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DHEA, Testosterone and Epitestosterone in Urine
for GC or GC/MS Confirmations
Using 200mg 10mL HyperSep Verify-CX Extraction Column
(Part Number: 60108-742)
Prepare Sample
Pipette 5mL of urine into borosilicate glass test tubes
Add internal standard* and adjust the sample pH to
5.5 to 6.5 using concentrated monobasic or dibasic
sodium phosphate
Mix sample
Centrifuge samples at 3,000rpm for 5 minutes
Condition Verify-CX Extraction Column
3mL of CH
3
OH
3mL of DI H
2
O
1mL of 100mM phosphate buffer (pH 6.0)
Apply Sample
Pour supernatant onto column
Allow to flow via gravity
Wash Column
3mL of DI H
2
O
Dry column (10 minutes at >10"Hg)
Elute Steroids
3mL of CH
3
OH
Collect eluate at 1 to 2mL/min
Enzymatic Hydrolysis
Dry eluate under a stream of nitrogen; add 2mL of 0.2M
phosphate buffer (pH 7.0) and 250 units of
β
-glucuronidase
Mix/vortex and allow to incubate at 50°C for 1 hour
Cool sample, cap and adjust the pH to 10-11 using a
1:1 mixture of NaHCO
3
/Na
2
CO
3
Additional Clean-up
Add 5mL of n-butyl chloride to each sample. Cap the
tubes and shake vigorously for 10 minutes and then
centrifuge at 3,000rpm for 5 minutes. Transfer the
organic layer to a clean test tube and dry under a
stream of nitrogen. Place the dried sample in a desiccator
and further dry under vacuum for 30 minutes.
Derivatize
Add 50µL of MSTFA***/NH
4
/dithioerythritol (1000:2:5,
v/w/w) and incubate at 70°C for 20 minutes
Centrifuge sample at 3,000rpm for 1 minute and transfer
directly to GC injector vials
Quantitate
Inject 1 to 2µL onto the gas chromatograph
For mass spectrometry, monitor the following ions:
Compound
Primary Ion** Secondary
Testosterone
432
417
Epitestosterone
432
417
DHEA
432
417
16
α
hydroxyl-testosterone*
520
259
* Suggested internal standard at 20ng/mL
** Quantitation ion
*** Part number TS-48910 (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
Forensic
Applications