June 29

Claudia Oellig


Institute of Food Chemistry, Department of Food Chemistry and Analytical Chemistry (170a), University of Hohenheim, 70599 Stuttgart, Germany

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Simple and fast screening methods are becoming more and more important for the analysis of residues and contaminants in food, since the number of samples and (un)known contaminants are steadily increasing. These methods are mainly aiming at fast and easy separation and detection techniques and the development of alternative sample clean-up procedures for a higher reliability and sensitivity of the analyses with high throughput.
In this respect, the potential of high-performance thin-layer chromatography (HPTLC) is perfectly suited which was used to develop the concept of planar solid phase extraction (pSPE) in 2011. In the beginning, pSPE was developed as an effective and efficient clean-up method for multi-residue analysis of pesticides in food [1, 2, 3]. Generally, the pSPE concept combines the separation of the target analytes of chemical groups from the matrix and focusing them in one or more target zone(s). Thus, the concept can be amended for a broad spectrum of groups of food contaminants. Successful examples are: (i) a pSPE screening for the sum of ergot alkaloids in rye with fluorescence detection (FLD) [4], (ii) pSPE–FLD screening approaches for the analysis of MOSH and MOAH in food packaging materials [5] and vegetable oils [6], (iii) a pSPE screening for the sum of nonylphenols in surface water [7] using the planar yeast estrogen screen [8] for detection, and (iv) a pSPE screening for the analysis of chlorinated paraffins in vegetable oils [9]. Further pSPE methods are currently under development, for example, a method for the analysis of pyrrolizidine alkaloids.

References
[1]  C. Oellig, W. Schwack, J. Chromatogr. A 1218 (2011) 6540-6547.
[2]  C. Oellig, W. Schwack, J. Chromatogr. A 1260 (2012) 42-53.
[3]  C. Oellig, W. Schwack, J. Chromatogr. A 1351 (2014) 1-11.
[4]  C. Oellig, T. Melde, J. Chromatogr. A 1441 (2016) 126-133.
[5]  M. Wagner, C. Oellig, J. Chromatogr. A 1588 (2019) 48-57.
[6]  M. Wagner, C. Oellig, J. Chromatogr. A 1662 (2022) 462732.
[7]  D. Schick, C. Oellig, Anal. Bioanal. Chem. 411 (2019) 6767-6775.
[8]  D. Schick, W. Schwack, J. Chromatogr. A 1497 (2017) 155-163.
[9]  C. Oellig, Y.-A. Hammel, J. Chromatogr. A 1606 (2019) 460380.

Mario Aranda¹, Karem Henriquez-Aedo², Jonathan Carrasco-Sandoval², Pedro Aqueveque<sup>2

1</sup>Pontificia Universidad Católica de Chile, Av. Vicuña Mackenna 4860, 7820436, Santiago, Chile
²Universidad de Concepción, Barrio Universitario s/n, 4070139, Concepción, Chile

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Chronic non-communicable diseases (CNCD) are responsible for 41 million of deaths per year, which represents 71% of all deaths worldwide. The most relevant ones are cardiovascular, cancer, diabetes, and chronic respiratory diseases accounting for around 80% of all CNCD deaths. These figures have increased the interest in searching bioactive molecules in natural sources with therapeutic potential over CNCD and neurodegenerative diseases.
Several analytical techniques have been applied to detect this kind of molecules but each one has critical drawbacks. In this scenario, high performance thin layer chromatography (HPTLC) emerges as the technique of choice for bioactive molecules detection and identification because is unique features like the capacity of performing effect-directed analysis (bioassay) straight on the HPTLC plate and the direct coupling with mass spectrometry (MS).
This presentation describes some examples of HPTLC/bioassay/MS approach to detect and identify bioactive molecules with therapeutic potential against CNCD and neurodegenerative diseases, in particular, those with inhibitory activity over key enzymes such as acetylcholinesterase, alpha-glucosidase and cyclooxygenase.
In this respect, a new HPTLC-bioassay to detect cyclooxygenase inhibitors is presented, which is based on the oxidation of N,N,N,N-tetramethyl-p-phenyl diamine (TMPD) during the reduction of prostaglandin G2(PGG2) to prostaglandin H2(PGH2), reaction monitored at 590 nm. Microplate bioassay conditions, i.e., arachidonic acid and TMPD concentration and incubation time, were optimized via design of experiments and then transferred to HPTLC showing promising results such as inhibitors detection at ug level.

Acknowledgments
Authors want to thank to Chilean National Agency of Research and Development (ANID) for doctoral scholarship granted. This work was financially supported by projects FONDECYT Nº1211803 and REDES Nº180178.

Ágnes M. Móricz

Plant Protection Institute, Centre for Agricultural Research, ELKH, Herman O. Str. 15, 1022, Budapest, Hungary

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HPTLC hyphenations enable fast and high-throughput screening for bioactive compounds of complex matrices as well as their characterization. However, in several cases the identification of the effective compounds is impossible due to their insufficient separation or low abundance. Here are three solutions.
Overpressured layer chromatography (OPLC) as a pump-driven forced flow technique enabled a longer development distance, thus the partial separation of two tansy root compounds, which were not separable by conventional HPTLC [1]. The OPLC-bioassays and OPLC-DART-HRMS showed six compounds responsible for different bioactivity and allowed the identification of four polyacetylenes. Moreover, using irregular particle layers, the separation time was approximately halved by OPLC, if compared to HPTLC.
Two closely related constitutional isomers, oleanolic acid and ursolic acid are co-migrated in normal phase HPTLC. These compounds are not distinguishable by HRMS and give a common inhibition zone in the HPTLC-bioassays. The two-dimensional liquid chromatographic hyphenation, the heart-cutting HPTLC-HPLC-DAD-MS via an elution head-based interface enabled the separation and assignment of both isomers in the lemon balm leaf [2]. Ursolic acid was more abundant and is mainly responsible for the bioeffects.
In bilateral band compression (BBC) the samples are applied in band, and after development, the lanes are compressed in a perpendicular direction from both sides with appropriate solvent. HPTLC-BBC resulted in more than six times higher peak height and area, however, the noise level and the RSDs were increased as well [3]. OPLC-BBC-bioassay allowed the bio-detection of the minor antibacterial components of thyme essential oil, which were not detectable by a conventional OPLC-bioassay process.

Acknowledgement
This work was partially funded by the National Research, Development and Innovation Office of Hungary (NKFIH K128921).

References
[1]  Á.M. Móricz, T.T. Häbe, P.G. Ott, G.E. Morlock, J. Chromatogr. A 1603 (2019) 355-360.
[2]  Á.M. Móricz, V. Lapat, G.E. Morlock, P.G. Ott, Talanta, 219 (2020) 121306.
[3]  Á.M. Móricz, E. Mincsovics, P.G. Ott, J. Liq. Chromatogr. Relat. Technol., 43 (2020) 300-304.

Eike Reich

HPTLC Association, Dianastrasse 10, 4310 Rheinfelden, Switzerland

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With the commercial introduction of plates coated with fine particles by MERCK in the late 1970s, the foundation for the evolution of HPTLC was laid. Over the last four decades, significant progress has been seen in the development of instruments. Most recently, even fully automatic systems became available. The available tools - instrument and software - have supported scientific research of all aspects of planar chromatography, stimulated curiosity, and expanded the range of what is possible.
Yet, it seems that it was the discussion of a rigorous concept of standardization, its subsequent implementation into pharmacopoeias, and the resulting enforcement by authorities, that made HPTLC a distinct and accepted technique for routine analysis, particularly in quality control of herbal materials. What will be next?
This presentation connects fundamental concepts, such as standardization and comprehensive HPTLC fingerprinting, with current ideas about the evaluation of system suitability, comparison of data across multiple plates, and the use of complementary developing solvents, leading to HPTLC 4.0. This vision may be developed into a global platform for collaboration of researchers and routine users of HPTLC. It has the potential of taking the inherent advantages of planar chromatography into a bright future.

Irena Maria Choma, Hanna Nikolaichuk, Ewelina Sobstyl

Faculty of Chemistry, Maria Curie-Skłodowska University, Maria Curie-Skłodowska Sq.3, 20 031 Lublin, Poland

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The plant kingdom is an inexhaustible source of biologically active compounds used as components of supplements and pharmaceutical preparations. Effect directed detection (EDD), i.e. thin-layer chromatography combined with direct bioautography (TLC-DB), and TLC-screening are convenient tools for bio-profiling of active constituents of plants. In TLC-DB, the effect observed and measured directly on a TLC plate results from an action of biologically active constituents of a sample, e.g., inhibition of bacteria growth related to antibacterial activity of separated plant constituents. The detected bioactive components can be further identified using spectroscopic techniques such as GC-MS, LC-MS, IR, NMR. TLC–bio-profiling can also be used to distinguish among plants from various species and origin, find the most active ones, and point to possible adulterations. We focused on Schisandraceae family and Rhodiola rosea L. (various species, supplements, and pharmaceutical preparations) [1-3]. The most interesting findings were the strong antibacterial properties of S. chinensis and S. rubriflora fruits, as well as acetylcholinesterase, glucosidase, tyrosinase, and lipase inhibition of all tested species. Strong glucosidase inhibition was observed for Kadsura japonica fruits, which generally did not reveal biological activities. NP-PEG assay and TLC-DPPH test showed that leaves of various Schisandra species are rich in polyphenolic compounds and have strong antioxidant properties (especially leaves of S. rubriflora and K. japonica). Strong antioxidant, antibacterial and enzyme-inhibiting properties were found for the root and rhizome of R. rosea L.. Various R.rosea pharmaceutical preparations and supplements differ in their biological properties as well as in the content of marker compounds: rosavin, salidroside, and tyrosol.

References
[1]  E. Sobstyl, A. Szopa, H. Ekiert, S. Gnat, R. Typek, I.M. Choma, J. Chromatogr. A, 1618 (2020) 460942.
[2]  H. Nikolaichuk, M. Studziński, I.M. Choma, J. Liq. Chromatogr. Rel. Technol., 43 (2020) 361-366.
[3]  H. Nikolaichuk, R. Typek, S. Gnat, M. Studziński, I.M. Choma, J. Chromatogr. A, 1649 (2021) 462217.

Hanna Nikolaichuk¹, Gertrud Morlock², Irena M. Choma<sup>1

1</sup>Department of Chromatography, Faculty of Chemistry, Maria Curie-Sklodowska University, Lublin, Poland
²Chair of Food Science, Justus Liebig University Giessen, Giessen, Germany

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High-performance thin-layer chromatography (HPTLC) is a suitable method for separating, searching, and evaluating biological samples with complicated matrices such as plant extracts. Moreover, the HPTLC is compatible with detecting samples' biological and biochemical activity. The great advantage of HPTLC is the possibility of analyzing many samples and comparing the composition of the samples in one run. The detection of antibiotics, antioxidants, enzyme inhibitors, genotoxic compounds, and estrogen and androgen activity can be performed directly on the HPTLC plate. Additionally, the advantage of HPTLC is the direct possibility of isolating and identifying the target substances using high-resolution mass spectrometry (HRMS) [1,2].
In our present study, HPTLC hyphenated HRMS was applied to determine bioactive substances in 15 supplement preparations of Rhodiola rosea L. root, in fruits and leaves of Akebia quinata Decaisne, and flower of Clitoria ternatea L.. For this purpose, the enzyme inhibition assays such as acetylcholinesterase, butyrylcholinesterase, tyrosinase, lipase, α/β-glucosidase, α-amylase, and β-glucuronidase were used. Antibacterial properties were investigated using Gram-positive bacteria Bacillus subtilis and Gram-negative bacteria Avilibrio fischeri assays. Additionally, the plants were tested for estrogen, antiestrogen, androgen, and antiandrogen activity. Moreover, samples were tested for genotoxic activity. The active bands detected by bioassays directly on the HPTLC plate were identified using the TLC-MS interface.

References
[1] Nikolaichuk, H.; Typek, R.; Gnat, S.; Studziński, M.; Choma, I.M., J. Chromatogr. A 1649, (2021).
[2] Morlock, G.E., In Encyclopedia of Analytical Science, (2019).

Ashis Kumar Goswami¹, Hemanta Kumar Sharma<sup>2

1</sup>Girijananda Chowdhury Institute of Pharmaceutical Science, Azara, Guwahati, Assam-781017, India
²Department of Pharmaceutical Sciences, Faculty of Science and Engineering, Dibrugarh University, Dibrugarh, Assam-786004, India

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Assam, a state in India, belongs to the eastern Himalayan biodiversity region, wherein the local population uses various herbs to treat different ailments; but because of the indiscriminate use, these herbs have become endangered in their natural habitat. Two such herbs are Coptis teeta and Homalomena aromatica; the high polarities of protoberberine alkaloids have made it difficult to quantify the alkaloids in C. teeta whereas, it is a tedious stuff to separate the various terpenoids in the volatile oil of H. aromatica. Two distinct TLC based densitometric methods were developed for identification, quantification and validation of linalool in the volatile oil of rhizomes of H. aromatica and berberine in the methanol extract of rhizomes of C. teeta. The separation for quantification of berberine in C. teeta was achieved in a solvent system consisting of Butanol: Ethyl acetate: Formic acid: Water on TLC aluminum plates precoated with silica gel 60 F₂₅₄. For quantification of linalool, the chromatographic plates were developed in Toluene: Ethyl acetate solvent system at a ratio of 9.5:0.5 and visualized with p-anisaldehyde reagent. The validated method gave compact bands of berberine at R_F of 0.70 whereas, compact bands of linalool was observed at Rf of 0.35. Both the methods developed were precise, accurate and reproducible with regards to the International Conference on Harmonization guidelines. Linear results were obtained for both berberine and linalool with correlation coefficients of 0.997±0.09 % (R±SD) in the concentration range of 90-210 ng/band and 0.980±0.05 % (R±SD) in the concentration range of 2-20 nL/band respectively. The concentration of berberine in C. teeta was found to be 30.97±0.55 mg in 100 mg of the crude drug whereas, the concentration of linalool in the volatile oil of H. aromatica was found to be 5.839±0.42 μL in 10.000 μL of the H. aromatica volatile oil. The method developed here in can be implemented in the analysis and routine quality control of herbal materials and formulations containing C. teeta and berberine as well as volatile oils containing linalool and quality control of rhizomes of H. aromatica.

Gertrud Morlock

Justus Liebig University Giessen, Heinrich-Buff-Ring 26-32, 35392 Giesssen, Germany

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Is it a dozen or even more? The more complex the samples, the more information is helpful in understanding about their composition. Chromatographic systems and detectors are coupled in hyphenated techniques to extract the maximum amount of information from a chromatographic run. Combining the best features of planar chromatography, effect-directed assays, column chromatography, and spectral detections results in powerful hyphenations [1]. It reveals how efficiently and effectively our natural plant-based food contributes to our homeostasis, and straightforwardly gives important answers [1-4]. Miniaturization and method greenness (eco-friendliness) are also important keys. To achieve this, the simulation of the coordinated processes of all digestion segments was miniaturized and combined with separation and effect-detection. [5] The potential of new technologies was used to create a miniaturized all-in-one open-source 2LabsToGo system [6].

References
[1]  G.E. Morlock, Anal. Chim. Acta 1180 (2021) 338644.
[2]  T. Schreiner, A. Ronzheimer, M. Friz, G.E. Morlock, Multiplex planar bioassay with reduced diffusion on normal phase: androgens and verified antiandrogens in botanicals, in submission.
[3]  I. Klingelhoefer, N. Hockamp, G.E. Morlock, Anal. Chim. Acta 1125 (2020) 288-298.
[4]  T. Schreiner, D. Sauter, M. Friz, J. Heil, G.E. Morlock, Front. Pharmacol. 12 (2021) 755941.
[5]  G.E. Morlock, L. Drotleff, S. Brinkmann. Anal Chim Acta 1154 (2021) 338307.
[6]  L. Sing, W. Schwack, R. Göttsche, G.E. Morlock, 2LabsToGo − Recipe for building your own chromatography equipment including biological effect-detection in complex samples, in submission.

Maricel Marin-Kuan, Emma Debon, Helia Latado, Bastien Gentili, Claudine Cottet, Patrick Serrant, Lucrecia Pons, Julie Mollergues, Karma Fussell, Amaury Patin

Société des Produits Nestlé, Vers-ches-les-Blanc, 1000 Lausanne Switzerland

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Assessing the safety of food is challenging as foods are complex mixtures containing thousands of known and unknown substances occurring at various concentrations. Theoretically, to thoroughly address safety of foods, all chemicals of concern should be identified and toxicologically tested. Standard safety assessment combines exposure data with toxicological information to make a statement on potential health impact of chemicals. Because of ethical and scientific reasons, the use of animal studies in toxicology is undesirable and must be restricted to a minimum. In addition, standard in vivo toxicological studies are very resource and time consuming and may constitute a bottleneck in new product development.
The key question is how to detect chemicals of most concern on which further toxicological investigations should be focused? The use of bioassays has been proposed as a breakthrough solution to address the unidentified and uncharacterized compounds present in mixtures. Bioassays were designed to detect hazards i.e chemicals with specific bioactivities/properties. Bioassays targeting toxicologically relevant endpoints (cytotoxicity, endocrine activity and genotoxicity) unravel the presence of toxicology-relevant chemicals in the mixture under investigation providing information for risk assessment (1).
Recent progress using Thin Layer Chromatography (HPTLC) coupled to bioassays has been proposed as a breakthrough solution to fill some of the gaps identified with current multi-well plate bioassays (2). HPTLC effect-direct analysis (EDA) for toxicologically relevant endpoints will be presented anchoring bioassays with HPTLC and chemical analysis in complex mixtures of unknown composition with the goal to optimize and speed up decision-making in Food Safety.

References
(1) Schilter, B., Burnett, K., Eskes, C. et al. (2019). Value and limitation of in vitro bioassays to support the application of the threshold of toxicological concern to prioritize unidentified chemicals in food contact materials. Food Addit Contam 36, 1903-1936.
(2) Daniel Meyer, Maricel Marin-Kuan, Emma Debon, et al., 2020. Detection of Low Levels of Genotoxic Compounds in Food Contact Materials using an alternative HPTLC-SOS UmuC Assay. ALTEX – Alternatives to animal experimentation

Etil Guzelmeric¹, Nisa Beril Sen¹, Ecesu Sezen², Rengin Reis³, Hande Sipahi⁴, Vesna Glavnik⁵, Irena Vovk⁵, Erdem Yesilada<sup>1

1</sup>Yeditepe University, Faculty of Pharmacy, Department of Pharmacognosy, Kayisdagi Cad., Atasehir, 34755, Istanbul, Turkey
²Yeditepe University, Faculty of Pharmacy, Kayisdagi Cad., Atasehir, 34755, Istanbul, Turkey
³Acıbadem Mehmet Ali Aydınlar University, Department of Toxicology, Kayisdagi Cad., No:32, Atasehir, 34755, Istanbul, Turkey
⁴Yeditepe University, Faculty of Pharmacy, Department of Toxicology, Kayisdagi Cad., Atasehir, 34755, Istanbul, Turkey
⁵Laboratory for Food Chemistry, National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia

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Turkey is located at the intersections of European-Siberian, Iranian-Turan and Mediterranean phyto-geographical regions harbouring a rich flora of over 1,750 plant taxa. Turkey links Asia to Europe and contributes to exchanging plant species between the two continents. Slovenia is situated in Central Europe at the midpoint of Mediterranean area, Alpine region, and Pannonian plain. Beekeeping is one of the oldest and most traditional agricultural activities in both countries. Among the bee products, bee pollen is composed of floral pollen with nectar or honey, enzymes, wax, and bee secretion. It is used in apitherapy and diet due to its highly health-promoting components. It is recognised as "the only perfectly complete food" because it includes all the essential amino acids required by people. However, its chemical composition is directly related to the plant source around the beehive.
This study aimed to comparatively investigate the chemical and bioactivity profiles of thirty bee pollen samples from Turkey and Slovenia. Initial palynological analysis demonstrated that twenty samples were monofloral bee pollen originating mostly from Castanea sativa, Salix sp., Bellis sp., and Hedera helix, and the rest were multifloral. Besides, thirty compounds were screened by HPTLC method and the most common phenolics in bee pollen samples were determined as caffeic acid, chlorogenic acid, rutin, kaempferol, quercetin, hyperoside and luteolin. HPTLC-MSⁿ analyses resulted also in identification of unknown compounds. In bioactivity testing, the monofloral bee pollen sample from Castanea sativa showed the highest antioxidant activity by DPPH, CUPRAC, and FRAP methods. Moreover, the bee pollen samples at non-cytotoxic concentrations showed concentration-dependent anti-inflammatory activity through the nitrite inhibition in LPS-activated RAW264.7 cells.

This study was supported by The Scientific and Technological Research Council of Turkey (TÜBİTAK; Project No: 119N569) and the Slovenian Research Agency (ARRS; research core funding No. P1-0005 and the bilateral project BI-TR/20-23-004).

Markus Flörs^1,2, Wolfram Seitz¹, Rudi Winzenbacher<sup>1

1</sup>Zweckverband Landeswasserversorgung, Am Spitzigen Berg 1, 89129 Langenau, Germany
²Eberhard Karls University of Tübingen, Department of Geoscience, Schnarrenbergstraße 94-96, 72074 Tübingen, Germany

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The discharge of anthropogenic substances into the environment causes a great number of chemicals to be ubiquitous in waterbodies and the aquatic environment [1]. Since these substances are sometimes toxic (e.g. pesticides) and due to their ubiquitous nature, they are often well researched and already closely monitored. By contrast, metabolites or especially transformation products of these substances are oftentimes unknown or only sparsely researched. Therefore, only little or no information about their toxicity is available. To monitor this huge number of unknown metabolites and transformation products and evaluate their toxicity effect-directed analysis is used [2].
Established bioassays in combination with HPTLC enable hormonal or neurotoxic compounds to already be monitored. Besides these endpoints, direct and indirect acting genotoxic substances are also of major concern for drinking water production [3]. Therefore, we investigated the combination of the HPTLC-umuC assay with a prior S9 metabolic activation step.
One of the goals was to adapt the media of the existing umuC microtiter plate bioassay [4] to the newly developed HPTLC-umuC assay [5]. Thus, the newly developed bioassay can easily be implemented by laboratories which already perform the umuC microtiter plate assay. The other goal of this study was to investigate the suitability of an external S9 metabolic activation step prior to HPTLC fractionation without interfering with fluorescence detection or HPTLC separation. As a result the developed S9 metabolic activation step could not only be used with the newly developed HPTLC-umuC assay but also with previously established HPTLC hyphenated bioassays (e.g. yeast estrogen screen or acetylcholinesterase inhibition assay) to detect metabolically induced effects.

References
[1]  R. Loos, R. Carvalho, D. C. António, et al., Wat. Res. 47 (2013) 6475-6487.
[2]  Z. Tousova, P. Oswald, J. Slobodnik, et al., Sci. Total Environ, 601 (2017) 1849-1868.
[3]  L. Stütz, P. Leitner, W. Schulz, et al., J. Planar Chromatogr. 32 (2019) 173-182.
[4]  DIN 38415-3:1996-12, Beuth Verlag, Berlin, 1996.
[5]  D. Meyer, M. Marin-Kuan, E. Debon et al., ALTEX,38 (2021) 387-397.