1. Basis: GB / T 22388—2008
2. Principle: The sample is extracted with trichloroacetic acid solution-acetonitrile, purified by a cation exchange solid phase extraction column, measured by high performance liquid chromatography, and quantified by external standard method.
3. Reagents and materials: Unless otherwise stated, all reagents are analytically pure, and the water is the first grade water specified in GB / T 6682.
3.1 Methanol: chromatographically pure;
3.2 Acetonitrile: chromatographically pure;
3.3 Ammonia: the content is 25% ～ 28%;
3.4 Trichloroacetic acid;
3.5 Citric acid.
3.6 Sodium octane sulfonate: chromatographically pure;
3.7 Methanol aqueous solution: accurately measure 50 mL of methanol and 50 mL of water, mix well and set aside;
3.8 Trichloroacetic acid solution (1%): accurately weigh 10 g of trichloroacetic acid in a 1 L volumetric flask, dissolve with water and bring to volume to the mark, mix well and set aside;
3.9 Ammonia methanol solution (5%): accurately measure 5 mL of ammonia water and 95 mL of methanol, mix well and set aside;
3.10 Ion pair reagent buffer: accurately weigh 2.10 g of citric acid and 2.16 g of sodium octane sulfonate, add about 980 mL of water to dissolve, adjust the pH to 3.0, and bring the volume to 1 L for later use.
3.11 Melamine standard product: CAS 108-78-01, purity is greater than 99.0%;
3.12 Melamine standard stock solution: Accurately weigh 100 mg (accurate to 0.1 mg) of melamine standard in a 100 mL volumetric flask, dissolve with methanol aqueous solution (3.7) and bring to volume to the mark, and prepare a standard with a concentration of 1 mg / mL Store the stock solution at 4 ° C protected from light.
3.13 Cation exchange solid phase extraction column: Mixed cation exchange solid phase extraction column with benzenesulfonated polystyrene-divinylbenzene polymer, 60 mg, 3 mL, or equivalent.
3.14 Qualitative filter paper.
3.15 Microporous filter membrane: 0.2 μm, organic phase.
3.16 Nitrogen: purity greater than or equal to 99.999%
4. Instruments and equipment
4.1 High performance liquid chromatography (HPLC) instrument: equipped with ultraviolet detector or diode array detector.
4.2 Analytical balance: the sense of quantity is 0.00001 g and 0.01 g.
4.3 Centrifuge: The rotation speed is not less than 10000 r / min.
4.4 Tianjin Hengao Ultrasonic Extractor. HS, HU series
4.5 Shanghai Hegong Solid Phase Extraction Plant. HGC-8
4.6 Tianjin Hengao nitrogen blowing instrument. HGC, HSC series
4.7 Tianjin Hengao vortex oscillator. HMS-350
4.8 Tianjin Hengao vacuum pump. HPD-25
4.9 Tianjin Hengao precision gas steady flow regulating valve.
4.10 Plastic centrifuge tube with plug: 50 mL.
5. Sample handling
5.1 Extract and weigh (liquid milk, milk powder, yogurt, ice cream and toffee, etc.) 2 g (accurate to 0.01 g) of the sample in a 50 mL plastic centrifuge tube with a stopper, add 15 mL of trichloroacetic acid solution (3.8) and 5 mL of acetonitrile, ultrasonic extraction for 10 min, and then shaking extraction for 10 min, centrifugation at not less than 10000 r / min for 30 min. After filtering the supernatant with filter paper moistened with trichloroacetic acid solution, dilute to 25 mL with trichloroacetic acid solution, remove 5 mL of the filtrate, add 5 mL of water and mix well to make the liquid to be purified.
Note: If the fat content in the sample is high, it can be degreased with n-hexane liquid-liquid distribution saturated with trichloroacetic acid solution and then purified by SPE column.
5.2 Activation Activate (3.13) cation exchange solid phase extraction column with 3 mL methanol and 5 mL water in sequence. The precision gas steady flow regulating valve in front of the rotating solid phase extraction device makes the flow rate of the washing liquid not exceed 1 mL / min
5.3 Load the sample and transfer the liquid to be purified in 5.1 to the solid phase extraction column (5.2).
5.4 Rinse with 3 mL water and 3 mL methanol successively, after pumping to near dryness,
5.5 Elution Elute with 6 mL of ammoniated methanol solution (3.9), and collect the eluent in a test tube. The flow rate of the entire solid phase extraction process does not exceed 1 mL / min. 5.6 The concentrated eluent was dried with nitrogen at 50 ° C. The residue (equivalent to 0.4 g sample) was made up to volume with 1 mL of mobile phase, vortexed for 1 min, and passed through a microporous filter membrane for HPLC determination.
6. High performance liquid chromatography
HPLC reference conditions
a) Chromatographic column: C8 column, 250 mm × 4.6 mm (id), 5 μm, or equivalent;
C18 column, 250 mm × 4.6 mm (id), 5 μm, or equivalent.
b) Mobile phase: C8 column, ion pair reagent buffer (3.2.10) -acetonitrile (85 + 15, volume ratio), mix well.
C18 column, ion pair reagent buffer (3.2.10) -acetonitrile (90 + 10, volume ratio), mix well.
c) Flow rate: 1.0 mL / min.
d) Column temperature: 40 ℃.
e) Wavelength: 240 nm.
f) Injection volume: 20 μL.
7. The analysis is based on the GB / T 22388-2008 standard detection method, and the recovery rate of the sample measured using Tianjin Hengao equipment is as follows:
Adding level (mg / Kg) recovery rate blank
2 116%
4 108%
6 92%
8 96%
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