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5.
Operation steps
(1) Sample extraction Weigh 2g (accurate to 0.
01g) of the sample into a 50mL plastic centrifuge tube with a stopper, add 15mL trichloroacetic acid solution and 5mL acetonitrile , ultrasonically extract for 10 minutes, and then shake for 10 minutes, and then extract at least 4000r Centrifuge for 10 min at /min
.
After the supernatant is filtered through filter paper moistened with trichloroacetic acid solution, the volume is adjusted to 25 mL with trichloroacetic acid solution, and 5 mL of the filtrate is removed, and 5 mL of water is added to mix well and used as the solution to be purified
Note: If the fat content in the sample is high, you can use the liquid-liquid partition of n-hexane saturated with trichloroacetic acid solution to degrease and then use the SPE column for purification
.
(2) Add 3mL methanol and 3mL water to the sample purification solid-phase extraction column in advance to adjust through the small column, discard the effluent, and then add the diluted solution to be purified.
After all the samples pass through the small column, use 3mL water and 3mL water in sequence.
Wash the solid phase extraction column with 3 mL methanol and pump it to near dryness.
Add 6 mL ammoniated methanol solution to the solid phase extraction column for elution.
Collect the eluate and blow dry with nitrogen at 50°C.
The residue (equivalent to 0.
4g sample) Use 1mL mobile phase to make the volume constant, vortex and mix for 1min, and pass through a microporous membrane for measurement
.
(3) Determination by liquid chromatograph
①Instrument conditions
Chromatographic column: C 8 column, 250mm×4.
6mm(id), 5um, or equivalent; C 18 column, 250mm×4.
6mm(id), 5um, or equivalent
.
Mobile phase: C 8 column, ion pair reagent buffer-acetonitrile (85+15, volume ratio), mix; C 18 column, ion pair reagent buffer-acetonitrile (90+10, volume ratio), mix well
②Standard linearity: Use mobile phase to dilute the melamine standard stock solution step by step to obtain a standard working solution with a concentration of 0.
8, 2, 20, 40, and 80ug/mL.
The concentration is from low to high for sampling detection, and the peak area-concentration is used as the peak area-concentration.
Figure, get the standard curve regression equation
.
③Quantitative determination: The response value of melamine in the sample solution to be tested should be within the linear range of the standard curve.
If it exceeds the linear range, it should be diluted and then injected for analysis
.
6.
Result calculation
The content of melamine in the sample is calculated by the chromatographic data processing software or the following formula
.
Where x——the content of melamine in the sample, mg/kg
A——The peak area of melamine in the sample solution, cm 2
p——The concentration of melamine in the standard solution, ug/mL
V——The final constant volume of the sample solution, mL
A s ——The peak area of melamine in the standard solution, cm 2
m——the mass of the sample, g
f-dilution factor
7.
Tips
(1) The flow rate of the solid phase extraction column should not be too fast, otherwise it will affect the purification effect.
(2) Recovered quality control samples should be added when necessary to monitor the quality of the test.
(3) C 18 and C 8 chromatographic columns are reversed phase columns.
