Nicotine-Related Compounds in Urine on Raptor Biphenyl by LC-MS/MS

	Peaks	t_R (min)	Precursor Ion	Product Ion
1.	Nornicotine	1.25	149.10	80.05
2.	Norcotinine	1.81	163.09	80.05
3.	trans-3'-Hydroxycotinine	1.88	193.12	80.05
4.	Nicotine	1.95	163.15	132.10
5.	Anabasine	2.20	163.13	91.63
6.	Cotinine	2.88	177.12	98.07

Column

Raptor Biphenyl (cat.# 9309512)

Dimensions:

100 mm x 2.1 mm ID

Particle Size:

5 µm

Pore Size:

90 Å

Guard Column:

Raptor Biphenyl EXP cartridges 5 mm, 2.1 mm ID, 5 µm (cat.# 930950252)

Temp.:

30 °C

Standard/Sample

Diluent:

Water

Conc.:

25 ng/mL (fortified human urine)

Inj. Vol.:

5 µL

Mobile Phase

0.1% Formic acid, 5 mM ammonium formate in water

0.1% Formic acid in methanol

Time (min)	Flow (mL/min)	%A	%B
0.00	0.4	90	10
1.00	0.4	90	10
2.00	0.4	70	30
3.00	0.4	30	70
3.01	0.4	90	10
5.00	0.4	90	10

Detector	MS/MS
Ion Mode:	ESI+
Mode:	MRM
Instrument	UHPLC
Sample Preparation	The urine from a non-tobacco user was fortified with analytes to a final concentration of 25 ng/mL and prepared by a liquid-liquid extraction method. A 250 μL urine sample was mixed with 50 μL of 5N NaOH and extracted with 1.5 mL of 50/50 methylene chloride/diethyl ether. A portion of organic phase (1.0 mL) was dried and reconstituted with 200 μL of water for analysis.

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