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Forensic
Applications
Buprenorphine and Norbuprenorphine in Blood and Urine
Using 500mg 6mL HyperSep Aminopropyl Extraction Column
(Part Number: 60108-519)
Sample Preparation
1mL of 100mM acetate buffer (pH= 5)
Add internal standard*
Mix/vortex
Add 1mL of blood, plasma/serum
Add 2mL of 100mM acetate buffer (pH= 5)
Mix/vortex
Sample pH should be 6.0±0.5
Adjust pH with 100mM monobasic or dibasic
sodium phosphate
Centrifuge
Enzyme Hydrolysis of Glucuronides
1mL of 100mM acetate buffer
Add internal standard*
Add 1 to 5mL of blood or urine
Mix/vortex
Add 2mL of 100mM acetate buffer (pH= 5)
Hydrolyze with helix pomatia (5,000 units/mL)
Heat for 3 hours at 60°C
Cool before proceeding
Condition HyperSep Aminopropyl Extraction Column
1 x 3mL CH
3
OH
1 x 3mL DI H
2
O
1 x 1mL 100mM acetate buffer (pH=5.0)
NOTE:
Aspirate at <3"Hg to prevent sorbent drying
Apply Sample
Load at 1 to 2mL/minute
Wash Column
1 x 2mL DI H
2
O
1 x 3mL 100mM acetate buffer (pH=5.0)
1 x 3mL methanol
Dry column (5 to 10 minutes at greater than 10"Hg/full
flow for positive pressure manifold)
Elute
1 x 3mL methylene chloride/iso-propano/ammonium
hydroxide (78/20/12) (make elution solvent fresh)
Collect eluate at 1 to 2mL/minute
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.
Evaporate to dryness at <40°C
Derivatize
Add 50µL ethyl acetate and 50µL BSTFA
(with 1% TMCS)**
React 20 minutes at 70°C
Remove from heat source to cool
NOTE:
Do not evaporate BSTFA
Analysis
Inject 1 to 2µL onto GC/MS
For mass spectrometry monitor the following ions:
Compound
Primary lon
Secondary
Tertiary
Buprenorphine-D4-TMS*
454
486
510
Buprenorphine-TMS
450
482
506
Norbuprenorphine-TMS
468
500
524
Norbuprenorphine-D3-TMS*
471
503
527
* Internal standards: Buprenorphine-D4-TMS and Norbuprenorphine-D3-TMS
** Part number TS-38831
Recommended GC Column
Part Number
TraceGOLD TG-5SilMS 30m x 0.25m x 0.25µm
26096-1420