CN105203661A - Method for detecting various phthalic acid ester plasticizers in vegetables, fruits and edible mushrooms at same time - Google Patents

Abstract

The invention provides a method for detecting various phthalic acid ester plasticizers at the same time. The method includes the steps that firstly, a sample is extracted and purified in an optimized QuEChERS pretreatment mode; secondly, analysis is carried out in a gas chromatography-tandem mass spectrometry mode; finally, accurate qualitative and quantitative analysis is carried out with an isotope internal standard method. The method can be applied to detecting twenty-three phthalic acid ester plasticizers in plant source character foods such as common Chinese cabbages, tomatoes, strawberries, grapes, shiitake mushrooms and agaric at the same time. By means of the method, the plasticizers introduced through external source ways are avoided to the maximum degree, the pretreatment step is simple, consumed time of solid-phase extraction and purification is shortened, and consumed solvents and argent compound losses of the solid-phase extraction and purification are reduced; the method has the advantages of being sensitive, accurate, good in stability, short in consumed time and easy to popularize; the method can be suitable for detecting mass samples at the same time.

Description

A kind of method simultaneously detecting multiple phthalic ester plasticizer in veterinary antibiotics, edible fungi

Technical field

The present invention relates to a kind of method detecting multiple phthalic ester plasticizer in veterinary antibiotics, edible fungi, belong to agricultural product security detection technique field.

Background technology

Phthalic ester (phthalicacidestersPAEs) is the important industrial chemicals of a class, belongs to large industrial goods, and according to ASSOCIATE STATISTICS display, the output of the annual phthalate material in the whole world is up to 6,000,000 tons.Its purposes is very extensive, almost all industrial products as agricultural chemicals, chemical fertilizer, cosmetics, medicine, lubricant, coating, etc. in the middle of all containing PAEs, but it is mainly used as the property-modifying additive of plastics, to increase plasticity and the pliability of product, improve the intensity of product, so be referred to as plastifier.Because PAEs is not combined with chemical bond form with polyolefins plastic matrix, therefore very easily free out, from plastics diffusion mobility to soil, the surrounding medium such as water body and air.PAEs compounds is also a kind of environmental hormone compounds simultaneously, its chemical constitution is made up of 1 rigid plane aromatic ring and 2 plastic, non-linear aliphatic side chainses, the feature such as there is large, water-soluble low, the difficult volatilization of mobility, solidifying point is low and be hydrolyzed in the environment, water splitting is extremely slow, easily long-term in surrounding medium exist and enter food chain by vegeto-animal bioconcentration, in interference blood, the normal level of hormone, affects biological reproduction, growth and behavior.Research shows that PAEs and metabolic product thereof have endocrine to animal used as test, Reproductive and developmental toxicity can be produced, immunotoxicity, neurotoxicity, there is teratogenesis, carcinogenic and mutagenic effect, and wherein the most troubling and the most serious harm be that it is on the endocrine of animal and on the phylogenetic impact of reproduction of male animal.In " priority pollutant list " is listed 6 kinds of phthalic esters such as repefral (DMP), diethyl phthalate (DEP), the positive dibutyl ester of phthalic acid (DBP) by ring Environmental Protection Agency of the U.S. (EPA), and DMP, DBP and phthalic acid two (2-ethyl) own ester (DEHP) are also classified as screen priority pollutants by China.

In recent years PAEs was originated to " plastifier " great food safety affair disturbance of Taiwan generation in 2011 in the concern of field of food, rise in May, 2011 in the food of Taiwan and successively detect 6 kinds of phthalic ester plasticizer compositions such as DEHP, DBP, DMP, DEP, to on June 8th, 2011, Taiwan announcement is detected plasticizer-containing food and has reached 961 kinds, relates to nearly 300 of manufacturer.Subsequently, domesticly also in the middle of white wine, beverage, edible oil, toy etc., PAEs class plastifier is detected.Whether current China defines " non-polluted farm product ", be just to remain or some heavy metal substances containing agricultural and veterinary chemicals, and ignores as this kind of environmental hormone class material human body being had to harm of PAEs.In view of taking place frequently of above-mentioned food safety affair, and along with the toxicity of researchers to PAEs class material and the further investigation of harm, in the past two years in agricultural product security field, also start gradually to pay close attention to PAEs class plastifier.

Hot house is a kind of important form of industrialized agriculture, utilizes hot house to carry out vegetable growing, have per unit area yield high, by advantages such as seasonal effect are little, special vital role is served to the disparities between supply and demand alleviating vegetables dull seasons.Hot house and plastic film coated a large amount of uses, make As time goes on, hydrogen bond weak between PAEs and plastic molecules or van der Waals interaction and very easily free out, migrate in the middle of air in booth, irrigation water or sedimentation, by soil absorption, add the content of PAEs in soil.Because PAEs belongs to semi-volatile organic compounds, the approach of vegetables uptake and accumulation PAEs in hot house, has two kinds of viewpoints in research collection of reports both domestic and external: in (1) soil, PAEs is absorbed by root system of plant and migrates to cauline leaf.PAEs class plastifier can enter xylem through root cortex, accumulates on axis by the effect of root hair cell, or arrives leaf portion by translocation and accumulate; (2) because the Henry'S coefficient of PAEs is low, boiling point is high and steam forces down, can only evaporate in air slowly, and the PAEs be present in air can be adsorbed on particle consumingly, then by greenhouse plants cauline leaf uptake and accumulation.According to the planting type of Some Fruits and edible fungi in current industrialized agriculture, such as current more widely used mulching planting strawberry, emerges strawberry, equal plastic-film-covered cultivation in growth cycle that SPRING WHEAT BEFORE AND AFTER FLOWERING is longer.The fruit such as grape, peach of bagging plantation (be mainly paper bag, part is containing the film of plastic components), general bagging time is longer, at young fruit 7-10 days before plucking in period of Fruit physiology shedding.By the impact of sunshine, temperature, PAEs class plastifier likely moves within its fruit growth cycle, enrichment; And edible fungus culturing adopts at present, and plastics are planted, the technology of plastic bottle cultivation is comparatively general.For mushroom plastic bag cultivation, moist heat sterilization is carried out subsequently with the packed culture medium raw material of filling out of acrylic plastering, the PAEs migration of plasticizer is likely entered culture medium raw material by the environment of long-time High Temperature High Pressure, after this thalline inoculation, mycelial growth, fruit body development, fruiting flower bud, each stage in maturity stage all absorb nutrient from nutrient culture media, the likely enrichment in its growth cycle of PAEs class plastifier; After de-bag, throughout the growing season there is covered rearing with plastic film; After gathering, most with plastics fresh-keeping packaging, in whole cultivation, recovery process, all likely there is migration in PAEs class plastifier.

At present, in agricultural product, the mensuration of PAEs class plastifier residual quantity there is no standard method, standard GB/T/T21911-2008 defines containing fatty foods sample with not containing the standard determination method of PAEs in fatty foods sample, specific aim is not strong, because PAEs class plastifier belongs to liposoluble substance, solubleness in water is very low, the effect of mechanical shaking extraction of adding water is inevitable not good, and sample is not purified, this is for improper the common Chinese cabbage etc. that matrix interference is more serious is rich in chlorophyllous vegetable sample.The pre-treating method that domestic researcher studies PAEs in vegetables is at present general with reference to EPA3550B standard, and instrumental method adopts gas chromatography list quadrupole rod mass spectrum (GC-MS) analytical approach, with reference to EPA8270C method.For GC-MS analytical approach, often plant compound due to PAEs class plastifier and substantially all contain identical ion m/z=149, if sample substrate is comparatively complicated, there is interference and make chromatographic resolution not good, with regard to likely only according to Selective ion mode scanning result and occur judge by accident situation.The method about mensuration PAEs class plastifier delivered on foreign periodical pays close attention to field of food more, such as, in the middle of edible oil, wine, beverage etc., little to the pre-treating method reference of the plant-derived agricultural product such as veterinary antibiotics and edible fungi.

Summary of the invention

The object of the present invention is to provide a kind of method simultaneously detecting multiple phthalic ester plasticizer, the method comprises: first extract sample and purify, then detect, finally by the residual quantity of plastifier in Internal standard Accurate Determining sample through gas chromatography tandem mass spectrum (GC-MS/MS) analysis.

This method is applicable to detecting multiple plastifier in the veterinary antibiotics such as common Chinese cabbage, tomato, strawberry, grape, mushroom and auricularia auriculajudae and edible fungi simultaneously.

Specifically, a kind of method simultaneously detecting multiple plastifier that the present invention describes, comprises the following steps:

(1) extraction of sample and purification:

Sample after homogeneous pulverizing adds example weight 1-2 acetonitrile doubly, quick vortex 15-30s, and wherein the unit of weight portion is g, and the unit of parts by volume is mL;

Add the anhydrous sodium sulfate of example weight 0.5-1.5 times weight and the sodium chloride of 0.1-0.5 times of weight more fast, violent jolting 20-40s, vortex 1-2min, centrifugal 15-25min under 1500-3000r/min rotating speed;

Then centrifuged supernatant certain volume is got in the tool plug glass centrifuge tube being weighed into certain proportion purification filler, vortex 1-2min, centrifugal 10-20min under 3000-6000r/min rotating speed; Wherein, described purification filler is at least two kinds of compositions of anhydrous magnesium sulfate, N-propyl group ethylenediamine (PSA) and Graphon (GCB), and its ratio is 1:4-8:0.1-0.5;

Then get centrifuged supernatant, add a certain amount of Isotopic Internal Standard solution, at 36-50 DEG C, nitrogen blows near dry;

Finally redissolve with normal hexane, load sample injection bottle after vortex mixing and treat machine analysis, on concrete Isotopic Internal Standard, machine concentration is the D4-DEHP of 50ng/mL;

(2) gas chromatography tandem mass spectrum (GC-MS/MS) is analyzed:

The condition that wherein gas chromatography is separated is: the low pole chromatographic column adopting ThermoTG-5MS (30m*0.25mm*0.25 μm) or same nature, temperature programme, Splitless injecting samples and constant current mode; Carrier gas is high-purity helium of pure degree≤99.99%, injector temperature 280 DEG C, heating schedule is initial column temperature 60 DEG C (1min), with 20 DEG C/min ramp to 220 DEG C (0min), again with 5 DEG C/min ramp to 280 DEG C, keep 3min;

Mass spectrum part by multiple-reaction monitoring pattern (SRM) piecewise acquisition and accurately qualitative, quantitative, qualitative, the quota ion of 23 kinds of phthalic ester plasticizers and interior mark D4-DEHP to parameters such as, the collision energies optimized in table 1;

(3) Isotopically labelled internal standard:

Adopt Internal standard to set up the typical curve of various phthalic ester plasticizer, calculated the concentration of each component to be measured to be measured by instrument software quantitative function, the content of each plastifier to be measured in right back-pushed-type (1) calculation sample.

$\mathrm{\ω}=\frac{C*V}{m}$ Formula (1)

In formula:

ω---the content of component to be measured in sample, unit is ng/kg, μ g/kg;

C---measure the concentration of component to be measured in liquid, unit is nanograms per milliliter, ng/mL;

V---extract liquor capacity, unit is milliliter, mL;

M---sample sample weighting amount, unit is gram, g;

The gas chromatography tandem mass spectrometer that wherein the present invention is used is GC-MS/MS, ThermoTSQ8000, other

Similar triple quadrupole bar tandem mass spectrometer is also suitable for.

The chromatographic retention of table 1:23 kind phthalic ester plasticizer and interior mark D4-DEHP, qualitative, quota ion to and the collision energy optimized

A kind of method simultaneously detecting multiple phthalic ester plasticizer of the present invention can be applied in the veterinary antibiotics such as common Chinese cabbage, tomato, strawberry, grape, mushroom, auricularia auriculajudae and edible fungi to be extracted while 23 kinds of plastifier, purify and detects.The present invention has short, sensitive and accurate feature consuming time compared with other detection method, detects while being applicable to batch samples.The material can introducing plastifier in environmental system is a lot, the method whole process avoids the pollution introducing plastic products, the QuEChERS pre-treating method improved is simple, the glass centrifuge tube adopted is after alkaline rinse soaking flushing is clean, can reuse in 180 DEG C of baking 3-4h, and such as the pollution of DBP, DIBP, DEHP of several plastifier the most easily detected can reduce to below 5ng/mL, make testing result more reliable, the situation introducing plastifier in full-range solvent blank and matrix blank sample investigation environmental system in process of the test, can be designed.The disposable extraction column cost costly that do not adopted disposable glass Solid-Phase Extraction column purification both to save, again saves solvent cost and time cost.

Embodiment

Embodiment one

Get common Chinese cabbage, tomato, the grape of plantation in the hot house of production base, the strawberry sample of covering with plastic film plantation, and with the mushroom of polybag cultivation and auricularia auriculajudae sample in Edible Fungi base, after paper bag sampling, carry out analysis according to following operating process respectively and detect.

Various sample is respectively charged in rustless steel container, at a high speed pulverizes homogeneous samples with handheld stirring machine, load preserve in refrigerator that vial is built in-20 DEG C to be measured.

After sample takes out and returns to room temperature, take 10g sample and be placed in 40mL point base thickness wall glass centrifuge tube (customized), add 10mL acetonitrile, quick vortex 15s.Add rapidly 5g anhydrous sodium sulfate and 1g sodium chloride (weigh up in advance and be placed in measuring cup) again, vortex 1min after hand quick oscillation 30s immediately.The object of this method of operating is: add salt again add organic solvent in sample after, can avoid exothermic effect better like this, thus avoids the recovery affecting test analyte.Centrifugal 15min under the rotating speed of 1000rpm/min;

Supernatant 5mL is pipetted in the 15mL spiral cover glass centrifuge tube being weighed into purification filler, vortex mixing 1min with glass pipet.Centrifugal 10min under the rotating speed of 4000rpm/min;

Supernatant 2mL is pipetted in glass test tube again with glass pipet, draw with 100 μ L liquid spectrum sample introduction needle, add the inner mark solution (D4-DEHP) of the 1.0mg/L of 0.1mL, after vortex mixing, nitrogen blows near dry, redissolve with 2mL normal hexane, proceed to sample injection bottle after vortex and treat the analysis of GC-MS/MS sample introduction, inside putting on machine concentration is 0.05mg/L.

Gas chromatography tandem mass spectrometer is Thermo, TSQ8000 triple quadrupole bar tandem mass spectrum.

Gas chromatography separation condition: the low pole chromatographic column adopting ThermoTG-5MS (30m*0.25mm*0.25 μm) or same nature, temperature programme, Splitless injecting samples and constant current mode; Carrier gas is high-purity helium of pure degree≤99.99%, flow rate of carrier gas is 1.2mL/min, injector temperature 280 DEG C, heating schedule is initial column temperature 60 DEG C (1min), with 20 DEG C/min ramp to 220 DEG C (0min), again with 5 DEG C/min ramp to 280 DEG C, keep 3min, sample working time is 24min.Sampling volume is 1 μ L;

Triple quadrupole bar tandem mass spectrum instrument parameter is: transmission line temperature 280 DEG C, ion source temperature 280 DEG C, filament current 50 μ A, by multiple-reaction monitoring pattern (SRM) piecewise acquisition also accurate qualitative, quantitative;

Further method is verified, comprises following a few partial content:

(1) typical curve: the 23 kinds of plastifier bought from German Dr.Ehrenstorfer company and a kind of deuterated-DEHP have the standard items normal hexane of certificate to be mixed with the standard reserving solution of 1000mg/L respectively, again with 23 kinds of plastifier mixed standard solution (wherein DNP of 25mL volumetric flask preparation 20-80mg/L, DINP, these 3 kinds of plasticizer concentrations of DIDP are 80mg/L, it is 4 times of all the other plasticizer concentrations 20mg/L), with this solution for mother liquor, 0.001 is made into respectively with the bare substrate gradient dilution of various sample, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, serial mixed standard solution (the wherein DNP of 1.0mg/L, DINP, these 3 kinds of plasticizer concentrations of DIDP are correspondingly 0.004, 0.008, 0.02, 0.04, 0.08, 0.2, 0.4, 0.8, 2.0, 4.0mg/L), the Isotopic Internal Standard solution D 4-DEHP consistent with adding concentration in sample test liquid is added in the mixed standard solution of each concentration, the machine concentration of making also is 0.05mg/L.Adopt the typical curve that matrix mark-on internal standard method is set up, in the corresponding range of linearity, all plastifier all reach well linear in different matrix, coefficient R ²>=0.99.

(1) sensitivity: by the sensitivity of hybrid standard product solution with the method assay method of above-mentioned vehicle solution gradient dilution, detecting of method is limited between 0.001 ~ 0.02mg/L.

(2) accuracy (TIANZHU XINGNAO Capsul): to add the recovery that target mode investigates method in each matrix sample, 6 concentration Pitch-based sphere are set, each concentration level 6 parallel sample, adding concentration is 0.005mg/kg, 0.01mg/kg, 0.02mg/kg, 0.05mg/kg, 0.2mg/kg, 0.5mg/kg (20 kinds), and other 3 kinds of DNP, DINP, DIDP are correspondingly 0.02mg/kg, 0.04mg/kg, 0.08mg/kg, 0.2mg/kg, 0.8mg/kg, 2.0mg/kg.Wherein front 4 low concentration levels are for investigating the quantitative limit of method, meet the qualitative requirement of method, quantitative and qualitative analysis ion pair all presents the peak reaching signal to noise ratio (S/N ratio) and require, the minimum interpolation concentration level that TIANZHU XINGNAO Capsul and precision meet the demands is the quantitative limit of method.All the other two is middle and high concentration, and the scope of test findings statistics TIANZHU XINGNAO Capsul is 70.5 ~ 120.3%.

(3) precision: the precision scope of 23 kinds of plastifier in common Chinese cabbage, tomato, strawberry, grape, mushroom, auricularia auriculajudae matrix is respectively 1.2 ~ 26.3%, 0.8 ~ 16.7%, 2.1 ~ 18.5%, 1.1 ~ 19.2%, 2.6 ~ 17.6%, 1.8 ~ 19.4%.

Method of the present invention can be disposable simultaneously to 23 kinds of accurate qualitative, quantitatives of phthalic ester plasticizer, and adopt QuEChERS pretreatment mode easy to be quick, reliable and stable, provides cost savings and analysis time.

Claims (6)

1. detect a method for multiple plastifier simultaneously, it is characterized in that: said method comprising the steps of: use paper bag collected specimens, hand-held mixer pulverizes homogeneous samples in rustless steel container, load freezen protective sample in vial; Extraction and cleaning is carried out by QuEChERS pretreatment mode after weighing sample; On scavenging solution, machine is by gas chromatography Tandem Mass Spectrometry Analysis; With multiple plastifier in Internal standard Accurate Determining sample.

2. the method simultaneously detecting multiple plastifier according to claim 1, is characterized in that:

(1) extraction of sample and purification:

Sample after homogeneous pulverizing adds acetonitrile, quick vortex; Add anhydrous sodium sulfate and sodium chloride fast again, violent jolting; Then centrifuged supernatant certain volume is got in the tool plug glass centrifuge tube being weighed into certain proportion purification filler, vortex, centrifugal; Then get centrifuged supernatant, add a certain amount of Isotopic Internal Standard solution D 4-DEHP, blow in nitrogen near dry; Finally redissolve with normal hexane, load sample injection bottle after vortex mixing and treat machine analysis;

(2) gas chromatography tandem mass spectrum (GC-MS/MS) is analyzed: mass spectrum part adopts multiple-reaction monitoring pattern (SRM) piecewise acquisition;

(3) Isotopically labelled internal standard: adopt Internal standard to set up the typical curve of various phthalic ester plasticizer, quantitatively calculate the concentration of each component to be measured, the content of each plastifier to be measured in calculation sample.

3. the method simultaneously detecting multiple plastifier according to claim 1 and 2, it is characterized in that: described purification filler is at least two kinds of compositions of anhydrous magnesium sulfate, N-propyl group ethylenediamine (PSA) and Graphon (GCB), and its ratio is 1:4-8:0.1-0.5.

4. the method simultaneously detecting multiple plastifier according to claim 1 and 2, is characterized in that: described plastifier is phthalate.

5. the method simultaneously detecting multiple plastifier according to claim 1 and 2, is characterized in that: the application of described method on veterinary antibiotics, edible fungi or other field crop.

6. the method simultaneously detecting multiple phthalic ester plasticizer according to claim 1 and 2, is characterized in that: described concrete detected parameters is

(1) extraction of sample and purification:

Sample after homogeneous pulverizing adds example weight 1-2 acetonitrile doubly, quick vortex 15-30s, and wherein the unit of weight portion is g, and the unit of parts by volume is mL; Add the anhydrous sodium sulfate of example weight 0.5-1.5 times weight and the sodium chloride of 0.1-0.5 times of weight more fast, violent jolting 20-40s, vortex 1-2min, centrifugal 15-25min under 1500-3000r/min rotating speed;

Then centrifuged supernatant certain volume is got in the tool plug glass centrifuge tube being weighed into certain proportion purification filler, vortex 1-2min, centrifugal 10-20min under 3000-6000r/min rotating speed; Wherein, described purification filler is at least two kinds of compositions of anhydrous magnesium sulfate, N-propyl group ethylenediamine (PSA) and Graphon (GCB), and its ratio is 1:4-8:0.1-0.5; Then get centrifuged supernatant, add a certain amount of Isotopic Internal Standard solution D 4-DEHP, at 36-50 DEG C, nitrogen blows near dry; Finally redissolve with normal hexane, load sample injection bottle after vortex mixing and treat machine analysis;

(2) gas chromatography tandem mass spectrum (GC-MS/MS) is analyzed: the condition that wherein gas chromatography is separated is: the low pole chromatographic column adopting ThermoTG-5MS (30m*0.25mm*0.25 μm) or same nature, temperature programme, is not separated sample introduction and constant current mode; Carrier gas is high-purity helium of pure degree≤99.99%, and mass spectrum part adopts multiple-reaction monitoring pattern (SRM) piecewise acquisition;

(3) Isotopically labelled internal standard: adopt Internal standard to set up the typical curve of various phthalic ester plasticizer, quantitatively calculate the concentration of each component to be measured, the content of each plastifier to be measured in calculation sample.