DETECTABUSE™ "NO VACUUM" GRAVITY SERIES GV-65 METHOD FOR THE ANALYSIS OF
HYDROLYZED OR "FREE" OPIATES IN URINE BY GC-MS
Although this method has performed well in our laboratory, it must be validated by your laboratory before it is used to report patient values. We would appreciate your comments on its performance and welcome your suggestions for improvements or enhancements.
Revised: January 2003 (* TEA Elution Solvent Stability)
SAMPLE PREPARATION- (Please see Notes and Supplemental Information before proceeding)
A. Acid Hydrolysis
1. Pipet 1.0 ml urine sample into a 16x100 mm pressure rated glass tube with a Teflon® lined screw cap.
2. Pipet 0.2 mL concentrated HCL into each tube.
3. Add 300 ng Morphine-d3, 300 ng Codeine-d3, and other appropriate deuterated
standards into each tube. (See note B at the end of the sample prep. section)
Optional Oxime Formation to Eliminate Interference From Codones and Morphones (See Derivatization note)
A. Add 100 µL (10% w/v) Hydroxylamine (1 g Hydroxylamine HCl/10
mL Deionized H2O)
B. Vortex and proceed to step 4.
4. Cap tubes and heat in autoclave or heating block. (See note C at the end of the sample prep. section)
5. Cool tubes.
6. Add 1.0 mL of a 12% aqueous Potassium Hydroxide (KOH) per mL of sample and mix. 12 g KOH (Assay 87%) is added to a 100 mL flask which is then carefully filled with Deionized Water. ( See precaution at the end of this section)
7. Add 1.0 mL of a 20% solution of monobasic Potassium Phosphate solution to each sample and mix (20 grams/100 mL H2O prepared monthly). Final pH should be between 2.5 and 3.5. If necessary, adjust the pH with a few drops of either 1N KOH or 1N HCl.
8. Samples should be centrifuged for 3 min. at 3000 RPM.
Precaution: Adsorption must be done at an acidic pH of approximately 2.5-3.5. The 1.0 mL of KOH and buffer added in steps 6 and 7 ordinarily sets up the optimum pH for adsorption. If however, the pH is too high, i.e. greater than 4.0, drug recoveries will decline with increasing pH. If the pH is too low, the column washes will not remove all of the hydrolysis acid remaining on the column. The excess acid may cause the basic elution solvent to become weakly basic or acidic which will lead to partial or complete hindrance of drug elution. A non-bleeding pH paper can be used to test the pH of the eluate by wetting the paper with the solvent followed by a water rinse which will enable the color reaction to take place. This should only have to be done for the first run to assure that pH conditions are correct throughout the run. If you encounter any problems with this procedure please contact us for assistance.
B. Enzymatic Hydrolysis Sample Preparation Without Hydrolysis - "Measurement of Free Opiates"
(See Note D at the end of the sample prep. section)
1. Pipet 1.0 ml urine sample into a 16x100 mm pressure rated glass tube
with a Teflon® lined screw cap. Notes:
1. Add 0.1 mL of 0.2M Acetate Buffer, pH 5.0 to 1 mL of urine containing
100 ng Morphine-d3 and 100 ng Codeine-d3 as internal standards. (pH should
be 4.5 - 5.0)
2. Add approximately 5000 units of Beta-Glucuronidase, Sigma type L-2,
from Patella Vulgata, to each sample.
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).
Optional Oxime Formation
A. Add 100 µL (10% w/v Hydroxylamine.
B. Vortex and incubate at 50 °C for 30 min. Proceed to step 4.
4. Add 1.0 mL of a 1% HCl in H2O and mix.
5. Samples should be centrifuged for 3 min. at 3500 RPM.
3. Add 100 ng Morphine-d3 and 100 ng Codeine-d3 into each tube.
Optional Oxime Formation (Cannot be used when testing for 6-MAM)
A. Add 100 µL Hydroxylamine
(10% w/v aqueous) to prepared samples.
B. Vortex and incubate at 50°C for 30 min. Proceed to Step 4.
4. Add 1.0 mL of a 1% HCl in H2O and mix.
5. If cloudy or precipitated, Centrifuge for 3 minutes at 3000 RPM.
A. Either enzymatic or acid hydrolysis may be used to when quantitating
urinary opiates.
B. 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.
C. 1. Autoclave at 150°C for 15 min. at 15 PSI.
2. Heating block at 120°C for at least 30 min. Complete hydrolysis
requires at least 2 hours.
D. A separate 6-MAM method is available which uses 4 mL of urine for greater
sensitivity.
HARDWARE SETUP - (Please refer
to the Detectabuse Hardware Setup Instructions)
Column Conditioning and Activation of Cation Function
1. Wash column with 1.0 mL of Methanol. Allow to flow by gravity. SAMPLE EXTRACTION - (Please see Notes at end of this section
before proceeding)
1. Pour samples onto preconditioned column. Hydrolyzed samples should
be carefully poured to prevent transfer of hydrolysis debris onto the
column. Allow to flow by gravity. Samples will flow through the column
at a rate of 1-2 mL/min. 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. DERIVATIZATION - Following are some of the more commonly used
derivatizing schemes. Others such as PFPA are available upon request.
Single TMS DERIVATIZATION - Using BSTFA or MSTFA with 1% TMCS
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.
2. Wash column with 3.0 mL of Deionized Water. Allow the columns to flow
by gravity.
3. Wash column 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.
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.
1. To each dried extract add 50 µL Acetonitrile, vortex mix, then add
50 µL BSTFA or MSTFA.
3. Mix the tube contents, flush with nitrogen or argon and cap the tube
or transfer contents into 100 µL reaction vials and seal.
4. Incubate the mixture @ 70°C for 20 min.
5. Allow the mixture to come to room temperature. Inject 2.0 µL.
Single DERIVATIZATION - Using MBTFA
1. To each dried extract add
50 µL Acetonitrile and 50µL 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.
Double Derivative - Oxime/TMS
1. Follow instructions for optional oxime formation.
2. Follow instructions for TMS derivatization.
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.
The use of the optional oxime derivative converts Morphone and Codone 6-Keto positions to their corresponding oximes which prevents direct silane formation via enolization at this site. The subsequent treatment of extracts with BSTFA or MSTFA forms the TMS derivative of Codeine and Morphine hydroxyl function and TMS/Oxime derivatives at the Keto-Oxime functions of the Codones and Morphones. This double derivative technique produces significantly different spectra and retention times for the Codones and Morphones without affecting Codeine and Morphine. The improved chromatographic separation produced by this technique results in complete separation of 6-Hydroxyl Opiates from 6-Keto Opiates on a 12 meter DB-1 or DB-5 column.
Many laboratories use the MBTFA derivative without the oxime step. Although several compounds overlap, separation is achieved by ion selection.
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%).
