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Benzodiazepines in Urine for GC or GC/MS Confirmations
Using 200mg 10mL HyperSep Verify-CX Extraction Column
(Part Number: 60108-742)
Prepare Sample – ß-Glucuronidase Hydrolysis
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% acetonitrile in 100mM phosphate
buffer (pH= 6.0)
Dry column (5 minutes at >10"Hg)
1 x 2mL hexane
Elute Benzodiazepines
1 x 5mL ethyl acetate containing 4% ammonium hydroxide
Collect eluate at 1 to 2mL/minute
Dry Eluate
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
Quantitate
Inject 1 to 2µL onto GC/MS
For mass spectrometry monitor the following ions:
Generic Name
Trade Name
Primary Ion***
Secondary
Tertiary
Nordiazepam -D5-TBDMS
332
334
333
Nordiazepam-TBDMS
327
328
329
Oxazepam-D5-TBDMS
462
519
462
Oxazepam-TBDMS
Serax
457
513
459
Temazepam-D5-TBDMS
362
390
288
Temazepam-TBDMS
Restoril
®
357
359
385
Lorazepam-TBDMS
Ativan
®
491
513
493
Clonazepam
Klonopin
®
372
374
326
7-Aminoclonazepam –TBM
456
458
513
Diazepam
Valium
®
256
283
221
Desalkylflurazepam-TBDMS
345
347
402
Prazepam*
269
241
324
α
-Hydroxymidazolam-TBDMS
Versed
®
398
400
440
Desmethylflunitrazepam-TBDMS
357
310
356
7-Aminoflunitrazepam-TBDMS
397
324
398
Alprazolam
Xanax
®
308
279
204
α
-Hydroxyalprazolam-D5-TBDMS
386
388
387
α
-Hydroxyalprazolam-TBDMS
383
384
381
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
Recommended GC Column
Part Number
TraceGOLD TG-5MS 30m x 0.25m x 0.25µm
26098-1420
Pharmaceutical/Biotech
Applications