DETECTABUSE™ "NO VACUUM" GRAVITY SERIES GV-65 METHOD FOR THE ANALYSIS OF
LSD AND METABOLITES IN URINE 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 5.0 mL of urine 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 urine or blood sample, the solvent volume must not exceed
5% 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 50 µL BSTFA and 15 µL TMS-imidazole. PRECAUTION: Derivatized LSD is subject to loss by adsorption
in the injection sleeve and on the capillary column as it ages. When sensitivity
begins to drop, inject a few 5 µL aliquots of Silyl-8 GC column conditioning
solution (Pierce Reagents) onto the capillary column with the oven set
to 150 °C and the purge set to off. If this does not restore sensitivity,
changing the injector sleeve and if necessary, cutting 6-8 inches off
the capillary column should restore it. We suggest that you first run an unextracted standard at the LOQ level
to make sure that the "system" is sensative enough to determine picogram
levels of LSD. 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 10 ng of Lysergic Acid Methylpropylamide (LAMPA) per mL of sample as internal standard. Mix.
3. Add 0.5 mL, 10% HCl in Deionized Water. Mix.
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 Deionized Water. Allow the columns to flow
by gravity.
3. Wash column with 2.0 mL of Methanol. Allow the columns to flow by gravity.
5. 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 2.0 mL of n-Butylchloride 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) 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.
3. Mix the tube contents, flush with nitrogen or argon and cap the tube
or transfer contents into 100 µL reaction vials and seal.
4. Incubate the mixture @ 70°C for 20 min.
5. Allow the mixture to come to room temperature. Inject 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%).
