Session 7

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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.

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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.

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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.

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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.