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Cyanuric Acid and Melamine in Food Materials for LC/MS/MS
Using 200mg 6mL HyperSep Verify-CX Extraction Column
(Part Number: 60108-722)
and 200mg 6mL HyperSep Retain-AX Extraction Column
(Part Number: 60107-412)
Prepare Sample
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 (CSDAU206 (BCX) column
Condition HyperSep Verify – CX 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
Column SPE
Wash Column
Wash HyperSep Verify-CX Column
1 x 1mL DI H
2
O
Collect wash for use with HyperSep Retain-AX Column
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
Evaporation
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 to LC/MS
HyperSep Retain-AX SPE Procedure
Adjust solution from Wash and Elute Steps to pH 7 **
Condition HyperSep Retain-AX 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 adjusted to pH7 at
1 to 2mL/minute
Wash HyperSep Retain – AX Column
1 x 3mL DI H
2
O
1 x 1mL CH
3
OH
Dry column (just enough to remove residual solvent)
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
Reconstitute
Reconstitute sample in 100µL of mobile phase
Add external standard*
Inject 5µL to LC/MS
Compound
MRM Transition
Melamine
127.1/85.1
2, 4 Diamino 6-hydroxy pyrimidine*
127.1/67.0
Cyanuric Acid
127.8/84.9
* External standard: 2, 4 Diamino 6-hydroxy pyrimidine
** Adjust pH with 100 to 200µL of 5% (v/v) (aq)NH
4
OH
Recommended HPLC Column
Part Number
Hypersil GOLD AX 3µm, 150 x 2.1mm
25503-152130
Pharmaceutical/Biotech
Applications