June 30

Jean-Luc Wolfender, Adriano Rutz, Arnaud Gaudry, Luis Quiros Guerrero, Olivier Kirchhoffer, Louis-Félix Nothias, Emerson Ferreira Queiroz, Pierre-Marie Allard

School of Pharmaceutical Sciences, University of Geneva, CMU – Rue Michel Servet 1, 1211 Geneva 11, Switzerland

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The recent rapid innovations made in metabolite profiling and bioassays have begun to drive a change of paradigm in natural products research. Indeed, having at hand full or partial of structure of possibly all metabolites in given natural extract at different quantitative levels open the possibility to perform pharmacognosy studies from a more holistic perspective.
The increasingly amount of accurate metabolome data that can be acquired on massive sample sets, notably through high resolution mass spectrometry data dependent MS/MS analyses (HRMS/MS), allows mapping of natural extracts at an unprecedented precision level [1]. While the acquisition of larger volumes of data is ongoing, contextualizing it is a lagging process and new initiative such as LOTUS contributes to this effort by linking chemical, taxonomical and bibliographical information [2].
In this context we push forward our applications and further development of UHPLC-HRMS/MS Molecular network (MN) approaches [3] to provide enhanced annotation confidence level. We have recently built massive MN on natural extracts libraries, including thousands of plant and microorganism extracts. This allows the creation of virtual libraries of NPs through an advanced automated annotation pipeline. We are developing computational methods and structural-taxonomic libraries to navigate this massive chemical space in order to rationally identify valuable bioactive NPs to focus on. For their full chemical and pharmacological characterization, we then deploy an advanced workflow that allows precise targeted isolation based on metabolite profiling data. Different recent applications will illustrate these aspects and give future prospects in natural products research.

Acknowledgements
The authors are grateful to Green Mission Pierre Fabre for establishing and sharing the unique library of extracts and the Swiss National Science Foundation (SNF N° CRSII5_189921 / 1).

References
[1]  J.L. Wolfender, J.M. Nuzillard, J.J.J. van der Hooft, J.H. Renault, Anal. Chem. 91 (2019) 704-742.
[2]  A. Rutz, M. Sorokina, J. Galgonek, D. Mietchen, E. Willighagen, A. Gaudry, J.G. Graham, R. Stephan, R. Page, J. Vondrášek, C. Steinbeck, G.F. Pauli, J.L. Wolfender, J. Bisson, P.M. Allard, bioRxiv (2021) 2021.2002.2028.433265.
[3]           A. Rutz, M. Dounoue-Kubo, S. Ollivier, J. Bisson, M. Bagheri, T. Saesong, S.N. Ebrahimi, K. Ingkaninan, J.L. Wolfender, P.M. Allard, Front. Plant Sci. 10 (2019) 1329.

Paola Dugo¹, Katia Arena¹, Francesco Cacciola², Luigi Mondello<sup>1

1</sup>University of Messina, Dipartimento CHIBIOFARAM, polo Annunziata, 98168 Messina, Italy
²University of Messina, Dipartimento BIOMORF, polo Annunziata, 98168 Messina, Italy

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Natural products and vegetable extracts are rich source of bioactive molecules such as flavonoids, alkaloids, saponins, and so on. Although many studies have been carried out on the chemical characterization of these samples, there is still much interest in the detection of novel bioactive compounds. Conventional one-dimensional liquid chromatography (1D-LC) does usually provide sufficient resolving power for their determination. However, in many cases, their complexity is so high that the separation capability offered by 1D-LC is not enough thus requiring more powerful analytical technologies for both their characterization and quantification. Comprehensive two-dimensional LC (LC×LC) involving the coupling of two or more orthogonal or quasi-orthogonal separation systems is an interesting alternative capable, through its selectivity and sensitivity, to detect minor components.
For the analysis of natural products and vegetable extracts, different LC×LC methodologies including NP×RP, RP×RP and HILIC×RP have been successfully investigated. In this contribution, applications of MicroLC×LC in the field of natural products are reported and discussed.

Anna Laura Capriotti, Andrea Cerrato, Carmela Maria Montone

Department of Chemistry, University of Roma La Sapienza, Piazzale Aldo Moro,5 00185 Rome, Italy

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Short peptides are of extreme interest in clinical and food research fields; nevertheless, they still represent a crucial analytical issue. This lecture aims to provide an overview of analytical solutions developed for enrichment, separation, and untargeted identification of short peptides in biological matrices (i.e.urine) and food matrices. Direct analysis of short peptides is not usually feasible due to the potential association with high abundant components, such as proteins, and the low abundance of peptides compared to other molecules, which can cause extensive ion suppression during electrospray ionization (ESI). A dedicated protocol for enrichment and clean-up of short peptides can overcome some of these issues. In particular, the use of carbonaceous material for the enrichment and separation will be discussed, highlighting the advantages of retaining polar, non-polar, acidic, and positively zwitterionic charged compounds due to strong hydrophobic and ion-exchange interaction [1]. However, sample clean-up is not straightforward either due to the heterogeneous nature of short peptides in polarity, hindering their pre-concentration from complex matrices and chromatographic separation. Alternative separation strategies will be considered to improve the coverage of short polar peptides. Another aspect that will be discussed in detail will be developing a database implemented in Compound Discoverer 3.0, a software dedicated to the analysis of short molecules, to create a data processing workflow specifically dedicated to short peptide tentative identification. Finally, an untargeted metabolomics approach of prostate cancer zwitterionic and positively charged compounds in urine will be presented [2].

Acknowledgements
The work was supported by the PRIN project Prot. 2017Y2PAB8, entitled “Cutting Edge Analytical Chemistry Methodologies and Bio-Tools to Boost Precision Medicine in Hormone-Related Diseases”, provided by the Italian Ministry of Education, Universities and Research.

References
[1]  S. Piovesana, A.L. Capriotti, A. Cerrato, C. Crescenzi, G. La Barbera, A. Laganà, C. M. Montone, C. Cavaliere, Anal Chem. 45 (2019) 11474-11481.
[2]  A. Cerrato, C. Bedia, A.L. Capriotti, C. Cavaliere, V. Gentile, M. Maggi, C. M. Montone, S. Piovesana, A. Sciarra, R. Tauler, A. Laganà, Anal. Chim. Acta, 1158 (2021) 338381.

Günther K. Bonn

Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University of Innsbruck, Innrain 80-82, 6020 Innsbruck, Austria. ADSI - Austrian Drug Screening Institute, 6020 Innsbruck

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Innovative approaches in analytical chemistry have become increasingly important for natural product research and new advances in separation science enable research into inaccessible areas of natural product isolation. Since herbal preparations like plant extracts are often multi-component mixtures with hundreds or thousands of small molecules at different concentrations, their separation and analysis is often difficult to perform. Thus, novel enrichment and purification methods based on advanced solid-phase extraction techniques have been developed to reduce the complexity of plant extracts, while HPLC has been used for separation, pre-concentration and fractionation. The ability to apply these techniques to robotic systems enables high throughput screening. Significant progress has been made in developing new stationary phases that can be tailored to a specific application and thus offer endless possibilities for optimizing selectivity. A further coupling to high-resolution mass spectrometry facilitates the identification and quantification of active ingredients in natural products. Additionally, the combination of separation science with spectroscopy offers the possibility to combine various technologies in phytopharmacy as well as food analysis. Near- and mid- infrared spectroscopy enable quick and simultaneous qualitative and quantitative analysis of raw plants and liquid extracts without destruction. Moreover, infrared imaging has been used to study the distribution of active substances in plant materials. All of these approaches offer new strategies for quality control in phytoanalysis and enable deeper insights into the biochemical background of medically relevant questions. In this presentation, novel approaches in analytical chemistry for various applications in the areas of phytopharmacy, phytocosmetics and phytonutrients are demonstrated and discussed.

 

 

Danilo Corradini
CNR, Institute for Biological Systems, Area della Ricerca di Roma 1, Via Salaria Km 29,300 - 00015 Monterotondo Stazione (Rome), Italy

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Plant secondary metabolites are not only an important part of plant defense system against pathogenic attacks and environmental stresses, but also a useful array of natural products with remarkable biological activities. Most of them are the therapeutic agents occurring in medicinal plants and play important roles in disease prevention and health-promoting effects of edible plants and plant-derived food products and dietary supplements. Moreover, a variety of plant secondary metabolites confer specific sensory characteristics to plant-based foods, processed or not, whose botanical and geographical origin may be related to their occurrence and amount. Thus, these compounds are also potential chemical markers for the evaluation of botanical and geographical origin of plant-derived food products and dietary supplements.
This communication describes the development of computer-assisted RP-HPLC methods for the separation, identification and quantification of phenolic compounds, which are a large class of plant secondary metabolites with health-promoting properties comprising a great number of heterogeneous structures. The study has been conducted by a Design of Experiments (DoE) approach that allows the simultaneous optimization of gradient time (tG), column temperature (T) and binary or ternary mobile phase composition on the basis of the retention times and peak areas of selected phenolic compounds, obtained by a restricted number of experiments. The resulting RP-HPLC methods have been used to investigate the occurrence and content of phenolic compounds in leaves and fruits of olive trees (Olea europaea), in monovarietal extra virgin olive oil of different cultivar and geographical origin, and in olive mill waste water produced by processing the same selected batches of olive fruits by a laboratory olive oil press machine. The different phenolic compound profiles and content determined in samples differing for botanical and geographical origin and/or for the technological transformation process are illustrated and discussed.

Acknowledgements
This work was supported by MSCA-RISE Action of the H2020 Programme, Project 734899 - Olive-Net.

Blanka Bucsella^1,2, Anika Hoffmann¹, Mathieu Zollinger¹, Kevin Pacios¹, Fabio Stephan^1,2, Martin Pattky^1,2, Ralph Daumke³, Reto Mühlemann³, Brian Frank³, Franka Kalman<sup>1

1</sup>HES-SO Valais-Wallis, Institute of Life Technology, Rue de l’Industrie 23, 1950 Sion, Switzerland
²Lonza AG, Quality Control Biopharma, Rottenstrasse 6, 3930 Visp, Switzerland
³FILTROX AG, Moosmühlestr. 6, 9001 St. Gallen, Switzerland

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Endotoxin (ET) testing in pharmaceuticals is a crucial requirement for patient safety. This paper presents a novel instrumental analytical ET quantification assay [1]. The Kdo-DMB-LC assay uses common analytical laboratory equipment ((U)HPLC-FLD) and allows the quantification of ETs in complex matrices from about 10e7 EU / mL down to about 30 EU / mL (RSE based). Test results are obtained in concentration units (e.g., ng ET / mL), which can then be converted to commonly used ET activity units (EU / mL). During mild acidic hydrolysis, the rare ET specific 3-deoxy-D-manno-oct-2-ulsonic sugar acid (KDO) is obtained quantitatively. After that, KDO is stoichiometrically reacted with DMB, which results in a highly fluorescent derivative. The mixture is separated using RP-(U)HPLC followed by KDO-DMB quantification by fluorescence detection. From the KDO content the ET content in a sample is calculated. The applicability of the Kdo-DMB-LC in applied research is demonstrated. ETs were quantified in partially purified bacterial biopolymers, which were produced by Gram-negative bacteria. Results were compared to LAL results of the same samples. A high correlation was found between the results of both methods. Further, the new assay was successfully utilized for the development of novel ET specific depth filters, which allow efficient, economic, and sustainable ET removal e.g., during DSP. In addition, the ET content was monitored in the supernatants of Escherichia coli K12 and Pseudomonas putida KT2440 cultivations from inoculation until harvest [2]. The Kdo-DMB-LC assay is an easy to install tool to optimize reactor sttings with respect to the ET content in dependence on cultivation time and conditions, a task difficult to achieve using the common LAL assay. It is economic, has a small error and it has the potential to complement the animal-based biological LAL pyrogenic quantification tests, which are accepted today by the health authorities worldwide for the release of commercial pharmaceutical products. The new Kdo-DMB-LC assay brings ET testing to the 21st century.

References
[1] B. Bucsella, A. Hoffmann, M. Zollinger, F. Stephan, M. Pattky, R. Daumke, F. Heiligtag, B. Frank, M. Bassas Galia, M. Zinn and F. Kalman, Analytical Methods, 12(38) (2020) 4621-4634.
[2] A. Hoffmann, Anika Hoffmann¹, K. Pacios, R. Mühlemann, R. Daumke, B. Frank and F. Kalman, in preparation.

Imre Molnár, Hans-Jürgen Rieger, Arnold Zöldhegyi

Molnár-Institute, Schneegloeckchenstrasse 47, 10407 Berlin, Germany

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First of all, we are thankful to Danilo Corradini, a former important coworker of Csaba Horváth and the chairwoman Irena Vovk to organize the Csaba Horváth session.

Csaba Horváth was not only a pioneer in the creation of a whole new revolutionary analytical technology, which he christened in 1967 High Pressure Liquid Chromatography or HPLC. He was also the first, using the computer in his research work. He purchased in the spring of 1976 a PDP-11 brand new computer which had the size of ca. 50x50x200 cm and looked like a cabin. He was developing research theories on band spreading with Fred Linn on this computer.
Consecutively, Lloyd Snyder, John Dolan, Tom Jupille and myself started to develop a program to model HPLC separations in 1985, which we also exhibited at PittCon 1986. Lloyd named this software DryLab.
In the last 36 years a vigorous development took place to model HPLC. The lecture will present some of the milestones in this work and report about the most recent achievement of this excellent technology.

Milan Hutta, Róbert Góra

Comenius University in Bratislava, Faculty of Natural Sciences, Department of Analytical Chemistry, Ilkovičova 6, 84215 Bratislava, Slovakia

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Various natural substances are too complex analytes even for recent highly efficient analytical methods. One research approach is to characterize and analyze such complex systems is based on operational definition. Operationally defined analytes isolated from complex matrices resulted in development of novel combined multidimensional methods based on liquid chromatography separation with potential extension to combinations with electroseparation methods (ESM) and multi-detection (UV-VIS spectrophotometry, spectrofluorimetry, electroanalytical detection) and mass spectrometry (MS). Liquid chromatography working under extreme separation conditions (pH, ionic strength, temperature, additives, solvents etc.) as first dimension separation method is investigated. Usually, tough separation conditions are not directly compatible with the other methods in second separation dimension – some modes of HPLC, capillary isotachophoresis (ITP), capillary zone electrophoresis (CZE) (with various detection principles) and MS.

Systematic study of orthogonality of novel combined techniques for characterization of operationally defined humic substances (HS) and/or lignin was done. Compatibility of used extreme extractive isolation and separation conditions in HPLC (ion exchange, reversed phase, size-exclusion) with common conditions of various end techniques was tested. The approach is valuable for solution of HS interferences elimination in soil samples containing pesticides. Comprehensive techniques of multidimensional liquid chromatography (under the conditions of extreme pH, I, temperature, large-volume injection, solid sample injection etc.) combined off-line or on-line with the other separation or identification methods are discussed. Application of methods combining extreme separation conditions in HPLC (pH within the range 0-2 or alternatively 10-14, ionic strength above 0.1 mol/l, temperature well above 40°C, non-common mobile phase solvents dimethylformamide) and normal separation conditions in ESM and MS in the field of environmental analysis, analysis of degradation products of soil humic substances.

Acknowledgements
The research was supported by the grant of projects APVV-17-0318, APVV-0462-20.

Modest Gertsiuk

State Institution “Institute of Environmental Geochemistry of the National Academy of Sciences of Ukraine”, Academician Palladin Avenue, 34а, 03142, Kyiv, Ukraine

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The city of Kalush, located in Western Ukraine, was a major center of the chemical industry in the former Soviet Union. In particular, at the production association "Chlorvinyl" were produced perchloroethylene, carbon tetrachloride by direct chlorination of hydrocarbons. Hexachlorobenzene was formed as a by-product of this synthesis, the content of which in production waste was over 90%. According to the Stockholm Convention, this compound belongs to the persistent organic pollutants, and waste with hexachlorobenzene - to the first class of danger. As there were no such waste disposal plants in the Soviet Union at that time, it was decided to bury of hexachlorobenzene waste at a landfill near the city of Kalush. The waste was placed in iron barrels with a capacity of 200 liters and buried in the trench. As of 2001, there were 11,352.5 tons of hexachlorobenzene wastes at the landfill. Over time, the barrels corroded and hexachlorobenzene migrated into soils, groundwater and surface water. This posed a great danger to the health of the population of Kalush and other settlements located nearby. In view of this, it was decided to remove waste from the landfill.

During the work on the removal of waste, the placement of waste and the state of contamination of soils, groundwater and surface water were monitored. Sampling was performed both from the surface and from wells located on the landfill. It was found by gas chromatography that the concentrations of hexachlorobenzene in the soil exceeded the maximum allowable concentrations by hundreds of thousands of times. Soil contamination reached a depth of 12 m and the maximum levels of contamination were at a depth of 2-3 meters, where waste barrels were placed. Significant exceeding of the maximum allowable concentrations of hexachlorobenzene was observed in groundwater and surface water in the area of the landfill. A map of waste location was compiled and used for their removal.

Danijela Ašperger¹, Bruna Babić¹, Dijana Cerić², Tomislav Bolanča<sup>1

1</sup>University of Zagreb, Faculty of Chemical Engineering and Technology, Department of Analytical Chemistry, Marulićev trg 19, 10000 Zagreb, Croatia
²Bergman Labora, Karlsrovägen 2D, 182 53 Danderyd, Sweden

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Pharmaceuticals are used in human and veterinary practice and are so-called emerging contaminants. Their occurrence in the environment is still not law regulated, while certain number of published articles record their negative impact on human health. From the literature overview, pharmaceuticals were determined in different environment samples, but little bit less in here examined sediment. Sediment acts like a potential receiver for many hazardous substances including pharmaceuticals who can be easily emitted to drinking water reservoirs depending on the sediment characteristics [1]. In this work pharmaceuticals who belong to different structure groups were determined in sediment sample and following five extractions were developed and optimized: extraction by agitation (EA), ultrasound solvent extraction (USE), microwave assisted extraction (MAE), matrix solid phase dispersion (MSPD) and pressurized solvent extraction (PSE). Chosen pharmaceuticals were tylosine as macrolide antibiotic, albendazole, febantel and levamisole as antihelmintics, lidocaine and procaine as anesthetics and hydrocortisone and dexamethasone as glucocorticoids. Pharmaceutical concentrations extracted from sediment sample were determined by high performance liquid chromatography using diode array detector. After chromatographic analysis, all methods were validated and the following optimal parameters were determined for each extraction method: sediment mass, solvent, contact time between sediment and solvent, extraction duration, agitation frequency and extraction temperature (EA); solvent, extraction duration, power and temperature of ultrasonic bath (USE); solvent, extraction duration, temperature, solvent volume (MAE); sorbent, solvent, solvent volume, sediment/sorbent mass ratio (MSPD); solvent (PLE) [2].

Acknowledgments
This paper was prepared as part of the project Advanced Water Treatment Technologies for Microplastics Removal (IP-2019-04-9661, AdWaTMiR).

References
[1]  M. S. Díaz-Cruz, M. J. López de Alda, D. Barceló, TrAC 22 (2003) 340-551.
[2]  D. Drljača, D. Ašperger, M. Ferenčak, M. Gavranić, S. Babić, I. Mikac, M. Ahel, Chromatographia, 79 (2016) 209-223.

Leonardo Checchini, Lapo Renai, Massimo Del Bubba

University of Florence, Department of Chemistry, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy

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Environmental monitoring of organic micropollutants (OMPs), even though based on advanced extraction and chromatographic analytical approaches, is indeed a rather laborious issue for several reasons. Among them, environmental analysis must address a wide range of matrices and is usually faced with the simultaneous analysis of a very large number of analytes, often characterized by different physicochemical properties. Furthermore, OMPs are often capable of exerting negative effects on the environment and humans, even if present at very low concentrations (i.e. ng/L and sub-ng/L levels), thus requiring highly sensitive analytical methods. The use of environmentally friendly analytical approaches is another important aspect that should not be overlooked, which involves the use of extraction solvents characterized by low or no toxicity and minimal sample handling. Last but not least, environmental monitoring requires a high number of data in order to allow a significant spatial-temporal evaluation, thus requesting methods characterized by a high throughput level, which is often obtained through method automation.
In this lecture, several examples of method optimization are illustrated, dealing with the increase of the overall analytical throughput. These case-studies focuses on the analysis of different classes of analytes of high environmental concern in various matrices, by means of the improvement of (i) the sample extraction and purification strategies [1], (ii) the chromatographic separations, and (iii) the instrumental detection, also using chemometrics tools [2,3]. Preliminary data regarding the unconventional extraction of emerging OMPs from solid samples are also given and discussed.

References
[1]  L. Ciofi, C. Ancillotti, U. Chiuminatto, D. Fibbi, B. Pasquini, M.C. Bruzzoniti, L. Rivoira, M. Del Bubba, Anal. Bioanal. Chem. 408 (2016) 3331-3347.
[2]  L. Ciofi, C. Ancillotti, U. Chiuminatto, D. Fibbi, L. Checchini, S. Orlandini, M. Del Bubba, J. Chromatogr. A 1362 (2014) 75-88.
[3]  L. Renai, C.V.A. Scordo, A. El Ghadraoui, S. Santana.Viera, J.J. Santana Rodriguez, S. Orlandini, S. Furlanetto, D. Fibbi, D. Lambropoulou, M. Del Bubba, J. Chromatogr. A 1649 (2021) 462225.

Hansjoerg Majer

Restek Corporation, 110 Benner Circle, Bellefonte. PA 16823, USA

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For some time now, polar contaminants have been experiencing increased interest because they are not easily accessible with existing analytical methods such as GC or reversed-phase LC.
Polar pesticides, for example, cannot be included into the large panels for screening methods in Food Safety analysis.
Polar, mobile, and toxic (PMT) components have long been missed by regulatory agencies because their detection has been particularly difficult. In the drinking water sector, with the advent of suitable analytical methods, calls for guideline and limit values have now been growing louder since 2018.
In addition to ion chromatography/MS coupling and SFC-MS coupling, novel HILIC or mixed-mode separation phases for LC-MS/MS coupling have proven to be advantageous as suitable methods.
Examples are polar poly- and perfluorinated substances (PFAS) and precursors of nitrosamines.
Various suppliers have now successfully applied novel mixed-mode separation phases to the analysis of polar pesticides and polar, short-chain PFAS.
In this presentation, we want to show, how such a mixed-mode column can be applied in mini-screenings even for larger groups of similar components.
Up to 17 polar pesticides can be detected in one run, which has led to a method based on this column recently being included in the QuPPe document of the European Reference Laboratory for Single Residue Analysis.
In addition, we want to show, how new very short chain PFAS and novel polar substitutes like alcoholic Telomers can be analyzed together with legacy PFAS compounds in Environment and Food.