LS/T 6138-2020 Grain and oil inspection. Determination of aflatoxin in grains. Immunomagnetic bead purification ultra-high performance liquid chromatography

This standard was drafted in accordance with the rules given in GB/T 1.

This standard was 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, National Grain and Material Reserve Bureau Standard Quality Center, Beijing Dongfu Jiuheng Instrument Technology Co.

The main drafters of this standard: Wang Songxue, Ye Jin, Xuan Zhihong, Yang Weimin, Li Keqiang, Zhang Zhihang, Zhou Minghui, Wang Pei, Wu Xianshao, Yang Jun, Ji Jiansheng, Li Xiaoming, Hu Bin, Liu Ying, Xie Tingting, Tian Guojun, Han Xiao , Xu Chunfeng, Li Jing, Shao Zhiling, Qiu Qingfeng, Li Weixiang, Lu Anxiang, Zhao Hongwei, Zhao Huanyu

Release time: January 21, 2020;

Implementation time: July 21, 2020

For more details, please click: " LS/T 6138—2020 Cereals and Oils Inspection.

1 Scope

This standard specifies the principles, reagents and materials for the determination of aflatoxins B ₁ , B ₂ , G ₁ , G ₂ (AFB ₁ , AFB ₂ , AFG ₁ , AFG ₂ ) in grain by immunomagnetic bead purification ultra-high performance liquid chromatography .

This standard is applicable to the determination of AFB ₁ , AFB ₂ , AFG ₁ and AFG ₂ in grain and oil samples such as wheat, corn, rice and vegetable oil

The detection limit of AFB1 in this standard is 0.

2 Normative references

The following documents are indispensable for the application of this document

GB/T 5490 General Rules for Grain and Oil Inspection

GB/T 5491 Inspection of Grain and Oilseeds: Sampling and Separating Method

GB/T 6682 Analytical laboratory water specifications and test methods

3 Principle

The aflatoxin in the sample was extracted by methanol-water mixture, and the supernatant was purified and enriched by immunomagnetic beads, then analyzed by ultra-high performance liquid chromatography and quantified by external standard method

4 Reagents and materials

Unless otherwise specified, the reagents used in this method are all analytically pure, and the experimental water should meet the first-level water requirements specified in GB/T 6682

4.

4.

4.
3 Sodium chloride
.

4.
4 Disodium hydrogen phosphate
.

4.
5 Potassium dihydrogen phosphate
.

4.
6 Potassium chloride
.

4.
7 Hydrochloric acid
.

4.
8 TritonX-100[C ₁₄ H ₂₂ O(C ₂ H ₄ O) _n ] (or Tween-20, C ₅₈ H ₁₁₄ O ₂₆ )
.

4.
9 Methanol-water mixture 70: 30, the volume fraction of 70 mL methanol is added to 30 mL water, and mix
.

4.
10 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 with 900 mL of water, adjust the pH to 7.
4 with hydrochloric acid, and dilute to 1000 mL with water
.

4.
11 0.
1% TritonX-100 (or Tween-20) PBS solution (hereinafter referred to as PBST): Take 1 mL of TritonX-100 (or Tween-20) and dilute to 1,000 mL with PBS
.

4.
12 Standard product

Aflatoxin B1 , B2, G1, G2 solid powder (purity>99%) or standard solution certified by the country and awarded with a certificate of standard material
.

4.
13 Preparation of standard solution

4.
13.
1  Mixed standard solution

Accurately pipette 4 standard aflatoxins (4.
12) of a certain volume or quality into a 10 mL volumetric flask to prepare aflatoxins B1, B2, G1, G2, and the concentrations are respectively 100 mg/L and 30 mg/L , 100 mg/L, 30 mg/L, dilute to the mark with methanol.
This solution is sealed and stored at -20 °C and protected from light.
The validity period is 6 months
.

4.
13.
2 Standard series of working solutions

Accurately pipet 25 μL, 50 μL, 100 μL, 250 μL, 500 μL, and 1,000 μL of 4 kinds of aflatoxin mixed standard solutions (4.
13.
1) into 10 mL volumetric flasks, and dilute to the mark with methanol.
The concentration of the prepared standard curve solution is shown in Table 1.
The solution is sealed and stored at 4°C and protected from light.
The validity period is 1 week
.

4.
14 Plastic centrifuge tubes (2.
0 mL, 10 mL, 50 mL)
.

4.
15 Glass inner cannula (400 μL)
.

4.
16 Chromatography injection bottle (2 mL)
.

4.
17 Volumetric flask (10 mL)
.

4.
18 Aflatoxin immunomagnetic beads pretreatment kit: recovery rate ≥85%
.

Note: For different batches of kits, quality verification is required before use
.

5 Apparatus and equipment

5.
1 Balance: Sensitivity 0.
1 mg and 0.
01 g
.

5.
2 Crusher: The motor speed is ≥1000 r/min, which can make all the samples pass through a 20-mesh sieve after being crushed
.

5.
3 Centrifuge: speed ≥6 000 r/min
.

5.
4 Vortex mixer
.

5.
5 Oscillator (oscillation frequency ≥50 times)
.

5.
6 A set of single-channel pipetting guns (the maximum volume is 20 μL, 100 μL, 200 μL, and 1,000 μL respectively)
.

5.
7 Dispenser (range 10~100 mL)
.

5.
8 Magnetic rod and magnetic rod cover or automatic purifier containing magnetic rod and magnetic rod cover
.

5.
9 Ultra performance liquid chromatography-fluorescence detector (with general volume flow cell or large volume flow cell)
.

Note: When a large-volume flow cell is used, no post-column derivatizer of any model or method is required
.

5.
10 Post-column derivatizer
.

6 Analysis steps

6.
1 Sampling and sub-sampling

According to GB/T 5491, during the sampling process, pay attention to prevent sample contamination, and take a representative sample of at least 1 kg
.

6.
2 Sample preparation

6.
2.
1 Liquid samples (vegetable oil)

Mix all liquid samples thoroughly in a container and store them in a sample bottle for later use
.

6.
2.
2 Solid samples (wheat, corn, rice, etc.
)

Use a pulverizer (5.
2) to crush the sample until it passes through a 20-mesh sieve, mix it thoroughly, and store it in a sample bottle for later use
.

6.
3 Extraction and purification

Accurately weigh 5 g (accurate to 0.
01 g) of the sample in a 50 mL centrifuge tube, add 20 mL of extraction solution (4.
9), vortex for 20 min, then centrifuge at 6,000 r/min for 5 min, and draw 0.
5 mL of supernatant Put the solution in a 10 mL reaction tube, add 4.
5 mL PBST (4.
11) and mix well, add 100 μL of aflatoxin immunomagnetic beads and oscillate the reaction for 5 min, separate by magnetic rod (5.
8) for 30 s, then transfer the immunomagnetic beads To the 2 mL reaction tube containing 1 mL PBST (4.
11), oscillate for 1 min.
After 30 seconds of magnetic separation by the magnetic rod, transfer the immunomagnetic beads to the 2 mL reaction tube containing 1 mL PBS (4.
10) and oscillate for 1 min.
After 30 s of magnetic separation, the immunomagnetic beads were transferred to a 2 mL reaction tube containing 0.
5 mL methanol (4.
2) and oscillated for 1 min.
After 30 s of magnetic separation, the immunomagnetic beads were discarded, and methanol was collected for sample injection.
The bottle is ready for injection
.

Note 1: When using immunomagnetic beads of different manufacturers, the operation may be different, and the operation should be carried out according to the requirements of the instructions for use
.

Note 2: The mycotoxin automatic purification instrument can be used after optimizing the operating parameters
.

7 Determination

7.
1 Reference conditions for liquid chromatography

7.
1.
1 Derivative-free method (direct detection with large flow cell)

Chromatographic reference conditions:

Chromatographic column: C18 column (column length 50 mm or 100 mm, column inner diameter 2.
1 rrm, packing particle size 1.
7 μm).
Or equivalent performance
.

Fluorescence detection wavelength: excitation wavelength 360 nm; emission wavelength 440 nm
.

Mobile phase: A: methanol, B: water
.
Isocratic elution, A: B = 40: 60
.

Flow rate: 0.
2 mL/min
.

Column temperature: 30 °C
.

Injection volume: 2 μL
.

7.
1.
2 Post-column Derivative Method

Chromatographic reference conditions:

Chromatographic column: C18 column (column length 50 mm or 100 mm, column inner diameter 2.
1 mm, packing particle size 1.
7 μm).
Or equivalent performance
.

Fluorescence detection wavelength: excitation wavelength 360 nm; emission wavelength 440 nm
.

Mobile phase: A: methanol, B: water
.
Isocratic elution, A: B=40:60
.

Flow rate: 0.
2 mL/min
.

Column temperature: 30 °C
.

Injection volume: 2 μL
.

Post-column derivative
.

7.
2 Preparation of standard curve

The standard working solution of aflatoxin (4.
13.
2) from low concentration to high concentration is injected into liquid chromatography for detection and analysis
.
Taking the concentration of aflatoxin standard working solution as the abscissa and the corresponding chromatographic peak area as the ordinate, the standard linear regression equation is obtained
.

7.
3 Determination of sample solution

The response value of the test compound in the test sample solution (6.
3) should be within the linear range of the standard curve, and the sample with a concentration exceeding the linear range should be diluted and re-injected for analysis
.

8 Result calculation

The content of aflatoxin in the sample is calculated according to formula (1):

Where:

X ——The content of AFB1, AFB2, AFG1 or AFG2 in the sample, in micrograms per kilogram (μg/kg);

c ——The content of AFB1, AFB2, AFG1 or AFG2 in the sample measured from the standard curve, the unit is nanogram per milliliter (ng/mL);

V1——The volume of the extract, the unit is milliliters (mL);

V2——The volume of the extract used for dilution, in milliliters (mL);

V3——Volume of diluent used for dilution, in milliliters (mL);

V4——Volume of eluent used for immunomagnetic beads, in milliliters (mL);

m——The weight of the sample, the unit is grams (g)
.

The calculation result is expressed as the arithmetic average of two independent measurement results obtained under repeatability conditions, with 3 significant figures reserved
.
When the measurement result does not meet the repeatability requirement, the measurement shall be re-measured according to the provisions of GB/T 5490 and the result shall be calculated
.

9 Precision

9.
1 Repeatability

The absolute difference between the two independent test results obtained under repeatability conditions is less than or equal to the repeatability limit (r), which should be greater than 95%.
The repeatability limit for aflatoxin B1, B2, G1, G2 content (r ) see Appendix
B .

9.
2 Reproducibility

The absolute difference between the two independent test results obtained under the reproducibility condition is less than or equal to the reproducibility limit (R) should be greater than 95%.
The reproducibility limit of the content of aflatoxin B1, B2, G1, G2 (R) ) see Appendix
B .

Appendix A (informative appendix)

Ultra performance liquid chromatogram of aflatoxin standard solution

Appendix B (informative appendix)

Statistical results of inter-laboratory comparison experiments

Organize 6 laboratories to conduct inter-laboratory comparison experiments on 6 different samples, and establish the precision of this method.
The results are statistically analyzed according to GB/T 6379.
1 and GB/T 6379.
2.
The statistical results are shown in Table A.
1 and Table A.
.
2
.