Analytica Chimica Acta 1180 (2021) 338644

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Analytica Chimica Acta

journal homepage: www.elsevier.com/locate/aca

Tutorial

High-performance thin-layer chromatography combined with effect-

directed assays and high-resolution mass spectrometry as an
emerging hyphenated technology: A tutorial review
Gertrud E. Morlock
Chair of Food Science, Institute of Nutritional Science, and TransMIT Center for Effect-Directed Analysis, Justus Liebig University Giessen, Heinrich-Buff-Ring
26-32, 35392 Giessen, Germany

h i g h l i g h t s g r a p h i c a l a b s t r a c t

Super-hyphenation suited to identify

multi-modulating compounds in
complex samples.

Important components are not over-
looked by minimalistic sample
preparation.

Simultaneous differentiation of
agonistic/antagonistic effects pro-
vides understanding.

On-surface metabolization systems
show (de)activation of conversion
products.

Open-source LabToGo development
miniaturizes status quo technology.

a r t i c l e i n f o a b s t r a c t

Article history: Among the analytical advances, hyphenated HPTLC offers great potential for solving pressing questions.
Received 9 February 2021 It provides straightforward information about effects arising from individual compounds in complex or
Received in revised form natural samples separated in parallel. The chromatographic separation is combined with effect-directed
11 May 2021
detection using enzymatic or biological assays. This helps to select from the thousands of compounds in a
Accepted 12 May 2021
Available online 18 May 2021
sample the important ones that need to be further characterized using high-resolution mass spec-
trometry. Unique benefits are discussed exemplarily arising from its super-hyphenation, minimum re-
Dedicated to the 75th birthday of Professor quirements for sample preparation, detection of multi-modulating compounds or agonistic versus
Dr. Rob Verpoorte, Leiden University, The antagonistic effects, and miniaturized on-surface metabolization. HPTLC stands for a versatile, creative
Netherlands and flexible open-format technique. As miniaturized open-source LabToGo system, it shows the potential
to be applied as Citizen Science.
Keywords: © 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND
Product quality and safety license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Effect profiling
Biotransformation or conversion product
nanoGITþactive digestive system
Open-source LabToGo system
Citizen science

Contents

E-mail address: Gertrud.Morlock@uni-giessen.de.

https://doi.org/10.1016/j.aca.2021.338644
0003-2670/© 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/
).
G.E. Morlock Analytica Chimica Acta 1180 (2021) 338644

1. Status quo of emerging technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

1.1. Targeted strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.2. Untargeted strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.3. Predictive strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2. Emerging hyphenated technologies driven by HPTLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.1. Non-target high-throughput 8D hyphenation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.2. Minimum requirements for sample preparation and comprehensive effect evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.3. Agonistic and antagonistic effect detection at a glance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.4. Identifying multi-potent or multi-modulating compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.5. Simulated on-surface digestion or metabolization for study of biotransformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3. Unique benefits and limitations of hyphenated HPTLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
4. Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Declaration of competing interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Supplementary data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

1. Status quo of emerging technologies [31], which is needed to reduce system contaminations and matrix-
effects [33]. Each generic sample preparation or simplified method
Emerging technologies are opening up new avenues with new (e.g., dilute and shoot) has to cope with the always differing
opportunities and potential. Inspiring analytical advances were complexity of natural samples due to the biodiversity of nature.
made with regard to faster strategies [1,2], higher peak capacities Multiclass methods are validated not necessarily for all urgent
[3], hyphenated systems [4,5], non-target analyses [6e8], auto- compounds that would be needed, but for those that worked well
mation by microfluidics [3,9], miniaturization to lab-in-syringe with the given method. The instability of the mixture of so many
[10], lab-on-valve [11], lab-on-a-chip [12e14], organ-on-a-chip standard compounds is another known obstacle in multi-methods
[15,16], 3D printed technologies [17e19], artificial intelligence [34]. Although the scope of analytes monitored within the global
[20] and open-source systems [21e23] to mention few. Nonethe- food supply chain is steadily expanding, even the most promising
less, further in-depth research often reveals inherent limitations to multiclass methods are always behind the facts. About 182 million
even the most promising new technology. On the one hand, substances are registered in Chemical Abstracts Service [35]. In
emerging technologies can be problem creators, especially if they 2019, the EU-27 production of chemicals hazardous to health and
raise a cascade of new unsolvable questions. On the other hand, environment were about 210 and 85 million tons, respectively [36].
emerging technologies can be problem solvers giving answers to Given the fact that China has chemical sales (1.2 billion Euro) higher
challenging analytical questions. Especially there is need to give than the EU-27 and USA combined [37], the global chemical output
answers with regard to the unidentified major compound portion is evident. If it is estimated that a few percent of chemicals are in
of a complex sample, whether it is harmful or safe. Hence, we need daily use, which is not overstated due to the huge tonnages
to widen the focus from known unknowns e the ones we do know mentioned, hazardous chemical contamination is a concern. Even if
we do not know e to unknown unknowns e the ones we do not all known target compounds, such as residues, contaminants,
know we do not know [24,25]. For progress in analytical science, it adulterations, metabolites and breakdown products, were detected
is far better to give an approximate answer to the right question using a powerful multiclass method, the major unidentified part of
than an exact answer to the wrong question [26]. Of the main unknown unknowns would still remain uncertain. The question
sample fraction of unknowns, we should at least know the therefore arises as to how this sample part can be handled and
important active compounds in it, not just what is analyzable. whether a full answer is given (harmless or harmful sample) or only
in the sense of what mainstream techniques can analyze in terms of
known compounds.
1.1. Targeted strategies

Most methods are tailored to the evaluation of target substances 1.2. Untargeted strategies
[27e29], all requiring strict regimens for compound analysis.
Powerful multiclass/-analyte methods aimed at reducing the time Routine quality control of products is based on the analysis of
required for the analysis and increasing the manageable number of main ingredients or known defects. This keeps most of the sample
analytes in a single chromatographic run [2,30,31]. The measure- components outside the analytical focus, as evidenced by the
ment itself can be quick [32], but the effort required to develop the thousands of components separated in complex samples by
method, inspect and manage the generated data, and proof the comprehensive chromatographic techniques. It is in discussion why
assignment of target compounds is considerable. The more current research and existing regulatory policies have failed
powerful analytical systems are, the more complex they are to [1,38,39] to address the global production chain [40,41], the
operate, requiring high-level expertise [31]. The increasing use of chemical complexity of our world and the diversity of natural
sophisticated ultra-high or high performance liquid chromatog- samples, calling for more comprehensive information and generic
raphy high-resolution mass spectrometry (UHPLC- or HPLC- or systematic agnostic technologies [42]. Detecting as many fea-
HRMSn) systems exposed the difficulties associated with the tures as possible is also limited in the outcome. Even for untargeted
increasing number of target compounds. It is not so easy to achieve strategies, the analyst has to select a sample preparation workflow,
always sufficient data points per peak and dwell time, to set instrumental settings and acquisition modes as well as parameters
correctly the acquisition parameters, to obtain an adequate linear for data preprocessing and data processing, e.g., to enable an
working range and acceptable sensitivity for highly diluted samples automated peak finding and prioritization of suggested hits. Such
2
G.E. Morlock
inputs have a great impact on the model result. The widespread use
of mass spectrometers does not mean that one will subsequently
see all relevant compounds in the molecular networks, since not all
compounds are ionizable. A portion of compounds in a complex
sample is always disadvantaged and discriminated, even for
comprehensive strategies [43]. The signal intensity of compounds
varies depending on sample matrix and given system performance.
Cutting-edge HRMSn systems and network science [32,44,45]
rapidly become expensive and complex for routine operation. Even
the best network is rather an illusion than a true and proper
simulation of the natural complexity of mechanisms. The transfer
to further laboratories and the verification of quantitative data is
another challenge [43]. Complex samples analyzed by sophisticated
UHPLC-HRMSn systems require an elaborate sample preparation
[46,47]. The more extensive the sample preparation, the more
targeted and the less comprehensive the analysis [48,49]. For non-
target analysis, even the most generic preparation of a solid sample,
based on a simple extraction step, results in at least several crude
extracts that differ in polarity and pH value, as every extraction is
selective. Even worse, toxicological data are generally not available
for most of the unidentified detected features of a complex sample.
Strategies to reduce the number of detected features, like
increasing the signal offset or threshold, may lead to erroneous
conclusions, feeding illusions. Low-signal, discriminated or non-
ionizable compounds can have important biological effects and
be important targets in a complex sample. If no mainstream tech-
nology is able to recognize them, there are no critical questions
about methodical bubbles. Such fallacies can be disregarded, but it
hinders progress, understanding and safety. Finally we need to
know.
1.3. Predictive strategies
The sophisticated omicsetechnologies including computational
network assessments brought even more complexity to light
[50,51]. Cutting-edge separation efficiency and high detectability
increases the data complexity and data management. There is no
point to separate as many compounds as possible in a sample, as
this endeavor can never be satisfactorily accomplished with com-
plex samples. It distracts from the essential question of what
matters. All detected features need to be evaluated thoroughly, but
how to deal with it, as it is not known in advance whether a feature
is relevant or not. Only a small portion of features can be assigned to
tentative compounds or be elucidated in their structure. It is
neither possible to identify them all nor to make a toxicological
evaluation of each one. In addition, natural samples do vary in its
composition. In other words, most separated compounds in a
complex sample are unknown in terms of structure and toxico-
logical profile. Why create complexity that then needs to be
simplified to make it manageable? With regard to the lack in
toxicological or bioactivity data of unidentified features, more and
more virtual estimations are made in analytical science. However,
extracting proper information out of large data sets remains a
challenge [44e52]. Predictions of small structures with bioactivity
toward a certain target is based on quantitative structureeactivity
relationships, cheminformatics, artificial neural networks, deep
learning, Bayesian modelling and further machine learning algo-
rithms applied to the experimental data. Science is being driven
toward a prediction-based scientific landscape. Machines already
exist today, without programmers completely understanding how
they learned to do it [53,54]. We can reflect on the powerful virtual
estimations to open up new perspectives, but to what extent shall
predictions be used to make decisions in the real world with real
consequences? Algorithms used for activity predictions cannot bear
responsibility, nor can an extract stand for the whole sample
3
Analytica Chimica Acta 1180 (2021) 338644
complexity, nor is a complex separation free of co-elution, nor is
there a panacea detector, nor can we properly simulate the un-
imaginable complexity of natural mechanisms. Taking into account
the given limitations and the fact that we only see what is stable,
extractable, separable and detectable, most knowledge based on
analytical results is approximate and preliminary. It is obvious that
minimal sample preparation (to better represent the complex
sample as a whole), orthogonal separations (to reduce matrix
interference or co-elution) and multi-detection (not to overlook
components) are important requirements for analytical methods.
However, this leads to the next challenge how to manage the
proper evaluation of the thousands of compounds in a complex
sample. Which component is important to be identified or eluci-
dated in its structure?
2. Emerging hyphenated technologies driven by HPTLC
Coming now to the crux why the interdisciplinary hyphenation
of chemistry and biology is so important for the analysis of complex
and natural samples. Knowing virtually what is supposed to be in a
sample does not mean knowing actually what to expect from a
sample in reality. Science is a never-ending cascade of surprises.
Given the thousands of unidentified compounds therein (harmful
or not, or even beneficial), this variety of compounds cannot
properly be mapped by even the best virtual prediction. The
strategy of focusing on relevant bioactive compounds in a complex
sample would substantially reduce the number of substances to be
identified as well as the amount of data to evaluate. It is far better to
use real biological facts than the best virtual activity prediction. If
most features in a sample obtained by powerful comprehensive
technologies are not identified, how can we be sure that a food or
environmental sample can be considered safe? Even if a hundred
harmful target compounds are quantitatively measured by a vali-
dated method, it can not be said that the sample is safe, if the
sample is estimated to consist of many thousands of compounds. In
contrast, the outcome includes and refers to all components of a
sample if a biological response is exploited, e.g., using a genotoxic,
cytotoxic or neurotoxic assay.
Hence, the fusion of chromatography with biological assays is
helpful in the evaluation and decision making process on a complex
or natural sample. However, HPLC combined online with assays for
effect-directed analysis (EDA) is not compatible with long-lasting
incubation times required for biological assays or with the moni-
toring of the biological response over a longer 30-min period as
used for luminescent bacteria in the environmental field [55].
Despite instrumental effort, HPLC-EDA is only working for instant
bioassays [56] or fast reacting enzyme assays [57]. For offline col-
umn chromatographic systems based on HPLC [58] or gas chro-
matography (GC) [59], the instrumental complexity and time-
consuming nature of such practices are obstacles to its routine use.
Research on high-performance thin-layer chromatography
(HPTLC) is a niche, which is proven by the overview on the publi-
cations in the field of chromatography over the last eight decades
(Fig. S1). However, this fact can not explain why the beneficial
potential of HPTLC combined with EDA and HRMS is ignored and
overlooked in almost all the reports on HPLC-EDA or GC-EDA or
inspiring analytical advances [60e62]. HPTLC-EDA is utmost effi-
cient and straightforward to directly draw the attention to the
bioactive hotspots of a sample [63e66]. Considered as proof, this
hyphenation confirms what is to be expected. At the same time,
unexpected surprises are revealed; the benefit gained is the
simultaneous evaluation of the unknown compound portion of a
complex sample in terms of biological and toxicological effects. This
would be important information in the risk assessment of chem-
icals and products, especially those that go through a global
G.E. Morlock
processing chain. It is obvious that the currently selected chemical
marker compounds used to evaluate product quality can not
represent the existing sample complexity. Effect-directed finger-
prints would complement the data for product quality and safety
assessment. It is evident that chromatography combined with an
effect-directed detection is superior to microtiter plate assays
providing a sum effect (sum parameter) only for complex samples.
The need for information on individual active compounds is inev-
itable. In addition, HPTLC-EDA workflows are faster and less
expensive, if compared to status-quo microtiter plate assays
[67,68], and provide the basis for full spectroscopy/spectrometry-
based identification of compounds [69e71]. Hence, the tutorial
review is focused on quantitative HPTLC combined with reliable
EDA (or modern bioautography) and HRMS as an emerging tech-
nology. Only together is it key as an emerging technology, as the
next questions will be: how much is the effect in reference to well-
known active compounds and which bioactive compound is it?
Nevertheless, the key question is how to obtain information about
unknown bioactive compounds in complex samples quickly and
inexpensively to properly evaluate the sample and to unveil valu-
able insights that otherwise remain hidden? In the following, five
selected strategies for becoming aware of important unknown ef-
fects in complex samples are outlined.
2.1. Non-target high-throughput 8D hyphenation
Comprehensive information on bioactive compounds in com-
plex samples is directly obtained by hyphenated techniques. These
use orthogonal separation and multiple detection principles to
maximize the information obtained by a single sample run [72].
The following strategy of a super-hyphenated HPTLC including a
biological evaluation (Fig. 1 [73]) stands for a method that is as
generic as possible, suitable for routine use, with minimal workload
and maximum compatibility to a wide range of analytes and sample
matrices. The 8D hyphenation allows the non-target high-
throughput screening of complex samples for individual bioactive
compounds. The separation efficiency of HPTLC is ideal for the
transfer to the second orthogonal dimension. Identification or
structure elucidation is only performed for the most bioactive
compound zones. Briefly, liquid samples or crude extracts of solid
samples are simultaneously subjected to the highly matrix-robust
Fig. 1. 8D hyphenation NP-HPTLC-UV/Vis/FLD-EDA-RP-HPLC-DAD-MS, shown for bioanalytical profiling of cinnamon spices in parallel on a plate, taking 4 h for 18 samples. Savings
are expected to be at least more than 60% in cost and analysis time compared to status quo analyses [73]. Modified with permission from Elsevier.
4
Analytica Chimica Acta 1180 (2021) 338644
normal phase (NP)-HPTLC separation, followed by multi-imaging
via ultraviolet (UV), visible (Vis) and fluorescence detection (FLD)
as well as EDA. The imaging HPTLC technique offers the compara-
tive evaluation of constituents of side-by-side separated samples.
This flatters our brain and mind, which is trained to evaluate im-
ages in a highly efficient way every second of every day. Out of the
bioautogram, active zones were heart-cut eluted into an orthogo-
nally aligned reversed phase (RP)-precolumn used as analyte trap
for desalting (salts from the bioassay medium). The subsequent
switch of the valve eluted the trapped analyte(s) to the main col-
umn. The subsequent photodiode array and MS detections were not
influenced by even highly salted bioassay media. As a result, the 8D
super-hyphenation was reliable and robust for routine use in a wide
range of fields (e.g., food and feed research, traditional medicines,
pharmaceutical sciences, drug discovery, environmental and life
sciences). Instead of the quantitative zone elution into the mass
spectrometer [74], surface scanning via direct analysis in real time
(DART)-MS [75,76] can also be applied. Both are quantitative
coupling techniques, although with different advantages and
limitations.
2.2. Minimum requirements for sample preparation and
comprehensive effect evaluation
Sample preparation must be minimalistic for a proper evalua-
tion of a sample. Since the sample needs to remain as native as
possible, a comprehensive non-target analysis must be matrix-
tolerant. The second strategy shows the analysis of estrogen-like
compounds in quite different food matrices (Fig. 2). Solid samples
need to be extracted to obtain a liquid crude solution to be sub-
jected to the analytical system [77]. However, liquid foods like beers
can be applied directly (possibly diluted, or degassed for quanti-
tative study). Being applied as native as possible, any loss or
discrimination of compounds by sample preparation steps is avoi-
ded. The same two estrogen-like compounds were identified in the
beer extracts (Fig. 2 [77]) as well as in the beers analyzed directly
(Fig. 3 [78]). The latter beers were applied as area to spread the load
of the inherent matrix on the adsorbent. The same area application
was advantageous for the application of surface and wastewater
crude extracts (Fig. 4 [79]). It is evident that matrix robustness is a
key feature of HPTLC with regard to complex samples, which
G.E. Morlock Analytica Chimica Acta 1180 (2021) 338644

Fig. 2. Discovery of estrogen-like hormones in propolis tincture, dill, beer, wine and waste water by HPTLC combined with the planar yeast estrogen screen (pYES): main steps of
the RP-HPTLC-UV/Vis/FLD-pYES-MS bioassay (15 min per sample calculated for several samples in parallel on a plate); Saccharomyces cerevisiae cells containing the human estrogen
receptor hRa and reporter gene encoding for b-D-galactosidase were applied on the chromatogram to detect estrogen-like hormones as 4-methylumbelliferon (MU)-blue fluo-
rescent zones (marked) after enzymatic cleavage of the substrate 4-methylumbelliferon-b-D-galactoside (MUG) upon hRa activation, followed by mass spectrometric character-
ization. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

should be analyzed as native as possible. In addition, the wide range
of possible application volumes (0.1e1000 mL) allows flexibility. The
enrichment factor during spray-on application (evaporation of
solvent along with sample concentration) improves the detect-
ability of compounds. Here, yeast cells containing the human es-
trogen receptor hRa and a reporter gene encoding for b-D-
galactosidase were applied as a biological detector to reveal
estrogen-like compounds. Using 4-methylumbelliferyl-b-D-galac-
toside as fluorogenic substrate, endocrine-disrupting compounds
like estrogens were detected as blue fluorescent zones in food
products. Instead, also red fluorescent bands can be generated us-
ing alternatively resorufin-b-D-galactoside as substrate [80]. Side
reactions of the substrate with sample components need to be
excluded [81]; if required, other substrates can be used that are
suited better [80,82].
2.3. Agonistic and antagonistic effect detection at a glance
The largely unknown chemical landscape is deeply rich in ef-
fects. In status-quo sum parameter assays, antagonists block the
detection of agonists, and cancel each other out which leads to
wrong conclusions. In order to differentiate both effects, samples
have to be studied tediously in separate microtiter plate assays
performed in parallel. In this respect, the following third straight-
forward strategy provides a deeper understanding by simulta-
neously revealing and differentiating the nature of the agonistic
and antagonistic properties of a complex mixture [83]. The concept
is valid and can be applied to any assay, although here it was shown
for the bioassay with the human androgen receptor to make it
different from the previously shown detection of estrogen-like
compounds. After sample application (here six different cos-
metics) and development, a stripe of a well-known agonist (here
5
the androgen testosterone) is oversprayed on the right side of each
separated sample track. After piezoelectric spraying of the planar
yeast androgen screen (pYAS) suspension and substrate solution as
well as intermediate incubation periods, blue fluorescent andro-
gens (agonists) were detected along the left sample track side and
fluorescence-reducing anti-androgens (antagonists) along the right
sample track side (Fig. 5 [83]). The resulting image illustrates the
planar yeast anti-/androgen screen (pYAAS), in which both effects
were revealed for each sample. The fluorescence or fluorescence
reduction as evident in the RP-HPTLC-pYAAS-FLD bioautogram was
also measured densitometrically. The respective densitograms for
each sample side at 366/>400 nm clearly showed that only
antagonistic effects were detected for the six different cosmetic
formulations. The concept provides not only the detection, differ-
entiation and quantification of individual agonistic versus antago-
nistic effects (for the latter inverse peak, the software evaluation is
restricted; however, a semi-quantification is possible), but also the
characterization of bioactive compound zones by super-
hyphenation to high-resolution mass spectrometry, as mentioned
in section 2.1. The planar agonist/antagonist assay strategy repre-
sents a non-target, versatile, universal and cost-efficient bio-
analytical tool, applicable for routine analysis in many fields. About
15 samples were analyzed in parallel within 5 h or 6 h, calculated as
20 or 24 min per sample, without the need for complex fraction-
ation and tedious compound isolation.
2.4. Identifying multi-potent or multi-modulating compounds
Pharmaceutical multidrug combination cocktails are known for
their side effects. Hence, network analysis [44,45] is explored for a
better understanding of complex biological systems. The same is
valid for most alternative approaches of traditional, herbal or
G.E. Morlock
Fig. 3. Bioprofiling of 14 beers, only degassed and diluted with methanol, subjected to
the RP-HPTLC-UV/Vis/FLD-pYES bioassay for detection of estrogen-like hormones:
chromatograms at Vis after rectangular application of 10 mm  30 mm, at FLD 366 nm
after focusing to a sharp start band and separation up to 70 mm as well as respective
bioautogram after the pYES bioassay [78] (same bands marked as in the extract of
Fig. 2).
Fig. 4. Ultratrace analysis of estrogen-like hormones in 8 different river water and wastewater crude extracts by the RP-HPTLC-UV/Vis/FLD-pYES bioassay: chromatograms at FLD
366 nm after rectangular application of 7 mm  25 mm, focusing to a sharp start band, and separation up to 65 mm as well as respective bioautogram after the pYES bioassay [79].
Modified with permission from Elsevier.
6
Analytica Chimica Acta 1180 (2021) 338644
phyto-medicine. Often the principle of action is not or not entirely
clear and cannot be explained in scientific terms [84], and also
computational bioactivity estimations are limited in informative
value [52]. In addition, good practices in manufacturing and pro-
cessing vary widely [82]. When the same kind of food is consumed
regularly, also foods are able to gently modulate our well-being (the
same applies for feeds). However, it is very difficult to identify the
compounds responsible for an activity among the thousands of
unknown compounds in a complex food sample. As a trend, func-
tional plant extracts are increasingly added to industrial food
products (known as nutraceuticals, functional food and health
food) to increase the health benefit for consumers [82,85e87].
Especially comminuted or powdery products are not only more
susceptible to oxidation, degradation and loss of activity (due to
larger surface), but also adulteration, falsification and counter-
feiting [39,88e91]. In addition, active contaminants and residues
do play a role. These are significant, even when only present at trace
levels, as it is known for endocrine disruptors [77e79,83]. It is
obvious that products with a global production and processing
chain are out of full control. Hence, a real (not virtual) bioanalytical
effect-directed strategy is highly important, which is able to prove
the authenticity (characteristic patterns), to discover product de-
fects or undesirable components by pattern recognition and multi-
detection (multi-imaging), and to point to individual known and
unknown active compounds (bioactivity profiles). Exemplarily
shown for ginger [92] or tea (Fig. 6 [82]), multi-potent and multi-
modulating compounds were discovered. It explains how specific
foods can affect health when consumed daily. Meanwhile an
effective battery of streamlined planar assays has been reported to
detect antibiotics [93,94], genotoxic compounds [67], neurotoxic
compounds [68], inhibitors of different enzymes [82,95e97],
different endocrine disruptor types at the same time [98] and
among others fungicides [99]. New bioanalytical tools open up new
G.E. Morlock Analytica Chimica Acta 1180 (2021) 338644

Fig. 5. Discovering of both agonistic and antagonistic compounds, exemplarily shown for the planar yeast anti-androgen/androgen screen (pYAAS): general scheme and RP-HPTLC-
pYAAS-FLD bioautogram at 366 nm of 6 cosmetics C1eC6 (each 0.7 mg/12 mm band), each on right side oversprayed with the vertical androgenic testosterone (T) stripe (each 230
ng/4 mm  70 mm area) before bioassay application: no blue fluorescent androgens were detected (left track side), but several fluorescence-reducing anti-androgens (right track
side revealed the parabens ME, EE, PE and BE; *application zone) as well as zoom on C1 with respective densitograms on both track sides at 366/>400 nm [83]. Modified with
permission from Elsevier. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

insights into complex samples. It is advisable to be prepared for
unexpected surprises in samples.
2.5. Simulated on-surface digestion or metabolization for study of
biotransformation
The understanding of the complex gastrointestinal metabolism
of nutrients and the activation or inactivation of food ingredients
with promising or harmful effects as a kind of multifunctional team
player is still at its infancy. For a straightforward study of the co-
ordinated processes of all digestion segments, a miniaturized
nanomolar simulated system was reported on the same adsorbent
surface (in situ) [100], based on a harmonized in vitro protocol [101].
On-surface metabolization, immediate separation, multi-imaging
and effect-directed detection was altogether demonstrated as a
compact all-in-one system termed nanoGITþactive (Fig. 7 [100]).
Among others, it was applied to basic food samples (casein, starch
and rapeseed oil) and plant extracts (ginger, tea and turmeric) to
investigate their interaction with digestive amylolytic, proteolytic
and lipolytic enzymes. Apart from simulation of the digestive tract,
the on-surface simulation of the metabolization in the liver with
regard to detoxification or toxification of compounds was also
shown [68,100]. The S9 liver system, an extract of the enzyme-rich
tissue from mammalian liver, was successfully applied on surface
for metabolic simulation of the activation or inactivation of xeno-
biotics, mainly through the phase 1 cytochrome P450 pathway. The
in situ results were successfully verified by comparison with state-
of-the-art in vitro results [68,100]. The side-by-side comparison of
metabolized and non-metabolized food (up to 16 samples in par-
allel) showed changes in the bioactivity of the conversion products
when an enzymatic assay for detection of neurotoxins or a bio-
logical assay for detection of genotoxins was applied. Notice was
also taken of non-eluted or front-eluted conversion products
because all non-volatile sample constituents remained stored on
the adsorbent surface. The separated conversion products were
accessible for further characterization, e.g., by multi-detection us-
ing a derivatization reagent sequence [82], or as mentioned in
section 2.1 by heart-cut hyphenation to orthogonal separation and
7
detection systems [73] suited for structure elucidation [69,71]. Such
versatile substance information obtained on the same surface is
very valuable in case of unidentified conversion products.
3. Unique benefits and limitations of hyphenated HPTLC
After 75 years of bioautography, Goodall and Levi [102] would
be delighted with the instrumental playground for planar hy-
phenations that we have today (Fig. 8 [63,66]). The terminology has
already been addressed [63], not to use terms incorrectly
[103e106]. Briefly, the umbrella term is effect-directed assays. The
subterm bioassay implies the use of microorganisms or cells, at
least cell organelles, as the abbreviation bio stands for biology ac-
cording to dictionaries. Hence, a mere enzyme assay is a
biochemical assay, and based on a chemical compound only, a
radical scavenging assay is a chemical assay. Using selected effect-
directed assays, thousands of compounds in a complex mixture can
be reduced to few crucial ones which is very helpful in the un-
derstanding of active principles and in the management of risk
assessment (Fig. 9 [63,66]). Although volatile minor constituents
may be lost or constituents may be altered by (photo)oxidation
which are clear limitations of HPTLC, most sample constituents
remain safely stored on the adsorbent. The versatile advantages
due to the open planar format in comparison to column-based
hyphenations were already summarized [65]. Only the fusion of
all three together  HPTLC, EDA and HRMS  is key to an emerging
technology. Eight benefits are highlighted in the following.
1. The samples are analyzed as native as possible, as the HPTLC
method is highly robust to the sample matrix load (high matrix
tolerance) due to the option for rectangular sample application
and the single use of the stationary phase. The more minimalist
the sample preparation (saves costs), the more comprehensive
the view on the sample (less is overlooked), which greatly
supports the understanding about a sample. The minimum re-
quirements for sample preparation allow for a comprehensive
effect evaluation.
G.E. Morlock
Fig. 6. Discovery of multi-potent or multi-modulating compounds in 20 different tea extracts (each 10 mg/band) by effect-directed profiling via HPTLCeEDAeUV/Vis/FLD; bioactive
compound mixture (M, 0.6 mg/band each) used as reference.
2. Many samples are investigated in parallel under the same
chromatographic conditions on always fresh adsorbent. Sample
volumes can easily be adjusted for application (from 0.1 to
1000 mL). This supports a cost-efficient, robust and less error-
prone (e.g., regarding cross-contamination) sample screening.
3. The chromatographic separation avoids false positive or false
negative results caused by matrix interferences or antagonistic
8
Analytica Chimica Acta 1180 (2021) 338644
or synergistic effects, which occur using high-throughput
in vitro systems (provide the sum parameter only).
4. Organic solvents (freely selectable for the best separation) are
readily evaporated before biological or enzymatic assay detec-
tion. The on-surface separated compounds are directly acces-
sible for the assay. Difficulties such as incompatibility of sample
G.E. Morlock
Fig. 7. Scheme of the miniaturized all-in-one nanoGIT þ active system for simulated on-surface digestion, separation and detection (1) food sample (e.g., starch, protein and fat): in
contact with the enzyme system (e.g., a-amylase, pepsin and pancreatin plus bile extract), start of metabolization by moistening and digestion over the respective incubation period,
(2) separation of the metabolization products formed, (3e6) multi-imaging and quantification by UV/Vis/FLD including effect-directed detection for bioactivity screening and (7e10)
orthogonal characterization by RP-column separation of a heart-cut bioactive compound zone, diode array detection and orbitrap HRMSn detection.
solvents or insolubility of compounds with regard to the polar
assay medium are non-existent.
5. The image is worth a thousand words. The side-by-side sample
comparison flatters our brain and mind, which are highly
trained to evaluate images. The HPTLC image can benefit from
recent advances in fundamental image analysis through artifi-
cial intelligence.
6. By revealing individual active ingredients in complex mixtures,
HPTLC reduces the number of substances to be analyzed to a
reasonable level and helps to make effective decisions about
which substances are relevant for further analysis and toxico-
logical assessment. The ability to detect compounds is typically
in the lower ng to pg range per band. Regulated limit values
down to the ultra trace level (ng/L, ng/kg) can directly be ach-
ieved (without concentration steps), depending on the assay
sensitivity and (higher) application volume [67,68,79].
7. The need for data storage and data handling is minimalistic. On
one plate, many multi-component mixtures can be screened,
but only at the point-of-care (most active compound zones) of
the planar assay format, respective zones are heart-cut eluted
and orthogonally analyzed further (Figs. 1, 6 and 7). The gener-
ation of the molecular formula and structure elucidation is
performed only for relevant active compound zones. For
recording of the exact mass spectra, the expensive HRMS system
is used highly targeted. This is quite in contrast to the recording
of the whole sample run including background and matrix for
non-target HPLC- or GC-HRMS sample screening.
8. HPTLC is no rigid and inflexible online system, but a highly
dynamic step-automated system. The actual data on the sample
analysis can be retrieved and evaluated intermediately. The
following analysis steps can thus be better adapted at any time
to be as efficient as possible. The inherent potential for versatile,
flexible and creative solutions is evident (Figs. 1, 3, 4, 7 and 10).
On a closer inspection, the immense unique advantages
outweigh the few disadvantages of hyphenated HPTLC. The true
potential of quantitative and hyphenated HPTLC is not trained in
9
Analytica Chimica Acta 1180 (2021) 338644
education. As a consequence, the analytical science is full of unused
opportunities for more efficiency. Ignoring the potential of HPTLC is
finally a waste of time and money for industries, authorities and
research in general, as the arguments for its efficiency and unique
possibilities [65] are obvious and proven.
4. Outlook
HPTLC stands for a versatile and flexible open-format technique
that can be exploited for new ideas. It offers many possibilities, and
thus challenges the analytical mind to be creative and think
differently. New potentials open up whether used as routine super-
hyphenation, biotransformation system (nanoGITþactive) or simul-
taneous profiling for agonistic versus antagonistic effects. Only the
fusion of HPTLC (optionally including heart-cut to HPLC [73]), EDA
and HRMS is key to an emerging hyphenated technology, as it has
the power to straightforwardly solve pressing analytical tasks. The
combined straightforward use of orthogonal separations and
multiple detection principles maximizes the information obtained
by a single chromatographic run for many samples in parallel. To
give a metaphorical comparison, edge science can also be made
taking the subway instead of driving a luxurious sports car (Fig. S2).
In addition, no special styling (sample preparation) is needed if you
go by subway or metro. In other words in HPTLC, many raw extracts
are efficiently transported in parallel and separated.
Even more advanced are miniaturized systems. Although
miniaturization scales down material consumption [106], it does
not necessarily mean that all the equipment needed for a minia-
turized system could not fill a laboratory or could be used outside a
laboratory. A true miniaturization of a chromatographic system can
also be used by Maker Communities or volunteers in any location to
screen samples. Citizen Science has given scientific access to highly
motivated volunteers, especially for sustainable development goals
[108]. Open-source developments are in many respects an inter-
esting and at the same time exciting field of interdisciplinary
research to which it is worthwhile to contribute. Similar to radical
chain reactions, open-source tools are exponential and highly
G.E. Morlock Analytica Chimica Acta 1180 (2021) 338644

Fig. 8. Versatility and classification of planar effect-directed assays reported in literature (A; list might not be complete); targeted characterization of important bioactive compound
zones by information on polarity, solubility, chromatographic behavior, spectral and chemical properties (B), all obtained by the same separation for many samples in parallel
[64,66]. Modified with permission from Elsevier and q&more.

dynamic in their progress due to crowd intelligence, which is a
powerful means to generate progress. A public domain open-source
analytical platform [109] will help improve product quality, if
highly motivated volunteers would perform a fast product check,
exemplarily by latest open-source versions of planar chromatog-
raphy. Such an open-source LabToGo system combining all planar-
chromatographic steps in the same all-in-one system [23,107]
miniaturizes the status-quo technology substantially. Exploiting
print and media technologies for chromatographic purposes, it was
termed Office Chromatography [110]. Instead of printing inks on
papers, chemical solutions are inkjet-printed on the adsorbent
layer for sample application, development and derivatization
(Fig. 10 [107]). Particularly miniaturized ultrathin layers
(10 cm  5 cm) are advantageous for integration in this concept
[111], as it minimizes difficulties arising from the larger HPTLC plate
format (20 cm  10 cm). Plate images were captured by inexpensive
light-emitting diodes in a comparable quality to status-quo
instrumentation [23,107]. Sophisticated open-source image evalu-
ation by pattern recognition, machine learning algorithms and
artificial neural networks was proven to be advantageous for
implementation [20,71,112e114]. As the open-source printing of
the layer material itself (printing the adsorbent slurry on a plain

10
G.E. Morlock Analytica Chimica Acta 1180 (2021) 338644

Fig. 9. Straightforward hyphenated bioanalytical imaging to reduce the thousands of compounds in a complex sample to the relevant active ones including active unknown
unknowns (A) and straightforward strategy for analysis of complex samples (B) [,64[66]]. Modified with permission from Elsevier and q&more.

Fig. 10. All-in-one LabToGo system for sample analysis: OCLab2 [107]. Modified with permission from Elsevier.

glass plate) was also shown [22,70], all analysis steps can be per-
formed in the miniaturized LabToGo system, meaning from layer
print to sophisticated image evaluation.
More than ever, consumers are worldwide concerned about the
quality of foods [115e117]. Food enters us, unites with us and
provides the building blocks of our body functions. Healthy food
supports the well-being of every individual, and in a wider
perspective, the prosperity of a society. The use of efficient and
straightforward tools accelerates the gain of knowledge. The above
mentioned open-source LabToGo system can be used to check the
product quality (foods, feeds, dietary supplements, cosmetics,
commodities, pharmaceuticals, environmental samples etc.). At any
site, the multi-imaging profiling and cloud-supported image eval-
uation by artificial intelligence can be a first check for irregularities
or defects in products, processed and traded worldwide. Non-
compliant samples, which do not match to reference samples in
the cloud-based database, may be reported by a simple click to the
respective authorities for a more detailed analysis as part of the
ongoing food inspection. Such open-source-based strategies can
support citizens, researchers, authorities and decision makers.
There is urgent need as mentioned [39,88e91] to check the product
quality (global processing chain), to reduce cases of adulteration
and falsification, to identify contamination or degradation, to
discover unknown effective compounds in the samples whether
dangerous or beneficial to health including metabolic in-/activa-
tion. Societies are especially open-minded to innovations when the
food quality strategies and official food control systems are
continuously improved [118e120]. Hyphenated HPTLC offers high
potential for solving urgent questions, and especially, it is able to
provide approximate answers to the right questions.

11
G.E. Morlock
Declaration of competing interest
The author declares that she has no known competing financial
interests or personal relationships that could have appeared to
influence the work reported in this paper.
Acknowledgement
Thank is owed to the Bundesamt für Ausrüstung, Information-
stechnik und Nutzung der Bundeswehr grant E/U2AD/KA018/IF565.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.aca.2021.338644.
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Compounds that Matter for Food Safety along the Global Food Chain,
Q&more, vol. 10, 2019. https://q-more.chemeurope.com/q-more-articles/
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[67] D. Meyer, M. Marin-Kuan, E. Debon, P. Serrant, C. Cottet-Fontannaz,
B. Schilter, G.E. Morlock, Detection of low levels of genotoxic compounds in
food contact materials using an alternative HPTLC-SOS-Umu-C Assay, ALTEX
(2021), https://doi.org/10.14573/altex.2006201.
[68] E. Azadniya, J. Mollergues, T. Stroheker, K. Billerbeck, G.E. Morlock, New
incorporation of the S9 metabolizing system into methods for detecting
acetylcholinesterase inhibition, Anal. Chim. Acta 1129 (2020) 76e84.
[69] I. Yüce, G.E. Morlock, Streamlined structure elucidation of an unknown
compound in a pigment formulation, J. Chromatogr. A 1469 (2016) 120e127.
[70] I. Yüce, M. Mayr, G.E. Morlock, Quantitative inkjet application on self-
printed, binder-free HPTLC layers for submicromole-scaled analytical 1H
NMR spectroscopy, Anal. Chim. Acta 1087 (2019) 131e139.
[71] D. Fichou, I. Yüce, G.E. Morlock, eicCluster software, an open-source in silico
tool, and on-surface syntheses, an in situ concept, both exploited for signal
highlighting in high-resolution mass spectrometry to ease structure eluci-
dation in planar chromatography, J. Chromatogr. A 1577 (2018) 101e108.
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13
Analytica Chimica Acta 1180 (2021) 338644
of column liquid chromatography and spectroscopy, Trends Anal. Chem. 26
(2007) 847e854.
[73] T. Schreiner, G.E. Morlock, Non-target bioanalytical imaging via eight-
dimensional orthogonal liquid chromatography-mass spectrometry,
including bioassay, heart-cut trapping and online desalting, J. Chromatogr. A
1647 (2021) 462154.
[74] G.E. Morlock, N. Brett, Correct assignment of lipophilic dye mixtures? Per-
formance data for the TLC-MS Interface and a case study for high-
performance thin-layer chromatography-mass spectrometry,
J. Chromatogr. A 1390 (2015) 103e111.
[75] T.T. H€abe, M. Jamshidi-Aidji, J. Macho, G.E. Morlock, Direct bioautography
hyphenated to direct analysis in real time mass spectrometry: chromato-
graphic separation, bioassay and mass spectra, all in the same bioautogram,
J. Chromatogr. A 1568 (2018) 188e196.
[76] T.T. H€abe, G.E. Morlock, Improved desorption/ionization and ion trans-
mission in surface scanning by Direct Analysis in Real Time mass spec-
trometry, Rapid Commun. Mass Spectrom. 30 (2016) 321e332.
[77] G.E. Morlock, I. Klingelho €fer, Liquid chromatography-bioassay-mass spec-
trometry for profiling of physiologically active food, Anal. Chem. 86 (2014)
8289e8295.
[78] G.E. Morlock, I. Klingelho €fer, Discover Estrogens by High-Performance Thin-
Layer Chromatography, Justus Liebig University Giessen, Video, 2015. www.
youtube.com/watch?v¼Q7AGuljcFvQ&feature¼youtu.be. accessed 5th
May 2021.
[79] I. Klingelho€ fer, G.E. Morlock, Bioprofiling of surface/wastewater and bio-
quantitation of discovered endocrine-active compounds by streamlined
direct bioautography, Anal. Chem. 87 (2015) 11098e11104.
[80] D. Schick, W. Schwack, Planar yeast estrogen screen with resorufin-b-D-
galactopyranoside as substrate, J. Chromatogr. A 1497 (2017) 155e163.
[81] K. Rhee, R.M. van Rijn, R. Verpoorte, Qualitative determination of false-
positive effects in the acetylcholinesterase assay using thin layer chroma-
tography, Phytochem. Anal. 14 (2003) 127e131.
[82] G.E. Morlock, J. Heil, A.M. Inarejos-García, J. Maeder, Effect-directed profiling
of powdered tea extracts for catechins, theaflavins, flavonols and caffeine,
Antioxidants 10 (2021) 117.
[83] I. Klingelho€fer, N. Hockamp, G.E. Morlock, Non-targeted detection and dif-
ferentiation of agonists versus antagonists, directly in bioprofiles of everyday
products, Anal. Chim. Acta 1125 (2020) 288e298.
[84] L. Shao-Hsiang, C. Po-Sheng, H. Chun-Chieh, H. Yi-Tu, L. Mei-Ying, L. Wei-
Hung, L. Yuan-Chuan, L. Alan Yueh-Luen, Unlocking the mystery of the
therapeutic effects of Chinese medicine on cancer, Front. Pharmacol. 11
(2021) 601785.
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(2013) 558e567.
[86] M. Younes, P. Aggett, F. Aguilar, R. Crebelli, B. Dusemund, M. Filipi c,
M.J. Frutos, P. Galtier, D. Gott, U. Gundert-Remy, C. Lambre
, J.C. Leblanc,
I.T. Lillegaard, P. Moldeus, A. Mortensen, A. Oskarsson, I. Stankovic,
I. Waalkens-Berendsen, R.A. Woutersen, R.J. Andrade, C. Fortes, P. Mosesso,
P. Restani, D. Arcella, F. Pizzo, C. Smeraldi, M. Wright, Scientific opinion on
the safety of green tea catechins. EFSA panel on food additives and nutrient
sources added to food (ANS), EFSA J 16 (2018), e05239.
[87] G.E. Morlock, J. Heil, V. Bardot, L. Lenoir, C. Cotte, M. Dubourdeaux, Effect-
directed profiling of 17 different fortified plant extracts by high-performance
thin-layer chromatography combined with six planar assays and high-
resolution mass spectrometry, Molecules 26 (2021) 1468.
[88] T. Rocha, J.S. Amaral, M.B.P.P. Oliveira, Adulteration of dietary supplements
by the illegal addition of synthetic drugs: a review, CRFSFS 15 (2016) 43e62.
[89] A.M. Inarejos-García, I. Helbig, P. Klette, S. Weber, J. Maeder, G.E. Morlock,
Authentication of commercial powdered Tea (Camellia sinensis L.) extracts by
gas chromatography, ACS Food Sci. Technol. (2021) (in press).
[90] C.M. Gilroy, J.F. Steiner, T. Byers, H. Shapiro, W. Georgian, Echinacea and truth
in labeling, Arch. Intern. Med. 163 (2003) 699e704.
[91] J. Sherma, F. Rabel, Advances in the thin layer chromatographic analysis of
counterfeit pharmaceutical products: 2008e2019, J. Liq. Chromatogr. Relat.
Technol. 42 (2019) 367e379.
[92] S. Krüger, A. Bergin, G.E. Morlock, Effect-directed analysis of ginger (Zingiber
officinale) and its food products, and quantification of bioactive compounds
via high-performance thin-layer chromatography and mass spectrometry,
Food Chem. 243 (2018) 258e268.
[93] M. Jamshidi-Aidji, G.E. Morlock, From bioprofiling and characterization to
bioquantification of natural antibiotics by direct bioautography linked to
high-resolution mass spectrometry: exemplarily shown for Salvia miltior-
rhiza root, Anal. Chem. 88 (2016) 10979e10986.
[94] C. Stiefel, T. Schubert, G.E. Morlock, Bioprofiling of cosmetics with focus on
streamlined coumarin analysis, ACS Omega 2 (2017) 5242e5250.
[95] I.A. Ramallo, S.A. Zacchino, R.L.E. Furlan, A rapid TLC autographic method for
the detection of xanthine oxidase inhibitors and superoxide scavengers,
Phytochem. Anal. 17 (2006) 15e19.
[96] M. Jamshidi-Aidji, G.E. Morlock, Fast equivalency estimation of unknown
enzyme inhibitors in situ the effect-directed fingerprint, shown for Bacillus
lipopeptide extracts, Anal. Chem. 90 (2019) 14260e14268.
[97] E. Mahran, M. Keusgen, G.E. Morlock, New planar assay for a streamlined
detection and quantification of b-glucuronidase inhibitors and application to
botanical extracts, Anal. Chim. Acta X 4 (2020) 100039.
[98] L. Moscovici, C. Riegraf, N. Abu-Rmailah, H. Atias, D. Shakibai, S. Buchinger,
G.E. Morlock Analytica Chimica Acta 1180 (2021) 338644

G. Reifferscheid, S. Belkin, Yeast-based fluorescent sensors for the simulta- Exploration and Classification of Thin-Layer Chromatograms, Talanta (2021).
neous detection of estrogenic and androgenic compounds, coupled with In press, https://doi.org/10.1016/j.talanta.2021.122460.
high-performance thin layer chromatography, Biosensors 10 (2020) 169. [115] F. De Canio, E. Martinelli, EU quality label vs organic food products: a
[99] A.M. Mo
ricz, D. Krüzselyi, P.G. Ott, Z. Gar

ni, G.E. Morlock, J. Bakonyi,
adi, S. Be multigroup structural equation modeling to assess consumers' intention to
Bioactive clerodane diterpenes of giant goldenrod (Solidago gigantea Ait.) buy in light of sustainable motives, Food Res. Int. 139 (2021) 109846.
root extract, J. Chromatogr. A 1635 (2021) 461727. [116] V. Barrere, K. Everstine, J. The
olier, S. Godefroy, Food fraud vulnerability
[100] G.E. Morlock, L. Drotleff, S. Brinkmann, Miniaturized all-in-one nano- assessment: towards a global consensus on procedures to manage and
GITþactive system for on-surface metabolization, separation and effect im- mitigate food fraud, Trends Food Sci. Technol. 100 (2020) 131e137.
aging, Anal. Chim. Acta 1154 (2021) (2021) 338307. [117] A. Zhang, A. Mankad, A. Ariyawardana, Establishing confidence in food
[101] M. Minekus, M. Alminger, P. Alvito, S. Ballance, T. Bohn, C. Bourlieu, et al., safety: is traceability a solution in consumers' eyes? J. Consum. Prot. Food
A standardised static in vitro digestion method suitable for food  an in- Saf. 15 (2020) 99e107.
ternational consensus, Food & Function 5 (2014) 1113e1124. [118] F. Wu, C. Lu, M. Zhu, H. Chen, J. Zhu, K. Yu, et al., Towards a new generation of
[102] R.R. Goodall, A.A.A. Levi, Microchromatographic method for the detection artificial intelligence in China, Nat. Mach. Intell. 2 (2020) 312e316.
and approximate determination of the different penicillins in a mixture, [119] J.M. Soon, Application of bayesian network modelling to predict food fraud
Nature 158 (1946) 675. products from China, Food Contr. 114 (2020) 107232.
[103] J. Zhou, Q. Tang, T. Wua, Z. Cheng, Improved TLC bioautographic assay for [120] D. Li, M. Zang, X. Li, K. Zhang, Z. Zhang, S. Wang, A study on the food fraud of
qualitative and quantitative estimation of tyrosinase inhibitors in natural national food safety and sample inspection of China, Food Contr. 116 (2020)
products, Phytochem. Anal. 28 (2017) 115e124. 107306.
[104] A. Urbain, C.A. Simo ~es-Pires, Thin-layer chromatography for the detection
and analysis of bioactive natural products, in: R.A. Meyers (Ed.), Encyclo-
pedia of Analytical Chemistry, JohnWiley & Sons, Hoboken, NJ, USA, 2020.
[105] S. Agatonovic-Kustrina, G. Ramenskaya, E. Kustrin, D.W. Morton, Charac-
terisation of a-amylase inhibitors in marigold plants via bioassay-guided
high-performance thin-layer chromatography and attenuated total
reflectanceeFourier transform infrared spectroscopy, J. Chromatogr. B 1173
(2021) 122676.
[106] E.V.S. Maciel, A.L. de Toffoli, E. Sobieski, C.E.D. Naza
rio, F.M. Lanças, Minia- Gertrud Elisabeth Morlock
turized liquid chromatography focusing on analytical columns and mass holds the Chair of Food Sci-
spectrometry: a review, Anal. Chim. Acta 1103 (2020) 11e31. ence at the Justus Liebig
[107] F. Schade, W. Schwack, Y. Demirbas, G.E. Morlock, Open-source all-in-one University Giessen, and is
LabToGo Office Chromatography, Anal. Chim. Acta (2021) in print. also Director of the TransMIT
[108] K. Shulla, W.L. Filho, J.H. Sommer, A. Lange Salvia, C. Borgemeister, Channels Center for Effect-Directed
of collaboration for citizen science and the sustainable development goals, Analysis. She was awarded
J. Clean. Prod. 264 (2020) 121735. with the Young Scientist
[109] D. Baker, M. van den Beek, D. Blankenberg, D. Bouvier, J. Chilton, N. Coraor, et Award (Kurt-T€aufel-Preis) of
al., No more business as usual: agile and effective responses to emerging the German Society of Food
pathogen threats require open data and open analytics, PLoS Pathog. 16 Chemistry in 2009 and with
(2020), e1008643. the Father of Stevia Award
[110] G.E. Morlock, Miniaturized planar chromatography using office peripherals in 2018. She has made about
e Office chromatography, J. Chromatogr. A 1382 (2015) 87e96. 170 peer-reviewed original
[111] S. Kirchert, M. Schulz, M. Oberle, G.E. Morlock, Development of a new par- research paper since 2006,
ticulate 4-mm adsorbent layer for ultrathin-layer chromatography (minia- 76 further scientific papers,
turized chromatogram), J. Chromatogr. A 1587 (2019) 247e255. 15 book chapters, 250 post-
[112] D. Fichou, P. Ristivojevic, G.E. Morlock, Proof-of-principle of rTLC, an open- ers and 280 oral presenta-
source software developed for image evaluation and multivariate analysis tions, conducted 70
of planar chromatograms, Anal. Chem. 88 (2016) 12494e12501. workshops and edited the
[113] C. Audoin, S. Holderith, K. Romari, O. Thomas, G. Genta-Jouve, Development online database CCBS con-
of a workflow for high-performance thin-layer chromatography data pro- taining ca. 11,000 abstracts
cessing for untargeted metabolomics, J. Planar Chromatogr. 27 (2014) for 17 years. She is active in
328e332. scientific committees, indus-
[114] Matthias Guggenberger, Josua T. Oberlerchner, Heinrich Grausgruber, t r i a l b o a r d s a n d ex p e r t
Thomas Rosenau, Stefan Bo €hmdorfer, Self-Organising Maps for the groups.

14