1. Add 1.0 mL of sample to a 16 x 100 mm disposable borosilicate glass tube with an inert screw cap top.
2. Add 500 ng of D-Amphetamine-d5 and 500 ng of D-Methamphetamine-d5 to each urine sample or 50 ng of D-Amphetamine-d5 and 50 ng of D-Methamphetamine-d5 to each serum or oral fluid sample (Include deuterated MDA, MDMA, and MDEA if analyzed).
3. Add 1.0 mL, 1% HCl in Deionized Water. If 2.0 mL of sample is used than use 2.0 mL of 1% HCl in Deionized Water.
4. The occasional cloudy or precipitated sample should be centrifuged for 3 minutes at 3000 RPM.

Note: When adding an internal standard dissolved in an organic solvent to a sample, the solvent volume must not exceed 5% of the buffered sample volume. Higher solvent concentrations may produce extraction losses.

WITH PERIODATE OXIDATION (OPTIONAL)

1. Add 2.0 mL of 0.25 M Phosphate Buffer, pH 9.1 to 1 mL of sample spiked with internal standard as above.
2. Add 0.4 mL of 10% Sodium Metaperiodate in H2O to sample spiked with internal standard (pH should be between 8-9). Mix well.

Preparation of Sodium Metaperiodate: Add 10 grams of Sodium Metaperiodate to 100 mL of deionized H2O, mix well until solution is clear.

3. Incubate at room temperature for 20 min.
4. Add 0.4 mL,10% HCl in Deionized Water. A very basic sample may require more acid (Final pH should be between 2-3).
5. The occasional cloudy or precipitated sample should be centrifuged for 3 minutes at 3000 RPM.
6. Proceed to sample extraction.

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. Wash column with 2.0 ml of 0.1M glacial Acetic Acid. Allow to flow by gravity. This wash may be eliminated if oxidation is not performed.
3. Wash column with 3.0 mL of Deionized Water. Allow to flow by gravity.
4. Wash column with 1.0 mL of Methanol. Allow to flow by gravity.
5. Wash column with 1.0 mL Ethyl Acetate. Allow to flow by gravity. Proceed to Sample Elution.

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 1.5 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. Add 100 µL of Tartaric Acid** in Ethyl Acetate (1 mg/mL) to each tube, followed by gentle mixing.
5. 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.
** This is a saturated solution. Allow the crystals to settle before pipetting 100 µL aliquots into the elution tubes.

DERIVATIZATION - Following are some of the more commonly used derivatizing schemes. Others such as PFPA are available upon request.

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. Heat at 70°C for 20 mins.
2. Add 100 µL of Tartaric Acid in Ethyl Acetate (1 mg/mL) to each tube after they have cooled, followed by gentle mixing.
3. Dry down the eluates at less than 50°C and reconstitute with 100 µL Ethyl Acetate.
4. Inject 1.0 µL, or transfer to an autosampler vial.

Using the 4-CB Derivative:
1. Add 100 µL of 4-Carbethoxyhexafluorobutyrl chloride which has been diluted 1:10 with n-Butyl Chloride to each dried column eluate, and cap tubes. Gently mix the contents and incubate at 65°C for 20 min.
2. Add 300 µL Absolute Ethanol (Pure or specially Denatured 3A Formula) and incubate at 65°C for 15 min.
3. Dry the eluate under N2 or argon at less than 50°C. Reconstitute with 100 µL Ethyl Acetate.
4. Inject 1-2 µL or transfer to an autosampler vial.

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%).