Differentiation of prototropic ions in regioisomeric caffeoyl quinic acids by electrospray ion mobility mass spectrometry

Article abstract of DOI:10.1002/rcm.7151

Rationale A series of dietary important regioisomeric chlorogenic acids were investigated by ion mobility mass spectrometry (IM-MS). The existence of prototropic isomers separated in the drift dimension was observed and investigated further using tandem m

Full text of DOI:10.1002/rcm.7151

Research Article
Received: 15 September 2014
Revised: 5 January 2015
Accepted: 17 January 2015
Published online in Wiley Online Library:
Rapid Commun. Mass Spectrom. 2015, 29, 675–680
(wileyonlinelibrary.com) DOI: 10.1002/rcm.7151
Differentiation of prototropic ions in regioisomeric caffeoyl
quinic acids by electrospray ion mobility mass spectrometry
1
1
1
1
*
Nikolai Kuhnert , Ghada H. Yassin , Rakesh Jaiswal , Marius F. Matei and
Christian H. Grün2
1School of Engineering and Science, Jacobs University Bremen, Campusring 8, 28759 Bremen, Germany
2Unilever R&D, Olivier van Noortlaan 120, 3133 AT Vlaardingen, The Netherlands
RATIONALE: A series of dietary important regioisomeric chlorogenic acids were investigated by ion mobility mass
spectrometry (IM-MS). The existence of prototropic isomers separated in the drift dimension was observed and
investigated further using tandem mass spectrometry (MS/MS) and compared with suitable synthetic analogues.
METHODS: Using a quadrupole ion mobility time-of-flight mass spectrometer, the IM-MS and IM-MS/MS spectra of
selected chlorogenic acids were recorded in the negative ion mode and compared with synthetic analogues.
RESULTS: Regioisomeric di- and monocaffeoyl quinic acids can be readily separated and investigated using IM-MS.
Comparison of drift times allows assignment of the regiochemistry of precursor ions as well as for fragment ions. For
5-caffeoyl quinic acid the existence of prototropic ions was suggested and probed using synthetic analogues, unable to
show this type of isomerism. These investigations suggest the presence of prototropic isomers with carboxylate and
phenolate sites, respectively.
CONCLUSIONS: We report on IM-MS measurements on regioisomeric mono- and dicaffeoyl quinic acids, which are
important dietary natural products. Both classes of compounds can be readily separated by IM-MS in the drift time
dimension and, following MS2 experiments, fragment ion regiochemistry unambiguously determined. 5-Caffeoyl quinic acid
shows two IM-MS signals, which we assign to prototropic isomers after comparison with suitable synthetic analogues, with a
negative charge located at the carboxalate or phenolate functionality, respectively. Copyright © 2015 John Wiley & Sons, Ltd.
The structural elucidation of complex natural products has
for more than three decades been the prerogative of nuclear
magnetic resonance (NMR) spectroscopy. In particular,
aspects of the regio- and stereochemistry of natural products
can be readily extracted from one- and two-dimensional
NMR spectra.[1]
Ion mobility mass spectrometry (IM-MS) constitutes a
second MS-based technique able to differentiate between
isomeric small molecules based on separation in the drift
time dimension. Isomeric compounds are separated
according to their collisional cross section, which can be
obtained experimentally and compared with theoretical
computed values, therefore allowing predictive structural
However, mass spectrometry (MS) offers a series of
advantages over NMR spectroscopy including superior
sensitivity, more facile coupling to separation methods, in
particular liquid chromatography, and the ability to
isolate and individually characterize precursor ions of
selected m/z ratios in the gas phase. In general, MS was for
decades considered to be isomer blind, failing to provide
valuable structural information on more complex molecules.
This picture has slightly changed over the last decade with
general methods based on tandem mass spectrometry arising
for selected classes of compounds. These methods are able
not only to differentiate between isomeric compounds,
but additionally provide unambiguous information for
structure elucidation based on specific hydrogen-bonding
motifs in gas-phase ions leading to predictable fragment
spectra ultimately allowing assignment of regio- and
stereochemistry.[2,3]
elucidation.[4] IM-MS has become
a routine tool to
investigate protein conformation in the gas phase.[5]
However, applications in small molecule chemistry are still
rare and not general. Rappaport published pioneering work
on IM-MS separation of E/Z pairs of isomers.[6] More recently,
IM-MS was applied to study a growing number of small
isomeric molecules, including diastereomeric disaccharides,[7]
the ephidrine/pseudoephedrine pair of diastereoisomers,[8]
carbamazepine hydroxylated regioisomeric metabolites,[9]
silver ion adducts of the regioisomeric pair of hesperidin and
neohesperidin,[10] regioisomeric phthalic acids,[11] E/Z isomers
of caffeic acid derivatives,[12] diastereomeric terphenyl
ruthenium complexes,[13] diastereomeric nickel complexes of
all naturally occurring amino acids,[14] regioisomeric
proanthocyanidines and theasinensins,[15] and stereoisomers
of curcubituril inclusion complexes.[16]
A second type of isomerism that occurs frequently is
prototropic isomerism and has been postulated for gas-
phase ions in mass spectrometry. This isomerism is a general
form of tautomerism, where isomers result as a consequence
of protons migrating to different functional groups within
the same molecule.[17] To our knowledge only a single
* Correspondence to: N. Kuhnert, School of Engineering and
Science, Jacobs University Bremen, Campusring 8, 28759
Bremen, Germany.
E-mail: n.kuhnert@jacobs-university.de
Rapid Commun. Mass Spectrom. 2015, 29, 675–680
Copyright © 2015 John Wiley & Sons, Ltd.

N. Kuhnert et al.
example of the actual presence of different prototropic
isomers has been probed by mass spectrometry.[18] In the
case of the quinolone antibiotic norfloxacin, a pH-dependent
change in fragment spectra has been observed that was
rationalized following ion mobility separation of two
protomeric [M+H]+ pseudomolecular ions combined with
suggested protonation sites at the amine nitrogen or
carboxylic acid moiety displaying distinguishable fragment
spectra.[19] However, when considering fragmentation
mechanisms in mass spectrometry, the equilibrium between
prototropic isomers by proton shifts must be assumed to
Ion mobility mass spectrometry conditions
CQA samples were diluted to 10 μg/mL in methanol/water
(1:1 v/v) for direct infusion experiments on a Waters Synapt
G2 HDMS mass spectrometer. The system was used in
negative ionization electrospray mode with the resolution
set to 20 kDa at m/z 556. The mass axis was calibrated using
sodium formate.
A solution of leucine-enkephaline at
2 ng/mL was used for the lock mass signal. Reference scans
were taken every 10 s. Source parameters were optimized
using solutions of CQA. The capillary voltage was 2.1 kV,
the cone voltage was 20 V, the source temperature was
100 °C, the desolvation gas flow was 500 l/min, and the
desolvation temperature was 350 °C. For ion mobility
experiments, the helium cell gas flow was 180 mL/min,
the IMS gas flow was 90 mL/min, the IMS wave velocity
was 650 m/s, and the IMS wave height was 35 V. Nitrogen
was used as carrier buffer gas. The scan time was 1 s with
an interscan time of 0.024 s.
For collision-induced dissociation (CID) tandem mass
spectrometry, a collision energy ramp was applied. For CID
in the trapping cell (prior to mobility separation), the collision
energy ramp was 15–30 eV for CQAs and 20–30 eV for
methylated CQAs. The collision energy was ramped from
10 to 20 eV for CID experiments in the transfer cell (post-
mobility separation).
allow for
a
satisfactory rationale of fragmentation
mechanisms.[20] Furthermore, ion yields in tandem mass
spectra can be unusually low, as well, suggesting that
fragmentation may occur from less populated prototropic
isomers.[21] In this contribution we report on the use of ion
mobility mass spectrometry to distinguish between different
regioisomers of caffeoyl and dicaffeoyl quinic acids
providing for the first time data on the differentiation of
multiple isomers (more than two) based on their mobility
drift time and additionally show for the first time the ability
of IM-MS to differentiate between prototropic forms of
individual isomers, giving evidence for their existence and
relevance in gas-phase chemistry.
Data were obtained and processed using Waters MassLynx
4.1 SCN 870 and DriftScope 2.1 software. Collisional cross
sections were obtained after geometry optimization using
Gaussian 09 using MOBCAL software.
EXPERIMENTAL
Chemicals
All the chemicals (analytical grade) were purchased from
Sigma-Aldrich (Bremen, Germany). All caffeoyl quinic acids
(CQAs) were purchased from Phytolab (Vestenbergsgreuth,
Germany) and their purity checked by LC/MS as previously
described.[2,3] The mixture of cis/trans-5-CQA was obtained
as previously described.[26]
UPLC/IM-MS conditions
Chromatographic separation was achieved using an
ACQUITY ultra-performance liquid chromatography (UPLC)
system (Waters) equipped with a ACQUITY BEH C18 column
(length 100 mm, diameter 2.1 mm, particle size 1.7 μm) with a
Figure 1. Structures of caffeoyl and dicaffeoyl quinic acids 1–7.
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Rapid Commun. Mass Spectrom. 2015, 29, 675–680

IM-MS and prototropism of chlorogenic acids
Table 1. MS and IM-MS data of compounds 1–7 and 11, 12 (for IM-MS spectra, see Supplementary Figs. S4 and S5, Supporting
Information)
Drift time fragment ions
Drift time
[ms]
from MS2 [ms] (m/z
Compound
Formula
C25H24O12
C25H24O12
C25H24O12
m/z value
515.1
fragment ion in brackets)
1 (3,4-diCQA)
2 (3,5-diCQA)
3 (4,5-diCQA)
6.23
3.95 (353, 4-CQA); 2.39 (191);
2.13 (161); 1.90 (135)
515.1
6.04
6.16
3.9 (3-CQA), 4.56 (353); 2.39
(191); 2.13 (161); 1.90 (135)
3.88 (4-CQA), 4.49 (353); 2.39
(191); 2.13 (161); 1.90 (135)
2.39 (191); 2.13 (161); 1.90 (135)
2.39 (353); 2.31 (173); 2.13 (161);
1.90 (135)
515.1
4 (3-CQA)
5 (4-CQA)
C16H18O9
C16H18O9
353.1
353.1
3.95
3.99, 4.60
6 (5-CQA)
C16H18O9
C16H18O9
C17H20O9
C18H22O9
353.1
353.1
367.1
381.1
3.91, 4.52
3.83, 4.48
2.39 (191); 2.13 (161); 1.90 (135)
2.39 (191); 2.13 (161); 1.90 (135)
7 (cis-5-CQA)
11 (5-CQA methyl ester)
12 (5-diOMeCQA)
4.22, 5.02 (100%)
4.67, 5.99 (100%)
linear gradient elution employing water (solvent A) and
acetonitrile (MeCN, solvent B) both fortified with 0.1% acetic
acid as follows: 0–3 min, 2% B; 10 min, 20% B; 30–35 min,
100% B; 36–45 min, 2% B. The column temperature was set
at 35 °C, the flow rate was 0.3 mL/min and the injection
volume was 1 μL.
Purified reference samples of 3,4-diCQA, 3,5-diCQA and
4,5-diCQA 1–3 were subjected to typical IM-MS conditions.
All three regioisomeric compounds showed a single peak in
the LC/MS run in the negative ion mode and a single peak
each in their ion mobilograms (not shown). All three
regioisomers displayed distinct drift time values confirming
the ability of ion mobility to distinguish between regioisomeric
natural products if chromatographically separated. Drift times
are respectively 6.23, 6.04, and 6.16 ms. After fragmentation of
the three diCQA regioisomers, an extracted ion mobilogram of
the main fragment ion at m/z 353 was obtained and was
compared with drift times of regioisomeric CQA derivatives,
discussed in the following section. By doing so, the
regiochemistry of the most abundant isobaric fragment ions
can be unambiguously established within the experimental
error of the IM-MS experiment. For example, the main
fragment ion of 3,4-diCQA 1 results from loss of a caffeoyl
moiety from the 3-position yielding a 4-CQA fragment ion;
3,5-diCQA 2 loses the caffeoyl moiety from the 5-position
yielding a 4-CQA fragment ion and 4,5-diCQA 3 loses a
caffeoyl moiety from the 5-position yielding a 4-CQA fragment
ion. This confirms our previous hypothesis that the ease of loss
of the caffeoyl moiety follows the order 1 > 5 > 3 > 4.[2,3] The
drift time of all further fragment ions at m/z 191, 173, 161 and
135 could also be recorded and are given in Table 1.
Synthetic procedures
Syntheses of 5-O-(3,4-dimethoxy) cinnamoylquinic acid 4 and
methyl 5-O-caffeoylquinate 8 including all spectroscopic data
are provided in the Supporting Information.
RESULTS AND DISCUSSION
Caffeoyl and dicaffeoyl quinic acids are ubiquitous natural
products that occur in many dietary plants and are consumed
by humans at an estimated level of 1–2 g per day, with coffee
being the richest source of these important natural
products.[22] These compounds exist in nature in the form of
a mixture of regioisomers with three isomers of caffeoyl
quinic acid and six isomers of dicaffeoyl quinic acid, all found
in green coffee beans.[3] Many other dietary sources contain
the same compounds including maté, plum, stevia, etc.[23–25]
All isomers can be readily distinguished based on their
elution profile in reversed-phase HPLC/MS and their
structure can be unambiguously elucidated using tandem
mass spectrometry.[2,3] For rationalization of fragmentation
mechanisms, we have suggested the existence of
prototropic forms of caffeoyl quinic acid anions in the gas
phase.[3] To add a second MS-based method for isomer
distinction and probe the presence of prototropic isomers,
we decided to investigate the behavior of these compounds
by IM-MS.
While the LC/MS runs in the negative ion mode of the
caffeoyl quinic acids 3-CQA, 4-CQA and 5-CQA 4–6 showed
a single chromatographic peak (not shown), in the ion
As model compounds for our investigation we chose the
three most abundant dicaffeoyl and monocaffeoyl quinic
acids 1–6 in coffee along with a green coffee extract and a
mixture of trans/cis-caffeoyl quinic acid isomers 6 and 7. All
chemical structures are given in Fig. 1.
Figure 2. Extracted ion mobilogram at m/z 353 showing two
distinct prototropic isomers of 5-caffeoyl quinic acid 6.
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N. Kuhnert et al.
Figure 3. Structure of prototropic isomers of 5-caffeoyl quinic acid as suggested
previously.[2,3]
mobilograms of 4-CQA 4 and 5-CQA 6 two peaks could be
observed, e.g. for 5-CQA at 3.91 and 4.52 ms, respectively,
shown in Fig. 2. Xie et al. reported the identical observation
on 5-CQA 6 and suggested that the two signals in the
mobilogram could be explained by the presence of trans-
and cis-caffeoyl quinic acids 6 and 7, which we have recently
tandem mass spectra are expected differing in intensities of
the fragment ions. Indeed, the tandem mass spectra of the
two isomers showed two distinct fragmentation patterns
differing in the intensity of their fragment ion at m/z 191.0578
(C7H11O6) compared to the precursor ion at m/z 353.0936
(Fig. 4). We propose that the spectrum showing the fragment
ion at higher relative abundance (Fig. 4(B)) corresponds to
prototropic isomer 10, because the caffeoyl moiety of this
isomer is easier cleaved due to hydrogen-bond activation of
the C–O single bond. This is in line with our previous
assumption that fragmentation of 5-CQA with loss of
162 amu (ÀC9H7O3) requires a negative charge on the phenolic
oxygen.[2,3] The fragmentation mechanism as suggested
previously[2] is shown in Fig. 5.
In order to further support this hypothesis we decided to
obtain 5-caffeoyl quinic acid methylester 11 and dimethyl
caffeoyl quinic acid 12 through chemical synthesis (Fig. 6,
see also Supporting Information). These two compounds
lack the potential for prototropic isomerism with compound
11 only able to furnish a phenolate ion and compound 12
only able to furnish a carboxylate ion. Consequently, for
each of the compounds a single drift time is expected.
The ion mobilograms of compounds 11 and 12 show single
high-abundance peaks at 5.02 and 5.66 ms, respectively,
corresponding to their respective pseudomolecular ions at
m/z 367 and 381 accompanied by minor peaks at 2% of the
intensity (data in Supplementary Figs. S2 and S3, see
Supporting Information). A second peak of high abundance
is absent, as found for the unmethylated derivative 5.
Methylated CQA derivatives 11 and 12 are unable to form
prototropic isomers. These data therefore support our
hypothesis that non-methylated 5-CQA indeed undergoes
shown to be
a product of photochemically induced
isomerisation or thermally induced isomerization in coffee
brewing.[26,27] However, when subjecting a sample containing
a 50:50 mixture of trans- and cis-5-CQA 7 to an UPLC/IM-MS
experiment, both geometric isomers could be readily
separated in the chromatographic dimension on reversed-
phase packing, yielding two signals in the ion mobilogram
each at non-identical drift times (see Supporting Information).
Accordingly, an alternative explanation for the observation of
two distinct drift times for caffeoyl quinic acid geometric
isomers is required. We propose that the two signals observed
should be assigned to two prototropic isomers of caffeoyl
quinic acid with the first isomer being a carboxylate anion 8
and the second isomer a phenolate anion 9 (Fig. 3).
To substantiate our hypothesis, we performed a CID MS2
experiment in which the individual isomers are fragmented
after ion mobility separation. If our assumption is correct,
prototropic isomerism forming structures
8 and 9. In
addition to photochemically or thermally induced
isomerization, Xie et al. also suggested that cis/trans
isomerization could be induced by the electrostatic fields in
the ion spray source during electrospray ionization.
However, if this would be correct, this cis/trans isomerization
Figure 4. Collision-induced dissociation tandem mass
spectra of two 5-caffeoyl quinic acid isomers separated by
ion mobility at drift times of (A) 3.91 ms and (B) 4.52 ms.
Figure 5. Mechanism of fragmentation of 5-CQA 6 as
suggested previously.[2]
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Rapid Commun. Mass Spectrom. 2015, 29, 675–680

IM-MS and prototropism of chlorogenic acids
Figure 6. Synthesis and structures of CQA derivatives 11–12 unable to display prototropic
isomerism.
would also occur with compounds 11 and 12 and 1–3,
leading to dual peaks in the ion mobilogram. However,
our data show only single peaks in the mobilograms of these
compounds, indicating that cis/trans isomerization is clearly
absent. Together, these data support our hypothesis that
prototropic isomerization occurs instead of cis/trans
isomerization. The ability of IM-MS to differentiate
prototropic isomers in CQA derivatives seems to be based
on the unusual fact that both prototropic isomers 8 and 10
exist in distinct conformations, one of them stabilized by
hydrogen bonding in an inverted cyclohexane chair, yielding
conformers of sufficiently distinct collisional cross sections.
This ability to stabilize an inverted chair conformation is
particularly pronounced for 5-CQA and completely absent
for 4-CQA.
Acknowledgements
The authors acknowledge funding from Unilever R & D
Bangalore and Jacobs University Bremen. We thank Mr K. I. Assaf
for assistance with CCS calculations and Ms A. Müller for
technical assistance.
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SUPPORTING INFORMATION
Additional supporting information may be found in the
online version of this article at the publisher's website.
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Copyright © 2015 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2015, 29, 675–680