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Forensic
Applications
Fentanyl/Norfentanyl in Oral Swabs
Using 200mg 10mL HyperSep Verify-CX Extraction Column
(Part Number: 60108-742)
Preparation of Standards
Separate tube add 0, 1, 5, 10ng of Fentanyl/Norfentanyl
in CH
3
OH
Evaporate solvent
Add 100µL of drug free oral fluid
Mix/vortex
Stand for 30 minutes
Take clean, dry swab and swab up the oral fluid
Allow to stand for 15 minutes
Remove oral swab
Sample Preparation
200µL of CH
3
OH (pH 6)
Add internal standard*
Insert oral swab into CH
3
OH and mix for 1 minute,
Add 100µL of CH
3
OH
Allow to stand for 10 minutes
Remove swab and 3mL of 100mM phosphate buffer (pH 6)
Mix/vortex
Centrifuge
Condition HyperSep Verify-CX Extraction Column
1 x 3mL CH
3
OH
1 x 3mL H
2
O
1 x 1mL 100mM phosphate buffer (pH 6)
NOTE:
Aspirate at <3"Hg to prevent sorbent drying out
Apply Sample
Load sample at 1 to 2mL/minute
Wash Column
1 x 3mL DI H
2
O
1 x 3mL 1% acetic acid
1 x 3mL methanol
Dry column (5 minutes at >10"Hg)
Wash Column
1 x 3mL ethyl acetate;
ACN:ammonia(78:20:2 v/v)
Collect eluate at 1 to 2mL/minute
Evaporate eluates under a gentle stream of nitrogen <40ºC
Analysis
Reconstitute sample in 20µL of methanol
Inject 5µL onto LC/MS
Compound
MRM Transition
Fentanyl
333.2/188.3
Fentanyl-D5*
342.2/188.2
Norfentanyl
233.2/84.1
Norfentanyl-D5*
238.3/84.1
* Internal standard: Fentanyl-D5 and Norfentanyl-D5
Recommended HPLC Column
Part Number
Hypersil GOLD aQ 5µm, 50 x 3mm
25305-053030