TN-1161

APPLICATIONS
A Sensitive and Fast LC-MS/MS Method for Measurement of Nicotine and
Metabolites in Human Urine
Shuguang Li1, Seyed Sadjadi1, Carrie J. Haglock2, Simon Lomas1 and Jeff Layne1
1
Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA
2
ARUP Laboratories, 500 Chipeta Way, Salt Lake City, UT 84108 USA

A method is illustrated for simultaneous analysis of nicotine, two Sample Preparation

of its major metabolites, cotinine and 3-hydroxycotinine, as well The individual deuterated internal standard (IS) stock solutions
as nornicotine and anabasine from human urine samples. The (1000 ng/mL) were prepared in acetonitrile and stored at 4 °C until
method described uses Strata®-X-C solid phase extraction (SPE) use. A working solution containing all IS at a final concentration of
cartridges for sample clean-up and concentration, followed by 100 ng/mL was prepared in acetonitrile. 100 μL of the IS working
fast (<6 min) LC/MS/MS analysis using a Gemini® NX-C18 column. solution was then added to 1 mL of diluted human urine speci-
men, yielding a final urine IS concentration of 100 ng/mL.
Introduction
Nicotine is the most abundant alkaloid present in all tobacco For calibrators, a stock solution of 1 mg/mL of each analyte was
products along with being a major tobacco-specific component prepared in acetonitrile and stored at 4 °C until use. The working
in both mainstream tobacco smoke and environmental tobacco solution of 25 μg/mL was prepared by dilution with methanol. A
smoke (ETS). Determination of nicotine metabolism / pharmaco- standard calibration curve was generated by spiking different ali-
kinetics provides a useful tool for estimating uptake of nicotine quots of the working stock solution into blank human urine speci-
and tobacco-related toxicants, understanding the pharmacologic men; yielding a nine-point calibration curve (1, 2.5, 5, 10, 25, 50,
effects of nicotine and nicotine addiction. (Xu et al 2004). 100, 250, and 500 ng/mL).

In addition to nicotine and its metabolites, tobacco products also Three human urine quality control (QC) samples were prepared
contain other alkaloids that can serve as unique markers of to- in triplicates with a different lot of working standard solution to
bacco use. Two such examples are anabasine and nornicotine, yield QC concentration of 4, 40 and 400 ng/mL, respectively. The
which are present in tobacco products but not in nicotine replace- samples were prepared for analysis using the SPE procedure.
ment therapies.
The urine sample was prepared by diluting 0.5 mL urine samples
Revision: 0

Our goal was to develop a sensitive, specific, accurate and fast with 0.5 mL of 20 mM ammonium acetate, pH 4, and adding
analytical method to simultaneously quantify nicotine and me- 100 μL internal standards.
tabolites in human urine using SPE for sample cleanup and con-
centration, and fast LC/MS/MS analysis using a Gemini NX-C18 Solid Phase Extraction (SPE)
column. Cartridge: Strata-X-C (60 mg/3 mL)
Part No.: 8B-S029-UBJ
Materials and Methods Condition: 2 mL Methanol (1-2 mL/min)
All reagents and solvents were HPLC or analytical grade. HPLC Equilibrate: 2 mL Ammonium acetate buffer
PHEN-RUO-00029

grade methanol and acetonitrile was purchased from Honeywell, Load: 0.5 mL Diluted urine sample
Burdick & Jackson (Muskegon, MI). Milli-Q Water was used for Wash 1: 2 mL Ammonium acetate buffer
reagents preparation, SPE, sample preparation and to prepare Wash 2: 2 mL 30 % Methanol in water
the LC mobile phase. Anabasine, Cotinine, Nicotine, Nornicotine, Dry: > 10” Hg for 5 min to remove residual water
and Ammonium Bicarbonate was purchased from J.T. Baker, Inc., Elute: 2 x 2 mL 1.5 % Ammonium hydroxide in methanol
(Phillipsburg, NJ). Ammonium hydroxide was purchased from Dry down: Nitrogen gas at 55 ºC
Sigma-Aldrich. Trans-3’-Hydroxycotinine-d3 was purchased from
Reconstitute: 500 μL of Acetonitrile/20 mM Ammonium
Toronto Research Chemicals, Inc. (North York, ON, Canada). Nic- bicarbonate (10:90)
otine-d4 was purchased from C/D/N Isotopes, (Quebec, Canada).
Cotinine-d3 was purchased from Cerilliant, (Round Rock, TX). LC/MS/MS
Nornicotine-d4 was purchased from C/D/N Isotopes, (Quebec, Column: Gemini 3 µm NX-C18
Canada). Dimensions: 50 x 2.0 mm
An Agilent 1200 Series HPLC (Agilent® Technologies Inc., Santa Part No.:00B-4453-BO
Clara, CA USA) was interfaced with API 4000™ MS/MS with ESI Mobile Phase: A: 20 mM Ammonium bicarbonate
TurboIonSpray® (AB SCIEX Foster City, USA) operated in positive B: Acetonitrile
ionization mode (ESI+). Gradient: Time (min) B (%)
0 10
3 75
3.1 10
5 10
Flow Rate: 0.5 mL/min
Temperature: 25 °C
Injection: 10 μL
Detection: AB SCIEX API 4000™ MS/MS (ESI+)

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MS/MS Conditions Figure 1.

Nicotine, Cotinine, 3-OH-cotinine, Nornicotine and Anabasine analysis
Ionization: ESI
(10 ng/mL urine extracted standard)
Polarity: Positive
Scan Type: MRM 1.6e5
4

Curtain Gas (CUR): 50 1.5e5 1) Nornicotine

Gas 1 (GS1): 50 1.4e5 2) 3-OH-Cotinine
Gas 2 (GS2): 50 1.3e5
3) Anabasine
IS: 5500 1.2e5
4) Cotinine
1.1e5
Collision Gas (CAD): 5 5) Nicotine
1.0e5
Interface Heater (Ihe): On

Intensity, cps
9.0e4
Temperature (TEM): 550 ˚C 8.0e4

Entrance Potential (EP): 10 7.0e4

5

App ID 22022
6.0e4 2
Table 1. 5.0e4
3
Mass Dependant Parameters 4.0e4
1
3.0e4

Q1 Mass Q3 Mass Dwell Time 2.0e4

Compounds DP CE CXP
(amu) (amu) (msec) 1.0e4

0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min
Nicotine 163.1 132.1 50 56 23 14

Nicotine-d4 167.1 136.0 50 56 21 14

Figure 2.
Cotinine 177.1 80.1 50 70 31 8 Extracted Ion Chromatograms for Nicotine, Cotinine, 3-OH-cotinine, Nor-
nicotine and Anabasine at a concentration of 10 ng/mL in human urine
Cotinine-d3 180.1 80.1 50 70 31 8
2.31
5.0e5
Nornicotine 149.1 80.1 50 71 29 8
0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min
Nornicotine-d4 153.1 84.1 50 70 29 8
1.73
1.00e6
3-OH-cotinine 193.1 80.1 50 70 31 8
0.00
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min
3-OH-cotinine-d3 196.1 80.1 50 66 35 8
1.16
4.9e5

Anabasine 163.1 120.1 50 70 31 8

0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min

1.08
2.7e5
2.0e5
Results and Discussion

App ID 22035
0.0

The use of the rugged pH stable Gemini® NX-C18 column al- 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min

lowed for fast elution of nicotine and its metabolites in less than 3.1e5
1.71

3 minutes (Figure 1). This fast separation allows for multiplexing 0.0
0.86
2.31

techniques to handle the analysis of large numbers of samples. In 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min

ESI positive mode, nicotine and its metabolites were detected by

monitoring the mass transitions and their deuterium-labeled inter-
nal standards listed in Table 1. The most abundant transition for
each analyte was used for quantification. The second mass tran-
sition (not listed) served as a confirmation for each analyte. Figure
2 shows the extracted ion chromatograms for nicotine, cotinine,
3-hydroxycotinine, nornicotine and anabasine at concentrations
of 10 ng/mL in extracted human urine. Note that anabasine and
nicotine have the same parent mass, accounting for the 3rd peak
in the anabasine XIC (Figure 2). Since the deuterium-labeled
internal standard was not available for anabasine, cotinine-d3
served as its internal standard because it has the nearest reten-
tion time (Figure 2). The pH of the mobile phase was adjusted so
that anabasine and cotinine elute as closely as possible. Anaba-
sine can be separated at a slightly higher pH (pH 9) if the deuter-
ated internal standard were to become available.

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APPLICATIONS

Results and Discussion (con’t)

Standard calibration curves were generated over the concentra-
tion range of 1.0 ng/mL to 500 ng/mL by plotting the relative re-
sponse (peak area of nicotine and its metabolites / peak area of
internal standards) versus concentration. The standard calibration
curves were linear over the calibration ranges with R2 values of
0.9997 for nicotine, 0.9979 for cotinine, 0.9997 for nornicotine,
0.9973 for 3-OH-cotinine and 0.9995 for anabasine, separately
(Figure 4).

Sensitivity of the method was evaluated by determining the low-

est level concentration with a signal to noise of at least 10:1
for limit of quantification (LOQ). At the lowest level standard
concentration (1.0 ng/mL) the signal-to-noise ratios were 46.1
for nicotine, 141.5 for cotinine, 16.1 for nornicotine, 50.7 for
3-OH-cotinine and 24.5 for anabasine, separately (Figure 3 and
Table 2). Therefore, the LOQs were estimated to be < 1.0 ng/mL
for nicotine, cotinine, nornicotine, 3-OH-cotinine and anabasine
(Table 2). Furthermore, the current method achieved additional
sensitivity by reducing specimen size from 1000 μL to 500 μL
urine. If greater sensitivity is required or if the intended detector
cannot meet the 1 ng/mL sensitivity, reducing the final elution vol-
ume by half is an easy approach. The upper limit of quantification
was 500 ng/mL for all the analytes (Table 2).

Table 2.
Statistical Data of Nicotine and Metabolites in Urine by LC/MS/MS
Analyte Y-intercept R2 Intra Assay Precision % (N = 3) S/N RT
LOQ ULOQ
4 ng/mL 40 ng/mL 400 ng/mL 1 ng/
min
ng/mL ng/mL Mean % CV Mean % CV Mean % CV mL
Nicotine 1 500 0.0495x 0.9997 96.13 3.72 105.67 2023 102.00 0.80 46.10 2.31
+0.0503
Cotinine 1 500 0.0386x 0.9979 94.84 3.06 100.00 0.99 101.37 3.98 141.50 1.73
+0.0372
3-OH-cotinine 1 500 0.0398x 0.9973 103.10 5.85 98.20 1.92 107.33 4.95 50.70 1.16
+0.044
Nornicotine 1 500 0.0285x 0.9997 101.53 5.94 100.67 1.66 105.43 6.09 16.10 1.08
+0.0233
Anabasine 1 500 0.0132x 0.9995 97.03 2.77 107.00 2.64 95.53 4.67 24.50 1.71
+0.0311

LOQ = Limit of quantification

ULOQ = Upper limit of quantification
CV = Coefficient of Variation (Standard Deviation / Mean)
S/N = Signal / Noise
RT = Retention time

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Figure 3. 3.4e4
1.05

Nicotine and metabolites analysis (1 ng/mL extracted standard) 3.2e4

3.0e4
2.30 2.8e4
1.04e4
1.00e4 2.6e4
9500.00 2.4e4
9000.00
2.2e4
8500.00

Intensity, cps
8000.00 2.0e4
7500.00 1.8e4
7000.00 Nicotine 1.6e4 Nornicotine
6500.00
Intensity, cps

1.4e4
6000.00
1.2e4

App ID 22038
5500.00
5000.00 1.0e4
0.86
4500.00 8000.0
4000.00 6000.0
3500.00 4000.0 0.66 1.23
3000.00 1.62

App ID 22036
2000.0
2500.00
2000.00 0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min
1500.00
1000.00 2.15 3.81 4.12
2.45 2.73 3.15 3.39
3.11 4.29
500.00
0.00
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min 1.08
6160
6000

5500

5000
2.0e4 1.70
1.9e4 4500
1.8e4
4000
1.7e4
Intensity, cps

1.6e4 3500
1.5e4
3000
3-OH-Cotinine
1.4e4
1.3e4
Cotinine
2500
Intensity, cps

1.2e4

App ID 22039
1.1e4 2000
1.0e4
1500
9000.0 0.99 1.851.95
1.35
8000.0 1000
7000.0
0.80 1.441.70 2.37
App ID 22037

6000.0 2.17 500 0.71

5000.0
0
4000.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min
3000.0
2000.0
1000.0
0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min 0.86
2.2e4
2.1e4
2.0e4
1.9e4
1.8e4
1.7e4 1.70
1.6e4
1.5e4
1.4e4
Intensity, cps

1.3e4
1.2e4
Anabasine
1.1e4
1.0e4
9000.0
8000.0

App ID 22040
7000.0
6000.0
5000.0
4000.0 2.31
2.01
3000.0 1.08
2000.0
1000.0
0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min

Page 4 of 8
TN-1161

APPLICATIONS

Figure 4. 18.9
Standard curves from 1 ng/mL to 500 ng/mL for Nicotine, Cotinine, Nor- 18.0
nicotine, 3-OH-cotinine and Anabasine 17.0
16.0
15.0
14.6
14.0 14.0

Analyte Area / IS Area

13.0
13.0
12.0
12.0 11.0
11.0 10.0 3-OH-Cotinine
9.0
R2 = 0.9973
Analyte Area / IS Area

10.0
8.0
9.0
Nicotine 7.0
6.0
8.0
R2 = 0.9997

App ID 22044
5.0
7.0
4.0
6.0 3.0
5.0 2.0
1.0

App ID 22041
4.0
0.0
3.0 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
Analyte Conc. / IS Conc.
2.0

1.0

0.0 6.9
0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 6.5
Analyte Conc. / IS Conc.
6.0

5.5

18.5 5.0
Analyte Area / IS Area

18.0
4.5
17.0
16.0 4.0
15.0
14.0
3.5
Anabasine
13.0
3.0
R2 = 0.9995
Analyte Area / IS Area

12.0 2.5
11.0 Cotinine

App ID 22045
2.0
10.0 R2 = 0.9979
1.5
9.0
8.0 1.0
7.0 0.5
6.0
0.0
App ID 22042

5.0 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
4.0 Analyte Conc. / IS Conc.
3.0
2.0
1.0
0.0
0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
Analyte Conc. / IS Conc.

14

13

12

11

10
Analyte Area / IS Area

7 Nornicotine
6 R2 = 0.9997
5
App ID 22043

0
0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
Analyte Conc. / IS Conc.

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Results and Discussion (con’t) We optimized the SPE extraction procedure to allow for the best
Three levels of QC samples were prepared at 4, 40 and 400 ng/ recovery of the urinary nicotine and its metabolites and to have
mL. These concentrations were selected to represent low, medi- the greatest potential for high-throughput sample preparation and
um, and high concentration across the calibration range for each automation. The Strata®-X-C sorbent represented the most durable
analyte. The three level QC samples were extracted in the same and selective material.
way as the actual sample described above and analyzed in trip-
licates to assess reproducibility. The mean expected recovery of
the lowest level QC samples at 4 ng/mL was 96.13 % for nicotine,
Conclusion
A rapid analysis of urinary nicotine and its metabolites was de-
94.80 % for cotinine, 103.10 % for 3-OH-cotinine, 101.53 % for
veloped. The application of the Gemini® NX-C18 column in this
nornicotine and 97.03 % for anabasine (Table 2).
method results in a shorter chromatographic analysis time, pro-
The percentage Coefficient of Variation (%CV) for the intra assay viding a productivity benefit for clinical testing laboratories with
precision were from 2.77 % to 5.85 % for levels of 4 ng/mL, 0.99 % a dramatic increase in efficiency while simultaneously reducing
to 2.64 % for levels of 40 ng/mL and 0.8 % to 6.09 % for levels costs due to solvent consumption. Sample preparation using
of 400 ng/mL, respectively (Table 2). No endogenous signal was Strata-X-C SPE concentrates the nicotine and metabolites and
found in 3 nonsmoker urine specimens, demonstrating the selec- removes potential sample matrix interferences, which coupled
tivity of the method. There were no carryovers observed by inject- with high efficiency Gemini NX-C18 column provides for the low
ing blank urine samples after the highest calibrator (500 ng/mL). level detection and quantitation of nicotine and its metabolites in
human urine.
The chromatogram in Figure 5 illustrates a urine/matrix suppres-
sion study when using an ESI source and represents a post-col- References
umn infusion of high concentration standards of nicotine, coti- 1. R.A. Davis, M. Curvall. Determination of nicotine and its metabolites in biologi-
nine, 3-OH-cotinine, nornicotine and anabasine, while a low level cal fluids: In vivo studies. In: J.W. Gorrod P. Jocob, eds. Analytical determina-
tion of nicotine and related compounds and their metabolites. Amsterdam:
urine extract is injected on column. The top trace contains the Elsevier Science, 1999, 583-643.
MRM transitions for all five analytes and the bottom trace is the
2. Kyerematen GA, Vesell ES. Drug Metab. Rev., 1991, 23, 3.
four internal standards channel (nornicotine-d4, 3-OH-cotinine-
d3, cotinine-d3 and nicotine-d4). As expected, this section of the 3. Kakajima M, Drug Metab. Dispos., 1996, 24, 1212.
chromatogram contains highly polar components of the extract 4. Matt GE. Biomarkers, 2006, 11, 507-523.
and is virtually un-retained. Nicotine, its metabolites and their in- 5. T. Tuomi, T. Johnsson, K. Reijula. Clin. Chem. 1999, 723, 185-94.
ternal standards elute between 1 to 2.5 min, in the region of stabil- 6. X Xu, M.M. Iba, C. P. Weisel. Clin. Chem., 2004, 50, 2323-2330.
ity between 0.8 – 2.8 min. The region where the mobile phase is
mostly organic, 2.9 – 4.5 min, produces the most dramatic signal
increase. This is to be expected as the ionization source becomes
more productive with a lower viscosity and lower boiling point
solvent.

Figure 5.
Urine matrix effect on Nicotine, its metabolites and their IS’s response
using an ESI source
1.4e6
1.2e6
Intensity, cps

1.0e6
8.0e5 Major Supression
6.0e5
4.0e5
2.0e5
Major Enhancement
0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min

2.8e5 3
2.5e5 1. Nornicotine-d4
2. 3-OH-Cotinine-d3
App ID 22046
Intensity, cps

2.0e5
3. Cotinine-d3
1.5e5 4. Nicotine-d4
2 4
1.0e5

5.0e4 1
0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min

Page 6 of 8
TN-1161

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Ordering Information

Gemini® HPLC Columns

3 μm Microbore, Minibore and Narrow Bore Columns (mm) SecurityGuard™ Cartridges (mm)
Phases 20 x 2.0 30 x 2.0 50 x 2.0 100 x 2.0 150 x 2.0 50 x 3.0 100 x 3.0 150 x 3.0 4 x 2.0*
NX-C18 00M-4453-B0 00A-4453-B0 00B-4453-B0 00D-4453-B0 00F-4453-B0 00B-4453-Y0 00D-4453-Y0 00F-4453-Y0 AJ0-8367
for ID: 2.0-3.0 mm

3 μm Analytical Columns (mm) SecurityGuard Cartridges (mm)

Phases 50 x 4.6 100 x 4.6 150 x 4.6 250 x 4.6 4 x 3.0*
NX-C18 00B-4453-E0 00D-4453-E0 00F-4453-E0 00G-4453-E0 AJ0-8368
for ID: 3.2-8.0 mm

5 μm Minibore and Narrow Bore Columns (mm) SecurityGuard Cartridges (mm)

Phases 30 x 2.0 50 x 2.0 150 x 2.0 50 x 3.0 100 x 3.0 150 x 3.0 250 x 3.0 4 x 2.0*
NX-C18 00A-4454-B0 00B-4454-B0 00F-4454-B0 00B-4454-Y0 00D-4454-Y0 00F-4454-Y0 00G-4454-Y0 AJ0-8368
for ID: 2.0-3.0 mm

5 μm Analytical Columns (mm) SecurityGuard Cartridges (mm) For Gemini Capillary or Preparative HPLC
Phases 50 x 4.6 100 x 4.6 150 x 4.6 250 x 4.6 4 x 3.0* Columns, Guards, and Adapter, contact your
NX-C18 00B-4454-E0 00D-4454-E0 00F-4454-E0 00G-4454-E0 AJ0-8368 Phenomenex technical consultant or local
for ID: 3.2-8.0 mm distributor.

Ordering Information
Strata®-X-C Solid Phase Extraction (SPE)
Format Sorbent Mass Part Number Unit
Microelution 96-Well
2 mg 8M-S029-4GA 1 Plate/Box
96-Well Plate
10 mg 8E-S029-AGB 2 Plates/Box
30 mg 8E-S029-TGB 2 Plates/Box
60 mg 8E-S029-UGB 2 Plates/Box
Tube
30 mg 8B-S029-TAK* 1 mL(100/box)
30 mg 8B-S029-TBJ 3 mL (50/box)
60 mg 8B-S029-UBJ* 3 mL (50/box)
100 mg 8B-S029-EBJ 3 mL (50/box)
100 mg 8B-S029-ECH 6 mL (30/box)
200 mg 8B-S029-FBJ 3 mL (50/box)
200 mg 8B-S029-FCH 6 mL (30/box)
500 mg 8B-S029-HBJ 3 mL (50/box)
500 mg 8B-S029-HCH 6 mL (30/box)
*Tab-less tubes available. Contact Phenomenex for details.

If Phenomenex products in this technical note do not provide at

least an equivalent separation as compared to other products of
the same phase and dimensions, return the product with com-
parative data within 45 days for a FULL REFUND

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