Diisopropylethylamine/hexafluoroisopropanol-mediated ion-pairing ultra-high-performance liquid chromatography/mass spectrometry for phosphate and carboxylate metabolite analysis: utility for studying cellular metabolism

Article abstract of DOI:10.1002/rcm.7667

Rationale: Mass spectrometric (MS) analysis of low molecular weight polar metabolites can be challenging because of poor chromatographic resolution of isomers and insufficient ionization efficiency. These metabolites include intermediates in key metabolic pathways, such as glycolysis, the pentose phosphate pathway, and the Krebs cycle. Therefore, sensitive, specific, and comprehensive quantitative analysis of these metabolites in biological fluids or cell culture models can provide insight into multiple disease states where perturbed metabolism plays a role. Methods: An ion-pairing reversed-phase ultra-high-performance liquid chromatography (IP-RP-UHPLC)/MS approach to separate and analyze biochemically relevant phosphate- and carboxylic acid-containing metabolites was developed. Diisopropylethylamine (DIPEA) was used as an IP reagent in combination with reversed-phase liquid chromatography (RP-LC) and a triple quadrupole mass spectrometer using selected reaction monitoring (SRM) and negative electrospray ionization (NESI). An additional reagent, hexafluoroisopropanol (HFIP), which has been previously used to improve sensitivity of nucleotide analysis by UHPLC/MS, was used to enhance sensitivity. Results: HFIP versus acetic acid, when added with the IP base, increased the sensitivity of IP-RP-UHPLC/NESI-MS up to 10-fold for certain analytes including fructose-1,6-bisphosphate, phosphoenolpyruvate, and 6-phosphogluconate. It also improved the retention of the metabolites on a C₁₈ reversed-phase column, and allowed the chromatographic separation of important isomeric metabolites. This methodology was amenable to quantification of key metabolites in cell culture experiments. The applicability of the method was demonstrated by monitoring the metabolic adaptations resulting from rapamycin treatment of DB-1 human melanoma cells. Conclusions: A rapid, sensitive, and specific IP-RP-UHPLC/NESI-MS method was used to quantify metabolites from several biochemical pathways. IP with DIPEA and HFIP increased the sensitivity and improved chromatographic separation when used with reversed-phase UHPLC.

Full text of DOI:10.1002/rcm.7667

Research Article
Received: 27 April 2016
Revised: 14 June 2016
Accepted: 15 June 2016
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2016, 30, 1835–1845
(wileyonlinelibrary.com) DOI: 10.1002/rcm.7667
Diisopropylethylamine/hexafluoroisopropanol-mediated ion-
pairing ultra-high-performance liquid chromatography/mass
spectrometry for phosphate and carboxylate metabolite analysis:
utility for studying cellular metabolism
Lili Guo1, Andrew J. Worth1, Clementina Mesaros1, Nathaniel W. Snyder2,
Jerry D. Glickson3 and Ian A. Blair1
*
1Penn SRP and Center for Excellence in Environmental Toxicology, Department of Systems Pharmacology and Translational
Therapeutics, University of Pennsylvania, Philadelphia, PA 19104, USA
2A.J. Drexel Autism Institute, Drexel University, Philadelphia, PA 19104, USA
3Department of Radiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
RATIONALE: Mass spectrometric (MS) analysis of low molecular weight polar metabolites can be challenging because of
poor chromatographic resolution of isomers and insufficient ionization efficiency. These metabolites include intermediates
in key metabolic pathways, such as glycolysis, the pentose phosphate pathway, and the Krebs cycle. Therefore, sensitive,
specific, and comprehensive quantitative analysis of these metabolites in biological fluids or cell culture models can
provide insight into multiple disease states where perturbed metabolism plays a role.
METHODS: An ion-pairing reversed-phase ultra-high-performance liquid chromatography (IP-RP-UHPLC)/MS
approach to separate and analyze biochemically relevant phosphate- and carboxylic acid-containing metabolites was
developed. Diisopropylethylamine (DIPEA) was used as an IP reagent in combination with reversed-phase liquid
chromatography (RP-LC) and a triple quadrupole mass spectrometer using selected reaction monitoring (SRM) and
negative electrospray ionization (NESI). An additional reagent, hexafluoroisopropanol (HFIP), which has been previously
used to improve sensitivity of nucleotide analysis by UHPLC/MS, was used to enhance sensitivity.
RESULTS: HFIP versus acetic acid, when added with the IP base, increased the sensitivity of IP-RP-UHPLC/NESI-MS up
to 10-fold for certain analytes including fructose-1,6-bisphosphate, phosphoenolpyruvate, and 6-phosphogluconate. It also
improved the retention of the metabolites on a C18 reversed-phase column, and allowed the chromatographic separation of
important isomeric metabolites. This methodology was amenable to quantification of key metabolites in cell culture
experiments. The applicability of the method was demonstrated by monitoring the metabolic adaptations resulting from
rapamycin treatment of DB-1 human melanoma cells.
CONCLUSIONS: A rapid, sensitive, and specific IP-RP-UHPLC/NESI-MS method was used to quantify metabolites from
several biochemical pathways. IP with DIPEA and HFIP increased the sensitivity and improved chromatographic
separation when used with reversed-phase UHPLC.
Ultra-high-performance liquid chromatography/selected
reaction monitoring mass spectrometry (UHPLC/SRM-MS)
is considered to be the “gold standard” for quantification of
relatively low abundance metabolites from complex biological
matrices.[1] Robust and reproducible UHPLC/SRM-MS
methods are dependent on several factors, including adequate
and consistent chromatography, particularly for isomeric
compounds, which are often indistinguishable by SRM/MS
analysis. Efficient ionization in the source of the mass
spectrometer is required for the detection of analytes. These
requirements are particularly important when analyses are
performed on complex biological samples, as co-eluting of
interfering compounds can suppress signal and diminish the
sensitivity, and potentially specificity, of a method.[2,3]
Cellular metabolism has received renewed interest in recent
years due to emerging applications of metabolomics in drug
discovery and precision medicine[4–6] and playing a causative
role in a range of diseases.[7–11] In particular, the metabolic
reprogramming that occurs in cancer has been proposed as
contributing to pathogenesis, disease progression, and has
sparked renewed interest in targeting altered metabolic
pathways as therapeutic interventions.[12,13] As such, an
improved understanding of cancer metabolism, as well as
the genetic or epigenetic factors that contribute to the
metabolic alterations, is critical to explore disease-specific
phenotypes or pathways for therapeutic purposes.
Unfortunately, many of these bioenergetic processes of
interest involve metabolites that are difficult to analyze by
* Correspondence to: I. A. Blair, Center for Cancer
Pharmacology, University of Pennsylvania, 854 BRB II/III,
421 Curie Boulevard, Philadelphia, PA 19104-6160, USA.
E-mail: ianblair@mail.med.upenn.edu
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L. Guo et al.
conventional
LC/SRM-MS
approaches.[14–16]
More
facilitate NESI and maximize interactions of the analyte with
the immobilized IP reagent. Increasing the organic solvent
content of the mobile phase (by gradient elution) reduces
the interaction of the IP base with the stationary phase and
allows the metabolites to elute from the column. The added
non-nucleophilic base also serves to reduce the sodium and
potassium adducts which can reduce the signal derived from
the deprotonated analyte molecule [M–H]À.[33,34]
specifically, glycolysis, the pentose phosphate pathway
(PPP), and the Krebs or tricarboxylic acid (TCA) cycle are
major metabolic pathways which can be differentially
regulated by disease states.[11] Many of the metabolites are
low molecular weight hydrophilic compounds, containing
phosphate, carboxylic acid, or amino acid moieties. Therefore,
retention on common reversed-phase C8 or C18 LC columns is
generally poor and many of the metabolites are not amenable
to sensitive analysis by positive mode electrospray ionization
(PESI). Metabolite concentrations can span a very wide
concentration range and there are numerous isomeric
metabolites. This means that accurate quantification requires
selective and reproducible chromatography coupled with
efficient generation of ions in the electrospray ionization
source for quantification of low-abundance metabolites.
Many LC/MS methods for the analysis of small molecule
polar metabolites have employed hydrophobic interaction
liquid chromatography (HILIC)-mediated separation.[17–20]
Advances in the materials for HILIC columns make this
approach much more reliable than the earlier generation of
columns. A drawback of this methodology is the necessity for
sample resuspension in an organic solvent, which decreases
sensitivity since some metabolites have very low solubility in
high-organic solvents thus causing selective undervaluation.
Using lower organic concentrations for the resuspension solvent
impairs the binding of the analytes to the stationary phase of
HILIC columns and causes peak shapes to be non-ideal, which
then adds to the difficulty of separating isobaric or isomeric
species. Moreover, relatively long equilibration times are
required when using HILIC methods in order to obtain a stable
retention time between runs.[21,22]
Oligonucleotide analysis by IP-LC/MS has generally
involved the addition of HFIP to the non-nucleophilic amine
base in the mobile phase.[35] By titrating the base to near neutral
pH with HFIP (pH 8), the phosphate-rich negatively charged
oligonucleotides undergo IP with the positively charged base,
resulting in increased chromatographic retention on reversed-
phase LC columns.[33,34] In addition, the low boiling point of
HFIP (58.2°C) facilitates its rapid evaporation from the surface
of liquid droplets in the source of the mass spectrometer (Fig. 1).
This process is hypothesized to rapidly increase the pH within
the droplets due to the effective concentration of base, making
this approach highly efficient in negative mode ionization.[35]
As this approach is very effective for oligonucleotide
analysis,[34,35] we reasoned that it should also be useful for the
analysis of the phosphate- and carboxylic acid-containing small
molecules that make up intermediary cellular metabolism. The
negatively charged phosphate and carboxylate moieties on the
metabolites should have similar chromatographic properties to
oligonucleotides. To extend the analytical coverage of carbon
metabolism, we also coupled IP with chemical derivatization
in order to analyze isomeric phosphate metabolites as well as
the unstable and poorlyionized[36,37] keto-acidsα-ketoglutarate,
pyruvate, and oxaloacetate metabolites.
There are several reports detailing the chromatographic
separation of polar metabolites using ion-pairing (IP)-LC/MS.[23–30]
The exact mechanism of IP-LC is still controversial even
though it was discovered over 30 years ago.[31,32] A widely
accepted mechanism involves an ion pair model in which
the hydrophobic moieties of an IP non-nucleophilic base
(such as tributylamine) interact with the hydrophobic groups
(such as C18) on the stationary phase. Polar, negatively
charged metabolites such as phosphates and carboxylates
then interact with the protonated nitrogen moiety of the
tributylamine base and are retained on the column.[25] The
mobile phase is normally maintained at pH 7.0 in order to
EXPERIMENTAL
Chemicals and reagents
Diisopropylethylamine (DIPEA), tributylamine (TBA),
1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), phenylhydrazine
HCl, dimethyl sulfoxide (DMSO), rapamycin, glucose 6-
phosphate, fructose 6-phosphate, mannose 6-phosphate,
glucose 1-phosphate, mannose 1-phosphate, galactose 1-
pohsphate, fructose 1,6-bisphosphate, glyceraldehyde 3-
phosphate, dihydroxyacetone phosphate, 2-phosphoglycerate,
Figure 1. Scheme showing how HFIP facilitates NESI.
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Copyright © 2016 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2016, 30, 1835–1845

Diisopropylethylamine/hexafluoroisopropanol IP-UHPLC/MS
phosphoenolpyruvate, pyruvate, 6-phosphogluconate, ribulose
5-phosphate, ribose 5-phosphate, sedoheptulose 7-phosphate,
erythrose 4-phosphate, glutathione, flavin adenine dinucleotide
(FAD), nicotinamide adenine dinucleotide (NAD), NADH,
nicotinamide adenine dinucleotide phosphate (NADP),
NADPH, acetyl-CoA, succinyl-CoA, citrate, isocitrate,
succinate, fumarate, malate, lactate, α-ketoglutarate,
citrate, 500 ng [13C3]-pyruvate, 2 μg [13C3]-lactate, 500 ng
[13C4,15N]-aspartate, 2 μg [13C5,15N]-glutamate and 500 ng
[13C6]-glucose 6-phosphate). Samples were pulse-sonicated
for for 30 s with a probe tip sonicator and centrifuged at
16,000 g for 10 min. The supernatant was transferred to two
new tubes: 50 μL were transferred to one tube and diluted 5
times with 50 mM ammonium carbonate for direct analysis
of the underivatized redox cycling metabolites (Fig. 2) and
700 μL were transferred to one tube containing 300 μL of
phenylhydrazine in methanol/water (4/1 v/v) (3 mg/mL)
for analysis of underivatized and derivatized metabolites
(Fig. 2). Derivatization was conducted by incubation at room
temperature for 2 h before evaporation to dryness under
nitrogen. Water (100 μL) was used to re-suspend the samples.
2-hydroxyglutarate,
hydroxybutyrate,
oxaloacetate,
mevalonate, hexosamine-6-phosphate,
acetoacetate,
β-
phosphoserine, serine, glutamate, aspartate, AMP (adenosine
monophosphate), ADP (adenosine diphosphate), ATP
(adenosine triphosphate), CTP (cytidine triphosphate), UTP
(uridine triphosphate), GTP (guanosine triphosphate),
phosphohydroxypyruvic acid, acetyl-CoA, succinyl-CoA,
[13C5,15N]-glutamate, [13C4]-succinate, [13C3]-lactate and
[13C6]-citrate were purchased from Sigma-Aldrich (St. Louis,
MO, USA). Optima LC-MS grade water, methanol, and
acetonitrile (ACN) were purchased from Thermo Fisher
Scientific (Waltham, MA, USA). [13C6]-Glucose 6-phosphate,
[13C6]-glucose, [13C5,15N2]-glutamine, [13C4,15N]-aspartate,
[13C3]-pyruvate, [13C4]-fumarate and glutathione ([13C2,15N]-
glycine) were purchased from Cambridge Isotope Laboratories
(Tewksbury, MA, USA).
Injection volume was
5 μL in both methods. The
phenylhydrazine-derivatized samples were run with gradient
1 and the underivatized samples were run with gradient 2.
Ultra-high-performance liquid chromatography-mass
spectrometry
An UltiMate 3000 quaternary UHPLC system equipped with a
refrigerated autosampler (6°C) and a column heater was used.
Both gradients used a Phenomenex Synergy Polar-RP column
(150 × 2 mm i.d., 4 μm, 80 Å; Phenomenex, Torrance, CA, USA)
at a flow rate of 0.2 mL/min. Solvent A was 5 mM DIPEA and
200 mM HFIP and solvent B was methanol with 5 mM DIPEA
200 mM HFIP. The linear gradient 1 was as follows: 100% A for
6 min, 98% A at 8 min, 86% A at 12 min, 50% A at 14 min and
10% A at 15 min. 10% A was held for 4 min, back to 100% A
over 1 min prior to a 5 min equilibration. Linear gradient 2
was shorter, in order to run the unstable redox cycling
metabolites (NAD+, NADH, NADP+ and NADPH) as quickly
as possible after the extractions: 100% A for 2 min, 80% A at
4 min, 10% A at 6 min, and 10% A at 8 min. 10% A was held
for 2 min, back to 100% A over 1 min prior to a 4 min
equilibration. The separations were performed at 55°C. MS
analysis was conducted on a TSQ Quantum Ultra AM mass
spectrometer (Thermo Scientific, San Jose, CA, USA) equipped
with a HESI II source operating in negative mode. The TSQ
Quantum operating conditions were as follows: spray voltage
4000 V; vaporizer temperature 200°C; capillary temperature
350°C; tube lens 90 V. The sheath gas (nitrogen) and auxiliary
gas (nitrogen) pressures were 45 and 10 (arbitrary units),
respectively. Both Q1 and Q3 resolutions were set at
0.7 amu. Scan width was 0.002 and the dwell time was
20 ms. Collision-induced dissociation used argon as the
collision gas at 1.5 mTorr. The collision energy was optimized
for each metabolite and the values are reported in Table 1. Data
analysis was performed with Xcalibur software (version 2.6).
Statistical analysis was performed in Microsoft Excel or Prism
v6 (GraphPad Software Inc. La Jolla, CA, USA).
Standard solutions
Each standard compound or internal standard was weighed
and dissolved by sonication in Optima pure water to stock
solutions of 1 μg/mL. Initially, each of the compounds was
run individually to check for interferences and determine the
order of elution for isomeric compounds. The final standard
solution mix contained 60 polar metabolites (Table 1) at a
concentration of 1000 ng/mL. For quantification, a 10-point
calibration curve was constructed by 1:2 serial dilutions from
the highest concentration of calibration mixture with water.
The stock solutions were kept at À20°C. Internal standard
mixtures were made for each experiment in order to match the
estimated amounts of the individual metabolites in the samples.
Cell culture, treatment and isotopic labeling
DB-1 melanoma cells were maintained in MEMα medium with
10% fetal bovine serum (FBS) and antibiotics (100 units/mL
penicillin and 100 mg/L streptomycin). For the labeling
experiment, DB-1 cells were cultured in 10 cm tissue culture
plates, and incubated overnight with glucose- and
glutamine-free DMEM containing 10% FBS as well as 5 mM
[13C6]-glucose and
2
mM [13C5,15N2]-glutamine or
corresponding unlabeled nutrients at the same concentrations.
Human diffuse large cell lymphoma DLCL2 cells were
maintained in RPMI medium containing 10% FBS and
antibiotics. DLCL2 cells (2 × 106), cultured in tissue culture
flasks, were incubated with 0.1% DMSO or 200 nM rapamycin
for 48 h. Cells were counted at the end of each treatment. All
treatments were performed in triplicate.
RESULTS AND DISCUSSION
Metabolite extraction and derivatization
Optimization of chromatographic separation and NESI
signal intensity
Cells were washed twice with phosphate-buffered saline
(Mediatech, Manassas, VA, USA) (1 mM pH 7.4) before being
scraped into 750 μL of ice-cold methanol/water (4/1 v/v). For
cells treated with rapamycin, samples were spiked with
internal standards (500 ng [13C4]-succinate, 500 ng [13C6]-
The main goal in developing the IP-UHPLC/SRM-MS method
was to achieve increased sensitivity for most metabolites and
good chromatographic separation for the isobaric or isomeric
Rapid Commun. Mass Spectrom. 2016, 30, 1835–1845
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L. Guo et al.
Table 1. Parent (Q1) and product ions (Q3), collision energies (CE) and internal standards (ISTDs) for 60 cellular metabolites
that were analyzed
#
Metabolite
Q1 (m/z)
Q3 (m/z)
CE (V)
RT (min)
ISTD
1
glutamine
serine
aspartate
glutamate
hexosamine 6-phosphate
hydroxybutyrate
ribulose 5-phosphate
lactate
glyceraldehyde 3-phosphate
glucose 6-phosphate
fructose 6-phosphate
GSH
145
104
132
146
258
103
229
89
127
74
15
18
15
13
40
20
40
15
40
20
20
20
25
40
15
40
20
20
40
40
17
25
12
25
15
15
40
40
40
40
40
20
40
15
40
40
20
20
20
20
20
20
40
40
40
40
40
40
20
15
40
15
15
15
40
20
30
20
40
1.7
1.8
[13C5,15N2]-glutamine
[13C3,15N]-serine
2
3
88
4.6
[13C4,15N]-aspartate
[13C5,15N]-glutamate
[13C3]-lactate
4
128
79
4.7
5
4.7
6
59
5.1
[13C3]-lactate
7
79
5.2
[13C4]-succinate
8
43
5.4
[13C3]-lactate
9
169
259
259
306
199
229
147
289
259
169
184
346
133
611
145
147
115
117
183
349
349
319
185
426
167
191
319
349
191
339
522
482
275
483
506
259
259
379
289
429
290
177
866
191
221
235
808
662
664
742
774
79
5.6
[13C4]-succinate
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
97
5.9
[13C6]-glucose 6-phosphate
[13C6]-glucose 6-phosphate
GSH ([13C2,15N]-glycine)
[13C4]-succinate
97
5.9
143
97
5.9
erythrose 4-phosphate
ribose 5-phosphate
mevalonate
6
6
79
[13C4]-succinate
59
6.1
[13C4]-succinate
sedoheptulose 7-phosphate
glucose 1-phosphate
dihydroxyacetone phosphate
phosphoserine
79
6.1
[13C4]-succinate
97
6.2
[13C6]-glucose 6-phosphate
[13C4]-succinate
97
6.8
79
7.1
[13C4]-succinate
AMP
134
115
306
101
129
71
7.2
[13C4]-succinate
malate
7.7
[13C4]-succinate
GSSG
7.8
GSSG ([13C2,15N]-glycine)
[13C4]-succinate
alpha-ketoglutarate
2-hydroxyglutarate
fumarate
7.9
7.9
[13C4]-succinate
8
8
[13C4]-fumarate
succinate
phosphohydroxypyruvate
glucose 6-P-PZ
mannose 6-P-PZ
ribulose 5-phosphate-PZ
2-phosphoglycerate
ADP
phosphoenolpyruvate
citrate
ribose 5-phosphate-PZ
fructose 6-P-PZ
isocitrate
fructose 1,6-bisphosphate
GTP
CTP
73
[13C4]-succinate
79
10.8
13.8
14.5
14.8
15
15.1
15.4
15.4
15.6
15.6
15.8
15.9
15.9
16
16.1
16.1
16.3
17.2
17.2
17.2
17.6
18.4
18.7
19.3
19.4
19.5
19.5
19.5
19.6
6.1*
9.4*
9.8*
12.2*
[13C4]-succinate
79
[13C6]-glucose 6-phosphate-PZ
[13C6]-glucose 6-phosphate-PZ
[13C6]-glucose 6-phosphate-PZ
[13C6]-citrate
79
97
79
328
79
[13C6]-citrate
[13C6]-citrate
111
97
[13C6]-citrate
[13C6]-glucose 6-phosphate-PZ
[13C6]-glucose 6-phosphate-PZ
[13C6]-citrate
79
73
97
[13C6]-citrate
424
384
97
[13C6]-citrate
[13C6]-citrate
6-phosphogluconate
UTP
ATP
[13C6]-citrate
385
159
79
[13C6]-citrate
[13C6]-citrate
glyceraldehyde 3-phosphate-PZ
dihydroxyacetone phosphate-PZ
sedoheptulose 7-phosphate-PZ
erythrose 4-phosphate-PZ
fructose 1,6-bisphosphate-PZ
flavin adenine dinucleotide (FAD)
pyruvate-PZ
[13C6]-glucose 6-phosphate-PZ
[13C6]-glucose 6-phosphate-PZ
[13C6]-glucose 6-phosphate-PZ
[13C6]-glucose 6-phosphate-PZ
[13C6]-glucose 6-phosphate-PZ
[13C6]-citrate
79
79
79
79
247
92
[13C3]-pyruvate-PZ
[13C6]-citrate
succinyl-CoA
408
92
92
92
408
540
408
620
408
acetoacetate-PZ
oxaloacetate-PZ
alpha-ketoglutarate-PZ
acetyl-CoA
[13C3]-pyruvate-PZ
[13C3]-pyruvate-PZ
[13C3]-pyruvate-PZ
[13C6]-citrate
NAD+
[13C4]-succinate
NADH
[13C6]-citrate
NADP+
[13C6]-citrate
NADPH
[13C6]-citrate
PZ: phenylhydrazone derivatives.
*Indicates the retention times obtained from gradient 2.
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Diisopropylethylamine/hexafluoroisopropanol IP-UHPLC/MS
Figure 2. Work-up flow for the extraction and analysis of polar metabolites by
IP-RP-UHPLC/NESI-MS for DLCL2 cells. Gradient 1 is 25 min whereas
gradient 2 is 15 min in order to quickly analyze unstable redox cycling
metabolites.
compounds. Here we tested phosphate and carboxylate
heated source than acetic acid, increasing the pH and
facilitating negative ionization (Fig. 1). TBA is commonly used
as an IP reagent at a concentration of 10 mM.[9,11] However,
this high concentration can only be reached with lower pH
when TBA is paired with a stronger acid, like acetic acid.
DIPEA has higher water solubility and allows up to 10 mM
concentrations when paired with HFIP in aqueous mobile
phases. Therefore, we tested DIPEA and TBA on four different
columns with different stationary phases from different
manufacturers with different concentrations of HFIP (data
not shown). Compared to DIPEA, TBA increased the retention
time of the analytes by 5–10 min. Most isomers (like
isocitrate/citrate and glucose 6-phosphate/fructose 6-
phosphate) were not baseline separated by the increased
metabolites from central energy metabolism, which are
important in studies such as cancer cell metabolism. The most
intense ion for all the carboxylic acids, sugar phosphates and
nucleotides corresponded to the loss of a proton from each
molecule [M–H]À (Table 1) as reported previously for ESI.[38]
In the presence of HFIP, the product ion with least background
for the sugar phosphates was 79 Da ([PO3]À). The loss of 97 Da
was also monitored and used as a qualifier. All carboxylic
acids gave decarboxylation products (loss of 44 Da) or
decarboxylation with loss of water. The most abundant
product ion from nucleotides was generated from the loss of
phosphate (loss of 98 Da) or the loss of both phosphates and
sugar moiety (Table 1). In the second case, the product ion is
the remaining nucleobase (e.g. adenine, 134 Da).
retention times. In contrast, DIPEA provided
a faster
We reasoned that the same stationary phases used in
achieving good separations with IP methods previously
reported[9,24–26] should also be useful with HFIP.
Tributylamine (TBA) has been used previously as one of the
IP reagents for the separation of small polar
metabolites.[23,25,28,29,36] It is often used in combination with
acetic acid[9,25] to separate carboxylic acids, sugar phosphates,
and nucleotides. We expected that, compared with acetic acid,
HFIP would improve the MS sensitivity, since the boiling
point of the HFIP (58.2°C) is much lower than the boiling point
of acetic acid (118°C). The HFIP evaporates more rapidly in the
chromatographic method that still achieved separation of
most of the isomeric species. As with all the parameters for
IP-based methods, there is no perfect IP base for all
metabolites. Due to a solubility issue of TBA in water, the
combination of TBA and HFIP can never create an ideal
gradient for separating all metabolites. TBA had generally less
sensitivity for most metabolites. For lactate, for example, the
sensitivity was 10-fold less. For most metabolites, TBA-
containing mobile phases were between 2–5-fold less
sensitive, but for fumarate and malate, they were
approximately twice as sensitive and the peak shapes were
Rapid Commun. Mass Spectrom. 2016, 30, 1835–1845
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L. Guo et al.
significantly better. Interestingly, a concentration of 1 mM
DIPEA gave the best sensitivity for pure standards. However,
this low concentration was insufficient for cell extracts because
some metabolites were not adequately retained on the column.
5 mM DIPEA improved the retention of all of the analytes on
the column and showed the highest retention time stability for
running samples for long periods of time. Under this
condition, the retention time shifts of the same metabolites
were within 8 s for different biological samples.
The four columns we examined are described next. (1)
Phenomenex Synergy Polar-RP (150 × 2 mm i.d., 4 μm, 80 Å;
Phenomenex, Torrance, CA, USA): This column has fast
elution times and good separation for early eluents. It provides
the residual IP reagent often causes intense interfering ions
that are difficult to remove. It is noteworthy that in contrast
to TBA, residual DIPEA did not result in strong interfering
ions when switching from negative to positive ion mode. This
is probably due to a balance between its lower boiling point
and higher solubility in the mobile phase. In addition, the
molecular weight of DIPEA (129 Da) is lower than that of
TBA (185 Da) and so the interference from DIPEA is less
problematic for metabolites that give rise to ions > m/z 130
in positive mode. This issue of interfering ions is of even
greater concern when the IP method is used on high-resolution
mass spectrometers.[18,19] With the column offline, two simple
washes with 0.1% formic acid in methanol coupled with high
capillary temperatures at the end of the day were found to
effectively prevent the buildup of DIPEA.
HFIP at a concentration of 200 mM was found to provide
the best sensitivity and robustness for the cellular extract
analysis (Fig. 3). For some key metabolites, especially those
with longer retention time with HFIP, HFIP increased the
sensitivity by more than 10-fold compared to that of DIPEA
alone, including fructose 1,6-bisphosphate (F 1,6-bP) and 6-
phosphogluconate (6-PG) (Fig. 3). The two isomeric products
from F 1,6-bP, glyceraldehyde 3-phosphate (GAP) and
dihydroxyacetone phosphate (DHAP), were baseline
separated within 7 min. The sensitivity for GAP was reported
to be 100 times lower than for DHAP,[23] but with the HFIP
modifier we observed the sensitivity for GAP to be only 10
times less than the sensitivity for DHAP. Interestingly,
compared to acetic acid, HFIP added to DIPEA significantly
improved the peak shapes and increased sensitivity by 10-
fold for analytes eluting towards the middle and the end of
the chromatogram.
maximum separation for late
eluents especially
phenylhydrazine-derivatized metabolites. (2) Xselect HSS
C18 (150 × 2.1 mm, 3.5 μm, 100 Å; Waters, Milford, MA,
USA): This column is excellent for DIPEA/HFIP analysis
and provides good separation of phenylhydrazine-
derivatized metabolites. Although it does not separate the
later eluting peaks as well as the Synergy Polar-RP, this
column is more robust so that more samples can be analyzed
without chromatographic degradation. (3) Atlantis T3
(150 × 1 mm, 5 μm, 100 Å; Waters, Milford, MA, USA): Peaks
on this column are sharp. However, the column does not
provide good separation for later eluting analytes when using
DIPEA/HFIP. (4) Phenomenex Luna C18 (250 × 2 mm i.d.,
3 μm, 100 Å; Phenomenex, Torrance, CA, USA): When using
a 45 min gradient, this column provides good separation for
both early and late eluting isomers, e.g. fructose 6-phosphate
versus glucose 6-phosphate and phenylhydrazine-derivatized
fructose 6-phosphate versus glucose 6-phosphate. However,
this separation can only be accomplished using long gradients
and the peaks are very wide.
A short chromatographic method is essential in order to
accommodate the analysis of a large number of samples, but
none of the columns that were tested would separate all
isomeric species in a short (under 30 min) chromatographic
run. Different columns were able to separate different isomeric
Conventional IP reagents such as TBA will contaminate the
LC/MS system so that cleaning procedures often involve
purging with solvents over long periods of time.[23] In
addition, when switching from negative to positive mode,
Figure 3. Representative IP-RP-UHPLC/NESI-MS chromatograms for separation
improvement and sensitivity increase due to HFIP. (A) Solvents used contained
5 mM DIPEA but no HFIP. (B) Solvents used contained 5 mM DIPEA and
200 mM HFIP. F 1,6-bP: fructose 1,6-bisphosphate; GAP: glyceraldehyde
3-phosphate; DHAP: dihydroxyacetone phosphate; 2-PG: 2-phosphoglycerate;
PEP: phosphoenolpyruvate.
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Diisopropylethylamine/hexafluoroisopropanol IP-UHPLC/MS
pairs, but we found it to be extremely challenging to separate
the hexose phosphates isomers. Notably, it has been
challenging to separate hexose-phosphate using regular LC
methods even with long gradient elutions.[25]
bisphosphate (F 1,6-bP), GAP, DHAP, ribulose 5-phosphate,
ribose 5-phosphate, sedoheptulose 7-phosphate and
erythrose 4-phosphate all formed mono-phenylhydrazone
derivatives. Loss of the aniline radical (92 Da) or the common
losses of 79 Da and 97 Da from the phenylhydrazone-
phosphate provided abundant product ions. In a similar
way to keto-acid phenylhydrazone derivatives, the
phenylhydrazone-phosphates were retained longer on the
reversed-phase column than the underivatized analytes
(Table 1). Importantly, the derivatization facilitated the
chromatographic separation of isomeric analytes. For
example, G 6-P and F 6-P eluted at similar retention times
before derivatization with phenylhydrazine (Fig. 5(A))
Derivatization of metabolites with a carbonyl group by
phenylhydrazine
Many α-keto acid metabolites including pyruvate,
oxaloacetate and α-ketoglutarate are unstable and difficult
to analyze by LC/MS. A previous report has described the
use of phenylhydrazine derivatization for analysis of α-keto
acids by IP-LC/MS.[36] We have also found that
phenylhydrazine derivatization stabilized the metabolites
and increased their chromatographic retention times. There
was also a significant enhancement in sensitivity due to the
increased mass and improved chromatographic resolution.
Carbohydrates exist in equilibrium between the closed ring
form and the open straight-chain form. At room temperature
phenylhydrazine can react with ketone or aldehyde groups
from open-chain carbohydrates such as fructose 6-phosphate
(F 6-P) and glucose 6-phosphate (G 6-P) to form
phenylhydrazone derivatives (Fig. 4). At higher temperatures
oxidation of the α-hydroxyl group occurs, which is followed
by formation of a bis-hydrazone derivative known as an
osazone.[39] UHPLC/MS analysis of reactions conducted at
room temperature revealed that F 6-P, G 6-P, fructose 1,6-
but were separated by more than
1
min after
formation of phenylhydrazone derivatives (marked with -PZ)
(Figs. 4 and 5(B)). Both analytes were also well separated from
mannose 6-phosphate (Table 1). Of note, hexose phosphates
including glucose 1-phosphate, mannose 1-phosphate and
galactose 1-phosphate are resistant to derivatization because
they cannot form open-chain keto or aldehyde forms. This
further contributed to the selective detection of hexose
phosphates, since underivatized hexose 1-phosphates could
be monitored in the same chromatographic run. Furthermore,
their analysis was not compromised by the phenylhydrazine
derivatization reaction (Fig. 6). Thus, all metabolites
(derivatized or not) could be quantified in
a single
chromatographic run.
Figure 4. Derivatization of fructose 6-phosphate and glucose 6-phosphate with
phenylhydrazine.
Figure 5. (A) Fructose 6-phosphate (F 6-P) and glucose 6-phosphate (G 6-P)
without derivatization have identical retention times. (B) After derivatization
with phenylhydrazine they are chromatographically separated by more than
1 min by IP-RP-UHPLC/NESI-MS.
Rapid Commun. Mass Spectrom. 2016, 30, 1835–1845
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L. Guo et al.
Figure 6. Quantification of isotopically labeled metabolites from DB-1 melanoma cells after derivatization with phenylhydrazine.
For comparison, the carbonyl-containing metabolites were quantified in parallel experiments with phenylhydrazine (PZ)
derivatization (red boxes) or without derivatization (blue boxes), using gradient 1. Metabolites without a carbonyl moiety
(grey boxes) were not affected by the derivatization reaction and were quantified in the same IP-RP-UHPLC/NESI-MS analysis.
Stable isotope tracer analysis and metabolite quantification
from mammalian cells
intermediates within the central carbon metabolism were
labeled by culturing DB-1 melanoma cells in glucose- and
glutamine-free medium containing [13C6]-glucose and
[13C5,15N2]-glutamine. As heavy-labeled internal standards
are not available for all metabolites, this experiment aimed
to verify the specific retention times for several metabolites
by comparing detected isotopologue distribution with
predicted isotopologue pattern. After overnight labeling, most
carbons in the metabolites from glycolysis and PPP were
Stable isotope labeling is an important technique for dissecting
metabolic pathways and analyzing metabolic fluxes by
quantifying the stable isotope enrichment in metabolites from
labeled nutrients.[11] To evaluate the analysis of sugar
phosphates with and without phenylhydrazine derivatization
using the newly developed IP-RP-UHPLC/NESI-MS method,
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Rapid Commun. Mass Spectrom. 2016, 30, 1835–1845

Diisopropylethylamine/hexafluoroisopropanol IP-UHPLC/MS
Figure 7. Levels of central energy metabolites in DLCL2 cells treated with
rapamycin. The amounts of metabolites were normalized first to cell number
and then to the relevant metabolites in DMSO controls. 6-PG:
6-phosphogluconate; AKG: α-ketoglutarate; OAA: oxaloacetate.
labeled. The TCA cycle metabolites had a different distribution
of the rapamycin (mTOR) signaling pathway, which is a
master regulator of cellular metabolism. Consistent with
previous studies,[41,42] levels of metabolites from glycolysis
and PPP were significantly decreased in rapamycin-treated
cells and intermediates in the TCA cycle also decreased to a
lesser extent (Fig. 7). Interestingly, intracellular glutamate
and aspartate levels were the same in the presence and absence
of rapamycin, which may be due to increased uptake from the
medium (Fig. 7).
of isotopologues (Fig. 6). For example, α-ketoglutarate had
>90% labeling in M + 5, which is likely derived from
[13C5,15N2]-glutamine (Fig. 6). In contrast, citrate has
around 30% labeling in M + 5, which could arise from
[13C5,15N2]-glutamine through reductive metabolism,
whereas the 20% labeling in M + 4 may be derived from both
[13C6]-glucose and [13C5,15N2]-glutamine through an oxidative
pathway. Citrate was also detected as M + 2 (first Krebs cycle
round) and M + 4 (second Krebs cycle round) from [13C6]-
glucose, since acetyl-CoA is formed as M + 2 from [13C6]-
glucose (Fig. 6). Reductive glutaminolysis is rare under most
metabolic conditions but would increase to higher percentage
in hypoxia conditions in the presence of inhibitors of
CONCLUSIONS
The results of metabolite quantification and stable isotope
tracer analysis confirmed that the IP-RP-UHPLC/NESI-MS
method was robust and accurate. NESI sensitivity was
increased by the use of HFIP as the counter-ion, and
phenylhydrazone derivatization facilitated the separation of
isomeric metabolites as well as stabilizing α-keto acid
metabolites. This derivatization procedure did not
affect analysis of those metabolites that were unable to form
open-chain ketone or aldehyde forms, thus allowing the
multiplexed measurement of derivatized and non-derivatized
analytes from the same sample.
mitochondrial
enzymes.
Of
note,
in
terms
of
cellular metabolite analysis, signal/noise ratio for labeled
and unlabeled metabolites was significantly higher
for phenylhydrazone derivatives when compared with non-
derivatized metabolites, resulting in less variation and more
accurate measurements (Fig. 6, sedoheptulose 7-phosphate
and ribulose 5-phosphate). We have successfully used the
method to identify metabolic targets of anti-cancer drugs, to
compare metabolic features of different tumor tissues, and to
build network models for metabolic fluxes.[11,40] Although
the detection of specific metabolites depends on the cell line
and tissue used, this method provides detection of a wide
range of analytes from central energy metabolites from cells,
tissues and serum when limited amounts are available.
The most common approach for metabolic analysis is to
analyze the levels of steady-state metabolites. Metabolic
profiling covering a wide spectrum of cellular metabolites
can quickly reveal pathways affected by drug treatment or
different biological conditions. The methodology was also
used to quantify metabolites from DLCL2 cells treated with
rapamycin (Fig. 7). Rapamycin inhibits the mechanistic target
The IP-RP-UHPLC/NESI-MS method provides accurate
analysis of metabolites in cellular central energy metabolism
and, especially when coupled with stable isotope tracing,
gives insight into the mechanisms that are involved in
metabolic adaptations.[43]
A limited number of major
metabolites, based on their relative abundance in
normal cellular state, were evaluated in the present study.
IP-RP-UHPLC/NESI-MS analyses were conducted on
the most common isomeric species such as G 6-P-PZ and
F 6-P-PZ, which could be separated within 25 min. However,
the number of analytes quantified in a chromatographic run
Rapid Commun. Mass Spectrom. 2016, 30, 1835–1845
Copyright © 2016 John Wiley & Sons, Ltd.
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L. Guo et al.
metabolism in the cytosol and mitochondria of mammalian
cells. Mol. Cell. 2014, 55, 253.
[11] L. Guo, A. A. Shestov, A. J. Worth, K. Nath, D. S. Nelson,
D. B. Leeper, J. D. Glickson, I. A. Blair. Inhibition of
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[12] N. V. Rajeshkumar, P. Dutta, S. Yabuuchi, R. F. de Wilde,
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S. K. Jain, M. Hidalgo, C. V. Dang, R. J. Gillies, A. Maitra.
Therapeutic targeting of the Warburg effect in pancreatic
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[13] P. Kucharzewska, H. C. Christianson, M. Belting. Global
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could be expanded to include more metabolites from other
metabolic pathways as well. The robustness of reversed-phase
separation is ideal for scheduled SRM analyses based on the
relative retention times, thus preserving sensitivity of analysis
when multiplexing additional analytes. For high-resolution
instruments[18,19] the ability of this method to
chromatographically separate a wide diversity of analytes
may be quite useful in quantifying a larger number of
metabolites without any loss in sensitivity or specificity from
isomeric or unstable species. This method will significantly
facilitate metabolic research on cancer, metabolic diseases,
and immune cell regulation.
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Quantification of plasma carnitine and acylcarnitines by
high-performance liquid chromatography-tandem mass
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Acknowledgements
This project was supported by NIH grants U54HL117798,
P42ES023720, P30ES013508, P30CA016520, K22ES26235,
T32ES019851, R01CA129544, and R01CA172820 and
Pennsylvania Department of Health CURE grant to NWS.
a
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