Profiling of Endogenous Metabolites
Using Time-of-Flight LC/MSwith Ion‑Pair Reverse Phase
Chromatography
Application Note
MetabolomicsAuthor

Abstract

Yuqin Dai

A high performance ion-pair reverse-phase (IP-RP) chromatographic method

Agilent Technologies, Inc.
5301 Stevens Creek Blvd,
Santa Clara, CA 95051 USA

has been developed for use with a time-of-flight (TOF) mass spectrometer (MS).
This negative ionization method provides comprehensive coverage of many
endogenous metabolite classes including amino acids, organic acids, sugars andsugar phosphates, nucleosides and nucleotides, energy and redox metabolites, andcoenzyme A derivatives. Optimization of mobile phase pH and the LC gradient was
critical in obtaining good chromatographic separation of isomers such as citricacid and isocitric acid, and impacted peak shape and optimal signal response.
The IP-RP LC/TOF MS method was evaluated with 19 representative metabolite
standards covering the entire chromatographic region. All metabolites showed
good retention time reproducibility.

Introduction
High resolution accurate mass
LC/TOF or Q-TOF MS is routinely usedin metabolomics for discovery work1-2.
TOF MS is preferred due to spectral
acquisition speed, high mass accuracy,high isotopic ratio fidelity, and sensitive
detection of metabolites. Some analyticalchallenges remain3-4, including retentionof ionic metabolites, reproducibility inretention time (RT), chromatographic
separation of biologically important
isomers, and broad coverage ofmetabolite classes in a single analyticalrun.
To address these challenges, a robust
and reproducible ion-pair reverse
phase (IP‑RP) LC/TOF MS method
was developed using tributylamine as
the ion-pairing agent. As part of thechromatographic optimization, theeffect of both pH and concentration oftributylamine in aqueous and organic
mobile phases was investigated. Thismethod enables simultaneous detectionof different classes of polar and anionicendogenous metabolites with separationof several pairs of biologically relevant
isomers such as citric acid and isocitricacid.

Experimental
MethodA set of metabolite standards covering
different chemical classes and several
pairs of biologically relevant isomers
was selected for IP-RP LC/TOF MS
method development. Using anonlinear water/methanol gradient,
the chromatographic separation wasperformed on an Agilent ZORBAX RRHDExtend 80Å C18, 2.1 × 150 mm, 1.8 µmcolumn (p/n 759700-902) with anAgilent ZORBAX SB-C8, 2.1 mm × 30 mm,3.5 µm (p/n 873700-936) guard column.
Tributylamine (Sigma, p/n 90781‑50mL)
was added to both aqueous andorganic mobile phases to ensure a
constant concentration during gradientelution. The pH of the aqueous mobile
phase was adjusted using acetic acid(Fluka, p/n 49199-50mL-F), and the sameamount of acetic acid was added to theorganic mobile phase. All analyses wereperformed using negative ion electrosprayon an Agilent LC/TOF MS system.

Instrumentation

LC/MS analysis was performed usingan Agilent 1290 Infinity LC coupled to an
Agilent 6230 TOF with an Agilent Dual ESI
Source. The LC consisted of an Agilent
binary pump with seal wash (G4220A), an
Agilent sampler (G4226A) with thermostat(G1330B), an Agilent thermostatted
column compartment (G1316C) and an

Agilent 1100 isocratic pump (G1310A)
with a 100:1 splitter (G1607-60000)
for reference mass addition. Dynamicmass axis calibration was achieved bycontinuous infusion of a reference masssolution. Tables 1 and 2 summarize theoptimized LC and MS conditions.
Data acquisition and analysis was doneusing the Agilent MassHunter software
suite. The TOF MS provided sensitive, full
spectrum data, which facilitated method
development of the chromatographicconditions.

Table 1. The optimal LC parameters.
Agilent 1290 Infinity LC System
Analytical column

Agilent ZORBAX RRHD Extend 80Å C18, 2.1 × 150 mm, 1.8 µm(p/n 759700-902)

Guard column

Agilent ZORBAX SB-C8, 2.1 mm × 30 mm, 3.5 µm (p/n 873700-936)

Column temperature

40 °C

Injection volume

5 µL

Autosampler temperature

4 °C

Mobile phaseA) 97 % water/3 % methanol containing 5 mM TBA with pH adjusted byacetic acidB) Methanol containing 5 mM TBA with addition of acetic acid

Seal wash solvent

Isopropanol : H2O (1:1 v/v)

Flow rate

0.25 mL/min

Gradient

A nonlinear gradient from 0–99 % B in 22 minutes

Stop time

22 minutes

Post time

5 minutes

Table 2. The optimal MS parameters.
Parameter

Agilent 6230 TOF system

Ion mode

NegativeSource

Agilent Dual ESI

Capillary voltage

3,500 VDry gas temperature

250 °CDry gas flow

13 L/min

Nebulizer pressure

35 psiFragmentor

130 VSkimmer

60 V

Oct 1 RF Vpp

600 V

MS range

60–1,600 m/z

MS acquisition rate

1.5 spectra/sec

Reference mass

62.0327 and 983.0347

Reference pump flow

0.5 mL/min

Reference delivery

Agilent 1100 isocratic pump with 100:1 splitter (p/n G1607-60000)

Instrument mode

Extended dynamic range (2 GHz)

2

Results and Discussion

Figure 1 illustrates that this method
enabled detection of 38 metabolites
including amino acids, organicacids, sugars and sugar phosphates,nucleosides and nucleotides, energy
and redox metabolites, and coenzyme Aderivatives with separation of biologicallyrelevant isomers (Figure 2) in a 22-minuteanalytical run. Table 3 summarizes
the chemical formula, RT, and m/z
values for these metabolites. The
chromatographically separated isomers
are highlighted with four different colors.

Sugar mono and di-phosphates

5.0
Mono, di, and tri-organic acids

4.5
4.0

Energy and redox metabolites (for example, FAD, NAD(P)(H))

Counts3.5
3.02.52.0

Coenzyme A derivatives

Nucleotides

Sugars andamino acids

Nucleosides

141.5
1.0
0.502356789 10 11 12 13Acquisition time (min)1415161718192021

Figure 1. Overlaid extracted ion chromatograms (EICs) of 39 metabolite standards using a mobile phaseat pH = 5.8.

×105
D-G-6-P

5.0
4.5
4.0
3.5Counts

A robust, high-performance IP-RP LC/TOF
MS method was developed using anAgilent Extend column designed forrobust performance at high pH. Theaddition of tributylamine improved
retention of anionic metabolites, allowing
separation of both anionic and morehydrophobic metabolites in a single
method. The separation of biologicallyrelevant isomers was achieved by a
combination of a stepwise nonlinear
gradient and pH optimization of themobile phase. This resulted in theseparation of citric acid and isocitricacid, D-glucose-6-phosphate andα-D-glucose‑1-phosphate, leucine andisoleucine, and maleic acid and fumaricacid. Maintaining the same concentrationof tributylamine in both aqueous andorganic mobile phases helped control
the pH. Furthermore, the concentrationof tributylamine was decreased by half
without causing any significant impact
on the signal or separation. Generally,a lower concentration of buffer inthe mobile phase will improve source
cleanliness and system robustness.

×105

3.0
2.5
2.0

Isoleucine

Citric acid

Leucine1.5

α-D-G-1-P

Fumaric acid
Maleic acid

1.0

Isocitricacid

0.5
02

345

67
8
910Acquisition time (min)1112131415

Figure 2. Overlaid EICs showed chromatographic separations of four pairs of biologically relevant isomers.

3Table 3. A list of 38 metabolites with chemical formula, RT, and (M-H)– information.
CompoundChemical formula(M-H)–

RT (min) (Ave) (n = 3)

L-Arginine

C6H14N4O2

173.1044

1.15

GlycineC2H5NO274.02423

1.42

L-Glutamine

C5H10N2O3

145.0619

1.45

D-Glucose

C6H12O6

179.0561

1.47

D-Fructose

C6H12O6

179.0561

1.50

Isoleucine

C6H13NO2

130.0874

2.53

Leucine

C6H13NO2

130.0874

2.72

Inosine

C10H12N4O5

267.0735

4.64

Guanosine

C10H13N5O5

282.0839

5.17

L-Phenylalanine

C9H11NO2

164.0717

5.48

Glyceric acid

C3H6O4105.0188

6.13Glyoxylic acidC2H2O372.9931

6.38

Lactic acid

C3H6O389.0244

6.49

NAD

C21H27N7O14P2

662.1013

6.55

D-Glucose-6-phosphate (D-G-6-P)

C6H13O9P

259.0224

6.67

D-Fructose-6-Phosphate (D-F-6-P)

C6H13O9P

259.0224

6.72

Pyruvic acid

C3H4O387.0088

7.00

α-D-Glucose-1-phosphate (α-D-G-1-P)

C6H13O9P

259.0224

7.25

AMP

C10H14N5O7P

346.0558

8.69

Maleic acid

C4H4O4115.0037

8.72

Succinic acid

C4H6O4117.0193

9.78

GMP

C10H14N5O8P

362.0507

9.79

S-Malate

C4H6O5133.0137

10.01

N-acetylglutamic acid

C7H11NO5188.0564

10.22

Fumaric acid

C4H4O4115.0037

10.29

α-Ketoglutarte

C5H6O5145.01373

10.31

Citric acid

C6H8O7191.0197

13.73

Salicylic acid

C7H6O3137.0239

14.21

Isocitric acid

C6H8O7191.0197

14.28

D-Fructose-1,6-bisphosphate (D-F-1,6-2P)

C6H14O12P2338.9888

14.33

GTP

C10H16N5O14P3521.9834

17.68

FAD

C27H33N9O15P2

784.1499

17.83

ATP

C10H16N5O13P3505.9885

18.19

NADPH

C21H30N7O17P3744.0828

18.60Succinyl CoA

C25H40N7O19P3S

866.124

19.06

Acetyl CoA

C23H38N7O17P3S

808.1185

19.08

Isobutyl CoA

C25H42N7O17P3S

836.1498

19.49

Glycocholic acid

C26H43NO6464.3018

20.72

4Isomer pair

Isomer pair

Isomer pair

Isomer pairEffect of mobile phase pH on RT,
selectivity, and signal intensity

Changes to the pH of the mobile phase byadjusting the concentration of acetic acidcan alter the RT and selectivity for sugar
×105

D-G-6-PA

phosphates and nucleotide triphosphates(Figure 3). However, the signal intensities
of a number of metabolite classes were
impacted with this pH change (Figure 4).

×105
1.0

pH = 55Counts

Counts

α-D-G-1-P

FAD

0.5

D-F-1,6-2PATPGTP

00
6.0

×105

7.0
6.5
Acquisition time (min)7.5

13×105C

1.0CountsCountspH = 5.8

5D-G-6-P

6.0

×105

7.0
6.5
Acquisition time (min)D

0.5

13Counts

1.0

6.0

19

pH = 5.8

7.5

pH = 7.5

α-D-G-1-P

018

FAD ATP

×105

D-G-6-P

1516
17
Acquisition time (min)

GTP0E

514

D-F-1,6-2P

α-D-G-1-P0Counts

B

pH = 57.06.5
Acquisition time (min)7.514151617Acquisition time (min)1819ATPF

pH = 7.5
FAD

0.5

D-F-1,6-2PGTP0
1314151617Acquisition time (min)1819

Figure 3. Effect of three mobile phase pH conditions on the RT and selectivity of some phosphorylated metabolites.

5

2.5

3

1.5

2

2

1.0

1

1

0.5

000AM

IsoosP3

leucine
L
Gl eu
yo ci
xy nelic
La acict d
Py ic aru cidvi
M c acale id
Su iccc acidi
Fu nicrm a
α- ar cid
Ke icto ac
glu idtaCi ratet
Iso ric acc
Gl itri idyc c aoc cihl dicacid

2.0eDFruc
DtoseGlucos
eD6FrPuc
toαse
D6GlPucDos
Freuc
1toP
se-1
,62P

4uc

pH = 5pH = 5.8
pH = 7.5

3.0

4

DGlC

Figure 4. Effect of three mobile phase pH conditions on the signal responses of broad classes of endogenous metabolites.

5ATNA P
Ac DPHet
Iso yl Cbu oAtylCoA

pH = 5pH = 5.8
pH = 7.5

6×105
3.5PFAD5BP

pH = 5pH = 5.8
pH = 7.5

6×1057GTAGM×1057

The magnitude of the signal intensitychanges and the direction of RT shifts
are highly dependent on the metabolite
chemical class. Results suggest thatanalytical selectivity and sensitivity fora specific class of metabolites can beimproved by controlling the pH of themobile phase in IP-RP chromatography.
In this study, a mobile phase at pH 5.8
provided the best overall results forchromatographic separation of theisomers, with acceptable sensitivity
among the different classes ofmetabolites.

Good RT reproducibility

Reproducibility is important for reliable
differential analysis and confident
identification in metabolomics
studies. To ensure that the developed
IP‑RP LC/TOF MS method is robust andreliable, 19 representative metabolites
covering the entire chromatographic
region were selected for further
evaluation. Table 4 shows that good
reproducibility was achieved for RT. The
Agilent UHPLC binary pump with seal
wash increases robustness when using
buffered mobile phases by flushing theback side of the seal, thus maintaining
the seal lifetime.

Table 4. Reproducibility of RTs for the 19 representative metabolites.
CompoundChemical formula(M-H)–

RT (Ave) (n = 3)

RT (%CV)) (n = 3)

D-Glucose

C6H12O6

179.0561

1.470.42

D-Fructose

C6H12O6

179.0561

1.50

0.29

Inosine

C10H12N4O5

267.0735

4.64

0.10

Guanosine

C10H13N5O5

282.0839

5.170.27

Glyoxylic acidC2H2O372.9931

6.38

0.08

Lactic acid

C3H6O389.02446.490.08

NAD

C21H27N7O14P2

662.1013

6.55

0.07

D-Fructose-6-phosphate

C6H13O9P

259.0224

6.720.02

Pyruvic acid

C3H4O387.0088

7.000.02

AMP

C10H14N5O7P

346.0558

8.69

0.11

GMP

C10H14N5O8P

362.0507

9.79

0.10

α-Ketoglutarte

C5H6O5145.01373

10.310.06

Citric acid

C6H8O7191.0197

13.73

0.09

GTP

C10H16N5O14P3

521.9834

17.68

0.09

FAD

C27H33N9O15P2

784.1499

17.83

0.03

ATP

C10H16N5O13P3

505.9885

18.19

0.03

NADPH

C21H30N7O17P3

744.0828

18.60

0.02

Acetyl CoA

C23H38N7O17P3S

808.1185

19.08

0.03

Isobutyl CoA

C25H42N7O17P3S

836.1498

19.49

0.01

6ConclusionsReferences

These results demonstrate that thisrobust and reliable IP-RP method on
the Agilent 6230 TOF LC/MS provides
excellent analytical performance formetabolite profiling across a broad
range of metabolite classes. Excellent
chromatographic separation wasachieved for four pairs of biologicallyrelevant isomers: citrate/isocitrate,
D-glucose-6-phosphate/α-D‑glucose‑1phosphate, fumeric acid/maleic acid,and leucine/isoleucine. The Agilent
1290 Infinity LC system combined withthe 6230 TOF system delivered excellent
performance, making it an ideal system
for metabolomics profiling.

1. Dai, Y.; et al. Metabolomics Batch Data
Analysis Workflow to Characterize
Differential Metabolites in Bacteria,
Agilent Technologies Application Note,publication number 5991-5706EN,
2015.
2. Jenkins, S.; et al. Compound
Identification, Profiling and Pathway
Analysis of the Yeast Metabolome
in Mass Profiler Professional,
Agilent Technologies Application Note,publication number 5991-2470EN,
2013.
3. Zhang, T.; et al. Anal. Chem. 2012, 84,1994-2001.
4. Lu, W.; et al. Anal. Chem. 2010, 82,3212-3221.

7

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This information is subject to change without notice.
© Agilent Technologies, Inc., 2017
Published in the USA, February 1, 20175991-7802EN