2024 SGMS Meeting

The 41st SGMS Meeting will take place in Beatenberg on 24-25 October 2024 high above Lake Thun in the Bernese Oberland, with a scenic view of the Swiss Alps! The meeting will be preceeded by the SGMS school on the days prior to the meeting. 

Invited speakersRegistration, Info, Deadlines, FeesAbstract submissionProgramPlenary lecturesOral presentationsPoster presentationsSponsors

Invited speakers for meeting 2024

• Irene Chetschik, Zurich University of Applied Sciences ZHAW
• Albert Heck, Utrecht University and Netherlands Proteomics Centre
• Anton Kaufmann, Kantonales Labor Zürich
• Philipp Weller, Mannheim University of Applied Sciences

2024 SGMS Meeting Registration, Deadlines & Fees

Registration

Registration is closed. 

Contact the organizers with questions on registration or to be added on the waiting list (e-mail). 

Deadlines

• Early abstract submission: August 1st (Poster/Oral acceptance will be notified by September 1st).
• Abstract submission: September 1st (Poster/Oral acceptance will be notified by September 21st).
• Standard registration: August 31st

General Information

All lectures and workshops will take place at the main lecture hall of the Dorint Hotel in Beatenberg, Switzerland.

There is no need to register personally at the Dorint Resort Blüemlisalp! The SGMS committee will manage the hotel reservation and payments. Only the “extras” will have to be paid directly at the hotel reception (e.g. extra nights, phone-calls, muh-bar, mini-bar).

We will follow a first come first serve policy for the distribution of the hotel rooms (~110 rooms available!). Students that want to make use of the student price will be asked to share a room. A roommate can be indicated on the registration form.

Registration procedure

After registration you will receive a confirmation email with attached bill for the meeting and where appropriate SGMS membership fees. You can opt for becoming a member and benefit of a reduced registration fee.

A surcharge of CHF 50 will be for payments submitted after or during the meeting. Please pay in advance. 

Only full board can be chosen! There is no attending the meeting without hotel accommodation. 

In case of food allergies and intolerances, contact us such that arrangements can be made with the hotel. 

Cancellations

Cancellation requests, received by e-mail before October 1st, will qualify for a refund. There will be a handling fee of 25%. After October 1st, no refunds will be made; however, substitute participant can attend.

Notification of all such changes must be sent by e-mail before October 15th. 

No show: No refund will be made. Fees remain due.

Family-friendliness

The rooms and facilities at the Dorint hotel in Beatenberg are well suited if you need or want to bring family members of all ages. Contact us by e-mail to find the best arrangement for you. For the registration of accompanying people, please write an e-mail. 

Abstract submission

Abstract submission is closed!

Early deadline for abstract submission is August 1st (notification by September 1st). Regular deadline is September 1st  (notification by September 21st). 

Posters: Poster size should not exceed 146 H x 118 W cm (size of pin wall). There will be a defined poster session. The number of posters is limited. 

Meeting fees

SGMS Annual Meeting (registration includes one night with breakfast at the Dorint hotel, Thursday and Friday lunch, apéro and Gala dinner on Thursday, and coffee breaks). Wednesday night accommodation and dinner on request.

SGMS School (registration includes one night with breakfast at the Dorint hotel, Wednesday lunch and dinner, and coffee breaks). Tuesday night accommodation and dinner on request.)

all prices in Swiss Francs, including VAT (update 13.06.2024)

Student support program

All students are hosted at a special rate; double room occupancy. If no roommate is indicated on registration form, it will be allocated by the organizers.

Meeting program

Plenary lectures

Irene Chetschik: Exploring the chemical sensory code of chocolate by Sensomics

Zurich University of Applied Sciences (ZHAW), Wädenswil, Switzerland

Chocolate, being made traditionally from the roasted beans of Theobroma cocoa, which were discovered thousands of years ago in the Amazonian rainforest, is beloved for its flavour all over the word. Although a lot of research has been conducted in the past with the aim to unravel the compounds that are responsible for the flavour of cocoa and chocolate, techniques that combine instrumental analysis with human sensory perception have been seldomly applied to identify the compounds that are predominantly contributing to the overall flavour of cocoa and chocolate. The use of the Sensomics approach, that can be considered as a sensory guided analytical approach, enables the decoding of flavour profiles by the application of methods such as Gas Chromatography - Olfactometry coupled with Mass Spectrometry. By these means, the huge diversity of cocoa flavours coming together as a result of bean variety, post-harvest treatment and last but not least technological processing can be analysed on a molecular level. In this presentation, the importance of the exploration of the chemical sensory code of cocoa and chocolate products by Sensomics methodologies will be highlighted, both in the field of future quality assessment of cocoa and chocolate and as a valuable tool for the development of new cocoa and chocolate products that convince the consumers with their flavour properties and contribute to sustainability and fairness in the cocoa sector.

Albert Heck: Single Molecule Mass Spectrometry

Utrecht University and Netherlands Proteomics Centre, Utrecht, The Netherlands

Around for more than a century the analytical technique of mass spectrometry is blooming more than ever and applied in nearly all aspects of the natural and life sciences. In the last two decades the analytes to be measured have become much larger, more complex, more heterogeneous, largely due to the developments in advanced drug therapies. Molecular entities like antibody-based drugs, mRNA-based drugs, genome delivery vehicles and virus or lipid particle-based vaccines are more and more entering the therapeutic arena, but they come with challenges in quality control, that require more and better analytical technologies. In this lecture I will discuss efforts that we undertake in our lab to take up this challenge describing results from single molecule techniques such as mass photometry and especially charge-detection mass spectrometry. 
Charge detection MS (CDMS) is quite unique as it can simultaneously measure the charge and m/z ratio of single particles. We demonstrated the power of Orbitrap-based CDMS applied to a variety of fascinating systems, assessing for instance the cargo load of recombinant AAV-based gene delivery vectors, the build-up of immune-complexes involved in complement activation, and quite accurate masses of highly glycosylated proteins, such as the SARS-CoV-2 spike trimer proteins. 
The single molecule nature of the analyses allows us to track the behavior of individual ions inside the Orbitrap, which provides unique, fundamental insights into mechanisms of ion dephasing and demonstrated the stability of high mass ions. By modifying an Orbitrap UHMR we trapped and monitored individual ions for up to 25 seconds, resulting in an unprecedented improvement in signal-to-noise, mass resolving power, and accuracy in charge and mass determination.

Anton Kaufmann: Active research in the function as an industrial or government chemist
(looking back at an eventful professional life)

Kantonales Labor Zürich, Zurich, Switzerland

Industrial or governmental chemists are busy analyzing samples, struggling hard to meet tight deadlines and writing reports. Frequently, they encounter interesting scientific topics, worth to investigate in some more depth. Yet, time constraints, but frequently also the lack of support from the employer prevents them from doing applied research and in a next step publishing their results.
Throughout my professional life, I have been trying to be productive in both fields. This led to some 80 publications appearing in renowned, peer reviewed scientific journals and a number of analytical chemistry book chapters. I would like to share with you some interesting or sometimes even funny scientific encounters, highlights but also failures. The topics cover the reason why expensive old wines contain the highest lead residue levels? How analytical data in combination with multivariate statistic beats professional wine tasters when it comes to the identification of the geographical origin of wines? The challenges encountered when replacing time proven tandem quadrupole technology by high resolution mass spectrometry in the field of residue analysis (e.g. veterinary drugs or pesticides in food).  This not only refers to users who were and still are reluctant to embrace a new technology, but also static regulations which are written in a way to prevent any evolution. Trying to push methodology forward is not always a smoot and risk-free process. Most of the expensive instruments I was permitted to evaluate and finally to buy, soon became powerful workhorses in my laboratory. Yet, there were also a few cases where the big promises I had to make to my superiors were not fulfilled. This frequently required the time-consuming redevelopment and consequently re-validation of existing analytical methods. But in one case, I even had to give up a technology which at that time was not yet ready to handle the samples and the analytical problems encountered in my laboratory.
In short, you are going the hear stories about the joys, but also the challenges and the frustrations you are going to encounter when you do something more, or occasionally even something else, than what is written in your job description. 

Philipp Weller: Untargeted benchtop volatilomics at trace levels in foods and fermenations – why the gas phase is much more than hot air

Mannheim University of Applied Sciences, Mannheim, Germany

The term “volatilomics“ addresses the comprehension of the volatilome, which depicts an  important, but yet scarcely understood part of the metabolome – in systems biology, but also in fields, such as food analysis. Here, such approaches are often also called “foodomic” strategies and meanwhile are an integral part in food authentication and quality control. Volatilomics are an elegant way of correlating volatile organic compounds (VOCs) via the gas phase from the matrix with specific properties of that product: authenticity, quality or provenance are just a few examples. This is in particular relevant, as a major part of the aroma-relevant compounds in foods, e.g. in roasted coffee, citrus oils or saffron belong to the VOC fraction, which can be analyzed without sample contact by using the headspace over the sample. 
A major challenge is the complexity of the enormous amount of different substances found, which often are not relevant as individual species, but rather their total “fingerprint”, resulting of all amenable substances. This high-dimensional spectral information cannot be interpreted without applying powerful machine learning algorithms or chemometrics, a strategy which is generally referred to as "omics" approaches. These data are combined with modern machine learning techniques to extract the maximum possible information from products to improve quality and confirm authenticity. 
VOCs are also an important source of information in fermentation processes. As integral part of modern biotechnology, they feature an enormously complex gas phase, which is so far not part of existing, molecule-specific monitoring strategies.
Typically, the required techniques are laboratory-based and not useable at the so-called “point-of-need”, which limits their use. This presentation will demonstrate principles and examples of benchtop "volatilomics" approaches in food and fermentation processes, consequently named as “fermentomics”, that in the future could be used directly at the location where they are needed.

Oral presentations

Studying Unfolding of Peptides Using Gas-Phase FRET upon Activation by Photons and Collisions 

Kim Greis, Linus Busse, Ri Wu, Lukas Benzenberg, Renato Zenobi 

ETH Zürich, Department of Chemistry and Applied Biosciences, Zürich, Switzerland 

One of the premises of native mass spectrometry is that biomolecular ions can be generated in the gas phase with only very little disturbance in their structure. To achieve this, such molecules are usually electrosprayed from buffered aqueous solutions using low needle voltages and gentle settings for transferring the ion within the mass spectrometer. Ideally these conditions prevent unfolding of the investigated species. The structural analysis of proteins often relies on tandem mass spectrometry workflows, where the analyte ions are fragmented either by collision-induced dissociation (CID), ultraviolet photodissociation (UVPD), or electron-based activation methods (ExD). These methods provide a rich number of fragments that allows one to assign the primary structure of a protein with high confidence. However, obtaining information on the three-dimensional structure of proteins is challenging with these methods. Additionally, the activation of the ions could lead to unfolding, which is hardly investigated. 

To understand how collisional and photon-based activation influences the structure of peptides, we hyphenated mass spectrometry (MS) with Förster resonance energy transfer (FRET). This allows us to record the time-resolved fluorescence emission of mass-to-charge selected, labeled peptides. In the presence of a fluorescence quencher, the signal is quenched. The degree of quenching is distance dependent, allowing one to estimate the distance between a quencher and the label. By studying the time-dependent fluorescence signal, it is possible to determine to what extent collisional and photon-based activation unfold the investigated peptide. 

First results indicate that collisions below the fragmentation threshold do not lead to significant unfolding of the investigated peptides at the probed timescales. Increasing laser energies, on the other hand, leads to increased fluorescence lifetimes, indicative for unfolding of the peptide. As a next step, we will observe how the fluorescence lifetimes change after increased periods of laser irradiation. These experiments will provide guidelines under which conditions deviations from the native three-dimensional structure of peptides can be expected during mass spectrometric analysis. 

Microsolvation of Charged Sites in the Gas Phase: Are Native Protein Structures Retained? 

Lukas Raphael Benzenberg, Elena Giaretta, Ri Wu, Despoina Svingou, Renato Zenobi 

ETH Zürich, Department of Chemistry and Applied Biosciences, Zürich, Switzerland 

Native mass spectrometry is frequently used to characterize proteins and their complexes, thus, seeking to find conditions under which native structural features are retained upon desolvation. However, charged residues are believed to compensate for their loss of solvation by engaging in non-covalent interactions with the backbone, consequently disrupting secondary structure hydrogen-bond networks and leading to distortion of protein structure. In this study, gas-phase conformational analysis using fluorescence spectroscopy and ion mobility-mass spectrometry (IM-MS) provided complementary structural insights into the role of microsolvation by crown ether variants in inhibiting backbone collapse of charged sites, thus preserving native-like helical protein structures after desolvation. 

Experimental findings suggest a greater unfolding of the alpha-helical scaffold for highly charged species, consistent with increased Coulombic repulsion. Specifically, [M+6H]⁶⁺, [M+7H]⁷⁺ and [M+8H]⁸⁺ species exhibit donor-acceptor distances of 73.5±1.3 Å, 92.5±3.0 Å and 105.5±9.5 Å, respectively, with corresponding CCS values of 1304±2 Å2, 1438±9 Å2, and 1499±3 Å2. Complexation of charged sites with 18-Crown-6 results in reduced distances values, showing compaction of the helical structure by 19.57±0.05 Å and 16.0±0.1 Å for [M+7H+(18C6)4]⁷⁺ and [M+8H+(18C6)5]⁸⁺ species, respectively, with a minor compaction of 2.91±0.02 Å for [M+6H+(18C6)2]⁶⁺. While CCS incrementally increased per crown ether bound, the increase was insignificant compared to the CCS of [Na(18C6)1]⁺ or [K(18C6)1]⁺, approximating the inherent CCS of 18-Crown-6. Thus, a minor CCS reduction by microsolvation was presumed to be offset by a more substantial increase in CCS from crown ether addition to the lysine side chain. These findings indicate that while microsolvation helps preserve more compact structural features by preventing the collapse of charged side chains onto the backbone in vacuo, it does not sufficiently shield charges to counteract Coulombic repulsion. Crown ether variants with different cavity sizes or electron-rich substituents were further employed to evaluate potential enhancements in charge shielding. Distance constraints and collision cross sections (CCS) values comparable to those of 18-Crown-6 indicate that all crown ether variants probed achieve sufficient solvation of charged sites, while exhibiting negligible charge shielding. Comparing donor-acceptor distances of gaseous and microsolvated species with constraints obtained in the solution phase revealed that despite extensive microsolvation in the gas phase, the helix still undergoes significant elongation after desolvation. These findings indicate that unfolding events in vacuo are predominantly dictated by charge repulsion rather than backbone collapse of charged sites (intramolecular solvation). When ionized from a 150 mM NH4OAc aqueous solution, the formation of less charged species [M+5H]⁵⁺ and [M+4H]⁴⁺ was also observed. These species exhibit donor-acceptor distances of 64.4±2.0 Å and 56.8±1.5 Å, respectively, indicating greater compaction and resembling near-native, solution-phase structures with donor-acceptor distances of 48.0±0.9 Å (91.6% of the species) and 88.1±7.3 Å. 

We show that gas-phase fluorescence spectroscopy is a valuable tool in native mass spectrometry, providing high-resolution distance constraints that complement ion mobility-mass spectrometry shape information. This study demonstrated its sensitivity in detecting subtle structural changes and its suitability for microsolvation studies, facilitating direct comparisons of experimental data between gas-phase and solution-phase environments. We advocate for the broader integration of fluorescence spectroscopy into native mass spectrometry to advance structural biology research. 

Proteomic Characterization of Zebrafish Embryonic PAC2 Cell Line 

Mihai-Ovidiu Degeratu, René Schönenberger, Nikolai Huwa, Ksenia Groh 

Eawag, Swiss Federal Institute of Aquatic Science and Technology, Environmental Toxicology, Dübendorf, Switzerland 

Chemical testing for aquatic risk assessment requires high numbers of fish and ample resources, which raises both ethical and economical concerns. Cell lines derived from fish tissues/organs represent promising alternative (i.e., animal-free) test models for predicting chemical toxicity in fish. However, the adoption of fish cell lines in regulatory settings has been lagging behind compared to its faster-growing use in academic research. This could in part be due to a significant knowledge gap in the functional characterization of fish cell line models as well as insufficient understanding of how closely they resemble their tissues/organ of origin. These questions can be addressed by analysing the molecular profiles of fish cell lines to identify the expressed genes/pathways and thus deduce the corresponding functional capacities, as well as to compare them to their respective counterparts in vivo. Moreover, improved molecular characterization of fish cell lines could facilitate the development of novel toxicity testing strategies based on molecular markers of toxicity.  

Here I present the case of the zebrafish (Danio rerio) embryonic cell line, PAC2. This cell line is better suited for performing molecular analyses compared to fish cell lines derived from other species, because zebrafish has been frequently used in molecular biology and life sciences research, including biomedicine and (eco)toxicology. Hence, the rich molecular information and bioinformatics resources available for this species can be employed to improve the understanding of various molecular processes underlying the toxicity mechanisms and effects inside fish cells. Consequently, PAC2 holds the promise of becoming a powerful in vitro fish model for toxicity testing strategies that rely on understanding the molecular mechanisms of toxicity or include the measurement of molecular markers. However, like other fish cell lines, PAC2 is currently not well characterized on the molecular level.  

To address this knowledge gap, I performed mass spectrometry-based global proteomics analysis of the PAC2 cell line. Cells were sampled at different cell culture growth phases, ranging from the early (lag) phase through exponential to the stationary and early decline phases. Tryptic peptides were separated by nano-scale liquid chromatography (nanoLC) and analysed by tandem mass spectrometry (MS/MS) on the Thermo Scientific™ Orbitrap Fusion™ Lumos™ Tribrid™ mass spectrometer, operated in a data-independent acquisition (DIA) mode. Data processing was performed using the Spectronaut® 18 software from Biognosys AG (2023). Data analysis was performed in RStudio and Perseus.  

Our method allowed measuring approximately 7000 protein groups in the PAC2 cells. The protein abundance profiles were found to be clearly distinct between the different cell culture growth phases. Ongoing work focuses on developing a data analysis pipeline to explore the proteins and pathways expressed in the different growth phases and to perform comparative analyses across growth phases as well as with proteomics data collected in embryonic zebrafish in vivo. Subsequently, the developed strategy will be applied to characterize further fish cell lines frequently used in aquatic risk assessment. 

Human and Environmental Exposure by Synthetic Antioxidants: Insights from Wastewater 

Corina Meyer, Mara Baer, Juliane Hollender 

Eawag - Swiss Federal Institute of Aquatic Science and Technology, Environmental Chemistry, Dübendorf, Switzerland 

Synthetic antioxidants (SAOs) are of widespread use in everyday life products, including polymeric materials, medicinal face masks, personal care products and food. Based on their structural functionalities, three classes of antioxidants are differentiated, comprising phenolic, amine and phosphite antioxidants. They act by preventing oxidative reactions and extending product shelf life. As antioxidants are additives and not chemically bound to their substrates, leaching from materials and detection in wastewater treatment plants (WWTPs) has been reported. Correspondingly, uptake by humans is expected through contact to materials. However, little is known about the human and environmental exposure of antioxidants. Therefore, this study aims (i) to identify SAO metabolites in untreated wastewater to get insights into human exposure and (ii) to analyze the abatement behavior of the parent compounds and their metabolites through the different treatment steps in WWTPs to understand the environmental exposure. Prior to sample analysis, method optimization was conducted to enhance sensitivity in electrospray ionization for the phenolic antioxidants by the addition of ammonium fluoride in the eluent or in a post-column infusion. This enables more efficient deprotonation of low molecular weight SAOs in the negative and detection of ammonium adducts for high molecular weight SAOs in the positive ionization mode. Combined with online-SPE-LC-HRMS/MS analysis, a MS/MS library of SAO metabolites was generated by human liver S9 incubation experiments. This library, containing about 1000 potential metabolites, was compared to influent samples of three Swiss WWTPs, leading to approximately 50 matches. Using machine learning and in silico fragmentation tools, structure elucidation was so far possible for one compound (fenozan), which was confirmed by a reference standard. This result gives first hints with regard to human exposure towards SAOs. Besides the detection of metabolites in untreated wastewater, knowledge about the abatement of parent SAOs through the different treatment steps in WWTPs is missing. Thus, the same three WWTPs, equipped with an advanced treatment step (ozonation or granular activated carbon filtration), were sampled and analyzed by online-SPE-LC-HRMS/MS after every treatment step to assess environmental exposure by WWTPs. Results indicate incomplete abatement of some SAOs. Overall, humans and the environment seem to be exposed to synthetic antioxidants and further research is required to understand the uptake of SAOs into the human body, their metabolism and their behavior in wastewater treatment. 

Transfer of chlorinated paraffins from a yoga mat during the use phase

Oscar Mendo Diaz^1,2, Adriana Tell^1,3, Urs Stalder², Laurent Bigler², Susanne Kern³, Davide Bleiner^1,2, Norbert V. Heeb¹

1) Swiss Federal Laboratories for Materials Science and Technology, Empa!
2) University of Zurich, UZH
3) Zurich University of Applied Sciences, ZHAW, Switzerland

Chlorinated paraffins (CPs) are anthropogenic polychlorinated n-alkanes with the general molecular formula CnH2n+2-xClx. CPs are commonly used in the polymer industry as plasticizers and flame retardants.[1] Depending on their carbon-chain length, CPs are classified as very short-chain (vSC-, C<10), short-chain (SC-, C10–C13), me-dium-chain (MC-, C14–C17), long-chain (LC-, C18–C21) and very long-chain (vLC-, C>21) CPs. The chlorine content can vary between 30% and 70% w/w. Since 2017, SCCPs are labeled as persistent organic pollutants by the Stockholm Convention and MCCPs are under evaluation since 2021. Due to that, the determination of levels of CPs in plastics, transfer behaviors and human exposure are of rising concern.[2] 

Technical CP mixtures can contain a broad range of carbon- (C-, nC=9–30) and chlorine- (Cl-, nCl=2–20) homo-logues with millions of constitutional isomers and stereoisomers. These complex mixtures are indiscriminately introduced in plastic consumer products such as yoga mats to obtain the desired physic-chemical proper-ties.[3] However, Swiss regulations do not allow more than 1.5 mg of SCCPs per 1 g of plastic. Yoga mats are very often manipulated during demanding physical activities. Depending on the pressure applied and the time, CPs could be transferred from the yoga to the human skin. Such process could intensify the undesired human exposure to SCCPs, the intake of SCCPs in the body and lead to multiorgan dysfunctions.[4] 

Herein, a yoga mat collected from the Swedish market in 2017 was extracted directly using Soxhlet and wiped with a cotton-gauze and artificial sweat. All samples were analyzed by a liquid chromatographic system cou-pled with a mass spectrometer. Chlorinated paraffins were monitored as adduct ion of [M+Cl]- and detected with an Orbitrap mass analyzer. Mass spectrometric data were evaluated by the newly developed data evalua-tion tool CP-Hunter.[5] Positive correlations in the transfer of CPs were found between the exposure time, the number of contacts and the presence of artificial sweat. 

[1] Chen, C. et al., Environ. Sci. & Technol., 2022 ; [2] Huang, J.-W. et al., Sci. Total Environ., 2023 ; [3] Mendo Diaz, O., et al., Chemosphere, 2023 ;[4] Shuangshuang, C. et al., Environ. Int., 2023 ; [5] Mendo Diaz, O., et al., Anal. Chem., 2024

Semi-quantification of triglycerides with resolved fatty acid composition using a targeted MS3 approach on a novel hybrid nominal mass instrument 

Hector Gallart-Ayala¹, Charles Maxey¹, Rahul Ravi Deshpande², Bashar Amer², Susan S. Bird², Charles Maxey², Philip Remes², Claudia P.B. Martins², Cristina C. Jacob², Julijana Ivanisevic¹ 

1) University of Lausanne, Lausanne, Switzerland
2) Thermo Fisher Scientific, San Jose, CA, United States 

Triglycerides (TAGs) are the most abundant lipids in human plasma made up of a glycerol backbone and three fatty acids which are connected via ester linkages. The measure of their total circulating level in plasma has been widely used as key clinical biomarker of metabolic and cardiovascular diseases, however, this lipid class comprises a wide variety of chemically diverse triglyceride species with varying fatty acid composition. Currently, these species can be measured in a high throughput manner only at the sum composition level. Yet, structurally closely related species can have different biological roles and be associated with distinct metabolic consequences. Measuring individual TAG species with high selectivity or fatty acid chain resolved composition, would lead to a better understanding of their physiological functions in health and disease.   

In this work we used the fragmentation data acquired in an untargeted lipidomics experiment on a HRAM mass spectrometer to generate the list of transitions for a targeted MS3 assay aiming at quantification of individual TAGs in human plasma using the new hybrid nominal mass instrument.  

Plasma lipid extracts were separated on a Thermo Scientific Accucore C30 column (2.1x150 mm) and analyzed using an untargeted LC-ddMSn lipidomics approach on Thermo Scientific Vanquish Horizon UHPLC system coupled to a Thermo Scientific Orbitrap IQ-X Tribrid mass spectrometer. In this method, TAGs were automatically targeted by specific product ion or neutral losses which enabled further CID MS2/MS3 characterization in the same ddMS2 acquisition. TAGs were annotated with LipidSearch 5.1 software. TAGs with highest confidence “Grade A” annotation level were selected to develop a targeted MS3 assay using the same 30 minutes LC separation on the new hybrid nominal mass instrument for their estimated quantification in human plasma. Estimated concentrations are measured via single-point calibration or spike with multiple stable isotope-labeled internal standards with varying fatty acid chain composition (to account for the differences in ionization and fragmentation efficiencies). Method performance was evaluated using isotopically labeled triglyceride standards spiked in NIST plasma at different concentrations.  

LipidSearch software was used to match the experimentally acquired MS2 and MS3 spectra of potential TAG species detected in NIST plasma against the in silico predicted spectral library (Lipidblast?). More than 100 TAGs annotated with high confidence (“grade A”) were the fed into a targeted MS3 assay on the new hybrid nominal mass instrument for their routine quantification in human plasma. 

Removing Current Barriers in Non-target GC/MS Analysis for a Comprehensive Identification of Unknowns 

Arnd Ingendoh¹, Sonja Klee², Steffen Bräkling², Marleen Vetter², Eliska Ceznerova², Marlene Moskowitz³ 

1) Bruker Daltonics GmbH & Co KG, Fahrenheitstrasse, Bremen, Germany
2) Tofwerk AG, Schorenstrasse, Thun, Switzerland
3) Bruker Inc., Manning Park, Billerica, MA, USA 

Aside of standard and regulated methods for targeted analysis, there is a growing interest in environmental, food or biochemical studies to get a more holistic overview of compounds of interest in complex matrices. While LC/MS/MS became a standard for non-target analysis, GC-HRMS using electron ionization (EI) for NIST library searches in non-target studies often suffers from an ambiguous identification of unknowns. Due to less specific fragmentation, missing molecular ion signals or absence in the reference library, insufficient scorings or false positives are generated. With the addition of the accurate molecular mass or even an elemental composition by CI (chemical ionization), NIST search results can be efficiently filtered, reduced and corrected. Presented here are non-target studies for wastewater analysis, human urinary metabolites, comparison of vegan food with non-vegan equivalents and material emission where the simultaneous acquisition of EI and CI spectra in the same GC run efficiently improves the quality of information and enables the identification of yet unseen or difficult compounds.  

An 8500 GC was coupled to the ecTOF (Bruker, Bremen, Germany), which uses a 70 eV EI source and a novel HRP CI source in parallel in single runs. Various GC methods and sampling procedures were employed depending on the analytical need of the study, including liquid injection of extracted samples, headspace sampling including SPME and thermal desorption using Tenax tubes. To ensure the generation of the molecular ion information and for a highly efficient CI process, different reactant ions (e.g., N2H+, H3O+ and NH4+) can be selected and switched in the HRP CI source. 

The confidence of the compound identification improves significantly by combing the EI and CI information achieved in the same GC run. False positives from EI-only approaches can easily be detected and sorted out. NIST hybrid searches for compounds not listed in libraries are highly supported by the knowledge of the elemental composition from the CI data. Tentative structure elucidation becomes feasible in many cases. The novel workflow has the potential to change the acceptance and usage of GC-HRMS for non-targeted analysis substantially. 

Integrating Mass Spectrometry and Droplet Microfluidics for High-Throughput Biocatalyst Engineering 

Berk Kocar, Denijel Latifovic, Tania M. Roberts, Sven Panke 

ETH Zürich, Zürich, Switzerland 

Biocatalyst engineering campaigns typically rely on high throughput screening (HTS). However, present HTS methods frequently require custom assay designs to connect performance readout to an analytical method that can be applied with high throughput. Mass spectrometry (MS) offers a highly flexible, label-free alternative, but typically requires extensive sample preparation and hence is not employable in a high-throughput manner. Recent developments in the design of interfaces between droplet microfluidics and MS, specifically ESI-MS suggest that it might become possible to integrate droplet microfluidics and MS to achieve an MS-based HTS workflow. With respect to application to the field of biocatalyst optimization, the main challenges that remain are analyte ionization in complex reaction media, the sacrificial nature of the MS analysis of a droplet and the dependence on custom interfaces between microfluidic platforms and MS. This talk will summarize the progress and challenges in this field so far and provide an overview of the topic. The subject will be discussed in the context of recent work from our group aiming at utilizing this method for directed evolution campaigns. 

NTSuisse: A Web Platform for Exchange and Comparison of Swiss LC-HRMS Surface Water Data 

Johannes Boog, Michele Stravs, Heinz Singer 

Eawag - Swiss Federal Institute of Aquatic Science and Technology, Environmental Chemistry, Dübendorf, Switzerland 

The NTSuisse project aims to develop a web platform for the analysis and management of high-resolution mass spectrometry (HRMS) data, accessible to participating cantons, Swiss water suppliers, expert bodies, and the Swiss Federal Ministry of Environment. Key features of the NTSuisse platform include user-friendly data upload and storage capabilities, centralized automatic processing, target and suspect screening. The platform will allow stakeholders to manage and analyze their own data independently while also offering batch-wise data processing. Integrated quality control measures, such as EIC-annotated peak plots, will ensure the reliability of processing results. Additionally, the platform will include advanced data search capabilities with filters for time periods, mass, substances, and geographic areas, as well as visualization tools such as geospatial maps. Users will have the flexibility to extend the target and suspect lists, reprocess data batches if needed, and utilize or modify predefined templates for processing and screening. By centralizing data management, the platform will enable consistent and comprehensive evaluations across different cantons and observation sites, supporting environmental agencies, water suppliers, and other stakeholders. Ultimately, NTSuisse aims to provide a collaborative and efficient data platform that enhances water quality monitoring and promotes better understanding and management of chemical pollution in Swiss waters. 

Identification of new persistent halogenated compounds in the atmosphere using the Aprecon-GC-ToF-MS 

Alina Begley, Stephan Henne, Martin K. Vollmer, Stefan Reimann 

Empa, Laboratory for Air Pollution / Environmental Technology, Dübendorf, Switzerland 

The Montreal Protocol to phase out ozone-depleting substances like chlorofluorocarbons (CFCs) was signed in 1989 and is considered one of the most successful environmental regulations.  Since then, the replacement products for CFCs are also being successively phased-out: hydrochlorofluorocarbons (HCFCs) since 2013, and hydrofluorocarbons (HFCs) since 2019, and today developed countries use fourth generation replacements, hydrofluoroolefins (HFOs).  

The climate gases group at Empa is a part of the international measurement network Advanced Global Atmospheric Gases Experiment (AGAGE), which monitors the spatial and temporal distribution of these and other anthropogenic compounds in the atmosphere. Extremely low concentrations (ppq-ppt) of these substances are routinely measured with high precision (<1%) using a well-established cryogenic pre-concentration unit (Medusa) coupled to gas-chromatography (GC) with quadrupole mass spectrometry (MS) detection.  

The number of regulated compounds is increasing and many new unregulated compounds are released into the atmosphere, which we aim to identify, quantify, and monitor.  However, quadrupole-MS limits the number of ions that can be detected, therefore we have developed instrumentation using a time-of-flight (ToF)-MS. We can now identify new persistent halogenated compounds by combining an advanced preconcentration unit (Aprecon) coupled to GC-ToF-MS with automated fragment formula annotation "ALgorithmic Process for Identification of Non-targeted Atmospheric Compounds" (ALPINAC). To date, we have identified an additional 70 halogenated compounds in the atmosphere, 22 of which we have confirmed the identity with reference standards. Here we present preliminary finding of two of the most interesting newly identified compounds, bromoethane, primarily of biogenic origin, and vinylchloride, from industrial processes. 

Poster presentations

Feasibility of Using Routine Forensic Toxicology Data for Metabolomics Studies Exemplified for Amphetamine 

Annina Bovens, Claudio Leu, Thomas Kraemer, Andrea E. Steuer 

University of Zürich, Department of Forensic Pharmacology and Toxicology, Zürich Institute of Forensic Medicine, Zürich, Switzerland 

Background and Aims
Metabolome studies focus on the measurable change in (endogenous) metabolites triggered by a certain stimulus. In forensic and clinical toxicology, it is of interest to find analytical biomarkers for xenobiotics like drugs of abuse (DOA) that can improve case interpretation or elucidate underlying pharmacological mechanisms. (Placebo-) controlled clinical studies represent the gold standard for metabolome investigations but are hardly possible for DOA in humans for ethical reasons. This raised the question of whether routine cases could be used for this purpose. In toxicology, untargeted workflows have become common, so there is the possibility of re-processing routine data files retrospectively with metabolomics-like workflows. However, several challenges have to be considered. For routine cases, drug doses and time of ingestion remain unknown and confounding factors cannot be controlled, leading to some variability. When re-evaluating data files, additional inter-batch differences are introduced during preparation and data acquisition. This is particularly important as untargeted metabolome comparisons are only made based on peak area differences (metabolites of interest are unknown before analysis omitting quantification). Therefore, it is necessary to evaluate whether using such (retrospective) routine data for metabolomics studies in a toxicological context is possible. Here, we aimed to compare (endogenous) metabolomic findings of a placebo-controlled amphetamine administration study in humans (A) to routine cases positive and negative for amphetamine, prepared and analyzed within a single analytical batch (“one-batch”, B) and after re-evaluation of data files prepared and analyzed over 6 months (“multiple-batch”, C).  

Methods
For the controlled study (A), plasma was collected from healthy volunteers (npos=18, nneg=18) 3.5 h after crossover administration of amphetamine (single 40 mg dose) and placebo (Holze 2019). For the routine data set, whole blood from selected cases was used (npos=36, nneg=35), matching in age and drug co-administration. This data set was once analyzed and evaluated as “one-batch” (B) and once as “multi-batch” (C), measured over 6 months. All samples were extracted via protein precipitation (acetonitrile, 1:3, v/v) and analyzed with an untargeted liquid chromatography-tandem mass spectrometric method (Bruker Elute UHPLC; Bruker Impact II TOF-HRMS; data-independent acquisition; ESI+). Peak picking and alignment were done simultaneously for A, B, and C with MS-DIAL (version 4.9). In MetaboAnalyst (version 5.0), PQN-normalization was conducted, while further statistical analyses were performed in R (version 4.3.2). Features defined by a specific combination of mass and retention time were evaluated for significance by comparing amphetamine-positive and negative cases for A, B, and C separately. Features fulfilling the following criteria were considered significant: signal to noise (sn) ≥ 3, foldchange (fc) ≤ 0.5 or fc ≥ 1.9, p-value ≤ 0.05, present in ≥ 50% of the cases per batch and peak area ≥ 500. 

Results and Discussion
When systematically comparing metabolome results between a controlled study (A) and routine cases analyzed under two different conditions (“one batch”, B, and “multi-batch”, C) we would expect significant features from A to be found in B and C as well. First, significant features in A were defined as the basis for comparison with B. All 31 significant features from A were also present in B; however, only 4 (12.9%) of them were significant. These matching and significant features are all suspected to be related to amphetamine (fragments) based on library matches. As for endogenous metabolites, most features in B showed much smaller differences between amphetamine-positive and -negative cases, as most p-values were between 0.05 and 0.1 and/or fcs around 0.95. Therefore, feature-based uni-variate statistical testing seems unsuitable for endogenous biomarker searches with small differences between treatment groups. However, while significantly changed features from A did not match the results from B, 71 other features still met the defined significance criteria. It cannot be excluded that changes in these additional B features were caused by amphetamine, considering presumably higher and repeated dosing in routine cases compared to the controlled study, or they simply occurred by chance. Secondly, significant B features were compared to C. All 75 significant B features were present in C, but only 16 (21.3%) were significant. Features insignificant in C showed p-values between 0.05 and 0.1 and/or fcs around 0.8, pointing out the relevance of inter-batch differences regarding data acquisition. 

Conclusion 
We aimed to evaluate the utility of (retrospective) routine data in metabolomics, in toxicology. The comparison between a controlled study (A) and routine cases (B and C) revealed several key findings. For the current dataset (n=71 routine cases), only few significantly changed features from A could be confirmed in routine cases (B). Larger sample sizes or more sophisticated multi-variate statistics might compensate for higher inter-individual variation in routine cases and presumably allow a better overlap. As expected, re-evaluating retrospective routine data introduces even greater variation (B vs. C). Nevertheless, drug metabolites could still be detected in routine cases (B and C). This confirms the general feasibility of (re)using routine cases or acquired HRMS datafiles for untargeted metabolome(-like) investigations if expected changes are high enough. 

Sweat analysis of medical opioids and metabolites in critically ill pediatric patients 

Max Polke¹, Florian Zapf², Tanja Restin³, Julia T. Scherer¹, Thomas Kraemer⁴, Tina M. Binz¹, Clarissa D. Voegel¹ 

1) Center for Forensic Hair Analysis, Zurich Institute of Forensic Medicine, University of Zurich, Zurich, Switzerlan
2) Department for Pediatric Intensive Care Medicine and Neonatology, University Children’s Hospital Zurich, Zurich, Switzerland
3) Institute of Physiology, University of Zurich, Zurich, Switzerland
4) Forensic Pharmacology and Toxicology, Zurich Institute of Forensic Medicine, University of Zurich, Zurich, Switzerland 

Background & Aims: 
In forensic toxicology, matrices such as blood, saliva, urine and hair are commonly tested for illicit substances and drugs. However, sweat remains a largely unexplored matrix in forensic toxicology, although it can be collected easily and non-invasively. In addition, sweat arguably contributes to the contamination of hair and thus poses a challenge in hair analysis. The objective of this research study is to investigate sweat samples from a cohort of 112 children aged 0 to 13 years who were treated in a clinical setting with fentanyl, alfentanil, sufentanil, remifentanil or other more traditional opioids such as morphine. With the data obtained, we wanted to gain insights into the concentrations of opioids in sweat samples. For this purpose, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated targeting the mentioned opioids and selected metabolites. 

Methods: 
This research study has a monocentric, non-interventional, prospective design. Sweat samples from patients (newborns, children and adolescents) admitted to the pediatric intensive care unit at the University Children’s Hospital Zurich and exposed to medically approved opioids were collected with forensic swabs. The samples were extracted using a two-step extraction procedure with methanol, followed by a targeted LC-MS/MS analysis (QTRAP® 7500+). The analysis was performed in multiple reaction monitoring mode screening for fentanyl and its medically used analogues (alfentanil, sufentanil, and remifentanil), selected metabolites (4-ANPP, norfentanyl, β-hydroxyfentanyl), as well as other more traditional opioids. The method used was fully validated in terms of selectivity, linearity, limit of detection (LOD), lower limit of quantification (LLOQ), accuracy, precision, matrix effect and recovery. 

Results & Discussion: 
The LC-MS/MS method developed showed good selectivity and sensitivity for all analytes. Extraction efficiencies were above 80% for both measured quality control concentration levels. Values were acceptable in terms of bias (within ±20%) and imprecision (<20%). The method was then used to analyze a total of 112 pediatric sweat samples. All opioids administered to the patients during their stay in the pediatric intensive care unit, as well as the targeted metabolite could be detected in the sweat samples. Out of 97 cases involving fentanyl treatment, fentanyl was detected in 95 cases (98%), at concentrations ranging from 1.0 to 3595 pg/swab. In some cases, the metabolites of fentanyl, 4-ANPP (14 cases), norfentanyl (8 cases) and β-hydroxyfentanyl (36 cases) were quantified. Sufentanil was quantified in 53 out of 83 (64%) cases involving treatment, in concentrations between 2.2-73.9 pg/swab. Alfentanil (detected in 1 out of 2 treated cases) and remifentanil (detected in 4 out of 19 treated cases) were measured at concentrations below the LOQ. Morphine was detected in 103 out of 106 cases (97%) treated with morphine.  

Conclusion: 
Overall, a sensitive and specific method was developed and validated for simultaneous quantification of clinically used synthetic and classical opioids in sweat samples by LC-MS/MS. Two administrated analytes were only detected below LOQ, demonstrating the need for even more sensitive methods. The results will help to understand the role of sweat in the incorporation mechanisms of opioids into hair. These findings are of great relevance not only for forensic but also for clinical applications. For instance, the method could be used for research studies focused on therapeutic drug monitoring. 

Investigation of Phyllobilins in Apple Leaves Affected by Four Fungal Diseases Using HPLC-QTOF 

Luca Vestrucci^1,2, Lisa Marie Gorfer², Urban Spitaler², Sabine Oettl², Peter Robatscher², Matteo Scampicchio¹, Michael Oberhuber² 

1) Faculty of Agricultural, Environmental and Food Sciences, Free University of Bozen-Bolzano, Piazza Università 5, 39100 Bolzano, Italy Bolzano, Italy
2) Laimburg Research Centre, Laimburg 6 – Pfatten (Vadena), 39040 Auer (Ora), BZ, Italy 

Chlorosis by degradation of chlorophyll is a plant response to physiological and pathological stress. In this work, we investigated senescence-related chlorophyll catabolites, known as phyllobilins (PBs), during fungal diseases and whether they could serve as biomarkers to monitor plant health. 

Apple leaves from three cultivars (Gala, Golden Delicious, and Red Delicious) infected by four fungal diseases (apple scab, Marssonina apple blotch, Alternaria leaf blotch, and powdery mildew) were collected at different times during the year. Healthy and diseased leaves were gathered predominantly in summer, while senescent leaves were collected in autumn and served as a positive control. For each pathology five healthy, five diseased, and five senescent leaves samples, each containing five representative leaves, were collected. All diseased leaves presented symptoms such as necrotic or brown spots, except for powdery mildew, where the presence of the fungus was confirmed by end-point PCR. 

Qualitative analysis of PBs was performed using HPLC-high-resolution QTOF-MS. PBs were detected in ESI positive mode. Analysis was conducted in full scan and MS/MS. The presence of PBs was confirmed using a data-dependent inclusion list of 79 hypothetical and previously identified PBs. 

For each disease, the highest number of PBs in healthy, diseased, and senescent leaves (including pheophorbide a) is reported as follows: 
Apple scab: 4 in healthy leaves, 1 in diseased leaves, and 27 in senescent leaves. 
Powdery mildew: 5 in healthy leaves, 3 in diseased leaves, and 26 in senescent leaves. 
Alternaria leaf blotch: 7 in healthy leaves, 8 in diseased leaves, and 29 in senescent leaves. 
Marssonina apple blotch: 7 in healthy leaves, 20 in diseased leaves, and 21 in senescent leaves. 

We observed a general reduction in PBs formation in diseased leaves compared to healthy leaves, except for those affected by Marssonina apple blotch, which showed an increased presence of these compounds due to induced leaf senescence. The majority of PBs appeared in senescent leaves, confirming a significant chlorophyll degradation during autumn. Some PBs were found in healthy leaves, suggesting a physiological turnover of chlorophyll during the summer. Surprisingly, PB DNCC-632 was more abundant in healthy leaves than in diseased ones, implying the endoplasmic reticulum as possible target of fungal pathogens.  

In conclusion, the number of PBs varies depending on fungal pathologies. It appears that fungal pathogens influence chlorophyll degradation, and PBs may serve as potential biomarkers for monitoring plants where chlorosis is evident, such as in Marssonina apple blotch. Further investigations are needed to understand the role of pathogens in chlorophyll breakdown. 

A single-sample workflow for joint metabolomic and proteomic analysis of clinical specimens 

Hagen M. Gegner^1,7, Thomas Naake², Karim Aljakouch³, Aurelien Dugourd⁴, Georg Kliewer³, Torsten Müller³, Dustin Schilling⁴, Marc A. Schneider⁵, Nina Kunze-Rohrbach¹, Thomas G.P. Grünewald⁶, Rüdiger Hell¹, Julio Saez-Rodriguez⁴, Wolfgang Huber², Gernot Poschet¹ & Jeroen Krijgsveld³ 

1) Centre for Organismal Studies (COS), Metabolomics Core Technology Platform, Heidelberg University, Heidelberg, Germany
2) Genome Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany; Faculty of Medicine, Heidelberg University, Heidelberg, Germany
3) Division Proteomics of Stem Cells and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany
4) Institute for Computational Biomedicine, Bioquant, Faculty of Medicine, Heidelberg University and Heidelberg University Hospital, Heidelberg, Germany
5) Translational Research Unit, Thoraxklinik at Heidelberg University Hospital, Röntgenstraße 1, 69126, Heidelberg, Germany and Translational Research Center Heidelberg (TLRC), Member of The German Center for Lung Research (DZL), Heidelberg, Germany
6) Division of Translational Pediatric Sarcoma Research, German Cancer Research Center (DKFZ), German Cancer Consortium (DKTK), Heidelberg, Germany and Hopp-Children’s Cancer Center (KiTZ), Heidelberg, Germany and Institute of Pathology, Heidelberg University Hospital, Heidelberg, Germany
7) National Center for Tumor Diseases (NCT), NCT Heidelberg, DKFZ and Heidelberg University Hospital, Heidelberg, Germany; 7) CTC Analytics AG, Zwingen, Switzerland 

Integrating proteomic and metabolomic data provides crucial insights into cellular regulation and disease pathways. However, traditional workflows require separate samples and labor-intensive manual processing, introducing variability and limiting throughput. Here, we present "MTBE-SP3", a streamlined single-sample workflow combining optimized methyl-tert-butyl ether (MTBE) metabolite extraction with automated single-pot solid-phase-enhanced sample preparation (autoSP3) for proteins. This approach reduces inter-sample and technical variability, enhancing the robustness and explanatory power of multi-omics analyses. MTBE-SP3 is compatible with diverse biological matrices, including formalin-fixed paraffin-embedded (FFPE) tissue, fresh-frozen tissue, plasma, serum, and cells, demonstrating broad applicability in clinical research. Further tested in a proof-of-concept study using a lung adenocarcinoma patient cohort, MTBE-SP3 coupled with a novel network analysis approach revealed consistent proteomic and metabolomic alterations between tumor and non-tumor adjacent tissue. Beyond its current capabilities, MTBE-SP3 holds the potential for further automation. Building on our experience automating sample preparation and injection in other workflows using x,y,z robotic platforms, we envision adapting this approach to streamline the MTBE extraction and injection steps in LC/GC-MS metabolomic analyses. This exploratory avenue could further enhance throughput, reproducibility, and standardization in multi-omics studies, solidifying the role of MTBE-SP3 as a valuable tool for clinical research and potential routine diagnostics. 

Identification of ozonation transformation pathways of cyanobacterial toxins by LC-HRMS/MS 

Anita Lopes Souto, Valentin Rougé, Urs von Gunten, Elisabeth M.L. Janssen 

Department of Environmental Chemistry, Swiss Federal Institute of Aquatic Science and Technology (EAWAG), Dübendorf, Switzerland 

Cyanobacteria produce a diverse group of bioactive cyanopeptides with significant environmental and health risks due to their potential toxicity and persistence. When these cyanopeptides occur in natural water bodies that serve as drinking water sources, it is essential to investigate their degradation during water treatment processes. Ozonation presents a powerful treatment option to oxidize organic pollutants. While microcystins represent one group of cyanopeptides that is most intensively studied thus far, there is a significant knowledge gap regarding the behavior of other cyanopeptides during ozonation. To address this gap, LC-HRMS/MS was used to identify ozonation kinetics and transformation products (TPs) of various cyanopeptides extracted from laboratory-grown cyanobacteria. 

Different classes of cyanopeptides, including microcystins, anabaenopeptins, cyanopeptolins, aeruginosins, and aerucyclamides were exposed to ozone and samples were analysed by LC-HRMS/MS (ExplorisTM 240, Thermo Scientific). Data-dependent acquisition performing sequential MS2 experiments at various HCDs focused on suspected TPs that were predicted by mechanistic information of O3 reactions. Retention time (Rt) and predicted logDow were used as a primary identification of matched suspects, especially in cases where isomeric peaks were found. Further identification was done by annotation of MS2 fragmentation spectra. Together with a trend analysis from the kinetics of formation and decay of cyanopeptides and TPs, the most plausible transformation pathways were suggested. 

We propose novel TPs for microcystins and, for the first time, for other cyanopeptide classes, with particular focus on phenol, amine and thiol moieties. This study enhances our understanding of the fate of cyanopeptides during ozonation, highlights the value of HRMS/MS for elucidating reaction pathways, and confirms that the fundamental knowledge on the ozonation of functional groups can be applied to predict the transformation of complex compounds.   

Lactobreath: A pilot study to diagnose lactose intolerance based on the exhaled breath metabolome 

A. Vadakkechira¹,*, K. Mallick¹,*, R. Guillod², G. Vergères³, R. Zenobi¹, D. Pohl⁴, K. Pimentel³, S. Giannoukos¹ 

*Equal contribution

1) Department of Chemistry and Applied Biosciences, ETHZ, Zurich, Switzerland
2) aha! Swiss Allergy Centre, Bern, Switzerland
3) Agroscope, Bern, Switzerland
4) Department of Gastroenterology and Hepatology, USZ, Zurich, Switzerland;

Background: Food intolerances affect 15-20% of the population, leading to physical discomfort, dietary restrictions, and psychosocial challenges. A significant cause is the impaired digestion and transport of short-chain fermentable carbohydrates known as FODMAPs. Diagnosis methods include FODMAP exclusion followed by controlled reintroduction and the hydrogen breath test, though the latter has limited correlation with symptoms. Food intolerance often links to functional gastrointestinal disorders, with FODMAPs acting individually or combined. More research on pathophysiology and interventions is needed for diagnosis improvement. 

Objective/Aims: The primary objective of the study is to identify postprandial metabolic profiles in human exhaled breath associated with gastrointestinal symptoms of lactose malabsorption (LM) (Lactobreath profiles). Additionally, the study aims to:  

a) Further analyze the Lactobreath profiles to understand the clinical traits linked to LM. This includes exploring genetic polymorphisms affecting lactase gene expression, measuring breath hydrogen, and identifying lactose-derived urinary metabolites. 

b) Mechanistically link these Lactobreath profiles to metabolic traits associated with LM. Key methods include using the Atmo Gas Capsule for colonic gas measurement and analyzing the urine metabolome to gain a comprehensive understanding of LM's metabolic implications. 

Methods: To address these gaps, this project employs exhaled breath's molecular composition as a source of potential biomarkers for lactose malabsorption, serving as a proof-of-concept for food intolerance diagnosis. It seeks to identify breath markers for lactose tolerance/intolerance and mechanistically link them to metabolic traits, including urine metabolomics. A real-time, non-invasive technique based on secondary electrospray ionization coupled with high-resolution mass spectrometry (SESI-HRMS) will analyze the postprandial exhaled breath metabolome, obviating the need for time-consuming offline analysis. Standardized questionnaires will assess lactose intolerance symptoms correlated with key metabolites. A solid-state sensor will measure breath hydrogen, and an ingestible gas sensor will monitor gastrointestinal gases and transit time. Urine metabolites will be studied using gas chromatography coupled with mass spectrometry (GC-MS) and genetic polymorphisms via saliva samples. 

Results: The anticipated outcomes include the identification of specific breath metabolites associated with lactose malabsorption and the development of a predictive, non-invasive breath test for food intolerances. 

Conclusions: This research aims to significantly impact food intolerance understanding, allowing clinicians to identify suitable candidates for low FODMAPs diets and offer predictive non-invasive breath tests. It will create breath metabolomics profiles indicating lactose malabsorption-associated clinical traits and provide mechanistic insights into postprandial lactose response. Furthermore, it will advance exhaled breath metabolomics as an analytical tool for personalized nutrition development. 

Comprehensive discovery lipidomic workflow which utilizes a novel, multi-reflecting TOF with integrated informatics, providing highly confident lipid characterization and quantification 

Nyasha Munjoma¹, Hirose Kenji², Baharak Vafadar⁵, Laura Goracci³, Paolo Tiberi⁴, Jayne Kirk¹, Lee A. Gethings¹, and Richard Lock¹ 

1) Discovery Characterization and Imaging, Waters Corporation, Wilmslow, SK9 4AX, United Kingdom
2) Nihon Waters, Tokyo, Japan
3) Department of Chemistry, Biology and Biotechnology, University of Perugia
4) Mass Analytica, Barcelona, Spain
5) Waters AG Baden-Dättwil Switzerland 

Despite developments in analytical technology the detection and identification of lipids remains a significant challenge. To streamline the process of data acquisition and analysis we have developed a workflow which combines the attributes of the Xevo MRT Mass Spectrometer with data analysis powered by the Mass Analytica Lipostar2 software. The Xevo MRT is a novel next generation QTof- combining multi-reflecting technology with a ESI source.  

In this poster we demonstrate that the combination of reversed–phase UHPLC, high-resolution mass spectrometry and an intelligent workflow driven software allows for the rapid lipidomic analysis of biological samples. Robust performance at speed in a compact footprint and excellent mass accuracy improves identification confidence. 

Furthermore, Lipostar2 software provides a streamline, flexible solution for handling Waters Connect LC-MS data sets (DDA or DIA) and extensive statistical analysis and pathway tools to enable biomarker discovery and novel biological interpretation. 

Large-scale biomonitoring of bisphenol analogues and their metabolites in human urine 

Baptiste Clerc¹, Joëlle Houriet¹, Alexandra Jaus¹, Judith Jenny-Burri² and Gisela Umbricht¹ 

1) Federal Institute of Metrology METAS, Bern, Switzerland
2) Federal Food Safety and Veterinary Office FSVO, Bern, Switzerland 

In the years 2022 and 2023, the Swiss Federal Food Safety and Veterinary Office conducted a study about salt intake in Switzerland. The study included a representative sample of adults from different regions, age groups, and genders across Switzerland. Participants were asked to provide 24-hour urine samples. Alongside natrium in urine samples, participants’ blood pressure, body mass index (BMI), and potassium intake were also determined. Participants completed detailed questionnaires about their dietary habits, knowledge about salt and health, and other lifestyle factors. 

In this elaborate framework, the Swiss Federal Food Safety and Veterinary Office mandated METAS to determine in these urine samples the amount of approximately 10 bisphenols, 20 phthalate metabolites, and iodine. 

Bisphenols (BPs) are used as monomer for the production of polycarbonates, polyvinyl chloride, epoxy resins and thermal paper. Due to their presence in common consumer products as well as their chemical structure, they can migrate into food, beverages, water, air, dust and soil, leading to human exposure by a variety of routes. BPs are suspected to have disrupting effects on the endocrine system of humans and animals. Endocrine-disrupting chemicals may mimic, block or interfere with the body's hormones and are associated with a wide array of health issues. Glucuronidation of BPs in the intestine and liver is considered the main metabolic pathway for most BPs while other metabolites only result when the glucuronidation pathway is saturated. These metabolites are mainly excreted through urine. Therefore, human biomonitoring in urine is a crucial method to assess the possible presence of these chemicals and their corresponding metabolites in various body fluids to determine the overall extent of exposure in both a qualitative and quantitative manner.   

For the analysis, urine samples are spiked with internal standards and treated with β-glucuronidase to hydrolyze the glucuronide conjugates and set free the BPs. Sample purification is evaluated by comparing a trap column (online), with an offline solid phase extraction (SPE) 96-wells cartridge and with no solid-phase extraction, e.g. precipitation without SPE. The substances are quantified using a UHPLC-MS/MS system with negative polarity electrospray ionization applying scheduled multiple reaction monitoring. 

Advances in quantification of substances in exhaled human breath using secondary electrospray ionization 

Timon Käser, Stamatios Giannoukos, Renato Zenobi 

ETH Zürich, Department of Chemistry and Applied Biosciences, Zürich, Switzerland 

The quantification of volatile organic compounds (VOCs) in human breath has attracted considerable interest due to its potential impact on medical diagnostics, environmental exposure assessment, and forensic applications. Secondary electrospray ionization (SESI) is a method capable of detecting thousands of VOCs. However, there has been limited research on the accurate quantification of these compounds in breath. In addition, most studies using SESI to analyse breath have relied primarily on MS1 measurements, using accurate mass measurements but lack additional confirmation.  

Therefore, we quantified several compounds (C5 to C10 aldehydes, limonene and pyridine) in exhaled human breath from 12 volunteers over several days. Quantification is performed using external gas standards prepared from a system using evaporation chambers [1]. Small flows of standards (1-10 cm3/min) are mixed with a stream of humidified nitrogen to simulate human breath containing analyte concentrations between 1 ppb and 50 ppm. Several methods using full scan (FS), targeted selected ion monitoring (t-SIM) and parallel reaction monitoring (PRM) for quantitative breath analysis are compared. The optimization, sensitivity and selectivity of these methods for breath analysis are presented and discussed. 

In-depth biologics characterization with TRD maps

Samuel Barteau¹, Blandine Denefeld¹, Manuel Diez¹, Julien Schmitt², François Griaud¹

1) Analytical Characterization CH, Analytical Development Biologics, Technical Research & Development, Novartis Pharma AG
2) Process Analytical Sciences CH, Analytical Development Biologics, Technical Research & Development, Novartis Pharma AG

The pharmaceutical development of novel therapeutic proteins has been expanding in the last three decades to address unmet medical needs with transformative therapies.

Monoclonal antibodies (mAb) and related formats such as bi- and tri- specific mAb have multiple quality attributes that can be characterized and monitored with intact mass spectrometry (MS).

While conventional MS data analysis focuses on the detection and deconvolution per peak, this poster shows the benefits of Time-Resolved Deconvolution (TRD) maps for in-depth characterization of biologics quality attributes.

Unexpected observation of ring-re-closure following initial acid-catalyzed ring cleavage of bromazepam by liquid chromatography-high-resolution mass spectrometry 

Jonas Malzacher, Lana Brockbals, Andrea Eva Steuer, Thomas Kraemer 

University of Zürich, Department of Forensic Pharmacology and Toxicology, Zürich Institute of Forensic Medicine, Zürich, Switzerland 

Introduction: In forensic toxicology, the benzodiazepine bromazepam represents an important analyte usually identified and quantified via targeted LC-MS/MS analysis. Analysis of a bromazepam reference in untargeted LC-QTOF-MS analysis interestingly revealed two bromazepam peaks with identical MS and MS/MS information but differing retention times. While acid-catalyzed ring cleavage is a well-known phenomenon for benzodiazepines, it usually leads to benzophenones with different chromatographic and MS behavior that cannot explain the occurrence of an additional “bromazepam-like” structure. We aimed to perform systematic studies to further elucidate this unexpected finding. 

Method: A solution of 10 benzodiazepines (alprazolam, bromazepam, clonazepam, clonazolam, diazepam, midazolam, nitrazepam, nordazepam, pyrazolam and triazolam; 100 ng/mL) and 2 internal standards (lorazepam-d4 and trimipramine-d3; 50 ng/mL) was prepared in a mixture of aqueous and organic solvent (H2O:ACN, 95:5 v/v) and analyzed by an HPLC-HRMS/MS (reversed-phase chromatography gradient elution, eluent A (10 mM ammonium formate:acetonitrile (99:1) + 0.1 % formic acid) and eluent B (acetonitrile:ultrapure water (99:1) + 0.1 % formic acid); Sciex ZenoTOF 7600, untargeted data acquisition in data-dependent acquisition mode, mass range MS1 100-1000 Da, MS2 50-1000 Da, respectively). Targeted data processing focused on the respective benzodiazepines, and their expected ring-opened benzophenones without retention time restriction. The following influencing parameters were tested across 17 timepoints over 50 hours, each in triplicates: pH of eluent mixture (pH 3.2 and 5.3) and source temperature (450 °C and 650 °C).   

Results: Benzophenones were formed for all tested benzodiazepines except for Pyrazolam. Only for bromazepam, a ring-closed form of the benzophenone, identical in MS and MS/MS to the parent compound could be observed at the same retention time as its benzophenone. The pH significantly impacted the ring cleavage rate, with increased cleavage rates at pH 3.2 compared to 5.3. At pH 5.3, only alprazolam and midazolam showed ring opening. Elevated source temperature markedly enhanced the ring re-closure for bromazepam.  

Discussion: The second alleged bromazepam peak in the chromatogram is most likely attributable to an in-source reaction, which is supported by the fact that the benzophenone and the ring-closed form occur at the same retention time. Following the ring opening of benzodiazepines in an acidic environment, it is plausible that a ring closure occurs in the ion source during ionization. Given that the signal rises after increasing the applied energy through the higher source temperature, it seems likely that the closure of the ring is energy-dependent. The same effect was not observed for any other of the tested benzodiazepine. It has not yet been possible to propose concrete rules about the relationship of the structure to the opening or closing of the ring. 

Gas-phase fluorescence spectrometry for investigating time-resolved unfolding mechanisms of cytochrome c after desolvation 

Linus Busse, Lukas Benzenberg, Ri Wu, Kim Greis, Renato Zenobi 

ETH Zürich, Zürich, Switzerland 

Native mass spectrometry (nMS) has emerged as a pivotal technique for elucidating stoichiometry, binding kinetics, homogeneity, and topology of mass-selected proteins and their complexes with high sensitivity. To correlate gas-phase data of biomolecules to data obtained in the native environment, conditions for nMS are chosen to conserve properties and native structural features after ionization and desolvation. However, the assumption that structures and function of gas-phase biomolecular ions produced by soft ionization techniques such as electrospray ionization (ESI) mirror those in the condensed phase remains a subject of debate, largely due to insufficient structural insights if only MS-based investigations are used. Especially, globular proteins are believed to follow a temporal evolution of intermediate conformational ensembles, which ultimately result in complete unfolding following an ‘inside-out’ geometry where the hydrophobic protein core is exposed to the vacuum.  

In this work, we combine mass spectrometry and fluorescence spectroscopy, i.e. Förster resonance energy transfer (FRET), to interrogate the time-resolved unfolding mechanisms of cytochrome c. A customized quadrupole ion trap is utilized, which allows for irradiation of the ion cloud by a pulsed laser. Measurements of intramolecular distances (by tmFRET) allow a fairly high-fidelity deduction of gas-phase structures. Previous studies utilizing ion mobility spectrometry (IM-MS) have shown already after a rather short time, the protein unfolds in the gas phase. However, the structural information obtained by IM-MS alone is limited, because IM-MS affords only a global shape parameter. Also, the time period reported focused on the early stages of conformational rearrangement, up to 250 ms. The present work sheds light on a longer time frame of up to 10 seconds. 

We designed a workflow to label the protein at the N-terminus with a fluorescent dye, which is compatible with the absorption range of the heme group inside cytochrome C, and utilizing the bound iron as the quencher. This novel approach reduces the need for modification of the molecule and maintains a close-to-native state of the structure. Since cytochrome C has 16 lysine residues in its sequence, we employ different labeling sites to obtain structural insights from multiple secondary structures. In combination with computational modeling and IM-MS data, we are able to propose a detailed unfolding mechanism for the protein in the gas phase. This workflow provides in-depth insight into the unfolding mechanism. 

Our findings demonstrate the potential of integrating MS and FRET to study biomolecular structures and their dynamics. The methodologies developed in this study can be applied to a wide range of proteins, providing a valuable tool for structural biology and analytical chemistry. This research not only advances our understanding of protein dynamics but also showcases the potential of combining mass spectrometry with fluorescence-based techniques to study biomolecules. The significance of this work lies in its ability to extend our understanding of protein behavior in the gas phase over extended time frames, offering new perspectives on their structural properties and unfolding mechanisms. 

Application-specific mass spectra re-calibration approaches 

Konstantin Nagornov, Anton Kozhinov, Yury Tsybin 

Spectroswiss, 1015 Lausanne, Switzerland 

Mass accuracy is a critical analytical parameter essential for effective mass spectral analysis. As mass spectrometry (MS) applications evolve, high mass accuracy becomes increasingly important for extracting detailed information from spectral data.  

Various data calibration methods are employed to achieve the required mass accuracy, including: (i) external calibration with single and multiple data points; (ii) internal calibration using a single data point, such as lock mass re-calibration during data acquisition; and (iii) internal calibration with multiple data points. Local space charge effects complicate re-calibration, necessitating non-linear calibration functions. Additionally, scan-to-scan variations in analyte ion numbers (total charge) affect re-calibration, particularly in MS with fluctuating ion sources like MALDI and LC/GC-based applications. Finally, isotopic fine structure can cause a significant shift in expected peak positions at certain resolution levels, particularly in small molecule analysis, which must be considered when calculating expected mass (m/z) values. 

We will discuss practical approaches to MS data re-calibration for various applications, including: (i) MALDI/DESI-based mass spectrometry imaging (MSI); (ii) LC-MS analysis of complex small-molecule mixtures, such as in metabolomics; and (iii) GC-MS. Each application requires specific re-calibration algorithms, with an emphasis on computational efficiency and speed in implementation. The advantages of accurate isotopic envelope simulations for determining expected peak positions are also highlighted. 

Unprecedented Reproducibility Achieved in the First High-Resolution Ion Mobility Inter-lab Lipidomic Study of Human Plasma 

Rachel Harris, Lucy Woods, Emanuel Zlibut, Sarah Stow, Allison Reardon, David Williamson, Jody May, Michelle English, Jennifer Krone, Komal Kedia, John McLean, Frederick Strathmann 

1) MOBILion Systems, Chadds Ford, PA, USA
2) Agilent, Santa Clara, CA, USA
3) Vanderbilt University, Nashville, TN, USA
4) Merck, West Point, PA, USA 

Broadscale in-depth plasma lipidomics is extremely challenging due to the underlying high lipid complexity and broad dynamic range. A wide arsenal of mass spectrometry (MS) methodologies have been developed in an attempt to dissect the human plasma lipidome. The precise composition remains unsettled due to insufficient resolving power to identify each individual isomeric and isobaric lipid species. We have previously shown that liquid chromatography based High-Resolution Ion Mobility (LC-HRIM) MS enables separation of sn-, double bond position, and cis/trans isomers, providing an improved characterization of complex extracts. Here, we further demonstrate the outstanding laboratory-independent reproducibility of this workflow in disentangling the human plasma lipidome. Initially, we benchmarked the performance by repeated analysis of six spiked internal standards and six endogenous lipid species, representing different lipid classes, in a NIST SRM 1950 lipid extract. The chromatographic separation was highly reproducible with variations in retention time (RT) about 0.2 RSD% in average when measuring the same NIST extract over five executive days in a single lab. In contrast, the variation in CCS values was extremely small, only 0.01% on average. Next, we asked three independent laboratories to perform the same analysis on their MOBIE® instruments. Expectedly, inter-lab measurements increased the overall variations, however, not dramatically in the case of CCS in particular. We observed a variation in RTs about 9% in average, whereas variation in CCS values was only 0.3% in average. Currently we are expanding the analyses to hundreds of lipids including isomeric and isobaric species. In summary, these results are highly intriguing, demonstrating the high data reproducibility between different laboratories, without significant loss in lipid identification confidence based on CCS values. The LC-HRIM-MS solution show high suitability towards rapid routine analyses of complex lipid extracts across multiple sites, without compromising all-inclusive and in-depth details.   

Application of gas-phase fluorescence spectroscopy and native ion mobility-mass spectrometry for characterization of a metalloenzyme 

Ri Wu, Renato Zenobi 

ETH Zürich, Department of Chemistry and Applied Biosciences, Zürich, Switzerland 

Mass spectrometry (MS) offers a versatile and powerful toolbox for the structural and functional characterization of biomolecules. However, relying on only a single MS method remains insufficient to accurately gauge the dynamic structure and interaction of biomolecular ions or complexes. To address this challenge, we have proposed a synergistic approach which utilizes Förster resonance energy transfer (FRET) and two types of ion mobility spectrometry (i.e., traveling wave and differential) to provide multiple constraints (i.e., intramolecular distance and shape) for structure refinement of gas-phase ions. The experimental collision cross-section (CCS) and donor-acceptor distance (rDA) values were used as constraints to reveal the structure of biomolecular ions. These endeavors have allowed us to uncover a much improved structural understanding on multiple systems in the gas phase, including polyalanine-based α-helical peptides [1, 2], a peptide forming a β-hairpin[3], “stapled” peptides [3], cyclic neuropeptides [4], neuropeptide-amyloid beta complexes [5]. Our work allows more stringent structural characterization of therapeutical peptide drugs and dynamic/transient protein complexes. 

In this poster, I will present the most recent work on studying the dynamical roles of metal ions and the intra-subunit disulfide bond in immature monomeric copper-zinc superoxide dismutase (SOD1) species, which is likely the source of aggregation. I will introduce the distinct conformational changes of monomeric SOD1 species upon metal binding and disulfide bond cleavage in four selective fluorophore-labeling SOD1 isomers. Further, vacuum ultraviolet photodissociation (UVPD) and electron-capture dissociation (ECD) provide structural insights by generating a wide array of informative fragment ions, enabling high-resolution mapping of metal binding at molecular level. 

References:  

Tiwari P., Wu R., Metternich J. Zenobi R.* J. Am. Chem. Soc. 143, 11291–11295 (2021). 
Wu R., Zenobi R.* et al. Nat. Commun. 14, 2913 (2023). 
Benzenberg L. R., Wu R.,* Zenobi R.* et al. J. Phys. Chem. Lett. 15, 5041–5046 (2024). 
Wu R., Zenobi R.* et al. J. Am. Chem. Soc. 144, 14441–14445 (2022). 
Wu R., Benzenberg L. R., Svingou D., & Zenobi R.* J. Am. Chem. Soc. 145, 10542–10547(2023). 
Wu R., Svingou D., Metternich, J. B., Benzenberg L. R., & Zenobi R.* J. Am. Chem. Soc. 146, 2102–2112(2024). 

A SICRIT ionization study for difficult components - from metal organics to perfluorinated aliphatics 

Jan Bucek¹, Ciara Conway^1,2, Markus Weber^1,2, Jan-Christoph Wolf², Christoph Haisch¹ 

1) Technical University of Munich, Chair for Analytical Chemistry, Garching, Germany
2) Plasmion GmbH, Augsburg, Germany 

Soft Ionization by Chemical Reaction In Transfer (SICRIT) is a soft ionization technology based on a dielectric barrier discharge ionization (DBDI). It allows for various kinds of sample introduction including GC, LC, SPME or Laser-ablation. Thus, it is employed for almost any kind of (small) molecule analysis covering mass range up to 2500 m/z and from polar to non-polar.  

In this study we investigate various compound classes with respect to sensitivity and ionization species formed. The investigated molecules are usually not, or only hardly, ionizable using conventional soft ionization techniques like ESI or CI but can be addressed using SICRIT. Here we cover PFAS, aliphatics, PAHs metalorganics and various synthetic and fragile molecules comprising interesting structure elements. Depending on the molecule SICRIT may offer alternative ionization mechanisms and/or soft ionization. The observed forms include the classical [M+H]+ [M+NH4]+ and M+ but also very uncommon ion species like [M-H]+, [M-F]- or [M+CO2]-, [M-Hn+Om]+ or even Nitrogen-Carbon exchange ions. Those may be formed depending on the operation conditions and gas phase chemistry involved. In this overview presentation we show how this unique ionization capabilities can be exploited to increase sensitivity but also selectivity for these compound classes, overcoming the low sensitivity in other ionization methods.  

Wherever possible, we complement these findings with new mechanistic insights into the underlying multiple ionization mechanisms in this cold plasma based ionization. 

TIMS-enabled 4D-Metabolomics^TM workflow for the automated analysis of derivatized analytes 

Jesper Havelund¹, Aiko Barsch², Stefanie Wernisch², Nikolas Kessler², Sofie Weinkouff², Heiko Neuweger², Matthew R. Lewis², Nils J. Færgeman¹ 

1) Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark
2) Bruker Daltonics GmbH & Co. KG, Bremen, Germany 

Chemical derivatization of polar metabolites, especially those with multiple charge sites, can help address limitations of traditional reversed-phase chromatography and electrospray-based mass spectrometry (LC-ESI-MS/MS) for metabolomics. Increased hydrophobicity and improved ionization can translate into improved separation of isobaric compounds, better signal-to-noise ratios, lower detection and quantitation limits, and reduced variability. However, derivatization often requires tracking of more than one reaction product per metabolite of interest in addition to tedious and error-prone, manual data evaluation and metabolite identification. In addition to the retention time and mass shifts, the biggest drawback is the lack of suitable metabolite databases for spectral matching, even for the most common derivatization agents. 

In this contribution, we present a software workflow that integrates in-silico derivatization, fragmentation, and prediction of collisional cross sections (CCS) for derivatized metabolites into an existing process for 4D-metabolomics on timsTOF instruments. 

For this work, metabolites extracted from a plasma sample (NIST Standard Reference Material 1950) were derivatized with 3-nitrophenylhydrazine (3-NPH) and subjected to 4D-metabolomics analysis in negative PASEF® mode on timsTOF HT (Bremen) and timsTOF Flex (Odense) instruments. 

For data analysis, we utilized the in-silico derivatization workflow included in Bruker MetaboScape 2025. Starting from a list of target compound structures, e.g., the metabolites of the tricyclic acid (TCA) cycle, all possible derivatization products are created in silico based on the chosen reactions of carboxy and carbonyl groups with 3-NPH. The list of potential products, supplemented by in-silico generated MS/MS spectra and predicted CCS values, is used to annotate the feature table generated by LC-TIMS-MS/MS using up to 5 matching criteria: retention time, accurate mass, isotopic pattern, MS/MS fragments, and CCS. 

This methodology can be used, as demonstrated by our work, to identify differentially expressed metabolites that represent biomarkers of disease with clinical relevance. 

Conflict of interest disclosure:
The contributing authors AB, NK, SW, StW, and MRL are employed by Bruker Corporation or one of its subsidiaries (“Bruker”). Bruker manufactures and sells analytical instruments including mass spectrometers and software. Bruker mass spectrometers and software were used in this study. 

Enhancing non-targeted analysis using comprehensive-two-dimensional gas chromatography coupled to a high-resolution mass spectrometry

Sebastiano Panto’¹, Dmitrii Rakov¹, Aous Khalefa¹, Nick Jones²

1) LECO European Application & Technology Centre, Berlin, Germany
2) LECO Corporation, St. Joseph, Michigan, USA 

Modern society produces, markets, and uses an increasing number of chemicals, which can be released into the environment through various pathways. The adoption of Non-Target Screening (NTS) workflows to identify these chemicals in the environment and organisms using high-resolution mass spectrometry has rapidly grown within the research community. The combination of comprehensive two-dimensional gas chromatography with high-resolution time-of-flight mass spectrometry (GCxGC-HR-TOFMS) is a powerful analytical technique for NTS of complex samples. This combination offers enhanced chromatographic separation and high-speed acquisition of accurate mass spectra across the full mass range. The generation of such rich data enables insightful retrospective review for vast numbers of unknown compounds. Additionally, target analysis can be performed simultaneously using the same data set obtained for NTS.

A systematic workflow is presented here for screening both target and non-target contaminants in various high-complexity samples. A combination of non-polar and polar stationary phases was used to enhance separation power of the GCxGC method adopted, resulting in better resolution between matrix and target compounds and, consequently, purer MS spectra for target POPs such as PCB, BDE, and Toxaphenes. Identification and confirmation were achieved through the injection of native standards and the application of dedicated targeted-processing methods. A novel Multi-Mode Source (MMS), capable of EI/CI (both positive and negative), was used to enhance identification confidence by providing library-searchable spectra, accurate mass, and molecular ion information for formula support and/or determinations. For this work, primarily EI and ECNI ionization modes were employed, with ECNI preferred for molecular ion information and formula support in cases where the identity of non-target substances could not be confirmed by EI ionization.

A list of target and non-target molecules is presented, along with information about identification methods (e.g., native standard, retention time, retention index, mass accuracy, etc.). EI high resolution mass spectra will be showed along with ECNI spectra used for confirmation. Formula computation approaches will be highlighted.

The benefits of GCxGC in providing superior separation capabilities and structured chromatograms are highlighted, along with the importance of the novel multi-mode source for HR-TOFMS, which allows for three ionization modes without hardware changes. The combination of ECNI spectra and EI information, such as retention time, retention index, and tentative identification through commercial databases, provided significant information that helped confirm the presence of halogenated POPs in the measured samples.

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