DETECTABUSE™ "NO VACUUM" GRAVITY SERIES GV-65 METHOD FOR THE ANALYSIS OF KETAMINE IN URINE 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: December 2004 (Improved recoveries using a lower sample pH prior to extraction)
SAMPLE PREPARATION - (Please see Notes and Supplemental Information before proceeding)
1. Add 1.0 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
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. SAMPLE ELUTION
1. Sample elution is done outside of the vacuum box. 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.
DERIVATIZATION (Only required if being assayed with Amphetamines)
Using HFBA: WITHOUT DERIVATIZATION (Preferred)
1. Add 100 µL of Ethyl Acetate to each tube. SUPPLEMENT - When using an automated robotic system
2. Add 50 ng of Ketamine-d4 to each sample.
3. Add 1.0 mL, 1% HCl in Deionized Water. If 2.0 mL of sample is used than
use 2.0 mL of 1% HCl in Deionized Water.
4.If cloudy or precipitated Centrifuge 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.
3. Proceed to Sample Extraction within 60 min. of column conditioning.
2. Wash column with 3.0 mL of Deionized Water. Allow to flow by gravity.
3. Wash column with 2.0 mL of Methanol. Allow to flow by gravity.
4. Wash column with 1.0 mL Ethyl Acetate. Allow 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.
1. Add 50 µL Ethyl Acetate and 50 µL Heptafluorobutyric Anhydride (HFBA)
to each dried column eluate, cap tubes and gently mix the contents.
2. Heat at 65°C for 20 min.
3. Dry down the eluates at less than 50°C and reconstitute
with 100 µL Ethyl Acetate.
4. Inject 1 µL or transfer to an autosampler vial.
2. Inject 1 µL or transfer to an autosampler vial.
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%).
