HyperSep Column Applications

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Forensic
Applications
Antidepressants/Painkillers in Urine and Blood
for LC/MS/MS Confirmations
Using 200mg 10mL HyperSep Verify-CX Extraction Column (Part Number: 60108-742)
Prepare Sample
To 1mL of 100mM phosphate buffer (pH 6.0) add
internal standard*
Add 1mL of urine or blood
Add 2mL of 100mM phosphate buffer (pH 6.0)
Mix/vortex
Sample pH should be 6.0±0.5
Adjust pH accordingly with 100mM of monobasic or
dibasic sodium phosphate
Mix/vortex
Centrifuge as appropriate
Condition Verify-CX Extraction Column
3mL of CH3OH then aspirate
3mL of DI H2O then aspirate
1mL of 100mM phosphate buffer (pH 6.0) then aspirate
NOTE: Aspirate at <3"Hg to prevent sorbent drying
Apply Sample
Load sample at 1 to 2mL/minute
Wash Column
3mL of DI H2O
3mL of 1% acetic acid
3mL of CH3OH
Dry column (5 minutes at >10"Hg)
Elute Antidepressants/Painkillers
3mL of ethyl acetate: acetonitrile: ammonia
(78:20:2, v/v/v)
Collect eluate at 1 to 2mL/minute
Evaporate
Evaporate eluate under a gentle stream of nitrogen
at <40°C
Reconstitute
Reconstitute sample in 100µL of methanol
Inject 5µL onto LC system
Quantitate
Mobile phase: acetonitrile: 0.1% formic acid (33:67, v/v)
Flow rate: 0.35mL/min
Recommended HPLC Column Part Number
Hypersil GOLD 3µm, 150 x 2.1mm 25003-152130
Compound Detection Ions
Amitriptyline 278.8/91.1
Amitriptyline-D3* 281.2/91.2
Diphenhydramine 256.2/167.1
Diphenhydramine-D3* 259.2/167.1
Doxepin 280.2/107.1
EDDP 278.2/234.2
EDDP-D3* 281.4/234.3
Methadone 310.2/105.1
Methadone-D9* 319.2/268.3
Nortriptyline 264.2/91.1
Norpropoxyphene 326.2/44.1
Norpropoxyphene-D5* 331.1/267.1
Propoxyphene 340.2/58.1
Propoxyphene-D11* 351.3/64.0
Sertraline 308.1/161.0
Tramadol 264.2/58.1
Tramadol-D3* 268.2/58.0
Venlafaxine 278.2/58.2
Zolpidem 308.2/235
* Suggested internal standards