This standard specifies the rapid test method for the determination of organophosphorus and carbamate pesticide residues in vegetables by enzyme inhibition method. This standard is applicable to the rapid screening and determination of organophosphorus and carbamate pesticide residues in vegetables. GB/T 5009.199-2003 Rapid detection of organophosphorus and carbamate pesticide residues in vegetables GB/T5009.199-2003 Standard download decompression password: www.bzxz.net

ICS67.040
National Standard of the People's Republic of China
GB/T5009.199-2003
Rapid determination for organophosphate andcarbamate pesticide residues in vegetables2003-08-11Issued
Ministry of Health of the People's Republic of China
Standardization Administration of the People's Republic of China
2004-01-01Implementation
GB/T5009.199—2003
The residues of organophosphate and carbamate pesticides in vegetables and the situations that have caused them are more prominent. The two test methods provided in this standard can quickly detect the residues of organophosphate and carbamate pesticides in vegetables, so as to find the problems in time, take measures, control the marketing of vegetables with high pesticide residues, and ensure the safety of people's food. This standard is proposed and managed by the Ministry of Health of the People's Republic of China. This standard was drafted by: Food Hygiene Supervision and Inspection Institute of the Ministry of Health, Beijing Product Quality Supervision and Inspection Institute, Guangdong Provincial Center for Disease Control and Prevention.
The main drafters of this standard are: Wang Lin, Wang Jing, Zhang Ying, Deng Feng, Yang Dajin. 570
1 Scope
Rapid detection of organophosphorus and carbamate pesticide residues in vegetables
GB/T5009.199—2003
This standard specifies the rapid detection method for the determination of organophosphorus and carbamate pesticide residues in vegetables by enzyme inhibition method. This standard is applicable to the rapid screening and determination of organophosphorus and carbamate pesticide residues in vegetables. Rapid test card method (paper strip method)bzxZ.net
2 Principle
Cholinesterase can catalyze the hydrolysis of indophenol acetate (red) into acetic acid and indophenol (blue). Organophosphorus or azomethine pesticides have an inhibitory effect on cholinesterase, which changes the catalytic, hydrolytic and discoloration processes. From this, it can be judged whether there is a high dose of organophosphorus or azomethine pesticides in the sample.
3 Reagents
3.1 Paper strips (rapid test cards) with cholinesterase and indophenol acetate reagents fixed on them. 3.2 pH 7.5 buffer solution: Take 15.0g of disodium hydrogen phosphate [NazHPO.·12H2O] and 1.59g of anhydrous potassium dihydrogen phosphate [KH,PO.] respectively, and dissolve them in 500mL of distilled water. 4 Instruments
4.1 Constant balance.
4.2 If conditions permit, equip with a 37℃±2℃ constant temperature device. 5 Analysis steps
5.1 Overall determination method
5.1.1 Select a representative vegetable sample, wipe off the soil on the surface, cut into pieces of about 1 cm square, take 5g and put it in a bottle with a lid, add 10mL buffer solution, shake 50 times, and let it stand for more than 2 minutes. 5.1.2 Take a rapid test card, dip the extract with a white tablet, and place it for more than 10 minutes for pre-reaction. If conditions permit, place it in a 37℃ constant temperature device for 10 minutes. The surface of the tablet after pre-reaction must be kept moist. 5.1.3 Fold the rapid test card in half, pinch it by hand for 3 minutes or use a constant temperature device for 3 minutes to make the red tablet and the white tablet overlap and react. 5.1.4 A blank control card with buffer solution should be set for each batch of determination. 5.2 Surface determination method (coarse screening method)
5.2.1 Wipe off the soil on the surface of the vegetable, drop 2 or 3 drops of buffer solution on the surface of the vegetable, and gently rub the dripping area with another piece of vegetable. 5.2.2 Take a rapid test card and drop the liquid on the vegetable onto the white tablet. 5.2.3 Place it for more than 10 minutes for pre-reaction. If conditions permit, place it in a 37℃ constant temperature device for 10 minutes. The surface of the tablet after the pre-reaction must be kept moist.
5.2.4 Fold the rapid test card in half, pinch it by hand for 3 minutes or use a constant temperature device for 3 minutes to make the red tablet and the white tablet overlap and react. 5.2.5 A blank control card with buffer solution should be set for each batch of determination. 571
CB/T5009.199-2003
6 Result determination
The result is expressed as whether the enzyme is inhibited by organophosphorus or carbamate pesticides (positive) or not inhibited (negative). Compared with the blank control card, the white tablet does not change color or is slightly light blue, which is a positive result. The white tablet turns sky blue or is the same as the blank control card, which is a negative result.
For samples with positive results, other analytical methods can be used to further determine the specific pesticide type and content. 7 Appendix
7.1 Technical indicators of rapid test cards
7.1.1 Sensitivity indicators: The detection limits of rapid test cards for some pesticides are shown in Table 1. Table 1 Detection limits of some pesticides
Pesticide name
Carbothion
Parathion
Isocarbophos
Malathion
Carbothion
Detection limit/(mg/kg)
Pesticide name
Acephate
Trichlorfon
Detection limit/mg/kg）
Pesticide name
Monocrotophos
Haoniandong
Furudan
Detection limit/(mg/kg）
7.1.2 Compliance rate: Among the more than 30 positive samples detected, the compliance rate of positive results should be more than 80% after verification by gas chromatography.
8 Instructions
8.1 Onion, garlic, radish, leek, celery, coriander, sunflower, fermented mushroom and tomato juice contain plant secondary metabolites that affect enzymes and are prone to false positives. When dealing with such samples, the whole plant (body) of vegetables can be extracted or the surface determination method can be used. For some vegetables with high chlorophyll content, the whole plant (body) of vegetables can also be extracted to reduce the interference of pigments. 8.2 When the temperature condition is lower than 37℃, the speed of enzyme reaction slows down accordingly. The time for the tablet to be placed in reaction after adding liquid should be relatively extended. The extension time should be determined by the blank control card. When it turns blue when pinched with fingers (body temperature) for 3 minutes, the operation can be carried out. Note that the sample placement time should be consistent with the blank control card placement time to be comparable. The reasons why the blank control card does not change color are: first, the buffer solution added to the tablet surface is small, the tablet surface after pre-reaction is not moist enough, and second, the suspension temperature is too low. 8.3 The time for the red tablet and the white tablet to overlap is 3 minutes. After 3 minutes, the blue color will gradually deepen, and after 24 hours, the color will gradually fade.
Enzyme inhibition rate method (spectrophotometry)
9 Principle
Under certain conditions, organophosphorus and carbamate pesticides have an inhibitory effect on the normal function of cholinesterase, and the inhibition rate is positively correlated with the concentration of the pesticide. Under normal circumstances, the enzyme catalyzes the hydrolysis of the neurotransmitter (acetylcholine), and its hydrolysis product reacts with the color developer to produce a yellow substance. The change in absorbance over time at 412nm is measured by a spectrophotometer to calculate the inhibition rate. The inhibition rate can be used to determine whether there is a high dose of organophosphorus or carbamate pesticides in the sample. 10 Reagents
10.1 pH 8.0 level flushing solution: Take 11.9g of anhydrous dipotassium hydrogen phosphate and 3.2g of potassium dihydrogen phosphate, respectively, and dissolve them in 1000mL of steamed stuffing water.
GB/T5009.199—2003
10.2 Color developer: Take 160mg dithiodinitrobenzoic acid (DTNB) and 15.6mg sodium bicarbonate, dissolve in 20mL buffer solution, and store in a 4℃ refrigerator.
10.3 Substrate: Take 25.0mg thioacetylcholine, dissolve in 3.0mL steamed stuffing water, shake well and store in a 4℃ refrigerator for later use. The shelf life shall not exceed two weeks.
10.4 Acetylcholinesterase: Dissolve in buffer solution according to the activity of the enzyme, and the absorbance change △A value in 3min should be controlled above 0.3. After shaking well, store in a 4℃ refrigerator for later use, and the shelf life shall not exceed four days. 10.5 The kit prepared with the above reagents can be used. The △A value of acetylcholinesterase should be controlled above 0.3. 11 Instruments
11.1 Spectrophotometer or corresponding measuring instrument. 11.2 Constant-weight balance.
11.3 Constant-temperature water bath or constant-temperature box.
12 Analysis steps
12.1 Sample processing: Select representative vegetable samples, rinse off the soil on the surface, cut into pieces about 1 cm square, take 1 g of sample, put it in a beaker or extraction bottle, add 5 mL of buffer solution, oscillate for 1 min to 2 min, pour out the extract, let it stand for 3 min to 5 min, and set aside. 12.2 Control solution test First add 2.5 mL of buffer solution to the test tube, then add 0.1 mL of enzyme solution and 0.1 mL of color developer, shake well and place at 37°C for more than 15 min (the control time for each batch of samples should be consistent). Add 0.1mL substrate is shaken evenly. At this time, the test solution begins to develop color reaction. It should be immediately placed in the instrument colorimetric cell and the absorbance change value △A after 3 minutes of reaction is recorded. 12.3 Sample solution test: First add 2.5mL sample extract to the test tube. Other operations are the same as the control solution test. Record the absorbance change value AA, after 3 minutes of reaction.
13 Result expression and calculation
13.1 Result calculation
See formula (1).
Inhibition rate (%) = [(AA.-AA,)/△A. J×100 Where:
AA. is the absorbance change value of the control solution after 3 minutes of reaction, and AA, is the absorbance change value of the sample solution after 3 minutes of reaction. 13.2 Result determination
The result is expressed as the degree of enzyme inhibition (inhibition rate). (1)
When the inhibition rate of the vegetable sample extract on the enzyme is ≥50%, it means that high doses of organophosphorus or carbamate pesticides are present in the vegetables, and the sample is positive. Samples with positive results need to be tested repeatedly for more than 2 times. For samples with positive results, other methods can be used to further determine the specific pesticide type and content. 14 Supplementary provisions
14.1 Technical indicators of the inhibition rate method
14.1.1 Sensitivity indicators: The detection limit of the enzyme inhibition rate method for some pesticides is shown in Table 2. 573
GB/T5009.199—2003
Pesticide name
Dichlorvos
Parathion
Phoxim
Methylamidophos
Malathion
Table 2 Technical indicators of the enzyme inhibition rate method for some pesticides Detection limit of pesticides Detection limit/(mg/kg)
Name of pesticide
Omethoate
Methyl isofenphos
Methomyl
Carbosulfan
Trichlorfon
Detection limit/(mg/kg)
14.1.2 Compliance rate: Among more than 30 samples with an inhibition rate of ≥50%, the compliance rate of positive results verified by gas chromatography should be above 80%.
15 Explanation
15.1 Onion, garlic, radish, leek, celery, coriander, sunflower, mushroom and tomato juice contain plant secondary metabolites that affect enzymes and are prone to false positives. When dealing with such samples, the whole plant (body) of vegetables can be extracted. For some vegetables with high chlorophyll content, the whole plant (body) of vegetables can also be extracted to reduce the interference of pigments. 15.2 When the temperature is lower than 37℃, the speed of enzyme reaction slows down. The reaction time after adding enzyme solution and color developer should be relatively extended. The extension time should be determined by the absorbance change △A of cholinesterase blank control test for 3 minutes. If the value is above 0.3, you can proceed. Note that the sample placement time should be consistent with the blank control solution placement time to be comparable. The absorbance change △A of cholinesterase blank control solution for 3 minutes is <0.3 because: first, the enzyme activity is not enough, and second, the temperature is too low. 574
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