76 / 144
Caffeine, Theophylline and Theobromine in Urine,
Blood and Plasma/Serum for LC/PDA Confirmations
Using 200mg 10mL HyperSep Verify-CX Extraction Column
(Part Number: 60108-742)
Prepare Sample
To 1mL of 100mM acetic acid add internal standard
Add 1mL of urine, blood or plasma/serum
Add 2mL of 100mM acetic acid
Vortex and centrifuge as appropriate
Condition Verify-CX Extraction Column
3mL of CH
3
OH then aspirate
3mL of DI H
2
O then aspirate
3mL of 100mM acetic acid
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
3mL of 100mM acetic acid
Dry column (5 minutes at >10"Hg)
Elute Beta Caffeine/Theophylline/Theobromine
3mL of ethyl acetate/methanol (90:10, v/v)
Collect eluate at 1 to 2mL/minute
Evaporate
Evaporate to dryness at <40°C
Reconstitute
1,000µL of 0.1% formic acid (aq)
Analysis
Inject 20µL of sample onto LC/PDA system
Mobile phase: acetonitrile:0.1% formic acid (10:90, v/v)
Flow rate: 0.1mL/min
Detector: diode array (200 to 350nm)
Column temperature: ambient
Recommended HPLC Column
Part Number
Hypersil GOLD 3µm, 150 x 2.1mm
25003-152130
76
Forensic
Applications
Carboxy-delta 9-THC in Urine
Using 500mg 6mL HyperSep Aminopropyl Extraction Column
(Part Number: 60108-519)
Sample Preparation
To 2mL of urine add internal standard*
Add 100µL of 10 M NaOH
Mix/vortex
Hydrolyze for 20 minutes at 60°C
Cool before proceeding
Adjust sample pH to 3.0 with approx. 1.0mL of glacial
acetic acid
Condition HyperSep Aminopropyl Extraction Column
1 x 3mL CH
3
OH
1 x 3mL DI H
2
O
1 x 1mL acetate buffer (pH=3.0)
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 2mL 100mM HCI/ACN (95:5)
Dry column (5-10 minutes at greater than 10"Hg)
1 x 200µL hexane then aspirate
Elute
1 x 3mL hexane/ethyl acetate (50:50)
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
Add 50µL BSTFA (with 1% TMCS)**
Mix/vortex
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 (TMS)
Primary Ion*** Secondary
Tertiary
Carboxy-delta 9-THC-D3*
374
476
491
Carboxy-delta 9-THC-D9*
380
479
497
Carboxy-delta 9-THC
371
473
488
* Suggested internal standard for GC/MS: -Carboxy-delta 9-THC-D9 and 9-THC-D3
** Part number TS-38831
*** Quantitation ion
Recommended GC Column
Part Number
TraceGOLD TG-35MS 30m x 0.25m x 0.25µm
26094-1420