Pathway Targeted Metabolomic Analysis in

Oral Cancer Cells Using High Performance

Ion Chromatography Coupling to a New
HR/AM Orbitrap Mass Spectrometry
Junhua Wang,1 Terri Christison,1 Krista Backiel, 2 Grace Ji, 3 Shen Hu, 3 Linda Lopez,1 Yingying Huang1
1
Thermo Fisher Scientific Inc, San Jose, CA; 2Cambridge Isotope Laboratories Inc, MA;
3
School of Dentistry and Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, CA

Overview

Purpose: Demonstrate ion chromatography (IC) coupling with high resolution and
accurate mass measurement (HR/AM) MS for targeted metabolomics analysis and the
possibility of HR/AM full scan to perform targeted quantitattion.
Methods: A high pressure Thermo Scientific Dionex ICS-5000+ HPIC IC system
coupled with Thermo Scientific Q Exactive HF mass spectrometer for nontargeted
profiling and targeted TCA cycle quantitative analysis in oral cell lysates using stable
isotope labeled standards as external calibration reference.
Results: Higher flow IC achieved short run time (18 min) and reproducible separation
with RT variation less than 2 sec during 150 real samples analysis. LOQ (CV<15%) at
0.1 pg/L for six stable isotope labeled TCA metabolites at five orders of magnitude
linear range from 0.2 pg to 20000 pg with R2 > 0.995. The absolute quantitative
analysis results agree well with untargeted differential analysis results, demonstrating
the capability of using HR/AM Orbitrap full mass scan for targeted quantitative analysis.

Data Processing

Differential analysis of profilin

2.2. Targeted analysis of TC
Tracefinder 3.2.
Mass Spectrometry

A new high field Thermo S

spectrometer was operated u
setting is from 15 000-240 00
with automatic gain control (A
time (IT) of 100 ms. Source
voltage 3.5kV; transfer tempe
C; Sheath gas 36; Aux gas 5
H2 O

Introduction

Mass spectrometry based metabolomics approach has obtained increasing attention in

oral cancer study. Recently, a highly analytically sensitive platform coupling capillary
ion chromatography (CapIC) with Q Exactive mass spectrometer has been
successfully developed for nontargeted metabolic profiling of head and neck cancer
cells [1]. The outstanding resolution of IC has led to the differentiation of many isobaric
and isomeric polar metabolites, and it has shown a broad coverage to glycolysis and
TCA intermediates. Interesting changes of TCA cycle metabolites in cancer stem cells
versus non-stem cancer cells were observed.
In this work, we utilized a higher flow IC system for higher throughput targeted analysis
to validate our previous observation. Isotopically labeled standards are ideal external
calibration references to spike into the metabolomic sample for targeted quantitative
analysis in LC/MS experiment because of their similar ionization effect and
chromatographic retention. Six stable isotope labeled standards available for TCA
cycle were used for the targeted quantitative analysis. The corresponding endogenous
metabolites in a large scale of samples were quantified easily against the standards by
using the software tool Thermo Scientific Tracefinder .

Methods

Sample Preparation
UMS1, UMS2, UMSCC5 and UMSCC6 head and neck cancer cells were cultured in
Dulbeccos modified eagle medium (DMEM) plus 10% fetal bovine serum, penicillin
(100 U/mL), and streptomycin (100 g/mL). The cells were maintained at 37C in a
humidified 5% CO2 incubator and passaged when they reached 90-95% confluence.
Cell number were counted and the representative numbers per plate were: UM1:3.46
millions, UM2: 2.88 millions, UMSCC5: 2.88 millions, MSCC6: 3.06 millions. The cells
on the remaining plates for IC/MS analysis were not counted but similar to these
numbers because they were cultured under the same conditions. Cellular metabolites
were extracted using liquid nitrogen snap-freezing method with methanol/water
according the literature [2]. Six isotope supplied by Cambridge Isotope Laboratories:
Sodium pyruvate (13C3, 99%), Malic acid (13C4, 99%), Fumaric acid (13C4, 99%),
Succinic acid (13C4, 99%), alpha-ketoglutaric acid (13C5, 99%) and Citric acid
(2,2,4,4-D4, 98%) were made into water at concentrations from 10000, 5000, 1000,
500, 100, 50, 10, 5, 1, 0.5, 0.1 pg/L (11 levels). 200 uL of such water with the
standards was used for cell lysate sample (dried) reconfiguration. The 11 calibration
level standards were randomly spiked into the samples in 3 replicates.

Inje
IC Pump
0.380 mL/min

Dionex ICS-5000+
HPIC Modular IC System

EG

Waste

FIGURE 1. Schematic diagra

for targeted and nontargeted

Results

IC/MS Optimization for High

We applied a high pressure IC

Higher speed separation. Thi

the pressure limit 5000 psi, wh
Higher reproducibility thanks
the capillary system).

The transfer line from autosam

i.d.) with a 14 L volume, whic
peak shape as compared to
volumes greater than 40 L.
which was found to reduce t
sample injection mode was us
and 5 L cut volume. This ac
which allows better suitability
CapIC running at a capillary f
Dionex ICS-5000+ system allo

Ion Chromatography
A Dionex ICS-5000+ HPIC ion chromatography was coupled to a new Q Exactive HF
mass spectrometers for the analysis. The IC was equipped with an anion electrolytic
suppressor (Thermo Scientific Dionex AERS 500) to convert the potassium
hydroxide gradient to pure water before the sample enters the MS. A 2 L partial loop
injection on a 5 L size loop of cellular metabolites were separated using a Thermo
Scientific Dionex IonPac AS11HC-4m, 2 250 mm column. IC flow rate was
0.38 mL/min supplemented post-column with 0.060 mL/min make-up flow of
MeOH/HOAc (2 mM).

FIGURE 2. Best separation o

using a linear gradient (amon
in 20 minutes on Dionex IonP

2 Pathway Targeted Metabolomic Analysis in Oral Cancer Cells Using High Performance Ion Chromatography Coupling to a New HR/AM Orbitrap Mass Spectrometry

ic Inc, San Jose, CA; Cambridge Isotope Laboratories Inc, MA; School of De

ICS-5000+ HPIC IC system

ss spectrometer for nontargeted
in oral cell lysates using stable
nce.

in) and reproducible separation

es analysis. LOQ (CV<15%) at
tes at five orders of magnitude
995. The absolute quantitative
analysis results, demonstrating
or targeted quantitative analysis.

obtained increasing attention in

itive platform coupling capillary
mass spectrometer has been
filing of head and neck cancer
e differentiation of many isobaric
oad coverage to glycolysis and
metabolites in cancer stem cells

her throughput targeted analysis

ed standards are ideal external
ample for targeted quantitative
similar ionization effect and
d standards available for TCA
The corresponding endogenous
easily against the standards by
.

k cancer cells were cultured in

% fetal bovine serum, penicillin
were maintained at 37C in a
ey reached 90-95% confluence.
mbers per plate were: UM1:3.46
MSCC6: 3.06 millions. The cells
t counted but similar to these
conditions. Cellular metabolites
method with methanol/water
ambridge Isotope Laboratories:
%), Fumaric acid (13C4, 99%),
(13C5, 99%) and Citric acid
ations from 10000, 5000, 1000,
200 uL of such water with the
onfiguration. The 11 calibration
in 3 replicates.

oupled to a new Q Exactive HF

ipped with an anion electrolytic
00) to convert the potassium
ters the MS. A 2 L partial loop
ere separated using a Thermo
0 mm column. IC flow rate was
60 mL/min make-up flow of

Differential analysis of profiling data was performed using Thermo Scientific SIEVE
2.2. Targeted analysis of TCA compounds was performed using Thermo Scientific
Tracefinder 3.2.

Mass Spectrometry
A new high field Thermo Scientific Q Exactive HF quadrupole-Orbitrap mass
spectrometer was operated under ESI negative mode for all detections. The resolution
setting is from 15 000-240 000. Full mass scan (m/z 67-1000) used resolution 120 000
with automatic gain control (AGC) target of 1x106 ions and a maximum ion injection
time (IT) of 100 ms. Source ionization parameters were optimized with the spray
voltage 3.5kV; transfer temperature, 320 C; S-Lens level, 50; heater temperature 325
C; Sheath gas 36; Aux gas 5.
H2 O

HPIC Modular IC System

H2 O
AXP Pump
0.80 mL/min

Injection from Autosampler

IC Pump
0.380 mL/min

EG

Trap Degas Guard

IC Column

Suppressor

Metabolite Name

Sodium pyruvate (13C3, 99%)

Succinic acid (13C4, 99%))

Malic acid (13C4, 99%)

alpha-ketoglutaric acid (13C5, 99%)

Fumaric acid (13C4, 99%)

Citric acid (2,2,4,4-D4, 98%)

RT: 0.00 - 20.01 SM: 7B
100

0
100

0
100

0.80 mL/min

H2O/Analytes
0.380 mL/min

50
0
100
50

Conductivity Detector
Waste

50

Methanol/
2 mM Acetic Acid
Dionex ICS-5000+
HPIC Modular IC System

3.27

50

Relative Abundance

upling with high resolution and

metabolomics analysis and the
itattion.

TABLE 1. Six TCA Cycle Stable Isoto

Data Processing

0
100

AXP-MS Pump
0.060 mL/min

50

To assist
desolvation

0
100
50

Q Exactive
Mass Spectrometer Plus

Groundin
g Union

Mixing
Tee

FIGURE 1. Schematic diagram of a High Performance IC-Q Exactive HF platform

for targeted and nontargeted metabolomic analysis.

11 sugar mo

RT: 4.11 - 16.79 SM: 7G

224.11 - 16.79 SM: 7G

RT:

We applied a high pressure IC system 5000+ in this work to take several advantages:
Higher speed separation. This system can operate at flow rate up to 380 L/min with
the pressure limit 5000 psi, while on ICS 4000 capillary system we ran 25 l/min.
Higher reproducibility thanks to the greater column capacity (2.0 mm vs. 0.4 mm on
the capillary system).
The transfer line from autosampler to injection valve was red PEEK tubing (0.13 mm
i.d.) with a 14 L volume, which was found to substantially improve the sensitivity and
peak shape as compared to a black (0.25 mm i.d.) or blue tubing (0.5 mm i.d.) with
volumes greater than 40 L. A black PEEK tubing was used from the waste valve,
which was found to reduce the carryover to negligible levels (<0.01%). PushPartial
sample injection mode was used on a 5 L loop (0.13 mm i.d., red) with 2 L injection
and 5 L cut volume. This achieved a total sample consumption volume of ~12 L,
which allows better suitability for the precise biological sample injection. Compared to
CapIC running at a capillary flow rates which used a very long gradient (45 min), the
Dionex ICS-5000+ system allows a short gradient of only 20 min shown in Figure 2.

20
18
24
16
22
14
20
12
18

Concentration
[mM]
10
10
85
85
10
10

FIGURE 2. Best separation of Glucose 6P and Fructose 6P isomer was achieved

using a linear gradient (among ~20 methods we tried) for metabolite separation
in 20 minutes on Dionex IonPac AS11HC-4m, 2 250 mm column.

23

10
16
148

126
104

8
5

82
60

4
24
2
22
0
20

(B)

9 sugar di-p

18
24
16
22

Fru

14
20
12
18

10
16
148

126
104
82
60
4

Retention
[min]
-3.0
0.20
17.0
19.0
19.1
20.0

7 9
11

24

IC/MS Optimization for Higher Throughput Analysis

FIGURE 3. Detection of six TCA is

concentration by IC-Q Exactive H

(A)

Results

2
0

(C) RTs of Peak 9

FIGURE 4. HPIC-Q Exactive HF d

Sugar Diphosphates from cancer
for 150 injection in (A).

Thermo Scientific Poster Note PN-64150-ASMS-EN-0614S 3

pe Laboratories Inc, MA; School of Dentistry and Jonsson Comprehensive C

TABLE 1. Six TCA Cycle Stable Isotope Standards

rmed using Thermo Scientific SIEVE

s performed using Thermo Scientific

ctive HF quadrupole-Orbitrap mass

mode for all detections. The resolution
(m/z 67-1000) used resolution 120 000
106 ions and a maximum ion injection
meters were optimized with the spray
Lens level, 50; heater temperature 325

Formula

AXP Pump
0.80 mL/min
Suppressor

RT (min)

Sodium pyruvate (13C3, 99%)

[13]C3H4O3

90.0188

[M-H]-

3.27

Succinic acid (13C4, 99%))

[13]C4H6O4

121.0328

[M-H]-

6.71

Malic acid (13C4, 99%)

[13]C4H6O5

[M-H]-

6.73

alpha-ketoglutaric acid (13C5, 99%)

[13]C5H6O5

137.0277
150.0310

[M-H]-

8.08

Fumaric acid (13C4, 99%)

[13]C4H4O4

119.0172

[M-H]-

8.63

Citric acid (2,2,4,4-D4, 98%)

C6H4[2]H4O7

195.0449

[M-H]-

11.90

RT: 0.00 - 20.01 SM: 7B

100

NL: 1.03E8
m/z= 90.0183-90.0193 F: FTMS - p
ESI Full ms [67.00-1000.00] MS
1ppm_isotopes_512_UM1B-3

3.27

50
6.70

NL: 1.55E8
m/z= 121.0322-121.0334 F: FTMS - p
ESI Full ms [67.00-1000.00] MS
1ppm_isotopes_512_UM1B-3

6.72

NL: 1.56E8
m/z= 137.0270-137.0284 F: FTMS - p
ESI Full ms [67.00-1000.00] MS
1ppm_isotopes_512_UM1B-3

50
0
100
50
0
100

Methanol/
2 mM Acetic Acid

NL: 1.38E8
m/z= 150.0302-150.0318 F: FTMS - p
ESI Full ms [67.00-1000.00] MS
1ppm_isotopes_512_UM1B-3

8.07

50

Conductivity Detector
H2O/Analytes
0.380 mL/min

Ion

0
100

H2 O

Obs. m/z

Relative Abundance

Metabolite Name

Study Design

0
100

AXP-MS Pump
0.060 mL/min

NL: 1.42E8
m/z= 119.0166-119.0178 F: FTMS - p
ESI Full ms [67.00-1000.00] MS
1ppm_isotopes_512_UM1B-3

8.63

50

To assist
desolvation

0
100

NL: 1.12E8
m/z= 195.0439-195.0459 F: FTMS - p
ESI Full ms [67.00-1000.00] MS
1ppm_isotopes_512_UM1B-3

11.89

50

Q Exactive
Spectrometer Plus

Groundin
g Union

Mixing
Tee

ormance IC-Q Exactive HF platform

nalysis.

(A)

7 9
11

224.11 - 16.79 SM: 7G

RT:

rate at flow rate up to 380 L/min with

apillary system we ran 25 l/min.

umn capacity (2.0 mm vs. 0.4 mm on

valve was red PEEK tubing (0.13 mm

ubstantially improve the sensitivity and
i.d.) or blue tubing (0.5 mm i.d.) with
bing was used from the waste valve,
egligible levels (<0.01%). PushPartial
(0.13 mm i.d., red) with 2 L injection
mple consumption volume of ~12 L,
ological sample injection. Compared to
sed a very long gradient (45 min), the
nt of only 20 min shown in Figure 2.

Concentration
[mM]
10
10
85
85
10
10

Fructose 6P isomer was achieved

we tried) for metabolite separation
m, 2 250 mm column.

12

14

16

18

18
24
16
22
14
20
12
18

23

10
16
148

126
104

(B)

10

9 sugar di-phosphate isomers

18
24
16
22

8 9

Fructose 1,6DP, 15.56 min

Cell lysates containing the sta

onto HPIC/Q Exactive HF
Scientific Tracefinder, the li

10
16
148

126
104

NL:
1.37E7
m/z=
338.9854338.9922
MS
NL:
50ppb_isoto
1.37E7
pes_512_UM
m/z=
1E-2
338.9854-

338.9922
MS
50ppb_isoto
pes_512_UM
1E-2

45

14
20
12
18

82
60

vs.

82
60

4
24
2
22
0
20

CSC

Calibration Curve for Stable

NL:
1.82E7
m/z=
259.0196259.0248
MS
NL:
50ppb_isoto
1.82E7
pes_512_UM
m/z=
1E-2
259.0196259.0248
MS
50ppb_isoto
pes_512_UM
1E-2

8
5

with isotope
label standard
at a certain
level

FIGURE 5. UM1 and UMSC

CSC was present in highly
in water at 11 cal levels. Th
lyophilized cell sample.

20

Fructose 6P, 7.94 min

20

Retention
[min]
-3.0
0.20
17.0
19.0
19.1
20.0

10
Time (min)

11 sugar mono-phosphate isomers

24

this work to take several advantages:

FIGURE 3. Detection of six TCA isotope label standards at 1 g/mL

concentration by IC-Q Exactive HF platform.

RT: 4.11 - 16.79 SM: 7G

nalysis

UMSCC1 and UMSCC2 cel

tongue cancer patient. Howev
less invasive. Similarly UMS
cells. Also, we found that the
invasive UM1 cells, the meta
(NSCC) is also very inter
metabolites in the TCA cycle
is very interesting that the f
oxoglutarate) had an increas
cycle (succinate/fumarate/m
versus NSCC. The study is
invasive cancer cells and
observation.

12
5

10

11

12

13

14

10

11

12

13

14

67

15

16

15

16

2
0

(C) RTs of Peak 9 (7.930.02 min, p-value=0.02, n=150)

FIGURE 4. HPIC-Q Exactive HF detected (A) 11 Sugar Monophosphates and (B)9

Sugar Diphosphates from cancer cell lysates. (C) RT reproducibility of Peak #9
for 150 injection in (A).

FIGURE 6. The HPIC/Q Exa

range from 0.1, 0.5, 1, 5, 10
injection) with R2 = 0.995 a

4 Pathway Targeted Metabolomic Analysis in Oral Cancer Cells Using High Performance Ion Chromatography Coupling to a New HR/AM Orbitrap Mass Spectrometry

Quantify Targeted Endogenou

Study Design

s. m/z

Ion

RT (min)

0188

[M-H]-

3.27

.0328

[M-H]-

6.71

.0277

[M-H]-

6.73

.0310

[M-H]-

8.08

.0172

[M-H]-

8.63

.0449

[M-H]-

11.90
NL: 1.03E8
m/z= 90.0183-90.0193 F: FTMS - p
ESI Full ms [67.00-1000.00] MS
1ppm_isotopes_512_UM1B-3
NL: 1.55E8
m/z= 121.0322-121.0334 F: FTMS - p
ESI Full ms [67.00-1000.00] MS
1ppm_isotopes_512_UM1B-3

UMSCC1 and UMSCC2 cells were initially established from the same tumor of a
tongue cancer patient. However, UM1 cells are highly invasive whereas UM2 cells are
less invasive. Similarly UMSCC5 cells are significantly more invasive than UMSCC6
cells. Also, we found that the cancer stem like cells (CSC) were present in the highly
invasive UM1 cells, the metabolic feature of CSC compared to nonstem cancer cells
(NSCC) is also very interesting. In previous nontargeted analysis, we found
metabolites in the TCA cycle show significant changes between CSCs and NSCCs. It
is very interesting that the first half cycle (pyruvate/citrate/cisaconitate/ isocitrate/2oxoglutarate) had an increasing upregulation in CSC cells, although the second half
cycle (succinate/fumarate/malate) showed progressive down-regulation in CSCs
versus NSCC. The study is to investigate the metabolic difference between highly
invasive cancer cells and less invasive cancers and to confirm the previous
observation.

Tracefinder software offers a m

compound to an external standa
target compound

NL: 1.56E8
m/z= 137.0270-137.0284 F: FTMS - p
ESI Full ms [67.00-1000.00] MS
1ppm_isotopes_512_UM1B-3
NL: 1.38E8
m/z= 150.0302-150.0318 F: FTMS - p
ESI Full ms [67.00-1000.00] MS
1ppm_isotopes_512_UM1B-3
NL: 1.42E8
m/z= 119.0166-119.0178 F: FTMS - p
ESI Full ms [67.00-1000.00] MS
1ppm_isotopes_512_UM1B-3
NL: 1.12E8
m/z= 195.0439-195.0459 F: FTMS - p
ESI Full ms [67.00-1000.00] MS
1ppm_isotopes_512_UM1B-3
14

16

18

with isotope
label standard
at a certain
level

114 pg 192 pg 2006 pg

n=18
n=22
n=12

CSC

vs. NSCC

UM1

vs. UM2

UM5

vs. UM6

Similar trend?
FIGURE 5. UM1 and UMSCC5, highly invasive. UM2 and UMSCC6, less invasive.
CSC was present in highly invasive UM1. Isotope label compounds were made
in water at 11 cal levels. The water with isotopes was used for reconstituting the
lyophilized cell sample.

20

ndards at 1 g/mL

2364 pg 1492 pg 5883 pg 2

n=20
n=22
n=10

Calibration Curve for Stable Isotope Label Standards

te isomers
NL:
1.82E7
m/z=
259.0196259.0248
MS
NL:
50ppb_isoto
1.82E7
pes_512_UM
m/z=
1E-2
259.0196259.0248
MS
50ppb_isoto
pes_512_UM
1E-2

6P, 7.94 min

Cell lysates containing the stable isotopes from low to high concentration were injected
onto HPIC/Q Exactive HF for analysis. The data was analyzed using Thermo
Scientific Tracefinder, the linear curve is shown in Figure 6.

292 pg
n=20

omers

8 9

15.56 min

NL:
1.37E7
m/z=
338.9854338.9922
MS
NL:
50ppb_isoto
1.37E7
pes_512_UM
m/z=
1E-2
338.9854-

Conclusion

12
12

13

14

12

13

14

67

904 pg
n=10

FIGURE 7. Calculated amounts o

replicate numbers in UM1, 2, 5, a
amount of glucose and secret a l
surprising that pyruvate and othe

338.9922
MS
50ppb_isoto
pes_512_UM
1E-2

45

190 pg
n=22

15

16

15

16

Based on our quantitative analys

TCA metabolites, including malate,
Similarly UMSCC5 cells express dr
UMSCC6 cells. These results sugg
more active TCA cycle than less in

min, p-value=0.02, n=150)

ugar Monophosphates and (B)9

) RT reproducibility of Peak #9

The targeted quantitation results

match exactly with previous differe

References
FIGURE 6. The HPIC/Q Exactive HF achieved five orders of magnitude linear
range from 0.1, 0.5, 1, 5, 10, 50, 100, 500, 1000, 5000,to 10000 pg/L (2 L
injection) with R2 = 0.995 and above (except for succinic acid-13C4, R2 = 0.987).

1. Wang, J. et al Anal. Chem., 2

2. Lorenz, M.A.et al Anal. Chem

All trademarks are the property of

This information is not intended to

might infringe the intellectual prop

Thermo Scientific Poster Note PN-64150-ASMS-EN-0614S 5

nter, UCLA, Los Angeles, CA

Quantify Targeted Endogenous Metabolites

ished from the same tumor of a

ly invasive whereas UM2 cells are
antly more invasive than UMSCC6
s (CSC) were present in the highly
compared to nonstem cancer cells
nontargeted analysis, we found
ges between CSCs and NSCCs. It
te/citrate/cisaconitate/ isocitrate/2SC cells, although the second half
essive down-regulation in CSCs
etabolic difference between highly
rs and to confirm the previous

Tracefinder software offers a method for quantitative analysis by linking the target
compound to an external standard reference and uses its calibration curve.
linked reference compound

target compound

183 pg
n=12

222 pg
n=20

104 pg
n=24

769 pg
n=12

686 pg
n=12

2364 pg 1492 pg 5883 pg 2591 pg

n=20
n=22
n=10
n=14

440 pg
n=16

419 pg 4118 pg
n=26
n=13

263 pg
n=17

114 pg 192 pg 2006 pg

n=18
n=22
n=12

vs. UM2

UM5

vs. UM6

Similar trend?

UM2 and UMSCC6, less invasive.

pe label compounds were made
s was used for reconstituting the

ards

to high concentration were injected

ata was analyzed using Thermo
Figure 6.

292 pg
n=20

190 pg
n=22

904 pg
n=10

333 pg
n=14

3809 pg 2372 pg 7779 pg 3103 pg

n=18
n=24
n=10
n=12

FIGURE 7. Calculated amounts of TCA metabolites per plate cells (avg. ~ 3M) and
replicate numbers in UM1, 2, 5, and 6. It is known that UM5 cells consume a very large
amount of glucose and secret a lot of lactate in cell culture. Therefore, it is not
surprising that pyruvate and other metabolites level are significantly higher in UM5.

Conclusion

Based on our quantitative analysis, UM1 cells display significantly higher levels of
TCA metabolites, including malate, fumarate, citrate, succinate and alpha-ketoglutarate.
Similarly UMSCC5 cells express dramatically higher levels of these metabolites than
UMSCC6 cells. These results suggests that highly invasive HNSCC cells possess a
more active TCA cycle than less invasive HNSCC cells.
The targeted quantitation results for the 6 TCA metabolites in CSC versus NSCC
match exactly with previous differential analysis (not shown here).

References

e orders of magnitude linear

5000,to 10000 pg/L (2 L
succinic acid-13C4, R2 = 0.987).

1. Wang, J. et al Anal. Chem., 2014, 86, 51165124.

2. Lorenz, M.A.et al Anal. Chem., 2011, 83, 34063414.

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries.
This information is not intended to encourage use of these products in any manners that
might infringe the intellectual property rights of others.
PO64150-EN 0614S

6 Pathway Targeted Metabolomic Analysis in Oral Cancer Cells Using High Performance Ion Chromatography Coupling to a New HR/AM Orbitrap Mass Spectrometry

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