1. Add 1.0 mL of urine to a 16 x 100 mm disposable borosilicate glass tube with an inert screw cap top.
2. Add 50 ng of Ketamine-d4 to each sample.
3. Add 3.0 mL of 0.25M Phosphate Buffer, pH 6.0.
4.If cloudy or precipitated Centrifuge for 3 minutes at 3000 RPM.

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.
2. Add 0.5 mL of a saturated Sodium Bisulfite solution to each column (50 grams/100mL H2O prepared weekly).
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 1.0 mL Water:Methanol (40:60) to each column. Allow to flow by gravity.
3. Add 1.0 mL n-Butyl Chloride 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 labeled screw cap 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. Add 100 µL of Tartaric Acid in Ethyl Acetate (1mg/mL) and dry under N2 or argon at less than 50⁰C.

*Elution Solvent without Triethylamine - Ethyl Acetate:Isopropanol (95:5), may be prepared and stored in a glass bottle with a Teflon or Polypropylene lined cap. Elution solvent with 2% TEA (2 mL TEA is added to 98 mL Ethyl Acetate:Isopropanol mix), is stable for at least one month. Close bottle tightly when not in use.

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 (Only required if being assayed with Amphetamines)

Using HFBA:
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 65⁰C for 20 min.
3. Add 100 µL of Tartaric Acid in Ethyl Acetate (1mg/mL) to each tube, followed by gentle mixing.
4. Dry down the eluates at less than 50⁰C and reconstitute with 100 µL Ethyl Acetate.
5. Inject 1 µL or transfer to an autosampler vial.

WITHOUT DERIVATIZATION

1. Add 100 µL of Ethyl Acetate to each tube.
2. Inject 1 µL or transfer to an autosampler vial.

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).