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Clonazepam and 7-Aminoclonazepam in Urine
for 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
Cool before proceeding
Condition HyperSep Verify-CX Extraction Column
1 x 3mL CH
3
OH
1 x 3mL deionized water
1 x 1mL 100mM phosphate buffer (pH 6.0)
NOTE:
Aspirate at <3"Hg to prevent sorbent drying
Apply Sample
Load at 1 to 2mL/minute
Wash Column
1 x 2mL deionized water
1 x 2mL 20% acetonitrile in 100mM phosphate
buffer (pH 6.0)
Dry column (5 minutes at >10"Hg)
1 x 2mL hexane
Elute Clonazepam/7-Aminoclonazepam
1 x 3mL ethyl acetate with 2% NH
4
OH
Collect eluate at 1 to 2mL/minute
NOTE:
Prepare fresh daily
Dry Eluate
Evaporate to dryness at <40°C
Derivatize
Add 50µL ethyl acetate and 50µL MTBSTFA (with 1%
TBDMCS)**
Mix/vortex
React 20 minutes at 90°C
Remove from heat source to cool
NOTE:
Do not evaporate MTBSTFA solution
Analysis
Inject 1 to 2µL of sample onto GC/MS
For mass spectrometry monitor the following ions:
Compound
Primary Ion*** Secondary
Tertiary
Clonazepam-TBDMS
372
374
326
7-Aminoclonazepam-TBDMS
342
344
399
Clonazepam-D4-TBDMS
376
378
377
7-Aminoclonazepam-D4-TBDMS 346
348
403
* Suggested internal standard for GC/MS: Clonazepam-D4, 7-aminoclonazepam-D4
** Part number TS-48927
*** Quantitation ion
Recommended GC Column
Part Number
TraceGOLD TG-5MS 30m x 0.25m x 0.25µm
26098-1420
Pharmaceutical/Biotech
Applications