1. Pipet 2.0 mL of urine to a 16 x 100 mm disposable borosilicate glass tube with an inert screw cap top.
2. Add 50 ng of Cotinine-d3 per mL of sample as internal standard..
3. Add 1.0 mL, 1% HCl in Deionized Water.Vortex mix.
4. If adjusted sample is turbid or precipitated centrifuge for 3 minutes at 3000 RPM.

Notes: 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.
2. Add 1.0 mL of a Sodium Bisulfite solution to each column. Prepare by dissolving 5 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.

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 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 40°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.

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.

RECONSTITUTION

1. To each dried extract add 100 µL Ethyl Acetate, vortex mix, then flush with nitrogen or argon.
3. Mix the tube contents, and cap the tube or transfer contents into 100 µL reaction vials and seal.
4. Inject 2.0 µL.

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%).