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Forensic
Applications
Benzodiazepine Screening in Blood, Serum, Urine and Tissue
Using 200mg 3mL HyperSep Diol Extraction Column
(Part Number: 60108-573)
Sample Preparation
To 1mL of 100mM phosphate buffer (pH= 6) add
internal standards*
Add 1mL blood/urine or 1g of (1:4) tissue homogenate
Mix/vortex
Add 3mL of 100mM phosphate buffer (pH= 6)
Sample pH should be 6.0±0.5
Adjust pH with 100mM monobasic or dibasic
sodium phosphate
Mix/vortex
Centrifuge
Procedure for Urine
To 1mL of acetate buffer (pH 5.0) containing 5,000 F
units/mL
β
-Glucuronidase
Add internal standards*
To this solution add 1mL of urine
Mix/vortex
Hydrolyze for 3 hours at 65°C
Allow to cool
Centrifuge for 10 minutes at 2,000rpm and discard pellet
Add 3mL of 100mM phosphate buffer (pH 6.0) and mix
Condition HyperSep Diol Extraction Column
1 x 3mL CH
3
OH
1 x 3mL 100mM phosphate buffer (pH 6)
NOTE:
Aspirate at <3"Hg to prevent sorbent drying out
Apply Sample
Load sample at 1 to 2mL/minute
Wash Column
1 x 3mL of 5% (v/v) ACN in 100mM phosphate
buffer (pH6)
Dry column (5 minutes at >10"Hg)
1 x 3mL of hexane
Dry column (5 minutes at >10"Hg)
Elute
1 x 3mL ethyl acetate; ammonia (98:2 v/v)
Collect eluate at 1 to 2mL/minute
Evaporate eluates under a gentle stream of nitrogen <40°C
Derivatize
Add 50µL ACN and 50µL BSTFA with 1% TCMS
Heat for 30 minutes at 70°C
Remove from heat source to cool
Analysis
Inject 1µL into GC/MS
Compound
Primary Ion
Secondary
Tertiary
Alprazolam
308
279
204
Alprazolam-D*
513
284
Alphahydroxyalprazolam
318
396
383
Alphahydroxyalprazolam-D5*
386
401
Diazepam
256
283
284
Diazepam-D5*
287
289
Lorazepam
429
430
347
Lorazepam-D4*
433
435
Nordiazepam
34
342
343
Nordiazepam-D5*
345
347
Oxazepam
429
313
430
Oxazepam-D5*
435
433
Temazepam
343
257
283
Temazepam-D5*
348
262
* Suggested internal standards: Alprazolam-D, Alphahydroxyalprazolam-D5, Diazepam-D5,
Lorazepam-D4, Nordiazepam-D5, Oxazepam-D5, Temazepam-D5
Recommended GC Column
Part Number
TraceGOLD TG-5SilMS 30m x 0.25m x 0.25µm
26096-1420