Talanta 219 (2020) 121306

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Talanta
journal homepage: www.elsevier.com/locate/talanta

High-performance thin-layer chromatography hyphenated to

high-performance liquid chromatography-diode array detection-mass
spectrometry for characterization of coeluting isomers
Agnes
� �ricz a, *, Vira
M. Mo �g Lapat a, Gertrud E. Morlock b, P�eter G. Ott a
a
Plant Protection Institute, Centre for Agricultural Research, Herman O. Str. 15, 1022, Budapest, Hungary
b
Chair of Food Science, Institute of Nutritional Science, and TransMIT, Center of Effect-Directed Analysis, Justus Liebig University Giessen, Heinrich-Buff-Ring 26-32,
35392, Giessen, Germany

A R T I C L E I N F O A B S T R A C T

Keywords: The effect-directed analysis on a planar chromatogram allows for fast non-target screening, multi-imaging
Two-dimensional liquid chromatography (2D detection of effects (bioprofiling) and highly targeted characterization and isolation of bioactive compounds.
LC) For direct characterization by high-resolution mass spectrometry (HRMS), however, the orthogonal hyphenation
Bioassay
of two different liquid chromatographic techniques (planar and column chromatography) is still underexplored.
Hyphenated technique
Antibiotics
In particular, it can be helpful in case of coeluting compounds. Exemplarily, lemon balm (Melissa officinalis L.)
Traditional medicine leaf extract was analysed by high-performance thin-layer chromatography in combination with bioactivity assays
Triterpenoids for antibacterial (against the Gram-positive Bacillus subtilis and the Gram-negative Aliivibrio fischeri) and
α-glucosidase-inhibitory compounds (HPTLC-UV/Vis/FLD-EDA). High-resolution mass spectra of two bioactive
compound zones were directly recorded via an elution head-based interface. By HPTLC-HESI-HRMS, the com­
pound in zone a inhibited A. fischeri and was identified as linolenic acid, whereas the two closely related
constitutional isomers oleanolic acid and ursolic acid were present in zone b. This was proven by two-
dimensional liquid chromatography. Heart-cutting HPTLC-UV/Vis/FLD-HPLC-DAD-MS allowed the separation
of the two isomers and proved both to be present in the bioactive zone with ursolic acid at a much higher
abundance.

1. Introduction HPTLC-FT-Raman [10] spectroscopy. In situ workflows for recording of

Chromatography combined with bioactivity assays (effect-directed
analysis, EDA) is a powerful hyphenation to detect/discover drug can­
didates from natural extracts [1]. In contrast to column chromatog­
raphy, the planar chromatography, especially high-performance
thin-layer chromatography (HPTLC), has several advantages for EDA.
HPTLC-EDA is (1) highly affordable in the running costs, (2) needs less
sample preparation steps, (3) has capacity for fast high-throughput
screening, (4) excludes cross-contamination (plate is used once), (5) is
easily combined with bioassays (in situ in the adsorbent), (6) allows for
non-target multi-imaging of effects (bioprofiling) and (7) highly tar­
geted characterization and isolation of bioactive compounds [2,3].
Compounds present in the bioactive zones were characterized by
HPTLC-(HR)MS using elution head-based [4] or desorption-based in­
terfaces (e.g., DESI and DART [5–7]), HPTLC-FTIR [8,9] or
spectra within the adsorbent were used for HPTLC-FTIR [8],
HPTLC-FT-Raman [10] or desorption-based MS interfaces [5–7]. How­
ever, mainly off-line workflows have been applied. On the one hand, the
adsorbent region of interest was scraped off the glass carrier plate, the
target zones were off-line eluted, the solvent evaporated, the residues
dissolved in an appropriate solvent and further analysed. For example,
off-line NMR has been applied for identification [11] and even for
evaluation of coeluting compounds [12]. Or, off-line HPLC-MS/MS was
used to characterize bioactive zones [13,14]. On the other hand,
elution-head based interfaces allowed for the online elution and less
contamination-prone collection of target zones in vials. For example,
off-line NMR has been applied for quantification [15]. Or, off-line
HPLC-MS/MS was used to analyse target zones containing pesticides
used for fruits, vegetables and tea (planar solid phase extraction
clean-up) [16,17].

* Corresponding author.
E-mail address: moricz.agnes@agrar.mta.hu (A.M.
� M�
oricz).

https://doi.org/10.1016/j.talanta.2020.121306
Received 3 April 2020; Received in revised form 15 June 2020; Accepted 17 June 2020
Available online 30 June 2020
0039-9140/© 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
A.M.
� M�
oricz et al.
The direct online elution of compound zones into the mass spec­
trometer emerged in the last 15 years [18]. Latest progress is a fully
automated elution head-based interface, made possible by open-source
technologies [4]. The mere integration of an additional column after
the elution head-based interface, and thus, online HPTLC-HPLC-MS/MS
has rarely been exploited so far. In one example, the intense background
signals of a with caffeine solution impregnated HPTLC plate were
separated from the Sudan dye analytes in the 2nd orthogonal HPLC
dimension [19]. The resulting analyte mass spectra were free from
caffeine and plate background interferences. This successful
HPTLC-HPLC coupling has been presented at many conferences since
2008 [41]. In another example, cholinesterase inhibiting alkaloids were
investigated by TLC-HPLC-DAD-TOF MS [20]. The main limitation of
HPTLC-EDA is the low peak capacity (resolution), and especially in case
of questionable coeluting compounds, such an online HPTLC-HPLC-MS
configuration is helpful [40]. It would minimize the additional effort
to prove the coeluting compounds in a bioactive zone. So far, the zone
resolution has been improved by automated multiple development [21],
two-dimensional HPTLC separation [22,23], or forced-flow layer chro­
matographic techniques, like overpressured layer chromatography [6].
Lemon balm (Melissa officinalis L., Lamiaceae family) is one of the
oldest and most popular medicinal plants. It is native to the eastern
Mediterranean basin, but widely cultivated also in Europe and the US.
The traditionally used, aromatic dried leaves exert antiviral and anti­
spasmodic activity [24], and so far no adverse effect has been described.
Alcoholic leaf extracts exhibited antioxidant, antibacterial,
anti-cholinesterase, antiviral and antiproliferative effects that could be
attributed to volatile monoterpenes (e.g., citral and citronellal), flavo­
noids (e.g., luteolin, kaempferol and quercetin) and phenolic acids (e.g.,
caffeic, chlorogenic and rosmarinic acids) [25,26]. In particular, the
characterization of antibacterial compounds and α-glucosidase in­
hibitors of lemon balm leaf extract are demanded to improve the un­
derstanding and therapy of infectious diseases and diabetes or obesity,
respectively [27,28].
The aim of this study is to develop a hyphenated HPTLC-UV/Vis/
FLD-HPLC-DAD-ESI-MS workflow that could be helpful for compound
characterization in case of coeluting compounds. First, an HPTLC-UV/
Vis/FLD-EDA screening was applied. The characterization and identifi­
cation of the most potent compounds followed by multi-imaging, heated
electrospray ionization high-resolution mass spectrometry (HESI-
HRMS) and hyphenated HPTLC-UV/Vis/FLD-HPLC-DAD-ESI-MS.
2. Experimental section
2.1. Materials
HPTLC plates silica gel 60 F254 were obtained from Merck Millipore
(Darmstadt, Germany). Oleanolic acid (�97%) and ursolic acid (�90%)
were purchased from Fluka (Buchs, Switzerland). Fast Blue Salt B (95%),
α-glucosidase solution (from Saccharomyces cerevisiae) were from Sigma-
Aldrich (Steinheim, Germany). 2-Naphthyl-α-D-glucopyranoside was
from Fluorochem (Karlsruhe, Germany). Acetonitrile (gradient grade)
was supplied by Fisher Scientific (Pittsburg, PA, USA). Other solvents
(analytical grade), formic acid and vanillin were delivered by Reanal
(Budapest, Hungary). Sodium acetate and 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) were purchased from Carl Roth
(Karlsruhe, Germany). The marine bacterium Aliivibrio fischeri (strain
DSM-7151) was bought from the Leibniz Institute DSMZ, German
Collection of Microorganisms and Cell Cultures (Berlin, Germany) and
the soil inhabitant Bacillus subtilis (strain F1276, gift from J�
ozsef Farkas,
Central Food Research Institute, Budapest, Hungary). Fully flowering
Melissa officinalis L. (lemon balm) were collected in Lenti, Hungary, in
July 2016.
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Talanta 219 (2020) 121306
2.2. Standard solutions and sample preparation
Oleanolic acid and ursolic acid were dissolved in methanol (1 mg/mL
each). Lemon balm leaves were dried at 25 � C in the dark and pulverized
in a coffee grinder (Bosch MKM6000, Stuttgart, Germany). The milled
leaves (100 mg) were macerated for 24 h with 1 mL methanol in a glass
vial. The supernatant was directly used for HPTLC.
2.3. HPTLC-UV/Vis/FLD-EDA
HPTLC plates were pre-washed by development with acetonitrile –
water (4:1, V/V) and dried for 30 min. The methanolic leaf extract (1–5
μL) and standards (1 and 2 μL) were applied onto HPTLC plates as 6-mm
bands with 8 mm distance from the bottom (ATS4 or ATS3, CAMAG,
Muttenz, Switzerland) and dried for 0.5 min (in a cold stream of air
using the hair-drier, if not stated otherwise). Chromatograms were
developed up to 7 cm in a Twin Trough Chamber (biostep, Bur­
khardtsdorf, Germany or CAMAG) with n-hexane – ethyl acetate 7:3, V/
V. After development the plate was dried for 2 min (cold stream of air)
and documented at UV 254 nm (UV), UV 366 nm (FLD) and after
derivatization with vanillin sulphuric acid reagent (40 mg vanillin, 10
mL ethanol and 200 μL concentrated sulphuric acid; heating at 110 � C
for 5 min) at white light illumination (Vis, transmittance mode, TLC
Visualizer, CAMAG or digital camera Cybershot DSC-HX60, Sony, Neu-
Isenburg, Germany).
Three additional chromatograms were prepared and the antibacte­
rial assays against B. subtilis [5] and A. fischeri [6] and the α-glucosidase
assay [29] were carried out as reported. Briefly, one chromatogram was
immersed into the B. subtilis suspension. After incubation (almost 100%
humidity, at 28 � C, for 2 h) the bioautogram was visualized by dipping
the layers into the aqueous MTT solution (1 mg/mL), followed by
another 30-min incubation until bright zones against a violet back­
ground were revealed. Another chromatogram was immersed into the
A. fischeri suspension and instantly monitored in real-time for 30 min
using 1 min exposure time in 5-min intervals (iBright™ FL1000 Imaging
System, Thermo Fisher Scientific, Budapest, Hungary). Dark (or bright)
zones against the bioluminescent background indicated active zones. A
third chromatogram was dipped into the α-glucosidase solution (10
units/mL in 0.1 M sodium acetate buffer adjusted to pH 7.5), incubated
(almost 100% humidity, at 37 � C, for 20 min), immersed into the sub­
strate solution (1.2 mg/mL 2-naphthyl-α-D-glucopyranoside in ethanol),
further incubated at room temperature for 10 min, visualized by im­
mersion into the aqueous Fast Blue Salt B solution (1 mg/mL) and dried
(hair-dryer, for 2 min). The enzyme inhibitors were indicated as bright
zones against a violet background documented at white light illumina­
tion (reflectance mode).
2.4. HPTLC-HESI-HRMS
Zones of interests were online eluted (MS-grade methanol at a flow
rate of 0.1 mL/min) using the oval elution head (4 mm � 2 mm) of the
PlateExpress (Advion, Ithaca, NY, USA) and a quaternary pump (Ulti­
mate LPG-3400 XRS, Dionex Softron, Germering, Germany) into the
HESI-Q Exactive Plus hybrid quadrupole-orbitrap mass spectrometer
(Thermo Fisher Scientific, Bremen, Germany). The spray voltage was
3.5 kV, capillary temperature 270 � C, and sheath and auxiliary nitrogen
gas 20 and 10 arbitrary units, respectively. A full scan was recorded in
the range of m/z 50–750 with a resolution of 280,000 in both negative
and positive ionization modes. The automatic gain control target was 3
� 106 and the maximum injection time 100 ms.
2.5. HPLC-DAD-MS and HPTLC-UV/Vis/FLD-HPLC-DAD-ESI-MS
The HPLC-DAD-ESI-MS system was expanded by installing a TLC-MS
Interface (CAMAG) with an oval elution head (4 mm � 2 mm) between
the pump and the column. With this hyphenation the compounds from
A.M.
� M�
oricz et al.
Fig. 1. HPTLC chromatograms (A–F) of lemon balm leaf extract (l) and the bioactive components ursolic acid (u), oleanolic acid (o), and the mixture of the acids (u/
o) developed with n-hexane – ethyl acetate 7:3 (V/V), documented at UV 254 nm (A), 365 nm (B) and white light illumination after derivatization with vanillin
sulphuric acid reagent (C) versus effect-directed detection using B. subtilis (D), A. fischeri (E) and α-glucosidase assay (F); HPTLC-HESI-HRMS spectra of zone b
obtained in the negative (G) and positive (H) ionization modes via an elution-head based interface and background subtraction.
the HPTLC zones can be eluted directly into the eluent for the HPLC
column. The zone elution time was 0.3 min. An HPLC-MS system (LC-
MS-2020, single-quadrupole, Shimadzu, Kyoto, Japan) equipped with
binary gradient solvent pump, vacuum degasser, thermostated auto­
sampler, column oven, diode array detector and electrospray ionization
mass spectrometer (ESI-MS) was used. The isocratic separation with
95% aqueous acetonitrile containing 0.1% formic acid was carried out
on a Reprospher 100 C18-DE column (150 mm � 3 mm ID, 5 μm particle
size, Dr. Maisch, Ammerbuch, Germany) with a C18 guard column (10
mm � 3 mm ID) at 35 � C using a flow rate of 0.2 mL/min and 1 μL in­
jection volume. ESI-MS parameters were desolvation line temperature,
250 � C; heat block temperature, 400 � C; drying gas flow (N2), 10 L/min;
nebulizer gas flow (N2), 1.5 L/min; negative ionization mode. Data was
Fig. 2. Densitometric evaluation at UV 195 nm (A), and after derivatization
with vanillin sulphuric acid reagent at 550 nm (B) of the HPTLC chromato­
grams of ursolic acid (u), oleanolic acid (o), the mixture of the acids (u/o) and
the lemon balm extract (l) developed with n-hexane – ethyl acetate 7:3 (V/V).
3
Talanta 219 (2020) 121306
acquired and processed using the LabSolutions 5.42v software
(Shimadzu).
3. Results and discussion
3.1. Effect-directed analysis of lemon balm by HPTLC-UV/Vis/FLD-EDA
The mobile phase was selected based on the results of the B. subtilis
assay (Figure S-1). This so selected chromatographic system was applied
to further antibacterial and enzyme inhibition assays for bioactivity
profiling. The lemon balm leaf extract was separated with n-hexane –
ethyl acetate, 7:3, and investigated for a potential activity against Gram
positive B. subtilis and Gram negative A. fischeri bacterial cells as well as
α-glucosidase (Fig. 1). Multi-imaging by UV/Vis/FLD showed that most
compounds remained at the application zone, except for the red fluo­
rescent chlorophylls (Fig. 1A–C). Nevertheless, two antimicrobial zones
a and b acting against B. subtilis were revealed at hRF 44 and 49,
respectively (Fig. 1D). In addition, zone a was active against A. fischeri
bacteria (Fig. 1E), whereas zone b inhibited α-glucosidase (Fig. 1F).
None of the zones a and b were detectable at UV 254 and 365 nm, first
after derivatization with the vanillin sulphuric acid reagent, these were
revealed as violet bands.
3.2. Characterization of bioactive compounds by HPTLC-HESI-HRMS
and HPLC-DAD-ESI-MS
The bioactive zones a and b were further characterized by HPTLC-
HESI-HRMS. Therefore, the compounds in the two bioactive zones
were online eluted into the mass spectrometer using an elution head-
based interface to record the full scan mass spectra. For zone a, one
intense mass signal (base peak) was obtained at m/z 277.21719 in the
negative ionization mode and assigned to the deprotonated molecule
[M–H]- of C18H29O-2. According to our previous data [30,31], linolenic
acid had antibacterial activity against both investigated strains, no
UV/Vis/FLD activity and a slight violet hue after derivatization with the
vanillin-sulphuric acid reagent. The identity of linolenic acid in zone a
A.M.
� M�
oricz et al.
Fig. 3. Scheme of the heart-cutting HPTLC-HPLC-DAD-MS configuration.
Fig. 4. Analysis of HPTLC zone b of the lemon balm extract by HPTLC-HPLC-
DAD-MS recording signals at UV 190 nm (A) and at m/z 455 and m/z 523 in
negative ionization mode (B).
was confirmed also by HPTLC-vanillin sulphuric acid derivatization
(Figure S-2) and HPLC-DAD-MS analysis (Figure S-3) using the authentic
fatty acid. The HPTLC-HESI-HRMS spectrum of zone b showed one
intense mass signal (base peak) in both negative and positive ionization
modes, i.e. the deprotonated molecule of C30H48O3 at m/z 455.35337
[M–H]- and the respective sodium adduct at m/z 479.34986 [MþNa]þ,
respectively (Fig. 1G and H). Thus, oleanolic acid and ursolic acid,
already described as lemon balm constituents [32,33], could be present
in zone b. These two isomeric triterpenoids of identical molecular
weight are difficult to separate because they differ only in the position of
one methyl group (Fig. 1) [34]. For these compounds, similar bio­
activities have been observed, such as antibacterial effect against
B. subtilis and Escherichia coli [35] as well as potent α-glucosidase
inhibitory activity [36]. Their co-chromatography showed that both
reference standards oleanolic acid and ursolic acid inhibited the
Gram-positive B. subtilis (Fig. 1D) and increased the bioluminescence
intensity in the Gram-negative A. fischeri bioassay (Fig. 1E). However,
they coeluted in this separation system. A densitometric evaluation of
the HPTLC chromatograms of oleanolic acid, ursolic acid, a mixture of
both and lemon balm extract at UV 195 nm and after derivatization with
vanillin sulphuric acid reagent at 550 nm clearly showed this coelution
(Fig. 2).
3.3. Characterization of coeluting bioactive compounds by HPTLC-UV/
Vis/FLD-HPLC-DAD-ESI-MS
The HPTLC separation of oleanolic acid and ursolic acid was not
satisfying. Although the separation of these compounds has been ach­
ieved using RP layers or pre-chromatographic derivatization with hal­
ogens on silica gel layers [37,38], a more universal hyphenated
technique was desired for solving the issue of coeluting compounds.
Hence, a two-dimensional LC system (HPTLC-HPLC) was configured.
4
Talanta 219 (2020) 121306
Fig. 5. Analysis of HPTLC zones of ursolic acid (u), oleanolic acid (o), co-
migrated acids (u/o) and zone b of the lemon balm extract by HPTLC-HPLC-
DAD-MS recording signals at UV 190 nm (A) and at m/z 455 and m/z 523 in
negative ionization mode (B).
First, an HPLC-DAD-MS method was developed using a low mobile
phase flow rate (0.2 mL/min) that allowed for the installation of an
elution-head based interface between the pump and the column. It was
observed that adding formic acid to the HPLC mobile phase was
necessary to achieve an appropriate separation and good peak shape
without tailing, however, in this case, next to the deprotonated molec­
ular ion at m/z 455 [M–H]-, sodium formiate adducts (at m/z 523
[MþHCOONa–H]-, m/z 591 and m/z 659) were generated in the nega­
tive ionization mode, in which these triterpenoids were masked (Figure
S-4).
Then, the TLC-MS Interface was installed into the HPLC-DAD-MS
system (Fig. 3). Zone elution from a dried HPTLC adsorbent inevitably
injected a gas phase portion at the start of the elution into the eluent, and
thus into the column. However, this was disturbing only during the UV
detection between 4 and 8 min, whereas the passing bubbles did not
interfere with the MS signals (Fig. 4 and S-5). It is obvious that UV
detection by HPTLC-UV/Vis/FLD-HPLC-DAD can also be used, when the
peak shapes (at 13 and 13.5 min) are not distorted. Similar to HPLC, the
demonstrated two-dimensional liquid chromatography, enabled the
separation of oleanolic acid and ursolic acid eluted from their coeluted
HPTLC zone (Fig. 5). Both compounds were obviously present in the
zone b. Ursolic acid was more abundant and thus more responsible for
the biological effects than oleanolic acid. HPLC-DAD-MS analysis
confirmed that oleanolic acid and ursolic acid are the ingredients of the
lemon balm leaf extract and the extract was significantly richer in the
A.M.
� M�
oricz et al.
latter one (Figure S-6). In case of bioactive minor components, bilateral
band compression (zone focussing) has recently been shown as an
effective tool to increase the signal abundance [39].
4. Conclusions
The two-dimensional liquid chromatographic hyphenation HPTLC-
UV/Vis/FLD-HPLC-DAD-MS was successfully demonstrated via an
elution head-based interface. Multi-imaging, DAD and (HR)MS detec­
tion enabled the characterization and identification of two closely
related constitutional isomers, oleanolic acid and ursolic acid. These
isomers were hardly separable and not distinguishable by HRMS, co-
migrated in HPTLC and gave a common inhibition zone in the HPTLC-
bioactivity assays. The heart-cutting HPTLC-UV/Vis/FLD-HPLC-DAD
hyphenation was demonstrated to enable the separation and assignment
of both isomers to be present. Ursolic acid was determined to be more
abundant in the lemon balm leaf extract and is mainly responsible for
the bioactivity effects observed.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
CRediT authorship contribution statement
Agnes
� M. Mo � ricz: Conceptualization, Supervision, Methodology,
Resources, Investigation, Data curation, Writing - original draft. Vira
�g
Lapat: Investigation. Gertrud E. Morlock: Conceptualization, Re­
sources, Writing - review & editing. Pe �ter G. Ott: Methodology, Re­
sources, Writing - review & editing.
Acknowledgements
This work was partially funded by the National Research, Develop­
ment and Innovation Office of Hungary (NKFIH K128921). We are
grateful to Department of Pharmacognosy, Faculty of Pharmacy, Sem­
melweis University to kindly provide oleanolic acid and ursolic acid
standards. Instrumentation was partially funded by the Deutsche For­
schungsgemeinschaft (DFG, German Research Foundation) - INST 162/
471-1 FUGG; INST 162/536-1 FUGG. The authors thank Maryam
Jamshidi-Aidji and Imanuel Yüce, Food Science, JLU Giessen, for their
support in the performance of the α-glucosidase assay and HESI-HRMS
experiments, respectively.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.talanta.2020.121306.
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