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Cyanuric Acid and Melamine in Food Materials Multiresidue
Analysis in Cereal Grains for LC/MS/MS or GC/MS
Using 200mg 6mL HyperSep Verify-CX and HyperSep Retain-AX Extraction Columns
(Part Number: 60108-722 and 60107-412)
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 HyperSep Verify-CX column
Condition HyperSep Verify-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
Collect effluent for use with HyperSep Retain-AX SPE
Wash Column
1 x 1mL DI H
2
O
Collect wash for use with HyperSep Retain-AX
Remove collection tubes from manifold and go to
HyperSep Retain-AX section
1 x 3mL 100mM HCl
1 x 1mL CH
3
OH
Dry column (5 minutes at >10"Hg)
Elute Melamine
Insert fresh collection tubes into manifold
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
Evaporate
Evaporate eluates under a gentle stream of nitrogen <40°C
Reconstitute
Reconstitute sample in 1,000µL of CH
3
CN
Add external standard*
Inject 5µL
HyperSep Retain-AX Extraction Procedure
Adjust solution from wash and elution steps to pH 7**
Condition HyperSep Retain-AX 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 which has been pH adjusted at 1 to
2mL/minute
Wash Column
1 x 3mL DI H
2
O
1 x 1mL CH
3
OH
Dry column
Elute Cyanuric Acid
Insert fresh collection tubes into manifold
1 x 3mL of CH
3
OH containing 1% HCl
1 x 2mL of CH
3
OH containing 1% HCl
Collect eluate at 1 to 2mL/minute
Evaporation
Evaporate eluates under a gentle stream of nitrogen <40°C
Analysis
To sample add 100µL of mobile phase
Add external standard*
Inject 5µL onto LC/MS
Flow rate: 0.50mL/minute
Column temperature: ambient
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*
127.1/67.0
pyrimidineCyanuric Acid
127.8/84.9
* Suggested external standard: 2, 4 Diamino 6-hydroxy
** Adjust pH with 100 to 200µL of 5% (v/v) (aq) NH
4
OH
Recommended HPLC Column
Part Number
Hypersil GOLD HILIC 3µm, 150 x 2.1mm
26503-152130
Food Safety
Applications