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Opiates in Human Urine – Propyl Derivatives
for GC or GC/MS Confirmations
Using 200mg 6mL 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
concentrated HCI
Add 200µL 10% hydroxylamine solution in DI H
2
O
Mix/vortex
Heat to 90°C for 40 min 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 50% ammonium hydroxide
Mix/vortex
Adjust sample pH 5 to 6 by drop wise addition with
50% ammonium hydroxide
Prepare Sample-enzymatic Hydrolysis of Glucuronides
To 2mL of urine, add internal standard(s), and 1mL of
ß-Glucuronidase solution
ß-Glucuronidase solution contains 5,000 F units/mL
Patella vulgata in 100mM acetate buffer (pH 5.0)
Hydrolyze for 3 hours at 60°C
Centrifuge for 10 minutes at 2,000rpm and discard pellet
Adjust sample pH to 5 to 6 with 1.0 N NaOH
Condition Clean Extraction Column
1 x 3mL CH
3
OH
1 x 3mL DI H
2
O
1 x 1mL 100mM phosphate buffer (pH 6.0) then aspirate
NOTE:
Aspirate at <3"Hg to prevent sorbent drying
Apply Sample
Load at 1 to 2mL/minute
Wash Column
1 x 3mL DI H
2
O then aspirate
1 x 3mL 100mM acetate buffer (pH 4.5) then aspirate
1 x 3mL CH
3
OH then aspirate
Dry column (5 minutes at >10"Hg)
Elute Opiates
1 x 3mL ethyl acetate/isopropanol/ammonium
hydroxide (84:12:4)
Dry Eluant
Evaporate to dryness at <40°C
Derivatize
Add 200µL of a 1:1 solution of proprionic
anhydride pyridine
Make this solution fresh daily
Mix/vortex
React for 60 minutes at 60°C in a heater block
Remove from heat source to cool
Evaporate to dryness at <40°C
Reconstitute the residue with 50µL of ethyl
acetate/methanol (70:30)
Quantitate
Inject 1 to 2µL onto GC/MS
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 standard for GC/MS: Codeine-D3 and Morphine-D3
** Quantitation ion
Recommended GC Column
Part Number
TraceGOLD TG-5MS 30m x 0.25m x 0.25µm
26098-1420
Pharmaceutical/Biotech
Applications