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Forensic
Applications
Fentanyl/Norfentanyl in Urine, Blood and Plasma/Serum
for LC/MS/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 standards*
Add 1mL of urine, blood, plasma/serum
Add 2mL of 100mM phosphate buffer (pH 6.0)
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) then aspirate
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 of CH
3
OH
Dry column (5 minutes at >10"Hg)
Elute Fentanyl/Norfentanyl
3mL of CH
2
Cl
2
/IPA/NH
4
OH (78:20:2)
Collect eluate at 1 to 2mL/min
NOTE:
Prepare elution solvent fresh daily; add IPA/NH
4
OH mix,
then add CH
2
Cl
2
(pH 11-12)
Evaporate
Evaporate to dryness at <40°C
Reconstitute sample in 100µL of methanol
Quantitate
Inject 5µL onto LC/MS/MS system
Monitor the following ions:
Compound
MRM Transition
Fentanyl
333.2/188.3
Fentanyl-D5*
342.3/188.2
Norfentanyl
233.2/84.1
Norfentanyl-D5*
238.3/84.1
* Suggested internal standards
Mobile phase: acetonitrile: 0.1% formic acid (50:50, v/v)
Flow rate: 0.35mL/min
Column temperature: ambient
Recommended HPLC Column
Part Number
Hypersil GOLD PFP 3µm, 150 x 2.1mm
25403-152130