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Opiates in Urine – Propyl Derivatives for GC or GC/MS Confirmations
Using 200mg 10mL HyperSep Verify-CX Extraction Column
(Part Number: 60108-742)
Prepare Sample – Acid Hydrolysis of Glucuronides
To 2mL of urine add internal standard(s)* and 400µL of
concentrated HCl
Add 200µL of 10% hydroxylamine solution
Mix/vortex
Heat to 90°C for 40 minutes in a heating block, or an
autoclave for 15 minutes on a liquid cycle
Cool before proceeding
Centrifuge for 10 minutes at 2,000rpm and discard pellet
Add 500µL of 50% ammonium hydroxide
Mix/vortex
Adjust sample pH to 5-6 by drop-wise addition of 50%
ammonium hydroxide
Prepare Sample – Enzyme Hydrolysis of Glucuronides
To 2mL of urine add internal standard(s)* and 1mL of
β
-Glucuronidase solution (
β
-Glucuronidase solution
contains 5,000 F units/mL)
Hydrolyze for 3 hours at 60°C
Centrifuge for 10 minutes at 2,000rpm and discard pellet
Adjust sample pH to 5-6 with 1.0 M NaOH
Condition Verify-CX Extraction Column
3mL of CH
3
OH then aspirate
3mL of DI H
2
O then aspirate
1mL of 100mM phosphate buffer (pH 6.0)
NOTE:
Aspirate at <3"Hg to prevent sorbent drying
Apply Sample
Load sample at 1 to 2mL/minute
Wash Column
3mL of DI H
2
O
3mL of 100mM acetate buffer (pH 4.5)
3mL of CH
3
OH
Dry column (5 minutes at >10"Hg)
Elute Opiates
3mL of CH
2
Cl
2
/IPA/NH
4
OH (84:12:4)
Collect eluate at 1 to 2mL/min
NOTE:
Prepare elution solvent fresh daily; add IPA/NH
4
OH mix,
then add CH
2
Cl
2
(pH 11-12)
Dry Eluate
Evaporate to dryness at <40°C
Derivatize
Add 200µL of a 1:1 solution of propionic
anhydride:pyridineNOTE:
Make this solution fresh daily
Mix/vortex
React for 60 minutes at 60°C in a heat block
Remove from the heat source to cool
Evaporate to dryness at <40°C
Reconstitute the residue with 50µL of ethyl
acetate/methanol (70:30, v/v)
Quantitate
Inject 1 to 2µL onto gas chromatograph
For mass spectrometry, monitor the following ions:
Compound
Primary Ion** Secondary
Tertiary
Hydrocodone
299
242
214
Codeine
355
282
229
Codeine-D3*
358
285
232
Oxycodone
371
314
298
Hydromorphone
285
341
228
6-Acetylmorphine
327
268
383
Oxymorphone
357
300
413
Morphine
341
268
397
Morphine-D3*
344
271
400
* Suggested internal standards
Recommended GC Columns
Part Number
TRACE TR-DoA5 Column, 15m x 0.25mm x 0.25µm
26AF130P
TRACE TR-DoA35 Column, 15m x 0.20mm x 0.33µm
26AC497P
108
Forensic
Applications