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Beta Agonist Analysis
Using 60mg 3mL HyperSep Retain-CX Extraction Column
(Part Number: 60107-303)
Compounds
Cimaterol, sulfamonomethoine,
clenbuterol hydrochloride, salbutamol
Sample Preparation
Extract 20g of pig liver sample in acetonitrile, dried and
spiked with standard chloric acid (10mmol) solution
containing 4 agonists
Condition Retain-CX Extraction Column
5mL of methanol
5mL of DI H
2
O
5mL of 30mmol/L of chloric acid
Apply Sample
Load 2mL samples
Wash Column
5mL of DI H
2
O
5mL of methanol
Dry column (5-10 minutes at >10"Hg/full flow for
positive pressure manifold)
Elute Beta Agonists
5mL of 4% ammonia/methanol
Dry Eluate and Reconstitute
Evaporate to full dryness at room temperature under a
stream of nitrogen
Beta Agonists in Urine for GC/MS Confirmations
Using 200mg 10mL HyperSep Verify-CX Extraction Column
(Part Number: 60108-742)
Prepare Sample
To 1mL of 100mM acetate buffer (pH 4.5) add 1mL of urine
Add 2mL of 100mM
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 4.7)
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 Agonists
1 x 1mL dichloromethane/isopropanol and ammonium
hydroxide (78:20:2)
Collect the eluate at 1 to 2mL/minute (or 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 (derivatized solution) on to GC/MS
Recommended GC Column
Part Number
TraceGOLD TG-5MS 30m x 0.25m x 0.25µm
26098-1420
Pharmaceutical/Biotech
Applications