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Nicotine and Continine in Urine or Serum for GC or GC/MS Confirmations
Using 200mg 10mL HyperSep Verify-CX Extraction Column
(Part Number: 60108-742)
Prepare Sample
To 2mL of urine or serum add internal standard(s)* and
2mL of 100mM of phosphate buffer (pH 6.0)
Sample pH should be 6.0±0.5
Adjust pH accordingly with 100mM monobasic or dibasic
sodium phosphate
Mix/vortex and centrifuge as appropriate
Condition Verify-CX Extraction Column
3mL of CH
3
OH then aspirate
3mL of DI H
2
O then aspirate
1mL of 100mM phosphate buffer (pH 6.0)
NOTE:
Aspirate at <3"Hg to prevent sorbent drying
Apply Sample
Load sample at 1 to 2mL/minute
Wash Column
3mL of DI H
2
O
2mL of 200mM HCl
Dry column (5 minutes at >10"Hg)
2mL of hexane
Wash Column
Remove rack of collection tubes to re-wash columns
3mL of CH
3
OH
Dry column (5 minutes at >10"Hg)
Elute Nicotine and Continine
Replace rack of collection tubes
3mL of CH
2
Cl
2
/IPA/NH
4
OH (78:20:2)
Collect eluate at 1 to 2mL/min
NOTE:
Prepare elution solvent fresh daily; add IPA/NH
4
OH mix,
then add CH
2
Cl
2
(pH 11-12)
Concentrate
Evaporate to dryness at <40°C
Take care not to overheat or over evaporate
Reconstitute with 100µL ethyl acetate
Quantitate
Inject 1 to 2µL onto gas chromatograph
Monitor the following ions (GC/MS):
Compound
Primary Ion** Secondary
Tertiary
Nicotine
84
133
162
Nicotine-D4*
88
137
166
Continine
98
119
176
Continine-D3*
101
122
179
* D
3
-Continine and D
4
-Nicotine are available as deuterated internal standards
** Quantitation ions
Recommended GC Columns
Part Number
TRACE TR-DoA5 Column, 15m x 0.25mm x 0.25µm
26AF130P
TRACE TR-DoA35 Column, 15m x 0.20mm x 0.33µm
26AC497P
106
Forensic
Applications
Methaqualone in Urine, Blood, Plasma/Serum for LC/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, whole blood, plasma/serum
Add 2mL of 100mM phosphate buffer (pH 6.0)
Vortex and centrifuge as appropriate
Condition Verify-CX Extraction Column
3mL of CH
3
OH then aspirate
3mL of DI H
2
O then aspirate
1mL of 100mM phosphate buffer (pH 6.0)
NOTE:
Aspirate at <3"Hg to prevent sorbent drying
Apply Sample
Load sample at 1 to 2mL/minute
Wash Column
3mL of DI H
2
O
Dry column (5 minutes at >10"Hg)
2mL of hexane
Elute Methaqualone
3mL of hexane/ethyl acetate (50:50)
Collect eluate at 1 to 2mL/min
Dry Eluate
Evaporate to dryness at <40°C
Reconstitute
Reconstitute with 100µL of methanol
Analysis
Inject 5µL of sample onto LC/MS system
Monitor the following ions:
Compound
MRM Transition
Methaqualone
251.2/132.1
Methaqualone-D7*
258.2/138.2
* Suggested internal standard
Mobile phase: acetonitrile: 0.1% formic acid (30:70, v/v)
Flow rate: 0.35mL/min
Column temperature: ambient
Recommended HPLC Column
Part Number
Hypersil GOLD aQ 3µm, 150 x 2.1mm
25303-152130