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Forensic
Applications
Methadone/EDDP in Urine, Blood, Plasma/Serum and Tissue
for GC or GC/MS Confirmations
Using 200mg 10mL HyperSep Verify-CX Extraction Column
(Part Number: 60108-742)
Prepare Sample
To 1mL of 100mM phosphate buffer (pH 6.0) add
internal standard*
Add 1mL of urine, whole blood, plasma/serum or 1g
(1:4 homogenate) of tissue
Add 2mL of 100mM phosphate buffer (pH 6.0)
Sample pH should be 6.0±0.5
Adjust pH accordingly with 100mM monobasic or dibasic
sodium phosphate
Mix/vortex
Centrifuge as appropriate
Condition Verify-CX Extraction Column
3mL of CH
3
OH then aspirate
3mL of DI H
2
O then aspirate
1mL of 100mM phosphate buffer (pH 6.0)
NOTE:
Aspirate at <3"Hg to prevent sorbent drying
Apply Sample
Load sample at 1 to 2mL/minute
Wash Column
3mL of DI H
2
O
1mL of 100mM acetic acid
3mL CH
3
OH
Dry column (5 minutes at >10"Hg)
Elute Methadone/EDDP
3mL of ethyl acetate/acetonitrile/ammonia (78:20:2, v/v/v)
Collect eluate at 1 to 2mL/min
NOTE:
Prepare elution solvent fresh daily
Evaporate
Evaporate to dryness at <40°C
Reconstitute
Reconstitute sample in 100µL of methanol
Analysis
Inject 5µL of sample onto LC/MS system
Monitor the following ions:
Compound
MRM Transition
Methadone
310.2/105.1
Methadone-D9*
319.2/268.3
EDDP
278.2/234.2
EDDP-D3*
281.4/234.3
* Suggested internal standards
Mobile phase:
Time (min)
% Acetonitrile
% 0.1% Formic Acid
0
25
75
5
25
75
14
90
10
15
25
75
20
25
75
Flow rate: 0.35mL/min
Column temperature: ambient
Recommended HPLC Column
Part Number
Hypersil GOLD aQ 3µm, 150 x 2.1mm
25303-152130