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Forensic
Applications
Gamma-Hydroxybutyrate (GHB) in Blood, Urine and Tissue
Using 200mg 6mL HyperSep Retain-AX Extraction Column
(Part Number: 60107-412)
Sample Preparation
GHB standard; 200µg/mL in H
2
O
GHB –D6 internal standard; 100µg/mL
Standard
Whole Blood
Concentration
10µL
200µL
10µg/mL
25µL
200µL
25µg/mL
50µL
200µL
50µg/mL
100µL
200µL
100µg/mL
Make calibration standards and pipet 200µL of QC and
unknown bloods into appropriately labeled 1.5mL
plastic centrifuge tubes
Add 25µL of internal standard*
Add 1mL of acetone
Mix/vortex 15 seconds
Centrifuge
Transfer acetone layer to culture tubes
Evaporate extracts with N
2
at 700C
Reconstitute extracts with 200µL of 100mM Phosphate
Buffer (pH 6.0)
Mix/vortex 15 seconds
Condition HyperSep Retain AX Extraction Column
1 x 3mL of CH
3
OH
1 x 3mL of DI H
2
O
1 x 1mL of 100mM Phosphate Buffer (pH 6.0)
Apply Sample
Add sample with Eppendorf pipette
Elute
Place clean test tubes into vacuum manifold
Add 1mL of CH
3
OH/NH
4
OH (99:1) to original sample
test tube
Mix/vortex
Decant onto column and collect extract
Aspirate ~1"Hg
Concentrate
Remove test tube from Vacuum Manifold
Evaporate to dryness at 70°C using N
2
Derivatize
Add 100µL of ethyl acetate and 100µL of BSTFA with
1% TCMS**
Mix/vortex
Heat at 70°C for 30 minutes
Analysis
Inject 1 to 2µL onto GC/MS
For mass spectrometry monitor the following ions:
Compound
Primary Ion *
Secondary
Tertiary
GHB-D6-di-TMS*
239
240
241
GHB-di-TMS
233
234
235
* Suggested internal standard for GC/MS: D6-GHB
** Part number TS-38831
Recommended GC Column
Part Number
TraceGOLD TG-35MS 30m x 0.25m x 0.25µm
26094-1420