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Opiates in Urine for GC/MS Confirmations
Using 200mg 6mL HyperSep Retain-CX Extraction Column
(Part Number: 60107-314)
Sample Preparation (Enzymatic Hydrolysis)
To 1mL of urine add internal standard(s) and 1.0mL
ß-Glucuronidase solution. (ß-Glucuronidase solution
contains 5,000 F units/mL Patella Vulgata in 100mM
acetate buffer, pH 5.0). Hydrolyze for 3 hours at 60°C.
Cool, then centrifuge for 10 minutes at high speed and
discard pellet
Adjust pH to 6.0±0.5 with 1.0N NaOH
NOTE:
For unconjugated (free) opiates; to 1mL urine, add
internal standard(s) and 1mL 100mM phosphate buffer
(pH 6.0). Proceed to next step.
Apply Sample
Load at a rate of 1 to 2mL/min
Was Column
1 x 1mL DI H
2
O
1 x 1mL 100mM acetate buffer (pH 4.5)
1 x 1mL MeOH
Dry column (3 mins at >10"Hg)
Elute Opiates
2 x 0.5mL CH
2
Cl
2
/IPA/NH
4
OH (78/20/2), collect eluate
at 1 to 2mL/min
Evaporate eluate to dryness at <40°C
Derivatization
Add 50µL ethyl acetate and 50µL BSTFA with 1%
TMCS**, then cap, mix/vortex
React for 20 mins at 70°C, allow to cool
NOTE:
Do not evaporate BSTFA solution
Analyze
Inject 1 to 2µL onto GC/MS
For mass spectrometry monitor the following ions:
Analyte (TMS)
Target (Quantitation) Ion Qualifier Ions
Codeine-TMS
371
234, 343
Codeine-D6-TMS*
377
237, 349
Morphine-TMS
429
401, 414
Morphine-D6-TMS*
435
404, 420
6-Acetylmorphine-TMS
399
400, 340
6-Acetylmorphine-D6-TMS
405
406, 343
* Suggested internal standards: Codeine-D6-TMS, Morphine-D6-TMS
** Part number TS-38831
Recommended GC Column
Part Number
TraceGOLD TG-5MS 30m x 0.25m x 0.25µm
26098-1420
Paroxetine in Blood, Plasma/Serum and Urine
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) add
internal standards*
Add 1mL whole blood, serum/plasma or urine
Add 2mL of 100mM phosphate buffer (pH= 6)
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 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 DI H
2
O
1 x 3mL 100mM acetic acid
1 x 3mL CH
3
OH
Dry column (5 minutes at >10"Hg)
Elute Paroxetine
1 x 3mL Ethyl
acetate:acetonitrile:ammonium
hydroxide (78:20:2)
Collect eluate at 1 to 2mL/minute
Evaporation
Evaporate eluates under a gentle stream of nitrogen <40°C
Dissolve residue in 100µL CH
3
OH
Injection Volume: 5µL onto LC/MS triple quad
Compound
MRM Transition
Paroxetine
330.0/190.1
Paroxetine-D6*
336.0/76.1
* Suggested internal standard: Paroxetine-D6
Recommended HPLC Column
Part Number
Hypersil GOLD C8 3µm, 50 x 4.6mm
25203-054630
Pharmaceutical/Biotech
Applications