Coral Barbas

Centre of Metabolomics and Bioanalysis (CEMBIO), Facultad de Farmacia, Universidad San Pablo-CEU, CEU Universities, Urbanización Montepríncipe, 28060 Boadilla del Monte, Spain

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Metabolomics, the comprehensive analysis of metabolites in a biological sample, is a huge analytical challenge due to the physico-chemical diversity of analytes, their concentration ranges, and properties of the biological matrices. Separation techniques greatly enhance the analytical capabilities of mass spectrometry (MS) where accurate annotation is vital for data interpretation; however, metabolite identification is still a major bottleneck in untargeted metabolomics.

Along this presentation, I will discuss different analytical challenges in metabolite identification that we have faced at CEMBIO and how we have approached them using CE-TOF-MS, LC-QTOF-MS, and LC-IM-QTOF-MS.

We have demonstrated the robustness of the in-source fragmentation (ISF) in CE-TOF-MS, which makes possible the annotation of compounds by means of their fragmentation pattern [1]. ISF together with the mechanisms of the major fragmentation reactions observed for 57 amino acid standard compounds allowed us to establish a workflow for targeted extraction of modified amino acids [2] with high biochemical value which led us to the identification of unknown epi-metabolites.

A more recent technique, ion mobility spectrometry (IMS) allows the separation of molecules on the gas phase based on their spatial configuration. Combined with LC-MS, stands out as an excellent tool to study different isomeric compounds. Oxylipins and esterified oxidized lipids are bioactive compounds that play crucial roles in physio-pathological processes, like infections, cancer, and Alzheimer’s. Their analysis, usually done by LC-MS, is hindered by the presence of multiple isomers which in some cases can be disease-specific. Therefore, its identification could reveal reliable diagnostic markers for the diseases they are involved in. We have used LC-IMS-MS to improve the annotation of this type of compounds both in standards and plasma samples of patients suffering different diseases. This allowed the confirmation of elevated oxidized lipids as well as the identification of new and previously unresolved species.

References

[1]  M. Mamani-Huanca, A. Gilde la Fuente, A. Otero, A. Gradillas, J. Godzien, C. Barbas, Á. López‐Gonzálvez, J. Chromatogr. A, 1635 (2021) 461758.
[2]  M. Mamani-Huanca, A. Gradillas, A. Gilde la Fuente, Á. López‐Gonzálvez, C. Barbas, Anal. Chem. 92 (7) (2020) 4848–4857.

Janusz Pawliszyn, Abir Khaled, Alexander Kasperkiewicz

FACILITATING HIGH THROUGHPUT FOOD DETERMINATIONS WITH THIN FILM SPME TECHNOLOGIES

Department of Chemistry, University of Waterloo, Waterloo, ON N2L3G1, Canada

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The current sample preparation techniques used in multiclass multiresidue analysis of pesticides in fruits and vegetables as well as veterinary drugs in animal tissue involve time-consuming procedures that are not always effective at minimizing matrix interferences. These methods often involve the use of large amounts of organic solvents which lead to hazardous waste. Moreover, they lack automation and high-throughput capabilities. Screening of contaminants present in food and origin of food is an important task. New developments in high throughput determinations facilitated by SPME in thin film format [1] have been demonstrated not only with GC/MS and LC/MS, but direct couplings to MS. These are critical advances which will impact effectiveness of public protection. In this presentation we will describe two alternative approaches for multiclass multiresidue analysis based on solid phase microextraction. The first approach is based on conventional liquid chromatography- tandem mass spectrometry methods (LC–MS/MS) [2]. The second approach is based on the emerging direct analysis MS techniques [3]. In both approaches, the main goal is aimed at minimizing matrix effects and organic solvent use, while maximizing sample throughput. The fully automated sample preparation workflow allows for total extraction time of less than 1 min per sample when 96 extractions are simultaneously conducted, while the direct to MS workflow allows for total analysis time of less than 1 min per sample with screening in both negative and positive ionization modes in the Coated Blade Spray (CBS) method. Strategies of determination of hydrophobic pesticides will be discussed as well [4]. All methods were able to achieve excellent accuracy and precision results [5].

 

References

[1]  E. Nazdrajić, K. Murtada, J. Pawliszyn, Anal. Chem. 93 (2021) 4764-4772.
[2]  A. Khaled, V. Singh, J. Pawliszyn, J. Agric. Food Chem. 67 (2019), 12663-12669.
[3]  A. Khaled, G. Gómez-Ríos, J. Pawliszyn, Anal. Chem. 92 (2020) 5937-5943.
[4]  A. Kasperkiewicz, J. Pawliszyn, Food Chem. 339 (2021), Article #127815.
[5]  A. Kasperkiewicz, S. Lendor, J. Pawliszyn, Talanta 236 (2022), Article #122825.

 

 

Luigi Mondello, Gemma De Grazia, Lorenzo Cucinotta, Danilo Sciarrone
ChiBioFarAm Department, University of Messina, Vaile Annunziata, 98168, Messina, Italy

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The collection of analytes from natural sources is the goal of each preparative system. Conventional GC analysis for preparative purpose presents different limitations when highly pure compounds have to be collected at milligrams level in a reasonable time. Although wide-bore columns (0.53 mm I.D.) are commonly used, providing an enhanced sample capacity, an excess of on-column sample amounts will result in skewed peaks and decreased resolution. On the other hand, the collection of pure components requires the injection of lower amounts in order to avoid co-elutions on the wide-bore column; as a consequence, the collection of highly pure components, at the milligram level, requires an increased total collection time. The higher is the injection volume, the lower is the total time required to collect a specific compound, thus the highest injection volume should be always used. Aiming to improve the productivity of the system a multidimensional prep-GC instrument is presented with the goal to reduce the total collection time and to improve the purity of the components collected.

To improve the capability of the system, an on-line 4D chromatographic system (prep LC-GC-GC-GC) instrument can be adopted enabling the injection of higher sample volumes from the separated HPLC components by exchanging the dirty sample matrix with a pure HPLC solvent containing the molecules of interest and acting as an additional automated sample preparation step.

Gérard Hopfgartner¹, David Ruskic¹, Patrick Mueller¹, Ron Bonner²

¹Life Sciences Mass Spectrometry, Department of Inorganic and Analytical Chemistry, University of Geneva, 24 Quai Ernest Ansermet, CH-1211 Geneva 4, Switzerland

²Ron Bonner Consulting, Newmarket, ON, Canada

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LC-MS/MS-based global metabolomics or forensic investigations with electrospray ionization rely on exact mass determinations, collision induced spectra and statistical analysis for compounds detection and structural identification. The use of large MS/MS libraries, in silico fragmentation and LC retention prediction tools plays more and more an important role to achieve that goal. Beside large ionization response range, metabolites do often not generate fragment rich CID spectra for protonated and deprotonated precursor ions. In addition, adduct precursor ions (Na, K,…) also do not fragment well which leaves many potential hits unexploited. This calls for alternative ionization approaches and additional fragmentation techniques such as photodissociation (PD) or electron induced dissociation (EID). As the analysis needs to be keep short and sample is limited, multimodal approaches are becoming essential to gain as much as possible qualitative and quantitative information in a single LC-MS run. Recent instrumental improvements in high-resolution mass spectrometry (HRMS) have enabled data independent information acquisition (DIA) schemes, such as SWATH [1]. With SWATH a collision induced dissociation MS/MS spectra can be generated for every precursor ion. Comparison with MS/MS database or the use of in-silico fragmentation tools can further improve compound identification, but improved selectivity and MS/MS information is needed. Implementing differential mobility spectrometry (DMS) for multidimensional separations (LCxDMS/DMS or SFCxDMS/MS) into the analytical workflow as the use of electron induced dissociation (EID), can significantly improve the performance of the analysis on singly charged precursor ions including adducts and multimers. Furthermore, under specific conditions atmospheric pressure ionization with micro-LC enables the selective generation either protonated molecules or radical cations. As for EID, radical fragmentation is of high interest as it opens the use of electron impact libraries for compound ID in LC-MS.

Reference

[1]  R. Bonner, G. Hopfgartner, TrAC - Trends Anal. Chem. 120 (2019) 115278 https://doi.org/10.1016/j.trac.2018.10.014.

Václav Kašička, Jan Bílek, Dušan Koval, Petra Sázelová, Filip Teplý

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo n. 2, 166 10 Prague 6, Czechia
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Recently, new diquats (DQs), derivatives of widely used herbicide diquat, were prepared [1]. These chiral N heteroaromatic dicationic compounds comprising 2,2’-bipyridine moiety are attractive for variable applications, e.g. as catalysts, dyes, redox indicators, DNA intercalators, and chiral selectors. For these applications, enantiopurity of (P)- and (M)-isomers of DQs has to be checked. For this purpose, a new capillary electrophoresis (CE) method was developed. CE analyses were performed on Agilent Technologies 7100 CE system (Waldbronn, Germany) in a hydroxypropylcellulose coated fused silica capillary (id/od 50/375 µm, total/effective length 385/300 mm), at –12 kV separation voltage and 25°C. The DQs (0.2 mM) were injected hydrodynamically (700 Pa × 10 s) and detected at 200 nm. (P)- and (M)-enantiomers of all 11 DQs were baseline or better resolved by CE using aqueous 22/35 mM sodium/phosphate buffer, pH 2.5, as background electrolyte (BGE) and 6 mM randomly highly sulfated α-, β- and γ-cyclodextrins (S-CDs) as chiral selectors. Interactions of dicationic DQs with highly sulfated CDs resulted in formation of strongly negatively charged fast migrating DQ-CD complexes [2] that were separated with high resolution within a short time of 5–8 min. In addition, strength of the complexes of DQ enantiomers with S CDs was evaluated. The apparent stability constants of the DQ-CD complexes were determined from the dependence of effective mobilities of (P)- and (M)-enantiomers of the DQs on concentration of S-CDs in the BGE by nonlinear regression analysis as described in [3]. The DQs enantiomers formed moderate to strong complexes with all three types of S-CDs with the apparent stability constants in the range (7.80–547.4) × 10^3 L/mol.

Acknowledgements
This work was supported by the GACR (project 20-03899S) and the CAS (project RVO 61388963).

References
[1]  H.R. Talele, D. Koval, L. Severa, P.E. Reyes-Gutiérrez, I. Císařová, D. Šaman, L. Bednárová, V. Kašička, F. Teplý, Chem.-Eur. J. 24 (2018) 7601-7604.
[2]  D. Koval, L. Severa, L. Adriaenssens, J. Vávra, F. Teplý, V. Kašička, Electrophoresis 32 (2011) 2683.
[3]  S. Štěpánová, V. Kašička, J. Sep. Sci. 38 (2015) 2708.

Bogusław Buszewski^1,2, Hussam Al-Saoud^2,3, Michał Szumski², Marek Potrzebowski⁴, Myroslav Sprynskyy^2
1Department of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicu University in Toruń, Gagarina7 str. Pl-87 100 Toruń (Poland)
²Interdyscyplinary Center of Modern Techniology, Nicolaus Copernicu University in Toruń, Gagarina7 str. Pl-87 100 Toruń (Poland)
³Departament of Materiale Engineering and Production, Faculty of Mechanical Engineering, Białystok University of Technology, Wiejska 45c, Pl-15 351 Białystok (Poland)
⁴Center of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza str. 112, Pl-90 001 Łódź (Poland)

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Silica has always been the object of interest of specialists and used for many purposes. Particularly for over 100 years, it has been used in chromatography and other separation techniques as an adsorbent or a carrier for stationary phases. Due to the high demands placed on chromatographic stationary phases, synthetic silica is used to provide high column efficiencies and avoid many adverse interactions. Recently, bio-silica obtained as a result of biogenic processes carried out by the selected strains of algae turned out to be an alternative to the synthetic silica [1]. This material, in contrast to the synthetic one, is characterized by a micro- or nano-hierarchical structure. Biosilica is an inorganic polymer formed by organisms such as diatoms or siliceous sponges of orthosilicate units in which two silanol groups are joined together to one bond or siloxane. Its low manufacturing cost as well as high quality in terms of chemical composition, mechanical stability and resistance are guaranteed by a good biosynthesis reproducibility. In this paper, the conditions of bio-silica biosynthesis, used as an adsorbent and carrier of stationary phases for liquid chromatography and related techniques, will be presented. The characteristics of bare and modified materials using porosimetric techniques, microscopic imaging, surface architecture characteristics via spectral and spectroscopic methods as well as chromatographic assessment will be discussed. Potential application possibilities (first proposals in the literature) in chromatographic separation and sample preparation will also be presented.

Acknowledgements
This work was financially supported by the Foundation for Polish Science co-financed by the European Union under the European Regional Development Found, project Advanced bio-composites for tomorrow’s economy BIOG-NET, FNP POIR.04.04.00-00-1792/18-00. The project is carried out within the TEAM-NET program.

Reference
[1]  M. Sprynskyy, P. Pomastowski, M. Horonowska, A. Król, K. Rafińska, B. Buszewski, Mater. Des., 132 (2017) 22-29.

Tony Edge¹, David Dunthorne¹, Mark Fever¹, Michael Donaldson¹, Lewis Couchman², Ken Tiong Ng³, Leon Barry³

¹VWR, part of Avantor, VWR International Ltd., Hichrom, 1-3 The Markham Centre, Station Road, Theale, Reading, UK
²Analytical Services International, St. George’s University of London, UK
³Environmental Research Group, School of Public Health, Imperial College London, UK

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Achieving fast LC analyses is essential in high sample throughput laboratories, such as clinical, drug discovery and environmental labs. Over the last two decades, UHPLC has enabled many labs to dramatically increase sample throughput, through use of shorter columns packed with sub-2 micron particles. However, at the same time, the performance of modern mass spectrometers has continued to evolve. Improved sensitivity and ultra-fast data acquisition capabilities, provide opportunities to further reduce analytical run times, using specially designed, high throughput columns.
This presentation will look at the use of 10 mm columns for the analysis of complex samples, either derived from biological or environmental sources. The presentation will initially outline the theoretical considerations of short columns, starting with van Deemter theory, before moving to a more detailed kinetic plot interpretation of their application. From here, practical considerations of the use of such short columns will be discussed, specifically looking at dwell volumes, tubing and data acquisition rates.
The analysis of complex samples requires a degree of sample preparation prior to application of high throughput LC-MSMS, due to matrix issues. The presentation will investigate the impact that the level of sample preparation has on the data integrity when applied to high throughput scenarios. Finally, the presentation will conclude with a series of applications detailing the benefits that the use of 10 mm length columns can have for high throughput analysis, without the loss of data integrity. The applications will include a series of therapeutic drugs, environmental pollutants and some samples from a hospital laboratory.

Jeremy D. Glennon¹, Alyah Buzid², Phyllis E. Hayes¹, Majidah Alsaeedi¹, Huda Alghamdi¹, F. Jerry Reen³, Gerard P. McGlacken¹, John H.T. Luong¹

¹School of Chemistry and the Analytical & Biological Chemistry Research Facility (ABCRF), University College Cork, College Road, Cork T12 YN60, Ireland
²Department of Chemistry, College of Science, King Faisal University, P.O. Box 380, Al-Ahsa, 31982, Saudi Arabia
³School of Microbiology, University College Cork, College Road, Cork T12 YN60, Ireland

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Combining the nanomolar detectability at solid electrodes with high efficiency chromatographic media can provide selective multianalyte trace determination of key electroactive compounds. In this work, the coupling of electrochemical detection at a boron-doped diamond electrode (BDD) with core-shell chromatography (LC-BDD) is applied for the rapid nanomolar detection of targeted solutes including phenolics, neurotransmitters and microbial metabolites.
Among the applications examined is the rapid detection of key catecholamine biomarkers elevated in traumatic brain injury (dopamine, epinephrine and norepinephrine) using hydrophilic interaction liquid chromatography (HILIC) on superficially porous (core-shell) and porous Z-HILIC columns [1]. Key microbial metabolites are also rapidly detectable by Halo C18 LC-BDD exemplified by signalling molecules or bacterial pheromones for cell-to-cell communication (quorum sensing (QS)) from Pseudomonas aeruginosa, an antibiotic-resistant human pathogen associated with chronic lung infections.
Application is extended to the detection of guaiacol (2-methoxyphenol), a food and beverage spoilage metabolite indicative of contamination by the Gram-positive thermophilic Alicyclobacillus spp. bacterium [2]. Additionally, studies into the profiling of essential phenolic flavouring compounds in whiskey samples [3] and to selected pharmaceutical compounds are described [4].

Acknowledgements
The authors wish to acknowledge the following funding bodies for project grants and individual scholarships: Irish Research Council, Enterprise Ireland, the Ministry of Education (Saudi Arabia) and the Cultural Bureau (Dublin) and previously Science Foundation Ireland.

References
[1]  M. Alsaeedi, H. Alghamdi, P. E. Hayes, A. M. Hogan and J. D. Glennon, Separations 8 (2021) 124.
[2]  P. E. Hayes, A. Buzid, J. H. T. Luong and J. D. Glennon, Electroanalysis 33 (2021) 766-773.
[3]  P. E. Hayes, J. H.T. Luong, E. S. Gilchrist, A. Buzid, J. D. Glennon, J. Chromatogr. A 1612 (2020) 460649.
[4]  H. Alghamdi, M. Alsaeedi, A. Buzid, J. D. Glennon, J.H.T. Luong, Electroanalysis 33 (2021) 1137-1142.

Kareem Yusuf¹, Osama Shekhah², Ahmad Aqel¹, Setah ALHarbi¹, Zeid ALOthman¹, Mohamed Eddaoudi²

¹Department of Chemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, KSA
²Advanced Membranes and Porous Materials Centre (AMPMC), King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, KSA
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Using Metal-Organic Frameworks (MOFs) in chromatography as a stationary phase with superior properties has already given very promising results [1]. However, the use of MOF-polymer composite stationary phase is still very limited, and even more scarce in Gas Chromatography. MOF-polymer capillary monolithic columns combine MOFs properties in terms of the very high surface area, controlled structure, and limitless diversity, with the high permeability and flexible structure of the polymeric monoliths.
The present work discusses the fabrication of a composite material of sodilite topology Zeolite-like Metal Organic Framework (sod-ZMOF) nanocrystals incorporated into Divinylbenzene (DVB) monolithic polymer (ZMOF@DVB) in a capillary column (18cm × 250μm i.d.) to enhance the separation efficiency of the DVB monolith using a conventional low-pressure gas chromatograph.
5mg of sod-ZMOF nanocrystals were prepared and dispersed into DVB polymerization mixture, then polymerized to form ZMOF 5mg @DVB composite monolith in situ. Two batches of columns were prepared to investigate the effect of ZMOF incorporation in comparison to DVB blank monolithic columns. Chromatographic performance, as well as separation efficiency of various Volatile Organic Compounds (VOCs), isomers, and gases, were investigated.
To better understand the role of incorporating sod-ZMOF into the polymer matrix in its monolithic form, a thermodynamic characterization was carried out using Inverse Gas Chromatography (IGC) at infinite dilution under 0.5 Mpa column pressure and various column temperatures [2]. The free energy of adsorption (ΔGA), enthalpy of adsorption (ΔHA), and entropy of adsorption (ΔSA) were determined. Dorris-Gray and Schultz et al. methods estimated the dispersive component of surface energy. The acidic, KA, and basic, KD, parameters for both materials were estimated using a group of polar probes.

Acknowledgments:
This work was funded by the Researchers Supporting Project Number (RSP2022R429) King Saud University, Riyadh, Saudi Arabia.

 References
[1]  K. Yusuf, A. Aqel, Z. ALOthman, J. Chromatogr. A 1348 (2014) 1–16.
[2]  K. Yusuf, O. Shekhah, Z. ALOthman, M. Eddaoudi, Appl. Sci. 1 (2021) 10243.

Ahmad Aqel, Kareem Yusuf, Zeid A. ALOthman, Khalid M. Alotaibi, Ahmed-Yacine Badjah-Hadj-Ahmed
Department of Chemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, KSA

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Capillary liquid chromatography has become one of the most important developments in separation technology. Capillary chromatography performed using columns with an internal diameter ≤ 500 μm. This technique carried out using fused silica capillaries and prepared with a variety of different stationary phases. However, the successful development of this technique is closely related to the technical challenges associated with the columns manufacturing. Monolithic media have rapidly become popular and attracted increasing interest as separation phases. They consist of a single rigid piece of porous material that possesses a unique pore structure distribution with micrometer sized macropores and nanometer sized mesopores. Unfortunately, naked monolith is lake of small pores, which does not provide sufficient interaction sites for separation of small molecules especially with isocratic modes. Several approaches have been proposed to enhance the efficiency of the monolithic columns. In this work, small amounts of micro/nanoparticles such as carbon nanotubes and metal organic frameworks have been added into the porous polymer monoliths under specific conditions to enhance the separation efficiency of small molecules. Hydrodynamic and morphological properties of the prepared materials and columns were thoroughly characterized. The columns were evaluated by separation mixtures of different compounds such as hydrocarbons, phenols, and ketones. The combination of monoliths and capillary chromatography offer several advantages that include fast and sensitive analysis, in addition to the consumption of much smaller amounts of solvents, samples, and stationary phases, which will reflect positively on the environment and cost.

Acknowledgements
This project was funded by the national plan for science, technology & innovation (MAARIFAH), King Abdulaziz City for Science & Technology, KSA, Award No. 2-17-01-001-0053.

References
[1]  A. Aqel, S. Alzahrani, A. Al-Rifai, M. Alturkey, K. Yusuf, Z. ALOthman, A. Badjah, Curr. Anal. Chem. 16 (2020) 223-233.
[2]  A. Al-Rifai, A. Aqel, L. Al Wahibi, Z. ALOthman, A. Badjah, J. Chromatogr. A 1535 (2018) 17-26.

Dalibor Satinsky¹, Ivona Lhotska¹, Martina Hakova¹, Slavomira Zatrochova¹, Anezka Adamcova¹, Pavlina Moravcova¹, Jiri Chvojka², Jakub Erben², Frantisek Svec<sup>1

1</sup>Department of Analytical Chemistry, Faculty of Pharmacy, Charles University, Hradec Králové, Heyrovského 1203, 50005, Hradec Králové, Czech Republic
²The Technical University of Liberec, Faculty of Textile Engineering, Department of Nonwovens and Nanofibrous Materials, Studentská 1402/2, 46001 Liberec 1, Czech Republic

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Miniaturization of sample preparation and extraction techniques is currently a hot topic and nanofiber extraction techniques belongs to highly challenging and fundamental research goals. Sample preparation is an integral and usually the most complicated part of the analytical procedure that includes removal of problematic parts of the sample (interfering matrix components, proteins, lipids), as well as the pre-concentration of target analytes. Nanofibrous polymers feature a good loading capacity and enhanced kinetics of the adsorption resulting from the high surface to volume ratio [1,2].
In our contribution, we will present the advanced approaches in application of direct current spinning and alternating current spinning for electrospun polymers for extraction of contaminants (mycotoxins, endocrine disruptors, pesticides, pharmaceutical residues) from various samples – food, soft drinks, wine, beer, milk, human serum and plasma, and river waters. Pros and cons of extraction techniques based on nanofibers coupled to column switching liquid chromatography systems will be discussed and presented. Preparation of new composite materials consisting of polymer fibers comprising different chemistries specifically developed for extraction, coating, and functionalization of nanofibers with graphen will be introduced. Nanofibers with restricted access material functionality for direct extraction of pharmaceuticals from proteinaceous matrix – human serum and bovine milk will be presented too.

Acknowledgements
This work was supported by the grant project no. 20-19297S from Grant Agency of the Czech Republic and by the EFSA-CDN project (no. CZ.02.1.01/0.0/0.0/16_019/0000841) co-funded by the ERDF.

References
[1]  M. Hakova, L. Chocholousova Havlíkova, P. Solich, F. Svec, D. Satinsky, Trac. Trends Anal. Chem. 110 (2019) 81–96.
[2]  M. Hakova, L. Chocholousova Havlikova, F. Svec, P. Solich, D. Satinsky, Anal. Chim. Acta 1121 (2020) 83–96.

Dušan Berek
Polymer Institute, Slovak Academy of Sciences, Dubravska cesta 9, 84541 Bratislava, Slovakia

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Block copolymers, BLC belong to the group of synthetic nonmetallic materials called complex polymers. They have valuable properties and found applications for example as flocculants, carriers of drugs and compatibilizers of polymer blends, etc. BLC are formed with two or several (only rarely with more than three) polymeric chains called blocks. Blocks have different chemical composition or physical architecture and they are mutually connected with a chemical bond. The structure of common diblock and triblock copolymers is schematically displayed as A-B, A-B-A or A-B-C. Most BLC contain their parent homopolymers, the “free blocks”, which may spoil their utility properties. Therefore, it is important to determine their amount and to adjust the synthesis of BLC to reduce their formation. The molecular characterization of BLC and determination of parent homopolymers is an analytical challenge. The limited separation selectivity of the most commonly polymer characterization method, size exclusion (gel permeation) chromatography, SEC/GPC is usually not sufficient to fulfill the task. Other, advanced methods of polymer liquid chromatography must be applied. Three of them will be presented in the contribution.

1. Liquid chromatography under critical conditions, based on unique mutual compensation of entropic partition (exclusion) and enthalpic interactions of macromolecules within column
2. Full adsorption of samples containing distinct macromolecules on a nonporous microparticulate sorbent, followed with their sectional, selective elution into an SEC/GPC column
3. Barrier methods, the emerging techniques, which are based on the action of slowly eluting, narrow liquid zones, barriers, which selectively decelerate fast elution of macromolecules of distinct nature.

Sunkyung Jeong, Doo Soo Chung
Department of Chemistry, Seoul National University, Seoul 08826, Korea

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Liquid extraction surface analysis (LESA) has the advantage of directly sampling analytes on a surface, thus avoiding unnecessary dilution by homogenization of the bulk sample commonly practiced in solid sample analysis. By combining LESA with capillary electrophoresis (CE), the additional advantage of separating analytes before detection can be accomplished. As a representative application of LESA-CE to a solid surface sample, non-infiltrative pesticides on the apple skin were analyzed. When non-infiltrative pesticides are applied to fruits such as apples, they remain on the fruit skin without being translocated to the fruit pulp. First, glufosinate-ammonium and glyphosate on the apple skin were directly extracted and injected into the separation capillary. After in-capillary derivatization with a fluorophore NBD-F, the pesticides were analyzed by CE-LIF. Secondly, three pesticides, chlorantraniliprole, kresoxim-methyl, and pyraclostrobin on the apple skin were analyzed by LESA-CE. For these water-insoluble neutral analytes, micellar electrokinetic chromatography combined with an on-line sample stacking method, analyte focusing by micelle collapse was employed. In addition, using a commercial CE instrument, the LESA process was performed much faster and more reliably compared to LESA-CE using a homemade CE setup. Thirdly, to analyze both cations and anions on a solid surface with a single run of a reasonable detection time, two contactless capacitively coupled conductivity detectors (C4D) were placed on a single capillary. After LESA using the inlet of a coated fused silica capillary having a weak but reversed EOF, the extracts were placed between the two C4Ds. When a reverse electric field is applied, the cations and anions migrated in the opposite directions, and were quickly detected with the C4D located in each direction. The ionic composition of a lithium battery anode surface was investigated by this LESA-CE/2C4D. The developed LESA-CE is promising for the analysis of biological surfaces of cells and tissue as well as technical surfaces.

Acknowledgements
The authors acknowledge the supports from the National Research Foundation of Korea (2021R1F1A1045947).

Matthias Guggenberger¹, Josua T. Oberlerchner¹, Heinrich Grausgruber², Thomas Rosenau¹, Stefan Böhmdorfer^1
1University of Natural Resources and Life Sciences, Vienna (BOKU), Institute of Chemistry of Renewable Resources, Konrad-Lorenz-Straße 24, 3430 Tulln, Austria
²University of Natural Resources and Life Sciences, Vienna (BOKU), Institute of Plant Breeding, Konrad-Lorenz-Straße 24, 3430 Tulln, Austria

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Chromatograms obtained by HPTLC are highly suited for visual comparison and classification of samples, but manual evaluation might suffer from operator bias or fatigue. Common chemometric methods for classification struggle with the non-uniform background noise of HPTLC plates and limited plate-to-plate reproducibility.
We employed the Self Organizing Map (SOM) algorithm to overcome these limitations.[1] It uses iterative and distance-dependent learning to prepare a map-like projection, on which chromatograms of high similarity are located close together, while less similar ones are placed further apart. The maps were used for direct visualization of similarity relations of the samples, implementation of multi-class and one-class classifications, determinations of signals that contributed to misclassification (which was also useful for validation of method robustness), and detection unusual samples for a detailed evaluation or quality control.
We analyzed a set of 495 HPTLC chromatograms of essential oils with conventional multiclass SOMs as well as one-class SOM Quality Control Index (SOMQC, [2]). Both approaches give convincing results (95% correct classification). SOMQC offers the exceptional advantage, that it assigns indistinct samples to several classes. This is a clear indicator that the sample is faulty or demands manual verification.
The classification is tolerant towards noise and inevitable variations among replicates. Only minimal data preprocessing is required. Grouping by plates was never observed, even though simple RGB data were used as input.

Acknowledgments
We are grateful for the essential oils provided as a gift by Sonnentor Kräuterhandelsgesellschaft mbH and for the support of the Austrian Biorefinery Center Tulln (ABCT).

References
[1]  M. Guggenberger, J. T. Oberlerchner, H. Grausgruber, T. Rosenau, S. Böhmdorfer, Talanta 233 (2021) 122460.
[2]  S. Kittiwachana, D.L.S. Ferreira, L.A. Fido, D.R. Thompson, R.E.A. Escott, R. G. Brereton, Anal. Chem. 82 (2010) 5972–5982.

Urh Černigoj
BIA Separations, a Sartorius company, Mirce 21, 5270 Ajdovščina, Slovenia

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Plasmid DNA (pDNA) was key to the development of biologic drug manufacturing. Today, it plays a crucial role in the production of cell and gene therapies and vaccines. HPLC based analytical assay is a powerful technique that can be used to efficiently monitor pDNA purity, quantity and isoform composition throughout the entire purification process. Elution of weak anion exchangers, for example commercially available CIMac^TM pDNA analytical column, with sodium chloride gradients is commonly employed for analysis of sample mixtures containing different isomers of plasmid DNA [1]. We have found that gradient elution of a CIMac pDNA in the presence of guanidine hydrochloride (Gdn) roughly doubles resolution between open-circular (oc) and supercoiled (sc) isomers [2]. It also improves resolution among sc, linear, and multimeric/aggregated forms. Selectivity of the improved method is orthogonal to electrophoresis, but with better quantification than agarose electrophoresis, better quantitative accuracy than capillary electrophoresis, and resolution approaching capillary electrophoresis. Unfortunately, Gdn elution does not decrease a reversible entrapment of the oc isoform in the restrictions within the pores of the monolithic material for ≥7 kbp pDNA on a CIMac pDNA columns with 1.4 um channel diameter. Therefore, a monolithic polymer with 6 um channels was developed to enable robust and reliable analytical separation of plasmid isoforms larger than 10 kbp. The novel material in combination with Gdn gradient enables a baseline separation between oc and sc pDNA as well as ≥85% recovery for oc, sc and linear isoforms of 15 kbp large pDNA even at 10 CV/min flow rate. Presented improvements were successfully implemented in PATfixTM pDNA analytical platform package, designed to give »at-line« reliable insight during process development and production of every possible pDNA.

References
[1]  B. Gabor, U. Černigoj, M. Barut, A. Štrancar, J. Chromatogr. A, 1311 (2013) 106-114.
[2]  U. Černigoj, J. Vidič, A. Ferjančič, U. Sinur, K. Božič, N. Mencin, A. Martinčič Celjar, P. Gagnon, A. Štrancar, Electrophoresis 42 (2021) 2619-2625.

Koen Sandra, Liesa Verscheure, Gerd Vanhoenacker, Tom Merchiers, Shauni Detremmerie, Julie Storms, Isabel Vandenheede, Pat Sandra

RIC group, President Kennedypark 26, 8500, Kortrijk, Belgium
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Biotech drugs, once considered a risky and nearly impossible route to explore, are today dominating the pharma landscape. In the footsteps of the biotech pioneers walking this bumpy road, our chromatographic community rose to the challenge and sharpened the analytical toolbox.
Throughout the years we have seen products become increasingly complex and witnessed analytics becoming more and more performant.
This talk will reflect on the recent advances in the analysis of biologics; from the coupling of historical incompatible chromatographic methods with mass spectrometry, to the combination of ever more separation modes in multidimensional set-ups and the implementation of chemical and enzymatic reactors in the flow path for primary and higher order structural assessment.

Živoslav Tešić¹, Milica Nešović², Stevan Blagojević²

¹Faculty of Chemistry, University of Belgrade, Studentski trg 12 – 16, 11000 Belgrade, Serbia
²Institute of General and Physical Chemistry, Belgrade, Studentski trg 12 – 16, 11000 Belgrade, Serbia

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In recent years, the botanical and geographical origin of food has become an important topic in the context of food quality and safety, as well as consumer protection, in accordance with international standards. In the case of honeys, Melissopalynology analysis is used as the method of choice for assessing botanical origin. When collecting nectar, bees add a certain amount of pollen to it, which is used to assess the botanical origin. By counting pollen particles of appropriate plant species, botanical origin is attributed to such honey. The problem arises when honey contains a large number of pollen particles from nectarless plants. In such cases, it is necessary to apply some additional methods to assess the botanical origin. Polyphenols, secondary metabolites of plants, can provide important information about the origin.
In this study, we analyzed buckwheat honey with different pollen content, which varied from 5 to 40% of pollen, nectar as well as pollen using the UHPLC-DAD system connected to triple-quadrupole mass spectrometer. The 31 polyphenols were quantified. Dominant content is found for propolis-derived flavonoids such as chrysin and pinocembrin. The significant contribution was also attributed to p-hydroxybenzoic acid and p-coumaric acid as others discovered and suggested as markers for the botanical origin of buckwheat honey. An excellent correlation was found between nectar and honey polyphenols regardless of the content of pollen particles [1]. In the cases of buckwheat honey with low pollen content, comparing the polyphenolic profile of honey and nectar provides important additional data necessary for assessment of the botanical origin.

Reference
[1]  M. Nesovic, U. Gasic, T. Tosti, N. Horvacki, N. Nedic, M. Sredojevic, S. Blagojevic, Lj. Ignjatovic, Z. Tesic, RSC Adv. 11 (2021) 25816-25829.

Virginia Coman¹, Simion Beldean-Galea<sup>2

1</sup>Babeş-Bolyai University, Raluca Ripan Institute for Research in Chemistry, Fântânele 30, 400294 Cluj-Napoca, Romania
²Babeş-Bolyai University, Faculty of Environmental Science and Engineering, Fântânele 30, 400294 Cluj-Napoca, Romania

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The extraction stage of the compounds from a matrix is very important for the results of the final analysis because it can generate many errors, waste and health problems. Therefore, the principles of green chemistry in analytical chemistry aim to significantly reduce the waste generated by the analyses [1]. Due to the toxicity of solvents, various extraction methods have been developed in recent years to significantly reduce or completely remove the amount of solvents used to extract the compounds from the matrix [2]. The miniaturized extraction (microextraction) methods offer certain advantages: low amounts (microliters) of solvents, small amount of sample to be analysed, reduced extraction time, high enrichment factor (order of hundreds), and high selectivity and precision. Due to these benefits, these methods have developed rapidly and are applied to various classes of organic and inorganic compounds in different liquid or solid matrices [3].
This review is a state-of-the-art of the applications of the liquid-phase microextraction (LPME) in the analysis of pharmaceuticals (anti-inflammatories, antidepressants, antibiotics, hormones, anti-cancer drugs, etc.) in biological fluids (serum, urine, saliva, etc.) and environmental (waters, sediments) samples. Our work is focused on the theoretical aspects of LPME and the applications of dispersive liquid-liquid microextraction, dispersive liquid-liquid microextraction based on solidification of floating organic droplet and hollow fiber microextraction. Aspects related to the analysis techniques (gas chromatography, liquid chromatography, capillary electrophoresis) coupled with different detectors following the microextraction stage are also presented. A critical comparison is made between microextraction techniques and extraction techniques used for routine analysis to provide a viable and greener alternative.

References
[1]  A. Spietelun, Ł. Marcinkowski, M. de la Guardia, J. Namieśnik, Talanta 119 (2014) 34-45.
[2]  F.R. Mansour, N.D. Danielson, Anal. Chim. Acta 1016 (2018) 1-11.
[3]  N. Campillo, K. Gavazov, P. Viñas, I. Hagarova, V. Andruch, Appl. Spectrosc. Rev. 55 (2020) 307-326.

Antonia Garcia¹, María Bajo¹, Gabriel Montero¹, Laura Mayo¹, Vanesa Alonso-Herranz¹, Clarissa Caroli², Elisa Zubeldia-Varela³, Tomás Clive Barker-Tejeda³, Alma Villaseñor³, David Rojo¹, Coral Barbas¹, Maria Fernanda Rey-Stolle<sup>1

1</sup>Center for Metabolomics, CEMBIO, Facultad de Farmacia, Universidad San Pablo-CEU, CEU Universities, 28660 Boadilla del Monte, Madrid, España
²Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy
³CEMBIO, Facultad de Farmacia, Universidad San Pablo-CEU, CEU Universities, 28660 Boadilla del Monte, Madrid, España &Departamento de Ciencias Médicas Básicas, Instituto de Medicina Molecular Aplicada

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GC-MS is a well-established technique. Just in one run in untargeted metabolomics, a large set of metabolites can be covered, such as amino acids, primary carbon metabolism, organic amines, free fatty acids, polyols, mono- and disaccharides, and sterols, among others. Typical advantages for metabolite annotations are retention time index (RI) and commercial spectrum libraries, with rich fragmentation [1]. Typical drawbacks are the required chemical derivatization, which converts the components of the samples into volatile and less polar molecules and the multiple derivatives. Methoxymation-silylation or alkylation are some of the most common in which acidic protons are converted into thermally stable derivatives. The former is more universal but reaction must take place after evaporation to dryness whereas the latter generates derivatives in milder-reaction conditions with higher stability [2] and allows the study of relevant microbiome metabolites, such as short-chain organic acids. After fifteen years of untargeted metabolomic studies based on low mass resolution GC-MS, we have concluded that at least 10% of the possible compounds remain unknown in the profile. The scenario is far worse with alkyl derivatives (after alkyl chloroformate [2]). Unfortunately, commercial libraries are unit mass resolution and besides, many of these derivatives are not included yet and that introduces a huge bias, especially for metabolites with multiple functional groups.
High-resolution and exact mass GC-MS can shed some light on the structural elucidation of metabolites and their multiple derivatives. This research aimed to take advantage of the new technology to open a new era for identification. Multiple derivatives covering all types of metabolites were investigated through standard solutions, samples, and spiked samples. Besides, a step-by-step approach for elucidation of unknowns will be presented here with examples from our ongoing CEMBIO studies in allergy, COPD, anorexia nervosa, Lynch cancer, and COVID-19, with plasma and stool samples.

References
[1]  F. Rey-Stolle et al., Anal. Chim. Acta (2021) 339043.
[2]           L. Zhao et al,. Anal. Chem. 89 (2017) 5565-5577.

Natasa P. Kalogiouri^1,2, Natalia Manousi^2,3, Antonio Ferracane^2,4, George A. Zachariadis³, Stephanos Koundouras⁵, Victoria F. Samanidou³, Peter Q. Tranchida⁴, Luigi Mondello^4,6,7, Erwin Rosenberg<sup>2

1</sup>Laboratory of Analytical Chemistry, Department of Chemistry, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
²Institute of Chemical Technology and Analytics, Vienna University of Technology, Getreidemarkt 9/164, 1060 Vienna, Austria
³Laboratory of Analytical Chemistry, Department of Chemistry, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
⁴Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Messina, Italy
⁵Laboratory of Viticulture, School of Agriculture, Aristotle University of Thessaloniki, Thessaloniki, 54124, Greece
⁶Chromaleont s.r.l., c/o Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Messina, Italy
⁷Department of Sciences and Technologies for Human and Environment, University Campus Bio-Medico of Rome, Rome, Italy

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Authentication is defined as the process that unquestionably verifies that food is genuine. In a global food market, Protected Designation of Origin (PDO) wines are highly appreciated for the period of aging that allows the evolution of unique organoleptic properties, and thus, the guarantee of the vintage age is a critical authenticity issue [1]. The coupling of SPME-Arrow to GCxGC/MS analysis in combination with chemometric techniques could establish advanced analytical methods for the investigation of the volatile fingerprint of wine and revolutionize the field of wine authenticity. In this work, an SPME-Arrow-GCxGC/MS method was optimized [2], and used for the determination of volatile markers in 24 monovarietal red wine samples belonging to the PDO Xinomavro Naoussa, produced during 4 different years (1998, 2005, 2008 and 2015), in Northern Greece. Overall, 258 volatile compounds were tentatively identified in all samples. The data matrix that constituted of 24 samples and 258 features was further processed with multivariate techniques to establish mathematical models and reveal volatile markers for each vintage age. A partial least square – discriminant analysis (PLS-DA) model was developed and successfully classified all the samples to the proper class according to the vintage age with an explained total variance of 85.1%. Variant Importance in Projection (VIP) algorithm was used to calculate the VIP scores of the determined volatiles and distinguish the most important features that affect the discrimination, revealing markers [3]. The developed prediction model was validated and the analyzed samples were classified with 100% accuracy according to the vintage age, on the basis of their volatile fingerprint. The established PLS-DA model provided satisfying clustering, revealing 15 markers as most important for the classification of the wine samples.

References
[1] N.P. Kalogiouri, V.F. Samanidou, Environ. Sci. Pollut. Res. Int. 28, 42 (2021) 59150-59164.
[2] I. Šikuten, P. Štambuk, J. Karoglan Kontic, E. Maletic, I. Tomaz, D. Preiner, Molecules 26, 23 (2021) 1-16.
[3] N.P. Kalogiouri, N. Manousi, E. Rosenberg, G.A. Zachariadis, A. Paraskevopoulou, V. Samanidou, Food Chem. 363 (2021) 130331.

Alice Ryckebusch
Advion Interchim Scientific

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Low to High pressure LC purification of small and large molecules with 3X detection UV, ELSD & MS: the right system and column for your needs:

• - Full range of puriFlash instruments from Flash Chromatography up to Preparative LC
• - More than 50 different selectivities available (from UPLC/HPLC up to Flash/prep)
• - Versatility of our CMS (Compact Mass Spectrometer): extensive range of innovative sample introduction systems (from fastest direct probe analysis requiring no sample preparation to High Performance compound separation by LC)

Introduction to 2 new instruments completing our range:

• - New Evaporator puriFlash XS-VAP: up to 90 samples in parallel, from a few mL up to 250mL per position with really low gas consumption
• - New TLC-FlashReader to visualize your TLC plates and archive them in your electronic notebook but not only! You can transfer the Data to your puriFlash system to get the best possible purification method in an instant

Daria Janiszewska¹, Małgorzata Szultka-Młyńska¹, Paweł Pomastowski², Michał Złoch², Bogusław Buszewski<sup>1,2

1</sup>Department of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, Gagarin 7, 87-100 Torun, Poland
²Centre for Modern Interdisciplinary Technologies, Nicolaus Copernicus University, Wilenska 4, 87-100 Torun, Poland

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Bacteria are known for both their beneficial and destructive properties. They cause spoilage of food products and cause many dangerous human, animal and plant diseases. Accurate identification of bacterial species is critical to the choice of antibiotic therapy and food quality control [1]. Currently, the most popular technique used to identify bacteria is matrix-assisted laser desorption/ionization with tandem time-of-flight analyser mass spectrometry - MALDI-TOF MS. Identification of micro-organisms is based on a comparison of the obtained mass spectra of ribosomal proteins with reference spectra available in the database [2]. Unfortunately, MALDI-TOF MS cannot distinguish between closely related species such as E. coli and Shigella or B. licheniformis and B. sonorensis. Misidentification or lack of identification can also be attributed to the still too small database of reference spectra [3].
In our study we tested the possibility of standard proteomic identification of bacteria using shortened incubation times (7, 9, 12h) and different sample preparation methods (bacterial extracts, whole cells, whole cells with formic acid). Besides, we performed 16S rDNA gene sequencing as a reference method. We also performed lipid profile analysis of the studied microorganisms using the MALDI-TOF MS technique, using two lipid extraction methods (Folch, Bligh and dyer) and two different MALDI matrices (HCCA and DHB) The analysis was performed in both positive and negative mode.
The results show that combined several ‘-omics’ methods, such as proteomics, genomics and lipidomics, are necessary toaccurately identify bacteria. The analyses also allowed us to add new reference spectra to the Bruker BioTyper database.

References
[1]  G. Black. Microbiology: principles and explorations, Nowy Jork: Wiley, 2008.
[2]  A.E. Clark, E.J. Kaleta, A. Arora, D.M. Wolk, Clin. Microbio.l Rev. 26 (2013) 547–603.
[3]  E. De Carolis, A. Vella, L. Vaccaro, R. Torelli, T. Spanu, B. Fiori, B. Posteraro, M. Sanguinetti, J Infect Dev Ctries 8 (2014) 1081–1088.

Carmela Maria Montone, Anna Laura Capriotti

Department of Chemistry, Università di Roma “La Sapienza”, Sapienza University of Rome, Piazzale Aldo Moro 5, Rome, 00185, Italy

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Protein tyrosine O-sulfation is an important post-translational modification, correlated to inflammation, virus infection, and signal pathways. Analytical methods for enrichment and detailed study of sulfopeptides, able to provide peptide sequence and site localization, are currently limited due to issues with low abundance and poor stability of the sulfate modification [1]. In this context, an enrichment method was developed for two commercial peptides, representative of mono- and di-sulfated peptides, by comparison of five sorbent materials, i.e. two commercial weak anion exchange mixed mode sorbents and three phosphopeptide enrichment materials [2]. Recoveries were studied by UHPLC-multiple reaction monitoring analysis. The Fe-IMAC kit provided recoveries >80% from spiked bovine serum albumin digests and good selectivity. The enrichment was tested on serum samples within a shotgun proteomics workflow with a protein dephosphorylation step and tryptic digestion. The recovery of the entire analytical workflow was 20%, which was compatible with previous data on TiO2 phosphopeptide enrichment.
The issue of sulfate detection and localization was studied by different fragmentation techniques (CID, HCD, ETD, EThcD, ETciD) and compared to the related phosphorylated counterpart sequences. All tested conditions were suitable for phosphopeptide analysis, but for intact sulfopeptides only the sequence could be obtained. However, the use of metal adduct precursors, especially potassium ones, improved the stability of the sulfate modification and provided information on both sequence and site localization of sulfate under ETD and ETD-hybrid strategies. In-source neutral loss of SO₃ and under EThcD provided diagnostic peaks suitable to distinguish the sulfopeptides from the nearly isobaric phosphopeptides.

Acknowledgements
This work was supported by PRIN project Prot. 2017Y2PAB8.

References
[1]  D.Virág, B. Dalmadi-Kiss, K. Vékey, L. Drahos, I. Klebovich, I. Antal, K. Ludányi. Chromatographia 83 (2020) 1–10.
[2]  A.L. Capriotti, A. Cerrato, A. Laganà, C.M. Montone, S. Piovesana, R. Zenezini Chiozzi, C. Cavaliere. Anal. Chem. 92 (2020) 7964–7971.

Andrea Cerrato, Anna Laura Capriotti, Aldo Laganà

Dipartimento di Chimica, Università degli Studi di Roma La Sapienza, Piazzale Aldo Moro 5, 00185, Rome, Italy

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Lipidomics, once considered a branch of metabolomics, has nowadays gained its proper analytical approaches that differ significantly from routinary methods for metabolomics. In untargeted lipidomics, the structural information is highly dependent on the analytical methods with a progressively higher degree of confidence from the individuation of the lipid classes and subclasses to the evaluation of the stereochemical properties. Data processing and identification by software programs are toughened by a large number of adducts and several isomeric mass overlaps, that occur any time different lipid species generate adducts with the same sum composition [1]. Phosphocholine-containing lipids (PCLs) that present a positively charged quaternary ammonium, undergo in-source fragmentation of a methyl group in negative ion mode. Moreover, these compounds can generate intense adducts with formate or acetate ions present in the chromatographic buffers. As a result, not only a multitude of ions are formed, but these adducts suffer from numerous isomeric mass overlaps. Buffer modification workflows (BMW) are based on the use of unlabeled and stable-isotopically labeled buffer modifiers and have been recently proposed in untargeted metabolomics for significantly facilitating feature annotation [2]. For the first time, a BMW is presented specifically for phosphocholine-containing lipids based on the simultaneous use of AcOH and d3-AcOH in the chromatographic buffers followed by a customized data processing workflow on Compound Discoverer software. The proposed methodology was optimized by the Box-Behnken design of experiments, and the overall performance was compared to several standard metabolomics- and lipidomics-based approaches. The optimized methodology was applied to the characterization of human plasma resulting in 135 PCLs extracted from the raw datasets and correctly annotated [3].

References
[1]  Y.H. Rustam, G.E. Reid, Anal. Chem. 90 (2018) 374–397.
[2]  W. Lu, X. Xing, L. Wang, L. Chen, S. Zhang, M. R. McReynolds, J. D. Rabinowitz, Anal. Chem. 92 (2020) 11573–11581.
[3]  A. Cerrato, S. E. Aita, A. L. Capriotti, C. Cavaliere, C. M. Montone, S. Piovesana, A. Laganà, Anal. Chem. 93 (2021) 15042–15048.

Adrian Golebiowski^1,2, Aleksandra Orzol², Malgorzata Szultka-Mlynska², Katarzyna Glowacka³, Mateusz Cichorek³, Pawel Pomastowski¹, Edith Cruzado-Tafur³, Boguslaw Buszewski<sup>1,2

1</sup>Centre for Modern Interdisciplinary Technologies, Nicolaus Copernicus University, Wilenska 4, 87-100 Torun, Poland
²Department of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, Gagarin 7, 87-100 Torun, Poland
³Department of Plant Physiology, Genetics and Biotechnology, University of Warmia and Mazury in Olsztyn, Oczapowskiego 1a, 10-719 Olsztyn, Poland

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The chemical element can be present at various states of oxidation or associated with compounds (organic or inorganic)- determining these forms is a speciation analysis. (Bio)ligands - metal properties determine the toxicity of compounds or determine bioaccumulation, translocation, transfer through membranes. The results of cadmium speciation in pea plants (Pisum sativum L.) were showed. The plants were grown in hydroponic cultivation in a study mimicking abiotic stress (heavy metal contamination – 50 µM CdSO₄) for the plant where the role of silicon (1 mM or 2 mM Na₂SiO₃) in counteracting the negative effects of contamination on plant development was investigated. After 21 days of Cd treatment or/and Si supplementation, roots, shoots and leaves were analyzed. The scanning electron microscopy (SEM) was used to visualize the morphology of the roots of pea plants. After the sample preparation process (homogenization, lysis, dissolution of compounds in an aqueous solution), coupled analytical techniques based on size exclusion chromatography with the use of UV, MALS and ICP-MS detectors were used to characterize the chemical forms of cadmium. The collected fractions from the column were subjected to more detailed characteristics in terms of organic deposit (ESI- MS, MALDI-MS) to give more information about these species. The presented results are the beginning of understanding and description of the phenomenon of transfer and bioaccumulation of cadmium and silicon through the plant cell wall.

Acknowledgments
This work was supported by research project Opus 18 No. 2019/35/B/ST4/02791 (2020-2024) from the National Science Centre, Kraków, Poland.

Taja Verovšek^1,2, David Heath¹, Ester Heath<sup>1,2

1</sup>Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia
²Jožef Stefan International Postgraduate School, Jamova 39, 1000 Ljubljana, Slovenia

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Wastewater-based epidemiology (WBE) is used to estimate drug use in a population by determining drug biomarkers (parent compound or metabolite) in raw wastewater. Selecting parent compounds as biomarkers, like in the case of amphetamine (AMP), methamphetamine (MAMP) and 3,4-methylenedioxymethamphetamine (MDMA), can lead to biased consumption estimates since the parent compound may originate from the consumption or the disposal of the unused drug. Fortunately, in the case of chiral biomarkers, such as amphetamines, enantiomeric profiling can complement WBE data by discriminating between disposal and consumption and offering information on the potency of drugs and their drug origin, i.e., licit or illicit [1]. So far, chiral liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been used for enantiomeric profiling of amphetamines in wastewater [1]. As an alternative, chiral derivatisation with (R)-(−)-α-Methoxy-α-(trifluoromethyl)phenylacetyl chloride followed by GC-MS/MS detection was explored in this study. The method showed good performance in terms of recovery (81–99%), accuracy (99–111%), repeatability (1–8 %RSD) and linearity (LOQ–1000 ng/mL), while LOD and LOQ were 120 ng/L and 400 ng/L, respectively. When applied to wastewater samples from two Slovene municipalities (Ljubljana: LJ1 – anomalously high MDMA load, LJ2 – random wastewater sample, and Velenje: VE1 – high AMP load), racemic MDMA in LJ1 (enantiomeric fraction, EF=0.511) indicated the disposal of the unused drug and consumption the enrichment of R-MDMA in LJ2 (EF=0.666). Racemic amphetamine (VE1: EF=0.514 and LJ2: EF=0.459) suggested that both racemic and more potent S-AMP are available, while MAMP is available only in its more potent, S-enantiomeric form (S-MAMP detected in LJ2: EF=0).

Acknowledgements
This work was supported by the Slovenian Research Agency (ARRS): Program group P1-0143 and Projects L1-9191 and N1-0143.

 References
[1]  B. Kasprzyk-Hordern, D.R. Baker, Environ. Sci. Technol. 46 (2012) 1681–1691.

Radik Mametov^1,2, Maciej Monedeiro-Milanowski², Wojciech Kupczyk³, Bogusław Buszewski<sup>1,2

1</sup>Department of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Gagarina 7, 87-100, Torun, Poland
²Interdisciplinary Centre for Modern Technologies, Nicolaus Copernicus University, Wilenska 4, 87-100, Torun, Poland
³Department of General, Gastroenterological and Oncological Surgery, CM, UMK, Torun, Poland

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After development and introduction of SPME by Arthur and Pawliszyn [1] new direction and trends in SPME is in progress. Different types of devices and materials were introduced. One of the versatile types of materials as SPME coatings are conductive polymers and their composites and modifications. Conductive polymers are materials with a highly π-conjugated polymeric chain, which have mechanical properties of organic polymers and both electronic properties of metals. The most widely used representatives are polypyrrole [2], polyaniline and polythiophene [3]. In this study polypyrrole-MOF coatings were directly electrodeposited onto metal wires in one step. The electropolymerization process was carried out under a constant deposition potential and applied to the corresponding aqueous/organic electrolyte containing pyrrole and MOF particles. Obtained composite materials were characterized by different methods. The influence of synthesis parameters on the extraction efficiency by new fibers was evaluated.  Obtained fibers were utilized for extraction a different types of VOCs emitted by bacteria. Parameters of extraction identification were optimized.

Acknowledgements
This work was supported by The National Science Centre (Poland) in a framework of the Preludium 18 project No. 2019/35/N/ST4/04363 (2020-2022).

References
[1]  C. L. Arthur, J. Pawliszyn, Anal. Chem. 62 (1990) 2145–2148.
[2]  R. Mametov, G. Sagandykova, F. Monedeiro, B. Buszewski, Talanta 232 (2021).
[3]  M. Lashgari, Y. Yamini, Talanta 191 (2019) 283-306.

Natalia Manousi¹, Natasa P. Kalogiouri¹, Peter Q. Tranchida², George A. Zachariadis¹, Luigi Mondello², Erwin Rosenberg³, Antonio Ferracane<sup>2

1</sup>Laboratory of Analytical Chemistry, Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece
²Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Messina, Italy
³Institute of Chemical Technologies and Analytics, Vienna University of Technology, 1060 Vienna, Austria

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Honey is widely consumed worldwide due to its high nutritional and medicinal value and its acceptance from the consumers is closely related to its organoleptic characteristics that depends on the volatile constituents of the samples [1]. The aim of this research was the development of an analytical method for the characterization of the volatile compounds released from honey samples of different botanical origin. As such, solid-phase microextraction (SPME) Arrow combined with comprehensive two-dimensional gas chromatography-mass spectrometry (GC×GC-MS) was used for the first time for the determination of volatile compounds in honey. SPME Arrow fibers are characterized by high extraction phase area and volume, resulting in high sensitivity, while they overcome the short-comings of conventional SPME including low mechanical durability [2]. The analytical protocol was optimized to obtain the maximum efficiency in terms of extraction and separation of the volatile constituents. Under the optimum extraction conditions, SPME Arrow was compared with conventional SPME regarding their performance using different types of honey samples. The utilization of SPME Arrow enabled the identification of a higher number of volatile compounds in the examined samples, while it resulted in higher sensitivity and reproducibility. Moreover, an in-depth sample characterization was made possible, due to the high separative power of GC×GC-MS equipped with a cryogenic modulator. A non-orthogonal set-up consisting of a polar stationary phase used in first dimension and an apolar micro-bore column (5%-phenyl-95%methylpolysiloxane) was selected. The tentative identification of the volatile compounds was performed by using a mass spectral database containing linear retention index information. All things considered, the combination of SPME Arrow and GC × GC serves as a powerful analytical tool for the monitoring of volatile components in honey samples.

References
[1]  L. Piasenzotto, L. Gracco, L. Conte, J. Sci. Food Agric. 83 (2003) 1037–1044.
[2]  J.S. Herrington, G.A. Gómez-Ríos, C. Myers, G. Stidsen, D.S. Bell, Separations 7 (2020).

Milica Nešović¹, Nebojša Nedić², Uroš Gašić³, Živoslav Tesić⁴, Stevan Blagojević⁵, Irena Vovk<sup>6

1</sup>Institute of General and Physical Chemistry, Studentski trg 12-16, Belgrade 11158, Serbia
²University of Belgrade - Faculty of Agriculture - Institute for zootehnics, Nemanjina 6, Belgrade - Zemun 11080, Serbia
³University of Belgrade - Department of Plant Physiology, Institute for Biological Research “Siniša Stanković”, Bulevar despota Stefana 142, 11060 Belgrade, Serbia
⁴University of Belgrade - Faculty of Chemistry, Studentski trg 12-16, Belgrade 11158, Serbia
⁵Institute of General and Physical Chemistry, Studentski trg 12 – 16, 11158 Belgrade, Serbia
⁶Laboratory for Food Chemistry, National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia

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The botanical and geographical origin of the honey could provide different phenolic profiles of honey. In this regard, the presented study analyzed monofloral, polyfloral, and honeydew honey from Tara Mountain in Serbia. Phenolic analysis of 27 honey samples was done using an ultra-high-performance liquid chromatography system with a diode-array detector and connected to a triple-quadrupole mass spectrometer (UHPLC-DAD MS/MS). In order to provide important information for the assessment of the botanical and/or geographical origin of honey samples, phenolic analysis of honey samples confirmed the presence of 19 phenolic compounds. The most prominent compounds were p-coumaric acid, caffeic acid, and pinocembrin. The dominant presence of kaempferol was also noted. Otherwise, other phenolic compounds such as quercetin 3-O-rhamnoside, quercetin 3-O-glucoside, and luteolin showed the lowest values. Results for antioxidant activity expressed through the total phenolic content (TPC) and relative scavenging activity (RSA) were in the range from 307.0 to 1273.8 mg GAE/kg, and 730.3 to 3888.7 µmol TE/kg, respectively. This study could provide insight into the contribution of phenolic compounds to the antioxidant activity [1], as well as to the origin [2] for honey from Tara. In addition, apparent significance were phenolic compounds, which could be used as the markers of the botanical and geographical origin of honey.

Acknowledgments
This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (contract Nos. 451-03-68/2022-14/ 200051, 451-03-68/2022-14/200007).

References
[1]  M. Nešović, U. Gašić, T. Tosti, J. Trifković, R. Baošić, S. Blagojević, Lj. Ignjatović, Ž. Tešić, RSC Adv. 10 (2020) 2462.
[2]  M. Nešović, U. Gašić, T. Tosti, N. Horvacki, N. Nedić, M. Sredojević, S. Blagojević, Lj. Ignjatović, Ž. Tešić, R. Soc. Open Sci. 7 (2020) 201576.

Cene Skubic¹, Eva Kočar¹, Petra Nassib¹, Kaja Blagotinšek Cokan¹, Irena Vovk², Mitja Križman², Damjana Rozman<sup>1

1</sup>Center for Functional Genomics and Bio-Chips, Institute of Biochemistry and Molecular genetics, Faculty of Medicine, University of Ljubljana, Zaloška cesta 4, 1000 Ljubljana, Slovenia
²Laboratory for Food Chemistry, National Institute of Chemistry, Ljubljana, Hajdrihova 19, 1000 Ljubljana, Slovenia

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Cholesterol synthesis is a housekeeping pathway and any abnormalities in the late part of synthesis usually lead to the accumulation of sterol intermediates which results in severe malformations in humans. There are 19 predicted sterol intermediates in cholesterol synthesis, at least 13 of them experimentally confirmed. Sterol intermediates, apart from being cholesterol precursors, have other important physiological functions, their accumulation can be toxic, but due to lack of accessible methodology knowledge is scarce. In order to study sterols synthesis abnormalities, their function and the effect of different pathologies on their concentration, the development of a simplified LC-MS method was necessary.
Some of the sterol intermediates have the exact same mass and MRM so good separation is necessary. We were able to develop a simple and robust LC-MS/MS for the quantitative analysis of 13 sterols from the late part of cholesterol synthesis using available standards (zymosterol, dehydrolathosterol, 7-dehydrodesmosterol, desmosterol, zymostenol, lathosterol, (dihydro-)FF-MAS, (dihydro-)T-MAS, lanosterol, 7-dehydrocholesterol and dihydrolanosterol). For separation we used pentafluorophenyl stationary phase and mobile phase in isocratic mode consisting of methanol, water, 1-propanol and formic acid (80/10/10/0,05%). We were able to apply and validate developed method on different biological samples, like HepG2 liver cell models with deletions in cholesterol synthesis genes, on human serum, mouse liver tissue and primary mouse hepatocytes.
We were able to detect majority of targeted sterols in biological samples. As expected, the largest concentrations of sterols were present in cell/liver models with deletion of cholesterol synthesis genes, where sterols above deleted enzymes are accumulating in large quantities. In some samples, especially the human serum, cholesterol concentration is several orders of magnitude larger than other sterols and due to a large chromatographic peak, can obstruct detection of lathosterol and both sterols have to be quantified using an adapted protocol. With minor changes in isolation procedure, our method can be used on a wide variety of biological samples, enabling us to decipher enigmatic roles of sterols in health and disease.

Monika Śmiełowska^1,2, Bożena Zabiegała<sup>1

1</sup>Department of Analytical Chemistry, Faculty of Chemistry, Gdańsk University of Technology (GUT), Narutowicza 11/12 St., 80-233 Gdańsk, Poland
²Department of Environmental Chemistry and Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, Gagarina 7, 87-100 Toruń, Poland

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Monitoring of polybrominated diphenyl ethers (PBDEs) in indoor environments involves determining their concentrations in air, airborne particles, and settled dust. Each of these factors is a source of human exposure to PBDEs. In this study, an attempt was made to model the concentrations of PBDEs in various typical indoor environments based on actual measurements of PBDEs in dust collected from them. The analytical procedure for the determination of eight PBDE representatives in the dust was based on the matrix solid phase dispersion (MSPD) technique. Based on the results obtained, the concentrations of the other components of the commercial mixtures of PBDEs in the dust were estimated. The resulting model of PBDEs distribution in dust and air was used to estimate adult exposure to PBDEs in the indoor environments studied. For each of the examined environments, dust ingestion was the predominant route of exposure. For congeners with log Koa > 12, this was virtually the only route of exposure. Comparison of total PBDE exposure and oral reference dose (RfD) values allowed assessment of the risk from PBDE exposure. No risk from exposure to PBDEs was noted. However, there is still a need to expand environmental monitoring of PBDEs in Poland.

Acknowledgements
This work was supported by National Science Centre, Poland under the Preludium 16 programme in the years 2019–2022, project no. 2018/31/N/ST10/03664.

Sieglinde Zelzer¹, Andreas Meinitzer², Dietmar Enko², Markus Herrmann², Walter Goessler<sup>3

1</sup>Institute of Chemistry, University of Graz, Graz, Austria
²Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Graz, Austria
³Institute of Chemistry, University of Graz, Graz, Austria

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Different foods are a source of vitamin D, whereby vitamin D2 is produced in plants and mainly in fungi [1]. Therefore, mushrooms are an important source for all people and the content of vitamin D2 in different mushroom species is of interest [2]. Most of the published methods use high performance liquid chromatography (HPLC) with spectrophotometric detection, which requires tedious sample preparation because of lacking detection selectivity. Highly variable vitamin D2 levels can be found in literature and method errors can not be excluded [3,4]. Detection using mass spectrometry ensures high analytical selectivity and sensitivity. Hence, a two-dimensional high performance liquid chromatography mass spectrometry (HPLC-MS/MS) method was developed for the determination of native vitamin D2 in mushroom samples. The final method comprises a one step extraction procedure provides a limit of detection of 0.01 μg vitamin D2/g dry mass (DM), a limit of quantification of 0.05 μg vitamin D2/g DM. A total run time including re-equilibration of the columns for the next injection was 7.5 min. Standard addition to extracts of Pleurotus ostreatus and Lentinula edodes showed excellent correlation coefficients (R2) of 0.996 and 0.999. Vitamin D2 concentrations in Pleurotus ostreatus and Lentinula edodes were determined to be 0.119 ± 0.004 (n=3; coefficient of variation (CV) 3.6%) and 0.078 ± 0.003 μg/g DM (n=3; CV 3.5%) respectively. Spiking experiments in mushroom samples resulted in recovery rates of about 90.4 ± 2.1%.
Our newly developed LC-MS/MS method comprises a fast and simple sample preparation and a short run time. Thus, accurate measuring of vitamin D2 in mushrooms is possible, which is helpful to improve the knowledge about vitamin D2 levels in mushrooms.

Acknowledgements
This study is part of the PhD of the first author SZ.

References
[1]  C. Proserpio, V. Lavelli, F. Gallotti, M. Laureati, E. Pagliarini, Nutrients 11 10 (2019) 2441.
[2]  L. O’Mahony, M. Stepien, M.J. Gibney, A.P. Nugent, L. Brennan, Nutrients 3 (2011) 1023–1041.
[3]  S.J. Huang, C.P. Lin, S.Y. Tsai, J. Food Compos. Anal.42 (2015) 38–45.
[4]           T.S. Keflie, N. Nölle, C. Lambert, D. Nohr, H.K. Biesalski, Saudi J Biol Sci. 26 (2019) 1724–1730.

Narmin Hamidli, Melinda Andrasi, Cynthia Nagy, Attila Gaspar

University of Debrecen, Egyetem ter 1, 4032, Debrecen, Hungary

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Proteins are the actuators of many vital biological processes. Understanding the structural characterization in their intact states entails the studies of cell biology, disease prevention and treatment [1]. Top-down mass spectrometric (MS) technique is sensitive to enable the studies for structural and dynamical identification of intact proteins when coupled with capillary zone electrophoresis (CZE) [2]. However, a serious concern is the analyte adsorption on the bare fused silica (BFS) capillary surface, which necessitates the application of extreme pH or the use of coatings to minimize the analyte-wall interactions [3]. Our study involves the use of BFS capillaries employing the background electrolytes with very low pH and compares the analytical performance with those coated with polybrene as a dynamic and linear polyacrylamide (LPA) as a static coating. The work presents the differences in the ideal operating conditions of each capillary. The results suggested the analysis in BFS capillaries with BGE of low pH (pH=1.8) resulted in good precision (0.56-0.78 RSD% and 1.7-6.5 RSD% for migration times and peak areas respectively) and efficiency values with minimum adsorption into the capillary wall. Coated capillaries showed higher resolving power for the separation of different forms (subunits of hemoglobin) of the protein. However, the separation performance in LPA coated capillary distinguished from others based on their stability, reproducibility over 25 runs and shorter analysis time in less than 10 min. The applicability of these methods was also supported by the analysis of protein rich snake venom. Thus, the application of BFS capillaries for the analysis of intact protein mixtures would be also an efficient choice compared to coated capillaries when ideal conditions are applied.

Acknowledgments
The research was supported by Stipendium Hungaricum (#242771) and the New National Excellence Program of the Ministry for Innovation and Technology (ÚNKP-20-3-I).

References
[1]  O. Skinner, N. Haverland, L. Fornelli, Nature Chem. Biol. 14 (2018) 36-41.
[2]  J.P. Williams, L.J. Morrinson, J.M. Brown, J.S. Beckman, V.G. Voinov, F. Lermyte, Anal. Chem. 92 (2020) 3674-3681.
[3]  N. Hamidli, M. Andrasi, C. Nagy, A. Gaspar, J. Chromatogr. A 1654 (2021) 462448.

Simion Beldean-Galea¹, Cătălina Herghelegiu¹, Vlad Pănescu¹, Clément Guillard², Virginia Coman<sup>3

1</sup>Babeş-Bolyai University, Faculty of Environmental Science and Engineering, Fântânele 30, 400294 Cluj-Napoca, Romania
²École Supérieure de Physique et de Chimie Industrielle de la Ville de Paris, Paris Sciences et Lettres University, 10 rue Vauquelin, 75231 Paris Cedex 05, France
³Babeş-Bolyai University, Raluca Ripan Institute for Research in Chemistry, Fântânele 30, 400294 Cluj-Napoca, Romania

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Triazines are herbicidal pesticides with a planar heterocyclic aromatic structure with benzene ring in which three carbon atoms are replaced by three nitrogen atoms. Recent studies have shown that triazines are widely used in agriculture for high crop yields, but their incorrect and abusive application can cause endocrine disrupting effects in both humans and animals [1]. According to the European Union legislation, the maximum residue limit for total triazines is 0.5 μg/L and for each individual triazine 0.1 μg/L [2].
The aim of this work is to develop a method for analysing seven triazines (prometon, propazine, atrazine, simazine, prometryn, ametryn, and terbutryn) in drinking water samples collected from 25 rural Roma communities in Transylvania, Romania, to estimate the risk to human health.
Solid phase extraction (SPE) was used to isolate the target triazines from aqueous matrices to analyse them by chromatographic methods. The efficiency of three SPE cartridges (C18-U, Strata X, and Strata SAX) was tested, with the best results for the C18-U cartridge with a recovery of more than 90% for all triazines analysed, except atrazine (mean value of 60%).
The coupled techniques, gas chromatography with mass spectrometry (GC-MS) and liquid chromatography with photodiode array detector (HPLC-PDA), were used in order to obtain a selective and a sensitive analysing method for the investigated triazines. Our GC-MS (TR-5MS, 30 m x 0.25 mm, ID 0.25 μm; He) results using selected ion monitoring (SIM) mode showed a limit of detection (LOD) of 0.02 μg/L and a limit of quantification (LOQ) of 0.06 μg/L, while those obtained by the HPLC-PDA method (Nova-Pak C18, 300 x 3.9 mm, 4 μm; gradient elution ACN–KH₂PO₄) a LOD of 0.15 μg/L and a LOQ of 0.45 μg/L.
The developed method was successfully applied to analyse the target triazines in 57 drinking water samples.

Acknowledgements
The research leading to these results has received funding from the Norway Grants 2014-2021 under Project contract no. 23 / 2020 (RO-NO-2019-0463).

References
[1] S. Zheng, M. He, B. Chen, B. Hu, J. Chromatogr. A 1614 (2020) 460728.
[2] Q. Yang, B. Chen, M. He, B. Hu, Talanta 186 (2018) 88-96.

Lapo Renai¹, Cristina Vanessa Agata Scordo¹, Ayoub El Ghadraoui¹, Sergio Santana-Viera², JoséJuan Santana Rodriguez², Serena Orlandini³, Sandra Furlanetto³, Donatella Fibbi⁴, Dimitra Lambropoulou⁵, Massimo Del Bubba<sup>1

1</sup>Department of Chemistry, University of Florence, Via della Lastruccia 3-13, 50019 Sesto Fiorentino (Florence, Italy)
²Instituto Universitario de Estudios Ambientales y Recursos Naturales (i-UNAT), Universidad de Las Palmas de Gran Canaria, 35017 Las Palmas de Gran Canaria, Spain
³Department of Chemistry “U. Schiff”, University of Florence, Via U. Schiff6, 50019 Sesto Fiorentino, Florence, Italy
⁴GIDA S.p.A. Via di Baciacavallo 36, 59100 Prato, Italy
⁵Department of Chemistry, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece

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This study focused on the Analytical Quality by Design (AQbD) optimization of the chromatographic separation and mass spectrometric detection of a wide group of structurally heterogeneous model pharmaceutical compounds (PhCs) and transformation products (TPs), chosen to cover the challenging issues of the co-presence of compounds characterized by (i) a wide range of physicochemical properties, (ii) the same mass transitions, and (iii) different ionisation modes [1]. Italian consumption of PhCs were also considered as election criteria of target analytes. C18 and PFP stationary phases, ACN/CH₃OH ratios and acidity of the eluents, column temperature, initial organic phase percentage, and elution gradient were investigated by AQbD, aiming at optimizing critical resolutions, sensitivities, and analysis time. Statistically significant models were obtained in most cases with fitting and cross validation coefficients in the ranges of 0.681-0.998 and 0.514-0.967, respectively. After optimization, the analysis of PhCs was performed in a single chromatographic run, adopting a mixed acquisition mode based on scheduled acquisition windows comprising both single polarity and continuous polarity switching. For most investigated PhCs, the method provided detection limits in the sub-ng/L to low ng/L range, meeting for macrolides the sensitivity requested by 2018/840/EU. The optimized method was applied to the direct injection analysis of PhCs and TPs in four wastewater treatment plant (WWTP) effluents and surface water (SW) samples collected in the receiving water bodies. Absolute values of matrix effect were found to be far higher than 20% for most target analytes in most samples. Seventeen PhCs and two TPs were quantified in at least one sample, at the wide concentration range of about 1-3200 ng/L [2].

References
[1]  S. Orlandini , S. Pinzauti , S. Furlanetto, Anal. Bioanal. Chem. 405 (2013) 443–450.
[2]           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.

Simion Beldean-Galea¹, Vlad Pănescu¹, Cătălina Herghelegiu¹, Zsolt Boros^1,2, Virginia Coman<sup>3

1</sup>Babeş-Bolyai University, Faculty of Environmental Science and Engineering, Fântânele 30, 400294 Cluj-Napoca, Romania
²University of Agriculture Sciences and Veterinary Medicine, Faculty of Veterinary Medicine, Calea Mănăștur 3-5, 400372 Cluj-Napoca, Romania
³Babeş-Bolyai University, Raluca Ripan Institute for Research in Chemistry, Fântânele 30, 400294 Cluj-Napoca, Romania

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Persistent organic pollutants (POPs), namely 20 organochlorine pesticides (OCPs), 12 polychlorinated biphenyls (PCBs) and 16 polycyclic aromatic hydrocarbons (PAHs) were analyzed in 22 agricultural soil samples collected from 13 rural Roma communities in Transylvania, Romania, using the chromatographic coupled techniques, GC-ECD for halogenated compounds and GC-MS for PAHs. These compounds were extracted from the collected soil samples by ultrasound-assisted extraction method, followed by open-column chromatography to purify the extract [1]. The total amount of each type of POP analyzed in the investigated soil samples and the prevalent POPs found, were as follows:
- OCPs, between 0.087 and 0.707 mg/kg soil; prevalent OCPs (number of samples): α-HCH (22), Heptachlor (21), Dieldrin (15), Heptachlor Exo (14), Methoxychlor (10), 4,4-DDT (9), 2,4-DDD (4), 2,4-DDT (2), Hexachlorbenzene (2), and Tecnazene, Endrin, Heptachlor Endo, Trans-chlordan, and Cis-chlordan (1 sample). Not found: γ-HCH, Quintozen, Aldrin, α-Endosulfan, 4,4-DDE and β-Endosulfan.
- PCBs, between 0.002 and 0.210 mg/kg soil; prevalent PCBs (number of samples): PCB-(138+180) (20), PCB-194 (12), PCB-52 (8), PCB-31 (7), PCB-153 (5), PCB-18 (3), PCB-28 (2), PCB-114 (1). Not found: PCB-44, PCB-101 and PCB-149.
- PAHs, between 2.88 and 501.77 mg/kg; prevalent PAHs (number of samples): Fluoranthene, Pyrene, Chrysene (22); Benzo[b]- and Benzo[k]fluoranthene (20), Benzo[g,h,i]perylene and Dibenzo[a,h]anthracene (19), Indeno[1,2,3-cd]pyrene (18), Benzo[a]pyrene (14). Not found: Acenaphthene, Anthracene and Phenanthrene.
The PAH diagnostic ratio between the sum of low molecular weights and of high molecular weights (ƩLMW/ƩHMW), respectively is under one, which suggests a pyrogenic source of PAHs in all analyzed soil samples.

Acknowledgements
The research leading to these results has received funding from the Norway Grants 2014-2021 under Project contract no. 23 / 2020 (RO-NO-2019-0463).

Reference
[1] B. Barhoumi, M.S. Beldean-Galea, A.M. Al-Rawabdeh, C. Roba, I.M. Martonos, R. Bălc, M. Kahlaoui, S. Touil, M. Tedetti, M. Ridha Driss, C. Baciu, Sci. Total Environ. 660 (2019) 660–676.

Anja Kristl¹, Matevž Pompe², Aleš Podgornik<sup>2

1</sup>Institute of Forensic Medicine, Faculty of Medicine, University of Ljubljana, Korytkova ulica 2, 1000 Ljubljana, Slovenia
²Faculty for Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, 1000 Ljubljana, Slovenia

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Separation of macromolecules such as peptides, proteins, DNA or RNA molecules can be more challenging than the separation of small molecules due to many influential parameters that affect retention [1]. In addition to the most common optimization tactics (change in column chemistry, mobile phase composition, temperature, flow rate), increasing the pressure has been shown to have a significant impact on macromolecule adsorption processes in reversed-phase chromatography [2]. Our study showed a similar increase in retention when performing separations of macromolecules on an anion exchange column [3]. An investigation of lnK values showed a decrease in the partial molar volume upon adsorption on the stationary phase [4]. The retention increase with pressure rise was more pronounced for isocratic separations of larger and more flexible molecules. Given these results, we then attempted to optimise the separation of IgG with intact and opened structure by using only the pressure increase. However, using the same elution buffer as in previous separations, the retention time decreased with the pressure increase, which is in contrast to all published pressure effect on retention. We will present additional studies on the effect of pressure using a model protein that has a significant dipole moment to allow separations on both anion and cation exchange columns. Based on this comprehensive understanding of the effects of pressure, we show a practical approach of optimising a separation method for seven therapeutic insulin variants [5] with increasing the separation pressure by as little as 130 bar. This study demonstrates how understanding adsorption processes is beneficial for method optimization.

Acknowledgments
The work was supported by the Slovenian Research Agency (ARRS) through programme P1-0153, P1-0201 and project J2-9440.

References
[1]  A. Astefanei, I. Dapic, M. Camenzuli, Chromatographia, 80 (2017) 665–687.
[2]  S. Fekete, J.-L. Veuthey, D.V. McCalley, D. Guillarme, J. Chromatogr. A, 1270 (2012) 127–138.
[3]  A. Kristl, P. Lokošek, M. Pompe, A. Podgornik, J. Chromatogr. A, 1597 (2019) 89–99.
[4]  A. Kristl, M. Lukšič, M. Pompe, A. Podgornik, Anal. Chem., 92 (2020) 4527–4534.
[5]  A. Kristl, A. Podgornik, M. Pompe, J. Chromatogr. B Biomed. Appl., 1171 (2021) 122557.

Sanling Zuo¹, Waldemar Studzinski², Katarzyna Stadnicka¹, Przemyslaw Kosobucki<sup>2

1</sup>PBS Bydgoszcz University of Science and Technology, Faculty of Animal Breeding and Biology, 28 Mazowiecka, 85-084, Bydgoszcz, Poland
²PBS Bydgoszcz University of Science and Technology, Faculty of Chemical Technology and Engineering, 3 Seminaryjna, 85-326 Bydgoszcz, Poland

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In poultry, probiotics and prebiotics are used to improve the gut health by modulating its microbiome. The role of metabolites extracted by probiotics, especially in early nutrition needs elucidation. The metabolomic protocol was optimized, using intestinal cell models, to analyse the footprint of probiotics. Co-culture of Caco-2 cells with the candidate probiotic is the chosen study model. 3 groups were used: 1/ the blank control: Caco-2 monolayer at >90% confluence, 2/ the negative control: candidate probiotic culture, 3/ the sample: co-culture of Caco-2 cells and the probiotic at a ratio of 30:1 (bacteria: cell), cultivated for 48h, n=3 replications. The medium from each group was collected and subject to Gas Chromatography- Mass Spectrometry. The pretreatment is a critical step that determines the quality of the analysis. The optimization was performed using standard solutions: acetic acid, formic acid, caproic acid, succinic acid and the other common intestinal metabolized fatty acids. Three major protocol components were compared: 1/ different drying methods and temperatures, 2/ different reconstitution reagents, 3/ different vaporization temperature programs. Regarding the drying method, the SpeedVac low temperature vacuum concentration (at -4℃) allowed for a higher recovery rate compared to the nitrogen blow drying and vacuum drying method at room temperature. The use of MeOH, KOH/MeOH, and Isooctane as reconstitution reagents lead to a severe loss of the sample components. Derivatization was found as a optimal method to stabilize volatile compounds with a good mass spectral responses. After optimizing the gasification program, the main peaks were separated and a good mass spectrum was obtained. Raw data was matched with the NIST17.L library for non-targeted metabolomics. Changes in short-chain fatty acids profiles, and specific organic acids were found in the biological samples with the proposed protocol. This protocol will be used in further metabolomic studies with a co-culture model.

Acknowledgments

This work was supported by: travel grant at PBS "Dzialania Naukowe Mlodych" and partially by NCN (Poland), 2019/35/B/NZ9/03186 (OVOBIOM).

Luca Menegoz Ursol¹, Ana Srbinovska¹, Chiara Conchione¹, Karolina Brkić Bubola², Olivera Koprivnjak³, Sabrina Moret<sup>1

1</sup>Department of Agri-Food, Environmental and Animal Sciences, University of Udine, via Sondrio 2/A, 33100, Udine, Italy
²Institute of Agriculture and Tourism, Deparment of Agriculture and Nutrition, K. Huguesa 8, 52440, Poreč, Croatia
³Faculty of Medicine, Department of Food Technology and Control, University of Rijeka, Brace Branchetta 20, 51000, Rijeka, Croatia

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Olive oils may be contaminated with mineral oil hydrocarbons (MOH) from different sources (on average 10-20 mg/kg), but only few researchers [1] investigated the presence of trace amounts (<2 mg/kg) of saturated hydrocarbons (MOSH) in oils obtained by olives directly hand-pickled from the tree. On the other hand, olive oils also contain endogenous n-alkanes which may provide information related to the olive’s variety, geographical origin, presence of leaves in the milling phase, etc. [2]
Different from endogenous n-alkanes, which show a typical distribution from n-C21 to n-C35, with the prevalence of odd terms over even ones, mineral oils give GC-FID traces characterized by humps of unresolved peaks, sometimes dominated by linear alkanes with an equal distribution of even and odd terms.
On-line HPLC-GC-FID represents the reference method for mineral oil determination in edible oils and, by simply adjusting the amount of sample injected, it can also be used to investigate on endogenous n-alkanes. When analysing MOSH amounts in the range between 0.5- 2.0 mg/kg, the analytical determination must be preceded by sample enrichment and a purification step to eliminate the interference by endogenous n-alkanes.
The first aim of this contribution was to propose simple and low solvent consumption protocols to investigate the distribution of endogenous and exogenous hydrocarbons in extra virgin olive oils. Subsequently, the optimized protocols were used to analyse oils from selected olive samples (2 different cultivars, Leccino and Bjelica) collected in Croatia and Italy. For the first time, the impact of the presence of leaves and the absence of stones during the milling phase, on both exogenous and endogenous hydrocarbons, was evaluated. The results obtained showed the presence of traces of MOSH in all the samples analysed and confirmed the great potential of endogenous n-alkanes in discriminating the cultivar and the different milling conditions.

References
[1]  L. Menegoz Ursol, C. Conchione, A. Srbinovska, S. Moret, Food Chem. 370 (2022) 130966.
[2]  A. Srbinovska, C. Conchione, L. Menegoz Ursol, P. Lucci, S. Moret , Foods 9 (2020) 1546.

Anže Pavlin, Drago Kočar

Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, 1000 Ljubljana, Slovenia

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Exact discrimination between honey types is necessary in the field of honey determination and for detecting counterfeits. In this study chestnut (C), linden (L), acacia (A), spruce (S) and silver fir (SF) honey types were investigated with intention of finding specific compounds and the most abundant compounds for each honey type. A simple method of solid phase micro-extraction (SPME) of headspace was developed with no special pretreatment of the samples. Analyzes were performed on gas chromatography system coupled with mass spectrometer.
Analysis resulted in determination of 12 specific compounds for linden, 6 specific compounds for chestnut, 4 specific compounds for acacia, 5 specific compounds for spruce and 9 specific compounds for silver fir honey, 36 in total of which concentration ranged from 11.5 ng/g to 2515.5 ng/g relative to internal standard benzophenone. Some of them were suggested as possible markers for corresponding honey type.
Study of most abundant compounds were also made. 5 compounds with highest content in each honey type were considered. Their concentration ranged from 346 ng/g to 31390 ng/g relative to internal standard benzophenone. As the most abundant compound in honey, methyl octanoate, is proposed. It was present in top 5 compounds with highest concentration in 4 different honey types (chestnut, acacia, spruce, silver fir) ranging from 346.0 to 1261.4 ng/g relative to internal standard benzophenone. Methyl octanoate was one of the most abundant compounds determined using SPME method also in cotton honey [1].

Acknowledgement
The authors thank Medex d.o.o. for providing honey samples and Mikro+Polo d.o.o., for providing laboratory material. This work was supported by the Slovenian research agency ARRS, grant number P1-153.

Reference
[1] E. Alissandrakis, A. C. Kibaris, P. A Tarantilis, P. C. Harizanis, M. Polissiou, J. Sci. Food Agric. 85 (2005) 1444–1452.

Jure Zekič^1,2, Mitja Križman<sup>1

1</sup>Laboratory for Food Chemistry, Department of Analytical Chemistry, National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia
²Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, SI-1000 Ljubljana, Slovenia

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Among hemp (Cannabis sativa L.) metabolites, cannabinoids and terpenes represent two of the most important groups. Both of them are interesting also from the analytical aspect, and are most often determined separately, due to the differences in their chemical and physical characteristics. Simultaneous determination of cannabinoids and terpenes thus represents a relatively demanding challenge. In our work, an original gas chromatographic method for simultaneous determination of major terpenes and cannabinoids in plant samples and their extracts has been developed. The main issues in the method development process were related to the large differences in polarity and volatility between both groups of analytes, but also to the need for an exhaustive decarboxylation of cannabinoid acidic forms. Extraction procedure has been optimised and acetone was found out as the most appropriate extraction solvent. For successful chromatographic separation, a medium polarity column was applied. Basic validation parameters of the method were tested and the linear range, LOD and LOQ were determined for main analytes. Parallel testing proved the results for cannabinoids are comparable to those obtained from established HPLC methods [1].

Acknowledgement
This research was supported by the Slovenian Research Agency (research core funding No. P1-0005).
Jure Zekič is a student of the doctoral programme in Chemical sciences at the Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia.

Rerefence
[1] J. Zekič, M. Križman, Molecules 25 (2020) 5872.

Črtomir Podlipnik, Matevž Pompe, Damjan Jan Pavlica

Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, 1001 Ljubljana, Slovenia

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Hydroperoxides are of great importance in atmospheric and biological chemistry. However, their determination is associated with several problems: unknown and usually low absorption coefficients, high reactivity, thermal instability, and a lack of available reference standards. Since no standards were available for α pinene hydroperoxides, we developed a GC-FID method with predicted relative response factors using the concept of effective carbon number.1
Hydroperoxides of α-pinene were synthesised with photochemically generated singlet oxygen. Nuclear magnetic resonance (NMR) was used to identify four hydroperoxide derivatives of α-pinene. NMR has the advantage of being an absolute detection method, which makes it the method of choice for accurate quantification of analytical reference standards. However, due to complexity and price, chromatographic methods are still more common for routine analysis.
Four hydroperoxide isomers were separated by liquid and gas chromatography with spectrophotometric and mass spectrometric detection (MS). Because the analytes had low absorption coefficients and weak MS ionization, we developed a GC-FID approach with prediction of response factors. The analytes were unstable in the injector and even on the column, so we performed a derivatization step. The linearity of the method was confirmed between 1 and 90 mg/L with correlation coefficients above 0.99. The method is reproducible with relative standard deviations below 5%.2
The method was used to study the aging of turpentine. The mass fraction of α-pinene hydroperoxides increased from 0.1 % to 5.1% after 20 days of air exposure. The formation of hydroperoxides was also detected in photochemical reactions with PM10 particles. Moreover, we believe that this new synthesis and analysis approach could be used for other unstable hydroperoxides.

References
[1] J. T. Scanlon, D. E. Willis, J. Chromatogr. Sci. 23 (1985) 333–340.
[2] D. J. Pavlica, Č. Podlipnik, M. Pompe, Acta Chim. Slov. 68 (2021) 728–735.

Acknowledgements
The study was carried out with financial support from the Slovenian Research Agency (P1-0153).

Justyna Piechocka, Natalia Litwicka, Rafał Głowacki

University of Lodz, Faculty of Chemistry, Department of Environmental Chemistry,163 Pomorska Str., 90-236 Łódź, Poland

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So far, the association between homocysteine (Hcy) and its metabolite Hcy thiolactone (HTL), and origination of some civilization diseases has been well documented. Among others, the rise of Hcy and HTL levels in plasma/urine is considered to be the risk predictor of morbidity and mortality of Alzheimer`s disease (AD), cardiovascular disease (CV) and serves as a predictor of some systemic non-fatal diseases, in particular diabetes mellitus (DM). Moreover, it was found that formaldehyde (FA) levels are elevated in urine and blood of patients with AD, CV events and DM. Additionally, Hcy, HTL and FA toxicity is well-known [1, 2]. In parallel, it is well established that Hcy and HTL are reactive towards aldehydes in aqueous environment, forming substituted thiazinane carboxylic acids. This report provides evidence that Hcy/HTL and FA adduct, namely 1,3-thiazinane-4-carboxylic acid (TCA) is formed in vivo in humans.
Here, we present a gas chromatography–mass spectrometry (GC–MS) based method which was elaborated to identify and quantify TCA in human urine [3]. The GC–MS assay involves chemical derivatization of the analyte with isobutyl chloroformate in the presence of pyridine, followed by ethyl acetate extraction of obtained isobutyl derivative of TCA. The validity of the method has been demonstrated based upon US FDA recommendations. The assay linearity was observed within 1–50 µM range for TCA in urine. The method was successfully applied to urine samples delivered by apparently healthy volunteers (n=15). The GC–MS assay may provide a new analytical tool for routine clinical analysis of the role of TCA in living systems in the near future.

Acknowledgements
The research was supported by University of Lodz Initiative of Excellence Research University (IDUB) grants for young researchers (project number B2111101000020.07). The study was approved by the Ethics Committee of the University of Lodz (decision identification code 4/KBBN-UŁ/III/2020-21).

References
[1]  M. Pietzke, J. Meiser, A. Vazquez, Mol. Metab. 33 (2020) 23.
[2]  P. Oliveira, F. Laurindo, Clin. Sci. 132 (2018) 1257.
[3]  J. Piechocka, N. Litwicka, R. Głowacki, Int. J. Mol. Sci. 23 (2022) 598.

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.

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.

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.

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.

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.