109 / 144
109
Forensic
Applications
Free (Unbound) Opiates in Blood, Plasma/Serum and Tissue
for GC or GC/MS Confirmations
Using 200mg 10mL HyperSep Verify-CX Extraction Column
(Part Number: 60108-742)
Prepare Sample
To 1mL of 100mM phosphate buffer (pH 6.0) add
internal standards*
Add 1mL of blood, plasma/serum or 1g (1:4) of tissue
homogenate
Mix/vortex and allow to stand for 5 minutes
Add 2mL of 100mM phosphate buffer (pH 6.0)
Mix/vortex
Sample pH should be 6.0±0.5
Adjust pH accordingly with 100mM monobasic or dibasic
sodium phosphate
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)
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 acetate buffer (pH 4.5)
3mL of CH
3
OH
Dry column (5 minutes at >10"Hg)
Elute Opiates
3mL of CH
2
Cl
2
/IPA/NH
4
OH (78:20:2)
Collect eluate at 1 to 2mL/min
NOTE:
Prepare elution solvent fresh daily; add IPA/NH
4
OH mix,
then add CH
2
Cl
2
(pH 11-12)
Dry Eluate
Evaporate to dryness at <40°C
Derivatize
Add 50µL of ethyl acetate and 50µL BSTFA
(with 1% TMCS)***
Overlay with N
2
and cap
Mix/vortex
React for 30 minutes at 70°C
Remove from the heat source to cool
NOTE:
Do not evaporate BSTFA solution
Quantitate
Inject 1 to 2µL onto gas chromatograph
For mass spectrometry, monitor the following ions:
Compound
Primary Ion** Secondary
Tertiary
Codeine-D3-TMS*
374
237
346
Codeine-TMS
371
234
343
Morphine-D3-TMS*
432
290
327
Morphine-TMS
429
287
324
* Suggested internal standard
** Quantitation ion
***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