DETECTABUSE™ GRAVITY SERIES GV-65 CATION EXCHANGE METHOD FOR THE ANALYSIS OF
TRICYCLICS 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: March, 2003
Supercedes May, 2001 - Revision addresses stability of TEA in sample elution section
SAMPLE PREPARATION - (Please see Notes and Supplemental Information before proceeding)
1. Add 2.0 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. 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.
1. Sample elution is done outside of the vacuum box. DERIVATIZATION - MBTFA
1. To each dried extract add 50 µL Acetonitrile, vortex mix, then add
50 µL MBTFA. 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.
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 the appropriate deuterated standards to each sample.
3. Add 0.5 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.
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:H2O (40:60). Allow the columns to flow by gravity.
4. 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).
5. Add 1.0 mL Hexane. 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, flush with nitrogen or argon and cap the tube
or transfer contents into 100 µL reaction vials and seal.
3. 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%).
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).
