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DHEA, Testosterone, and Epitestosterone in Urine
for GC or GC/MS Analysis
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*, adjust sample pH to 5.5 to 6.5
using concentrated sodium phosphate monobasic
or dibasic
Mix sample
Centrifuge samples at 3,000rpm for 5 min
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)
Apply Sample
Pour supernatant onto column
Allow to flow via gravity
Wash Column
1 x 3mL DI H
2
O
Dry column (10 minutes at >10mm Hg)
Elute Steroids
1 x 3mL of CH
3
OH
Collect at 1 to 2mL/minute
Enzymatic Hydrolysis
Dry eluate under a stream of nitrogen; Add 2mL of
200mM 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 to 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. The tubes and
shake vigorously for 10 minutes and then centrifuge at
3,000rpm for 5 min. Transfer the organic layer to
clean test tubes and dry under a stream of nitrogen.
Place dried sample in a desiccator and further dry
under vacuum for 30 minutes.
Derivatize
Add 50µL of MSTFA/NH
4
l/dithioerythritol
(1,000:2:5, V/W/W) and incubate at 70°C for 20 min
Centrifuge sample at 3,000rpm for 1 min and transfer
directly to GC injector vials
Quantitate
Inject 1 to 2µL onto GC/MS
For mass spectrometry monitor the following ions:
Compound
Primary Ion** Secondary
Testosterone
432
417
Epitestosterone
432
417
DHEA
432
417
16
α
Hydroxytestosterone*
520
259
* Suggested internal standard at 20ng/mL: 16
α
Hydroxytestosterone
** Quantitation ion
Recommended GC Column
Part Number
TraceGOLD TG-5MS 30m x 0.25m x 0.25µm
26098-1420
Pharmaceutical/Biotech
Applications