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Forensic
Applications
Buprenorphine and Norbuprenorphine in Urine,
Blood and Plasma/Serum for HPLC Analysis
Using 200mg 10mL HyperSep Verify-CX Extraction Column
(Part Number: 60108-742)
Prepare Sample
To 1mL of 100mM phosphate buffer (pH 5.0) add
internal standards*
Add 1mL of urine, whole blood, plasma/serum
Add 2mL of 100mM phosphate buffer (pH 6.0)
Mix/vortex and centrifuge as appropriate
Total (sum of free and conjulated
Buprenorphine/Norbuprenorphine)
To 1mL of acetate buffer (pH 5.0) containing 5,000 F
units/mL of
β
-Glucuronidase, add internal standards*
To this solution, add 1mL of urine or blood
Mix/vortex
Hydrolyze for 3 hours at 65°C
Allow to cool, then add 3mL of 100mM phosphate
buffer (pH 6.0) and mix
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) 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 acetic acid
3mL of methanol
Dry column (5 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, v/v/v)
or
3mL of ethyl acetate/acetonitrile/ammonia (78:20:2, v/v/v)
Collect eluate at 1 to 2mL/minute
Evaporate
Evaporate to dryness at <40°C
Reconstitute
Add 50µL of methanol
Quantitate
Inject 5µL of sample onto the LC/MS system
For mass spectrometry, monitor the following ions:
Compound
Detection Ions
Buprenorphine
468.4/55.1
Buprenorphine-D4*
472.4/59.1
Norbuprenorphine
414.3/83.1
Norbuprenorphine-D3*
417.4/55.1
* Suggested internal standards
Mobile phase: acetonitrile: 0.1% formic acid (50:50, v/v)
Flow rate: 0.35mL/min
Recommended HPLC Column
Part Number
Hypersil GOLD Phenyl 3µm, 50 x 2.1mm
25903-054630