Session 5

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.