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Forensic
Applications
Benzodiazepines in Serum or Plasma
Using 200mg 10mL HyperSep Verify-CX Extraction Column
(Part Number: 60108-742)
Sample Preparation
To 1mL H
2
O add 1.0mL of 100mM phosphate
buffer (pH 6.0)
Add internal standard(s)*
Add 1mL of serum or plasma
Mix/vortex
Sample pH should be 6.0
Adjust pH accordingly with 100mM monobasic or
dibasic sodium phosphate
Centrifuge
Condition HyperSep Verify-CX Extraction Column
1 x 3mL CH
3
OH
1 x 3mL DI H
2
O
1 x 1mL 100mM phosphate buffer (pH= 6.0)
NOTE:
Aspirate at <3"Hg to prevent sorbent drying
Apply Sample
Load at 1mL/minute
Wash Column
1 x 2mL DI H
2
O
1 x 2mL 20% ACN in 100mM phosphate buffer (pH 6.0)
Dry column (10 minutes at >10"Hg)
1 x 2mL hexane
Elute
1 x 5mL ethyl acetate containing 2% ammonium hydroxide
Collect eluate at 1 to 2mL/minute.
Evaporate to dryness at <40°C
Reconstitute
Reconstitute in mobile phase
Analysis
Inject sample onto HPLC
* Suggested internal standards: Alprazolam-D5, Alphahydroxyalprazolam-D5, Diazepam,
Lorazepam-D4, Oxazepam-D5, Temazepam-D5
Recommended HPLC Column
Part Number
BETASIL Phenyl/Hexyl 5µm, 150 x 4.6mm
73005-154630
Benzodiazepines in Whole Blood
Using 500mg 6mL HyperSep Diol Extraction Column
(Part Number: 60108-575)
Sample Preparation
To 1mL of pH 6 buffer add internal standards*
Add 2mL of whole blood
Mix/vortex
Add 5mL of pH 6 buffer
Sonicate for 10 seconds
Centrifuge at ~2700rpm for 15 minutes
Condition HyperSep Diol Extraction Column
1 x 3mL ethyl acetate
1 x 3mL CH
3
OH
1 x 3mL DI H
2
O
1 x 3mL 0.1M phosphate buffer (pH 6.0)
Apply Sample
Load sample by gravity
Wash Column
1 x 3mL DI H
2
O
1 x 3mL 5% ACN in 0.1M phosphate buffer (pH 6.0)
Dry columns 5 minutes at full vacuum or >10"Hg
1 x 3mL Hexane
Elute
2 x 3mL ethyl acetate
Evaporate to dryness under nitrogen at ~55°C
Add external standards*
Derivatize
Add 100µL ACN and 100µL MTBSTFA w/1% t-BDMCS
Heat for 30 minutes at 70°C
Remove from heat source to cool
Inject 1µL into GC/MS-NCI
NOTE:
Do not evaporate MTBSTFA solution
Analysis
Inject onto GC/MS
* Suggested standards: Diazepam-D5 and Lorazepam-D4.
Recommended GC Column
Part Number
TraceGOLD TG-5SilMS 30m x 0.25m x 0.25µm
26096-1420