Revised: May 2006
Note: This revision includes: use of Helix Pomatia enzyme in place of Patella Vulgata, changes in the derivatization (elimination of acetonitrile which greatly increases the reagent reactivity), changes in ion selection when using MBTFA.

B. Enzymatic Hydrolysis
1. Pipette 1.0 mL of sample and 0.1 mL of 0.2M Acetate Buffer, pH 5.0 into a 13x100 mm pressure rated borosilicate glass tube with a Teflon^® lined screw cap (pH should be 4.5 - 5.0). For urine analysis add 1000 ng/mL of appropriate deuterated internal standard to each sample tube. Serum and/or oral fluid internal standard levels should be close to the cutoff level requirements of the assay.
2. Add approximately 5000 units of Beta-Glucuronidase, Sigma type HP-25, from Helix Pomatia, to each sample. Typical preparations contain approximately 100,000 units/mL. In this case 50 µL contains 5000 units.
3. Mix gently and incubate at 50°C for 2 1/2 hours or 37°C for 4 hours. Complete hydrolysis is also achieved in 16 hours at room temperature (15 - 30°C).
4. Add 1.0 mL of 1% HCl in H2O and mix.
5. Samples should be centrifuged for 3 min. at 3500 RPM.
Note: If methoxylamine is used to form the oxime derivative 100 mg should be added per mL of the Acetate buffer.

Sample Preparation Without Hydrolysis - "Measurement of Free Opiates" (See Note B at the end of the sample prep. section)

Measurement of Free Opiates in Urine

Notes:
A. 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.
B. A separate 6-MAM method is available which uses 3 mL of sample for greater sensitivity.

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

Column Conditioning and Activation of Cation Function

1. Wash columns 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 25 grams of Sodium Bisulfite in 100 mL of a (1:1) mixture of H2O:0.25M Phosphate Buffer, pH 6.0. Prepare monthly (Store refrigerated).
3. Proceed to Sample Extraction within 60 min. of column conditioning.

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

1. Pour samples onto preconditioned columns. Hydrolyzed samples should be carefully poured to prevent transfer of hydrolysis debris onto the columns. Allow to flow by gravity. Samples will flow through the columns at a rate of 1-2 mL/min.
2. Wash columns with 3.0 mL of Deionized Water. Allow the columns to flow by gravity.
3. Wash columns with 2.0 mL of Methanol. Allow the columns to flow by gravity.
5. Add 1.0 mL Ethyl Acetate. Allow the columns to flow by gravity.

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. 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.

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 - Following are two of the more commonly used derivatizing schemes for extended opiates. They are superior to TMS derivatives because the compounds are well separated by either retention time or Ions, eliminating the need to form oximes to achieve the separation required for quantitation.

Anhydride Derivatization - Using Propionic Anhydride, 99+%  (Aldrich Chemical Company)
1. To each dried extract add 50 µL Pyridine, vortex mix, then add 50 µL Propionic Anhydride.
3. Mix the tube contents, flush with nitrogen or argon and cap the tube.
4. Incubate the mixture @ 100°C for 45 min.
5. Allow the mixture to come to room temperature.Add 1.0 mL of Hexane. Mix.
6. Dry under Argon or Nitrogen @ 65°C.
7. Add 100 µL Ethyl Acetate. Mix and inject 1-2 µL.

DERIVATIZATION - Using MBTFA

1. To each dried extract add 75µL of N-Methyl-bis(trifluoroacetamide) (MBTFA).
2. Mix the tube contents, cap and incubate at 65°C for 30 mins.
3. Transfer contents into 100 µL reaction vials and seal.
4. Inject 1-2.0 µL.

Note: