

Compared with prior art, the advantage of detection method of the present invention is: easy, quick, accurate, highly sensitive, detection time is short, shortens sense cycle, reduces testing cost.

Accompanying drawing explanation

Fig. 1 is fluopicolide 0.01mg/kg collection of illustrative plates

Fig. 2 is wild cabbage-blank collection of illustrative plates

Fig. 3 is that wild cabbage adds 0.01mg/kg collection of illustrative plates

Fig. 4 is the blank collection of illustrative plates of grape

Fig. 5 is that grape adds 0.01mg/kg collection of illustrative plates

Embodiment

The present invention is further illustrated below in conjunction with specific embodiment.Following examples are only illustrative examples, do not form inappropriate limitation of the present invention, and the multitude of different ways that the present invention can be limited by summary of the invention and cover is implemented.Unless stated otherwise, the present invention adopts reagent, compound and equipment are the art conventional reagent, compound and equipment.

1. instrument condition

(1) Mass Spectrometry Conditions: ESI source holotype ionizes; Multiple-reaction monitoring (MRM); Sheath temperature degree: 350 DEG C; Sheath airshed: 12L/min; Atomization gas pressure: 40psi; Capillary voltage: 3000V (ESI+), 3500V (ESI-); Dry gas temperature: 300 DEG C; Dry gas flow: 7L/min; Mass spectrum acquisition parameter is as shown in table 1:

The lower 6 kinds of agricultural chemicals mass spectrum acquisition parameters of table 1MRM monitoring pattern

(2) liquid-phase condition: AgilentProshell120EC-C18 (3.0mm × 100mm, 2.7 μm) chromatographic column; Mobile phase A: 0.1% aqueous formic acid; Mobile phase B: methyl alcohol; Flow velocity: 0.4mL/min; Column temperature: 30 DEG C; Sample size: 2 μ L, quantivative approach: external standard method (peak area); Condition of gradient elution is as shown in table 2:

Table 2 condition of gradient elution

2. sample preparation

Accurately take 20g and be accurate to the sample of 0.1g in 250mL triangular flask, add 40mL acetonitrile, homogenate 1min in high-speed homogenization machine, collect filtrate in the 200mL tool plug graduated cylinder that 5-7gNaCl is housed, after cover lid, thermal agitation 1min, left at room temperature 1h, get supernatant 10mL, evaporating solvent is near dry, residue 2mL methylene chloride-methanol dissolves, be transferred in solid-phase extraction column, use methylene chloride-methanol washing sample bottle again, each 4mL, wash 2 times, and cleansing solution is all proceeded in solid-phase extraction column, collect whole efflux, after evaporating solvent near doing, redissolve with 5mL methyl alcohol, and cross 0.22 μm of organic phase filter membrane, obtain sample solution to be detected.

3. standard solution preparation

By the same kind matrix blank sample of not containing fluopicolide by " 2. sample preparation " step process, obtain the residue after sample extraction purification, add appropriate fluopicolide standard solution, vortex mixes, and is mixed with the fluopicolide hybrid standard working fluid of variable concentrations.

4. measure

Under above-mentioned operation condition of chromatogram, after instrumental baseline is stable, continuous injection number pin mark sample solution, calculate each pin relative response, until the response of adjacent 2 pin fluopicolides changes when being less than 1.2% relatively, measure according to the order of standard specimen solution, sample solution, sample solution, standard specimen solution.

5. the result recorded is substituted into following formula to calculate, draws the content of fluopicolide:

$\mathrm{Ci}=\frac{\mathrm{Ai}}{\mathrm{As}}\×\mathrm{Cs}$

In formula: the concentration of fluopicolide in Ci-sample;

Ai-sample chromatographic peak area;

As-fluopicolide hybrid standard working fluid chromatographic peak area;

Cs-fluopicolide hybrid standard working fluid concentration.

The above embodiment only have expressed embodiments of the present invention; it describes comparatively concrete and detailed; but therefore can not be interpreted as the restriction of the scope of the claims of the present invention; in the ken that those of ordinary skill in the art possess; can also make various changes and modifications under the prerequisite not departing from present inventive concept, these changes and improvements all fall in the protection domain of application claims.

Claims (6)

1. the detection method of fluopicolide in vegetable and fruit, comprises the steps:

(1) sample preparation: carry out extracting and purifying again after with an organic solvent being extracted by the sample of containing fluopicolide;

(2) standard solution preparation: by the same kind matrix blank sample of not containing fluopicolide by step (1) process, obtain the residue after sample extraction purification, add appropriate fluopicolide standard solution, vortex mixes, and obtains the fluopicolide hybrid standard working fluid of variable concentrations;

(3) solution detects, and sample solution is carried out liquid chromatograph with standard solution respectively in turn by following condition and is separated and Mass Spectrometer Method:

(3.1) liquid chromatograph is separated: AgilentProshell120EC-C18 (3.0mm × 100mm, 2.7 μm) chromatographic column; 0.1% aqueous formic acid is mobile phase A, methyl alcohol is that Mobile phase B carries out linear gradient elution; Flow velocity is 0.4mL/min; Column temperature is 30 DEG C; Sample size is 2 μ L, and quantivative approach is external standard method (peak area);

(3.2) Mass Spectrometer Method: ESI source holotype ionizes; Multiple-reaction monitoring (MRM); Sheath temperature degree is 350 DEG C; Sheath airshed is 12L/min; Atomization gas pressure is 40psi; Capillary voltage: 3000V (ESI+), 3500V (ESI-); Dry gas temperature is 300 DEG C; Dry gas flow is 7L/min-1;

(4) fluopicolide content in test liquid is calculated as follows:

$\mathrm{Ci}=\frac{\mathrm{Ai}}{\mathrm{As}}\×\mathrm{Cs}$

In formula: the concentration of fluopicolide in Ci-sample;

Ai-sample chromatographic peak area;

As-fluopicolide hybrid standard working fluid chromatographic peak area;

Cs-fluopicolide hybrid standard working fluid concentration.

2. detection method as claimed in claim 1, it is characterized in that: described extraction, its concrete steps are: accurately take 20g and be accurate to the sample of 0.1g in 250mL triangular flask, add 40mL acetonitrile, homogenate 1min in high-speed homogenization machine, collect filtrate in the 200mL tool plug graduated cylinder that 5-7gNaCl is housed, after cover lid, thermal agitation 1min, left at room temperature 1h, get supernatant 10mL, evaporating solvent is near dry, and residue 2mL methylene chloride-methanol dissolves.

3. detection method as claimed in claim 1, it is characterized in that: described extraction adopts florisil silica solid-phase extraction column, and concrete steps are: be transferred in solid-phase extraction column by sample, then use methylene chloride-methanol washing sample bottle, each 4mL, wash 2 times, and cleansing solution is all proceeded in solid-phase extraction column, collect whole efflux, after evaporating solvent near doing, redissolve with 5mL methyl alcohol, and cross 0.22 μm of organic phase filter membrane, obtain sample solution to be detected.

4. the method as described in any one of claim 2-3, is characterized in that: described methylene chloride-methanol volume ratio is 95: 5.

5. the method as described in any one of claim 2-3, is characterized in that: described evaporating solvent near dry, can be under nature volatilization, heating volatilization, water-bath with high-purity Nitrogen evaporator gentleness blow near dry in any one.

6. detection method as claimed in claim 5, it is characterized in that: described water-bath, temperature is 40-55 DEG C.