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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
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 Enzyme Hydrolysis of Glucuronides
To 2mL of urine add internal standard(s)* and enzyme
preparation in buffer
Mix/vortex
Heat to 60°C for sufficient time in a heating block
(depends on analytes and enzyme)
Add 200µL 10% hydroxylamine solution
Heat to 60°C for 30 min in a heating block
Adjust pH to 5 to 6
Centrifuge for 10 minutes at 2,000rpm and discard pellet
Condition Extraction Column
1 x 3mL CH
3
OH
1 x 3mL DI H
2
O
1 x 3mL 100mM phosphate buffer (pH 6.0)
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
1 x 3mL 100mM acetate buffer (pH 4.5)
1 x 3mL CH
3
OH
Dry column (5 minutes at >10"Hg)
Elute Opiates
1 x 3mL CH
2
Cl
2
/IPA/NH
4
OH (76:20:4)
Collect eluate at 1 to 2mL/minute
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 ethyl acetate and 100µL BSTFA
(with 1% TMCS)**
Overlay with N
2
and cap
Mix/vortex
React 45 minutes at 70°C in a heat block
Remove from heat source to cool
NOTE:
Do not evaporate BSTFA solution
Quantitate
Inject 1 to 2µL onto GC/MS
For mass spectrometry monitor the following ions:
Compound
Quant Ion Secondary
Tertiary
Meperidine-D4
251
222
250
Meperidine
247
218
246
Normeperidine-D4 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
Codeine-D3 TMS*
374
359
346
Codeine-D6 TMS*
377
349
316
Codeine TMS
371
356
343
Norcodeine TMS
429
414
356
Dihydrocodeine TMS
373
315
358
Morphine-D3 TMS*
432
417
404
Morphine-D6 TMS*
435
420
404
Morphine TMS
429
414
401
Normorphine TMS
487
472
414
Diacetylmorphine
369
327
268
Hydrocodone Oxime-D3 TMS
389
300
374
Hydrocodone Oxime-D6 TMS
392
303
377
Hydrocodone Oxime TMS
386
297
371
Hydromorphone Oxime-D3 TMS
447
432
358
Hydromorphone Oxime TMS
444
429
355
Oxycodone Oxime-D3 TMS
477
462
420
Oxycodone Oxime-D6 TMS
480
465
420
Oxycodone Oxime TMS
474
459
417
Oxymorphone Oxime-D3 TMS
535
520
290
Oxymorphone Oxime TMS
532
517
287
* Suggested internal standards for GC/MS: D4-Meperidine, D4-Normeperidine,
D3-Codeine, D3-Morphine D6-Hydrocodone D6-Oxycodone. Suggest trying D6-Codeine,
and D6-Morphine for lowest LOD/LOQ.
** Part number TS-38831
Pharmaceutical/Biotech
Applications