July 1

Erwin Rosenberg¹, Maria Antoniadou¹, Chrysoula Kanakaki¹, Bernhard Klampfl¹, Robert D. Müller¹, Jürgen Kahr<sup>2

1</sup>Vienna University of Technology, Institute of Chemical Technologies and Analytics, Getreidemarkt 9/164 A-1060 Vienna, Austria
²AIT Austrian Institute of Technology GmbH, Center for Low-Emission Transport, Electric Vehicle Technologies, Giefinggasse 2, 1210 Vienna, Austria

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Gas chromatography is the most powerful technique to separate mixtures of volatile compounds; however, doing so normally requires a significant amount of time, and imposes thus limitation to the speed and the time resolution that can be achieved with chromatographic measurements. Many technologically relevant processes do, however, require measurements with a time resolution that is at the scale of one minute, or potentially even less. From the van Deemter equation it is obvious that it is impossible to maximise separation speed beyond the optimum separation velocity without losing efficiency [1]. A closer inspection of this equation reveals, however, various possibilities to gain separation speed.
We will discuss various approaches to speed up chromatographic separation and to gain time resolution between measurements that we have developed and applied in response to the need of monitoring the volatile compounds formed by the degradation of the organic electrolyte of lithium ion batteries during various use conditions. As extreme operation conditions or misuse can lead to catastrophic degradation of the electrolyte in the lithium ion battery, fast responding chromatographic techniques are required [2]. These include the use of vacuum outlet conditions, multiplexing, and of fast (positive or negative) thermal gradients. Their individual benefits and limitations will be presented and critically discussed here, with a particular view to their practical applicability.

Acknowledgements
The authors gratefully acknowledge financial support of this work by the Austrian Research Promotion Agency (FFG) under Project numbers 835790 (“SiLithium”), 858298 (“DianaBatt”) and 879613 (“OPERION”).

References
[1]  J.J. van Deemter, F.J. Zuiderweg, A. Klinkenberg, Chem. Eng. Sci. 5 (1956) 271-289.
[2]  Y.P. Stenzel, F. Horsthemke, M. Winter, S. Nowak, Separations 6 (2019) 26.

Wojciech Filipiak¹, Karolina Zuchowska¹, Robert Wlodarski<sup>2

1</sup>Department of Pharmacodynamics and Molecular Pharmacology, Faculty of Pharmacy, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, Jurasza 2 street, 85-089 Bydgoszcz, Poland
²Department of Anaesthesiology and Intensive Care, 10th Military Research Hospital and Polyclinic, Powstancow Warszwy 5 street, 85-681 Bydgoszcz, Poland

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 Piperacillin and imipenem are a common choice for empirical and targeted treatment of critically ill suffering from Ventilation-Associated Pneumonia (VAP), but their effective dosing is challenging and may result in sub-therapeutic concentrations that can lead to therapy failure and even promote antimicrobial resistance. Given that distribution of both β-lactams is altered by multiple factors and that only the free fraction of antibiotics is responsible for bacteria eradication, it is of utmost importance to directly determine only the unbound instead of the total drug concentration.
Therefore, we present the novel method utilizing Thin-Film Microextraction (TFME) based on an equilibrium partition of the free analyte between the sample matrix and solid phase, while the selection of appropriate adsorbents ensures a highly reproducible analysis of even trace levels of analytes in a complex matrix. All TFME blades used in this study were self-made, and both the extraction and LC-MS/MS conditions were optimized for piperacillin and imipenem allowing the high-throughput preparation of 96 samples at once, followed by rapid and selective LC-MS/MS analysis. The established method was validated according to the Guidelines of US Food and Drug Administration (FDA) in bioanalytical method validation.
The validation results confirm that proposed method is suitable for the selective, sensitive, accurate and precise analysis of the sole biologically active fraction of piperacillin and imipenem in the blood (n=18) and BAL (n=9) specimens collected from VAP patients hospitalized at intensive care unit. The measured unbound concentrations of β-lactams in BAL differ substantially from the values estimated by recalculation from total concentrations using published protein binding data. Observed high variability in nonlinear plasma-protein binding, along with drug-specific dependence on creatinine clearance and poor permeability to the site of infection highlight the need for personalized therapeutic drug monitoring in critically ill for tailored dosing strategy.

Acknowledgements
This research was funded by the National Science Centre, Poland under the Project No.: 2017/26/D/NZ6/00136.

Petr Cesla, Jana Vanova, Tomas Hajek, Barbora Blazickova

Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Studentska 573, CZ-53210 Pardubice, Czech Republic

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Two-dimensional comprehensive LC has become a commonly used technique suitable for the separation of highly complex samples. The application of this technique theoretically allows a higher number of compounds to be separated and quantified in a single analysis compared to that of unidimensional LC. However, the analytes are diluted during the analysis due to the passage of two separation dimensions and due to the contribution of the interface for the transfer of analyzed fractions. In the case of separation of biological samples, only a limited amount of sample is usually available. Therefore, it is beneficial to scale-down the separation system using narrow-bore or capillary columns, providing generally higher mass sensitivity of the developed methods, i.e. by application of capillary columns with monolithic stationary phases.
In this work, the benefits and difficulties of the application of different gradient profiles in both separation dimensions of LC×LC system are addressed. The factors affecting the dilution of the fractions transferred between the dimensions for combination of reversed-phase systems and hydrophilic interaction LC mode. The combination of conventional packed columns and organic/silica gel monolithic columns in both separation dimensions prepared with tailored chemistry and permeability provided an efficient way of minimizing the dilution during two-dimensional LC separation. The benefits of microcolumn separations are compared with the application of conventional columns connected using focusing interfaces. Different types of focusing interfaces have been tested and compared.

Acknowledgements
The financial support by the Czech Science Foundation, project No. 22-09556S is gratefully acknowledged.

Alen Albreht¹, Elena Chekmeneva^2,3, Humma Hussain^2,4, Beatriz Jiménez^2,3, Luke Whiley², Matthias Witt⁵, Matthew R. Lewis<sup>2,3

1</sup>Laboratory for Food Chemistry, Department of Analytical Chemistry, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia
²National Phenome Centre, Department of Metabolism, Digestion and Reproduction, Imperial College London, London W12 0NN, United Kingdom
³Section of Bioanalytical Chemistry, Department of Metabolism, Digestion and Reproduction, Imperial College London, London SW7 2AZ, United Kingdom
⁴Division of Anaesthetics, Pain Medicine and Intensive Care, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, London SW7 2AZ, United Kingdom
⁵Bruker Daltonics GmbH & Co. KG, MRMS Solutions, 28359 Bremen, Germany

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Metabolomics comprehensively studies an entire set of metabolites within cells, biofluids, tissues or organisms, also known as the metabolome, and has been rapidly gaining interest through the last 10 – 15 years. Variations in the normal metabolite concentration levels or absence/appearance of certain metabolites can indicate the onset of a disease or can merely reflect a response to a particular treatment. Thus, there are great aspirations for metabolomics to be used in future clinical environments for the prognosis, diagnosis and treatment response in personalized medicine and in public healthcare.
The simultaneous study of the metabolome, which contains thousands of different compounds, has been made possible mainly through technological advancements of analytical techniques and data mining. Nuclear magnetic resonance (NMR) and liquid-chromatography mass spectrometry (LC-MS) are two most common and powerful techniques used in metabolomics, however, they are not without their drawbacks. Endogenous interferences can compromise the quality of metabolic profiling assays by negatively impacting their specificity, detection capability, sensitivity, precision and quantitation accuracy.
Here, we identify two chemical interferences based on NMR and LC-MS data as N,N,N-trimethyl-L-alanine-L-proline betaine (L,L-TMAP), which was previously detected in human plasma and N,N-dimethyl-L-proline-L-proline betaine (L,L-DMPP), which occupy an unexpectedly large portion of available chromatographic space in LC-MS profiling of human urine. These species have exceptionally broad and asymmetrical peak shapes in RP as well as HILIC chromatographic profiles, presenting an anomaly that causes significant ion suppression of many co-eluting compounds in positive MS acquisition mode. We demonstrate that this issue can be effectively tackled by fine tuning chromatographic separation through pH and/or temperature adjustments.

Luka Mihajlović

Analysis doo, Japanska 4 lok 49, 11070, Novi Beograd, Serbia

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With the meteoric rise in instrument performance that we have been witnessing in the last 15 years, single-cell applications of various proteomic approaches, reliant on extremely sensitive and accurate instruments, are being developed. Single-cell proteomics have the potential to provide insights into various processes of disease development, treatment effects, as well as biomarker screening for disease diagnosis and prognosis. This lecture aims to shortly present modern advancements in the field of single-cell proteomics and explain the instrumentation, workflows and statistical approaches to data interpretation that are currently being developed.

Rainer Turek

Waters Corporation

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During the presentation of Waters, you will learn about the latest development in chromatography technology.
The evolution of stainless-steel instruments to bioinert systems and finally to our latest addition, the ACQUTIY Premier system, featuring nonmetallic surfaces in the entire flow path of the chromatographic system including the column used. Obtain a new level sensitivity and reproducibility for a plenty of compound classes, without the burdens of passivation, coatings, use of additives or peek material that all come along with consequences on user-experience and lab efficiency.