GB 31658.18-2022National Food Safety Standard - Determination of Triazine Residues in Animal Foods - High Performance Liquid Chromatography (English PDF)

5.1 Reagents

5.1.1 Methanol (CH3OH). chromatographically pure.

5.1.2 Formic acid (HCOOH). chromatographically pure.

5.1.3 Ammonium formate (CH5NO2). chromatographically pure.

5.1.4 Ammonia (NH3.H2O).

5.1.5 Glacial acetic acid (CH3COOH).

5.2 Solution preparation

5.2.1 60% acetonitrile solution. Take 60mL of acetonitrile, add 40mL of water, and mix well.

5.2.2 0.5% acetic acid in acetonitrile solution. Take 2.5 mL of glacial acetic acid, dilute to 500 mL with acetonitrile, and mix well.

5.2.3 5% ammonia solution. take 5mL of ammonia water, add 95mL of water, and mix well.

5.2.4 Acetic acid methanol solution (pH 7.0). Take 6 mL of glacial acetic acid, add 94 mL of methanol, mix well, adjust the pH to 7.0 +/- 0.05 with ammonia water.

5.2.5 0.02mol/L ammonium formate solution (pH4.0). take 1.26g of ammonium formate, add 900mL of water to dissolve, adjust the pH to 4.0+/-0.0 with formic acid

0.05, dilute with water to 1000mL.

5.3 Standards

Diacetamide triazine amidine (Diminazene acetic acid, C14H15N7.2C4H7NO3, CAS number. 908-54-3), content >= 91%.

5.4 Preparation of standard solution

5.4.1 Standard stock solution. Take an appropriate amount of diacetamide triazine standard (equivalent to about 10 mg of triazine), weigh it accurately, add water to dissolve

and dilute to a 10mL brown volumetric flask, and prepare a standard stock solution of triazine with a concentration of 1mg/mL. Store in the dark at 4°C, and the validity period is

5.5 Materials

5.5.1 Weak cation exchange solid phase extraction column. 60mg/3mL, or equivalent.

5.5.2 Microporous nylon filter membrane. 0.22 µm.

6.2 Analytical balance. Sensitivity 0.00001g and 0.01g.

6.3 pH meter.

6.4 Vortex mixer.

6.5 Vortex shaker.

6.6 High-speed refrigerated centrifuge. speed >= 10000r/min.

6.7 Solid phase extraction device.

7.1 Preparation of samples

7.1.1 Organization

Take an appropriate amount of fresh or thawed blank or test tissue, mince it, and homogenize it.

a) Take the homogeneous test sample as the test sample;

b) Take a homogeneous blank sample as a blank sample;

c) Take a homogeneous blank sample, add a standard solution of appropriate concentration, and add a sample as a blank.

7.1.2 Milk

Take an appropriate amount of fresh or thawed blank or test milk sample, and mix well.

a) Take the uniformly mixed test sample as the test sample;

b) Take a uniformly mixed blank sample as a blank sample;

c) Take a uniformly mixed blank sample, add a standard solution of appropriate concentration, and add a sample as a blank.

7.2 Storage of samples

Store below -18°C.

8.1 Extraction

8.1.1 Organization

Take 2g of the sample (accurate to +/-0.05g), put it in a 50mL polypropylene centrifuge tube, add 18mL of 60% acetonitrile solution, vortex for 1min, and store it on ice.

Adjust the pH to 5.5+/-0.1 with acetic acid, shake for 10 min, centrifuge at 10,000 r/min for 5 min, take the supernatant, and dilute it with 60% acetonitrile solution to

20.0mL, mix well, take 2.0mL (muscle samples take 10.0mL), set aside.

8.1.2 milk

Take 5g of the sample (accurate to +/-0.05g), put it in a 50mL polypropylene centrifuge tube, add 10mL of 0.5% acetic acid acetonitrile solution accurately, vortex

1 min, shake for 10 min, centrifuge at 10000r/min for 10 min, remove all the supernatant, and set aside.

8.2 Purification

Take a weak cation-exchange solid-phase extraction column, and activate it with 3mL of methanol and 3mL of water in turn. Take the spare liquid to pass through the column, and then dissolve it with 5% ammonia water in turn.

Wash with 3 mL of methanol solution and 3 mL of methanol, drain, add 1.0 mL of acetic acid methanol solution (pH 7.0), elute, drain, collect eluate, pass through microporous nylon

filter membrane for determination by high performance liquid chromatography.

8.3 Preparation of standard curve

Precisely measure an appropriate amount of standard working solution, dilute it with acetic acid-methanol solution (pH 7.0), and prepare a concentration of 0.05 µg/mL, 0.1 µg/mL,

0.2µg/mL, 0.5µg/mL, 1µg/mL, 2µg/mL, 5µg/mL series of standard solutions, passed through microporous nylon filter membrane, for HPLC

Chromatographic determination. With triazine chromatographic peak area as the ordinate and standard solution concentration as the abscissa, draw a standard curve. Find the regression equation and

correlation coefficient.

8.4 Determination

8.4.1 Reference conditions for liquid chromatography

a) Chromatographic column. C18 column (250mmx4.6mm, particle size 5µm), or equivalent;

b) Mobile phase. A is 0.02mol/L ammonium formate solution (pH4.0); B is methanol;

c) Gradient elution. see Table 1 for elution conditions;

d) Column temperature. 30°C;

e) Injection volume. 20 µL;

f) Detection wavelength. 370nm.

8.4.2 Determination method

Take the sample solution and the corresponding standard solution for single-point or standard curve calibration, and calculate the peak area according to the external standard method. Standard working solution and sample

The response value of triazine in the solution should be within the linear range of instrument detection. The retention time of triazine in the test sample and the retention time of triazine in the standard solution

The relative deviation of the retention time should be within +/-2.5%. The chromatogram of the standard solution is shown in Appendix A.

8.5 Blank test

Take the blank sample, except that no drug is added, the exact same determination steps are used for determination.

10.1 Sensitivity

The detection limit of this method in beef and mutton is 0.05 mg/kg, and the limit of quantification is 0.1 mg/kg. In bovine liver, bovine kidney, goat liver and goat kidney

The limit of detection was 0.2 mg/kg, the limit of quantification was 0.5 mg/kg, the limit of detection in cow and goat milk was 0.01 mg/kg, the limit of quantification was

0.02mg/kg.

10.2 Accuracy

This method is effective in beef and mutton 0.1mg/kg~1mg/kg, beef liver and sheep liver 0.5mg/kg~24mg/kg, beef kidney and sheep kidney

0.02mg/kg~0.3mg/kg for cow's milk and goat's milk, the recoveries were 60%~110%.

10.3 Precision

In this method, the relative standard deviation within the batch was <=10%, and the relative standard deviation between batches was <=10%.