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Forensic
Applications
Free and Conjugated Silocin in Urine
Using 200mg 10mL HyperSep Verify-CX Extraction Column
(Part Number: 60108-742)
Sample Preparation
1mL of 100mM phosphate buffer (pH 6)
Add internal standard*
Add 1mL of urine sample
Add 2mL of 100mM phosphate buffer (pH 6)
Mix/vortex
Centrifuge
Urine Hydrolysis
1mL of urine
Add internal standard* and 1mL of ß-glucuronidase solution
(ß-glucuronidase solution contains: 5,000 F units/mL
Patella vulgata in 100mM acetate buffer (pH=5.0))
Mix/vortex
Hydrolyze for 3 hours at 65°C
Centrifuge for 10 minutes at 2,000rpm and discard pellet
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)
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
1 x 3mL CH
3
OH
Dry column (5 minutes at >10"Hg)
Elute
1 x 3mL ethyl acetate;
ACN:ammonia(78:20:2 v/v)
Collect eluate at 1 to 2mL/minute
Evaporate eluates to dryness under a gentle stream
of nitrogen
Analysis
Reconstitute sample in 50µL of CH
3
OH
Inject 5µL onto LC/MS
Mobile phase:
Time
% ACN
%0.1% Formic Acid
0
20
80
5
20
80
Flow rate: 0.20mL/minute
Column temperature: ambient
Detector: Thermo Scientific TSQ Triple Quadrupole
Compound
MRM Transition
Psilocin
205.2/58.2
Psilocin-D10*
215.2/68.2
* Suggested internal standard: Psilocin-D10
Recovery >90% (n=10)
LOD: 10ng/mL
Recommended HPLC Column
Part Number
Hypersil GOLD PFP 3µm, 100 x 2.1mm
25403-102130