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Benzodiazepine Screen: Blood, Serum, Urine and Tissue for LC/MS/MS
Using 200mg 6mL HyperSep Verify-CX Extraction Column
(Part Number: 60108-722)
Prepare Sample
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 accordingly with 100mM monobasic or
dibasic sodium phosphate
Mix/vortex
Centrifuge as appropriate
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 Verify-CX Extraction Column
1 x 3mL MeOH
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) acetonitrile 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 Benzodiazepines
1 x 3mL ethyl acetate; ammonia (98:2 v/v)
Collect eluate at 1 to 2mL/minute
Evaporation
Evaporate eluates under a gentle stream of nitrogen <40°
Reconstitute
Reconstitute sample in 50µL of 0.02% formic acid (aqueous)
Instrumental Conditions: LC/MS/MS
Mobile phase 30:70 (acetonitrile:0.02% aq. formic acid)
Flow rate: 0.35mL/min
Column temperature: Ambient
Injection volume: 5µL on to MS triple quad
Compound
MRM Transition
Alprazolam
309.1/281.2
Alprazolam-D5*
314.1/286.2
Alphahydroxyalaprazolam
325.1/242.9
Alphahydroxyalprazolam-D5*
330.1/302.2
Chlordiazepoxide
300.1/227.0
Diazepam
285.5/192.5
Diazepam*
292.2/198.2
Lorazepam
321.1/275.1
Lorazepam-D4*
325.1/279.0
Nordiazepam
271.1/140.1
Nordiazepam-D5*
275.6/140.1
Oxazepam
287.1/241.1
Oxazepam-D5*
290.2/198.2
Temazepam
301.1/255.1
Temazepam-D5*
306.1//260.1
* Suggested internal standards: Alprazolam-D5, Alphahydroxyalprazolam-D5, Diazepam,
Nordiazepam-D5, Oxazepam-D5, Temazepam-D5
Recommended HPLC Column
Part Number
Hypersil GOLD PFP 3µm, 150 x 2.1mm
25403-152130
Pharmaceutical/Biotech
Applications