Book of Abstracts 16. ASAC JunganalytikerInnen Forum 11. & 12. Juni 2021 Salzburg - Virtuell - JAF 2021
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Book of Abstracts
16. ASAC JunganalytikerInnen Forum
11. & 12. Juni 2021
Salzburg – Virtuell
Day 1 Friday June 11th 2021
Targeted LC-MS/MS for the analysis of polyphenol exposure in humans
Ian Oesterle1,2, Dominik Braun1, Annette Rompel2, Benedikt Warth1
1
University of Vienna, Department of Food Chemistry and Toxicology, 1090 Vienna, Austria
2
University of Vienna, Department of Biophysical Chemistry, 1090 Vienna, Austria
ian.oesterle@univie.ac.at
Polyphenols are bioactive phytochemicals known for their beneficial impact on health,
exhibiting antioxidant, antibacterial and anti-inflammatory properties. Therefore, being able to monitor
their presence, both short and long-term, in humans would be beneficial to aid individualized medicine
and influence health outcomes. However, this proves to be a challenge as polyphenols are a highly
complex class of molecules, and they are extensively metabolized in the human body yielding
numerous biotransformation products. Currently, multiple techniques exist for human biomonitoring
(HBM) of polyphenol exposure, yet many of these assays have certain limitations. A major limitation
of these analytical methods is that tend to only look at a few specific polyphenol classes, typically
phenolic acids, and they tend to only monitor a low number of polyphenol metabolites, usually under
40, even though far more polyphenols exist. Another limitation is that some of the main
biotransformation processes that occur in humans are conjugation reactions, such as glucuronidation
and sulfation, yet many of these methods tend to omit conjugated metabolites and instead use
enzymatic deconjugation which presents its own disadvantages. Therefore, the development of a
comprehensive multi-analyte method including various polyphenol classes and their biotransformation
products would be valuable.
Hence, the aim of this work was to develop and validate a targeted LC-MS/MS method for the
analysis of approximately 100 different polyphenol metabolites that represent different polyphenol
classes, including some conjugated metabolites. The extraction of polyphenols was optimized and
validated in-house for several human matrices, including urine, serum, and plasma. Finally, a short
proof-of-principle was performed to analyze the change of the different polyphenols over 24 hours after
the ingestion of a high polyphenol diet.Novel HPLC-MS approaches for intact proteoform characterization of therapeutic
glycoproteins
Fiammetta Di Marco 1, 2, Maximilian Lebede1,2, Thomas Berger1,2, Wolfgang
Esser-Skala1,2,3, Therese Wohlschlager 1, 2, Christian G. Huber1, 2
1 Department of Biosciences, Bioanalytical Research Labs, University of Salzburg,
Hellbrunner Straße 34, 5020 Salzburg, Austria
2 Christian Doppler Laboratory for Innovative Tools for Biosimilar Characterization,
University of Salzburg, Hellbrunner Straße 34, 5020 Salzburg, Austria
³ Department of Biosciences, Computational Systems Biology Group, University of
Salzburg, Hellbrunner Straße 34, 5020 Salzburg, Austria
fiammetta.dimarco@sbg.ac.at
The predominant active substances in the biopharmaceutical formulations are
glycoproteins, e.g. monoclonal antibodies (mAbs), protein hormones, or lysosomal
enzymes. During manufacturing and/or storage, proteins may undergo to a plethora of
different post-translational modifications (PTMs), resulting in the existence of numerous
co-occurring proteoforms. Typical PTMs that may occur are enzymatic glycosylation of
Asn, enzymatic C-terminal Lys clipping, chemical deamidation or glycation of Asn and
oxidation of Met and Trp. Characterization of proteoforms at intact level is crucial to
ensure the safety and efficacy of the drug products.
In the last few years, interest in automated application of different liquid
chromatographic techniques compatible with MS (HPLC-MS) has grown in the field of
intact/native glycoprotein characterization. Among the different approaches,
reversed-phase (RP) HPLC-MS has proved to be useful to separate glycoforms of small
proteins (≤ 25 kDa) while strong anion and strong cation exchange (SAX and SCX)
HPLC-MS have emerged as valuable technique for the separation and identification of
large protein variants (≥ 100 kDa).
Here, we report the use of different HPLC-MS (RP, SCX, SAX) techniques to characterize
three glycoproteins covering different levels of PTM complexity and MW: the
recombinant human chorionic gonadotropin (r-hCG, ≈34 kDa), the mAb Rituximab
(≈150 kDa) and the lysosomal recombinant enzyme human acid α-glucosidase (r-hGAA,
≈110 kDa). Using these novel HPLC-MS approaches, we were able to separate different
types of protein variants and to detect their masses with direct hyphenation to MS.Micro-droplet-based calibration for quantitative elemental bioimaging by LA-ICPMS
Andreas SCHWEIKERT1,2, Sarah THEINER1, Anna SCHOEBERL1, Gunda KOELLENSPERGER1
1
Institute of Analytical Chemistry, Faculty of Chemistry, University of Vienna, Waehringer
Strasse 38, 1090, Vienna, Austria
2
Institute of Inorganic Chemistry, Faculty of Chemistry, University of Vienna, Waehringer
Strasse 42, 1090, Vienna, Austria
andreas.schweikert@univie.ac.at
A novel standardization strategy for quantitative elemental bioimaging by laser ablation (LA)
ICP-TOFMS will be presented.[1] Gelatine micro-droplet standards are produced by spotting
pL volumes of gelatine solutions spiked with elemental standards onto glass slides. The small
dimensions of these droplets and the use of a low-dispersion laser ablation ICP-TOFMS
setup allows measuring a droplet in only a few minutes. Therefore, as a key advance, high-
throughput analysis is enabled to establish imaging measurement sequences with quality
control- and standardization samples comparable to solution-based quantification exercises
by ICP-MS.
This is necessary because no commercial standards are available for bioimaging applications
and standardisation methods published so far are either very sample specific, limited to a
small number of analytes, often only one point calibrations, laborious to produce and/or
optimised for quadrupole based ICP instruments.[2,3]
As a proof-of-concept application, the tool-set was employed to investigate the
accumulation of metal-based anticancer drugs in multicellular tumor spheroid models at
clinically relevant concentrations.
[1] A. Schweikert, S. Theiner, D. Wernitznig, A. Schoeberl, M. Schaier, S. Neumayer, B.K. Keppler, G.
Koellensperger, Micro-droplet-based calibration for quantitative elemental bioimaging by LA-
ICPMS, Anal Bioanal Chem, (2021).
[2] M.T. Westerhausen, T.E. Lockwood, R.G. de Vega, A. Röhnelt, D.P. Bishop, N. Cole, P.A. Doble, D.
Clases, Low background mould-prepared gelatine standards for reproducible quantification in
elemental bio-imaging, Analyst, 144 (2019) 6881 6888.
[3] M. ala, V.S. elih, J.T. van Elteren, Gelatin gels as multi-element calibration standards in LA-ICP-
MS bioimaging: fabrication of homogeneous standards and microhomogeneity testing, The
Analyst, 142 (2017) 3356 3359.Exploring the Chemical Space of Protein Glycosylation in Noncovalent Protein
Complexes: An Expedition Along Different Structural Levels of Human Chorionic
Gonadotropin Employing Mass Spectrometry
Maximilian Lebede1,2, Fiammetta Di Marco1,2, Wolfgang Esser-Skala1,2,3, René Hennig4,5,
Therese Wohlschlager1,2, Christian G. Huber1,2
1Department of Biosciences, Bioanalytical Research Labs, University of Salzburg
2Christian
Doppler Laboratory for Innovative Tools for Biosimilar Characterization,
University of Salzburg
³Department of Biosciences, Computational Systems Biology Group, University of
Salzburg
4glyXera
GmbH
5Max Planck Institute for Dynamics of Complex Technical Systems
Maximilian.lebede@sbg.ac.at
Protein glycosylation is a major obstacle in bio-analytical structure elucidation. Here, we
combined high resolution mass spectrometry (MS) with subsequent bio-informatics to
assess a plethora of co-existing glycoforms present in recombinant human chorionic
gonadotropin (hCG, Ovitrelle®). Combination of established techniques, such as released
glycan or glycopeptide analysis and novel approaches featuring HPLC-MS for the
characterization of hCG subunits, as well as native MS for in-depth analysis of the heavily
glycosylated hCG heterodimer allowed us to take protein characterization to the next
level. Systematic transfer of data across structural levels, allowed us to gain insight into
the complexity hiding behind roughly 50 signals observed in the deconvoluted mass
spectrum of dimeric hCG. Despite this rather limited amount of signals an astonishing
number of over 1000 glycoforms was uncovered to constitute the chemical space of the
hCG dimer.A sensitive and fast multi steroid LC-MS/MS assay for the routine clinical use with
a one-step sample preparation: comparison to immunological methods
Valentin BRAUN1,2, Christian TIMM2, Martin RISCH2,3, Christoph SEGER1,2
1Universitiät Innsbruck, Austria, 2Dr. Risch Ostschweiz AG, Buchs SG, Switzerland,
³Kantonsspital Chur, Chur GR, Switzerland
valentin.braun@student.uibk.ac.at
Steroid analysis in clinical laboratories is dominated by immunoassays (IA) that have a high
sample turnover but are inherently limited in trueness, precision and sensitivity. ID-LC-
MS/MS has proved as far more capable tool delivering better sensitivity, specificity and the
possibility of parallel measurements of multiple steroids and their metabolites providing
the endocrinologist with more reliable and comprehensive diagnosis information.
An in-house developed LC-MS/MS assay (Sciex 6500+ MS coupled to an Agilent 1290 Infinity
II UHPLC, Restek Biphenyl column (2.7 µm, 2.1x100 mm), gradient elution (50 to 94% MeOH
in H2O with 0.2 mM NH4F, 7.8 min), allowing quantification of cortisol, cortisone,
testosterone, androstenedione, 17-OH-progesterone, DHEA, DHEAS, progesterone,
estradiol, corticosterone, 11- and 21-deoxycortisol and 11-deoxycorticosterone in human
serum. One-step sample preparation combined protein precipitation (100 µL sample,
200 µL ZnSO4 62 mM) with phospholipid removal using HybridSPE® filter cartridges to
reduce coeluting phospholipids. Inter-day precision was better than 10 % and bias less than
8 % in the clinical range of interest. LLOQ meeting biological reference ranges were set to
0.1 nM for all analytes except E2 (15 pM), cortisone (1 nM), DHEA (5 nM), cortisol (15 nM)
and DHEAS (140 nM). Trueness was proven by recovery experiments of ISO17034 reference
materials (Cerriliant), proficiency testing (UKNEQAS, 14 rounds) and measuring higher order
serum reference standards (ERM, BCR). Comparison against IVD-CE certified IAs in routine
(Roche, Siemens, IDS) showed that none of these assays were meeting the standards of LC-
MS/MS. Insufficient overall comparability was found for androstenedione (-66 % bias) and
17-OH progesterone (-80 % bias). Strong accuracy limitations at lower concentrations were
present for cortisol, progesterone, estradiol, DHEAS and testosterone. Especially if
laboratory service providers are changed, the observed bias based on metabolite cross-
reactivities might impact patient care. If low analyte levels are the biological norm (e.g. sex
hormones in children or elderly patients), clinical decision making might be compromised
by the limited reproducibility of IA results.
We have demonstrated that a reliable, sensitive, and robust multi-steroid method is
possible without the need for extensive multi-step sample preparation, complex 2D-
chromatography, or on-line SPE systems. Although steroid analysis by LC-MS/MS is still a
challenging task, especially when clinically relevant sample throughput is desired, the poor
assay performance of IAs makes it inevitable to address this task.Influence of extraction parameters in high throughput tissue extraction for
lipidomics
Thomas BÖGL1, Franz MLYNEK1, Markus HIMMELSBACH1, Wolfgang BUCHBERGER1
1Institute of Analytical Chemistry, Johannes Kepler University, 4040 Linz, Austria 2
thomas.boegl@jku.at
Lipidomics extraction protocols are manifold due to the lack of standardization. Although
there are initiatives trying to handle this issue, the high interest and fast development in
this field leads towards a high variety of preparation procedures [1]. Autoxidation and
hydrolysis are the most significant problems during the sample pretreatment. The high
number of measurements that come along with lipidomics studies demands therefore a
simple but extensive method. One-phase extractions have gained a high popularity because
of the time efficiency and easier sample handling. However, established extraction methods
typically include an evaporation step of collected extracts. Subsequently, lipids are
re-suspended in an appropriate solvent for further analysis. The composition of the used
suspension solvent is rarely discussed in literature. The purpose of our study is to figure out
if especially complex matrices of tissues influence not only the efficiency of the extraction,
but also the ability of the re-suspending of extracted lipids. Our investigation compared the
well-established Folch method with a popular one-phase extraction usually used for bio-
fluids, called MMC-method (Methanol-MTBE-Chloroform). For each extraction method four
different suspension solutions were used in a total of four different porcine tissues (brain,
liver, muscle, white adipose tissue). This enabled us to compare resuspension and
extraction efficiency in tissues with a different composition and lipid content. Our results
indicate a dependency upon used re-suspension solution, particularly in high fat content
tissues like white adipose tissue.
1. Liebisch G. et al., Lipidomics needs more standardization. Nature Metabolism,
2019. 1(8): p 745–747.Mass spectrometry-based lipid quantification by reversed phase chromatography utilizing a
counter gradient
Felina Hildebrand 1, Harald Schoeny1, Evelyn Rampler 1,2,3, Gunda Koellensperger 1,2,3
1
Department of Analytical Chemistry, Faculty of Chemistry, University of Vienna, Waehringer Str. 38, 1090
Vienna, Austria; 2 Vienna Metabolomics Center (VIME), University of Vienna, Althanstr. 14, 1090 Vienna,
Austria; ³ Chemistry meets Biology, University of Vienna, Althanstr. 14, 1090 Vienna, Austria
felina.hildebrand@univie.ac.at
Reversed phase liquid chromatography (RPLC) is the most common separation technique used for
mass spectrometry (MS)-based lipidomics. The major advantage of this method is the separation of
lipids on their species level, which reduces the co-elution of lipids and leads to a high selectivity
and sensitivity. However, lipid quantification using RPLC is challenging, when using a small number
of internal standards (ISTDs), because analyte lipids are separated from their corresponding ISTD.
When analyte and ISTD do not co-elute/-ionize, both are affected by different matrix effects during
the ionization process and the assumption, that all lipids within a polar lipid class have the same
ionization efficiency is not valid.
To overcome different solvent effects in RPLC-MS-based lipid quantification we suggest to utilize a
counter gradient, which is applied parallel to the gradient elution with a second UHPLC pump. The
flow of both gradient and counter gradient is combined before ionization leading to constant solvent
composition during the ionization for the whole gradient elution. Because two flows are combined,
dilution effects can be expected. Therefore, the RPLC was downscaled to a C18 column with an inner
diameter of 1 mm. Previous experiments with flow injection analysis confirmed that the ionization
efficiency of lipids is dependent on the solvent composition. The RPLC-MS setup utilizing the
counter gradient was used to quantify lipids in the standard reference material (SRM) 1950 (NIST,
USA), which is a pooled human plasma. Lipids were quantified either with a small number of ISTDs
with a known concentration (EquiSPLASH® Mix, Avanti Polar Lipids, USA) or by external calibration
normalized to 13C labeled lipids of LILY (lipidome isotope labeling of yeast) 1. The accuracy of both
quantification approaches was assessed by comparing absolute lipid concentrations to consensus
values of SRM 1950 2.
1. Schoeny, H. et al. A combined flow injection/reversed-phase chromatography–high-resolution
13
mass spectrometry workflow for accurate absolute lipid quantification with C internal
standards. The Analyst 146, 2591–2599 (2021).
2. Bowden, J. A. et al. Harmonizing lipidomics: NIST interlaboratory comparison exercise for
lipidomics using SRM 1950–Metabolites in Frozen Human Plasma. J. Lipid Res. 58, 2275–2288
(2017).Detailed Analysis of Parchment Degradation by ATR-FTIR and Raman Spectroscopy
Antonia MALISSA1,2, Federica CAPPA2, Manfred SCHREINER2, Martina MARCHETTI-DESCHMANN1
1
Institute of Chemical Technologies and Analytics, TU Wien, Getreidemarkt 9, 1060 Vienna, Austria
2
Institute of Natural Sciences and Technology in the Arts, Academy of Fine Arts Vienna,
Schillerplatz 3, 1010 Vienna, Austria
antonia.malissa@tuwien.ac.at
Parchment has been used as support material for manuscripts, scrolls and book covers since
Late Antiquity. Unlike papyrus or paper, it is obtained from animal skins primarily of sheep,
calves, and goats, basing its structure mainly on the fibrous protein collagen. Detailed
knowledge of the degradation mechanisms and the chemical modifications of collagen
induced by the environment are therefore essential for the appropriate conservation and
preservation of such precious objects because UV light, moisture, or atmospheric pollutants
can induce irreversible collagen modifications.
With this aim, three sets of samples, obtained from six different sheep parchments, were
subjects of artificial ageing with UV radiation at 155 W/m2, three different relative humidity
levels ranging from 30 to 80 %RH, and sulfur dioxide (SO2). The latter was furthermore tested
in combination at different %RH. Analysis was performed using a multi-spectroscopic
approach employing micro-invasive Fourier-transform infrared spectroscopy in attenuated
total reflection mode (ATR-FTIR) and Raman spectroscopy. Results were studied in detail
utilizing the second derivative of the FTIR and Raman spectra, followed by band fitting of the
amide I, II and III bands as well as of the amide A and B bands. Results showed that the
triple-helical structure of collagen changed which was mainly deduced from the amide I and
III bands in the FTIR and Raman spectra as well as by the amide II band in the FTIR spectra.
Lipid and water content changes were observed in the spectral region of 3600-2800 cm-1 in
ATR-FTIR spectra. [1] Additionally, information about amino acid changes, e. g. for proline
and hydroxyproline, was gained by the analysis of the fingerprint region between
1100-200 cm-1 in the Raman spectra. [2] A first correlation of the conformational changes of
collagen with different environmental conditions showed that the exposure to UV light and
SO2 has the greatest impact on collagen s secondary structures, inducing significant
reductions of ordered structures (e. g. α-helices) and an increase in unordered structures.
1. Olsztyńska-Janus, S., et al., ATR-IR study of skin components: Lipids, proteins and
water. Part I: Temperature effect. Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy, 2017. 188: p. 37-49.
2. Martinez, M. G., et al., Characterisation of structural changes in collagen with Raman
spectroscopy. Applied Spectroscopy Reviews, 2009. 54(6): p. 509-542.Monitoring mycotoxin-mixtures in infant stool by LC-MS/MS
Magdaléna Krausová1 , Dominik Braun1 , Lukas Wisgrill2 , Benedikt Warth 1
1 University of Vienna, Faculty of Chemistry, Department of Food Chemistry and
Toxicology, Währinger Straße 38, 1090 Vienna, Austria.
2 Division of Neonatology, Pediatric Intensive Care and Neuropediatrics,
Comprehensive Center for Pediatrics, Department of Pediatrics and Adolescent
Medicine, Medical University of Vienna, 1090 Vienna, Austria.
magdalena.krausova@univie.ac.at
Infants, especially those born prematurely, belong to a highly susceptible population.
Compared to the adults they differ in metabolic activities and have lower tolerance for
toxic exposures due to low body weight. Breastmilk, infant formula or complementary
food represent the major nutritional source. However, these foodstuffs might contain
natural contaminants such as mycotoxins. Exposure to mycotoxins is a worldwide issue,
but occurrence data on mycotoxin-mixtures in human stool is lacking. An important tool
for measuring the internal exposure to xenobiotics and contaminants including
mycotoxins, is human biomonitoring. To date, analytical methods for human matrices
have mainly focused on single exposures or classes of mycotoxins.
Here, we present a first targeted multi-mycotoxin LC-MS/MS method for term and
preterm infant stool. Using a quick and easy sample clean-up method involving dilution
followed by filtration, we have developed a highly sensitive LC-MS/MS method that
covers more than 20 mycotoxins including some of their phase I metabolites. Recoveries
of the infant stool samples, fortified in a concentration range of 0.075 – 675 µg/g dried
stool, were in the range of 70 – 112% with standard deviations below 20% for most
analytes. The filtration step worked for the majority of analytes, such as aflatoxins,
ochratoxins, zearalenone and its metabolites. The method was not effective for extracting
enniatins and beauvericin. Limits of detection using matrix-matched calibration curves
were in range of 0.03 – 7.4 ng/g dried stool. Pilot experiments showed no mycotoxin
contamination in Austrian premature infant stool (n=12) using the new LC-MS/MS
method. In addition to the sample preparation protocol, the optimization of the
LC-MS/MS method and data from the in-house validation will be presented.Establishing antibody labelling procedures for the imaging of in vivo samples using
LA-ICP-TOF-MS
Martin Schaier1, Sarah Theiner1, Andreas Schweikert1, Gunda Koellensperger1
1Institute of Analytical Chemistry, University of Vienna, Waehringer Strasse 38, 1090 Vienna,
Austria
martin.schaier@unive.ac.at
Studies on the uptake of metal-based anticancer drugs within different cell types in the
complex tumour microenvironment are still fragmentary to this date. The interaction with
specific cell types is of particular interest, as studies have shown significant dependence of Pt
uptake of metallodrugs on the proliferation state of the tumour. [1] Therefore, state-of-the-
art analytical methods are required to observe the accumulation of these compounds on
single cell level in tumour tissue. Recent advances in laser ablation and ICP-TOF-MS technology
made the simultaneous imaging of a wide range of elements (m/z = 14-254) with a resolution
(down to 1 µm), where single cells are visible, possible. In addition to the measurement of
physiologically relevant elements (e.g., Fe, Cu, Zn), metal-conjugated antibodies and
metallodrugs can be measured. In contrast to immunofluorescence, the number of usable
antibodies is not as limited, as there are no spectral overlaps. Moreover, using micro-droplet-
based calibration for LA-ICP-TOF-MS imaging enables multi-element quantification. [2] The
aim of this study was to establish antibody labelling procedures for measurements with LA-
ICP-TOF-MS, with a focus on in vivo samples. In particular, the method was optimized for the
labelling of cryosections and FFPE tissues. For this purpose, a total of 5 metal conjugated
antibodies (αSMA, CD3, CD11b, CD44, KI67) were tested on tumour sections of mice, which
were treated with anticancer drugs.
1. Theiner, S., et al., Fast High-Resolution Laser Ablation-Inductively Coupled Plasma Mass
Spectrometry Imaging of the Distribution of Platinum-Based Anticancer Compounds in
Multicellular Tumor Spheroids. Anal Chem, 2017, 89(23): 12641-12645.
2. Schweikert, A., et al., Micro-droplet-based calibration for quantitative elemental
bioimaging by LA-ICPMS. Anal Bioanal Chem, 2021.Analysing Poly- and Perfluorinated Alkyl Substances in
Paper-based Food Contact Materials
Milica JOVANOVIC1, Erich LEITNER1
1Technical University of Graz; Institute for Analytical Chemistry and Food Chemistry, Austria
milica.jovanovic@tugraz.at
Poly- and perfluorinated alkyl substances are attractive due to their stability and water/lipid
repellent properties for various applications, including in paper and board food contact
materials (FCMs). However, these substances have been found to be very persistent, bio
accumulative and toxic.
Recently some PFASs have begun being regulated or phased out. [1] Only few of the
substances have been risk assessed by the European Food Safety Authority (EFSA). From July
1st, 2020, in Denmark it is prohibited to place on the market paper and board FCMs in which
PFASs have been used. Usually PFAS are not applied in products from virgin fibre, but they
could potentially be found in recycled paper. Therefore, there is a need to quantify PFAS levels
in paper and board food contact materials (FCM) based on (partially) recycled material.
In this work, we present an overview of different analytical methods to analyse PFAS.
Additionally, we demonstrate our approach for targeted analysis of 24 PFAS, which can occur
in paper and board FCM. Our work consists of fortifying and extracting PFAS from paper,
followed by quantification with high-performance liquid chromatography coupled with triple
quadrupole mass spectrometry.
The progress that we present here is leading to an establishment of simple and efficient
method for quantification of PFAS that could be used for quick and easy monitoring of PFAS
in paper based FCM that are currently available in the market.
[1] Trier, D. X., Taxvig, C., Rosenmai, A. K., Pedersen, G. A., PFAS in Paper and Board for
Food Contact: Options for Risk Management of Poly- and Perfluorinated Substances,
Nordic Council of Ministers, Copenhagen K 2018.Determination of the amavadin concentration in different parts of Amanita muscaria
using HPLC-ICPMS
Martin WALENTA1, Simone BRAEUER1, Walter GOESSLER1
1Institute of Chemistry – Analytical Chemistry, University of Graz
Universitätsplatz 1/I, 8010 Graz, Austria
Amanita muscaria, also known as fly agaric, is probably one of the most iconic mushrooms in the
world. With its red cap and white dots, which are typical for the species mainly found in the forests
of Eurasia, it even made it into the gaming industry. From a chemical view this mushroom is quite
interesting for its ability to accumulate vanadium (V). It can easily take up more than 100 mg V/kg dry
mass (dm), whereas other mushrooms typically contain less than 0.5 mg V/kg dm. [1] The main V
species in the fruit body of this mushroom was identified around half a century ago as a complex
called amavadin [2]. But until now no method that allows a systematic examination of this V-
compound and confirm the hypotheses that amavadin is the only V species present in Amanita
muscaria is available.
Hence, we developed a new element-selective method by utilizing high performance liquid
chromatography (HPLC) coupled to inductively coupled mass spectrometry (ICPMS). The
chromatography was done using a strong anion-exchange column in combination with an aqueous
mobile phase containing an ammonium citrate buffer and ethylenediaminetetraacetate (EDTA). [3]
4000
3200
CPS (m/z = 51)
2400
1600
?
800 ?
0
0 2 4 6 8 10 12 14 16 18 20
Time [min]
Figure 1 HPLC-ICPMS chromatogram of aqueous extract of an Amanita muscaria gill sample.
After optimizing the method, the extraction efficiency of fruit-body samples was 74 ± 12 % and the
two isomers of amavadin eluted in less than 17 minutes. Surprisingly, other vanadium species than
amavadin were also detected at significant concentrations (Figure 1). Our results demonstrate that
the V-speciation in Amanita muscaria is more complex than thought until now. Further investigation
of the V uptake, metabolism and distribution of vanadium is required to elucidate the accumulation
and role of vanadium in this mushroom.
1. Řanda, Z. and Kučera, J., Trace elements in higher fungi (mushrooms) determined by
activation analysis. J. Radioanal. Nuclear Chem., 2004. 259: p.99–107.
2. Bayer, E. and Kneifel, H., Isolation of amavadin, a vanadium compound occuring in Amanita
muscaria. Z. Naturforsch. B, 1972. 27: p.207.
3. Braeuer, S., et al., Determination of the naturally occurring vanadium-complex amavadin in
Amanita muscaria with HPLC-ICPMS. J. Anal. At. Spectrom., 2021. Advance ArticleMicro-droplet-based calibration for quantitative elemental bioimaging by LA-ICPMS
Andreas SCHWEIKERT1,2, Sarah THEINER1, Anna SCHOEBERL1, Gunda KOELLENSPERGER1
1
Institute of Analytical Chemistry, Faculty of Chemistry, University of Vienna, Waehringer
Strasse 38, 1090, Vienna, Austria
2
Institute of Inorganic Chemistry, Faculty of Chemistry, University of Vienna, Waehringer
Strasse 42, 1090, Vienna, Austria
andreas.schweikert@univie.ac.at
A novel standardization strategy for quantitative elemental bioimaging by laser ablation (LA)
ICP-TOFMS will be presented.[1] Gelatine micro-droplet standards are produced by spotting
pL volumes of gelatine solutions spiked with elemental standards onto glass slides. The small
dimensions of these droplets and the use of a low-dispersion laser ablation ICP-TOFMS
setup allows measuring a droplet in only a few minutes. Therefore, as a key advance, high-
throughput analysis is enabled to establish imaging measurement sequences with quality
control- and standardization samples comparable to solution-based quantification exercises
by ICP-MS.
This is necessary because no commercial standards are available for bioimaging applications
and standardisation methods published so far are either very sample specific, limited to a
small number of analytes, often only one point calibrations, laborious to produce and/or
optimised for quadrupole based ICP instruments.[2,3]
As a proof-of-concept application, the tool-set was employed to investigate the
accumulation of metal-based anticancer drugs in multicellular tumor spheroid models at
clinically relevant concentrations.
[1] A. Schweikert, S. Theiner, D. Wernitznig, A. Schoeberl, M. Schaier, S. Neumayer, B.K. Keppler, G.
Koellensperger, Micro-droplet-based calibration for quantitative elemental bioimaging by LA-
ICPMS, Anal Bioanal Chem, (2021).
[2] M.T. Westerhausen, T.E. Lockwood, R.G. de Vega, A. Röhnelt, D.P. Bishop, N. Cole, P.A. Doble, D.
Clases, Low background mould-prepared gelatine standards for reproducible quantification in
elemental bio-imaging, Analyst, 144 (2019) 6881 6888.
[3] M. ala, V.S. elih, J.T. van Elteren, Gelatin gels as multi-element calibration standards in LA-ICP-
MS bioimaging: fabrication of homogeneous standards and microhomogeneity testing, The
Analyst, 142 (2017) 3356 3359.Silica nanoparticles as plastic antibodies for Human Serum Albumin recognition
produced by an integration of solid phase synthesis and Ostwald Ripening
Wisnu Arfian A. SUDJARWO 1, Soad S. ALZAHRANI1, Julia VÖLKLE1,2, Nathalie RUNGE1,
Peter A. LIEBERZEIT 1
1Universität Wien, Institut für Physikalische Chemie, Währinger Straße 42, 1090 Wien,
Austria
2CEST Centre for Electrochemistry and Surface Technology, Victor-Kaplan-Strasse 2, 2700
Wiener Neustadt, Austria
wisnu.arfian.anditya.sudjarwo@univie.ac.at; peter.lieberzeit@univie.ac.at
First introduced by Piletsky group[1][2], the combination of solid phase imprinting and
Ostwald Ripening succeeded to generate imprinted silica nanoparticles for several small
molecules. Herein, we describe how this synthesis approach leads to imprinted silica
nanoparticles (Si-MIP) for Human Serum Albumin (HSA) recognition. Starting by
anchoring HSA on silica surface, silica nanoparticles deposited around those template
molecules followed by restructuring the diameters of silica particles via Ostwald Ripening
in phosphate buffer. Eluting silica particles with warm water resulted in particles
comprising specific cavities with high affinity to HSA. DLS and SEM revealed the size of
120 nm, 242 nm, 296 nm, 157 nm and 196 nm for raw silica nanoparticles (Si-NIP), Si-MIP
3h, Si-MIP 5h, CS Si-MIP 3h and CS Si-MIP 5h, respectively. We examined the binding
properties of every silica MIP using fluorescence spectroscopy. All imprinting silica
nanoparticles capture HSA sensitively; among those CS Si-MIP 3h (slope of the
Stern-Volmer plot at k=0.0036) showed highest binding compared to Si-NIP (k=0.0001).
Imprinted silica nanoparticles also bind selectively toward HAS: binding affinity is 4 times
higher, than for lysozyme, and 2.8 times higher, than for trypsin. Direct sensor
measurement on QCM revealed highest frequency shifts for Si-MIPs 5h after the injection
of 250 ppm with -290 Hz while Si-NIP had only -15 Hz. This study demonstrates that the
method is feasible for assay development toward either small molecules or big molecules.
The difference between fluorescence and QCM data most probably results from the
different sizes of the two Si MIP particles.
1. Piletska E, Yawer H, Canfarotta F, Moczko E, Smolinska-Kempisty K, Piletsky SS,
Guerreiro A, Whitcombe MJ, Piletsky SA (2017) Biomimetic Silica Nanoparticles
Prepared by a Combination of Solid-Phase Imprinting and Ostwald Ripening. Sci Rep
7: . https://doi.org/10.1038/s41598-017-12007-0
2. Canfarotta F, Poma A, Guerreiro A, Piletsky S (2016) Solid-phase synthesis of
molecularly imprinted nanoparticles. Nat Protoc 11:443–455 .
https://doi.org/10.1038/nprot.2016.030Quantification of minor abundant glycosylation variants of monoclonal
antibodies
Katharina BÖTTINGER1, Christian G. HUBER1
1 University of Salzburg, Department of Biosciences, Bioanalytical Research Labs,
Hellbrunnerstrasse 34, 5020 Salzburg, Austria
katharina.boettinger@sbg.ac.at
N-glycosylation variants affect folding and thus the functionality and mechanisms of
action of monoclonal antibodies (mAbs). Despite only making up to 5% of the mass of a
mAb, glycosylation is responsible for the major part of a mAb’s heterogeneity. Due to the
nature of manufacturing, cells do not secrete one single product, but rather a mAb-pool
with different glycovariants of which a small fraction is afucosylated 1. Afucosylated
species have antibody-dependent cellular cytotoxicity (ADCC)-promoting effects:
increasing afucosylation levels by 10% resulted in 1.89-fold increase in ADCC, increasing it
to 62% already resulted in 5.93-fold ADCC-effect2. Therefore, minor abundant
glycosylation variants like afucosylation need to be accurately determined. N-glycans are
frequently analyzed at the released glycan or peptide level. However, the combination of
glycosylation pairs is lost using these approaches and artificial protein modifications
might be introduced. Thus, a thorough characterization at the intact or subunit level
would allow for paired-glycan analysis.
Here, we aim to quantify minor abundant glycosylation variants – specifically
afucosylated proteoforms – of a mAb at the intact protein level supported by subunit
analysis using high-performance liquid chromatography hyphenated to mass
spectrometry. For analysis, we chose trastuzumab as a model mAb as it shows a rather
high relative abundance of afucosylation of approximately 7%. Upon enzymatic dissection,
we sequentially simplified the glycan structures and antibody complexity. For this
purpose, we employed Endo S, a glycosidase eliminating the glycoform heterogeneity
leaving a mAb with and without core fucosylation. IgdE cleaves the mAb above the hinge
region keeping the Fc domain intact, thus allowing determination of glycan pairing.
Another approach employed was to enzymatically remove fucose to obtain a completely
afucosylated mAb. These approaches and combinations of them should allow for accurate
quantification of afucosylation.
1. Beck, A., Wagner-Rousset, E., Ayoub, D., Van Dorsselaer, A. & Sanglier-Cianférani, S.
Characterization of Therapeutic Antibodies and Related Products. Anal. Chem. 85,
715–736 (2013).
2. Thomann, M., Reckermann, K., Reusch, D., Prasser, J. & Tejada, M. L.
Fc-galactosylation modulates antibody-dependent cellular cytotoxicity of
therapeutic antibodies. Mol. Immunol. 73, 69–75 (2016).Mental health challenges in the PhD research community –
from awareness to action
Dr. Silvia Wehmeier
University of Graz
silvia.wehmeier@uni-graz.at
In academia mental health issues have come to the foreground, and the literature
surrounding student mental health continues to grow, as well as universities try to
implement services for the mental health of their students. Although, there is still a
growing concern regarding the increase of the mental health problems among PhD
students worldwide.
Mental health affects everyone and chemists are no exception, still it can be a difficult
subject to discuss. Efforts have been made to understand mental health in the research
environment and an exploration of the existing evidence base found that mental health
problems in PhD students are associated with high levels of work demands, work-life
conflict and that poor wellbeing can impact the productivity of researchers.[1] One study
reported that studying for a PhD can come at a cost, because PhD students were more
than twice as likely to have mental-health difficulties than the general highly educated
population.[2] Studies vary greatly in their evidence base, though recently, it has become
more common to focus on raising the awareness of mental health in the research
community. This talk will focus on: How can we start to cultivate awareness about mental
health in our research community? And, how can we detect stress sources, and cope with
uncertainty and anxiety?
1. Guthrie, S., et al., Understanding mental health in the research environment: A Rapid Evidence
Assessment. Santa Monica, CA: RAND Corporation, 2017.
2. Levecque, K., et al., Work organization and mental health problems in PhD students. Research Policy,
2017. 46(4): p. 868-879.Day 2 Saturday June 12th 2021
A KNIME workflow for processing data from targeted lipidomics approaches
Nina TROPPMAIR1, Robert AHRENDS1
1 Department of Analytical Chemistry, University of Vienna, Währinger Strasse 38,
1090 Vienna, Austria.
nina.troppmair@univie.ac.at
Recent advances in the field of mass spectrometry enable high-throughput analyses of
numerous lipids. However, lipidomics approaches further require reliable procedures for
data processing. The large amount of data obtained from targeted lipidomics experiments,
makes the use of software tools inevitable. The Konstanz Information Miner (KNIME) [1]
is a free and open-source environment, allowing the visual assembly and possibly
stepwise execution of a data pipeline. The herein described KNIME workflow supports
the comprehensive liquid chromatography-mass spectrometry-based analysis of lipids in
biological samples, calculating the lipid concentration in the samples and visualizing the
identified lipids based on their building blocks. As main input the workflow requires
results from signal integration. Depending on the data set, the biological and technical
replicates, the cell number or protein concentration for normalization, as well as the
amount of the internal standard may be entered. As quantitative analyses of lipids by
mass spectrometry necessitates the use of structurally similar internal standards to
compensate for variations in the extraction and differences in the analytical responses of
the analytes of interest, multiple internal standards are required for the analysis of a
wide range of lipids. Provided by the user, the respective internal standards are assigned
to the endogenous lipids using the KNIME workflow. If species, not following the
structure-retention time trends, should be excluded from the final results, the expected
retention times need to be defined as well. The outputs include several tables as well as
graphs, which are created using R [2] with the package ggplot2 [3]. Applying this
automated data processing approach reduces the burden of work, not requiring any
additional tasks once the input files are created. The KNIME workflow has proven suitable
for processing data from targeted sphingolipid analyses in biological samples, comparing
the quantitation results to the manually obtained outcome of the same data set.
1. Berthold, M.R., et al., KNIME: The Konstanz Information Miner. 2008, Springer
Berlin Heidelberg: Berlin, Heidelberg. p. 319-326.
2. R Core Team, R: A language and environment for statistical computing. 2021, R
Foundation for Statistical Computing: Vienna, Austria.
3. Wickham, H., ggplot2: Elegant Graphics for Data Analysis. 2016: Springer-Verlag
New York.Non-targeted mass spectrometry for elucidating the exposome and its impact on
the endogenous metabolome
Mira Flasch1, Veronika Fitz2, Evelyn Rampler2, Gunda Koellensperger2, Benedikt Warth1
1University of Vienna, Department of Food Chemistry and Toxicology, 1090 Vienna,
Austria
2University of Vienna, Department of Analytical Chemistry, 1090 Vienna, Austria
mira.flasch@univie.ac.at
Exposure to xenobiotics through the environment, food, and consumer goods are
ubiquitous and diverse. The totality of all these non-genetic exposures from conception
onwards is defined as exposome, but broader definitions also encompass associated
biological responses. Liquid chromatography-high resolution mass spectrometry
(LC-HRMS) enables the simultaneous analysis of external stressors (xenobiotics) and
internal responses (endogenous metabolites) to these chemicals. Yet, the chemical
diversity and the wide concentration range in human samples complicated the
simultaneous measurement of exposures and effects in the past. Furthermore, highest
sensitivity is required to detect and quantify low abundance environmental
contaminations. Human biomonitoring typically utilize tailored sample preparation and
targeted LC-MS/MS methods for this purpose. However, only a limited number of
analytes is covered and, unlike in non-targeted approaches, screening for emerging and
unexpected chemicals is not feasible.
We present first steps toward a comprehensive high throughput LC-HRMS workflow to
analyze endogenous metabolites and exogenous environmental exposures simultaneously
in urine. To demonstrate the capability of this novel approach, the reproducibility,
linearity and limit of detection of about 100 endogenous metabolites and 100 xenobiotics
were evaluated. Moreover, we compared the limit of detection (LOD) of the xenobiotics
between an established targeted (LC-MS/MS) and a non-targeted approach (LC-HRMS)
using the same LC-setup to examine the applicability of non-targeted screening for the
discovery of environmental contaminants. A Vanquish (Duo) UHPLC system equipped
with a reverse-phase (RP) column and a hydrophilic interaction liquid chromatography
(HILIC) column running with two independent pump systems was coupled to a Q Exactive
HF Quadrupole-Orbitrap mass spectrometer for non-targeted measurements and a QTrap
6500+ combined with an Agilent 1290 Infinity II system was applied for the targeted assay.
For the LOD comparison of xenobiotics the RP column was used exclusively. The
preliminary data evaluation revealed promising results for the dual measurement of
metabolites and environmental contaminants. We could further estimate an on average
approximately ten-times lower LOD for the targeted analysis compared to the LC-HRMS
approach.Trace Analysis of D-Amino Acids in Mouse Organs by 1D & 2D nanoHPLC – MS
Alexander Wenger, Roland Reischl
Bioanalytical Research Labs, University of Salzburg, Hellbrunner Strasse 34, 5020 Salzburg,
Austria
alexander.wenger@sbg.ac.at
Amino acids are not only the building blocks of proteins, but also fulfil important tasks in
their free form as gene expression regulators, neurotransmitters and within the
protein-phosphorylation cascade. Whereas long believed that only the L-enantiomer is of
importance, this thesis has been proven wrong, and new insights in the last few decades
revealed the utmost importance of D-AAs in all species, especially in mammals.
We present an analytical approach to successfully separate named proteinogenic amino
acid enantiomers, utilizing a two-dimensional nano HPLC method, employing Stationary
Phase Assisted Modulation (SPAM) in a single run.
In the first dimension, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQ) tagged
amino acids (stronger retention of amino acids on reversed phase column, offers
possibility for UV detection and improved ionization yields) were separated
chemo-selectively by reversed phase chromatography (self-packed C18 columns, ~ 240
mm x 0.1 mm, 3 μm dP) and the detection was carried out via UV absorption at 254 nm.
During this first dimension separation, amino acids were successfully pre-separated
(especially isoleucine and leucine) and excessive AccQ-Tag has been cut off. Eluents of the
first dimension were then modulated via SPAM, employing two trap columns, which were
loaded and backflushed alternatingly. “Trapped” amino acids were then sequentially
transferred to the enantioselective second chromatographic dimension (self-packed
ZWIX+/QN-AX columns, ~ 200 mm x 0.2 mm, 3 μm/ 1.8 μm d P). Nano ESI-MS Detection of
the analytes was carried in SRM mode in combination with positive electrospray
ionization.
In addition to the two-dimensional approach, a 1D method was developed, analyzing the
influence of various mobile phase compositions and column temperatures (below
ambient), on enantiomer separation performance. For method development,
aa-standards and certain mixtures thereof have been used. Successfully implemented
methods were utilized to analyze the L/D-AA contents of various self-prepared
mouse-organ samples.Global analysis of protein arginine methylation
Fangrong Zhang1, Jakob Kerbl-Knapp1, Maria J. Rodriguez Colman2, Andreas Meinitzer3, Therese
Macher1, Nemanja Vujić1,4, Sandra Fasching5, Evelyne Jany-Luig5, Melanie Korbelius1, Katharina
B. Kuentzel1, Maximilian Mack4,6, Alena Akhmetshina1, Anita Pirchheim1, Margret Paar7, Beate
Rinner8, Gerd Hörl7, Ernst Steyrer1, Ulrich Stelzl4,5 , Boudewijn Burgering2, Tobias Eisenberg4,6,9,
Brigitte Pertschy4,6,9, Dagmar Kratky1,4, Tobias Madl*1,4,10
1
Gottfried Schatz Research Center for Cell Signaling, Metabolism and Aging, Molecular Biology and
Biochemistry, Medical University of Graz, 8010 Graz, Austria
2
Oncode Institute and Department of Molecular Cancer Research, Center for Molecular Medicine,
University Medical Center Utrecht, 3584 CX, Utrecht, The Netherlands
3
Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, 8010 Graz,
Austria
4
BioTechMed-Graz, 8010 Graz, Austria
5
Institute of Pharmaceutical Sciences, University of Graz, 8010 Graz, Austria
6
Institute of Molecular Biosciences, NAWI Graz, University of Graz, 8010 Graz, Austria
7
Otto-Loewi Research Center, Physiological Chemistry, Medical University of Graz, 8010 Graz, Austria
8
Division of Biomedical Research, Medical University of Graz, 8036 Graz, Austria
9
Field of Excellence BioHealth – University of Graz, Graz, Austria
10
Lead contactLipidome analysis of murine megakaryocytes during maturation
Bianca de Jonckheere, Asst. Prof. Dr. Robert Ahrends
Institute of Analytical Chemistry
University of Vienna
bianca.de.jonckheere@univie.ac.at
Megakaryocytes (MKs) are large polyploid and highly specialized precursor cells localized within the
bone marrow, that function to produce and release blood platelets into the circulation [1]. They
originate from pluripotent hematopoietic stem cells via a multi-step differentiation process
followed by complex maturation. A MKs mature, they lose the ability to divide but continue to
replicate their DNA in a process called endomitosis, resulting in a polyploid and lobulated nucleus
that is 16N on average [2]. In addition, they form an elaborate invaginated membrane system and
undergo rapid cytoplasmic expansion, leading to the accumulation of a massive amount of
cytoplasmic proteins and granules [3]. This huge reorganization process of MKs requires highly
regulated signaling pathways in which proteins and lipids as structural components or signaling
molecules are intertwined. In platelets, lipids are essential for platelet integrity and play a
fundamental role in platelet lifespan, senescence, shape change and aggregation. In a full lipidome
analysis of platelets from 2018, our lab identified that roughly 20 % of the lipidome is changing
upon platelet activation [4]. We hypothesize that the lipidome of MKs will be converted towards a
platelet like lipidome and that the overall lipid amount increases while the cells grow and major
rearrangements in membrane structure occur. Indeed, a first quantification of the major
phospholipid classes and glycerolipids showed that the overall amount of triacylglycerol (TAG) and
phosphatidylglycerol (PG) increases, while the abundance of phosphatidylcholine ether lipids (PC -
O) decreases about 7 % over MK maturation.
1. Nakeff, A. and B. Maat, Separation of megakaryocytes from mouse bone marrow by velocity
sedimentation. Blood, 1974. 43(4): p. 591-5.
2. Penington, D.G., K. Streatfield, and A.E. Roxburgh, Megakaryocytes and the heterogeneity
of circulating platelets. Br J Haematol, 1976. 34(4): p. 639-53.
3. Italiano, J.E. and J.H. Hartwig, Chapter 2 - Megakaryocyte Development and Platelet
Formation, in Platelets (Third Edition), A.D. Michelson, Editor. 2013, Academic Press. p. 27-49.
4. Peng, B., et al., Identification of key lipids critical for platelet activation by comprehensive
analysis of the platelet lipidome. Blood, 2018. 132(5): p. e1-e12.Comparing Chromatographic Techniques for Targeted LC/MS-MS Method
Development Focused on the Livestock Metabolome
Dimitrios J. Floros, Kangkang Xu, Franz Berthiller, Heidi Schwartz-Zimmermann
Institute of Bioanalytics and Agro-Metabolomics
Department of Agrobiotechnology (IFA-Tulln)
University of Natural Resources and Life Sciences (BOKU), Vienna
Konrad-Lorenz-Straße 20, 3430 Tulln, Austria
dfloros@boku.ac.at
The metabolome, the total set of small molecules present in a biological sample, is the
product of host nutrition and genetics as well as microbial and environmental inputs. [1]
As such it remains the most challenging of the ‘omics’ fields, with its application in
agriculture and livestock still growing. [2] While untargeted or shotgun approaches excel
at sample profiling and hypothesis generation, they are hampered by the lack of absolute
quantification and difficulty with metabolite identification. On the other hand, targeted
approaches are intensive to establish and validate, but can be used as the quantitative
biomarkers so useful in clinical settings. To this end, we have compiled a unique library
of over three hundred compounds of interest in livestock metabolomics, providing for
each compound multiple optimized and curated selected reaction monitoring transitions
allowing the rapid establishment of targeted MS/MS methods. However, not all
metabolite classes are accessible under any one chromatographic separation technique.
[3] Here, we also present the retention patterns of our several metabolite classes when
subjected to high performance reversed-phase, HILIC, and lipidomic liquid
chromatography conditions as well as anion-exchange liquid chromatography techniques.
This information provides a starting point from which optimized LC-MS/MS methods
can be developed.
1. Patti G.J., et al., Metabolomics: the apogee of the omics trilogy. Nat Rev Mol Cell Biol.,
2012. 13: 263–269.
2. Goldansaz, S.A., et al., Livestock metabolomics and the livestock metabolome: A
systematic review. PLoS One, 2017. 12: e0177675.
3. Wernisch, S., Pennathur, S. Evaluation of coverage, retention patterns, and selectivity of
seven liquid chromatographic methods for metabolomics. Anal Bioanal Chem. 2016. 408:
6079–6091.Investigation of non-metallic inclusions in steel samples based on LA-ICP-MS.
Mukhametzianova Gulnaz 1, Christoph Walkner1, Jörg C. Korp2, Andreas Graf 3, Johanna
Irrgeher1, Thomas Meisel1, Thomas Prohaska1
1 Montanuniversität Leoben, Lehrstuhl für Allgemeine und Analytische Chemie, Franz
Josef-Straße 18, 8700 Leoben, Österreich
2 Dr. Korp Technological-Consulting, Buchengasse 17, 8793 Trofaiach, Österreich
3 Breitenfeld Edelstahl AG, Breitenfeldstraße 22, 8662 St. Barbara im Mürztal, Österreich
gulnaz.mukhametzianova@unileoben.ac.at
The need for high quality steel is steadily increasing, and imperfections of the material
especially the formation of non-metallic inclusions is a significant problem in manufacturing
and further application of steel. These non-metallic inclusions are undesirable compounds
that incorporate into steel during production. The entire batch of steel or product can be
rejected because non-metallic inclusions impact mechanical properties of the product.
Therefore, defining their origin during production process, disposition, and formation cause
are the keys to obviate these problems in the steel production stage.
This study presents an analytical approach to determine the elemental and isotope
fingerprint and visualization of macroscopic non-metallic inclusions based on Laser
Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS) and 3D microscopy.
LA-ICP-MS was used to examine the trace element mass fractions in the inclusions, such as
the rare earth elements (REE). The elemental composition can serve as fingerprint to trace
the origin of these inclusions. As additional parameter, the Sr isotopic composition was
measured using Laser Ablation coupled to Multi-Collector ICP-MS. Comparative samples of
excipients added during steel production were analysed in the form of powder after
dissolution and analyte/matrix separation. The ablation craters after LA-ICP-MS were
visualized and evaluated using a 3D microscope with a polarization filter. The images were
used to measure the parameters of the ablation craters including depths, size, and volume.
The volume measurement option demonstrated a significant variation of volumes of the
ablation craters in the material depending on the position and composition.
The results should serve to trace the origin of macroscopic inclusions in order to optimize
the production process and the quality of the steel by reducing the occurrence and the
quantity of non-metallic inclusions.Analysis of macrophages at the single cell level by LA-ICP-TOFMS
Anna Schoeberl1, Michael Gutmann2, Sarah Theiner1, Martin Schaier1, Andreas Schweikert1,3,
Walter Berger2, Gunda Koellensperger1
1Institute of Analytical Chemistry, University of Vienna, Waehringer Str. 38, 1090 Vienna, AUT
2Institute of Cancer Research, Med. University of Vienna, Borschkegasse 8A, 1090 Vienna, AUT
3Institute of Inorganic Chemistry, University of Vienna, Waehringer Str. 42, 1090 Vienna, AUT
anna.schoeberl@univie.ac.at
Macrophages comprise the biggest part of immune cells present in the tumor microenvironment,
which plays an important role in tumor progression. [1] Macrophages, which derive from
monocytes, can be divided into two subgroups, the classically activated M1 macrophages and the
alternatively activated M2 macrophages. M1 cells show tumor suppressing effects while M2 cells
lead to tumor progression. [2] Although previous studies have proven that clinically approved
chemotherapeutic drugs (e.g., cisplatin) have an impact on the interaction between macrophages
and tumor cells, the effect of different subtypes of macrophages on the tumor cells and the
chemotherapeutic treatment demands further investigation. [3] Hence, in this work we
investigated the platinum uptake after cisplatin treatment of different types of macrophages (and
monocytes) including THP-1 monocytes and M0, M1 and M2 macrophages. Cytospin samples
were measured using a laser ablation system coupled to an ICP-TOF mass spectrometer. Imaging
was performed at the single-cell level and the platinum concentration in the cells was determined
using gelatin micro-droplet standards. [4] The results show an increasing uptake rate of cisplatin
starting with THP-1 cells with the lowest Pt concentration, followed by M0, M1 and M2
macrophages, which exhibit the highest content. The highest Pt level in M2 macrophages was in
agreement with the hypothesis, that M2 cells represent the tumor promoting species and are
consequently expected to be more sensitive to chemotherapeutic drugs.
1. Dijkgraaf, E. M., et al., Chemotherapy Alters Monocyte Differentiation to Favor Generation
of Cancer-Supporting M2 Macrophages in the Tumor Microenvironment. Cancer Res.,
2013. 73(8): p. 2480–2492.
2. Larionova, I., et al., Interaction of Tumor-Associated Macrophages and Cancer
Chemotherapy. OncoImmunology, 2019. 8(7): e1596004.
3. Englinger, B., et al., Metal Drugs and the Anticancer Immune Response. Chem. Rev., 2019.
119(2): p. 1519–1624.
4. Schweikert, A., et al., Micro-Droplet-Based Calibration for Quantitative Elemental
Bioimaging by LA-ICPMS. Anal. Bioanal. Chem, 2021.You can also read
