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Forensic
Applications
Carboxy-delta-9-THC, Delta-9-THC (parent), Delta-9-Hydroxy THC in
Whole Blood for GC or GC/MS Confirmations
Using 200mg 6mL HyperSep Verify-AX Extraction Column
(Part Number: 60108-730)
Prepare Sample
To 1 to 2mL of whole blood add internal standard(s)*
Mix/vortex
Vortex and add dropwise 1mL of ice cold acetonitrile
Centrifuge and transfer acetonitrile to a clean tube
Adjust sample pH to 3.0±0.5 with approximately
2.0mL of 100mM sodium acetate buffer (check pH of
buffer to ensure that the pH value is
≈
3.0)
Condition Verify-AX Extraction Column
3mL of CH
3
OH then aspirate
3mL of DI H
2
O then aspirate
1mL of acetate buffer (pH 3.0) then aspirate
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
2mL of 100mM HCl/acetonitrile (95:5)
Dry column (5 to 10 minutes at >10"Hg/full flow for
positive pressure manifold)
200µL of hexane then aspirate (Additional step to remove
any residual moisture. Could substitute
200µL MeOH for hexane.)
Optional:
Dry column (5 minutes at >10"Hg/full flow for
positive pressure manifold)
NOTE:
The delta-9-THC (parent) will elute in hexane, so special
attention must be paid to not use more than 200µL of hexane in
the wash/dry step (the 200µL hexane wash step can be eliminated
if the column is allowed to dry longer under vacuum or by positive
pressure gas flow)
Elute THC (metabolites)
2mL of hexane (optional, contains delta-9-THC)
3mL of hexane/ethyl acetate (50:50)
Collect eluate at 1 to 2mL/minute
NOTE:
Before proceeding, ensure that there are no water droplets
at the bottom of the collection tube (this may increase drying
time and decrease BSTFA derivatizing agent efficiency)
Dry Eluate
Evaporate to dryness at <40°C
Derivatize
Add 50µL of ethyl acetate and 50µL of BSTFA
(with 1% TMCS)***
Mix/vortex
React for 20 minutes at 70°C
Remove from heat source to cool
NOTE:
Do not evaporate BSTFA
Quantitate
Inject 1 to 2µL onto gas chromatograph
For mass spectrometry, monitor the following ions:
Compound (TMS)
Primary Ion Secondary Tertiary
D
3
-Carboxy-delta-9-THC*
374
476
491
D
9
-Carboxy-delta-9-THC*
380
479
497
Carboxy-delta-9-THC
371
473
488
D
3
-Hydroxy-delta-9-THC*
374
462
477
Hydroxy-delta-9-THC
371
459
474
D
3
-delta-9-THC*
374
389
Delta-9-THC
371
386
(303, 315, 330, 343)**
*
Suggested internal standard
** Ions common to deuterated delta-9-THC and non-deuterated compounds
*** 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