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Buprenorphine and Norbuprenorphine in Urine and Blood
for GC/MS Confirmations
Using 200mg 10mL HyperSep Verify-CX Extraction Column
(Part Number: 60108-742)
Prepare Sample
To 1mL of 100mM acetate buffer (pH 5.0) add internal
standard*
Mix/vortex and add 1mL of urine or blood
Add 2mL of 100mM acetate buffer (pH 5.0)
Mix/vortex
Sample pH should be 6.0±0.5
Adjust pH accordingly with 100mM monobasic or dibasic
sodium phosphate
Centrifuge as appropriate
Enzyme Hydrolysis of Glucuronides
To 1mL of 100mM acetate buffer add internal standard*
Add 1 to 5mL of urine or blood
Mix/vortex then add 2mL of 100mM acetate buffer
(pH 5.0)
Hydrolyze with Helix Pomatia (5,000 units/mL), heat for
3 hours at 60°C
Cool before proceeding
Condition Verify-CX Extraction Column
3mL of CH
3
OH then aspirate
3mL of DI H
2
O then aspirate
3mL of 100mM acetate buffer (pH 5.0) then aspirate
NOTE:
Aspirate at <3"Hg to prevent sorbent drying
Apply Sample
Load sample at 1 to 2mL/minute
Wash Column
2mL of DI H
2
O
3mL of 100mM acetate buffer (pH 5.0)
3mL of methanol
Dry column (5 to 10 minutes at >10"Hg/full flow for
positive pressure Manifold)
Elute Buprenorphine/Norbuprenorphine
3mL of CH
2
Cl
2
/IPA/NH
4
OH (78:20:2)
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)
NOTE:
Before proceeding, ensure there are no water droplets at
the bottom of the collection tube (this may increase drying time
and decrease BSTFA derivatizing efficiency)
Dry Eluate
Evaporate to dryness at <40°C
Derivatize
Add 50µL of ethyl acetate and 50µL of BSTFA
(with 1% TMCS)***
React for 20 minutes at 70°C
Remove from heat source to cool
NOTE:
Do not evaporate BSTFA
Quantitate
Inject 1 to 2µL of sample onto the gas
chromatograph/mass spectrometer
For mass spectrometry, monitor the following ions:
Analyte
Primary Ion Secondary Tertiary
Buprenorphine-D4-TMS*
454
486
510
Buprenorphine-TMS
450
482
506
Norbuprenorphine-TMS
468
500
524
Norbuprenorphine-D3-TMS*
471
503
527
* Suggested internal standard
** Suggested quantitation ion
*** Part number TS-38831 (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
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Forensic
Applications