DETECTABUSE™ "NO VACUUM" GRAVITY SERIES GV-65 METHOD FOR THE ANALYSIS OF COCAINE,COCAINE METABOLITES AND COCAETHYLENE IN URINE, SERUM AND ORAL FLUIDS 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: MAY 2004
SAMPLE PREPARATION - (Please see Notes and Supplemental Information before proceeding)
1. Add 1.0 mL of sample to a 16 x 100
mm disposable borosilicate glass tube with an inert screw cap top. Note: 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.
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.
DERIVATIZATION
1. To each dried extract add 75 µL Acetonitrile, vortex mix, then add
25 µL MTBSTFA w/ 1% TBDMCS or BSTFA w/ 1% TMCS. (See SIM Table). Note: Polar solvents commonly used for derivatization
such as Acetonitrile or Ethyl Acetate will pick up moisture over time.
Because moisture will inhibit or prevent derivatization it is important
to keep a supply of solvents used for this purpose stored as protected
from moisture as possible. Storing these solvents in a desiccator or flushing
with Nitrogen or Argon after use is recommended.
Note: When using MTBSTFA for derivatization, it is a good
idea to inject 2 µL of Dichloromethane followed by 2 µL of Methanol after
every 20-30 injections. This will prevent the shortening of column life
often associated with the use of this very useful derivatizing reagent.
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 150 ng of Benzoylecgonine-d3 and Cocaine-d3 to each sample.
3. Add 1.0 mL, 1% HCl in Deionized Water.
4. The occasional cloudy or precipitated sample should be centrifuged for 3 minutes at 3000 RPM.
2. Add 1.0 mL of a Sodium Bisulfite solution to each column. 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).
3. Proceed to Sample Extraction within 60 min. of column conditioning.
2. Wash column with 3.0 mL of 0.01 HCl in Deionized Water. Allow the columns
to flow by gravity.
3. Wash column with 0.5 mL of Methanol. Allow the columns to flow by gravity.
4. Add 1.0 mL Ethyl Acetate. Allow the columns to flow by gravity.
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, flush with nitrogen or argon and cap the tube
or transfer contents into 100 µL reaction vials and seal.
3. Incubate the mixture @ 70°C for 20 min.
4. Allow the mixture to come to room temperature. Inject 1.0 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%).
