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Forensic
Applications
Anabolic Steroids 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 5mL of urine add suitable internal standards and 2mL
of
β
-Glucuronidase
β
-Glucuronidase: 5,000 F units/mL Patella vulgate in
100mM acetate buffer (pH 5.0)
Mix/vortex
Hydrolyze for 3 hours at 65°C
Cool before proceeding
Centrifuge for 10 minutes at 2,000rpm and discard pellet
Adjust sample pH to 6.0±0.5 with approximately
700µL of 1.0N NaOH
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 10% (v/v) CH
3
OH in DI H
2
O
Dry column (5 minutes at >10"Hg)
1mL hexane or hexane/ethyl acetate (50:50)
Elute Anabolic Steroids
Option a:
3mL of CH
2
Cl
2
/IPA/NH
4
OH (78:20:2); collect
eluate at 1 to 2mL/minute
NOTE:
Prepare elution solvent fresh daily; add IPA/NH
4
OH mix,
then add CH
2
Cl
2
(pH 11-12)
Option b:
3mL CH
2
Cl
2
/IPA (80:20)
Option c:
3mL ethyl acetate
Option d:
3mL CH
3
OH
Dry Eluate
Evaporate to dryness at <40°C
Derivatize
Add 50µL ethyl acetate and 50µL MSTFA (with 3%
trimethylsilyliodide)
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 MSTFA solution
Quantitate
Inject 1 to 2µL onto gas chromatograph
Monitor the ions (GC/MS) in the table below:
Compound
Primary Ion*
Secondary
Tertiary
Other
Testosterone-TMS
432
301
209
19-noretiocholanone-TMS
405
315
225
Oxymethalone
640
52
462
370,143
Dehydroepiandosteronw-2TMS
432
327
297
10-nortestosterone-2TMS
418
287
194
Oxymethalone metabolite #1
640
52
462
143
Oxymethalone metabolite #2
625
462
370
143
11-
β
-hydroxyandosterone
522
417
158
Methandienone
409
313
281
19-norandosterone-2TMS
405
315
225
Alpha-hydroxyetiocholanone
504
417
17-
α
-epitestosterone-TMS
432
341
327
209
Stanazolol
472
381
342
149
* Quantitation ion
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