1. Add 2.0 mL of urine to a 16 x 100 mm disposable borosilicate glass tube.
2. Add 250 ng of the appropriate deuterated standards to each sample.

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.

SAMPLE HYDROLYSIS

1. Add 0.5 mL of 0.2M Acetate Buffer, pH 5.0 to each prepared sample (pH should be 4.5 - 5.0).
2. Add 10,000 units of Beta-Glucuronidase, Sigma type H-2S from Helix Pomatia (or equivilent) to each sample.
3. Mix gently and incubate at 55^°C for two hours or 37^°C for 4 hours. Complete hydrolysis is also achieved in 16 hours at room temperature (15 - 30^°C).
4. Add 5.0 mL of 0.25M Phosphate Buffer, pH 6.0.
5. Centrifuge for 3 minutes at 4000 RPM.

HARDWARE SETUP - (Please refer to the Detectabuse Hardware Setup Instructions)

COLUMN CONDITIONING

1. Wash column with 1 mL of Methanol. Allow to flow by gravity.
2. Add 0.5 mL of Sodium Bisulfite (50 grams/100mL H2O prepared weekly) to each column.
3. Proceed to Sample Extraction within 60 min. of column conditioning.

SAMPLE EXTRACTION - (Please see Notes at end of this section before proceeding)

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.
2. Add 2 mL of Water:Methanol (60:40). Allow the columns to flow by gravity.
3. Add 1.0 mL Hexane to each column and proceed immediately to Step 4.
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).

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. Any columns that have not emptied within 5 or 6 min. may be induced with a low vacuum from a small vacuum pump.

SAMPLE ELUTION

1. Sample elution is done outside of the vacuum box.
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 basic Elution Solvent (Ethyl Acetate:Isopropanol 95:5) w/2% 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 55^°C.

* Elution solvent with 2% TEA, (2 mL TEA is added to 98 mL of Ethyl Acetate:Isopropanol 95:5 ) 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.

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.

DERIVATIZATION

1. To each dried extract add 70 µL Acetonitrile, vortex mix, then add 30 µL MTBSTFA and 15 µL Trimethylsilylimidizole (TMSI).
Mix the tube contents, flush with nitrogen or argon and cap the tube or transfer contents into 100 µL reaction vials and seal.
2. Incubate the mixture @ 70^°C for 30 min.
3. Allow the mixture to come to room temperature. Inject 1.0 µL.

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.
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).