The principle is that the sample is extracted with trichloroacetic acid solution-acetonitrile, cleaned up by cation-exchange solid-phase extraction column, and then determined by high-performance liquid chromatography and quantified by external standard method.
Specific steps for the determination of melamine in milk by hplc.
(1) Sampling
①Liquid milk, milk powder, yoghurt, ice cream, milk sugar, etc.
Weigh 2 g (accurate to 0.01 g) of sample in a 50 mL stoppered plastic centrifuge tube, add 15 mL of trichloroacetic acid solution and 5 mL of acetonitrile, ultrasonic extraction for 10 min, and then shake extraction for 10 min, centrifuged for 10 min at not less than 4000 r/min. The supernatant was filtered through the trichloroacetic acid solution-moistened filter paper and then fixed to 25 mL with a trichloroacetic acid solution, and then 5 mL of filtrate was removed, and 5 mL of filtrate was added to 5 mL of trichloroacetic acid solution. mL of filtrate, add 5 mL of water and mix well to make the solution to be purified.
②Cheese, cream chocolate, etc.
Weigh 2 g (accurate to 0.01 g) of the sample in the mortar, add an appropriate amount of sea sand (4 to 6 times the mass of the sample) ground into dry powder, transfer to a 50 mL plug plastic centrifuge tube, with 15 mL of trichloroacetic acid solution to wash the mortar several times, the cleaning solution was transferred to the centrifuge tube, and then to the centrifuge tube to add 5 mL of acetonitrile, and then the rest of the operation of the same as in the “ultrasonic extraction”. 10 min, …… add 5 mL of water and mix to make the purified solution.
Note: If the fat content in the sample is high, you can use a liquid-liquid partition of hexane saturated with trichloroacetic acid solution to remove the fat before proceeding to the next step.
(2) Purification
Transfer the cleanup solution to the solid phase extraction column. Wash with 3 mL of water and 3 mL of methanol sequentially, pump to near dryness and elute with 6 mL of ammoniated methanol solution.
The flow rate of the whole solid phase extraction process did not exceed 1 mL/min. The eluate was blown dry with nitrogen at 50 ℃, and the residue (equivalent to 0.4 g of the sample) was fixed with 1 mL of mobile phase, vortexed and mixed for 1 min, and then passed through microporous filtration membrane for HPLC determination.
(3) Determination by HPLC
①HPLC reference conditions
a) Column: C8 column, 250 mm×4.6 mm (i.d.), 5 μm, or equivalent; C18 column, 250 mm×4.6 mm (i.d.), 5 μm, or equivalent.
b) Mobile phase: C8 column, ion-pairing reagent buffer (3.2.10)-acetonitrile (85+15, v/v), mixing.
Column C18, ion-pairing reagent buffer (3.2.10)-acetonitrile (90+10, v/v), mix well.
c) Flow rate: 1.0 mL/min.
d) Column temperature: 40°C.
e) Wavelength: 240 nm.
f) Injection volume: 20 μL.
② Plotting of standard curve
The concentration of 0.8, 2, 20, 40, and 80 μg/mL of melamine standard working solution was obtained by diluting the melamine standard stock solution step by step with the mobile phase. The concentration of the standard working solution was injected into the sample from the lowest to the highest, and the peak area-concentration graph obtained the standard curve’s regression equation. The HPLC chromatogram of the matrix-matched spiked melamine sample is shown in Figure A.1 in Appendix A.
③ Quantitative determination
The response value of melamine in the sample to be measured should be within the linear range of the standard curve; beyond the linear range, the sample should be diluted and analyzed again.
④ Calculation of results
The limit of quantification of this method is 2 mg/kg.