LS/T 6139-2020 Grain and oil inspection Rapid qualitative detection of organophosphorus and carbamate pesticide residues in grains and their products

This standard was drafted in accordance with the rules given by GB/T1.

This standard is proposed by the State Bureau of Grain and Material Reserves

This standard is under the jurisdiction of the National Cereals and Oils Standardization Technical Committee (SAT/TC 270)

Drafting organizations of this standard: National Grain and Material Reserve Bureau Scientific Research Institute, Guangdong Provincial Grain Science Research Institute, Shandong Grain and Oil Testing Center, Shaanxi Grain and Oil Product Quality Supervision and Inspection Center, China Grain Reserve Management Group Co.

The main drafters of this standard: Ye Jin, Wang Songxue, Wang Yajun, Wang Pei, Jiang Guozhen, Li Keqiang, Guqin, Ji Lanyang, Hu Bin, Qiu Qingfeng, Sun Tinglin, Kang Ran, Niu Mengyu, Li Hua

Release time: January 21, 2020;

Implementation time: July 21, 2020

For more details, please click "LS/T 6139-2020 Grain and Oil Inspection Rapid Qualitative Detection of Organophosphorus and Carbamate Pesticide Residues in Grain and Its Products "

1 Scope

This standard specifies the principles, reagents and materials, instruments and equipment, sample preparation and result analysis of the rapid qualitative detection of organophosphorus and carbamate pesticide residues in grains and their products

This standard applies to the rapid qualitative detection of organophosphorus and carbamate pesticide residues in grains and their products

The detection limit of this standard, see Appendix A

2 Normative references

The following standards are indispensable for the application of this document

GB 2763 National Food Safety Standard Maximum Residue Limits of Pesticides in Food

GB/T 5491 Grain and oilseed inspection samples and sampling method

GB/T6682 Analytical laboratory water specifications and test methods

3 Principle

The organophosphorus and carbamate pesticide residues in the sample inhibit the reaction of insect esterase and enzyme receptor, the fluorescent substrate is activated and the fluorescence signal is weakened, and the organophosphorus and carbamate in the sample are calculated by the change of the fluorescence signal The enzyme inhibition rate of the pesticide is compared with the corresponding limit enzyme inhibition rate, so as to quickly and qualitatively determine whether the organophosphorus and carbamate pesticides in the sample exceed the standard

4 Reagents and materials

Unless otherwise specified, the reagents used in this method are all analytically pure-water is secondary water specified in GB/T 6682

4.

4.

4.
3 Anhydrous sodium sulfate
.

4.
4 Ethyl acetate and acetone solution (95:5 volume ratio): Take 5 mL of acetone and add 95 mL of ethyl acetate, and mix well
.

4.
5 Sodium sulfate solution: Take 200 g of anhydrous sodium sulfate, dissolve in water and dilute to 1,000 mL, and mix
.

4.
6 Phosphate buffer solution (hereinafter referred to as PBS): Weigh 8.
00 g sodium chloride, 1.
20 g disodium hydrogen phosphate (or 2.
92 g disodium hydrogen phosphate dodecahydrate), 0.
20 g potassium dihydrogen phosphate, 0.
20 g potassium chloride, dissolve it with 900 ml water, adjust pH to 7.
4 ± 0.
1 with hydrochloric acid, and dilute to 1000 ml with water
.

4.
7 1% TritonX-100 (or Tween-20) in PBS: Take 10 ml TritonX-100 (or Tween-20) and dilute to 100 ml with PBS
.

4.
8 Insect esterase solution ¹⁾ : Add 30.
0 mg of insect esterase to PBS and mix well
.
It is provided as a complete set of pesticide residue rapid detection products, or prepared according to its instructions
.

4.
9 Luciferase receptor solution ¹⁾ : Take 0.
5 mg of luciferase receptor into 10 mL of water, and mix
.
It is provided as a complete set of pesticide residue rapid detection products, or prepared according to its instructions
.

4.
10 Fluorescent substrate solution ¹⁾ : Take 1.
0 mg of fluorescent substrate and add 10 ml of ¹ % TritonX-100 (or Tween-20) PBS, and mix well
.
It can be provided as a complete set of pesticide residue rapid detection products, or formulated according to its instructions
.

5 Apparatus and equipment

5.
1 Balance: a sense of 0.
01 g
.

5.
2 Crusher: The motor speed is ≥1 000 r/mn, which can make all the samples pass through a 20 mesh (0.
850 mm) sieve after being crushed
.

5.
3 Drying instrument: temperature ≥ 4 5 ℃
.

5.
8 Vortex oscillator
.

5.
5 Centrifuge: Speed ​​A 4 5 0 0 o/mn o

5.
6 Centrifuge tube: 10 ml 
.

5.
7 Detection microtube with screw cap: 1.
5 mL 
.

5.
8 Incubator: temperature ≥ 35 ℃
.

5.
9 Fluorescence detector: excitation wavelength 460 nm ~ 550 nm, emission wavelength 590 nm 
.

6 Sample preparation

6.
1 Sampling and sub-sampling

Execute in accordance with GL/T 5491
.

6.
2 Sample crushing

Use a crusher (5.
2) to crush the sample until it passes through a 20-mesh sieve and mix well
.

6.
3 Sample extraction

Weigh 0.
5 g of the sample (accurate to 0.
01g) into a 10 mL centrifuge tube (5.
6), add 2 mL of sodium sulfate solution (4.
5), and then add 2 mL of ethyl acetate-acetone solution (4.
4) ), mix well
.
Centrifuge at 4 500 r/min for 2 min, transfer 100 μL of supernatant to a 2 mL centrifuge tube, and blow at 45 ℃ until nearly dry
.
1 mL of water reconstituted solution test solution A
.

Note: The sample extraction of different manufacturers may be slightly different, and the operation should be carried out according to the method specified in the product instruction
.

6.
4 Measurement procedure

Accurately pipet 100 μL of test solution A (6.
3) into the detection microtube (5.
7), then add 50 μL of insect esterase solution (4.
8), and mix well
.
Place the microtube in a 35° incubator (5.
8) and incubate for 10 min
.
Add 50 μL luciferase receptor solution (4.
9) to the bottom of the microtube and mix well
.
Place the microtube in an incubator (5.
8) at 35°C and incubate for 5 min
.
Then add 1 mL of fluorescent substrate solution (4.
10) to the microtube, and read the data with a fluorescence detector (5.
9)
.

Note 1: The addition of the fluorescent substrate solution (4.
10) is completed within 30 seconds to avoid air bubbles affecting the detection
.

Note 2: The sample measurement of different manufacturers may be slightly different, and the operation should be carried out in accordance with the method specified in the product instruction
.

¹⁾ Obtained from Charm Sciences, Inc.
This information is provided only to facilitate the use of this standard, not to recognize the brand.
Any product with the same technical parameters and performance can be used
.

6.
5 Blank test

Except that no sample is added, perform parallel operations in accordance with the provisions of 6.
3 ~ 6.
4
.

7 Result analysis

7.
1 Result calculation

The calculation result is expressed in terms of enzyme inhibition rate, calculated according to formula (1):

Where:

y ——enzyme inhibition rate, %;

I ₀ ——Blank test response value;

I ₁ ——Response value of the sample to be tested
.

The calculation result retains 3 significant digits
.

7.
2 Judgment of results

7 .
2 .
1 Select the target pesticide type

Determine the type of target pesticides based on information such as the purpose of the test or the type of sample matrix, source, and production area pesticide residue monitoring and pesticide application guidelines
.

7 .
2 .
2 Determine the inhibitory rate of the judgement enzyme

According to the limit value of the target pesticide and the corresponding limit enzyme inhibition rate, the lowest limit enzyme inhibition rate is selected as the judgement enzyme inhibition rate
.

7 .
2 .
3 Judgment method

7.
2.
3.
1 When the enzyme inhibition rate of the sample is higher than the judgement enzyme inhibition rate, it means that there may be high-dose organophosphorus or carbamate pesticide residues in the sample, and the sample is judged to be a suspected positive sample; for suspected positive For samples, reference methods should be used to determine the types and contents of specific pesticides
.

7.
2.
3.
2 When the enzyme inhibition rate of the sample is lower than the judgement enzyme inhibition rate, it means that the organophosphorus and carbamate pesticide residues in the sample are low, and the sample is judged as a negative sample
.

Note: The report should indicate which pesticides are suspicious or negative
.

Appendix A (Normative Appendix)

Method detection limit

Appendix B (informative appendix)

Calculation of limited enzyme inhibition rate

Based on the maximum residue limit of pesticides in GB 2763 food, the blank matrix limit concentration was added to determine the inhibition rate of each sample.
Six parallel samples were tested every day for 3 d.
The limit enzyme inhibition of the pesticide under the matrix The rate is calculated according to formula (B.
1)
.

Where:

y _{MRL ——Limited} enzyme inhibition rate, %;

y _m ——the average value of the inhibition rate of the blank matrix limit concentration spiked;

S—standard deviation
.

The intraday relative standard deviation should be less than 15%, and the intraday relative standard deviation should be less than 25%
.

Appendix C (informative appendix)

Limit enzyme inhibition rate of some common pesticide residues