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Forensic
Applications
Opiates in Urine – Oxime TMS Procedure
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 enzyme
preparation in buffer
Mix/vortex
Heat to 60°C for a sufficient time in a heating block
(The time depends upon analytes and enzyme)
Add 200µL of 10% hydroxylamine solution
Heat to 60°C for 30 minutes in a heating block
Adjust pH to 5-6
Centrifuge for 10 minutes at 2,000rpm and discard pellet
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 (78:20:2)
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 100µL of ethyl acetate and 100µL of BSTFA
(with 1% TMCS)***
Overlay with N
2
and cap
Mix/vortex
React for 45 minutes at 70°C in a heat block
Remove from the heat source to cool
NOTE:
Do not evaporate BSTFA solution
Quantitate
Inject 1 to 2µL onto gas chromatograph
For mass spectrometry, monitor the following ions:
Compound
Primary Ion** Secondary
Tertiary
D
4
-meperidine*
251
222
250
Meperidine
247
218
246
D
4
-normeperidine TMS*
308
280
309
Normeperidine TMS*
305
276
304
Tramadol TMS
335
245
290
O-desmethyltramadol TMS
393
378
303
N-desmethyltramadol TMS
393
378
116
Pentazocine TMS
357
342
289
D
3
-codeine TMS*
374
359
346
D
6
-codeine TMS*
377
349
316
Codeine TMS
371
356
343
Norcodeine TMS
429
414
356
Dihydrocodeine TMS
373
315
358
D
3
-morphine TMS*
432
417
404
D
6
-morphine TMS*
435
420
404
Morphine TMS
429
414
401
Nomorphine TMS
487
472
414
Diacetylmorphine
369
327
268
D
3
-hydrocodone Oxime TMS
389
300
374
D
6
-hydrocodone Oxime TMS*
392
303
377
Hydrocodone Oxime TMS
386
297
371
D
3
-hydromorphone Oxime TMS 447
432
358
Hydromorphone Oxime TMS
444
429
355
D
3
-oxycodone Oxime TMS
477
462
420
D
6
-oxycodone Oxime TMS*
480
465
420
Oxycodone Oxime TMS
474
459
417
D
3
-oxymorphone Oxime TMS 535
520
290
Oxymorphone Oxime TMS
532
517
287
* Suggested internal standards for GC/MS; suggest trying D
6
-codeine and D
6
-morphine for
lowest LOD/LOQ
** Quantitation ion
*** Part number TS38831 (10 x 1mL ampules)
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