DETECTABUSE™ GRAVITY SERIES GV-65 METHOD FOR THE ANALYSIS OF URINARY ANABOLIC STEROIDS AND METABOLITES BY GC/MS
This method is a preliminary procedure for investigational use only. Although it has performed well in our laboratory it must be validated by your laboratory before it is used to report patient values. We would appreciate your comments on it's performance and welcome your suggestions for improvements or enhancements.
Revised:September, 2003
This revision includes an improved derivatizing scheme and a modified methanol wash to improve recovery of Stanozolol.
SAMPLE PREPARATION - (Please see Notes and Supplemental Information before proceeding)
1. Add 3.0 mL of urine to a 16 x
100 mm disposable borosilicate glass tube. SAMPLE HYDROLYSIS
1. Add 0.5 mL of 0.2M Acetate Buffer, pH 5.0 to each prepared sample.
(pH should be 4.5 - 5.0) HARDWARE SETUP - (Please refer to the Detectabuse Hardware Setup
Instructions)
COLUMN CONDITIONING
1. Wash column with 1.0 mL of Methanol. Allow to flow by gravity. SAMPLE EXTRACTION - (Please see Notes at end of this section
before proceeding)
1. Pour samples onto preconditioned columns. Allow to flow by gravity.
Samples will flow through the columns at a rate of 1-2 mL/min. Note: If liquids do not elute freely by gravity flow, there
is either air trapped within the column bed or frits, or there is aqueous
phase remaining on the column because of a weak vacuum during the column
drying step. Tapping the column mounting plate onto the vacuum box should
initiate flow. If this does not do the job, use a rubber bulb to
gently push a few drops of elution solvent and trapped air into the collection
tube. Allow the remainder of solvent to flow by gravity. SAMPLE ELUTION
1. Sample elution is done outside of the vacuum box. Note: If a sample does not elute freely by gravity flow, there
is probably air trapped within the column bed or frits. In most cases,
tapping the column will initiate flow. If this does not do the job, use
a rubber bulb to gently push a few drops of elution solvent and trapped
air into the collection tube. Allow the remainder of solvent to flow by
gravity.
DERIVATIZATION
1. To each dried extract add 70 µL Acetonitrile, vortex mix, then add
15 µL Trimethylsylimidizole (TMSI) and 15 µL MBTFA.
SUPPLEMENT - When using an automated robotic system GC/MS ANALYSIS
2. Add the appropriate deuterated standards to each sample.
Note: When adding an internal standard dissolved in an organic solvent
to a urine or blood sample, the solvent volume must not exceed 3% of the
buffered sample volume. Higher solvent concentrations may produce extraction
losses.
2. Add 10,000 units of Beta-Glucuronidase, Sigma type H-2S from Helix
Pomatia (or equivilent) to each sample.
3. Mix gently and incubate at 55°C for two hours or 37°C
for 4 hours. Complete hydrolysis is also achieved in 16 hours at room
temperature (15 - 30°C).
4. Add 3.0 mL of 0.25M Phosphate Buffer, pH 9.1.
5. Centrifuge for 3 minutes at 4000 RPM.
2. Proceed to Sample Extraction within 30 min. of column conditioning.For
longer periods follow the Methanol wash with 3.0 mL of 0.25M Phosphate
Buffer, pH 9.1 to prevent column from drying out.
2. Add 2.0 mL of 0.1 N NaOH. Allow columns to flow by gravity.
3. Add 3.0 mL of deionized H20. Allow to flow by gravity.
4. Add 2.0 mL of Water:Methanol (80:20). Allow the columns to flow by
gravity.
5. Dry the columns by applying vacuum adjusted to at least 7" Hg for 5
minutes (Test by momentarily placing the heel of hand over the column
top. A strong pull should be felt through the column).
2. Place the column mounting plate on the elution rack loaded with an
appropriate number of 12 x 75 mm or 15 x 85 mm borosilicate glass test
tubes. Make sure that the hole pattern on the plate matches the hole pattern
on the rack.
3. Add 1.5 mL of n-Butyl Chloride to each column and allow solvent to
flow through the column by gravity into test tubes.
4. Dry under N2 or argon at 55°C.
Mix the tube contents, flush with nitrogen or argon and cap the tube or
transfer contents into 100 µL reaction vials and seal.
2. Inject 1-2.0 µL.
All liquids may be allowed to flow unassisted through the column or may
be pulled through the column with vacuum or pushed through with positive
pressure.
Assisted flow parameters may be set as follows:
Column Conditioning - Pass through column in approximately 20 seconds
(± 20%).
Sample, Sample Washes and Elution Solvent - Pass through column in approximately
60 seconds (± 20%).
Column Drying Steps - Use 12-15 PSI of positive pressure for 40 seconds
or vacuum set at 15" Hg for 30 seconds (These drying parameters are for
individual columns).
GC/MS: Hewlett-Packard equipped with Mass Selective Detector
GC Column: H.P. Ultra 2 Capillary Column (or equivalent), 15 m x 0.25
mm, 0.25 rn film thickness.
Acquisition Mode: SIM
Injector Temp.: 280°C
Detector Interface Temp.: 305°C
Temperature Program:
Initial: 150°C, program at 25°C/min. to
300°C
Final Time: Hold for 1.5 min.
Equil. Time: 1.0 min.
Splitless
He Flow: 1.0 mL/min. @ 200°C
Septum Purge: 3 mL/min.
Purge Off Time: 1.0 min.
Solvent Delay: 4.0 min.
Dwell: 20
Start Acq.:4.0 min.
Stop Run: 7.5 min.
