Available online at www.sciencedirect.
com
The overshadowed role of electron ionization–mass
spectrometry in analytical biotechnology
Deyber Arley Vargas Medina 1,2,
Edvaldo Vasconcelos Soares Maciel 1,2,
Natalia Gabrielly Pereira dos Santos 1,2 and
Fernando Mauro Lancas 1
Target and untargeted analysis of several compounds are
crucial methods in important areas such as omics sciences.
Gas chromatography coupled to mass spectrometry (GC–MS)
is widely used for volatile and thermally stable compounds. In
this case, the electron ionization technique (EI) is preferable as it
produces highly fragmented and reproducible spectra
comparable to spectral libraries. However, only a fraction of
target compounds is analyzable by GC without chemical
derivatization. Therefore, liquid chromatography (LC) coupled
with MS is the most used technique. Contrary to EI,
electrospray ionization does not produce reproducible spectra.
That is why researchers have been working on interfaces
between LC and EI–MS to bridge the gap between those
techniques. This short review will discuss advancements,
applications, and perspectives on biotechnological analysis.
Addresses
1
Laboratory of Chromatography, Institute of Chemistry at Sao Carlos,
University of Sao Paulo, P.O Box 780, 13566590 Sao Carlos, Brazil
2 ions are separated based on their mass-to-charge ratio (m/
Clemens Schöpf Institute, Department of Chemistry, Technical
University of Darmstadt, 64287 Darmstadt, Germany
Corresponding author: Lancas, Fernando Mauro (flancas@iqsc.usp.br)
Current Opinion in Biotechnology 2023, 82:102965
This review comes from a themed issue on Analytical
Biotechnology
Edited by Alexander Gruenberger, Janina Stephanie Bahnemann
and Christian Dusny
Available online xxxx
https://doi.org/10.1016/j.copbio.2023.102965
0958–1669/© 2023 Elsevier Ltd. All rights reserved.
Electron ionization–mass spectrometry: a
brief description
Electron ionization–mass spectrometry (EI–MS) is a
widely employed technique for the analysis of molecular
structure and composition in organic compounds. It in­
volves the vaporization and ionization of sample mole­
cules, followed by their separation and detection based
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on their mass-to-charge ratios. In EI–MS, gas-phase
analyte molecules are bombarded with high-energy
electrons within a vacuum chamber [1•].
The EI source comprises a tungsten filament, an ion
generation compartment, and a series of lenses for ion
manipulation. The filament generates an electron beam
that interacts with the sample, following a spiral path
guided by a cathode and small permanent magnets. Upon
collision with the electron beam, the gas-phase molecules
absorb energy and undergo ionization by losing an elec­
tron, resulting in the formation of odd-electron ca­
tion radicals (M+•). These species are inherently unstable
and readily undergo consecutive fragmentation through
intramolecular reactions, generating more stable species.
The positively charged species generated are subse­
quently ejected from the ionization source by applying a
positive potential to a repeller. Within the mass analyzer,
z) and recorded in the EI mass spectra [2].
EI–MS operates under vacuum conditions, with only ap­
proximately 1 in 10 000 molecules in the ionization source
being ionized. Fragmentation occurs exclusively through
intramolecular reactions, leaving other sample constituents
unaffected. Each EI–MS spectrum serves as a unique
fingerprint for a specific molecule, and the multitude of
recorded fragment ions provides valuable structural in­
formation for molecular identification and elucidation.
Besides, EI spectra are highly reproducible, so numerous
EI spectra libraries have been established, with some in­
tegrated into MS software packages for automated spectra
comparison and annotation of unknown analytes. The
capability for analyte identification remains the primary
advantage of EI–MS over other MS ionization sources.
The unparalleled role of electron ionization in
volatilomics
During the last decades, tremendous progress in bio­
technology and related fields has been made. A suc­
cessful example is human genome sequencing and the
consequent appearance of important areas such as
genomics, proteomics, lipidomics, and so on [3••].
Omics techniques aim to systematically analyze large
Current Opinion in Biotechnology 2023, 82:102965
2 Analytical Biotechnology
Abbreviations
API Atmospheric pressure ionization
DIP Direct inlet probe
EI Electron impact or ionization
E-LEI-MS Extractive-Liquid Sampling Electron
Ionization-Mass Spectrometry
ESI Electrospray ionization
FIA Flow injection analysis
cohorts of biological-related molecules to get informa­
tion about an organism’s state, function, and dynamics
[4••]. Volatilomics studies the emission of volatile or­
ganic compounds (VOCs) produced by metabolic path­
ways, easily found in noninvasive matrices (e.g. saliva,
urine, exhaled breath, and flowers) [5]. VOCs are en­
dogenous or exogenous compounds (< 1500 Da). No­
tably, the analysis of VOCs can aid in the detection of
diseases in humans, animals, and plants, as well as the
presence of microorganisms and other pathogenic agents
[5,6]. Approximately 2746 VOCs have been identified in
humans so far [7]. Regardless of the technique, un­
targeted or nontargeted analyses are challenging, often
demanding several steps, including experimental design,
data acquisition, and processing [6]. Because of the un­
ique characteristics of the compounds, their variable
concentration, and the complexity of the matrices where
they are found, powerful analytical techniques are re­
quired for their determination [6].
Gas chromatography coupled to mass spectrometry
(GC–MS) is widely used, especially in the case of
VOC analysis. This technique has several advantages,
such as high reproducibility, efficiency, and sensitivity
below ppt levels [6]. Concerning MS, EI is the most
reliable, efficient, and used ionization technique in
combination with GC [8]. The robust ionization process
of EI (70 eV) produces highly fragmented and re­
producible spectra, which, combined with its well-un­
derstood fragmentation rules, allows comparison against
available spectral libraries [8]. EI provides each analyte
with a spectrum that resembles a fingerprint, which is
reproducible across different MS instruments. Ad­
ditionally, matrix effects are rarely observed, as the
highly energetic process extinguishes most molecular
interactions [9]. The reproducible fragmentation pattern
combined with the GC retention time helps identify
known and unknown compounds. Although the re­
cognized efficiency of EI, omics studies often generate a
large set of data containing hundreds of compounds,
making the MS spectra interpretation difficult [10].
To overcome this downside, gas chromatography–tandem
mass spectrometry (GC–MS/MS) can be used to analyze
Current Opinion in Biotechnology 2023, 82:102965
GC Gas chromatography
LC Liquid chromatography
LEI Liquid electron ionization
MS Mass spectrometry
m/z Mass-to-charge ratio
MOI Microfluidic open interface
SPME Solid-phase microextraction
SIDS Sudden infant death syndrome
VOCs Volatile organic compounds
specific ionic fragments from each target compound sepa­
rately [10]. GC–MS/MS, utilizing high-resolution detectors
such as time-of-flight and Orbitrap, is an ideal technique
for accurate mass determination across the entire m/z
(mass-to-charge ratio) range [11]. Therefore, GC–MS and
GC–MS/MS with low-resolution (e.g. quadrupoles or ion
traps) or high-resolution detectors have been successfully
used in bioanalytical-related applications, including medi­
cine [8], natural products [12], drug discovery [13], foo­
domics [14], and biotechnology [15–17].
Summarizing, GC–EI–MS plays a crucial role in various
biotechnological applications, currently contributing i)
the diagnosis cancers, respiratory disorders, and in­
fectious diseases, ii) drug development based on volatile
compounds released by microorganisms, plants, or other
biological sources, iii) monitoring of plant health, asses­
sing the ripeness or quality of fruits, vegetables, and
other agricultural products, iv) bioprocess optimization
by monitoring VOCs as markers of the efficiency, per­
formance, and productivity of fermentation, microbial
growth, and other biotechnological processes, and v) in
microbiome studies, among many other areas [3].
Given recent advancements in GC, including compre­
hensive GCxGC, and the increasing affordability of high-
resolution mass spectrometers, it is widely acknowledged
that GC–MS(MS) satisfactorily meets the requirements
for analyzing VOCs. On the other hand, it is widely re­
cognized that GC is suitable for the straightforward ana­
lysis of volatile and thermally stable compounds.
However, for compounds that are not inherently volatile
or thermally stable, chemical derivatization is often ne­
cessary to enhance their thermal stability and volatility,
thus enabling their analysis by GC [18]. It should be
noted that derivatization procedures are typically labor­
ious, time-consuming, and susceptible to sample losses.
Moreover, it is estimated that only a narrow fraction of
compounds of current interest are analyzable by GC–MS
without any previous procedure (Figure 1) [19••]. Of
course, this is a problem for many researchers.
For this reason, the main technique currently chosen for
nonvolatile compound analysis is liquid chromatography
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 Electron impact mass spectrometry in biotechnology Vargas Medina et al.
Figure 1
Recommended chromatography-based methods according to the classes of compounds and polarity.
Reprinted with permission from [51]. Copyright (2020) Elsevier.
coupled to MS using electrospray ionization (LC–ESI–MS).
LC has become a more versatile technique than GC, with
similar or even better sensitivity and efficiency [19••].
Moreover, LC can analyze target compounds in a broader
range of molecular weights, with different polarities and
without thermal stability or volatility issues (Figure 1) [17].
However, one drawback is the incapability of comparing
ESI-generated spectra against spectral libraries due to the
soft-ionization process and nonreproducible data [20].
Therefore, the optimal approach involves coupling EI with
GC and LC, enabling the analysis of GC- and LC-amenable
compounds using EI. During the last 22 years, several works
focused on the topic have been published, reinforcing its
importance. Here, we bring to the journal audience an
overview of how much progress has been made, high­
lighting interesting applications and what to expect soon.
Can EI finally reach the same popularity as its counter­
part ESI?
The potential of electron ionization–mass
spectrometry for the analysis of nonvolatile
compounds and analytical biotechnology
Before the emergence of modern ESI sources, EI–MS
was widely used for peptide sequencing, proteomic, and
genomic studies [21]. Through derivatization, peptides
were converted into volatile polyaminoalcohols analyz­
able by GC–MS. Although it was a confident strategy
until before the emergence of modern atmospheric
pressure ionization sources, the process was tedious, la­
borious, and time-consuming. However, is it possible to
exploit the structural elucidation advantages of EI–MS
in analyzing nonvolatile or large molecules without de­
rivatization stages? That achievement depends on the
possibility of transferring the heavier analytes from a
condensate phase (liquid or solid) to the vacuum
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Current Opinion in Biotechnology
ambient of EI–MS in an efficient way. Two alternatives
could be explored for that purpose: i) the coupling of
EI–MS to LC and ii) the development of probes for
directly introducing samples into the source.
The coupling of electron ionization–mass spectrometry
with nano-liquid chromatography
Although in the past, the possibility to obtain EI–MS
spectra by coupling with LC was strongly limited by the
considerable amount of used mobile phase (mL/min), this
is a problem almost completely superseded by the modern
nano-LC [19,22••]. At nL/min flow rates, the impact of the
mobile phase over the EI–MS process drastically di­
minishes, and some standard EI–MS spectra can be ob­
tained from the liquid effluent. At the moment, three nano-
LC–EI–MS-interfacing strategies have been introduced: i)
Direct EI, ii) liquid electron ionization (LEI), and iii) Cold
EI. Although still with limitations, those interfaces are ex­
tending the range of the applicability of nano-LC–EI–MS
toward heavier and more polar compounds.
Direct EI relies on introducing the nano-LC effluent
into the ionization source through a fine capillary tube
(20–25-µm i.d.). This strategy can provide reproducible
mass spectra for several types of molecules and provides
the first demonstration of the absence of matrix effects
in the analysis of biological samples via nano-
LC–EI–MS [22••]. Direct EI has facilitated important
achievements, such as developing portable nano-
LC–EI–MS instruments for forensic analysis [23] and
determining free fatty acid profiles without needing es­
terification stages [24]. This previous study reveals par­
ticular importance since it sets the bases for future
lipidomic studies based on nano-LC–EI–MS. Although
Direct EI is still quite limited, this interfacing strategy
Current Opinion in Biotechnology 2023, 82:102965
4 Analytical Biotechnology
could bring important insights exploiting narrow-bore
(< 25) open tubular columns (OT) [25•]. OTs can be
directly coupled to the EI source without additional
interfacing devices, avoiding extra column void volume
and improving separation/detection efficiency [26•].
Recently, a series of OT columns with 2.0-µm i.d has
demonstrated outstanding efficiency and peak capacity
for digested protein analysis [27,28]. The future cou­
pling of those columns in nano-LC–EI–MS could bring
essential contributions to the development of the tech­
nique and enlarge its applicability range by reduction of
the mobile- phase flow rate until the pL/min order.
LEI is a more advanced nano-LC–EI–MS-interfacing
technology than Direct EI and provides a more ex­
tended applicability range, less noised spectra, and im­
proved detection limits [29]. Those improvements result
from incorporating a tailored vaporization chamber that
efficiently separates the stages of mobile-phase eva­
poration and analytes’ ionization. The merits of LEI
have been mainly demonstrated in the targeted analysis
of small molecules of diverse polarity, such as pesticides,
polycyclic aromatic hydrocarbons, hormones, and phe­
nols [30]. However, this interface also could allow the
performance of untargeted metabolic studies via nano-
LC–EI–MS. For example, during the study of sudden
infant death syndrome (SIDS), a newborn victim brain
sample was analyzed using LEI in full-scan mode. With
the aid of deconvolution software, the technique allowed
the easy and unequivocal identification of markers un­
derneath the matrix peak [31].
Furthermore, Cold EI is an interface developed for the
obtention of EI spectra with enhanced molecular ions.
This interface relies on the ionization of cold molecules in
supersonic beams and works with both GC and micro-/
nano-LC [32]. Cold EI is also practically free of matrix
effects and provides superior identification capabilities
than standard EI. The assignment of molecular formulas
is more confident from the enhanced molecular ion and
its isotopic pattern [33]. LC–Cold EI–MS has demon­
strated applicability to a more extended range of polarity
than LC–ESI–MS and superior separation/detection ef­
ficiency — providing narrower chromatographic peaks —
than GC–EI–MS [34]. Currently, Cold EI has made
possible the coexistence of GC–MS and LC–MS in a
single instrument [33]. A recent instrument introduced by
the developers of the Cold EI can easily switch between
GC–EI–MS and LC–EI–MS via software and without
instrumental modification. That unified instrument even
can record GC and LC effluents in the same chromato­
gram. Although still enduring important limitations, the
future improvement of unified chromatography with Cold
EI–MS could allow the complementary GC–MS/LC–MS
information for more broad exploration of unknown
samples. Figure 2 summarizes some examples of im­
portant applications of nano-LC–EI–MS.
Current Opinion in Biotechnology 2023, 82:102965
The potential of modern nano-liquid
chromatography–electron ionization–mass
spectrometry in analytical biotechnology
Although significant advances have been made in nano-
LC–EI–MS in recent years, the literature on analytical
biotechnology primarily relies on LC–EI–MS with lim­
ited reports. This scarcity can be attributed to the un­
derdeveloped nature of the technique and its restricted
accessibility, with only a few research groups worldwide
actively engaged in this field.
However, some interesting biotechnological-related
applications of nano-LC–EI–MS have been explored.
For instance, nano-LC–EI–MS has been tested for the
determination of pharmaceutical drugs used in treating
psychiatric disorders and pain management [36]. No­
table publications include the determination of ben­
zodiazepines in alcoholic beverages [37], and the study
of trans-cinnamaldehyde absorption in dermal tissues
using a nano-LC–EI–MS with a Direct-EI interface
[38]. Additionally, the analysis of potential genotoxic
impurities in active ingredients for drug formulation
has demonstrated the time-saving benefits of nano-
LC–EI–MS by avoiding extensive sample preparation
and derivatization processes [39]. In lipidomics-related
studies, nano-LC–EI–MS has shown promise with
studies profiling free fatty acids in mussels [24] and
determining nonesterified free fatty acids (FFAs) in
human plasma as a health indicator in diabetic patients
[40]. Another area of interest where LC–EI–MS has
been applied is in the analysis of dietary supplements.
Studies have quantified soy-content isoflavones in
dietary supplements and analyzed urine samples from
patients treated with these products [41]. LC–EI–MS
has also been employed for the quantification of
naturally occurring ephedrine alkaloids in standard
reference materials [42] and the analysis of poly­
phenols and caffeine in Camellia sinensis extracts
certified as NIST reference materials for dietary sup­
plement quality control [43]. Overall, these applica­
tions demonstrate the potential of LC–EI–MS in
analytical biotechnology, highlighting its advantages
and providing insights for further development in
the field.
Direct input techniques in electron
ionization–mass spectrometry
Bio-solid-phase microextractio–electron
ionization–mass spectrometry
Advances in the area of detection, mainly in MS, have
allowed the direct coupling of sample preparation tech­
niques such as solid-phase microextraction (SPME) with
MS, making it a tool for rapid screening and quantifi­
cation of analytes. Considering that SPME requires low
volumes of solvents to desorb the fiber, it ends up being
a great option for direct coupling with EI–MS. Cappiello
and collaborators coupled the SPME fibers to the
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 Electron impact mass spectrometry in biotechnology Vargas Medina et al. 5

Figure 2

Current Opinion in Biotechnology

Some examples of recent applications of nano-LC–EI–MS. (a) Comparative scheme of the mass spectra of C18 fatty acid obtained by nano-LC–EI–MS
with Direct-EI interface, and the spectra in the NIST database [22•]. (b) Cromatogram of the extract obtained from a SIDS victim’s brain sample
analyzed via nano-LC–EI–MS with an LEI interface [28•]. (c) Cromatogram of unified GC–EI–MS and LC–EI–MS obtained in a unified instrument
equipped with a Cold-EI interface [35].

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6 Analytical Biotechnology

Figure 3

Current Opinion in Biotechnology

Direct introduction techniques to EI–MS. (a) SPME device used for matrix analyte extraction. Soon after, use of the MOI to desorb the analytes and
introduce them into the LEI–MS. (b) FIA device for direct introduction of the sample into the Direct EI–MS. (c) DIP probe used for direct introduction of
solid samples into the EI ionization chamber for subsequent MS detection.
(a) Reprinted with permission from [44]. Copyright (2020) American Chemical Society. (b) Adapted from [45].

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 Electron impact mass spectrometry in biotechnology Vargas Medina et al.
LEI–MS through a microfluidic open interface (MOI)
(Figure 3a) [44]. MOI is a desorption chamber into
which the analytes extracted on the SPME fiber are
desorbed by a solvent at atmospheric pressure and
subsequently pumped to an LEI–MS interface.
Flow injection analysis direct electron ionization–mass
spectrometry
Samples can be directly introduced into an EI–MS source
via flow injection analysis (FIA). Analysis time becomes
even faster, bringing highly reliable information about the
nature of the analyte, considering the accuracy of the EI
spectra. Injection via FIA consists of boosting the sample
through a valve, and a carrier liquid constantly transports
the sample. With the help of a peristaltic pump, the
sample can be driven to Direct EI–MS (Figure 3b) [45].
Analytes together with the mobile phase are converted
into an aerosol under high temperature and vacuum. In­
side the ionization chamber, there is a hot vaporization
surface, which allows the vaporization of the sample and
subsequent ionization with electrons.
Extractive liquid sampling–electron ionization–mass
spectrometry
Those abovementioned Direct EI–MS sample in­
troduction systems rely on the use of FIA or HPLC
(High Performance Liquid Chromatography) pumps.
However, bear in mind that those systems dispense the
use of the chromatographic column, and work very re­
duced back pressures, in a previous review, we asked
and raise the possibility to develop straightforward
EI–MS ambient sampling systems exploiting the MS
vacuum pump to propel the sample into ionization EI
source [1•]. A first attempt to materialize that idea was
recently introduced by the Cappiello research group.
They developed a novel analytical strategy called ex­
tractive liquid sampling–electron ionization–mass spec­
trometry (E-LEI–MS), which enables direct analysis of
samples without the need for separation or pretreatment
steps [46••]. E-LEI–MS combines a device for solvent
release and ambient sampling with an EI source in a
single-quadrupole mass spectrometer (Figure 3c). This
system has demonstrated successful applications in
analyzing active ingredients in pharmaceutical tablets,
detecting pesticides on fruit peel, determining drugs of
abuse (such as cocaine) on banknotes, and analyzing
unknown components on painting surfaces.
Direct inlet probe
The direct inlet probe (DIP) probe is one way to in­
troduce solid or liquid samples directly into the EI–MS
source, currently available in many commercial GC–MS
instruments. This probe consists of a stainless-steel rod
designed, so that a quartz cup (i.d. 1 mm, length 7 mm)
can be fitted at the tip into which a solid sample of
∼0.2 mg is introduced [47,48]. In this approach, the
sample contained in the DIP is introduced into the
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7
ionization chamber, where the sample is vaporized and
ionized by electron impacts, and then, the generated ions
are detected in the MS [49]. This technique is very fast
and can be used for the direct obtention of EI–MS spectra
of nonvolatile solid and liquid samples. For instance,
DIP–MS has been successfully employed as a rapid and
straightforward tool for identifying heavy and ultraheavy
crude oils, asphaltenes, and tar sands in field applications,
eliminating the requirement for prior separation or treat­
ment. By programming the probe temperature ramp,
DIP–MS enabled the detection of individual components
within the mixture up to masses of 950 m/z [50]. Despite
its promising potential, DIP has not been extensively
explored in the context of biotechnological analysis, and
there are few or no reports describing its applicability in
this field found in the literature. Conducting additional
studies to explore the use of DIP for obtaining EI–MS
spectra of proteins, other biomolecules and even viruses
or bacteria could yield valuable insights into the field of
biotechnological analysis.
Techniques for direct sample introduction into the
EI–MS source are excellent alternatives to conventional
methods, such as GC–MS, for quick and easy identifi­
cation of compounds and subsequent decision-making.
Rapid detection techniques are promising in the area of
biotechnology, or other fields such as chemistry, biology,
and clinical and forensic analysis, as they can be the
initial step within analytical studies. Fields such as the
discovery of biomarkers (whether metabolites or pro­
teins) for disease prognosis or diagnosis; development of
new drug candidates; analysis of metabolites and mar­
kers in plants; control of toxins and xenobiotics in
plants; and proteins in tissues, are some possibilities that
the authors believe that these techniques have potential
to be explored.
Conclusions and future perspectives
EI is an ionization technique with an unparalleled ability
to provide highly reproducible and comparable mass
spectra. Along with GC, EI–MS is the chief technique in
the targeted and untargeted determination of volatile
compounds in several research fields. However, only less
of the 20% of molecules of current interest are analyz­
able by GC–EI–MS. Developing technology that allows
exploiting the advantages of EI–MS in the analysis of
nonvolatile compounds could bring new critical insights
into biotechnological research. Hence, the coupling with
nano-LC, through Direct EI, LEI, and Cold-EI inter­
faces, is expanding the range of applicability of the
EI–MS and opening the door for the development of
unified GC/LC instruments. Although important ad­
vances have been achieved in the last 20 years, nano-
LC–EI–MS still endures significant limitations.
Therefore, direct sample introduction strategies are a
promising alternative for obtaining EI–MS spectra of
Current Opinion in Biotechnology 2023, 82:102965
8 Analytical Biotechnology
heavy compounds. While probes for direct sample in­
troduction into EI–MS have been commercially avail­
able for several years, those have been little explored.
New practical demonstrations of their potential could
provide a bunch of new possibilities for biotechnology
research.
CRediT authorship contribution statement
Deyber Arley Vargas Medina: Conceptualization,
Visualization, Writing – original draft. Edvaldo Vasconcelos
Soares Maciel: Conceptualization, Visualization, Writing –
original draft. Natalia Gabrielly Pereira dos Santos:
Conceptualization, Visualization, Writing – original draft.
Fernando Mauro Lancas: Conceptualization, Visualization,
Funding acquisition, Project administration, Supervision,
Writing – review & editing.
Data Availability
Data will be made available on request.
Declaration of Competing Interest
The authors declare that they have no competing in­
terests or personal relationships that could have influ­
enced this work.
Acknowledgements
This work was supported by the São Paulo Research Foundation, FAPESP
[Grants 2017/02147-0, 2019/22724-7]; the National Council for Scientific
and Technological Development, CNPq [Grant 307293/2014-9]; the
Coordination for the Improvement of Higher Education Personnel
— Brazil, CAPES [Finance Code 001]; and the Alexander von Humboldt
Foundation.
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