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Melamine in Milk Products
Using 60mg 3mL HyperSep Retain-CX Extraction Column
(Part Number: 60107-303)
Sample Preparation
Weigh 5g of milk powder (or measure 10mL of milk) into
a 250mL flask
Add 50mL of 1% trichloroacetic acid (TCA)
Mix/vortex
Add 2mL of 2% lead acetate/water solution into the
mixture then sonicate for 20 minutes
Transfer part of the final mixture into a 10mL centrifuge tube
Centrifuge for 10 minutes at 8,000rpm
Condition Retain-CX Extraction Column
3mL of methanol
3mL of DI H
2
O
Apply Sample
Load 6mL of the sample extract onto the Retain-CX SPE
product 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/methanol
Collect eluate at 1 to 2mL/min
Dry Eluate and Reconstitute
Evaporate to dryness at <50°C using nitrogen
Reconstitute sample using 2mL of mobile phase
Melamine in Food Materials
Using 200mg 3mL HyperSep Retain-CX
(Part Number: 60107-304)
Sample Preparation
To 1 to 5g of sample add 10 to 25mL of CH
3
CN/DI H
2
O
(50:50)
Shake for 5 minutes
Centrifuge
Transfer 5mL of supernatant to clean glass screw top tube
Add 1mL of 100mM HCl
Add 1mL of CH
2
Cl
2
Shake for 5 minutes
Centrifuge
Transfer upper layer to clean glass tube
Add 2mL of DI H
2
O to CH
2
Cl
2
Shake for 5 minutes
Centrifuge
Add upper layer to previous aqueous portion
Apply to Conditioned SPE column
Condition HyperSep Retain-CX Extraction Column
1 x 3mL CH
3
OH
1 x 3mL DI H
2
O
NOTE:
Aspirate at <3"Hg to prevent sorbent drying out
Apply Sample
Load sample at 1 to 2mL/minute
Wash Column
1 x 3mL 100mM HCl
1 x 1mL CH
3
OH
Dry column (5 minutes at >10"Hg)
Elute Melamine
1 x 2mL of CH
3
OH containing 5% NH
4
OH
1 x 3mL 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 <40°C
Analysis
Reconstitute sample in 1,000µL of CH
3
CN
Add external standard*
Inject 5µL on to LC/MS
Instrument: Thermo Scientific TSQ Triple Quadrupole
Flow rate: 0.50mL/minute
Mobile phase:
Time:
% ACN %
0.1% Formic Acid
0
90
10
3
20
80
3.5
90
10
10
90
10
Compound
MRM Transition
Melamine
127.1/85.1
2, 4 Diamino 6-hydroxy Pyrimidine*
127.1/67.0
* Suggested internal standard: 2, 4 Diamino 6-hydroxy Pyrimidine
Recommended HPLC Column
Part Number
Hypersil GOLD HILIC 3µm, 150 x 2.1mm
26503-152130
Food Safety
Applications