CN103969363A - Detection method for plant growth regulator residues in fruits - Google Patents

Abstract

The invention relates to a detection method for plant growth regulator residues in fruits. The detection method comprises the following steps: the QuEChERS method is adopted to extract and purify the plant growth regulator residues in fruits, so as to enable the adding standard recovery rate of the target compound to reach 71%-98%; the RSD ( relative standard deviation) is not higher than 6%; the sensitivity and selectivity are favorable and the recovery rate is stable; the liquid chromatograph tandem mass spectrogram detection method is adopted to simultaneously detect forchlorfenuron, ethephon, 4-sodium chlophenoxycetate and 2,4- sodium acetate-dichlorobenzene oxygen; the detection limits range from 0.5 microgram/kg to 100 microgram/kg respectively; the sensitivity requirement of national limited standard is met. The detection method has favorable sensitivity and selectivity and can serve as a method for detecting the plant growth regulator residues in fruits.

Description

Plant growth regulator method for detecting residue in a kind of fruit

Technical field

The present invention relates to plant growth regulator method for detecting residue in a kind of fruit, the method detecting particularly to CPPU, ethephon (CEPHA)，2-(chloroethyl) phosphonic acid, 4-chlorophenoxyacetic acid sodium and 2,4-Dichlorophenoxyacetic acid sodium time.

Background technology

Along with social development, the diversity of product and rich in bringing people's material enjoyment, various drawbacks have also been walked close to our life unconsciously.The strawberry of " large as table tennis ", the Kiwi berry of " table tennis grape ", " potato size ", the tomato of outward appearance " bright-coloured ", mango, the banana of " ripener accelerates the ripening " are seen everywhere on market; But, in the behind of these phenomenons, but under cover " run counter to the secret of the natural law ".Here it is because at plantation or the later stage plant growth regulator such as for example having used " large fruit spirit ", " swelling agent ", " ethephon (CEPHA)，2-(chloroethyl) phosphonic acid ", " tomato spirit " that accelerates the ripening in process, plant growth regulator (plant growth regulators) is the material that a class and plant hormone have similar physiology and biological effect, be a class agricultural chemicals of growing for coordinate plant growth, comprise artificial synthetic compound and the natural phytohormone extracting from biology.Having found to have regulating plant growth and having grown functional mass has auxin, gibberellin, ethene, the basic element of cell division, abscisic acid, brassinosteroid, salicylic acid, jasmonic and polyamines etc., is mainly front 6 large classes and be used in agricultural production as plant growth regulator.

" large fruit spirit " or " swelling agent " chemical name are the chloro-4-pyridines of 1-(2-) 3-phenylurea, another name is again CPPU, forchlorfenuron; Shi Te is excellent; No. CAS: 68157-60-8.Molecular formula: C12H10ClN3O.Relative molecular weight or atomic weight: 247.7.Fusing point (DEG C): 165～170.White is to near-white crystalline powder.Solubleness 39mg/l(pH6.4 in water, 21 DEG C).

Tomato spirit chemical name is p-chlorophenoxyacetic acid; To chlorobenzene oxygen acetic acid, another name Tomatotone, tomato is logical, drop-proof agent, anti-fall strong fruit agent, (4-chlorobenzene oxygen) acetic acid.Molecular formula: C8H7ClO3.Molecular weight: 186.59.No. CAS: 122-88-3.White crystals.Fusing point 157-159 DEG C.Be dissolved in ethanol, acetone and benzene, be slightly soluble in water.There is fragrant.

Ethephon (CEPHA)，2-(chloroethyl) phosphonic acid is a kind of plant growth regulator of ethene releasing agent class, chemical name is 2 chloroethyl phosphoric acid, separately have the name ethephon (CEPHA)，2-(chloroethyl) phosphonic acid such as ethephon, an examination spirit as defoliant, pluck fruit agent, emasculation agent, be widely used in the production and storage of the vegetable and fruits such as tomato, apple, pears, strawberry, grape, banana.

April calendar year 2001, the Ministry of Agriculture started " pollution-free food action plan ", in nuisanceless production standard, explicitly point out, the use of plant growth regulator should be so that environment not be polluted, to health without harm, do not reduce fruit quality as principle, and emphasize " must not use the phenylurea class basic element of cell division to dip in fruit ".

In China's " the law of food safety ", there is hundreds of item state health standards, but omnifarious growth hormone is lacked to index of correlation regulation and detection method.And shortage operative norm detects difficultly, also bring puzzlement to the administration for industry and commerce law enforcement.GB 2763-2008 " maximum residue limit of Pesticide ", makes regulation to " ethephon (CEPHA)，2-(chloroethyl) phosphonic acid " maximum residue limit on crops surface.Standard is listed in the fruit such as some torrid zones and subtropics, and the maximum residue limit of " ethephon (CEPHA)，2-(chloroethyl) phosphonic acid " is 2mg/kg, but there is no corresponding detection method standard.And be the chloro-4-pyridine of 1-(2-for " large fruit spirit " or " swelling agent ") 3-phenylurea, " tomato spirit " be that p-chlorophenoxyacetic acid does not have corresponding limit standard regulation and detection method standard.In addition, 2,4-D is usually used in the preservation and freshness of oranges and tangerines, but does not also specify residue limits standard.

China's national standard is to lack detection method and limit standard to these plant growth regulator one, even if there is detection method in indivedual correctivess, also often that formulation is of the remote past, complex operation, accuracy is not high, the shortcomings such as single treatment can only detect a plant growth regulators, and detection efficiency is not high.Set up several frequently seen plants growth regulator detection method and development plant growth regulator multiple while detection technique, to effectively promote the raising of my institute's detection technique ability, be also of great significance for ensureing with the daily closely bound up food security tool of people simultaneously.

Up to the present, about CPPU, the residual assay method in fruits and vegetables rarely has report both at home and abroad.The people such as Hu Jiangtao adopt anionite propyl group ethylenediamine (PSA) to disperse Solid-Phase Extraction (DSPE) to purify the impurity in matrix, have set up high performance liquid chromatography (HPLC) detection method of CPPU.Main process is accurately to take (5.00 ± 0.05) g Kiwi berry homogenate sample.Be placed in 50 mL tool plug centrifuge tubes, add 5.0 mL acetonitriles, vortex 2 min that vibrate, after ultrasonic extraction 20 min, add the anhydrous MgSO of 2 g and 0.5 g NaC1, and vortex 2 min that vibrate, with the centrifugal l0min of 8 000 r/min.Then carry out purification run, extract 1.00 mL after accurate absorption is centrifugal are in 2 mL Teflon centrifuge tubes, add 100mg PSA and the anhydrous MgSO of 100 mg, vortex 1 min, with centrifugal 2 min of 3 000 r/min, get supernatant and cross the organic filter membrane of 0.22 m in sample injection bottle, measure by reversed-phase liquid chromatography UV-detector, detection limit reaches 0.005mg/kg.People's CPPUs such as Li Ruijuan are cleared up dynamic and residual mensuration in Kiwi berry, with the CPPU in methylene chloride extraction Kiwi berry sample, with after anhydrous sodium sulfate dehydration, after rotary evaporation is concentrated, by methanol constant volume, use C18 chromatographic column, UV-detector reverse-phase chromatography is measured its content, detects and is limited to 0.01mg/kg.

About p-chlorophenoxyacetic acid analytical approach, mainly contain at present thin-layered chromatography, vapor-phase chromatography.TLC method detection sensitivity is lower, and the loaded down with trivial details and condition of sample making course is difficult to control, therefore the recovery is lower, reappearance is poor; GC method is to derive as propyl ester after CPA-Na is extracted, and then separates and detects with GC-FID. but method is subject to the impact of FID detection sensitivity and cause sampling amount large, and sample pre-treatments step is comparatively numerous and diverse, is unfavorable for express-analysis.There is no at present national standard assay method.Huang is defended equality people and is adopted sodium hydroxide solution to extract, and removes impurity with chloroform solution extraction at night, then uses ether-sherwood oil (1:1) extraction, strip with sodium bicarbonate solution, use extracted with diethyl ether, with after anhydrous sodium sulfate dehydration, reversed-phase liquid chromatography UV-detector is measured again.The people such as Yan Jinliang adopt the chromatography of ions to detect the 4-chlorophenoxyacetic acid in bean sprouts.Sample is smashed to pieces, accurately take sample that 10 g evenly smash to pieces in 50 ml centrifuge tubes, add 20 ml deionized waters, soak 4 h above or spend the night, shake well, high speed centrifugation (8000 r/min) 10 min, get supernatant, and filtrate is crossed C18 pillar, direct injected after the filter membrane of 0.45 μ m.Select IonPac AS11 chromatographic column, the gradient elution of KOH leacheate, electric conductivity detector detects.The people such as Shi Qinghong detect the 4-chlorophenoxyacetic acid in soil with chromatography of ions electric conductivity detector.

The people such as Yin Guihao adopt diazomethane derivatization, and gas chromatography accurately detects the ethephon (CEPHA)，2-(chloroethyl) phosphonic acid content in tomato.Take tomato test portion approximately 10 g (accurately to l0 mg) in tygon beaker, add 0.5 mL methanolic hydrochloric acid solution and 50mL methyl alcohol, sonic oscillation extracts 5 min, filtering in the polyethylene volume bottle of 100 mL. residue extracts 1 time with 30 mL methyl alcohol again, merge extract in polyethylene volume bottle, be settled to 100 mL.Draw the above-mentioned constant volume solution of 10.0 mL, put into l5 mL tygon graduated centrifuge tube, flow down in 30～40 DEG C of water-baths and be concentrated into approximately 1.5 mL at drying nitrogen, add 0.5 mL methanolic hydrochloric acid solution and 8 mL absolute ethers, fully mix, place 10 min.Clear liquid is moved in another tygon graduated tube, Liquid Residue divides 2 extractions with 2 m1 absolute ethers, in the above-mentioned clear liquid of extract people, in 30～40 DEG C of water-baths, be concentrated into about l mL. in vent cabinet, in concentrate, drip diazomethane solution, until yellow does not take off. cover tightly stopper, placing 15 min. flows down and in 30～40 DEG C of water-baths, is concentrated into approximately 0.8 mL at nitrogen, be diluted to 2.00-5.00 mL with ether, its content of gas Chromatographic Determination, detects and is limited to 0.01mg/kg.The people such as Li Lihua adopt solid-phase microextraction-gas chromatography coupling technology to set up the method for measuring ethephon (CEPHA)，2-(chloroethyl) phosphonic acid residual quantity in mango juice.In 7ml head space bottle, add the standard solution 0.5ml that prepared or the KOH of 0.25g sample box 1ml 5mol/L, sealing immediately, after mixing, be placed in the gas chromatograph column oven that is preheated to 60 DEG C isothermal reaction 10min, taking-up is put on the solid phase micro extraction heating device that is preheated to approximately 60 DEG C, insert cooling extracting head, enrichment 5min, inserts rapidly gas chromatograph vaporizer by extracting head, thermal desorption 5min, the circumstances in which people get things ready for a trip of going forward side by side analysis of spectrum.The people such as Xu Maosheng adopt chromatography of ions electric conductivity detector, KOH and NaCO3 mixing leacheate, and AS9 separating column separates detection, and detectability reaches 0.1887ug/ml.The people such as Yan Jinliang adopt chromatography of ions electric conductivity detector, KOH leacheate, and AS18 chromatographic column detects the ethephon (CEPHA)，2-(chloroethyl) phosphonic acid in melon and fruit.Melon and fruit sample is through smashing to pieces, accurately take sample that 10 g evenly smash to pieces in 50 ml centrifuge tubes, add 20 ml deionized waters, ultrasonic extraction 10 min, or after soaked overnight, through shake well, high speed centrifugation (8000 r/min) 15 min, draw supernatant, filtrate is crossed C18 pillar, after the filter membrane of 0.45um, direct injected detects.Retention time and the baseline separation of sample experimental result and each ion are good.The people such as Zhou Yanming adopt headspace gas chromatography to detect ethephon (CEPHA)，2-(chloroethyl) phosphonic acid.

The detection method of above-mentioned these plant growth regulator or process loaded down with trivial details, consuming timely, detection limit is higher again, is difficult to adapt to more and more stricter food safety requirements.

Summary of the invention

The object of the invention is to for overcoming above-mentioned the deficiencies in the prior art, provide a kind of method easy, more fast, plant growth regulator method for detecting residue in the fruit of one-time detection multiple components.

In order to solve the problems of the technologies described above, the present invention adopts following technical scheme: plant growth regulator method for detecting residue in a kind of fruit, is characterized in that comprising following steps:

(1) sample pretreatment: take fruit sample, get edible part and put into beaker, homogenate 3min;

(2) extraction and cleaning: take 15g sample after homogenate in 50mL centrifuge tube, add the 15mL acetonitrile (being adjusted to pH=3.0 with formic acid in advance) containing 1% acetic acid, after violent jolting 1min, add 6.0g anhydrous magnesium sulfate and 1.5g anhydrous sodium acetate, after violent jolting 1min, the centrifugal 3min of 4000rpm, gets 1mL supernatant and adds 150mg anhydrous magnesium sulfate and 50mg PSA, after violent jolting 1min, the centrifugal 3min of 4000rpm, get supernatant, cross after 0.22um filter membrane, obtain extract;

(3) extract purifying step being obtained enters Liquid Chromatography-Tandem Mass Spectrometry instrument and detects;

The present invention is by QuEChERS method, employing adds acetonitrile containing acetic acid to the residual extraction and cleaning that carries out of plant growth regulator in fruit, make the recovery of standard addition of target compound reach 71%-98%, relative standard deviation is not higher than 6%, there is good sensitivity and selectivity, the recovery is stable, and adopt UPLC-MS/MS to realize CPPU, ethephon (CEPHA)，2-(chloroethyl) phosphonic acid, 4-chlorophenoxyacetic acid sodium and 2, when 4-Dichlorophenoxyacetic acid sodium, detect, method is easy, and the detection limit of four kinds of target compounds reaches 0.5-100.0 μ g/kg.

Below in conjunction with embodiment, the invention will be further described.

Brief description of the drawings

Fig. 1 is ethephon (CEPHA)，2-(chloroethyl) phosphonic acid chromatogram mass spectrogram;

Fig. 2 is 4-chlorophenoxyacetic acid sodium chromatogram mass spectrogram;

Fig. 3 is 2,4-Dichlorophenoxyacetic acid sodium chromatogram mass spectrogram;

Fig. 4 is forchlorfenuron chromatogram mass spectrogram;

Fig. 5 is the chromatogram mass spectrogram of 4 plant growth regulators;

Fig. 6 is the blank matrix standard model of concentration 1 chromatogram mass spectrogram in table 1;

Fig. 7 is the blank matrix standard model of concentration 2 chromatogram mass spectrogram in table 1;

Fig. 8 is the blank matrix standard model of concentration 3 chromatogram mass spectrogram in table 1;

Fig. 9 is the blank matrix standard model of concentration 4 chromatogram mass spectrogram in table 1;

Figure 10 is the blank matrix standard model of concentration 5 chromatogram mass spectrogram in table 1;

Figure 11 is the blank matrix standard model of concentration 6 chromatogram mass spectrogram in table 1;

Figure 12 is that Kiwi berry detects forchlorfenuron chromatogram mass spectrogram.

Embodiment

One, the instrument of practicality of the present invention and reagent

1) liquid chromatograph and chromatographic column

The Acquity UPLC of Waters company of U.S. Ultra Performance Liquid Chromatography instrument, is furnished with automatic sampler; Acquity UPLC BEH C18 liquid-phase chromatographic column (50 × 2.1 mm, 1.7 μ are m);

2) mass ion source

Select ESI ion gun to detect analysis.

3) mass spectrometer

The Premier XE tandem mass spectrometer of Waters company of the U.S..

4) other hardware

Millipore company of U.S. ultrapure water machine (Milli-Q).

Germany IKA company's T 18 homogenizers

Eppendorf company of Germany 5418 hydro-extractors

Germany Sai Duolisi CPA225D electronic balance

The solid-phase extraction device of Agilent company, joins the vacuum pump of company from all directions.

N-EVAP 111 Nitrogen evaporators of U.S. Organomation. Associates. Inc..

The R-215 Rotary Evaporators of BUCHI company of Switzerland.

5) reagent and consumptive material

CNWBOND LC-C18 500mg/6mL solid-phase extraction column.

DSPE purifying pipe (AOAC 2007.01) 150mgMgSO4,50mgPSA, 2mL

The residual grade sodium chloride of agriculture

Anhydrous magnesium sulfate, 98.0%, extra pure

Carbon-GCB Graphon, 120-400 order

The special super clean filler 40-60um of PSA QuEChERS, 60A

DSPE extracting tube (AOAC 2007.01) 6g magnesium sulfate, 1.5g sodium acetate, 50mL

two, standard items preparation

CPPU, 99.2%, Dr.Ehrenstorfer company produces;

Ethephon (CEPHA)，2-(chloroethyl) phosphonic acid, 98.0%, Dr.Ehrenstorfer company produces;

4-chlorophenoxyacetic acid sodium, 98%, reagent.

2,4-Dichlorophenoxyacetic acid sodium, 98.5%, Dr.Ehrenstorfer company produces.

Standard inventory solution: take 4 kinds of each 10.0mg(of standard sample of pesticide and be accurate to 0.01mg), dilute with methyl alcohol respectively and be settled to 100mL, being mixed with mass concentration is the single standard storing solution of 100mg/L, in Refrigerator store.

Standard operation solution: draw standard inventory solution 10.0mL in the volumetric flask of 100mL, dilute with water is settled to scale, and concentration is 10.0mg/L.Draw respectively CPPU 0.5mL, 4-chlorophenoxyacetic acid 5mL, 2,4-dichlorphenoxyacetic acid 5mL, ethephon (CEPHA)，2-(chloroethyl) phosphonic acid 50mL, in 100mL volumetric flask, are diluted with water to scale.Join to such an extent that hybrid standard working solution concentration is concentration 6.Draw respectively hybrid standard working solution 2.0,1.0,0.2,0.1,0.02mL in the volumetric flask of one group of 10mL, water is settled to scale, obtains concentration and is the mixed standard solution of concentration 5, concentration 4, concentration 3, concentration 2, concentration 1, as shown in table 1:

Table 1

Three, detect

(1) sample pretreatment:

In plant growth regulator retention analysis, must take suitable extracting method, effectively to extract and not cause the residual variation of plant growth regulator in sample, extraction agent does not cause interference to follow-up mass spectrophotometry simultaneously.

Because the physicochemical property that variety classes plant growth regulator is residual is widely different, the selection of extracting solvent is the most important link of pretreatment process.Relatively methyl alcohol, acetone, acetonitrile be as the extraction efficiency of extracting solvent, and experimental result shows, the pigment in fruits and vegetables is soluble in acetone, cause certain interference, and methyl alcohol and acetonitrile has higher extraction efficiency to testing compound, extract pigment relatively less to analyzing.Take acetonitrile/water (50/50, V/V), acetonitrile/water (75/25, V/V), acetonitrile and methanol/water (50/50, V/V), methanol/water (75/25, V/V), methyl alcohol to extract, and its extraction efficiency is studied.Because ethephon (CEPHA)，2-(chloroethyl) phosphonic acid is stable below pH=3.0, more than pH=4.0, easily decompose and emit ethene, all extracts all regulate pH=3.0 with formic acid solution in advance.Compare by test, the present invention preferably uses acetonitrile as solvent.

In this specific embodiment, choose respectively at random from the market the each 200g of fruits and vegetables such as Kiwi berry, strawberry, loquat, mango, little orange, large orange, banana, red little tomato, yellow little tomato, large tomato, grape, bean sprouts, longan, apple, imperial crown pears, snow pear, cucumber, grape, get edible part and put into respectively beaker, homogenate 3min;

(2) extraction and cleaning:

Take respectively 15g sample after homogenate in 50mL centrifuge tube, add the 15mL acetonitrile (being adjusted to pH=3.0 with formic acid in advance) containing 1% acetic acid, after violent jolting 1min, add 6.0g anhydrous magnesium sulfate and 1.5g anhydrous sodium acetate, after violent jolting 1min, the centrifugal 3min of 4000rpm, gets 1mL supernatant and adds 150mg anhydrous magnesium sulfate and 50mg PSA, after violent jolting 1min, the centrifugal 3min of 4000rpm, get supernatant, cross after 0.22um filter membrane, obtain extract;

(3) extract purifying step being obtained enters Liquid Chromatography-Tandem Mass Spectrometry instrument and detects;

Testing result is as shown in table 2:

Table 2

In described step (3) Liquid Chromatography-Tandem Mass Spectrometry instrument instrumental analysis condition as follows:

Chromatographic separation condition is as follows:

Ammonium acetate+0.01% formic acid of mobile phase A: 5mmol/L; PH=5

Mobile phase B: methyl alcohol;

Flow velocity: 0.25mL/min

Gradient program is as shown in table 3:

Table 3

Mass spectrum condition is as follows:

Ion gun: electric spray ion source (ESI);

Detection mode: multiple-reaction monitoring (MRM);

Capillary voltage: 3.0kV;

Secondary taper hole voltage: 2V;

Lens voltage: 0.2V;

Ion source temperature: 110 DEG C;

Taper hole blowback air flow velocity: 50L/h;

Desolventizing temperature degree: 380 DEG C;

Desolventizing gas velocity: 600L/h;

Inferior quality is counted resolution: 13;

High-quality is counted resolution: 13;

Ion energy: 2.2;

Photomultiplier transit tube voltage: 650V;

Collision pond pressure: 1.0e-3;

The monitoring ion pair (m/z) of each plant growth regulator, taper hole voltage, collision energy, ionization mode are in table 4:

Table 4

Four, the range of linearity of method and detection limit analysis

Get the standard reserving solution of 4 plant growth regulators, with strawberry blank sample as matrix, be mixed with respectively mass concentration typical curve as shown in table 1, selected Liquid Chromatography-Tandem Mass Spectrometry instrument instrumental analysis condition under measure, measurement result is referring to Fig. 6-11, taking the peak area of testing compound as ordinate, the concentration of testing compound is that horizontal ordinate carries out regretional analysis, obtains equation of linear regression, the range of linearity and related coefficient in table 5.As shown in Table 3, the linear relationship of each compound is good, and correlation coefficient r value is all not less than 0.999.According to 3 times of signal-noise ratio computation method detection limits.Visible related coefficient is 0.999, is greater than 0.995, linear good.Detection limit is respectively 0.5 μ g/kg to 100 μ g/kg, meets the sensitivity requirement of national limit standard.

Table 5

。

Claims (1)

1. a plant growth regulator method for detecting residue in fruit, is characterized in that comprising following steps:

(1) sample pretreatment: take fruit sample, get edible part and put into beaker, homogenate 3min;

(2) extraction and cleaning: take 15g sample after homogenate in 50mL centrifuge tube, add the 15mL acetonitrile containing 1% acetic acid, be adjusted to pH=3.0 with formic acid in advance, after violent jolting 1min, add 6.0g anhydrous magnesium sulfate and 1.5g anhydrous sodium acetate, after violent jolting 1min, the centrifugal 3min of 4000rpm, get 1mL supernatant and add 150mg anhydrous magnesium sulfate and 50mg PSA, after violent jolting 1min, the centrifugal 3min of 4000rpm, gets supernatant, cross after 0.22um filter membrane, obtain extract;

(3) extract purifying step being obtained enters Liquid Chromatography-Tandem Mass Spectrometry instrument and detects;

In described step (3) Liquid Chromatography-Tandem Mass Spectrometry instrument instrumental analysis condition as follows:

Chromatographic separation condition is as follows:

Ammonium acetate+0.01% formic acid of mobile phase A: 5mmol/L; PH=5

Mobile phase B: methyl alcohol;

Flow velocity: 0.25mL/min

Gradient program:

Mass spectrum condition is as follows:

Ion gun: electric spray ion source (ESI);

Mass Spectrometer Method mode: multiple-reaction monitoring (MRM);

Capillary voltage: 3.0kV;

Ion source temperature: 110 DEG C;

Taper hole blowback air flow velocity: 50L/h;

Desolventizing temperature degree: 380 DEG C;

Desolventizing gas velocity: 600L/h;

Photomultiplier transit tube voltage: 650V;

The monitoring ion pair (m/z) of each plant growth regulator, taper hole voltage, collision energy, ionization mode:

。