CN104316632B - The detection method of remaining pesticide in animal derived food - Google Patents

Abstract

The invention belongs to technical field of food detection, disclose the detection method of remaining pesticide in a kind of animal derived food, step comprises: is mixed with by pesticide standard substance and singly marks solution, then need to be configured to mixed standard solution according to mensuration, carry out analyzing mensuration drawing standard curve in online gel permeation chromatography-Gas-phase acidity instrument; Get animal derived food sample homogenization, with acetonitrile and normal hexane mixed dissolution, supersound extraction; By extracting solution through column extractor, collection and purification liquid and elutriant, thickening filtration must treat sample measuring liquid; Get and treat that sample measuring liquid is infused in line gel permeation chromatography-Gas-phase acidity instrument, according to treating pesticide content situation choice criteria working fluid in sample measuring liquid, to standard working solution with treating that sample measuring liquid equal-volume ginseng injects sample and measures, contrast qualitative according to retention time, quantified by external standard method. Detection method of the present invention can sharp separation interfering substance, reduce detection limit, detected result is accurate, applied widely.

Description

The detection method of remaining pesticide in animal derived food

Technical field

The invention belongs to technical field of food detection, the detection method being specifically related in a kind of animal derived food remaining pesticide.

Background technology

" in food, remaining pesticide regulates scheme " that on August 1st, 2014, Hong Kong was formally implemented is Hong Kong is remain pesticide in nutrient fortified food to manage the New Measure taked. This is regulated scheme and relates to 360 kinds of pesticides altogether, residue limits (EMRL) of 7086 maximum residue limit(MRL) (MRL)/again, residue limits (EMRL) of 1735 group maximum residue limit(MRL) (MRL)/again, does not relate to the pesticide of residue limits (EMRL) of maximum residue limit(MRL) (MRL)/again and performs without exception " set(ting)value ". The limitation of 18 kinds of wherein common pesticides in animal derived food is as shown in table 1:

Table 1.18 kind pesticide regulates the limitation requirement in scheme in Hong Kong

The enforcement of " in food, remaining pesticide regulates scheme " will increase the residue detection project of import and export food, the examination criteria that simultaneously can highlight interior ground is unsound, the increase greatly of the workload of government regulation and monitoring department can be caused, also the inspection and quarantine cycle can be caused to lengthen, clearance speed slows down, and export cost increases. Excessive risk adds the operational cost of enterprise, high cost also loses the price advantage of existing product outlet, a large amount of interior ground food and agricultural-food finally can be caused can not normally to supply port, even can have influence on enough supplies of the food of Hongkong market, then have influence on the stable of entire society. At present, the inspecting standard that in animal derived food, the detection of turzolon residual quantity is also not relevant at home, the method of the remaining pesticide of existing inspection generally adopts GC, GC-MS, GC-MSMS, ultra-high efficiency liquid chromatography-tandem mass spectrometry method, the HPLC method of inspection and off-line tlc etc., there is the restriction that detection speed is slow, detected result is not accurate enough or subject range is less, therefore a kind of method that pesticide residual quantity remaining in animal derived food carries out mensuration fast is provided, to reinforcement food safety, significant.

Summary of the invention

In order to overcome the shortcoming of prior art and deficiency, it is an object of the invention to provide a kind of detection method detecting remaining pesticide in the animal derived food that speed is fast, accuracy of detection height, subject range are big.

The object of the present invention is achieved through the following technical solutions:

In animal derived food, the detection method of remaining pesticide, comprises the steps:

(1) preparation of standardized solution: pipette mixing pesticide standardized solution 1.000mL, by acetone diluted and be settled to 10mL, shake even, be prepared into the standard reserving solution that mass concentration is 1.000mg/L; Accurately draw standard reserving solution 3.0mL, 5.0mL, 10.0mL, 30mL, 50mL, be placed in 100mL volumetric flask respectively, by acetone diluted to scale, shake even, obtained mass concentration is 0.03,0.05,0.10,0.30, the standard working solution of 0.50mg/L; Get 10 �� L respectively and inject online gel permeation chromatography-Gas-phase acidity instrument, carry out analyzing mensuration; Taking the peak area of component as ordinate zou, mass concentration is X-coordinate, and drawing standard curve, with quantified by external standard method;

Preferably, the compound method of described mixing pesticide standardized solution is: use acetone that pesticide standard substance are mixed with the list mark solution of 100mg/L, is then acetone by volume ratio: the Compound mixed solution of hexanaphthene=3:7 becomes desired concn mixing pesticide standardized solution; It is mixed with extraction standard solution, to reduce matrix interference after process for preparation using blank sample process;

Preferably, described pesticide standard substance are cycloate, carbofuran, simazine, diazinon, Diacloden, methidathion, oxyfluorfen, Buprofezin, azoles grass ketone, dirty bacterium ester, norflurazon, propargite, pyrrole fluorine first standing grain spirit, carbosulfan, R-1582, ethyl quizalofop, fluridone or fenvalerate;

Preferably, the described condition measured of analyzing is:

Chromatographic separation condition: chromatographic column: gel chromatographic columns shodexclnpakev-200; Moving phase: volume ratio is acetone: the mixed solution of hexanaphthene=3:7; Flow velocity: 0.1mL/min; Sample size: 10 �� L; Post temperature: 40 DEG C;

GC-MS separation condition: analytical column: Rxi-5silMS (30m �� 0.25mm �� 0.25um); Chromatogram column temperature: 82 DEG C keep 5min, then with 8 DEG C/min temperature programming to 300 DEG C, keeps 7.75min; Injector temperature: 120 DEG C keep 5min, then with 80 DEG C/min temperature programming to 250 DEG C, keeps 33.7min; Temperature programming large volume sample injection; Carrier gas: helium, purity >=99.999%, flow velocity 1.75mL/min; Electron bombardment ionization source: 70eV; Ion source temperature: 200 DEG C; GC-MS interface temperature: 300 DEG C; Mass spectrometric detection mode: Select ion monitor pattern;

(2) extract: getting animal derived food sample, high-speed stirring cuts into homogenate; Weigh 3.0g homogenised sample in 50mL centrifuge tube, add 5mL water, 15mL acetonitrile and 3mL normal hexane, high-speed homogenization 2min, mixing whirlpool 1min, supersound extraction 30min, add 4g sodium-chlor, mixing whirlpool 1min, the centrifugal 10min of 8000rmp, robs with shifting liquid and pipettes acetonitrile layer 5mL, be liquid to be clean;

Described animal derived food sample comprises the fat, edible offal, meat, milk or the egg that obtain from domestic birds and animals;

(3) purify: activate column extractor with 3mL acetonitrile, then liquid to be clean for 5mL is passed through column extractor, use 6mL acetonitrile again, collection and purification liquid and elutriant are in the concentrated bottle of 25mL, 40 DEG C of rotary evaporated to dryness, adding 1mL acetonitrile dissolved residue, solution obtains treating sample measuring liquid after crossing 0.22 ��m of organic phase filter membrane;

Preferably, described column extractor is strataNH2 solid-phase extraction column;

(4) sample determination: get gained and treat sample measuring liquid 10 �� L, inject online gel permeation chromatography-Gas-phase acidity instrument, according to treating pesticide content situation in sample measuring liquid, the standard working solution that Response to selection value is close, standard working solution and treat that the response value of pesticide content in sample measuring liquid all should in the linearity range of instrument, to standard working solution with treating that sample measuring liquid equal-volume ginseng injects sample and measures, contrast qualitative according to retention time, quantified by external standard method;

(5) blank test: except not adding and treating sample measuring liquid, carries out blank test by said determination step.

In above-mentioned animal derived food in the detection method of remaining pesticide, pesticide content results is calculated as follows: $X=\frac{A\×C\×V}{\mathrm{As}\×m},$

In formula:

Pesticide residual quantity in X animal derived food sample, unit is mg/kg;

A treats the peak area of pesticide in sample measuring liquid;

The concentration of pesticide in C standard working solution, unit is mg/L;

The constant volume of the final sample liquid of V, unit is mL;

The peak area of pesticide in AS standard working solution;

M treats the quality of final sample representated by sample measuring liquid, and unit is g;

Note: calculation result need to deduct blank value.

Detection method of the present invention has the following advantages relative to prior art tool:

(1) decontamination effect improving of animal derived food testing sample is remarkable, can effectively remove macromole chaff interference;

(2) pesticide rate of recovery height, when interpolation concentration is within the scope of 0.030��0.30mg/kg, the rate of recovery is 60%��110%;

(3) to common pesticide, detectability is all low to moderate 0.030mg/kg, detects more sensitive;

(4) sensing range being suitable for is big, can effectively detect the residual quantity of multiple common pesticide in animal derived food.

Accompanying drawing explanation

Fig. 1 is the standard total ion chromatogram of pesticide;

Fig. 2 is sample blank total ion chromatogram;

Fig. 3 is that sample adds mark total ion chromatogram.

Embodiment

Below in conjunction with embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited to this.

Embodiment 1

The detection method of remaining pesticide in a kind of animal derived food, instrument and equipment used is: Shimadzu online gel permeation chromatography-Gas-phase acidity instrument, Sigma whizzer, BUCHI Rotary Evaporators, Waters solid-phase extraction device, strataNH2 (500mg/3cc), IKA refiner, filter membrane, analytical balance (sense amount 0.0001g and 0.01g), vortex oscillator, ultrasound bath pot;

Unless otherwise specified, agents useful for same is analytical pure, and water is the one-level water that GB/T6682 specifies, acetone, that hexanaphthene is high performance liquid chromatography is pure.

Described detection method specifically comprises the steps:

(1) preparation of standardized solution: pipette mixing pesticide standardized solution 1.000mL, by acetone diluted and be settled to 10mL, shake even, be prepared into the standard reserving solution that mass concentration is 1.000mg/L; Accurately draw standard reserving solution 3.0mL, 5.0mL, 10.0mL, 30mL, 50mL, be placed in 100mL volumetric flask respectively, by acetone diluted to scale, shake even, obtained mass concentration is 0.03,0.05,0.10,0.30, the standard working solution of 0.50mg/L; Get 10 �� L respectively and inject online gel permeation chromatography-Gas-phase acidity instrument, carry out analyzing mensuration; Taking the peak area of component as ordinate zou, mass concentration is X-coordinate, and drawing standard curve, with quantified by external standard method;

The compound method of described mixing pesticide is: use acetone that pesticide standard substance are mixed with the list mark solution of 100mg/L, is then acetone by volume ratio: the Compound mixed solution of hexanaphthene=3:7 becomes desired concn mixing pesticide standardized solution; It is mixed with extraction standard solution, to reduce matrix interference after process for preparation using blank sample process;

Described pesticide standard substance are cycloate, carbofuran, simazine, diazinon, Diacloden, methidathion, oxyfluorfen, Buprofezin, azoles grass ketone, dirty bacterium ester, norflurazon, propargite, pyrrole fluorine first standing grain spirit, carbosulfan, R-1582, ethyl quizalofop, fluridone and fenvalerate, totally 18 kinds;

The described condition measured of analyzing is:

Chromatographic separation condition: chromatographic column: gel chromatographic columns shodexclnpakev-200; Moving phase: volume ratio is acetone: the mixed solution of hexanaphthene=3:7; Flow velocity: 0.1mL/min; Sample size: 10 �� L; Post temperature: 40 DEG C;

GC-MS separation condition: analytical column: Rxi-5silMS (30m �� 0.25mm �� 0.25um); Chromatogram column temperature: 82 DEG C keep 5min, then with 8 DEG C/min temperature programming to 300 DEG C, keeps 7.75min; Injector temperature: 120 DEG C keep 5min, then with 80 DEG C/min temperature programming to 250 DEG C, keeps 33.7min; Temperature programming large volume sample injection; Carrier gas: helium, purity >=99.999%, flow velocity 1.75mL/min; Electron bombardment ionization source: 70eV; Ion source temperature: 200 DEG C; GC-MS interface temperature: 300 DEG C; Mass spectrometric detection mode: Select ion monitor pattern;

18 kinds of pesticides standard substance Select ion monitor pattern (SIM) monitor ion and retention time as shown in table 2:

Table 2.SIM monitors ion and retention time

Sequence number	Chinese	Quota ion	Retention time	Qualitative ion
					1	Cycloate	154.2	17.15	215.00-155.00
2	Carbofuran	164.1	18.71	149.10-221.10
					3	Simazine	201.1	18.83	186.10-173.10
4	Diazinon	304.2	19.40	137.10-179.20
					5	Diacloden	212.1	22.68	182.10-247.00
6	Methidathion	145.0	23.55	125.00-302.00
					7	Oxyfluorfen	252.0	24.65	300.00-361.10
8	Buprofezin	105.1	24.68	172.10-305.20
					9	Azoles grass ketone	312.1	26.10	340.20-330.10
10	Dirty bacterium ester	116.1	26.21	131.10-222.10
					11	Norflurazon	303.1	26.30	145.00-173.00
12	Propargite-1	135.1	26.83	173.20-350.20
						Propargite-2	135.1	26.86	173.10-350.20
13	The spirit of pyrrole fluorine first standing grain	02.1	26.98	272.10-316.10
					14	Carbosulfan	118.1	27.39	160.20-323.30
15	R-1582	132.1	28.62	160.00-104.00
					16	Ethyl quizalofop	299.1	31.34	372.20-243.10
17	Fluridone	328.2	31.89	329.20-310.20
					18	Fenvalerate-1	167.1	32.26	225.10-419.20
	Fenvalerate-2	167.1	32.53	225.10-419.20

As shown in Figure 1, sample blank TIC schemes as shown in Figure 2 the standard of 18 kinds of pesticides total chromatography of ions (TIC) figure, and sample adds mark TIC schemes as shown in Figure 3;

The linear relationship that step (1) measures gained 18 kinds of pesticides is as shown in table 3:

The linear relationship of table 3.18 kind pesticide

(2) extract: getting animal derived food sample, high-speed stirring cuts into homogenate; Weigh 3.0g homogenised sample in 50mL centrifuge tube, add 5mL water, 15mL acetonitrile and 3mL normal hexane, high-speed homogenization 2min, mixing whirlpool 1min, supersound extraction 30min, add 4g sodium-chlor, mixing whirlpool 1min, the centrifugal 10min of 8000rmp, robs with shifting liquid and pipettes acetonitrile layer 5mL, be liquid to be clean;

Described animal derived food sample comprise obtain from domestic birds and animals fat, edible offal, meat, milk or egg, such as the fat of poultry, goat, pig, ox, horse, the meat of Mammals, liver, milk, eggs etc. can be eaten;

(3) purify: activate column extractor with 3mL acetonitrile, then liquid to be clean for 5mL is passed through column extractor, use 6mL acetonitrile again, collection and purification liquid and elutriant are in the concentrated bottle of 25mL, 40 DEG C of rotary evaporated to dryness, adding 1mL acetonitrile dissolved residue, solution obtains treating sample measuring liquid after crossing 0.22 ��m of organic phase filter membrane;

Owing to animal derived food containing fat usually, then to be carried out Solid-Phase Extraction column extracting. Select the column extractor of OasisHLB60mg/3cc, FlorisilPR1000mg/ml, Sep-PakVacC18500mg/3cc, Sep-PakVacAluminaN500mg/3cc, strataNH2500mg/3cc five kinds of opposed polarities, interpolation concentration is 0.3mg/kg, carrying out extraction experiments and measure the rate of recovery, the rate of recovery of different solid-phase extraction column is shown in Table 4:

The different solid-phase extraction column of table 4. is the rate of recovery of 0.3mg/kg in interpolation concentration

From table 3 rate of recovery comparative analysis it will be seen that the rate of recovery that obtains of solid-phase extraction column strataNH2 is higher and stable, the pesticide being suitable in extraction animal derived food of the present invention most;

(4) sample determination: get gained and treat sample measuring liquid 10 �� L, inject online gel permeation chromatography-Gas-phase acidity instrument, according to treating pesticide content situation in sample measuring liquid, the standard working solution that Response to selection value is close, standard working solution and treat that the response value of pesticide content in sample measuring liquid all should in the linearity range of instrument, to standard working solution with treating that sample measuring liquid equal-volume ginseng injects sample and measures, contrast qualitative according to retention time, quantified by external standard method;

(5) blank test: except not adding and treating sample measuring liquid, is undertaken by said determination step.

In above-mentioned animal derived food in the detection method of remaining pesticide, pesticide content results is calculated as follows: $X=\frac{A\×C\×V}{\mathrm{As}\×m},$

In formula:

Pesticide residual quantity in X animal derived food sample, unit is mg/kg;

A treats the peak area of pesticide in sample measuring liquid;

The concentration of pesticide in C standard working solution, unit is mg/L;

The constant volume of the final sample liquid of V, unit is mL;

The peak area of pesticide in AS standard working solution;

M treats the quality of final sample representated by sample measuring liquid, and unit is g;

Note: calculation result need to deduct blank value.

Getting the sample of dove meat, fresh hen egg, milk and pork fat, carry out the detection operation of aforesaid method, obtain various pesticide TIANZHU XINGNAO Capsul in the sample to which, result is as shown in table 5:

The TIANZHU XINGNAO Capsul (unit: %) of table 5.18 kind pesticide in different media

Sequence number	Chinese	Dove meat	Fresh hen egg	Milk	Pork fat
						1	Cycloate	73.33	66.67	70.00	64.00
2	Carbofuran	80.00	56.67	66.67	63.33
						3	Simazine	83.33	96.67	100.00	83.33
4	Diazinon	73.33	93.33	96.67	70.00
						5	Diacloden	80.00	100.00	76.67	93.33
6	Methidathion	116.67	120.00	130.00	116.67
						7	Oxyfluorfen	93.33	100.00	96.67	80.00
8	Buprofezin	76.67	86.67	96.67	63.33
						9	Azoles grass ketone	93.33	96.67	110.00	90.00
10	Dirty bacterium ester	80.00	90.00	86.67	86.67
						11	Norflurazon	83.33	90.00	90.00	86.67
12	Propargite	103.33	93.33	116.67	83.33
						13	The spirit of pyrrole fluorine first standing grain	76.67	90.00	93.33	80.00
14	Carbosulfan	60.00	73.33	76.67	73.33
						15	R-1582	53.33	86.67	90.00	83.33
16	Ethyl quizalofop	86.67	100.00	100.00	86.67
						17	Fluridone	86.67	83.33	86.67	86.67
18	Fenvalerate	76.67	70.00	106.67	70.00

As can be seen from Table 5, in interpolation concentration within the scope of 0.030-0.30mg/Kg, the rate of recovery is between 60%-110%.

Getting same food samples, the absolute difference of the twice independent measurement result obtained under repeatability condition must not exceed the 10% of arithmetical av.

Above-described embodiment is that the present invention preferably implements mode; but embodiments of the present invention are not restricted to the described embodiments; the change done under the spirit of other any the present invention of not deviating from and principle, modification, replacement, combination, simplification; all should be the substitute mode of equivalence, it is included within protection scope of the present invention.

Claims (2)

1. the detection method of remaining pesticide in animal derived food, it is characterised in that comprise the steps:

(1) preparation of standardized solution: pipette mixing pesticide standardized solution 1.000mL, by acetone diluted and be settled to 10mL, shake even, be prepared into the standard reserving solution that mass concentration is 1.000mg/L; Accurately draw standard reserving solution 3.0mL, 5.0mL, 10.0mL, 30mL, 50mL, be placed in 100mL volumetric flask respectively, by acetone diluted to scale, shake even, obtained mass concentration is 0.03,0.05,0.10,0.30, the standard working solution of 0.50mg/L; Get 10 �� L respectively and inject online gel permeation chromatography-Gas-phase acidity instrument, carry out analyzing mensuration; Taking the peak area of component as ordinate zou, mass concentration is X-coordinate, and drawing standard curve, with quantified by external standard method;

The compound method of described mixing pesticide standardized solution is: use acetone that pesticide standard substance are mixed with the list mark solution of 100mg/L, is then acetone by volume ratio: the Compound mixed solution of hexanaphthene=3:7 becomes desired concn mixing pesticide standardized solution; It is mixed with extraction standard solution, to reduce matrix interference after process for preparation using blank sample process;

Described pesticide standard substance are cycloate, carbofuran, simazine, diazinon, Diacloden, methidathion, oxyfluorfen, Buprofezin, azoles grass ketone, dirty bacterium ester, norflurazon, propargite, pyrrole fluorine first standing grain spirit, carbosulfan, R-1582, ethyl quizalofop, fluridone or fenvalerate;

The described condition measured of analyzing is:

Chromatographic separation condition: chromatographic column: gel chromatographic columns shodexclnpakev-200; Moving phase: volume ratio is acetone: the mixed solution of hexanaphthene=3:7; Flow velocity: 0.1mL/min; Sample size: 10 �� L; Post temperature: 40 DEG C;

GC-MS separation condition: analytical column: Rxi-5silMS30m �� 0.25mm �� 0.25 ��m; Chromatogram column temperature: 82 DEG C keep 5min, then with 8 DEG C/min temperature programming to 300 DEG C, keeps 7.75min; Injector temperature: 120 DEG C keep 5min, then with 80 DEG C/min temperature programming to 250 DEG C, keeps 33.7min; Temperature programming large volume sample injection; Carrier gas: helium, purity >=99.999%, flow velocity 1.75mL/min; Electron bombardment ionization source: 70eV; Ion source temperature: 200 DEG C; GC-MS interface temperature: 300 DEG C; Mass spectrometric detection mode: Select ion monitor pattern;

(2) extract: getting animal derived food sample, high-speed stirring cuts into homogenate; Weigh 3.0g homogenised sample in 50mL centrifuge tube, add 5mL water, 15mL acetonitrile and 3mL normal hexane, high-speed homogenization 2min, mixing whirlpool 1min, supersound extraction 30min, add 4g sodium-chlor, mixing whirlpool 1min, the centrifugal 10min of 8000rmp, pipettes acetonitrile layer 5mL with shifting liquid rifle, is liquid to be clean;

(3) purify: activate column extractor with 3mL acetonitrile, then liquid to be clean for 5mL is passed through column extractor, use 6mL acetonitrile again, collection and purification liquid and elutriant are in the concentrated bottle of 25mL, 40 DEG C of rotary evaporated to dryness, adding 1mL acetonitrile dissolved residue, solution obtains treating sample measuring liquid after crossing 0.22 ��m of organic phase filter membrane;

Described column extractor is strataNH2 solid-phase extraction column;

(4) sample determination: get gained and treat sample measuring liquid 10 �� L, inject online gel permeation chromatography-Gas-phase acidity instrument, according to treating pesticide content situation in sample measuring liquid, the standard working solution that Response to selection value is close, standard working solution and treat that the response value of pesticide content in sample measuring liquid all should in the linearity range of instrument, to standard working solution with treating that sample measuring liquid equal-volume ginseng injects sample and measures, contrast qualitative according to retention time, quantified by external standard method;

(5) blank test: except not adding and treating sample measuring liquid, carries out blank test by said determination step.

2. the detection method of remaining pesticide in animal derived food according to claim 1, it is characterised in that: the described animal derived food sample of step (2) is the fat, edible offal, meat, milk or the egg that obtain from domestic birds and animals.