Session 1
Centre of Metabolomics and Bioanalysis (CEMBIO), Facultad de Farmacia, Universidad San Pablo-CEU, CEU Universities, Urbanización Montepríncipe, 28060 Boadilla del Monte, Spain
This email address is being protected from spambots. You need JavaScript enabled to view it.
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.
FACILITATING HIGH THROUGHPUT FOOD DETERMINATIONS WITH THIN FILM SPME TECHNOLOGIES
Department of Chemistry, University of Waterloo, Waterloo, ON N2L3G1, Canada
This email address is being protected from spambots. You need JavaScript enabled to view it.
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.
ChiBioFarAm Department, University of Messina, Vaile Annunziata, 98168, Messina, Italy
This email address is being protected from spambots. You need JavaScript enabled to view it.
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.
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
This email address is being protected from spambots. You need JavaScript enabled to view it.
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.
Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo n. 2, 166 10 Prague 6, Czechia
This email address is being protected from spambots. You need JavaScript enabled to view it.
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.
1Instituto de Carboquímica/CSIC, Miguel Luesma Castán 4, 50018, Zaragoza, Spain
2Aragon Institute of Nanoscience/Universidad de Zaragoza, Campus Rio Ebro, 50018, Zaragoza, Spain
3Analytical Chemistry Department/Universidad de Zaragoza, Campus Río Ebro, 50018, Zaragoza, Spain
4CEQMA/CSIC-Universidad de Zaragoza, Campus San Francisco, 50009, Zaragoza, Spain
5Analytical Chemistry Department/Universidad de Zaragoza, Campus San Francisco, 50009, Zaragoza, Spain
6Instituto Aragonés de Ciencias de la Salud/Fundación ARAID, Campus San Francisco, 50009, Zaragoza, Spain
This email address is being protected from spambots. You need JavaScript enabled to view it.
The current scientific paradigm to understand the role of lipids in human diseases is based on the hypothesis that some diseases have distinctive lipid profiles (biomarkers) with respect to the respective healthy status. HPTLC separation of sample, followed by densitometry and MS coupling provides a simple but powerful approach for determining lipid classes and obtaining a detailed structural elucidation of lipids present in biological extracts, allowing their exact recognition by their m/z, and confirmation by their collision-induced dissociation MS/MS data [1]. The application of HPTLC to identification in human plasma of 19 molecular species related to globotriaosylceramides (Gb3) which are considered biomarkers of Fabry disease, a lysosomal storage disorder, will be first discussed [2]. Another example is the open question of whether exosome lipids can be considered as potential cancer biomarkers. This faces our current limited knowledge of their composition. A procedure to isolate exosomes and obtain a phospholipid (PL)-class determination and identification of their molecular species will be also discussed, from embryonic murine fibroblasts (NIH-3T3 cell line), and metastatic murine skin melanoma cells with different degrees of malignancy [3]. For both examples, a detailed structural characterization of molecular species of each concerned lipid class was performed directly from the chromatographic plate, thanks to an elution-based interface, using ion-trap technology by different approaches that involve simultaneous positive and negative ESI-MS, MS/MS, or APCI.
Acknowledgements
This work was supported by DGA-FEDER (E25_20R, N&SB), AES PI21/00036, CSIC 202180E076, ISCIII PI19/01007, ERC Advanced Grant CADENCE (grant no. ERC-2016-ADG-742684)
References
[1] V.L. Cebolla, C. Jarne, J. Vela, R. Garriga, L. Membrado, J. Galbán, J. Liq. Chromatogr. & Rel. Technol. 44 (2021) 148-170.
[2] C. Jarne, L. Membrado, M. Savirón, J. Vela, J. Orduna, R. Garriga, J. Galbán, V.L. Cebolla, J. Chromatogr. A 1638 (2021), 461895.
[3] M. Sancho-Albero, C. Jarne, M. Savirón, P. Martín-Duque, L. Membrado, V.L. Cebolla, J. Santamaría, Int. J. Mol. Sci. 2022, 23(3), 1150.
- Venue:
- Faculty of Chemistry and Chemical Technology