Notes: When adding an internal standard dissolved in an organic solvent to a sample, the solvent volume must not exceed 3% of the buffered sample volume. Higher solvent concentrations may produce extraction losses.

SAMPLE PREPARATION (Following Hydrolysis)

1. Add Glacial Acetic Acid (200µL /mL urine) and 1 mL 0.2M Acetate buffer, pH4.0 to each hydrolyzed sample. Mix. (The acid and buffer may be combined in these proportions as a single reagent).
2. If adjusted sample is turbid or precipitated Centrifuge for 5 minutes at 3000 RPM.

Note: Heating accelerates the hydrolysis and causes changes in the urine matrix which almost always results in clear samples after the pH is adjusted with Acetic Acid. Without heating, 1-2% of samples will require centrifugation to achieve normal flow rates through the column. A normal flow is particularly important when using a robotic sample processor with timed steps. It is a good practice to inspect all hydrolyzed samples before adding them to the column. Samples with obvious cloudiness or sediment should be centrifuged at 3000 RPM for 5 min. before continuing the procedure.
Note: Acetate Buffer is prepared as follows:
Weigh 27.2 gm of Sodium Acetate Trihydrate into a one liter volumetric flask. Add 800 mL of D.I. Water. Mix and dissolve. Bring the pH down to 4.0 with Glacial Acetic Acid. Finally, adjust the volume to one liter with D.I.Water.

SERUM SAMPLE PREPARATION (WITHOUT HYDROLYSIS)

1. Add 3.0 mL of 0.2M Acetate Buffer pH 4.0 to each sample (typically 0.1-2.0 mL), mix.

2. Add 15 ng each of deuterated Delta-9-THC-COOH-D9 and Delta-9-THC-D3 (25µL of a 6.0 µg/10mL Methanol) per mL of sample as internal standard.

3. If adjusted sample is turbid or precipitated Centrifuge for 5 minutes at 3000 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. Proceed to Sample Extraction within 30 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 1.0 mL Acetonitrile:Water (15:85) containing 2% Triethylamine (TEA)* to each column. Allow to flow by gravity.
3. Add 3 ml of Deionized H20. Allow to flow by gravity.
4. Add 1.0 mL of 70% methanol containing 0.5% Glacial Acetic Acid.to each column. Allow the columns to flow by gravity.
5. Remove residual acidic methanol wash from the column by momentarily applying vacuum adjusted to at least 7" Hg for approximately 15 seconds.

*Acetonitrile:Water (15:85) with 2% TEA 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 solvent 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 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.
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 1.0 mL Ethyl Acetate:Isopropanol (85:15) 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 60^°C.

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. 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

A. Using BSTFA

1. To each dried extract add 50 µL Acetonitrile, vortex mix, then add 50 µL BSTFA containing 1% Trimethylchlorosilane (TMCS).
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. Incubate the mixture @ 70^°C for 20 min.
4. Allow the mixture to come to room temperature. Inject 1.0 µL.

B. Using N-Methyl-N (tert-butyldimethylsilyl)-trifluoroacetamide (MTBSTFA) containing 1% TBDMCS

1. To each dried extract, add 75 µL n-Butyl Chloride and 25 µL MTBSTFA containing 1% TBDMCS.
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. Incubate the mixture @ 75^°C for 30 min.
4. Allow the mixture to come to room temperature. Inject 1.0 µL.