2022 SGMS Meeting

The 39th SGMS Meeting will take place in Beatenberg at 27-28 October 2022 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 (MS/MS: Fundamentals & Applications) on October 25-26 2022. 

RegistrationInfo, Deadlines, FeesProgramPlenary lecturesOral presentationsPoster presentationsSponsors

Confirmed speakers for meeting 2022

• Joseph A. Loo, UCLA (abstract)
• Luc J.C. van Loon, Maastricht University Medical Centre (abstract)
• Anneli Kruve, Stockholm University (abstract)
• Juan Zhang, Novartis (abstract)

SGMS School: MS/MS - Fundamentals & Applications

Information on the topic, the involved trainers and the program is on the 2022 SGMS School page. Register is closed.  

2022 SGMS Meeting and SGMS School Registration, Deadlines & Fees

The registration is now closed (17Oct2022).

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

The registration is via online form, see link above. You can register for Meeting and School or only Meeting and only School. 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 no attending the meeting without Hotel accommodation. 

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.

Abstract submission

Abstract submission is closed.

Please submit your abstracts for short talks and posters together with the registration for the meeting (here). 

Abstract submission for presentation

Short Oral Contributions: Early deadline for abstract submission for both talks and posters is August 1st. The extended deadline is September 1st. Abstracts submitted before August 1st will have priority. You can provide a concise flat text abstract with your registration on the online form. You will be contacted if your abstract has been selected to provide a rich text version that can include figures for the abstract book.

Posters: The same rules apply for the abstracts to be submitted. Poster size should not exceed 146 H x 118 W cm (size of pin wall). There will be a defined poster session. 

SGMS Annual Meeting (registration includes one night with breakfast at the Dorint hotel, Thursday lunch, Apéro and SGMS dinner, Friday lunch, and coffee breaks).

School & SGMS Annual Meeting (registration includes three nights with breakfast at the Dorint hotel, and all of the additional items included for School and SGSM meeting registration). Monday night accommodation and dinner on request.

Students support program

All students are hosted at a special rate of CHF 100; double room occupancy. If no roommate is indicated on registration form, it will be allocated by the organizers.

Meeting Program

Plenary lectures

Joseph A. Loo: Towards Illuminating the Dark Matter of Top-Down Mass Spectrometry

Department of Chemistry & Biochemistry and the Department of Biological Chemistry, David Geffen School of Medicine at the University of California, Los Angeles (UCLA)

Native mass spectrometry (MS) of proteins and protein assemblies reveals size and binding stoichiometry. But elucidating their structures to understand their function is more challenging. We show that native MS and native top-down MS, i.e., fragmentation of the gas-phase protein, can be effective for deriving structural information for soluble and membrane protein complexes, and much of this information can be correlated to the solution-phase structure. However, much more information (I believe) on protein structure can be derived if every peak in a tandem mass spectrum can be assigned – but why is this difficult? Non-MS experts would be surprised to learn that well over 50% of the peaks in a protein tandem mass spectrum are not assigned often, and thus not used, i.e., ignored. What analytical method allows 50% of data to be thrown away? A few examples will be shown to demonstrate that attempting to assign every peak in a tandem mass spectrum could lead to new information related to a protein’s structure, and potentially its function. 

Luc J.C. van Loon: You are what you eat–the study of post-prandial protein handling in vivo in humans

Department of Human Biology, Maastricht University Medical Centre

Skeletal muscle protein is constantly being synthesized and broken down, with a turnover rate of about 1-2% per day. The rate of skeletal muscle protein synthesis is regulated by two main metabolic stimuli, food intake and physical activity. Food intake, or more specifically protein ingestion, directly elevates muscle protein synthesis rates. The dietary protein derived essential amino acids act as signaling molecules activating anabolic pathways and provide precursors for muscle protein synthesis. Ingestion of a meal-like amount of dietary protein elevates muscle protein synthesis rates for several hours, providing evidence that ‘you are what you just ate’. When food is ingested after a bout of physical activity the post-prandial muscle protein synthetic response is augmented, with higher muscle protein synthesis rates sustained over a more prolonged period of time. In other words, when you ingest protein following a bout of physical activity ‘you become even more of what you just ate’. In contrast, when protein is ingested following a period of inactivity the post-prandial muscle protein synthetic response is blunted, coined anabolic resistance. Therefore, disuse makes you ‘become less of what you just ate’. These concepts play a key role in the prevention and management of age-related muscle loss and the development of chronic metabolic diseases. 

Anneli Kruve: Risk-based Prioritization of LC/HRMS Peaks: Predicting Concentration and Toxicity

Department of Environmental Science, Stockholm University, Stockholm, Sweden

A small fraction of chemical mixture toxicity can be explained by known chemical contaminants monitored by targeted analytical methods as only a few thousand chemicals have ever been monitored in the environment. The non-targeted analysis with LC/HRMS allows detecting hundreds or thousands of features simultaneously; however, the relevance of the features depends on the potential effect (toxicity) of the compound and on the concentration of that compound. Therefore, one of the most significant obstacles for non-targeted LC/HRMS screening has been the inability to provide quantitative and toxicity related information. To overcome this issue, we have developed a quantification approach based on in silico predicted electrospray ionization efficiencies. 

The proposed method has been tested for quantification of contaminants in different surface water, wastewater, as well as food and on EPA’s Non-Targeted Analysis Collaborative Trial (ENTACT) samples. In all of the applications, an average concentration prediction accuracy of ~3× has been observed. This means that the reported concentration of 3 ppm is likely to be in the range of 1 to 9 ppm (3 ppm/3 times to 3 ppm x 3 times). This accuracy is suitable for prioritization purposes, e.g., combining the concentration prediction with the toxicity predictions and identifying the most relevant contaminants. Additionally, recently different quantification approaches for non-targeted screening were compared head-to-head in NORMAN interlaboratory comparison. The preliminary results show that the predicted ionization efficiency clearly outperforms other approaches, such as the close-eluting standard approach, the structurally similar standard approach, or the parent-transformation product approach.  

Furthermore, to evaluate the total risk of the mixtures, toxicity also needs to be evaluated. LC/HRMS spectra carry information about the polarity, size, and functional groups present in the detected compounds, which also affect the toxicity of the compounds. This suggests that mixture toxicity could be predicted from the non-targeted LC/HRMS data directly. To explore this possibility, we use estimated physicochemical properties and empirical spectral information acquired in LC/HRMS analysis, and subsequent machine learning to predict the lethal concentration for 50% of the population (LC50) for rainbow trout, bluegill, fathead minnow, algae, and water flea retrieved from CompTox Chemical Dashboard (>800 compounds) alongside the concentration (exposure). Machine learning approaches like gradient boosting, random forest, and support vector machines are used. For model training, the experimental LC50 values and theoretical structural fingerprints were calculated from SMILES representation of the compound. The range for LC50 varies from 57000 to 0.0001 mg/L with the average experimental repeatability of 2.5x (max >100x). Root-mean-square error (RMSE) for training and test set are 4x and 10x on the concentration scale. For validation, MS/MS data from MassBank are used alongside spectra measured in-house with LC-Orbitrap. The fingerprints are calculated with SIRIUS+CSI:FingerID from experimental MS/MS spectra and used for predicting the LC50 values. The RMSE of 10x is observed, which agrees with the RMSE obtained with theoretical fingerprints. The analysis of the model reveals that the most important features are the exact mass of the compound, the presence of aromatic rings and/or sulfur, halogens, and oxygen. The exact mass is inversely correlated with the polarity of the chemicals and, therefore, baseline toxicity. 

Juan Zhang: Understanding the crosstalk between immunity and metabolism using targeted metabolomics

Novartis Institutes for BioMedical Research, Basel, Switzerland

Metabolomics as one of the omics technologies has become a key technology with increasing applications in life sciences and biomedical research. Metabolomics examines the products of biochemical pathways of living organism at specific points in time.  These characteristic metabolic profiles of a living system under certain condition can differ from its reference system in normal or "healthy" states. Activated macrophages or T-cells, for example, undergo metabolic reprograming to support their cellular functions and to provide energy for their rapid proliferation. Their distinct metabolic profile in return also defines their effector functions and phenotypes. From cellular immunometabolic phenotype to systemic interplay between aberrant metabolic homeostasis and immune dysfunction, understanding the crosstalk between immunity and metabolism has many implications on elucidating disease mechanisms and delivering useful tools for therapeutic intervention, for example translational metabolite biomarkers of diseases or translational pharmacodynamic markers as a guide to the effects of a therapy. While the scientific significance of biochemicals remains so important, their extreme diverse molecular properties and wide range of endogenous concentrations pose considerable analytical challenges for a robust and reliable application performance of the metabolomics technology. The utilization of combining adequate chromatographic separation techniques and tandem mass spectrometry can help to overcome some of the analytical challenges and provide unique solutions for applying targeted metabolomics in biomedical research to deliver timely impacts. A few examples of applying targeted metabolomics in understanding the crosstalk of immunity and metabolism will be presented and discussed.  

Oral presentations

Placebolomics; Breath metabolomics of placebo effects

Mélina Richard¹, Dilan Sezer², Sarah Bürgler², Luana Palermo², Yannick Schulz², Jens Gaab², Pablo Sinues¹ 

1. University Children's Hospital Basel (UKBB), Department of Biomedical Engineering; University of Basel, Institute for Biomedical Ethics; University of Basel  
2. Faculty of Psychology; University of Basel

Understanding the biological mechanisms of placebo effects have a great and overarching impact into the broad field of clinical interventions. Amongst others, placebo effects obscure the efficacy of drugs. Any tool to reduce this unwanted data variability, would benefit clinical trials and practice as well as patient care.  
The existing very limited literature addressing this topic at the metabolomic level suggests that placebo effects induce specific metabolic alterations. Additionally, numerous studies suggest that placebos can alter the experience of pain.  
This pilot study is addressing the question of whether placebo effects induce any measurable changes at the metabolic level. Our hypothesis is that placebo effects exist, and that they modulate certain metabolic pathways.  
We tested this hypothesis by collecting the exhaled breath metabolome using real-time mass spectrometry of 20 healthy subjects while using an objective method of measurement for placebo effects in pain analgesia (i.e. Cold Pressor Test; CPT) before and after the administration of a placebo (i.e. crossover design). Univariate and multivariate methods are currently being used to analyze the data. 
CPT pain threshold time showed no significant difference (p=0.1) between placebo and no placebo experiments. At the breath metabolomics level, we found ~240 features with a FDR < 0.05 for the pain vs. no-pain group. For the placebo effects, we found ~200 features that differ significantly (FDR < 0.5) from the placebo vs. no-placebo group. Ongoing work aims to unveil the main metabolic pathways governed by the pain and by the placebo effects. 

High-throughput identification of isomeric species by combining high-resolution ion mobility, Hadamard transform and infrared spectroscopy in a cryogenic multitrap

Vasyl Yatsyna, Ali H. Abikhodr, Ahmed Ben Faleh, Stephan Warnke, and Thomas R. Rizzo 

Laboratoire de Chimie Physique Moléculaire, École Polytechnique Fédérale de Lausanne, EPFL SB ISIC LCPM, Station 6, CH-1015 Lausanne, Switzerland

Separation and identification of isomers in complex mixtures is a long-standing problem in analytical chemistry, and high-throughput approaches for isomer analysis are especially crucial in the areas of glycomics, metabolomics and lipidomics. Even though high-resolution ion mobility spectrometry can rapidly separate many kinds of molecular isomers, it is particularly challenging to identify them unambiguously using traditional techniques such as tandem mass spectrometry. Alternatively, infrared (IR) spectra of molecular isomers measured directly in the mass spectrometer can provide reliable fingerprints for their unambiguous identification. Nevertheless, since measuring the IR spectra comes at the cost of increased acquisition time, developing various multiplexing schemes to speed up the spectroscopic measurements are required to be able to achieve high-throughput identification in the complex mixtures. In this work, we have developed a high-throughput approach that employs high-resolution IMS separation with subsequent Hadamard transform multiplexing [1] combined with multiplexed measurements of IR fingerprints in the cryogenic ion trap having multiple trapping regions [2]. For example, using two separate cryogenic trap sections that are interrogated simultaneously with the tunable IR laser light, we can speed up the spectroscopic measurements twice, which is by no means the limit. Moreover, using Hadamard transform multiplexing for each of the trapping regions of the cryogenic trap allows analyzing a large variety of species having a broad range of ion mobilities present in the complex mixture in the single laser scan. To demonstrate the combined multiplexing approach, we separate the human milk oligosaccharides extracted from pulled human milk, measure their IR fingerprints in a multiplexed manner in a single laser scan, and identify their isomeric forms by comparing the measured fingerprints to the database acquired using commercially available standards.  

[1] Vasyl Yatsyna, Ali H. Abikhodr, Ahmed Ben Faleh, Stephan Warnke, and Thomas R. Rizzo, Anal. Chem. 2022, 94, 6, 2912–2917  
[2] Stephan Warnke, Ahmed Ben Faleh, and Thomas R. Rizzo, ACS Meas. Sci. Au 2021, 1, 3, 157–164 

EXPECTmine pipeline: Data mining and machine learning for linking environmental exposures to toxic effects via a hazard driven prioritisation during non-targeted screening

Katarzyna Arturi, Juliane Hollender 

1. Department of Environmental Chemistry, Swiss Federal Institute of Aquatic Science and Technology, Eawag, Switzerland  
2. Institute of Biogeochemistry and Pollution Dynamics, ETH Zurich, Switzerland 

Non-targeted screening (NTS) of high-resolution mass spectrometry (HRMS) data has been rapidly gaining popularity and confidence in the putative characterization (identification and quantification) of complex samples in disciplines such as environmental, food, and health-based exposomic. State-of-the-art NTS workflows aim to convert thousands of molecular 'features' into quantified chemical structures. However, even the most sophisticated tools rely on manual validation of the results, thus making prioritization necessary. The traditional prioritization approaches are based on signals' abundance or frequency, outlying environmental exposures but lacking toxicological relevance so critical in the context of pollution. In this work, we propose a more comprehensive method combining features' exposures obtained from MS with their potential toxicity derived by machine learning from MSMS, thus assessing overall risks (risk = hazard [toxicity] x exposure [concentration]). For that purpose, we have developed MLinvitroTox, a machine learning (ML) tool for the prediction of toxicity fingerprints (ToxFps) for thousands of unknown HRMS features based on their MS and MSMS characteristics as well as existing ML NTS tools (CSI:FingerID, Dührkop et al. Nature Methods 16, no. 4 (2019): 299-302.). MLinvitroTox was trained on ToxCast and Tox21 high-throughput screening toxicity databases from EPA (Richard et al. Chemical Research in Toxicology 29, no. 8 (2016): 1225-1251.). It joins under one roof a plethora of optimized supervised classification algorithms (regression, decision trees, and deep learning) to predict over 500 toxic molecular endpoints used to compile ToxFps. Since each endpoint represents a cellular-level perturbation caused by a chemical/target interaction, the methodology is suitable for ML-derived linking of structural moieties with molecular-level disturbances in pathways that can be subsequently linked through adverse outcome pathways (AOPs) to specific toxic outcomes in living organisms. MLinvitroTox, coupled with the existing sophisticated NTS tools, results in a novel data mining pipeline (EXPECTmine), incorporating, in addition to hazard-driven prioritization, also tentative identification of the prioritized signals via various molecular structure annotation packages (MetFrag, and SIRIUS) and tentative quantification via ML-derived ionization efficiency prediction (Liigand et al. Scientific Reports 10, no. 1 (2020): 1-10.). So far, excellent ML results have been produced for 138 molecular endpoints representing 34 major toxic mechanistic targets. The performance was also validated with MassBank spectral records, exhibiting recall and precision values exceeding 0.9. Here, we present an example of retrospective deployment of EXPECTmine on digitally frozen samples for which HRMS/MS and bioassay results are available (Neale et al. Science of the Total Environment 576 (2017): 785-795.). The predictions of MLinvitroTox confirmed molecular toxicity mediated via androgen (AR), estrogen (ER), and aryl hydrocarbon (AhR) receptors, as well as oxidative stress (OS) for the majority of the wastewater targets. Furthermore, an additional 92 (AR), 456 (ER), 137 (AhR), and 13 (OS) features (cutoff 95 \% probability) from the pool of 4274 detected features for which molecular fingerprints could be predicted with CSI:FingerID, were tagged as potentially toxic, thus significantly increasing the explained mixture toxicity. The ongoing work focuses on the identification and analytical confirmation of the features of interest with the final goal of a more comprehensive mapping of pollution.

Using suspect screening approach to guide the toxicity assessment of metabolites produced by Microcystis aeruginosa

Mariana de Almeida Torres¹, Martin R. Jones², Colette vom Berg³, Ernani Pinto⁴ and Elisabeth M.-L Janssen³

1. School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil  
2. School of Biosciences, University of Birmingham, United Kingdom  
3. Swiss Federal Institute of Aquatic Science and Technology (Eawag)  
4. Centre for Nuclear Energy in Agriculture, University of São Paulo, Piracicaba, Brazil 

Cyanobacteria are a diverse and widespread group of photosynthetic prokaryotes that synthesize a variety of toxic and bioactive metabolites, from low molecular weight compounds to non-ribosomal peptides (so-called cyanopeptides). Such variety is illustrated in a recent collaboration of researchers, which compiled a comprehensive database containing structural information of more than two thousand cyanobacterial secondary metabolites, the CyanoMetDB (Jones et al, 2021). The most notorious metabolites are cyanotoxins – well-studied compounds in terms of their toxicity and mode of action for different taxa. However, the risk associated with the presence of most of these compounds in aquatic environments remains an open question, as only a few classes have their toxicological potential elucidated. Given this background, our study aims to use mass spectrometry to investigate the production and guide the toxicity assessment of peptides produced by a Microcystis aeruginosa strain (NPCD-01), previously isolated in a bloom event in Brazil.  
Cyanobacterial extracts were fractioned by HPLC into 11 fractions and analysed by LC-HRMS (LumosFusion Orbitrap, ThermoFisher Scientific) in a data-dependent acquisition of MS2 spectra triggered by an inclusion list of m/z values of the cyanopeptides compiled in CyanoMetDB as target ions. During the data processing, the confirmatory elements of the suspects were accurate mass (m/z), accurate isotopic pattern, retention time, and evidence found in the MS2 data (such as diagnostic fragmentation ions). After identifying the different sets of peptides present in each fraction, a toxicity screening of the combined (pool) and isolated fractions was first assessed with the sensitive grazer Thamnocephalus platyurus (ThamnoToxKit FTM, Microbiotests). The choice of the fractions to be tested in the grazer toxicity assays was guided by suspect screening. In turn, the Fish Embryo Toxicity tests (FET) were performed with zebrafish (Danio rerio) embryos (following the OECD guideline 236, 2013), guided by the suspect screening and toxicity results observed with the grazers.  
The suspect screening matched 17 cyanobacterial metabolites listed in CyanoMetDB with intensities higher than 0.1% (total content). The peptide profile of the cyanobacterial crude extract was dominated by 9 microginin variants (microginin741A being the most abundant), and cyanopeptolin959. The highest purity of mig741A was identified in fraction #3 (97%), and of the cyp959 in fraction #1 (87%). The mortality results of the whole extract for the crustaceans pointed to 60% mortality in the highest concentration (2.7 mg dw/mL). Surprisingly, the fraction screening with the grazer showed that fractions 9 and 10 (combined) prompted 100% mortality to the grazers at the same concentration tested in the pool, and no mortality was observed in the other fractions. Fractions 9 and 10 showed a similar peptide profile in the suspect screening, with matches for two linear peptides with m/z 504.3432 and 833.4893 as the most abundant compounds in the given fractions.  
The acute toxicity results of the combined fractions pointed to dose-dependent mortality for the zebrafish embryos, with the two highest concentrations tested (5.97 and 2.39 mg dw/mL) prompting 100% death. As also observed for the crustaceans, fractions #9+10 (combined) prompted 100% mortality in the same concentration range tested. On the other hand, fractions #1 and #3 did not point to an acute lethal effect in the zebrafish embryos. The tentative identification of the compounds present in fractions #9 and #10 is in progress, and such results might shed light on the promoter of the toxicity of the strain. To conclude, high-resolution mass spectrometry showed to be a powerful tool in conjunction with the toxicological assays to explore natural products from cyanobacteria and their potential impact on aquatic environments. 

Mass Spectrometry: Who invented it?

Marc Gonin 

TOFWERK AG

Some little known facts about the history of mass spectrometry. 

Uncovering urine adulteration attempts in forensic toxicology: An LC-MS based proteomics approach 

Tom D. Schneider, Thomas Kraemer, Andrea E. Steuer 

Zurich Institute of Forensic Medicine, University of Zurich, Zurich, Switzerland 

Aim: Sample adulteration aims at circumventing positive drug-testing results in a clinical, forensic or doping context by means of dilution, sample replacement or chemical alteration. Approaches employing liquid-chromatography mass-spectrometry (LC-MS) based metabolomics analysis showed promising results to identify manipulated samples, although identification and verification of actual biomarkers remains a challenging task. With this study we wanted to expand further to detect sample adulteration with hydrogen peroxide (H2O2) in human urine by LC-MS based protein-analysis.  
Methods : Urine samples from ten donors were split into two groups. One group was left as is, the other was treated with 10% H2O2 for 30 minutes. Urinary proteins were isolated, purified and enzymatically digested overnight with trypsin. 5 µg of digested protein were injected and separated on a Dionex LC system equipped with an Acclaim PepMap column (1.0 mm x 150 mm, 2 µm) running a gradient from 3% ACN, 3% DMSO and 0.1% formic acid to 28% ACN, 3% DMSO and 0.1% formic acid over 60 minutes at a flow-rate of 50 µl/min and measured by high-resolution quadrupole time-of-flight mass spectrometry (Sciex 6600). The acquired data was analysed using FragPipe (v17.1), where retention-time alignment, normalization, identification and label-free quantification of peptides and proteins was performed. Protein and peptide abundances were further evaluated and visualized with Prism (v8).  
Results and Discussion: Across all urine samples, we were able to reliably identify and quantify a total of 1250 peptides corresponding to 180 different proteins. Protein and peptide abundances showed distinct differences in samples treated with 10% H2O2 compared to untreated urine samples. Several proteins and peptides were significantly affected by the H2O2 treatment and either increased or decreased in quantity. For instance, uromodulin (UROM_HUMAN), cytoskeletal keratin (K2C1_HUMAN) and immunoglobulin lambda (IGLC7_HUMAN) showed a more than 5-fold decrease after oxidative treatment (p < 0.005) and alpha-1-acid glycoprotein (ORM1) could not be detected anymore after treatment with H2O2 in all measured samples.  
Beyond that, alterations to the primary amino-acid sequences could also be observed. H2O2 should mainly introduce oxidative changes to the primary amino-acid sequence of proteins and peptides. Methionine, tryptophan, lysine and tyrosine could be found in either oxidized (15.9949 Da) or dioxidized (+31.9898 Da) variants. These modified peptides, such as QM_dioxPGKGLEWM_oxGR (HV5X1_HUMAN) or VSLKTALQPM_dioxVSALNIR (UROM_HUMAN) could proof themselves as ideal biomarker peptides for exposing oxidative adulteration, as they could only be observed after aggressive oxidative treatment and are not present under normal physiological conditions in human urine.  
Further, the analysis of the human urinary proteome bears another advantage: It is likely very hard to fool the forensic toxicologist using synthetic urine as a substitute in urine drug-testing by using the approach presented here. The inherent complexity of the human physiology that ultimately results in the secreted urinary proteome is too complex to be recreated by any means other than substituting it with the urine of another, preferably drug-negative, healthy person. Consequentially, analysing the urinary proteome can already expose two common adulteration approaches: Using synthetic urine and chemical adulteration with H2O2.  
Conclusion: Sample adulteration aims at producing false-negative drug-testing results. With the study presented here, we explored the possibility of using LC-MS based proteomics analysis to reveal chemically altered urine samples and ultimately avoid false-negative results. We found distinct quantitative changes as well as possible marker peptides that are only present in oxidatively treated urine samples. Even though by no means standard in a forensic context, a method such as this could be employed when in doubt about the integrity of an authentic urine sample prior or after routine toxicological analysis and might, eventually, extent the toolbox that is available to the forensic toxicologist in routine casework.  

Real-time in vitro and in vivo metabolomics of bacterial pathogens using SESI-HRMS

K. Arnold^1,2, A. Gómez-Mejia³, J. Bär³, K. Dev Singh^1,2, T. Scheier³, S. Brugger³, A. Zinkernagel³, P. Sinues^1,2

1. University Children’s Hospital Basel (UKBB), 4056 Basel, Switzerland  
2. Department of Biomedical Engineering, University of Basel, 4123 Allschwil, Switzerland  
3. Department of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich University of Zurich, 8091, Zurich, Switzerland 

Bacterial respiratory infections (e.g. pneumonia) are a leading cause of hospitalization and death. Timely diagnosis and treatment is crucial for the outcome of therapy. Current diagnostic methods are partially invasive, time-consuming, expensive and not optimal for routine clinical practice. The objective of our research is to develop a diagnostic tool (e.g. breath test) to speed up bacterial pneumonia diagnosis. Therefore, we firstly aimed to characterize specific volatile metabolites present in the culture headspace of two common pneumonia pathogens, Streptococcus pneumoniae and Staphylococcus aureus. Secondly, metabolites present in exhaled breath of mice inoculated with the aforementioned pathogens were investigated and compared to metabolites observed in the first step. Bacterial cultures or mice were placed in an airtight box pervaded with 0.5 L/min of medical grade air. Volatile metabolites emitted by growing bacterial cultures or by mice during the course of infection were analyzed in real-time using secondary electrospray ionization high-resolution mass spectrometry (SESI-HRMS) over a period of 15 and 24 hours, respectively. Data were evaluated by applying different univariate and multivariate statistical models within MATLAB. SESI-HRMS was capable to detect relevant metabolites within the first minutes of measurement with a minimal amount of bacterial colonies. Furthermore, we identified distinct mass spectral features in bacterial cultures’ headspace enabling the discrimination of the bacterial pathogens on species and strain level. Similar temporal metabolic profiles were successfully captured during the infection of mice. Cross-referencing in vivo against in vitro data should therefore enable us to identify specific biomarkers linked to bacterial pneumonia infection.

Quantitative determination of phosphatidylethanol in dried blood spots for monitoring alcohol abstinence

Stefan Gaugler^1,2, Marc Luginbühl¹, Wolfgang Weinmann³ 

1. CAMAG, DBS Labor, Sonnenmattstrasse 11, 4132 Muttenz  
2. Fachhochschule Nordwestschweiz, Hofackerstrasse 30, 4132 Muttenz  
3. Institut für Rechtsmedizin der Universität Bern, Murtenstrasse 26, 3008 Bern

The quantitative analysis of substances in dried blood spots (DBS) has gained vast popularity in the past decade. The World Anti-Doping Agency (WADA) also recently committed to implementing DBS.  
Here, we present an automated solution that makes the necessity of volumetric sampling for quantitative DBS analysis obsolete. Combining automated reflectance-based HCT correction in combination with fully automated DBS LC-MS/MS analysis, the novel strategy permits high-throughput analysis in combination with HCT independence. Studying the model compound phosphatidylethanol 16:0/18:1, which is HCT-dependent due to incorporation into red blood cells, an implementation of DBS HCT normalization is presented. First, the performance of the automated HCT module with DBS is demonstrated compared to standardized HCT analysis from whole blood using a centrifuge. Second, the HCT dependency of fully automated PEth analysis from DBS is evaluated. Third, a solution to correct for the HCT dependency of PEth using the HCT scanner is presented.  
The study demonstrates that as soon as the HCT dependence of an analyte is known, a correction factor can be applied for the normalization of HCT levels. In the context of PEth, a linear increase in PEth concentration was observed, as the analyte is primarily located within the cellular fraction. Based on the obtained results, the use of a common correction factor for PEth DBS is possible.

The Digital Botanical Gardens Initiative

The DBGI Consortium¹, Emmanuel Defossez^2,3, and Pierre-Marie Allard³

1. The DBGI Consortium (https://www.dbgi.org/dendron-dbgi/)  
2. Institute of Biology, University of Neuchâtel, Neuchâtel, Switzerland  
3. Department of Biology, University of Fribourg, 1700 Fribourg, Switzerland

The recently launched Digital Botanical Gardens Initiative (DBGI) ambitions to explore innovative solutions for the collection, management and sharing of digital information acquired on living botanical collections. A particular focus will be placed on the large scale characterization of the chemodiversity of living plants collections through mass spectrometric approaches. The acquired data will be structured, organized and connected with relevant metadata through semantic web technology. The gathered knowledge will then inform ecosystem functioning research and orient biodiversity conservation projects. The DBGI initially aims to take advantage of the readily available living collections of Swiss botanical gardens to establish robust and scalable chemo- and biodiversity digitisation workflows. The ultimate goal is to apply these approaches in the field and at the global scale in wild ecosystems. Here, we will expose how the latest computational mass spectrometry developments are harnessed to collect, organize, interpret and share with community the botanical gardens' digitized chemodiversity.

Poster presentations

Effect of sample transportation on the proteome of human circulating blood extracellular vesicles

Anne-Christine Uldry¹, Natasha Buchs², Sophie Braga-Lagache³, Nicolas Bonadies⁴, Manfred Heller⁵

1. Proteomics and Mass Spectrometry Core Facility, DBMR, University of Bern, Bern, Switzerland
4. Inselspital, Clinical Hematology, University Hospital Bern, Bern, Switzerland

Objective: To measure transport metrics impacting on blood during transport from clinical ward to laboratory by a human carrier (C) or the pneumatic transport system (PTS) of the hospital and to correlate them to the proteome composition of circulating extracellular vesicles (cEV) isolated from the very same blood samples.

Introduction: cEV are released by cells and play an important role in cellular communication, signaling, inflammation modulation, coagulation and tumor growth. cEV are of growing interest, not only as biomarkers, but also as potential treatment targets. PTS and C are both routinely used for transport of blood samples from the clinical ward to the diagnostic laboratory. However, very little is known about the effect of the different ranges of mechanical forces on the protein composition of cEV.

Methods: Peripheral blood samples from six hematologically healthy volunteers as well as six patients with hematological malignancies were collected. Mechanical forces on samples were measured during PTS and C and recorded by a set of sensors connected to a minicomputer. Blood cells were counted by Sysmex, and samples processed as platelet-poor (PPP) and platelet-free (PFP) plasma. Particle size distribution was determined for all samples. cEV were isolated and analyzed by label-free, semi-quantitative proteomics. The determination of cell-type specific patterns and biological functions imprinted by the two transport methods on the cEV proteome were performed by means of differential protein abundances tests, protein annotations, gene ontology terms analyses, and Spearman Rank correlation analyses, together with the Lasso feature selection method. String.db with community clustering was used to visualize transport mediated effects.

Conclusion: Proteomics reveals new insights into transport mediated effects on blood cells with C stimulating the release of cEV by influencing plasma membrane reorganization and release of EV by an ectosomal pathway. In contrast, PTS rather activated endosomal pathways due to the transport metric correlations with proteins of organelle origin. Lasso depicts proteins acting as surrogates of network clusters.

A single-step sample extraction approach for LC-MS/MS quantification of nicotine, cotinine and 3’-hydroxycotinine in serum and saliva

Vera van der Velpen

Clinical Pharmacology and Toxicology, Inselspital

Smoking alternatives, such as electronic nicotine delivery systems (ENDS) are increasingly used. While these are generally regarded as less harmful than combustible tobacco products, the discussion regarding their safety is ongoing. At the same time, newer ENDS that deliver higher doses of nicotine might be helpful to smokers seeking to quit. Quantifying nicotine and its metabolites at low concentrations in biofluids is crucial for future discussions and decisions, but challenging due to abundance of nicotine in the environment and elaborate sample preparation techniques.

We aimed to develop an LC-MS/MS method with a simple sample extraction method for the quantification of samples from clinical ENDS studies often containing low concentrations of nicotine and its two metabolites cotinine and 3’-hydroxycotinine in saliva and serum.

Firstly, mobile phases at pH 10, at which nicotine is unionized, were successfully used to improve retention on a pH-stable reversed phase C18 column. Secondly, the same type of RP C18 column was used as a delay column to separate environmentally abundant nicotine and cotinine in the solvents from the sample peaks. Thirdly, a simple, single-step sample preparation was achieved by the addition of 80% methanol (+ internal standards) in a 1:10 dilution for saliva and a 1:1 dilution for serum with the aid of 20mM zinc sulfate. This LC-MS/MS method achieved LLOQs of 1ng/ml and 0.5ng/ml in saliva and serum, respectively, with linear ranges up to 1000ng/ml (R>0.99) for all three compounds in both biofluids. The inter-assay accuracy ranged from 92-112% with a precision of ≤ 8.3%.

Fast on-site determination of volatile organic compounds in newly produced cars with SPME including Formaldehyde on a mobile GC/MS device

Pascal Looser

PerkinElmer (Schweiz) AG

Many people love the smell of a brand-new car. However, that smell, or better the volatile organic compounds, that you are inhaling are potentially toxic. Analytical methods have been established to determine the components and their concentrations within the car interior, but all of these are time-consuming. The study presented here, focuses on rapid on-site analysis, that delivers results on specific targeted components in about 15 minutes. This was achieved using the Torion, a robust, portable, and lightweight GC/MS system. It features an inbuilt gas and power supply allowing you to take the Torion to your samples, wherever they are, eliminating the need to transfer samples to the lab

Novel analytical strategies for the characterization of peptide complexes by temperature-controlled cyclic ion mobility mass spectrometry

Philipp Bittner, Valdrin Islami, Tomáš Fiala, Helma Wennemers^*, Renato Zenobi^*

Department of Chemistry and Applied Biosciences, ETH Zurich, CH-8093 Zurich, Switzerland

Over the last few decades, electrospray ionization mass spectrometry (ESI-MS) has evolved into a gold standard for the analysis of biomolecules in the process of drug discovery and drug development. Despite the availability of very high-resolution MS instruments, the analysis of isomeric/isobaric compounds such as peptides or peptide complexes, glycosylated proteins or micro-heterogeneities in antibody formulations remains challenging and time-consuming. With the recent development of high-resolution ion mobility (IM) devices, the experimental possibilities have been brought to another level. Here we combine this technology with our home-built temperature-controlled nano-ESI (TCnESI) source to develop novel analytical strategies for the above-mentioned challenges [1,2]. The combination of our TCnESI source with a cyclic IM-MS instrument (cIM, Waters) gives us unique access to thermodynamic and kinetic information of complex mixtures, such as the co-formation and stability of isomeric/isobaric peptide complexes, which is currently not possible by any other technique. In an initial approach, we used our method for the label-free analysis of isomeric collagen model peptides (CMPs). Preliminary results show that cIM allows the separation of three isomeric CMPs with the same ionization profile, differing only in the position of an aspartic acid. Applying more passes in the cIM is expected to give a clear separation of these monomers and allow analyses of other functionalized isomeric/isobaric peptides at same m/z ranges. We plan to apply this method to fully characterize the specificity of heterotrimer formation in complex mixtures of three or even more label-free peptides. The expansion of this CMP toolbox and characterization by cIM-MS is expected to afford a much deeper understanding in the controlled formation of collagen triple helices.

Vapor-based gas standard system for secondary electrospray ionization-high resolution mass spectrometry

Cedric Wüthrich, Zhiyuan Fan, Guy Vergères, Renato Zenobi, Stamatios Giannoukos

Department of Chemistry and Applied Biosciences, ETHZ, Zurich, Switzerland

Secondary electrospray ionization (SESI) coupled to high-resolution mass spectrometry (HRMS) is a powerful tool for the analysis of gaseous samples, particularly exhaled breath.[1] To standardize this technology in terms of reproducibility and quantification, a dynamic gas standard generation system was developed and employed. The system consists of an array of evaporation chambers, wherein an equilibrium between an aqueous solution and the gas phase of a compound of interest is achieved. A certain concentration of a gas standard is obtained by flowing a carrier gas stream (mL/min) through the chamber, which is further diluted through a larger gas flow (L/min). As a test case, standards of short chain fatty acids were used to investigate the reproducibility and robustness of the developed system, as well as to measure the limits of detection and limits of quantification of the SESI-HRMS for the individual compounds in both dry and humid conditions. Low concentrations in the magnitude of parts-per-trillion were experimentally reached. The integration of this vapor generation system with SESI-HRMS will be one stepping stone to standardizing breath measurements with this technique.

[1] T. Bruderer, T. Gaisl, M.T. Gaugg, N. Nowak, B. Streckenbach, S. Müller, A. Moeller, M. Kohler, R. Zenobi, On-Line Analysis of Exhaled Breath, Chem. Rev. 119 (2019) 10803–10828. https://doi.org/10.1021/acs.chemrev.9b00005.

A fast UHPLC-MS/MS screening method for the analysis of DBS in an anti-doping context

Alessandro Musenga¹, Tobias Langer^1,2, Olivier Salamin¹, Raul Nicoli¹, Tiia Kuuranne¹

1. Swiss Laboratory for Doping Analyses, University Center of Legal Medicine Lausanne-Geneva, Lausanne University Hospital and University of Lausanne, Switzerland
2. University Center of Legal Medicine Lausanne-Geneva, Lausanne University Hospital, University of Geneva, Switzerland

Currently, doping control analyses are mostly performed in urine, since sample collection is non-invasive and can be easily performed in large numbers. The analysis of doping agents in urine samples mainly targets the metabolites of the related prohibited substances. On the other hand, the collection of blood samples requires trained personnel (e.g. a phlebotomist) and is more invasive. Furthermore, transport and storage of blood samples have special requirements for the temperature to preserve the quality of the samples. However, blood samples are more representative of the physiological conditions and in most cases, the intact drug compound can be found rather than its metabolites. An easier alternative for the collection of blood samples are dried blood spots (DBS), where a small amount of blood (10-20 ?L) is collected on a porous support material and dried afterwards. Samples are collected either with a finger prick or with the aid of a needle device placed on the upper arm. This collection method is minimally invasive and does not require a physician to collect the samples. The process of drying stops the degradation processes so that samples can be transported and stored at room temperatures as long as the humidity is kept to a minimal level.

Widely used for the screening of new-borns, the analysis of DBS has recently gained interest in the anti-doping community, since it can solve some of the issues of the blood samples. To address the use of this matrix, the World Anti-Doping Agency (WADA) has issued a technical document [1], which commands that the analysis can only be conducted on substances without threshold or reporting limits. Therefore, DBS qualitative analysis can be applied to the classes of substances that are prohibited in sports at all time, like anabolic agents, ?2-agonists, diuretics and hormone modulators [2]. Nevertheless, the DBS analysis of substances belonging to other classes such as stimulants or narcotics is feasible [3], even though the small sample volumes require sensitive techniques.

A fast and comprehensive method for the detection of over 100 substances in DBS was developed, using methanolic extraction combined with reversed phase ultra-high pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. The method has been validated according to ISO 17025 and the international standard for laboratories from WADA.

[1]: Collaborative DBS working groups WADA. WADA Technical Document - TD2021DBS: Dried Blood Spots (DBS) for Doping Control - Requirements and Procedures for Collection, Transport, Analytical Testing and Storage. 1.0. Montreal, Quebec; 2021. https://www.wada-ama.org/sites/default/files/resources/files/td2021dbs_final_eng.pdf
[2]: Thevis M, et al. 2016, Sports drug testing using complementary matrices: advantages and limitations. J Pharm Biomed Anal. 130, 220-230
[3]: Salamin O, et al. 2020, Is pain temporary and glory forever? Detection of tramadol using dried blood spot in cycling competitions. Drug Test Anal. 12 (11-12), 1649-1657

Targeted phosphoproteomics for the mTOR pathway in zebrafish PAC2 cells

Nikolai Huwa, René Schönenberger, Ksenia Groh

Eawag, Swiss Federal Institute of Aquatic Science and Technology, 8600 Dübendorf, Switzerland

Monitoring the activity of molecular signalling networks has potential applications in predictive ecotoxicology, e.g. to assess chronic toxicity outcomes such as effects on growth in fish. A crucial signalling pathway known to regulate cell growth and proliferation is governed by the mechanistic target of rapamycin (mTOR) kinase protein. Signal transduction and regulation within the mTOR pathway occur through protein phosphorylation, which has been traditionally studied with antibody-based methods. However, suitable antibodies are not always available for non-mammalian species, such as zebrafish (Danio rerio), an important model organism in human health and environmental toxicology fields. Therefore, we set out to develop an alternative approach using mass spectrometry-based targeted proteomics, which we first applied in the zebrafish PAC2 cell line. Sample preparation for phosphoproteomics typically includes an enrichment step due to the low abundance of phosphopeptides and needs to be optimized to ensure reliable quantification of phosphorylation dynamics. Our previous sample preparation approach involved multiple steps, including protein precipitation, resolubilisation, quantification and in-solution digestion, which was very labour-intensive and hence not suitable for high-throughput analyses. By using suspension trapping (S-TrapTM, Protifi) filters, we were able to simplify sample preparation steps and simultaneously increase the signal intensity of our peptide targets by up to 12-fold. This new approach also allowed the use of SDS (5%) in the lysis buffer, which enabled a fast cell lysis (1 min) and highly efficient solubilisation of proteins. For the enrichment of phosphopeptides, a TiO2-based method was applied and two different buffer compositions were compared with respect to their enrichment efficiency. The multiple reaction monitoring (MRM) assay that we have developed using synthetic peptides can now be applied to monitor changes in protein abundance and phosphorylation of 15 protein targets along the mTOR pathway, represented by 31 unique peptides. Zebrafish PAC2 cells sampled at different growth stages, i.e. the exponential and stationary phases, showed differential protein abundance and phosphorylation levels of proteins involved in proliferation, growth and energy regulation within the mTOR pathway. Using the developed assay, we will further investigate the mTOR-mediated signalling pathway and its role in growth regulation in zebrafish cells after exposure to pharmacological mTOR modulators or growth-impacting chemicals, or after nutrient deprivation.

A novel workflow to determine over 1000 pesticide residues in compliance with SANTE 11312/2021 guidelines in various food matrices

Holger Stalz¹, Susan Baumeister², Peter Kornas², Marcus Chadha³

1. Agilent Technologies, Switzerland
2. Agilent Technologies, Waldbronn, Germany
3. Agilent Technologies, Cheadle, UK

Pesticides play an import role in agriculture and food industry to improve the crop and food production. Residues of pesticides remaining in or on commodities such as fruits, vegetables or cereals can cause adverse health effects as well as environmental concerns. Regulatory agencies have set maximum residue levels (MRLs) for hundreds of pesticides and their metabolites. Most MRLs are set at low ppb levels, which poses significant challenges especially if hundreds of analytes are screened and quantified simultaneously in complex food matrices. In Europe, pesticide testing laboratories adhere to the SANTE/ 11312/2021 Guideline. This Guideline ensures a consistent approach controlling MRLs legally permitted in food or animal feed. Due to the huge number of pesticides, the analysis is very elaborate. Very often multiple analytical approaches and laboratory intensive workflows are involved. Both lead to high operating costs and slow turnaround times.

We present a comprehensive, joint LC-MS and GC-MS workflow for the simultaneous quantitation of >1000 pesticide residues in fruits and cereals of varying water content. Workflow performance was verified according to SANTE/ 11312/2021. During initial validation parameters like limit of detection (LOD) and limit of quantification (LOQ), linearity, recovery and precision were evaluated using the method performance criteria described in this Guideline. Details of sample preparation procedures and instrumentation set up will be discussed in conjunction with the data analysis parameters enabling the quantification and confirmation of pesticide residues.

Comparing different mass spectrometric approaches to detect oral testosterone undecanoate administration

Tobias Langer^1,2, Raul Nicoli¹, Olivier Salamin¹, Tiia Kuuranne¹, Alessandro Musenga¹

1. Swiss Laboratory for Doping Analysis, University Center of Legal Medicine Lausanne-Geneva, Lausanne University Hospital and University of Lausanne, Switzerland
2. University Center of Legal Medicine Lausanne-Geneva, Lausanne University Hospital, University of Geneva, Switzerland

Doping with endogenous anabolic androgenic steroids (EAAS) like testosterone (T) has always been difficult to detect, as they appear naturally in the human body and the discrimination between the natural state and drug administration is necessary. The individual longitudinal urinary analysis of endogenous steroids in the course of Athlete Biological Passport (ABP) can flag suspicious samples. Afterwards, the steroids origin in the relevant urine samples has to be confirmed by isotope ratio mass spectrometry (IRMS) to differentiate between endogenous and exogenous. As IRMS is a laborious and time-consuming technique, only urine samples with a suspicious steroid profile are analysed. However, in some samples, the urinary T concentrations are below the limit of detection. Moreover, certain individuals bear mutations that can influence the excretion of steroids, sometimes invalidating the evaluation of the urinary steroid profile. IRMS analysis has its limitations as well, such as the sensitivity and complex result interpretation.

To support urinary analysis and provide further proof of doping with EAAS, direct analysis of steroids in blood has been proposed [1]. It was shown that this approach improves the detection capability after transdermal T administration in women [2]. Medications for oral and parenteral T application contain T esters, which can be directly detected in blood as unequivocal proof of doping, removing the need for origin confirmation by IRMS. We developed a method capable of detecting the endogenous steroids T, androstenedione (A4) and dihydrotestosterone (DHT) as well as T esters in the same sample aliquot, using simple UHPLC-MS/MS instrumentation [3].

Serum samples from an oral testosterone undecanoate (TU) administration study [4] were analysed with this method to detect TU and demonstrate its influence on the endogenous steroids in blood. The corresponding urinary steroid profiles were also determined and several urine samples were selected for IRMS confirmation. The goal of this study is to compare the timeframe where oral TU administration is clearly detectable by the two different approaches: urinary IRMS analysis and TU detection in serum.

[1]: Ponzetto F, et al. (2016) Longitudinal monitoring of endogenous steroids in human serum by UHPLC-MS/MS as a tool to detect testosterone abuse in sports. Anal Bioanal Chem. 408 (3), 705-719
[2]: Salamin O, et al. (2022) Longitudinal evaluation of multiple biomarkers for the detection of testosterone gel administration in women with normal menstrual cycle. Drug Test Anal. 14 (5), 833-850
[3]: Langer T, et al. A comprehensive UHPLC-MS/MS method for the analysis of endogenous and exogenous steroids in serum for anti-doping purposes. submitted to Drug Test Anal.
[4]: Badoud F, et al. (2013) Profiling of steroid metabolites after transdermal and oral administration of testosterone by ultra-high pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. J Steroid Biochem Mol Biol. 138, 222-235

Comparison of dairy and dairy-free cheese flavor profiles using the newly developed GC-ecTOF

Michael Grössl, Marleen Vetter, Steffen Bräkling, Sonja Klee

TOFWERK AG

An increased awareness to health, environmental and animal welfare issues and a growing acceptance of vegan products, especially amongst the younger population, has led to an increase in non-dairy products. To attract new customers, vegan foods often try to imitate more established non-vegan products. Hence, characterization of flavor and aroma profiles is of particular interest.

The development of cheese flavor is the result of a complex combination of microbial and biochemical activities throughout the storage period, leading to the formation of a heterogeneous mixture of volatile and nonvolatile compounds. A newly developed time of flight mass spectrometer operating an electron ionization and a chemical ionization source in parallel (ecTOF) is used to characterize and compare the cheese flavor profiles of vegan-cheeses to those of non-vegan equivalents. By coupling a gas chromatograph (GC) to the ecTOF, target and suspect screening analysis is improved as well as non-target analysis enabled. Concurrent structural as well as accurate mass molecular ion information is generated, which helps in the identification of compounds of interest: flavor dominant compounds such as ketones, esters, aldehydes and organic acids and potential off-flavor compounds.

High-throughput residual solvent analysis according to USP 467 using direct mass spectrometry

Kara Merkle-Gams¹, Arnd Ingendoh¹, Christopher Pfaff¹, Ann-Sophie Lehnert¹, Mark Perkins², Vaughan Langford³

1. Syft Technologies GmbH, Berliner Allee 65, 64295 Darmstadt/D
2. Anatune Ltd., Girton Road, Cambridge, UK, CB3 0NA
3. Syft Technologies Ltd, 68 St Asaph St, Christchurch 8011, New Zealand

Volatile impurities occur frequently in pharmaceutical products and packaging and may be of concern due their toxicity. The United States Pharmacopeia’s Residual Solvents USP <467> chapter defines the approach to residual solvent analysis for most of the global pharmaceutical industry. Described are GC techniques using static headspace (HS) for sample introduction and FID (flame ionization detection). There are alternative procedures possible according to <467> which are permitted when they are fully validated and produce comparable results to the compendial methods or procedure. SIFT-MS its validation is described as an alternative procedure for USP <467> residual solvent analysis.

SIFT-MS uses eight reagent ions (H3O+, NO+, O2+, O-, O2-, OH-, NO2-, NO3-), generated by plasma microwave discharge from moist air to provide various ion-molecule reactions. Compounds of interest often interact differently with each reagent, and thus allow detection of a wide range of VOCs with high specificity, including the separation of isomers. The reagent ion selection is done in a first quadrupole with switching times in the millisecond range. After interaction with the sample in a gas flow tube under well-defined conditions, the product ions and unreacted reagent ions are detected in a second quadrupole. Rapid reagent ion switching provides high selectivity in the absence of chromatographic separation or high-resolution mass spectrometric analysis. Utilizing a compound library with known rate coefficients (k), the software instantaneously calculates each analyte’s absolute concentration from the specific reagent and product ion count rates. Typical run times for individual samples are in the range of less than one minute.

Here, quantitative testing of 28 Class 2 solvents listed in USP <467> was performed. Linearities were R > 0.93 for all compounds, with only four product ions having R < 0.99. Repeatability was generally RSD < 10% with elevated values for solvents with higher water affinity. Similar behaviours were observed for accuracy and recovery, with acceptance criteria being met more consistently for compounds with lower water affinity.

In addition to reporting the successful procedure validation for determination of most of the residual solvents evaluated, it is also discussed how direct MS might support quality-by-design and continuous manufacturing by on-line analysis of residual solvents. It demonstrates that SIFT-MS has the high potential of being used as an alternative procedure in USP <467> in compliance with acceptance criteria.

Acknowledgements: Study was generated and guided by Edmond Biba, PhD, United States Pharmacopeia, Rockville, MD, USA, exb(at)usp(dot)org

Simultaneous measurement of short and long chain lipid aldehydes by UHPLC-high resolution mass spectrometry to evaluate the oxidation of plasmalogens

Rodrigo.L. Faria, S. Miyamoto

1. Departamento de Bioquímica, IQ, USP, SP, Brazil

Introduction: Aldehydes modify biomolecules such as DNA, proteins and amino acid, resulting in a variety of diseases and cytotoxic effects. Lipid peroxidation is an endogenous source of aldehydes and can be described as a process of radical reactions that cause oxidation and chain breakage of unsaturated fatty acids, especially polyunsaturated fatty acids (PUFAs). Plasmalogens are glycerophospholipids with a vinyl-ether bond at the sn-1 position of the glycerol backbone and sn-2 enriched in PUFAs. Several studies support the hypothesis that plasmalogens act as antioxidants due to the high reactivity of their vinyl ether groups. Supposedly, the vinyl ether group is sacrificed to protect the PUFA in the sn-2 position and preventing lipid peroxidation. However, oxidation of the vinyl ether group produces long-chain aldehydes that can also modify biomolecules. In addition, long-chain aldehydes are much more hydrophobic compared to the sort-chain aldehydes produced by lipid peroxidation, making simultaneous analysis of these lipid aldehydes difficult. Objectives: In the present study, we developed a sensitive and specific method for the simultaneous detection of lipid aldehydes that supported the antioxidant action of plasmalogens.
Materials and methods: Aldehydes are not easily ionizable groups and may have isomers, such alcohol or ketones, that can lead to a misinterpretation of these species. Then, the derivatization of the aldehydes was carried out with 7-(diethylamino)coumarin-3-carbohydrazide (CHH) a fluorescent derivatization reagent and also an ionization enhancer. We optimized the reaction conditions of aldehydes with CHH. We checked the required excess of CHH, amount of acid for catalysis and reaction time and analyzed by UHPLC-Florescence and UHPLC-ESI-QTOF-MS/MS. Next, we analyzed the photo oxidized bovine brain plasmalogen by mass spectrometry.
Results: All of the derivatized aldehydes gave the corresponding [M+H]+ mass ions, as well as the common product ion with an m/z value of 244.0974 which is assigned to CHH. In the samples of photooxidized plasmalogens, only long-chain aldehydes were found, but no aldehyde from lipid peroxidation, despite the presence of PUFAs in the sn2 position of the glycerol backbone, supporting that plasmalogens are acting as sacrificial antioxidants.
Conclusion: Here we describe a method of rapid analysis of lipid aldehydes derivatized with CHH. Thus, it can be analyzed by UHPLC coupled to a mass or fluorescence detector.

Acknowledgments: This work was supported by FAPESP [CEPID-Redoxoma 2013/07937-8, FAPESP DD 2017/16140-7 to Faria, R.L.], CNPq [Miyamoto, S. 424094/2016-9], Capes and Pró-Reitoria de Pesquisa da USP.

Sicrit® Exploris™ Orbitrap setup: a Smart tool for MS facilities to expend the range of covered applications

Natalia Gasilova, Daniel Ortiz, Laure Menin

1. MSEAP, ISIC-GE-VS, SB, EPFL Valais

The Mass Spectrometry and Elemental Analysis Platform (ISIC-MSEAP, EPFL) is providing analyses for more than 100 laboratories in Switzerland, covering a very broad range of applications from the analysis of small organic molecules to large biomolecules and metals. In addition to the most common ionization techniques (ESI, APCI, APPI, MALDI, EI/CI and ICP), implementation of Cold-Spray ionization (CSI) allowed the analyze of sensitive supramolecular structures. Despite all of this, a gap remained for high-resolution routine analyses of small polar compounds that are normally only ionized by EI. Being equipped with low resolution GC-MS on one hand and high-resolution ESI-APCI/APPI-FTMS instruments on the other hand, investing in a high-resolution EI-MS instrument was not possible. Implementation of the Soft Ionization by Chemical Reaction in Transfer source (Sicrit®, Plasmion) on our Orbitraps provided the ideal solution, being fast, easy and sensitive for both direct introduction or GC-coupling mode. By now, more than 300 users' samples could be analyzed using this technique on our MS platform. Selected examples will be given in this poster.

Combination of native MS and ECD top-down fragmentation on an IMS-QTOF system

Marcus Macht¹, Jerre van der Horst², Osmond Rebello², Valery Voinov³, Joseph Beckmann³

1. MS Vision GmbH, Germany
2. Spectrometry Vision BV/ MS Vision, The Netherlands
3. e-MSion Inc., USA

Collision induced dissociation (CID) fragmentation is based on vibrational activation and will thus intrinsically destroy non-covalent interactions. In comparison, electron capture dissociation (ECD) only provides electronic activation and the dissociation does not necessarily interfere with the noncovalent interactions. Therefore we investigated the fragmentation of the tetrameric non-covalent 100kDa Concanavalin A complex by ECD. This complex is known to bind metals (Ca2+ and Mn2+) as well as glycans. The analysis was performed on a Waters Synapt G2 system modified in the pressure regime to promote native MS analysis (MS Vision) and also equipped with an ExD fragmentation cell from e-MSion between the IMS and the transfer part of the TWave device.

We performed experiments based on collision-induced unfolding in combination with ECD as well as ECD based fragmentation of the intact 100kDa complex with and without added glycans.

The CIU data clearly show the benefit of the pressure modifications, providing clearer data and separation between differentially folded states. In the fragmentation experiments, where CID did no show indication of bound metal ions, under ECD fragmentation Ca2+ as well as Mn2+ adduct ions could be observed. Also, glycan addition changes the ECD fragmentation pattern of the protein chain while the nature of this change is not yet fully understood.

Sensitivity improvement for the detection of steroid hormones and endocannabinoids in keratinized matrices

Clarissa D. Voegel^1,2, Maja E. Keller³, Patrick Graefling¹, Bruno Vogt², Thomas Kraemer³, Pierre Negri⁴, Tina M. Binz¹

1. Center for Forensic Hair Analytics, Zurich Institute of Forensic Medicine, University of Zurich, Zurich, Switzerland
2. Department of Nephrology and Hypertension, Inselspital, Bern University Hospital, University of Bern, Switzerland
3. Department of Forensic Pharmacology and Toxicology, Zurich Institute of Forensic Medicine, University of Zurich, Zurich, Switzerland
4. SCIEX, USA

Steroid hormones and endocannabinoids are important endogenous regulators of the human stress response. For the measurement of chronic stress, hair and nail analysis has been found to be the method of choice as it enables a long-term and retrospective determination of endogenous stress markers. The aim of this work was to compare an LC-MS/MS method for the analysis of endogenous steroids and endocannabinoids from keratinized matrices on two different mass spectrometers (SCIEX QTRAP 6500+ and SCIEX 7500) in order to determine sensitivity improvements.
For this purpose, analyte extraction followed by a SLE-based sample preparation was implemented for hair and nail samples. Surrogate analytes were used for the quantification of five steroid hormones (13C3-cortisone, 13C3-cortisol, 13C3-androstenedione, 13C3-testosterone, 13C3-progesterone) and four endocannabinoids (anandamide-D4, 2-arachidonylglycerol-D5, oleoylethanolamide-D4, palmitoylethanolamide-D4) in the sub pg/mg range. Peak areas and signal-to-noise ratios (S/N) of the calibrators were compared between the SCIEX QTRAP 6500+ and SCIEX 7500. Average peak area gains ranged from 9.9x to 71x and 3.7x to 42x for compounds extracted from hair and nails, respectively. The average S/N ratio gains ranged from 0.62x to 13.8x and 0.68x to 19.0x for compounds extracted from hair and nails, respectively.
The results demonstrate that the use of the Sciex 7500 system provided significant increases in peak area and S/N ratios for all steroid hormones and endocannabinoids in the hair and nail matrix. This system has the ability to routinely and robustly detect very low levels of analytes extracted from challenging biological matrices. The presented workflow provides the sensitivity levels required for the long-term retrospective measurement of endogenous biomarkers in keratinized matrix.
 

Operating regular LC in microflow mode to enhance sensitivity and metabolome coverage

Sergey Girel^1,2, Víctor González-Ruiz^1,2,3, Serge Rudaz^1,2,3

1. School of Pharmaceutical Sciences, University of Geneva, Rue Michel-Servet 1, Geneva, Switzerland
2. Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, Rue Michel-Servet 1, Geneva, Switzerland
3. Swiss Centre for Applied Human Toxicology (SCAHT), Switzerland

Exploratory non-targeted studies require the benefits of high-resolution mass spectrometry (HRMS) to pinpoint chemical entities behind biological interplay. On the other hand, metabolite analysis at very low concentrations could be mainly accomplished by using triple quadrupole detectors. The choice of capillary columns enabling micro-/nano-flow regimes is a rather common alternative to provide increased ion yield for less sensitive HRMS detectors. However, the need for dedicated, expensive instrumentation able to steadily handle such working conditions is a limitation in the widespread adoption of this alternative.

In this work, we demonstrate how a regular LC device (i.e. Waters I-Class system) can be easily tuned to operate in the microflow regime (20 µL/min) using a 0.5 mm ID capillary column to achieve more sensitivity and coverage for untargeted analysis of human biofluids. The increased sensitivity afforded by micro-LC is obtained through two main mechanisms. First, the reduced in-column band volume increases metabolite concentration at the column outlet. Second, ESI process is improved thanks to the decreased mobile phase flow, reducing chemical noise from solvents and buffers, and facilitating more efficient desolvation and ionization of the analytes.

Our measurements on two different binary pumps confirmed the possibility of performing microflow gradients at a limit of flow rate of 20 uL/min (down to 0.92 μL/min on channel B at start). Lower delivery rates resulted in detrimental retention time variability. Such low flow regimes required a thorough re-plumbing of the system to minimize delay volumes that would otherwise extend analysis time up to unpractical levels. This strategy allowed to use the 0.5mm ID capillary column and achieve significant sensitivity improvements in untargeted metabolomic analysis. The current setup was applied to steroid and lipid profiling, providing an about 20-fold increase of collected MS/MS spectra in a data-dependent acquisition mode.

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