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Beta Blockers in Blood, Urine for GC/MS Confirmations
Using 200mg 10mL HyperSep Verify-CX Extraction Column
(Part Number: 60108-742)
Prepare Sample
To 1mL of acetate buffer (pH= 4.5) add 1mL of blood
or urine
Add 2mL of acetate buffer (pH= 4.5)
Mix/vortex
Centrifuge as appropriate
Condition HyperSep Verify-CX Extraction Column
1 x 3mL CH
3
OH
1 x 3mL DI H
2
O
1 x 3mL 100mM acetate buffer (pH= 4.5)
NOTE:
Aspirate at <3"Hg to prevent sorbent drying
Apply Sample
Load at 1 to 2mL/minute
Wash Column
2 x 1mL acetone/methanol (1:1) aspirate
Dry column (5 minutes at >10"Hg)
Elute Beta Blockers
1 x 1mL dichloromethane/isopropanol/ammonium
hydroxide (78:20:2)
Collect the eluate by gravity
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
Derivatization solution: Methaneboronic acid at 5mg/mL
prepared in dry ethyl acetate (use molecular sieve)
Store this solution at –20°C (freezer conditions) until use
Reaction mixture
Add 100µL of the methaneboronic acid solution (see above)
Mix/vortex
React 15 minutes at 70°C
Remove from heat source to cool
NOTE:
Do not evaporate this solution
Analysis
Inject 1 to 2µL sample on to GC/MS
Recommended GC Column
Part Number
TraceGOLD TG-5MS 30m x 0.25m x 0.25µm
26098-1420
Caffeine, Theophylline and Theobromine in Blood,
Plasma/Serum, and Urine
Using 200mg 10mL HyperSep Verify-CX Extraction Column
(Part Number: 60108-742)
Prepare Sample
To 1mL of 100mM acetic acid add internal standard*
Add 1mL blood, serum/plasma, or urine
Add 2mL of 100mM acetic acid
Mix/vortex and centrifuge as appropriate
Condition HyperSep Verify-CX Extraction Column
1 x 3mL CH
3
OH
1 x 3mL DI H
2
O
1 x 1mL 100mM acetic acid
NOTE:
Aspirate at <3"Hg to prevent sorbent drying out
Apply Sample
Load sample at 1 to 2mL/minute
Wash Column
1 x 3mL DI H
2
O
1 x 3mL 100mM acetic acid
Dry column (5 minutes at >10"Hg)
Elute Caffeine/Theobromine/Theophylline
1 x 3mL ethyl
acetate:methanol(90:10)
Collect eluate at 1 to 2mL/minute
Evaporation
Combine eluates
Evaporate eluates under a gentle stream of nitrogen <40°C
Reconstitute
Reconstitute sample in 1,000µL of 0.1% formic acid (aq)
Inject 20µL on to LC
* Suggested internal standard: 8-Chlorotheophylline
Recommended HPLC Column
Part Number
Hypercarb 3µm, 50 x 2.1mm
35003-052130
Pharmaceutical/Biotech
Applications