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Forensic
Applications
Benzodiazepines in Urine
Using 200mg 10mL HyperSep Verify-CX Extraction Column
(Part Number: 60108-742)
Sample Preparation
To 2mL of urine add internal standard(s)* and 1mL of
ß-glucuronidase solution
ß-glucuronidase solution contains: 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 HyperSep Verify-CX Extraction Column
1 x 3mL CH
3
OH
1 x 3mL DI H
2
O
1 x 1mL 100mM phosphate buffer (pH= 6.0)
NOTE:
Aspirate at <3"Hg to prevent sorbent drying
Apply Sample
Load at 1mL/minute
Wash Column
1 x 2mL DI H
2
O
1 x 2mL 20% ACN in 100mM phosphate buffer (pH= 6.0)
Dry column (5 minutes at >10"Hg)
1 x 2mL hexane
Elute
1 x 5mL ethyl acetate containing 4% ammonium hydroxide
Collect eluate at 1 to 2mL/minute
Evaporate to dryness at <40°C
Derivatize
Add 50µL ethyl acetate and 50µL BSTFA**
(with 1% TMCS)**
Overlay with Nitrogen and cap
Mix/vortex
React 20 minutes at 70°C
Remove from heat source to cool
NOTE:
Do not evaporate BSTFA solution
Analysis
Inject 1 to 2µL onto GC/MS
For mass spectrometry monitor the following ions:
Generic Name
Trade Name
Primary Ion***
Secondary
Tertiary
Alprazolam
Xanax
®
308
279
204
α
-Hydroxyalprazolam-TMS
381
396
383
Chlordiazepoxide
Librium
®
282
283
284
Clonazepam
Klonopin
®
387
352
306
Diazepam
Valium
®
256
283
221
Desalkylflurazepam-TMS
359
341
245
Hydroxyethylflurazepam-TMS
288
360
389
Lorazepam-TMS
Ativan
®
429
430
347
Nordiazepam-TMS
341
342
343
Oxazepam-TMS
Serax
429
430
313
Prazepam*
269
241
324
Temazepam-TMS
Restoril
®
343
283
257
Triazolam
Halcion
®
313
314
342
α
-Hydroxytriazolam-TMS
415
417
190
* Suggested internal standard for GC/MS: Prazepam or Oxazepam-D5
** Part number TS-38831
*** Quantitation ion
NOTE: Flurazepam does not extract under these conditions; However metabolites such as desalkyflurazepam and hydroxyethylflurazepam will extract with high recovery.
Recommended GC Column
Part Number
TraceGOLD TG-5SilMS 30m x 0.25m x 0.25µm
26096-1420