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Determination of Melamine in Egg
Using 60mg 3mL HyperSep Retain-CX Extraction Column
(Part Number: 60107-303)
Sample Preparation
To 10mg of melamine add 100mL ACN 10mM/L citric
acid +10mM/L heptane sulfonic acid (sodium salt
buffer solution (pH 3.0))
Prepare standard standard solutions; 1mg/L, 5mg/L,
10mg/L, 15mg/L, 20mg/L with water and filter
solutions with a 0.45µm syringe filter (F2513-1)
Add 1g of egg sample into a 10mL-centrifuge tube
Prepare 1.0mg/kg, 2.0mg/kg, 10.0mg/kg sample
respectively by adding 10µL, 20µL, or 100µL of
melamine standard in to the 10mL centrifuge tube
Add 10mL of 1% trichloroacetic acid and 2mL of
5% lead acetate and shake
Ultrasound for 20 min
Centrifugate at 8,000rpm for 10 min
Condition HyperSep Retain-CX Extraction Column
1 x 3mL CH
3
OH
1 x 3mL H
2
O
Apply Sample
Load supernate into extraction column
Wash Column
1 x 3mL H
2
O
1 x 3mL CH
3
OH
Elute Melamine
1 x 5mL of CH
3
OH containing 5% NH
4
OH
Collect eluate at 1 to 2mL/minute
Evaporation
Evaporate eluates under a gentle stream of nitrogen 50°C
Analysis
Reconstitute sample in CH
3
CN
Inject 5µL on to LC/MS
Recommended HPLC Column
Part Number
Hypersil GOLD HILIC 3µm, 150 x 2.1mm
26503-152130
Melamine in Animal Feed
Using 60mg 3mL HyperSep Retain-CX Extraction Column
(Part Number: 60107-303)
Sample Preparation
To 5g of animal feeds, add 50mL of 0.1% trichloroacetic
acid aqueous solution and internal standard if desired
Vortex for 1 minute, then add 2mL of 2% lead acetate
aqueous solution. Sonicate for 20 minutes, then transfer
a portion of the mixture to a 10mL centrifuge tube.
Centrifuge at 8,000rpm for 10 minutes, then take
3mL of the upper layer solution for SPE clean-up.
Condition Retain-CX Extraction Column
3mL of methanol
3mL of DI H
2
O
Apply Sample
Load sample at 1 to 2mL/minute
Wash Column
3mL of DI H
2
O
3mL of methanol
Dry column (5 to 10 minutes at >10"Hg/full flow for
positive pressure manifold)
Elute Melamine
5mL of 5% ammonia in methanol
Dry Eluate and Reconstitute
Evaporate to full dryness at 50°C under a stream
of nitrogen
Reconstitute the sample to 2mL with 20% methanol
aqueous solution
Food Safety
Applications