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Forensic
Applications
Flunitrazepam and Metabolites in Urine for GC or GC/MS Confirmations
Using 200mg 10mL HyperSep Verify-CX Extraction Column
(Part Number: 60108-742)
Prepare Sample –
β
-Glucuronidase Hydrolysis
To 2mL of urine add internal standard(s)* and 1mL of
β
-Glucuronidase
β
-Glucuronidase: 5,000 F units/mL Patella vulgata in
100mM acetate buffer (pH 5.0)
Mix/vortex
Hydrolyze for 3 hours at 65°C
Centrifuge for 10 minutes at 2,000rpm and discard pellet
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
2mL of 20% acetonitrile in 100mM phosphate buffer
(pH 6.0)
Dry column (5 minutes at >10"Hg)
2mL hexane
Elute Flunitrazepam, 7-Aminoflunitrazepam and
Desmethylflunitrazepam
3mL of ethyl acetate with 2% NH
4
OH
Collect eluate at 1 to 2mL/min
NOTE:
Prepare elution solvent daily
Dry Eluate
Evaporate to dryness at <40°C
Derivatize
Add 50µL of ethyl acetate and 50µL MTBSTFA (with 1%
TBDMCS)***
Overlay with N
2
and cap
Mix/vortex
React for 20 minutes at 70°C
Remove from heat source to cool
NOTE:
Do not evaporate MTBSTFA solution
Quantitate
Inject 1 to 2µL onto gas chromatograph
For mass spectrometry, monitor the following ions:
Compound
Primary Ion** Secondary
Tertiary
Flunitrazepam
312
286
266
7-aminoflunitrazepam
283
255
254
Desmethylflunitrazepam
356
357
310
D
5
-oxazepam*
462
464
463
* Suggested internal standard
** Quantitation ion
*** Part number TS-48927 (10 x 1mL ampules)
Recommended GC Columns
Part Number
TRACE TR-DoA5 Column, 15m x 0.25mm x 0.25µm
26AF130P
TRACE TR-DoA35 Column, 15m x 0.20mm x 0.33µm
26AC497P