DETECTABUSE™ "NO VACUUM" GRAVITY SERIES GV-65 METHOD FOR THE ANALYSIS OF
6-MONOACETYLMORPHINE IN URINE, SERUM OR ORAL FLUID BY GC-MS
This method is a preliminary procedure for investigational use only. Although it has performed well in our laboratory, the method must be validated by your laboratory before it is used to report patient values. We would appreciate your comments on its performance and welcome your suggestions for improvements or enhancements.
Revised: January 2003 (* TEA Elution Solvent Stability)
SAMPLE PREPARATION - (Please see Notes and Supplemental Information before proceeding)
1. Add 3.0 mL of sample to a
16 x 100 mm disposable borosilicate glass tube with an inert screw cap
top. Note: a. When adding an internal standard dissolved in an
organic solvent to a sample, the solvent volume must not exceed 3% of
the buffered sample volume. Higher solvent concentrations may produce
extraction losses.
Note: b.
The Sodium Bisulphite reduction step, when combined with the unique derivatization
step, eliminates the interference from Codones and Morphones without affecting
morphine, codeine or 6-MAM . If this interference is not a concern, this
reduction step may be eliminated. The column still must be pretreated
with Sodium Bisulphite to activate the cation exchange moiety on the resin.
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. 1. Pour samples onto preconditioned column. Allow to flow by gravity.
Samples will flow through the column at a rate of 1-2 mL/min. Note: If liquids do not elute freely by gravity flow, there
is probably air trapped within the column bed or frits. Tapping the column
mounting plate onto the vacuum box should initiate flow.
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 - Using TMSI/MBTFA
1. To each dried extract add 70 µL Acetonitrile and 15 µL Trimethylsilylimidizole (TMSI).
SUPPLEMENT - When using an automated robotic system 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.
2. Add 50 ng of 6-MAM-d3 to each sample (see note a).
3. Pretreat samples by adding 1.0 mL of a saturated solution of Sodium Bisulphite
(see note b)
. Prepare by dissolving 25 grams
of Sodium Bisulfite in 100 mL of a (1:1) mixture of H2O:0.25M
Phosphate Buffer, pH 6.0. Prepare monthly (Store refrigerated). ).
4. Mix gently and incubate samples at 55°C for 15 min.
5. Add 1.0 mL, 1% HCl in Deionized Water.
6. If cloudy or precipitated Centrifuge for 3 minutes at 3000 RPM.
2. Add 1.0 mL of a Sodium Bisulfite solution to each column. Allow to
flow by gravity.Prepare by dissolving 25 grams of Sodium Bisulfite
in 100 mL of a (1:1) mixture of H2O:0.25M Phosphate Buffer,
pH 6.0. Prepare monthly.
3. Proceed to Sample Extraction within 60 min. of column conditioning.
2. Wash column with 3.0 mL of Deionized Water.
3. Wash column with 2.0 mL of Methanol. Allow the columns to flow by gravity.
4. Wash column with 1.0 mL Ethyl Acetate. Allow the columns to flow by
gravity. Proceed to Sample Elution.
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-Butylchloride:Ethyl Acetate (80:20) w/4% Triethylamine
(TEA)* to each column and allow solvent to flow through the columns by
gravity into the test tubes.
4. Dry under N2 or argon at less than 50°C.
* Elution solvent with 4% TEA, (4 mL TEA is added to 96 mL of n-Butyl
Chloride:Ethyl Acetate 80:20) is stable for approximately one week stored
in a glass bottle with a Teflon or polypropylene lined cap. Close bottle
tightly when not in use. A white residue begins to appear in the dried
down eluate when the TEA begins to deteriorate. Artifacts from this process
may interfere with "fast" GC/MS methods.
2. Mix the tube contents, cap and incubate at room temp. for at least
5 mins.
3. Add 15 µL N-Methyl-bis(trifluoroacetamide) (MBTFA), mix the tube contents,
flush with nitrogen or argon, cap the tube or transfer contents into 100
µL reaction vials and seal.
4. Inject 1-2.0 µL.
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%).
