Preferential alkali metal adduct formation by cis geometrical isomers of dicaffeoylquinic acids allows for efficient discrimination from their trans isomers during ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry

Article abstract of DOI:10.1002/rcm.7526

Rationale Caffeoylquinic acid (CQA) derivatives are a group of structurally diverse phytochemicals that have attracted attention due to their many health benefits. The structural diversity of these molecules is due in part to the presence of regio- A nd geometrical isomerism. This structural diversity hampers the accurate annotation of these molecules in plant extracts. Mass spectrometry (MS) is successfully used to differentiate between the different regioisomers of the CQA derivatives; however, the accurate discrimination of the geometrical isomers of these molecules has proven to be an elusive task. Methods UV-irradiated methanolic solutions of diCQA were analyzed using an ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/QTOFMS) method in negative ionisation mode. An in-source collision-induced dissociation (ISCID) method was optimized by varying both the capillary and cone voltages to achieve differential fragmentation patterns between UV-generated geometrical isomers of the diCQAs during MS analyses. Results Changes in the capillary voltage did not cause a significant difference to the fragmentation patterns of the four geometrical isomers, while changes in the cone voltage resulted in significant differences in the fragmentation patterns. The results also show, for the first time, the preferential formation of alkali metal (Li⁺, Na⁺ and K⁺) adducts by the cis geometrical isomers of diCQAs, compared to their trans counterparts. Conclusions Optimized QTOFMS-based methods may be used to differentiate the geometrical isomers of diCQAs. Finally, additives such as metal salts to induce adduct formation can be applied as an alternative method to differentiate closely related isomers which could have been difficult to differentiate under normal MS settings.

Full text of DOI:10.1002/rcm.7526

Research Article
Received: 28 September 2015
Revised: 21 January 2016
Accepted: 1 February 2016
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2016, 30, 1011–1018
(wileyonlinelibrary.com) DOI: 10.1002/rcm.7526
Preferential alkali metal adduct formation by cis geometrical
isomers of dicaffeoylquinic acids allows for efficient
discrimination from their trans isomers during ultra-high-
performance liquid chromatography/quadrupole time-of-flight
mass spectrometry
Mpho M. Makola1, Paul. A Steenkamp1,2, Ian A. Dubery1, Mwadham M. Kabanda3 and
Ntakadzeni E. Madala1
*
1Department of Biochemistry, University of Johannesburg, P.O. Box 524, Auckland Park 2006, South Africa
2CSIR Biosciences, Natural Products and Agroprocessing Group, Pretoria 0001, South Africa
3Department of Chemistry, North-West University (Mafikeng Campus), Private Bag x2046, Mmabatho 2735, South Africa
RATIONALE: Caffeoylquinic acid (CQA) derivatives are a group of structurally diverse phytochemicals that have attracted
attention due to their many health benefits. The structural diversity of these molecules is due in part to the presence of regio-
and geometrical isomerism. This structural diversity hampers the accurate annotation of these molecules in plant extracts.
Mass spectrometry (MS) is successfully used to differentiate between the different regioisomers of the CQA derivatives;
however, the accurate discrimination of the geometrical isomers of these molecules has proven to be an elusive task.
METHODS: UV-irradiated methanolic solutions of diCQA were analyzed using an ultra-high-performance liquid
chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/QTOFMS) method in negative ionisation mode.
An in-source collision-induced dissociation (ISCID) method was optimized by varying both the capillary and cone voltages
to achieve differential fragmentation patterns between UV-generated geometrical isomers of the diCQAs during MS analyses.
RESULTS: Changes in the capillary voltage did not cause a significant difference to the fragmentation patterns of the four
geometrical isomers, while changes in the cone voltage resulted in significant differences in the fragmentation patterns.
The results also show, for the first time, the preferential formation of alkali metal (Li+, Na+ and K+) adducts by the cis
geometrical isomers of diCQAs, compared to their trans counterparts.
CONCLUSIONS: Optimized QTOFMS-based methods may be used to differentiate the geometrical isomers of diCQAs.
Finally, additives such as metal salts to induce adduct formation can be applied as an alternative method to differentiate
closely related isomers which could have been difficult to differentiate under normal MS settings. Copyright © 2016 John
Wiley & Sons, Ltd.
Caffeoylquinic acid (CQA) derivatives, members of the
chlorogenic acid (CGA) family of plant polyphenolics, are
widely occurring in plants and have been the subject of
interest for decades due to their assumed health benefits.[1,2]
These phytochemicals are amongst the most ubiquitous
polyphenolic compounds in plants and are constituents
of most food and medicinal plants.[1,3,4] The interest in
these compounds lies in their health benefits which include
anti-oxidant,[5] anti-HIV1[6] and anti-inflammatory[7] activities.
The cis-trans isomerization of these molecules as a result of
ultraviolet (UV) irradiation of natural plant material and
authentic standards has been reported elsewhere.[8–10] These
cis-trans photo-isomerization reactions combined with the
natural occurrence of positional (regio-) isomers add to the
structural diversity of these phytochemicals and it is this
diversity that poses an undisputed analytical challenge that
hampers the accurate identification and quantification of
these compounds in plant extracts.[8,10] The development of
accurate techniques for the annotation of these molecules is,
therefore, of importance due to the growth in the field of
ethno-pharmacology where various teas and herbal extracts
are marketed for their health benefits which are dependent
on the presence and abundance of molecules such as CQA
derivatives.[11]
Analytical techniques such as nuclear magnetic resonance
(NMR) spectroscopy have been used to distinguish between
various regio- and geometrical (cis-trans) isomers of some
natural products.[12] However, challenges due to signal
overlapping and inadequate sensitivity associated with this
technique have led to the wider application of mass
spectrometric analyses in plant studies.[13–15] Mass
spectrometry (MS) is not only selective and sensitive, but
* Correspondence to: N. E. Madala, Department of
Biochemistry, University of Johannesburg, P.O. Box 524,
Auckland Park 2006, South Africa.
E-mail: emadala@uj.ac.za
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M. M. Makola et al.
can also be interfaced with gas chromatography (GC)[16] and
liquid chromatography (LC)[17] separation instruments with
relative ease. Liquid chromatography coupled with mass
spectrometry (LC/MS) has been the method of choice for
the analyses of plant extracts, especially CGA analyses.[3,18,19]
In the analyses of CQA and other CGAs, ion-trap mass
spectrometry (IT-MS) has been successfully used to
discriminate between the different classes of CGAs, resulting
in the development of hierarchical keys that can be used to
unambiguously distinguish between the various regioisomers
thereof.[3,8,18–20] However, IT-MS-based techniques have not
been able to distinguish between the geometrical isomers of
the mono- and di-CQA.[8,20] Increasingly, other MS methods
such as quadrupole time-of-flight (QTOF)MS are being used
for the analyses of plant extracts.[9,13,21] Recently, Madala
et al.[22] developed and optimized an in-source collision-
induced dissociation (ISCID) ultra-high-performance liquid
chromatography/quadrupole time-of-flight mass spectrometry
(UHPLC/QTOFMS)-based method to distinguish between the
cis and trans isomers of 4-acylated CGAs in three Momordica
plant species. This ability of ISCID-based QTOFMS methods
over the multistage fragmentation approach on the IT-MS
platform to distinguish between the cis and trans isomers of 4-
CQA may possibly be due to the fact that unlike IT-MS which
operates tandem mass spectrometry (MS/MS) fragmentation
solely in time, QTOFMS and other hybrid MS instruments
operate MS/MS fragmentation in time and space.[23] Moreover,
QTOFMS methods give a holistic view of the fragmentation
patterns of a compound as opposed to IT-MS which excludes
other ions and focuses on pre-selected ions in MSn methods.
In the current study, various QTOFMS settings (capillary and
cone voltage) were optimized to induce any underlying
fragmentation patterns between UV-generated geometrical
isomers of 3,5-diCQA.
MA, USA). The mobile phase consisted of milli-Q water
containing 0.1% formic acid (FA) (eluent A) and acetonitrile
containing 0.1% FA (eluent B). The gradient elution
and separation were achieved at a constant flow rate of
0.6 mL/min as follows: 15% B was kept constant for14 min
followed by a rapid gradient of 15–100% B over 1 min, and
kept at 100% B for 2 min and another gradient of 100–15% B
over 1 min and a final equilibration step with 15% B was
carried out for 2 min. For the alkali metal studies, a 10 mM
aqueous solution of alkali metal chloride salts was infused
into the main eluent stream through the on-board syringe
drive at a constant flow rate of 2 μL/min before entering the
ion source of the mass spectrometer. For MS detection, a
SYNAPT G1 QTOF mass spectrometer (Waters Corp.) was
used. The mass spectrometer was operated in V-optics mode
and all the analyses were carried out in negative electrospray
ionization (ESI) mode utilizing the following conditions:
extraction cone voltage, 4 V; source temperature, 120 °C;
desolvation temperature, 450 °C; cone gas flow rate, 50 L/h;
desolvation gas flow rate, 550 L/h. The mass range for
detection was from m/z 50 to 1000 with a scan time of 0.1 s
and an inter-scan time of 0.02 s. For ISCID, sampling cone
voltages of 10, 25, 40 and 60 V were used and the capillary
voltages were 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5 kV. Further
fragmentation of these isomers was achieved by ramping the
collision energy levels from 3 to 40 eV.
Statistical analysis
For statistical analyses of the initial optimization of ESI-MS
conditions needed for efficient discrimination between the
geometrical isomers of 3,5-diCQA, Statistica 12 software
(Dell, USA) was used. Here, responsive surface models
(RSM) and Pareto charts were computed and used to show
the statistical significance of the tested MS parameters,
namely cone and capillary voltages.
EXPERIMENTAL
RESULTS AND DISCUSSION
Chemicals
Authentic standards of trans-mono-caffeoylquinic acids (3-, 4-
and 5-CQA) and dicaffeoylquinic acids (1,3-diCQA, 1,5-
diCQA, 3,4-diCQA, 3,5-diCQA and 4,5-diCQA) were
purchased from Phytolab (Vestenbergsgreuth, Germany).
Analytical grade methanol and acetonitrile were obtained
from Romil (Cambridge, UK). Deionized milli-Q water was
generated using a Milli-Q Gradient A10 system (Millipore,
MA, USA). All other chemicals, such as formic acid (FA)
and leucine enkaphalin, were purchased from Sigma-Aldrich
(St. Louis, MO, USA).
Currently, the tentative identification of geometrical isomers
of CGAs is achieved through the use of chromatographic
retention profiles on reversed-phase (RP) columns.[8] Though
a useful method for CGA characterization, inconsistencies
in the elution order of the geometrical isomers have resulted
from the lack of standardized chromatography and
experimental conditions. Work done elsewhere, where
CQA samples were irradiated at 366 nm[24] instead of the
short wavelength of 245 nm normally used, showed
differences in the chromatographic elution profile from
what has been previously reported.[8,20] As such, an ISCID
UHPLC/QTOFMS method was optimized to discriminate
between the geometrical isomers of diCQA. ISCID allows
for CID to occur in the ion source of the mass spectrometer
to give similar fragmentation patterns to those obtained with
MS/MS methods without the need for pre-selection of
desired ions.[25,26] Initial chromatographic conditions were
optimized to result in efficient separation of the four
geometrical isomers of 3,5-diCQA (Scheme 1, Fig. 1). It should
be mentioned that other diCQA regioisomers (1,3-diCQA,
1,5-diCQA, 3,4-diCQA and 4,5-diCQA) were also studied
and they were all shown to result in a similar trend. For
Sample preparation
To obtain the cis isomers of the caffeoylquinic and
dicaffeoylquinic acids a 1 mg/mL stock solution was
prepared in 100% methanol. The samples were then irradiated
for 5 h at 245 nm using a UV box (Spectroline, USA).
UHPLC/ESI-QTOFMS analyses
During chromatographic separation, 3.0 μL of the sample was
injected and separated using an HSS T3 C18 reversed-phase
column (150 mm × 2.1 mm, 1.8 μm; Waters Corporation,
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Sodium adducts of cis geometrical isomers of dicaffeoylquinic acids
Scheme 1. Structures of the dicaffeoylquinic acid regioisomers.
consistency, most of the results presented here were achieved
with 3,5-diCQA isomers. The results for the other
regioisomers are also discussed throughout the manuscript
with most of the results in the Supporting Information.
UV-irradiated samples of 3,5-diCQA were subjected to
varying conditions of cone voltage and capillary voltage in
order to induce differential MS fragmentation patterns. Only
the optimized conditions, i.e. MS conditions that best showed
the differences in alkali metal adduct formation between the
geometrical isomers, were used for the analyses of other
regioisomers of diCQA. To distinguish between the cis and
trans isomers, peaks that increased in abundance/intensity
as a result of UV irradiation compared to the non-irradiated
control sample (which contains only the all-trans isomer),
were assigned as cis isomer peaks (Fig. 1; Supplementary
Figs. S1–S4, see Supporting Information). To distinguish
Rapid Commun. Mass Spectrom. 2016, 30, 1011–1018
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M. M. Makola et al.
molecules with acylation on the 5’ position of the quinic acid
such as 1,5-diCQA and 4,5-diCQA. One of the mono cis isomers
of these diCQA molecules (1,5-diCQA and 4,5-diCQA) elutes
later than its trans counterpart (Supplementary Figs. S2 and
S4), suggesting that the same phenomenon of cis isomerism at
the 5’ position of quinic acid may also play a role in increasing
the hydrophobicity of these mono-cis diCQAs.
During IT-MS, 3,5-diCQA (with [M–H]– of m/z 515)
consistently produces diagnostic markers at m/z 353
[M–H–162]– due to the loss of one caffeoyl moiety which
produces a base peak ion at the MS2 level. At the MS3 level,
a base peak at m/z 191[quinic acid–H]– and a secondary peak
at m/z 179 [caffeic acid–H]– with a consistent ratio of
approximately 2:1 are produced.[8,20] Similarly, in the current
study, the same ions were observed for all four geometrical
isomers using our optimized QTOFMS method (Figs. 2 and
3; Supplementary Fig. S5). Optimization of the in-source
QTOFMS conditions by changing the capillary and cone
voltages was carried out, and, interestingly, there were
minimal fragmentation differences between the isomers due
to changes in the capillary voltage but significant differences
were noted due to changes in the cone voltage
(Supplementary Figs. S6 and S7). From the results, it was
observed that at a capillary voltage of 3.5 kVand cone voltage
of 60 V, the three cis isomers (Fig. 1; peaks I, III and IV) had an
intense peak at m/z 375.065 [M–2H–162+Na]– as compared to
the trans isomer which showed an intense peak at m/z 353.082
[M–H–162]– due to the loss of one caffeic acid moiety (Fig. 2).
For the trans isomer the peak at m/z 353.082 [M–H–162]–
was much more intense than that of the sodium adduct at
m/z 375.065.[28] Between the cis isomers, the cis-cis
isomer was observed to form more of the sodium adduct
(m/z 375.065) relative to the deprotonated caffeoylquinic acid
fragment (m/z 353.082) than the other two mono-cis isomers
Figure 1. A representative UHPLC/QTOFMS base peak
intensity (BPI) chromatogram showing the separation of
different geometrical isomers of 3,5-diCQA generated
during UV exposure. Peak I: di-cis isomer, peak II: di-trans,
peak III: mono-cis 1 and peak IV: mono-cis 2.
between the generated cis isomers of 3,5-diCQA, peak I
(Fig. 1), which was produced in minor concentration, was
designated as the cis-cis (di-cis) isomer since it has also been
reported to be produced in trace amounts.[8,10] Furthermore,
the other two peaks, III and IV (Fig. 1), were designated as
mono-cis 1 (3cis,5trans-diCQA) and mono cis 2 (3trans,5cis
-
diCQA). The two mono-cis isomers, however, could not be
precisely differentiated as they were produced in relatively
equal abundance, contrary to the 1:4 ratio that was reported
elsewhere.[10] However, an inference can be made based on
the available information on the hydrophobicities of the
mono-acyl CGAs. It has previously been shown that the
cis isomers formed due to UV radiation change the
hydrophobicity of the mono-acylated CGAs significantly.[8]
For instance, an increase in hydrophobicity of cis-5-monoacyl
CGAs has been noted and, as such, the cis geometrical
isomers of 5-monoacyl CGAs elute later than their trans
counterparts in RP chromatography.[8,27] However, an
opposite case is observed for 3- and 4-monoacyl CGAs where
the cis isomers elute earlier than their trans counterparts.[8,27]
The increase or decrease in hydrophobicity of these
compounds has been linked to their ability or inability to
form intramolecular hydrogen bonds (IHB).[8] Therefore, if
an assumption is made that the hydrophobicity effects seen in
the mono-acyl CGAs are additive, then we would expect the
mono-cis isomer with the greater retention time to have the cis
configuration on the caffeoyl moiety acylated on the 5’ position
of the quinic acid, thus the 3trans,5cis-diCQA. Interestingly, our
preliminary quantum theoretical calculations using the
B3LYP/6-31G(d, p) method (both in methanol and in vacuo)
indicated the 3trans,5cis-diCQA mono cis isomer to be slightly
more energetically favourable than the 3cis,5trans-diCQA mono
cis isomer, due to stronger intramolecular hydrogen bonds
found in the former isomer. Furthermore, the 3trans,5cis-diCQA
mono cis isomer was also found to have a smaller dipole
moment than all the geometrical isomers of 3,5-diCQA,
suggesting that it is more hydrophobic, which could be the
reason for its late elution compared to the other geometrical
isomers (peak IV, Fig. 1). By extension, the same phenomenon
can also be supported by the elution profiles of other diCQA
Figure 2. Representative QTOFMS spectra for (from top
to bottom): di-trans, mono-cis 1, mono-cis 2, and di-cis
geometrical isomers of 3,5-diCQA with the differentiating
diagnostic sodium adduct [M–2H–162+Na]– peak seen at
m/z 375.066.
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Sodium adducts of cis geometrical isomers of dicaffeoylquinic acids
spiked with other alkali metal ions (lithium and potassium)
were also investigated for all the diCQA regioisomers. When
the chromatographic mobile phase was spiked with lithium
(Li+), similar results to those observed with Na+ were
achieved; here the cis-cis isomer of 3,5-diCQA (Fig. 3) and of
4,5-diCQA (Fig. 4) which were found to bind preferentially
to the Li+ ion compared to the trans-trans isomer as seen with
the peak at m/z 359.091 [M–2H–162+Li]–. The other two
mono-cis isomers also showed preference for the Li+ ion, with
the 3trans,5cis mono-cis isomer (peak IV, Figs. 1 and 3) showing
slightly greater affinity than the 3cis,5trans mono-cis isomer
(peak III, Figs. 1 and 3). The same phenomenon was
also observed with other positional isomers (Supplementary
Figs. S12–S14). The preferential metal adduct formation by
the cis geometrical isomers of the various regioisomers can
also be seen with the peaks of unfragmented adduct ions
([M–2H+Metal ion]–) (Figs. 2–5; Supplementary Figs. S5 and
S8–S17). Closer visual inspection of the mass spectra of the
geometrical isomers also shows another noteworthy trend
where detection of two independent MS occurrences seems
to exist in one spectrum. Here, the deprotonated molecule
ions at m/z 515 [M–H]– fragment to give a product ion at
m/z 353 [M–H–162]– whilst, on the other hand, the adduct
ions [M–2H+Na]– at m/z 537, [M–2H+Li]– at m/z 521 and
[M–2H+K]– at m/z 553 give rise to product ions at m/z 375,
359 and 391, respectively. As seen throughout the manuscript
thus far, the peak intensity ratio of the adducted precursor ion
after losing one caffeoyl moiety ([M–2H–162+Metal ion]–)
over the intensity of the non-adducted precursor ion after
losing one caffeoyl moiety ([M–H–162]–) was used to assign
metal preference for the cis isomers over their trans isomer
counterparts. Our results further show other higher order
adduct ions which also clearly show the adduct formation
preference of the cis isomers over their trans counterparts.
Figure 3. Representative QTOFMS spectra for (from top
to bottom): di-trans, mono-cis 1, mono-cis 2, and di-cis
geometrical isomers of 3,5-diCQA with the differentiating
diagnostic lithium adduct [M–2H–162+Li]– peak seen at m/z
359.090.
(Fig. 2). Comparing the two mono-cis isomers it was found that
one of them, 3trans,5cis-diCQA (Fig. 2(B)), displayed a higher
tendency to form the sodium adduct than 3cis,5trans-diCQA
(Fig. 2(C)), even though the difference is very minimal.
Our preliminary tandem mass spectrometry (MS2) results
on the deprotonated sodium adduct [M–2H+Na]– did not
show any differences between the isomers. As mentioned
above, the differences between the geometrical isomers
were more pronounced at elevated cone and capillary
voltages (Supplementary Fig. S6); however, changes in cone
voltages were found to have a more significant effect than
changes in capillary voltage (Supplementary Fig. S7). This
could be attributed to the fact that changes in the MS cone
voltage causes an increase in the internal energy of the
molecular ions which induces greater fragmentation while
the capillary voltage is concerned with the production of
the ion plume in front of the sample cone.[27,28] By
selecting/trapping a specific ion for further fragmentation,
the nuances are lost and fragmentation patterns are the
same, whereas when all ions are allowed to reach the
detector, a more holistic view is achieved. Analyses in space
allow ’real-time’ analysis since a continuous beam of ions
reaches the detector without filtering others (such as the
metal adduct in this case).[23,29,30] As such, our ISCID
method can therefore be regarded as superior in showing
the underlying differences between the geometrical isomers
of diCQA, a phenomenon that has been deemed impossible
hitherto.
To further confirm these metal-binding preferences by the
cis isomers, and to eliminate any possibility that our
observations are limited to one regioisomer (3,5-diCQA in
this case), we also investigated other diCQA regioisomers
(1,3-diCQA, 1,5-diCQA, 3,4-diCQA and 4,5-diCQA)
(Supplementary Figs. S8–S11). Furthermore, mobile phases
Figure 4. Representative QTOFMS spectra for (from top
to bottom): di-trans, mono-cis 1, mono-cis 2, and di-cis
geometrical isomers of 4,5-diCQA with the differentiating
diagnostic lithium adduct [M–2H–162+Li]– peak seen at m/z
359.088.
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M. M. Makola et al.
on the flavonoid. The metal binding of catechol-containing
molecules has also been known for a long time with serious
biological and chemical implications.[34] In MS studies, the
metal binding/chelation by catechol-containing flavonoids
has been noted and used to discriminate between closely
related molecules.[35] In the current study, the molecules
under investigation also possess catechol units (Scheme 1)
and this is believed to play a role in the chelation of the alkali
metals. In the past, depending on the metals used, different
stoichiometric ratios between the ligands and alkali metals
have also been noted.[36] From a close visual inspection of
the different geometrical isomers of diCQA, it can be seen
that the catechol unit of these isomers is very similar and, as
such, the differences in metal preference are not obvious.
However, our preliminary quantum theoretical calculations
using the B3LYP/6-31G(d,p) method have indicated more
stable adducts formed by cis isomers of 3,5-diCQA owing to
multiple coordinating groups around the alkali metal due to
a bent configuration which is absent in the trans configuration
(results not shown). Therefore, the cis isomers are expected to
retain the metals even at elevated cone and collision energies
before and after fragmentation.
From our findings, it is evident that the consistent alkali
metal adduct preference by cis geometrical isomers of the
diCQA regioisomers, in conjunction with ISCID-QTOFMS,
may be used as a means of differentiating these molecules
from one another, a phenomenon which has been deemed
impossible with multi-stage IT-MS-based approaches.[8,10]
The current results can be expanded by investigating
other metal adducts of higher orders as they are expected to
result in ions of different intensities which can further be
exploited for the discrimination of these geometrical isomers.
The use of other forms of additives or chemical modifications
other than metals has recently been applied for proper
annotation of diCQAs elsewhere.[36] Our results are expected
to further buttress the already established method of
discriminating between these isomers which, to date, is based
only on the chromatographic elution profile.[8] This MS-based
method allows for the annotation of these complex molecules
in the case where different chromatographic conditions
are applied.
Figure 5. Representative QTOFMS spectra for (from top
to bottom): di-trans, mono-cis 1, mono-cis 2, and di-cis
geometrical isomers of 3,4-diCQA with the differentiating
diagnostic potassium adduct [M–3H+2K]– peak seen at m/z
591.028.
Here for instance, when considering 4,5-diCQA, its cis
isomers can be discriminated from their trans counterparts
by looking at the intensities of the double deprotonated and
triple deprotonated metal adducts ions containing one or
two metal ions, respectively (Fig. 4). Furthermore, the
existence of these higher order adducts such as [M–4H+3Li]–
at m/z 533.142 and [M–3H+2K]– at m/z 591.027 (Figs. 4 and
5) can be exploited in future using more directed MS/MS or
MSn for alternative methods which can be used for definite
identification of geometrical isomers of these phenolics and
other related compounds.
Studies done on the mono-acyl CQAs showed a similar but
less pronounced trend as seen for the diCQAs (Supplementary
Figs. S18–S20), suggesting possible differences in the chelation
ability or coordination mechanism between the two structural
hierarchies of the CQAs, with the diCQA being superior. Here,
our results show that the cis isomers of the mono-acyl CQAs
formed the sodium adduct (m/z 375.066) more than their trans
counterparts. Moreover, amongst the mono-acyl CQAs, the
5-CQA regioisomer had a greater tendency to form the sodium
adduct than the 3- and 4-CQA regioisomers. This further
highlights the differences in alkali metal binding preferences to
be more pronounced on the cis-geometrical isomers of CQAs
regardless of the structural hierarchy (mono- and di-acylated
CQA in this case). However, as previously stated, to achieve
these underlying differences, MS optimization should be carried
out by either varying cone voltage or capillary voltage as
indicated herein.
From our results, it is evident that more research in the
field of phytochemical analysis is needed, especially for
the characterization of plant metabolites with health benefits.
The biological activity of such metabolites depends not
only on their functional groups, but also on the spatial
structural arrangement of these groups.[37] Thus, isomerization
of biologically active metabolites may affect their potency.
For example, Chen et al.[38] found that the photo-isomerization
of trans-cinnamic acid to cis-cinnamic acid affected its
anti-mycobacterial activity. Therefore, the accurate distinction
of such isomers using analytical techniques such as MS is very
important for quality control purposes.
CONCLUSIONS
The use of metal additives has previously been shown to be
a valuable tool for MS-based studies of isomeric and isobaric
compounds.[31–33] Zhang and Brodbelt,[31] using silver ions as
an additive, were able to not only distinguish between
various isomers of flavonoid glycosides, but were able to
obtain structural information such as the site of glycosylation
An ISCID-QTOFMS-based method has been developed for
the discrimination of geometrical isomers of diCQAs. The
results of our study have demonstrated, for the first time,
recognisable differences in fragmentation patterns of
geometrical isomers of CGA compounds. Here, cis
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Sodium adducts of cis geometrical isomers of dicaffeoylquinic acids
[8] M. N. Clifford, J. Kirkpatrick, N. Kuhnert, H. Roozendaal,
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geometrical isomers of the diCQAs were shown to have
greater affinity for alkali metals (Na, Li and K cations)
compared to their trans counterparts during MS analyses in
negative ionization mode. The observation of this same trend
in the mono-acyl CQAs shows that this preferential alkali
metal binding of the cis isomers is not limited by structural
hierarchy or to specific regioisomers. This also suggests that
other molecules in the CGA class of compounds may exhibit
the same trend. Under well-optimized conditions, an
interesting trend between the cis isomers was noted, where
some molecules had greater affinity for the alkali metal ions
in comparison to the other cis isomers. Our results, therefore,
encourage the use of different MS technologies to achieve a
holistic view of the mechanistic fragmentation chemistry
which can further be exploited for efficient and precise
metabolite identification purposes. To achieve the above,
other adduct ions of higher orders can be studied using more
directed tandem mass spectrometry methods (MS/MS or
MSn) in combination with quantum theoretical calculations.
Thus our results encourage the advancement of the well-
known hierarchical key methods of CGA annotations to
include the annotation of geometrical isomers thereof, based
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Acknowledgements
The University of Johannesburg and the South African
National Research Foundation (NRF) are thanked for
fellowship support (MM) and financial support (NEM, Grant
No. TTK1306111889).
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SUPPORTING INFORMATION
[32] A. M. Cirigliano, G. M. Cabrera. Differentiation of
Additional supporting information may be found in the
cyclosporin
A
from isocyclosporin
A
by liquid
chromatography/electrospray ionization mass spec-
online version of this article at the publisher’s website.
wileyonlinelibrary.com/journal/rcm
Copyright © 2016 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2016, 30, 1011–1018