Solid phase extraction (SPE) method for melamine HPLC detection

1. Basis: GB/T 22388 — 2008
2.
Principle: The sample is extracted with trichloroacetic acid solution - acetonitrile, purified by cation exchange solid phase extraction column, and determined by high performance liquid chromatography, and quantified by external standard method.
3.
Reagents and materials: Unless otherwise stated, all reagents were of analytical grade and the water was the first grade water specified in GB/T 6682 .
3.1
methanol: chromatographically pure;
3.2
acetonitrile: chromatographically pure;
3.3
ammonia water: the content is 25 % ~ 28 %;
3.4
trichloroacetic acid;
3.5
citric acid.
3.6
sodium octane sulfonate: chromatographically pure;
3.7
Aqueous methanol solution: accurately measure 50 mL of methanol and 50 mL of water, mix and reserve;
3.8
Trichloroacetic acid solution ( 1 %): Accurately weigh 10 g of trichloroacetic acid in a 1 L volumetric flask, dissolve in water and dilute to volume, mix and set aside;
3.9
ammoniated methanol solution ( 5 %): accurately measure 5 mL of ammonia water and 95 mL of methanol, mix and reserve;
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 dilute to 1 L for use .
3.11
melamine standard: CAS 108-78-01 , the purity is greater than 99.0% ;
3.12
melamine standard stock solution: Accurately weigh 100 mg (accurate to 0.1 mg ) melamine standard in a 100 mL volumetric flask, dissolve it with methanol aqueous solution ( 3.7 ) and dilute to volume to prepare a standard of 1 mg/mL . Store stock at 4 °C protected from light.
3.13
Cation Exchange SPE: Mixed cation-exchange solid phase extraction column, benzenesulfonic matrix is polystyrene - divinyl benzene polymer, 60 mg, 3 mL, or equivalent person.
3.14
qualitative filter paper.
3.15
microporous membrane: 0.2 μ m , organic phase.
3.16
Nitrogen: purity greater than or equal to 99.999 %
4.
Instruments and equipment
4.1
High Performance Liquid Chromatography ( HPLC ): equipped with a UV detector or a diode array detector.
4.2
Analytical balance: The sensitivities are 0.00001 g and 0.01 g .
4.3
Centrifuge: The speed is not less than 10000 r/min .
4.4
Tianjin Hengao ultrasonic extractor. HS, HU series
4.5
Shanghai Wogong solid phase extraction device. HGC-8
4.6
Tianjin Heng Ao 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
stoppered plastic centrifuge tube: 50 mL .
5.
Sample processing
5.1
extraction
Weigh 2 g (accurate to 0.01 g ) sample in a 50 mL stoppered plastic centrifuge tube, add 15 mL of trichloroacetic acid solution ( 3.8 ) and 5 mL of acetonitrile. Ultrasonic extraction for 10 min , and then shaking for 10 min , centrifuged at not less than 10000 r/min for 30 min . After the supernatant was filtered through a filter paper moistened with trichloroacetic acid solution, the volume was adjusted to 25 mL with a trichloroacetic acid solution , 5 mL of the filtrate was removed , and 5 mL of water was added to mix and then to be purified.
Note: If the fat content of the sample is high, it can be degreased with the hexane liquid - liquid saturated with trichloroacetic acid solution and then purified by SPE column.
5.2
Activation
A cation exchange solid phase extraction column was activated ( 3,13 ) with 3 mL of methanol and 5 mL of water in that order . Precision gas steady flow control valve before rotating solid phase extraction device to make the washing liquid flow rate not exceed 1 mL/min
5.3
Loading
5.1 liquid to be purified is transferred to a solid phase extraction column (5.2) in.
5.4
rinse
Wash with 3 mL of water and 3 mL of methanol in turn, and draw to near dryness.
5.5
elution
Elute with 6 mL of ammoniated methanol solution ( 3.9 ) and collect the eluate in a test tube. The flow rate of the whole solid phase extraction process does not exceed 1 mL/min . 5.6 Concentration
The eluate was dried with nitrogen at 50 ° C. The residue (corresponding to 0.4 g of sample) was made to volume with 1 mL of mobile phase, vortexed for 1 min , and passed through a microporous membrane for HPLC .
6.
High performance liquid chromatography
HPLC
reference conditions
a)
Column: C8 column, 250 mm × 4.6 mm ( id ), 5 μ m , or equivalent;
C18
column, 250 mm × 4.6 mm ( id ), 5 μ m , or equivalent.
b)
Mobile phase: C8 column, ion pair reagent buffer ( 3.2.10 ) - acetonitrile ( 85+15 , volume ratio), mix.
C18
column, ion pair reagent buffer ( 3.2.10 ) - acetonitrile ( 90+10 , volume ratio), mix.
c)
Flow rate: 1.0 mL/min .
d)
Column temperature: 40 °C.
e)
Wavelength: 240 nm .
f)
Injection volume: 20 μ L.
7.
Analysis
With GB / T 22388 - 2008 standard test method for analysis of a sample measured using the device of constant Tianjin Otis recovery results were as follows:
Addition level ( mg/Kg ) recovery
blank
2 116%
4 108%
6 92%
8 96%
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b)
Mobile phase: C8 column, ion pair reagent buffer ( 3.2.10 ) - acetonitrile ( 85+15 , volume ratio), mix.
C18
column, ion pair reagent buffer ( 3.2.10 ) - acetonitrile ( 90+10 , volume ratio), mix.
c)
Flow rate: 1.0 mL/min .
d)
Column temperature: 40 °C.
e)
Wavelength: 240 nm .
f)
Injection volume: 20 μ L.
7.
Analysis
With GB / T 22388 - 2008 standard test method for analysis of a sample measured using the device of constant Tianjin Otis recovery results were as follows:
Addition level ( mg/Kg ) recovery
blank
2 116%
4 108%
6 92%
8 96%

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