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Forensic
Applications
Cocaine and Benzoylecgonine in Oral Fluid
Using 60mg 6mL HyperSep Retain-CX Extraction Column
(Part Number: 60107-308)
Sample Preparation
Add 100 to 500µL of neat oral fluid sample to a clean tube
Add internal standard(s)* and let sit for 10 minutes at
room temperature
Add 800µL of 100mM phosphate buffer (pH= 6.0)
Mix/vortex for 10 seconds
Sample pH should be 6.0
Adjust pH with 100mM monobasic or dibasic
sodium phosphate
Condition HyperSep Retain-CX Extraction Column
1 x 200µL CH
3
OH
1 x 200µL DI H
2
O
1 x 200µL 100mM HCl
Apply Sample
Do not exceed 1mL/minute
Wash Column
1 x 500µL DI H
2
O
1 x 500µL 100mM HCl acid
1 x 500µL CH
3
OH/DI H
2
O (50:50)
Dry column (5 minutes at >10"Hg)
Elute
1 x 800µL CH
2
Cl
2
/IPA/NH
4
OH (70:26:4)
Do not exceed 1mL/minute
NOTE:
Prepare elution solvent fresh daily; add IPA/NH
4
OH mix,
then add CH
2
Cl
2
(pH 11-12)
Evaporate at <40°C under a stream of N
2
Derivatize
Fluoroalkylate:
Add 100µL PFPA (PFAA) or HFIP
Overlay with N
2
and cap
React 20 minutes at 70°C
Evaporate to dryness at <40°C
Reconstitute with 50µL ethyl acetate
TMS:
Add 25µL BSTFA (w. 1% TMCS)**
Add 25µL ethyl acetate
Overlay with N
2
and cap
Mix/vortex
React 30 minutes at 70°C
Remove from heat and allow to cool
NOTE:
Do not evaporate BSTFA solution
Analysis
Inject 2µL onto GC/MS
For mass spectrometry monitor the following ions:
Compound
Target (Quantitation) Ion
Qualifier Ions
Cocaine
182
198, 303
Cocaine-D3*
185
201, 306
Benzoylecgonine-TMS
240
256, 361
Benzoylecgonine-D8-TMS*
243
259, 369
* Internal standards: Cocaine-D3, Benzoylecgonine-D8-TMS
** Part number TS-38831
Recommended GC Column
Part Number
TraceGOLD TG-5MS 30m x 0.25m x 0.25µm
26098-1420