university of copenhagen

Non-aqueous CE-MS of cinchona alkaloids - characterizationof a novel CE-ESI-MS

interface

Hansen, Frederik André; Hansen, Steen Honoré; Petersen, Nickolaj J.

Publication date:
2016

Citation for published version (APA):

Hansen, F. A., Hansen, S. H., & Petersen, N. J. (2016). Non-aqueous CE-MS of cinchona alkaloids -
characterizationof a novel CE-ESI-MS interface. Abstract from CSSS2016, Copenhagen, Denmark.

Download date: 03. jan.. 2023

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One- and two-dimensional LC strategies for separating very complex mixtures

Peter Schoenmakers, University of Amsterdam, Science Park 904, 1098 XH

Amsterdam, The Netherlands

[email protected]

Liquid chromatography (LC) has progressed a long way toward the separation of very
complex mixtures of non-volatile analytes. Separations with a peak capacity
exceeding 1,000 have been published, using one-dimensional column LC with (very)
long columns and (very) long analysis times. Roughly, such high-resolution
separations may provide one peak per minute.
Comprehensive two-dimensional liquid chromatography (LC×LC) is attractive,
because it provides higher peak capacities in a reasonable time (e.g. 5000 peaks at
about one peak per second) and the combination of two different retention
mechanisms (selectivities). As a result, is increasingly used for the separation of
complex samples. Examples of the separation of polymers, surfactants, dyes and
peptides will be shown.
One significant bottleneck is the development of LC×LC methods, which even for
experts tends to require a good deal of time and effort. Optimization software, such
as the Program for Interpretive Optimization of Two-dimensional Resolution (PIOTR)
may help reduce the time required to develop an optimal method from typically a few
months to a few days.
Despite the progression of LC and LC×LC, many samples are too complex to be fully
separated and even the most fantastic mass spectrometers struggle with the
identification and quantitation of analytes that are introduced simultaneously in vastly
different concentrations. Thus, there is a need for even better separations. Spatial
three-dimensional LC promises peak capacities up to one million in the future.

8
Lipid and steroid hormone screening in a clinical setting using high resolution
separations and mass spectrometry
Jonas Bergquist

Analytical Chemistry, Department of Chemistry Biomedical Centre and Science for Life
Laboratory, Uppsala University, Box 599, 751 24, Uppsala, Sweden

Abstract

Steroids are natural lipid compounds with a perhydro-1, 2 cyclopentanophenanthrene ring

system. Steroids can be divided into four major classes: progestagens, corticosteroids,
androgens and oestrogens. Steroids and their metabolites play important roles in human
physiology and are useful biomarkers for the diagnosis of several pathological conditions.
Effectiveness of the diagnostic testing depends on the appropriateness of the exploited
markers of diseases; hence they are controlling human body functions as a part of the
endocrine, neuronal and immune systems. Therefore, the measurements of endogenous
steroid levels in the body fluids are widely employed to evaluate the androgenic status of the
pathological processes of the subject under investigation.

We have developed a routine ultra performance convergence chromatography (UPC2)-

MS/MS based diagnostic test for steroids/biomarkers of endocrine and metabolic diseases.
The sample preparation procedure consisted of four steps: lipid extraction from plasma,
enrichment of steroids by SPE, derivatization, and analysis of steroids by a UPC2-MS/MS. A
robust, sensitive and selective method for the determination of steroids in both plasma and
tissues has been established and evaluated. The method showed excellent precision,
accuracy, quantification limits, detection limits, and robustness.

In summary, this newly developed method is a valuable tool to evaluate action of steroids in
human diseases.

9
Analysis of scarce biofluids: electromembrane extraction (EME) and capillary
electrophoresis coupled to mass spectrometry (CE-MS) are a good match

Nicolas Drouin, Serge Rudaz, Julie Schappler*

Scarce biofluids are biological fluids, which are either difficult to obtain because the
collection is invasive (e.g. cerebrospinal fluid) or available in minute volume (e.g.
tissues, cell cultures, lachrymal fluid). These biofluids contain a large number of
potential biomarkers such as amino acids, neurotransmitters, neuropeptides, and
nucleobases involved in several pathologic processes.
In this context, electromembrane extraction (EME) appears an attractive sample
preparation approach, featuring high selectivity towards low molecular weight (LMW)
ionizable analytes with high recovery, fast extraction, and green chemistry. According
to the low recovered volume, the nature of the extracted compounds, and the
sensitivity goal, CE-MS/MS is particularly adapted to analyze the extracts obtained by
EME.
In this study, a new parallel EME (Pa-EME) device was developed, based on a
commercially PAMPA 96 well-plate and adapted in-house to be conductive and
reusable. The design and the initial experimental parameters (applied voltage,
extraction time, and agitation speed) were optimized with a second degree design of
experiments (DoE) for the extraction of model basic LMW compounds. The device was
further used for the extraction of numerous polar cationic metabolites (>50) from CSF
and various supported liquid membranes composition (organic solvents and carriers)
were evaluated. Due to the high number of experiments performed, the 96 well-plates
format was perfectly adapted to the DoE and the screening studies. In order to improve
the throughput of the analysis, a multisegment injection (MSI) approach enabling 4
analyses in one single run was performed. Results demonstrated that the combination
of Pa-EME and CE-MS/MS is an efficient approach for the selective analysis of cationic
metabolites from scarce biofluids.

10
LC-MS based metabolomics - strategies towards selectivity and high metabolite coverage

K. Ortmayr1,2, M. Schwaiger1, T. Causon2, S. Hann2, G. Koellensperger1

1
Institute of Analytical Chemistry, Faculty of Chemistry, University of Vienna, Währingerstr. 38, 1090
Vienna
2
Department of Chemistry, University of Natural Resources and Life Sciences-BOKU, Muthgasse 18,
1190 Vienna

The term untargeted metabolomics implies global and unbiased analysis. One key aspect is to find
differential metabolic fingerprints related to different biological conditions. This task is followed by
the complex and yet not routine task of annotating molecular formulas from mass spectrometric
features (denotes unique mass and retention time). As a matter of fact, in practice the generality or
comprehensiveness of the untargeted approach is compromised by the chemical diverse nature of
the constituents of the metabolome, yet it is crucial to obtain selectivity for a high coverage of the
metabolome, most desirable within one analytical run. It is commonly accepted that selectivity
provided by high mass resolution solely is not sufficient, as identical masses are generated by
structural isomers and/or in-source fragments. Hence, chromatographic selectivity is conceived as
essential in untargeted metabolomics regarding both aspects, i.e. accurate differential quantification
and feature annotation. Recently, in order to increase the throughput of analysis, novel on-line
combinations of orthogonal chromatographic LC separations have been introduced and will be
discussed regarding non-targeted metabolomic applications.

11
The influence of packing geometry on chromatographic peak capacity

During the past decade, the pillar array column format has come into consideration as an
promising alternative for the packed bed and the monolithic column in the field of liquid
chromatography. Where the flow resistance in the pillar column is comparable or even lower
than that of monoliths, the ordered nature of the pillars allows for theoretical plate heights
that are a factor of two smaller than (disordered) particulate columns with equally sized
particles.

The use of lithographic and etching techniques offers the ability to conceive virtually any flow-
through channel. In the present contribution, the impact of the support structures on
dispersion and permeability will be discussed. The distance between the support structures
and the concomitant optimal channel length will furthermore determine which sample
complexity can be handled for a given pressure limitation.

Because concentration loadability of the non-porous silicon structures is for most applications
insufficient we developed 2 strategies to render the pillars porous without losing the ordered
nature of the array. The principle, layer properties and some separation results of a an
additive sol-gel procedure will be presented, as well as for an electrochemical anodization
approach.

Because of the 2D (or often referred to as 2.5D to emphasize that all structures have the
same depth) nature of the pillar array one would intuitively expect that at large depths the
performance becomes closer and closer to that of channel without channel walls.
Unfortunately this is not the case. The situation is even more dramatic when non-verticality of
the pillars is taken into account as well. Increasing volume loadability therefore occurs at the
expense of inherent 2 D separation performance. For given micromachining limitations the
depth should be chosen such that a compromise is reached between volume loadability and
separation performance, which is dictated by the application. Also true 3D shapes are
possible, which generally result in a similar trend as for a rectangular channel. One shape
however (the I–shape) has the potential to optimize both features simultaneously. A number
of bench marking results and applications will be shown using rectangular channels, revealing
e.g. that a peak capacity of 800 can be reached by separating a phenone mixture on a 0.5 m
long channel with a spacing between the structures of 2.5 µm and a depth of 18 µm.

12
Chromatographic separation under HDX-MS quench conditions

Rune Salbo, Ph.D., Senior Research Scientist

Protein Structure and Interaction, Novo Nordisk A/S, Denmark

Hydrogen-Deuterium Exchange (HDX) Mass Spectrometry (MS) is a

technique for structural characterization of proteins in solution
where the labelling of the backbone amides with deuterium is
followed over time. It is essential for the experiment that the
hydrogen-exchange reaction is quenched during the analysis in
order to preserve the label. Therefore, disulphide bond reduction,
digestion with pepsin, and chromatographic separation of the
peptides must take place under suboptimal conditions at pH 2.5
and 0°C using very short gradients. The poor chromatographic
separation can be counter-acted by implementation of ion mobility
mass spectrometry, which adds another degree of separation. In
this study the structural and dynamic changes of a protein upon
binding of antibodies is probed and the antibody epitopes are
identified.

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 HPLC column technology in 2016 –
a state-of-art review and a critical appraisal

Stefan Lamotte, BASF SE, 670056 Ludwigshafen, Germany

Klaus K. Unger, Johannes Gutenberg-Universität, 55099 Mainz, Germany

Abstract
HPLC columns and stationary phases have reached a mature high-tech status. The
most common of about four million annually worldwide sold columns are reversed
phase (most of them n-octadecyl bonded silica) columns of 2 mm to 4.6 mm I.D. and
50 mm to 250 mm length packed with 2 µm to 5 µm size fully porous particles, 3 µm
size core-shell particles or monolithic silica columns (1). When implemented in a
suitable HPLC instrument and run at optimized conditions they fulfill the requirements
in pharmaceutical and chemical analysis resolving the main product and its impurities
at an abundance ratio of 1 : 1,000 in a short time at defined and certified conditions.
While the run to highly efficient columns and short analysis time was dominating the
field for many years the chromatographic community has now discovered the huge
potential of LC to adjust and to control the selectivity of separation for complex
mixtures of hundreds constituents and for those containing more than thousand
entities such as in bio fluids. In addition improved detection capabilities over the last
decade enable us to detect more than the tip of the iceberg, making former easy
separations much more complex. This will lead to multicolumn approaches, i.e.
coupled selectivities in contrast to columns for higher plate counts. The concepts
applied are: blending of adsorbents with different surface chemistries into so called
mixed mode phases, coupling of columns with different surface chemistries in series,
and column switching as a step into multidimensional LC. Next to separation in
multidimensional separations generating quantitative results with the same precision
as established for one dimensional HPLC will be the major task

(1) K.K.Unger, St. Lamotte, and E. Machtejevas, Column technology in LC, in

Handbook of separation science: Liquid chromatography, S. Fanali, P.R.
Haddad, C.F. Poole, P. Schoenmakers,, and D.Lloyd, editors, Vol. 1,
Fundamentals and instrumentation, Elsevier, Waltham, MA, US, 2013, 41 – 86

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 Signal processing - The missing link in analysis of XC, XC-MS and
XC×XC data
Jan H Christensen1,*, Nikoline J. Nielsen1, Søren Furbo1, Giorgio Tomasi1
1
Analytical Chemistry group, Section of Environmental Chemistry and Physics, Department of Plant
and Environment Sciences, University of Copenhagen Denmark
*
Corresponding author e-mail: [email protected]

Keywords: Oil analysis, two-dimensional chromatography, curve resolution, feature detection and
extraction, pixel-based analysis

Abstract
Non-targeted LC- and GC-High-Resolution-MS (XC-MS) experiments generate enormous
amounts of data. Each data matrix comprises more than 107 data points, complicated by the presence
of electronic- and chemical noise, artefacts and data redundancy.
We design analytical platforms that can be used for comprehensive analysis of petroleum samples.
Sample preparation should therefore be non-selective or with complementary selectivity such that the
entire range of compound properties can be covered by few analytical platforms. We aim to find
column combinations that provide the most orthogonal separations (i.e., providing different relative
retention times for the same compounds).
We construct custom-made signal processing procedures and develop generic functions for import
and cropping of different types of analytical data and develop new strategies for feature detection and
pixel-based analysis of multidimensional data. We work on solving the problems that arise for
multidimensional separation systems (e.g., LC×LC and GC×GC) such as retention time shifts in
multiple dimensions, complex baselines and the very large number of variables compared to samples.
We develop new scaling procedures using the analytical variation of each signal in replicate
analyses to reduce the importance of non-chemical information and allow robust modelling of data
with many more variables than samples. To do this, we also exploit the structure of data (sample × rt1
× rt2 × m/z) by employing multi-way models such as PARAFAC that can resolve and quantify even
grossly overlapping peaks.
In this presentation, I will give an overview of different strategies for signal processing that we
have developed and use in our group for analysis of petroleum samples.

15
 Intact protein glycoform profiling using CE–MS and HILIC–MS

Govert W. Somsena, Elena Domínguez Vegaa, Jordy van Angerena, Klára Petrůb, Sara
Tengattinic, Rob Haselberga

a
Division of BioAnalytical Chemistry, Vrije Universiteit Amsterdam, The Netherlands,
[email protected]; bDepartment of Analytical Chemistry, Faculty of Pharmacy in Hradec
Kralove, Charles University Prague, Czech Republic; cDepartment of Drug Sciences and
Italian Biocatalysis Center, University of Pavia, Italy

Mass spectrometry (MS)-based methodologies are implemented throughout all stages of

biopharmaceutical production, providing information on molecular mass, primary sequences,
post-translational modifications (PTMs), and higher order structures. Despite advances in MS
resolution and accuracy, the attainable structural information on intact biopharmaceuticals –
often mixtures of highly similar glycoforms – is limited by sample complexity and/or
ionization suppression by major sample components. Therefore, separation prior to MS
detection is mandatory to achieve reliable assignment of protein variants.

Capillary electrophoresis (CE) has the intrinsic capacity to produce narrow peaks for
macromolecules as well as the selectivity to separate closely-related proteoforms under MS-
compatible conditions. It will be shown that intact glycoprotein separation can also be
achieved by hydrophilic interaction liquid chromatography (HILIC), with protein resolution
being mainly governed by carbohydrate content. Comparison of CE–MS and HILIC–MS
indicates a remarkable complementarity of the two techniques with respect to glycoform
selectivity. Examples will highlight the performance of the developed CE–MS and HILIC–
MS systems with focus on the analysis of glycoproteins of pharmaceutical interest, such as
erythropoietin, antigen glycoconjugates, and monoclonal antibodies (mAbs).

16
 A Look Back on the Fascinating History of Chromatography
Thomas Welsch
Institute for Analytical and Bioanalytical Chemistry, Ulm University,
Albert-Einstein-Allee 11, D-89081 Ulm, Germany

Chromatography influences our everyday life as no other analytical technique. In the

second half of the last century, gas and liquid chromatography developed to the most
important analytical tools for the separation and determination of components in
simple and complex mixtures and matrices. This success story is based on the
contributions of innumerable researchers and multifaceted progress in column
technology and instrumentation.
The roots of chromatography date back to ancient Greece and Middle Ages, were
filtration- and purification operations included frontal chromatographic processes. The
investigation of “adsorption” and “capillarity” at the end of the 19 th century using
different materials e.g. paper and cotton were important for the discovery of elution
chromatography by Tswett in 1903.
In my presentation I will show some examples for this early chromatography and I will
highlight outstanding papers, which substantially contributed to the development of
Gas Chromatography, High Performance Liquid Chromatography and Electrochro-
matography. Unfortunately, some of the pioneering papers were misread in order to
become recognized by the chromatographic community.

17
Non-aqueous CE-MS of cinchona alkaloids - characterization of a novel
CE-ESI-MS interface

Frederik André Hansen, Steen Honoré Hansen and Nickolaj J. Petersen*

Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark

We have recently in our group at the University of Copenhagen developed a

robust and simple sheathless CE-ESI-MS interface (capillary electrophoresis
– electrospray ionization-mass spectrometry). In this presentation the
interface is characterized and compared with HPLC-MS for studying the
composition of alkaloids in Cinchona bark.
One common problem for sheathless interfaces for CE-MS has been
establishing a stable electric contact at the end of the separation capillary that
does not induce band broadening or affect the spray stability. In our device
the electric contact is generated through a submicron fracture in the capillary
close the ESI tip. The fracture provides a zero dead volume and excellent
conducting properties due to the large amount of ions in the electric double
layer. Electric current exceeding the upper limit of CE instrumentation of up to
300 µA can easily be obtained. Furthermore, the increased conductivity of the
buffer in the fracture generates field free pumping of the analytes towards the
ESI spray tip.
In this study the device was used to analyze the four major alkaloids
(diastereomeric pairs of quinine/quinidine and cinchonine/cinchonidine) in
Cinchona bark samples, in a non-aqueous BGE system without using a chiral
selector.
The separation was achieved in less than 15 min with a 75 cm capillary and a
detection limit of 1 ng/mL, approximately 150-2000 times lower than the
occurrence in the samples after standard sample preparation was achieved.
The sheathless interface showed a 40-fold improvement of the detection limit
compared to an Agilent® sheath liquid interface employing a similar non-
aqueous BGE system.

18
 The performance of stationary phases for fast liquid chromatography

Attila Felinger

Department of Analytical and Environmental Chemistry, University of Pécs,

Ifjúság útja 6, H-7624 Pécs, Hungary, e-mail:[email protected]

During the recent years, a number of chromatographic columns with various types of packing materials
dedicated for UHPLC instrumentation have been introduced. The most frequently used columns in this
area are narrow and short (2.1 mm ID and 50 mm length), and are packed with sub-3-µm or sub-2-µm
particles. Besides the use of the well-known and widely used fully-porous or core-shell packing
materials, another potential is in the development of monolithic silica columns, where improved
efficiency can be achieved and moderate column pressures are sufficient owing to the small skeleton
size and large (through-pore size)/(skeleton size) ratios.

The kinetic performance of reversed phase columns packed with fully porous and core-shell particles
with various particle diameters and the performance of a monolithic silica columns can be rather
different. We also compared the kinetic performance of the same type monolithic column mounted by
the producer or by the user. In addition to the results obtained with van Deemter plots and kinetic plots,
we suggest a column-reversal method to examine the bed heterogeneity at the column ends and explain
the reduction of column efficiency for early eluting solutes.

The monolithic column shows systematically better efficiency for early eluting compounds than the
packed columns, therefore an additional band broadening effect is suspected for the packed columns.
The effect of the presence of the frits and the bed heterogeneity of the columns near the frits can
characterized by a column-reversal method. It has been shown that significant differences can exist
between the two ends of the packed columns, while the monolithic column shows rather similar
performance at either column end.

The flow reversal method is useful to characterize the sample band broadening in the immediate vicinity
of the column ends. Although flow reversal has a peak compression effect, and the peaks observed with
reversed flow are always narrower and more symmetrical than the peaks obtained without reversing
the flow, flow reversal can be a useful tool to show the differences between the intrinsic plate heights of
the columns and also for showing the difference between the two respective column ends. The local
plate height was found much smaller in every case than the one we get for the unretained thiourea after
a simple injection without arrested and reversed flow.

The local efficiency at the two respective column ends differ from each other, however the difference is
negligible for the monolithic column. It cannot be guaranteed whether the inlet or the outlet of the
columns perform better. That varies from one manufacturer to the other.

19
Title: Over 10 000 peptide identifications from the HeLa proteome using single-shot capillary zone
electrophoresis tandem mass spectrometry

Authors: Liangliang Sun1, Guijie Zhu1, Xiaojing Yan1, Yimeng Zhao1, Alexander Herbert2, Michael
Westphall2, Matthew Rush2, Matthew Champion1, Josh Coon2, Norman Dovichi1
1. Department of Chemistry & Biochemistry, University of Notre Dame, Notre Dame, IN, United States.
2. Department of Chemistry, University of Wisconsin, Madison, WI, United States.

Abstract: Capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) has recently attracted
attention as a tool for shotgun proteomics. However, its performance for this analysis has fallen far below
that of reversed phase liquid chromatography (RPLC)-MS/MS. Here, we report the use of a CZE method
with a wide separation window (up to 90 min) and high peak capacity (~300).(1) This method is coupled to
an Orbitrap Fusion mass spectrometer via an electro-kinetically pumped sheath flow interface for analysis
of complex proteome digests. Single-shot CZE-MS/MS identified over 10,000 peptides and 2 100 proteins
from a HeLa cell proteome digest in ~100 min. This performance is nearly an order of magnitude superior
to earlier CZE studies and is within a factor of 2 to 4 of state-of-the-art nano ultrahigh pressure LC system.

(1) Sun L, Hebert AS, Yan X, Zhao Y, Westphall MS, Rush MJ, Zhu G, Champion MM, Coon JJ, Dovichi
NJ. Angew Chem Int Ed Engl. 2014; 53: 13931-3.

20
 Distribution constants by liposome electrokinetic chromatography

Susanne K. Wiedmer

Department of Chemistry, University of Helsinki, POB 55, 00014 University of Helsinki, Finland

Different techniques have been developed to quantify molecular interactions and measure the
interactions between drugs and lipid vesicles. Methods applied to interaction studies in pharmaceutical
and biomedical sciences include equilibrium dialysis, ultrafiltration, ultracentrifugation, filtration,
calorimetry, microdialysis, spectroscopic and chromatographic methods, and capillary electromigration
(CE) techniques. Among the used methodologies, CE has proven to be an attractive tool because
detailed information about the dynamics of molecular interactions under physiological conditions with
minimal sample consumption can be obtained. Nowadays a common set up for investigating analyte-
liposome interactions is liposome electrokinetic chromatography (LEKC).
In LEKC the capillary is filled with a liposome-containing background electrolyte (BGE) which serves
as a pseudostationary phase. We have used LEKC to investigate interactions of local anesthetics or
glaucoma drugs with liposomes comprising phosphatidylcholine and phosphatidylglycerol with and
without cholesterol at different temperatures. In addition, lipids and sterols extracted from human red
blood cells (RBC) and liposomes made from these were used as pseudostationary phase as well. The
interactions between the compounds and the liposomes will cause a change in the electrophoretic
mobilities of the compounds compared to conventional CE. By comparing these methods and
measuring the electrophoretic mobilities of the liposomes, the retention factors can be calculated.
Retention factors are used to elucidate the interactions between the drugs and a given composition of
the pseudo-stationary phase. Distribution constants, on the other hand, are used to quantify the
interactions between drugs and liposomes and these can be estimated from the retention factors and the
phase ratio of the system. Comparison between theoretically determined partitioning coefficients and
experimentally determined distribution constants were made.

21
 PSP –CEC
Future & Past

Main Challenges

R. Blom, C. Nilsson, I. Harwigsson,, S. Birnbaum, D. Chandrasekaren,

K. Shea & S. Nilsson

New insights in biomedicine and related areas require the parallel development of new
analytical methods.
The development of nanoparticle-based capillary electrochromatography (CEC) in our lab will
be reviewed. The nanoparticles, suspended in the electrolyte for use as pseudostationary phase
(PSP) in CEC. In PSP-CEC, the stationary phase is used only once allowing fast column
regeneration and circumventing carry-over effects.
Nanoparticles possess a favorable surface-to-volume ratio allowing highly efficient separations.

Used Nanoparticles including; Lucite-based, “Cubisomes”, Gadolinium based, MIP-particles.

Mode; Partial- and Full-filling PSP.
Detection ?
Playmates; GFP, hGH, Lyz, Pepsin and small molecules
Why Future?

[1] C Nilsson, S Nilsson Electrophoresis 2006, 27, 76-83

[2] C Nilsson, S Birnbaum, S Nilsson J. Chromatogr. A 2007, 1168, 212-224
[3] C Nilsson, P Viberg, P Spégel, M Jörntén-Karlsson, P Petersson, S .Nilsson Anal. Chem. 2006, 78, 6088-
6095
[4] F Priego-Capote, L Ye, S Shakil, S Shamsi, S Nilsson Anal. Chem. 2008, 80, 2881-2887
[5] C Nilsson, K Becker, I Harwigsson, L Bulow, S Birnbaum, S Nilsson Anal. Chem. 2009, 81, 315-321
[6] C Nilsson, I Harwigsson, K Becker, JP Kutter, S Birnbaum, S Nilsson, Electrophoresis 2010, 31, 459-464
[7] P. Ohlsson, , O. Ordeig, C. Nilsson, I. Harwigsson, J. P. Kutter, S. Nilsson, Electrophoresis 2010, 31,
3696-3702.
[8] C. Nilsson, S. Birnbaum, S. Nilsson, Electrophoresis, 2011, 32, 1141-1147.
[9] R. Blom, Shea, C. Nilsson, S. Birnbaum, Depack, S. Nilsson, Manus 2016
[10] R. Blom, C. Nilsson, S. Birnbaum, S. Nilsson, Manus 2016

22
 Enantio-/Stereo-selective Liquid Chromatographic Separation:
State of the Art, Quo Vadis.

Wolfgang Lindner
Institute of Analytical Chemistry, University of Vienna, Austria

Nowadays a large arsenal of chiral stationary phases (CSPs) and so-called “chiral columns”
thereof is commercially available associated with the advertisement that for practically any
“chiral” problem there will be a column available to solve the individual enantiomers of a
chiral compound. However, there remain some dilemmas:
1. Which column will be suitable to solve the enantioselective separation task?
2. Is there a remaining group of chiral analytes (selectands, SAs) for which no adequate
solution can be offered?

As the elucidation of the stereoselective molecular recognition principles of the CSPs for a
given SA remains a difficult task and as enantioseparation can hardly be predicted a column
screening remains still the first choice to be undertaken, externally or internally. In this
context the polysaccharide type CSPs proved to be most often the first choice followed by the
Pirkle type CSPs, macrocyclic CSPs and protein type CSPs. For very polar and chargeable
SAs it turned out that the chiral ion exchangers developed by our group are most promising.

In this lecture special emphasis will be given also on the enantioselective LC-
chromatographic resolution of free and N-protected amino acids and of short peptides as these
SAs fall into the category of quite difficult analytical and preparative challenges. Related
molecular recognition principles between the anionic, cationic and zwitterionic chiral
selectors (SOs) and our CSPs and the given SAs will be discussed. It can serve as basis for
further developments of highly dedicated CSPs in the frame of Quo Vadis aspects.

23
 Opportunities and challenges of LC - high resolution – MS in clinical & forensic toxicology

Hans H. Maurer
Department of Experimental and Clinical Toxicology, Saarland University
D-66421 Homburg (Saar), Germany, [email protected]

High-resolution mass spectrometry (HRMS) using double focusing mass analyzers was
established in the 1960ies and revolutionized the elemental analysis of organic compounds. It
became a powerful tool also in bioanalytical research and practice providing high selectivity
and specificity. After developing modern LC-MS interfaces using electrospray ionization and its
wide application in bioanalytical as well as in “omics” research and practice, the manufacturers
realized a broad market for HRMS. Since the last few years, robust LC-MS apparatus mainly
using time-of-flight or Orbitrap mass analyzers are getting more and more affordable to many
laboratories. This is also represented by the remarkable increase of publications and review
articles. Therefore, the aim of the presentation will be to give an overview on the current use
of HRMS in general and in particular for drug screening and quantification, and drug
metabolism studies. Limitations and further perspectives will close the presentation.

References
[1] H.H. Maurer. Perspectives of liquid chromatography coupled to low and high resolution mass spectrometry
for screening, identification and quantification of drugs in clinical and forensic toxicology [review]. Ther.
Drug Monit. 2010, 32, 324.
[2] M.R. Meyer, H.H. Maurer. Current applications of high-resolution mass spectrometry in drug metabolism
studies [review]. Anal. Bioanal. Chem. 2012, 403, 1221.
[3] H.H. Maurer. What is the future of (ultra)high performance liquid chromatography coupled to low and high
resolution mass spectrometry for toxicological drug screening? [review]. J. Chromatogr. A 2013, 1292, 19.
[4] M.R. Meyer, A.G. Helfer, H.H. Maurer. Current position of high-resolution mass spectrometry for drug
quantification in clinical & forensic toxicology [review]. Bioanalysis 2014, 6, 2275.
[5] A.G. Helfer, J.A. Michely, A.A. Weber, M.R. Meyer, H.H. Maurer. Orbitrap technology for comprehensive
metabolite-based liquid chromatographic-high resolution-tandem mass spectrometric urine drug
screening - exemplified for cardiovascular drugs. Anal. Chim. Acta 2015, 891, 221.

24
 "Advances in metabolic profiling - miniaturisation and metabolic phenotyping"

Ian D Wilson

Metabolic phenotyping (metabotyping) holds the promise of improving our understanding of the
underlying biology of normal physiological processes such as growth and aging as well as defining
mechanisms of disease, drug action, metabolism and toxicity. UPLC-MS, employing sub 2 micron
particles and 2.1 mm i.d. columns of varying lengths from 5 to 15 cm, is now a standard platform for
both global and targeted metabolite profiling. However, whilst this configuration has proved to be
both robust and efficient, moving to UPLC-based microbore chromatography, with 1 mm i.d.
columns, offers the possibility of making large gains via reduced sample and solvent consumption
(thereby maximising the use of often limited samples and reducing consumable costs) and
environmental impact. However, to be accepted, microbore systems have to be shown to be capable
of providing efficient, repeatable and robust methods suitable for analysing large numbers of
samples. An example of the successful transfer of a UPLC profiling method using a “conventional”
2.1 mm. i.d column-based to a 1 mm i.d. format will be provided with only minor modifications to an
existing UPLC system will be provided. Further gains in productivity can be achieved via the
modification of the gradient profile and compressing the separation into a shorter time. This
approach using rapid microbore metabolic profiling (RAMMP) via UPLC-MS provides a high-
throughput analytical platform for metabolic profiling and can enable that analysis of large numbers
of samples in a single run, eliminating the need to analyse them in multiple batches. Reducing the 12
min analysis time of our standard method to 2.5 min lead to a reduction in peak capacity, from 150
to 50, and in the number of features detected, from ca. 19, 000 to ca. 6000. However, the RAMMP
method achieved similar levels of group discrimination to that of “conventional” UPLC-MS when
applied to rat urine obtained from studies on the effects of 2-bromophenol and acetaminophen.
Despite the reduction in peak resolution the same features were responsible for discrimination of
the different sample groups whether the conventional or RAMMP data was examined. The
combination of reduced column diameter and length together with increased linear velocity and
high pressures thus provided a five-fold reduction in analysis time, a ten-fold reduction in solvent
consumption and the potential to also reduce sample consumption by ten-fold. Whilst we believe
that such microbore methods could become the new routine we are also beginning to see the
emergence of the next generation of capillary UPLC-MS and examples of the application of these
new systems will be also shown, perhaps providing a glimpse of the future.

25
26
Characterization of culture media containing non-ionic detergents by HILIC-ESI-TOF-MS
Eleni Lazaridi1, Nikoline J. Nielsen1, Lea Giørtz Johnsen2
1
Analytical Chemistry Group, Department of Plant and Environmental Sciences, Faculty of Science,
University of Copenhagen, Thorvaldsensvej 40, Frederiksberg 1871, Denmark
2
MS-Omics, Thorvaldsensvej 40, T644, Frederiksberg 1871, Denmark

Detergents are being widely used in cosmetics, drugs, food, and in production of microorganisms
due to their amphipathic nature (a nonpolar “tail” and a polar “head”) that allows them to act as
solubilizers, emulsifiers and stabilizers [3]. Non-ionic detergents, such as Poloxamer 188 and
Polysorbate 80, in culture media may interfere with the analyte separation and dete ction, and in
particular with the analyte quantification when using liquid chromatography - mass spectrometry
(LC-MS). It is very likely that the presence of detergents also gives rise to ion-suppression, and hereby
interferes with quantification of co-eluting compounds. Moreover, Polysorbate 80 can cause challenges
either to compound quantification due to its high ionisability or in case of lipid quantification fatty
acids can be produced when Polysorbate 80 is methylated [2]. Poloxamer 188 can interfere with
lipid extraction and recovery due to its emulsifier qualities [2].
Methods based on size-exclusion chromatography have been used for detergent removal, however
these methods cannot be used for quantification of large molecules i.e. metabolites, or
compounds that have similar molecular weight as the non -ionic detergent [1]. An alternative
strategy was suggested by Jäpelt et al (2016) where Polysorbate 80 was at least partly removed by
zinc-complexation followed by thiocyanate precipitation.
In this work it will be shown that this is also the case for another non-ionic detergent (Poloxamer 188),
accompanied by a full characterization of the two non-ionic detergents as well as by testing the
precipitation procedure in real samples. During the preliminary investigation the two non-ionic
surfactants, as well as the precipitation solution: zinc nitrate and ammonium thiocyanate were
characterized by HILIC-ESI-TOF-MS. Preliminary results showed that 1) both Poloxamer 188 and
Polysorbate 80 give rise to chromatographic peaks originating from multiple homologues series, 2)
detergents induce massive ion suppression, 3) the precipitation solution give rise to several
chromatographic peaks and 4) the metal ions in combination with thiocyanate can precipitate both
detergents tested.

References
1. Jäpelt, K., Johnsen, B., & Christensen, L. (2016). Removal of polysorbate 80 by complexation prior to
LC–MS analysis. Analytical and Bioanalytical Chemistry, 408(9), 2303-2307.
2. Shen, C., Hawari, J., & Kamen, A. (2004). Micro-quantitation of lipids in serum-free cell culture media: A
critical aspect is the minimization of interference from medium components and chemical
reagents. Journal of Chromatography B, 810(1), 119-127.
3. Zhang, R., Wang, Y., Tan, L., Zhang, H.Y., & Yang, M. (2012). Analysis of polysorbate 80 and its related
compounds by RP-HPLC with ELSD and MS Detection. Journal of Chromatographic Science, 50(7), 598-
607.

27
Two simple cleanup methods combined with LC-MS/MS for
quantification of steroid hormones in in vivo and in vitro assays
Johan JuhlWeisser & Cecilie Hurup Hansen & Rikke Poulsen & LizetteWeber Larsen & Claus Cornett & Bjarne Styrishave
Analytical BioSciences, Department of
Pharmacy, Faculty of Health and Medical Science, University of
Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark

Abstract Measuring both progestagens, androgens, corticosteroids as well as estrogens with a single method makes it
possible to investigate the effects of endocrine-disrupting chemicals (EDCs) on the main pathways in the mammalian
steroidogenesis. This paper presents two simple methods for the determination of the major steroid hormones in biological
matrixes using liquid chromatography tandem mass spectrometry (LC-MS2). A novel method was developed for the
determination of 14 steroids in the H295R in vitro assay without the need for solid phase extraction (SPE) purification prior
to LC-MS2 analysis. The in vitro assay was validated by exposing H295R cells to prochloraz for inhibiting steroid hormone
secretion and by exposing cells to forskolin for inducing steroid hormone secretion. The developed method fulfills the
recommendations for the H295R assay suggested by the OECD. Furthermore, a simple off-line SPE methodology
was developed for the necessary clean-up of in vivo assays. Samples, such as gonad tissue, plasma and serum, are
complex biological matrixes, and the SPE methodology was optimized to remove salts and proteins prior to elution of
target analytes. At the same time, lipophilic compounds were retained on the SPE cartridge during elution. This, combined
with the multi-steroid LC-MS2 method, made it possible to determine 10 steroids in male Sprague-Dawley rat gonad
tissue. Furthermore, it was possible to quantify 6 steroids in the plasma. In general, the observed concentration of steroid
hormones in plasma, testes, and H295R cell medium corresponded well with previous studies. The off-line SPE
method was validated using spiked charcoal-stripped serum. Method recovery, accuracy, precision and robustness were all
good. Instrument sensitivity was in the range of 55–530 pg/mL (LLOQ).

28
Selenium as an elemental detection label for quantification of Peptides
A comparison to fluorophore labelled peptide
Møller, L. H. , Bahnsen, J. S.a, Nielsen, H. M.a, Østergaard, J.a, Stürup, S.a & Gammelgaard, B.a
a

a
University of Copenhagen, Department of Pharmacy

Peptides are involved in many physiological processes and are increasingly used as drug
candidates. Peptide drugs most often offer high efficacy, selectivity and specificity and
additionally low toxicity due to their similarity to natural occurring compounds in the body. In
the development of new drugs, parameters as stability, pharmacokinetics and metabolism
must be monitored and thus sensitive quantitative analytical methods are of great importance
but can be a challenge in complex biological matrices.
In this study, selenium was introduced as an elemental detection label. By exchange of the
naturally occurring amino acid Met with the selenium containing analogue, Se was introduced
as an inherent label in the peptide structure and allowed for quantification by inductively
coupled plasma mass spectrometry (ICP-MS). The impact on peptide properties of labelling
peptides with the fluorophore TAMRA or the selenium (Se) containing amino acid SeMet was
evaluated. The cell-penetrating peptide (CPP) penetratin (Pen) was used as a model peptide
and three differently labelled variants of were synthesized; one labelled with Se (PenMSe), one
labelled with TAMRA (TAMRA-Pen) and one labelled with both Se and TAMRA (TAMRA-PenMSe).
The labelled peptides were characterized in terms of structural and physicochemical properties
as well as uptake efficiency in HeLa cells. Furthermore, stability of the labelled peptides and
identities of degradation products in cell media and cell lysates were evaluated by LC-ICP-MS,
LC-FLD and LC-ESI-MS.
Selenium labelling caused minimal change in hydrodynamic radius and the physicochemical
properties of the peptide and allowed for absolute quantitative determination of cellular uptake
by ICP-MS. Selenium is thus proposed as a promising alternative label for quantification of
peptides in general, altering the properties of the peptide to a minor extent as compared to
commonly used peptide labels.

29
Comparison of three sample preparation procedures for the quantification of L-Arginine and
asymmetric dimethylarginine in human plasma

Authors: A.M.V. Schou-Pedersen, J. Lykkesfeldt, Department of Veterinary Disease Biology, Faculty of

Health and Medical Sciences, University of Copenhagen.

Background: Asymmetric dimethylarginine (ADMA), an L-Arginine derived metabolite, is an endogenous

competitive inhibitor of endothelial Nitric Oxide Synthase (NOS), which generates the vasodilating NO. An
increased concentration of ADMA has been correlated with the possible development of coronary heart
diseases and ADMA is therefore considered to be an important early biomarker for endothelial dysfunction.
The most widely used analytical method for the quantification of L-Arginine and ADMA rely on laborious
solid-phase extraction (SPE), derivatization with a fluorescent tag followed by HPLC separation and
fluorescence detection.

Aim: The aim of the current study was to investigate if simple and robust protein precipitation (PP) could
replace SPE sample preparation. 2 types of PP procedures: (A) PP using cold TCA, 1 M, followed by
neutralization and (B) PP using a mixture of cold ammonia, 25 %, and acetonitrile (10:90, v/v) were
compared to procedure (C) consisting of Oasis MCX SPE clean up described in several publications. The
three sample preparation procedures were evaluated with regard to recovery, precision, resolution,
stability of samples and simplicity.

Methods: Separation of the ortho-phtaldialdehyde-mercaptoethanol derivatives of L-Arginine and ADMA

was performed at 40 °C on a Phenomenex Gemini (150 x 4.6 mm, 5 µm) column. The mobile phase
consisted of 50 mM potassium phosphate (pH 6.5) containing 14 % (v/v) acetonitrile. Detection was
obtained at excitation wavelength of 340 nm and emission wavelength of 455 nm. Recovery of the three
sample preparation procedures was determined by spiking human plasma with standard solutions of L-
Arginine and ADMA (n=3). The precision was estimated from the recovery experiment. The stability of the
samples was tested at 4 °C after 24 and 48 hours.

Results: The recovery (mean ± SEM) obtained with procedure (A) was found to 109 ± 1.75 % for L-Arginine
and 98.0 ± 5.84 % for ADMA. In comparison, procedure (B) showed low recoveries of 68.4 ± 3.18 % for L-
Arginine and 75.4 ± 7.59 % for ADMA. Regarding procedure (C), recovery for L-Arginine was 118 ± 2.02 %
and for ADMA 94.7 ± 4.89 %. The precision was acceptable for all three procedures. Figure 1a and 1b shows
examples of chromatograms obtained from either procedure (A) or procedure (C), respectively. Resolution
was considered to be acceptable, although the resolution of procedure (A) was slightly better. The stability
of the obtained supernatants was for all three procedures found to be acceptable (>90 % of parent) within
48 hours of storage at 4 °C.

Conclusion: Sample preparation procedure (A) and (C) performed similarly well regarding recovery,
precision, resolution and stability of samples. Procedure (B) was disregarded due to low recoveries. Taking
the simple and robust sample preparation of procedure (A) into account, this procedure was selected for
further validation studies. The validated analytical method has proven useful in quantifying L-Arginine and
ADMA in human plasma from clinical studies.

30
Fig. 1 Chromatograms of a human plasma sample extracted with either a) procedure (A) or b) procedure (C). The
inserts show the resolution of ADMA to the similar, but inactive symmetric dimethylarginine (SDMA).

31
32
The use of metabonomics to detect cases of food fraud in chicken eggs

Amy Johnson and Dr David Thompson, Keele University

Food fraud is a centuries old problem that has been increasing in occurrence over recent years due
in part to the globalisation of food supply chains. One particular aspect of food fraud that has
recently experienced an increase in prevalence is the misrepresentation of conventional food
products as organic, due to increasing consumer demand for organic produce. Chicken eggs are one
of the more popular food products affected by this type of food fraud, with conventional eggs being
misrepresented as organic, and thus are the focus of this research. This fraudulence of eggs, as well
as being morally ambiguous, can also have an economic impact. To avoid these negative implications
of food fraud, there are various laws and regulations in place to try to control the issue.

However, these laws and regulations rely on having suitable methods of detection for instances of
food fraud. Many current methods used to authenticate food products are based on detecting single
compounds in the samples, which makes it relatively easy for fraudsters to adulterate the products
with these compounds. The main aim of this research is to use metabonomic technologies and
chemical analysis methods, including the analysis of quality control samples, to develop metabolic
profiles of both conventional and organic eggs. This will result in a highly robust method of
discrimination between these eggs, using various different biomarkers and their concentrations,
making it much more difficult for fraudsters to adulterate their products.

This research has thus far obtained some preliminary results showing the metabolic differences
between cage and organic eggs, following HPLC-MS and statistical analysis.

33
34
Rapid Conformational Analysis of Protein Drugs in Formulation by
Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS)

Zeinab E. Nazari 1, Marco van de Weert 1, George Bou-Assaf 2, Damian Houde 2, Andrew Weiskopf 2, Kasper D.
Rand *1
1
Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen,
Universitetsparken 2, Co-penhagen 2100, Denmark
2
Department of Protein Pharmaceutical Development, Biogen, 225 Binney St., Cambridge Massachusetts
02142, USA

ABSTRACT:

Higher order structure characterization of protein therapeutics is of considerable importance for validating
function and safety, as well as in comparability studies of innovator biologics and their biosimilars. Hydrogen
Deuterium Exchange coupled to Mass Spectrometry (HDX-MS) has become an established method for
characterization of higher-order protein structure and dynamics. Here, we present a customized HDX-MS
methodology that uses manual Solid-Phase Extraction (SPE) to allow fast and simplified conformational analysis
of proteins under pharmaceutically relevant formulation conditions. Of significant practical utility, the
methodology allows global HDX-MS analyses to be performed without refrigeration or external cooling of the
setup. Real-world applicability was demonstrated by HDX-MS analysis of interferon-β-1a in formulation as well
as the model compounds insulin and angiotensin II. Using the customized SPE-HDX-MS setup, extraction of
labeled protein from the pharmaceutical formulation was optimized while monitoring the loss of deuterium
during analysis (back exchange). Two modes of operation where identified. In Mode 1, we used DMSO-
containing solvents for SPE, allowing the HDX-MS analysis to be performed at acceptable back exchange levels
(<30%) without the need for cooling any components of the setup. In mode 2, SPE and chromatography was
performed using a fast isocratic elution at 0 °C resulting in back exchange levels of 10-30%. Key advantages of
the methodology include easy implementation, low sample use, possibility to use solvents of choice for
optimized excipient removal from individual formulations, and fast data acquisition. Our results indicate that
the SPE-HDX-MS system can be used as a reliable and robust method for fast conformation analysis of proteins
in their intended formulations and thus could help to unleash the potential of HDX-MS methodology to address
several current analytical challenges in pharmaceutical development research.

35
 Flow-Induced Dispersion Analysis for Probing Anti-dsDNA Antibody Binding
Heterogeneity in Systemic Lupus Erythematosus Patients
Nicklas N. Poulsen1, Morten E. Pedersen1, Jesper Østergaard1, Nickolaj J. Petersen1,
Christoffer T. Nielsen2, Niels H. H. Heegaard2,3 and Henrik Jensen1
1
Department of Pharmacy, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark;
2
Department of Autoimmunology & Biomarkers, Statens Serum Institut, Artillerivej 5, 2300 Copenhagen, Denmark;
3
Department of Clinicial Biochemistry, Odense University Hospital, University of Southern Denmark, Sdr.
Boulevard 29, 5000 Odense C.

Detection of immune responses is important in the diagnosis of many diseases. For

example, the detection of circulating autoantibodies against double-stranded DNA
(dsDNA) is used in the diagnosis of Systemic Lupus Erythematosus (SLE). It is,
however, difficult to reach satisfactory sensitivity, specificity and accuracy with
established assays. Also, existing methodologies for quantification of autoantibodies
are challenging to transfer to a point-of-care setting.
Here we present the use of Flow-Induced Dispersion Analysis (FIDA)1 for rapid
(minutes) measurement of autoantibodies against dsDNA. A schematic presentation of
the assay is given in figure 1.
Figure 1. The automated FIDA protocol is
performed using commercial equipment
for capillary electrophoresis. The inlet
vials contain sample, indicator solution
and washing solutions. A hydrodynamic
flow is employed giving rise to a parabolic
flow profile (insert). The dispersion of the
indicator measured at the detector
increases if antibodies in the sample have
bound the indicator

Short fluorescently marked dsDNA sequences of approximately 30 base pairs were used
as indicators (ligands). The diffusivity of the indicator is measured by Taylor Dispersion
Analysis (TDA). A narrow indicator zone is injected in a capillary prefilled with sample
and the zone is eluted with sample. If the sample contains antibodies against double-
stranded DNA the indicator (dsDNA) will be part of a larger complex and the apparent
diffusivity decreases. The assay is fully automated with the use of standard CE based
equipment employing fluorescence detection.
FIDA is robust towards matrix effects as demonstrated by the binding curves obtained
in up to 85% human blood plasma. The binding curves were obtained with a model
monoclonal antibody against dsDNA. FIDA allows for flexible exchange of the DNA
sequences used to probe for the autoantibodies. Plasma samples from SLE positive
patients were analyzed using the new FIDA methodology as well as by standard indirect
immunofluorescence and solid-phase immunoassays. Interestingly, the patient
antibodies binds DNA sequences with different affinities, suggesting pronounced
heterogeneity amongst autoantibodies produced in SLE.
In conclusion, the FIDA based methodology is a new approach for autoantibody
detection, and holds promise for being used for patient stratification and monitoring of
disease activity.
(1) Nicklas N. Poulsen, Nina Z. Andersen, Jesper Østergaard, Guisheng Zhuang, Nickolaj J. Petersen and Henrik
Jensen, Analyst, 2015, 140, 4365-4369.

36
 Simple and versatile thiol-ene - based microfluidic devices for chromatographic separations

Alexander Jönsson, Unai Orruño, Jörg P. Kutter and Josiane P. Lafleur

Department of Pharmacy, University of Copenhagen, DENMARK

Porous polymer monoliths are a new category of materials which have attracted lots of interest as alternatives to
packed beds in the last 20 years. They take advantage of the ease of processability associated with polymers to
generate monoliths, films, and beads, often with well-defined porosities and high specific surface areas. These
materials are prepared using a very simple molding process carried out within the confines of a closed mold, such
as the microfluidic chip channel. We have recently reported a novel, rapid and simple method for the preparation
of emulsion-templated monoliths in microfluidic channels based on thiol–ene chemistry. The method allows
monolith synthesis and anchoring inside thiol–ene microchannels in a single photoinitiated step. These monoliths
offer promise as stationary phases for on-chip separations thanks to their narrow size distribution and the
possibility to easily modify their surfaces with chemical groups for various retention modes. The cross-linked
beads of the monolithic structure closely resembles a packed column but with a higher degree of packing. This
makes the on-chip microcolumn a convenient replacement for the, often difficult, packing of columns in
microchannels. For reverse-phase separations, alkyl mercaptans or 1-alkenes can be covalently bonded to the
surface of the monolith via thiol-ene click chemistry. We will present preliminary results demonstrating on-chip
solid phase extraction (SPE) of model analytes using these novel high surface area materials.

Additionally, the thiol-ene microfluidic channels support electroosmotic flow, allowing for separation via
Microchip Capillary Electrophoresis (MCE) or ElectroChromatography (MCEC). For example, chiral molecules
can be separated within a thiol-ene based microfluidic device by the addition of cyclodextrins to the running
buffer or by immobilizing the cyclodextrins directly to the channel walls via thiol-ene chemistry. We will present
preliminary data on the separation of chiral amino acids in thiol-ene microfluidic chips using these principles.

37
Detecting the differences between dead on arrival and normally slaughtered
chickens using metabonomic profiling

Kate Sidwick and David Thompson

Keele University

Food fraud is a challenge within the global food industry that affects the public on
an everyday basis. This was apparent in 2013 when horse meat was discovered in
processed beef products. The confidence in the meat industry has subsequently
decreased, causing a need for reliable methods for meat authentication to be
developed.

Methods for detecting food fraud usually involve searching for the presence or
absence of a certain substance within the product to confirm its authenticity, but this
can be easily adulterated. This project aims to use metabonomic methods to create a
metabolite fingerprint of the product, leading to a more reliable technique using a
combination of markers, and their concentrations, to authenticate the product, which
is much more difficult to falsify. This will involve using liquid chromatography-
mass spectrometry based protocols, including established quality assurance
methodology.

Specifically, animals that have deceased before slaughter are not allowed to be sold
as food produce, so this is an area that can be targeted by food fraud. The Animal
By-Products (Enforcement) Regulations 2013 states that all animals found to be dead
on arrival to the slaughterhouse must be removed and stained with a colouring
agent in order to distinguish it as a product not fit for human consumption. Large
portions of poultry are dead on arrival to the slaughterhouse, which decreases the
amount of sellable products for that company. This gives an economic reason for
fraudulent activity to occur where the dead on arrival poultry is processed normally
and allowed to continue into the human food chain. At present, there are no
methods to detect this specific food fraud. With the use of metabonomic methods,
markers could be found to determine whether the product was in fact dead on
arrival and should not be in the food chain.

38
 Analysis of pyrolysis oils on GC-qTOF and GCxGC-qTOF
Mette Kristensen1*, Peter Christensen1, Jan H. Christensen1
1
University of Copenhagen, Department of Plant and Environmental Sciences, Frederiksberg,
Denmark
*
Corresponding author e-mail: [email protected]

Keywords: PetroPhase 2016, Denmark, Comprehensive Gas Chromatography, Pyrolysis Oils.

Abstract
Gas chromatography coupled to mass spectrometry (GC-MS) has for a long time been the preferred
analytical tool in the area of petroleomics, but not all petroleum components are easy to measure with
this analytical platform and focus has mainly been on hydrocarbons and PAHs. However, the most
surface active compounds are expected to be some of the heavier oxygen- and nitrogen containing
compounds, and especially the interest in analysing carboxylic acids has increased. This compound
class is also suspected to be responsible for wettability in oil reservoirs [1] and for being potentially
toxic to aquatic organisms [2]. In this study the focus will be on analysing pyrolysis oil. Pyrolysis oil
is made by an anaerobic distillation of biomass and contains a higher fraction of carboxylic acids,
alcohol, ketones, aldehydes and other water-soluble compounds compared to crude oils. To analyse
some of the oxygen- and nitrogen containing compounds in pyrolysis oil, derivatization is necessary
to make them GC compatible. Four derivatization agents are tested in this study: BSTFA + 1%
TMCS, BF3•MeOH, MCF and HCl:MeOH. With the use of online derivatization, gas
chromatography and high resolution mass spectrometry (GC-qTOF), we aim to describe a
comprehensive method for fingerprinting of oxygen- and nitrogen-containing compounds in pyrolysis
oils. Different emission current and acquisition rates of the GC-qTOF system are tested and the
optimal settings are found. For further separation of this complex mixture, comprehensive two
dimensional gas chromatography coupled to high resolution mass spectrometry is applied (GCxGC-
qTOF). The final methods (GC-qTOF and GCxGC-qTOF) are applied to characterize oxygen- and
nitrogen-containing compounds in pyrolysis oil.

[1] Standal et al., Journal of Petroleum Science and Engineering 24, 131–144 (1999)

[2] Aeppli et al., Environ. Sci. Technol. 46, 8799−8807 (2012)

39
40
LC-MS/MS-based monitoring of in vivo protein biotransformation: quantitative
determination of trastuzumab and its deamidation products in human plasma
Peter Bults1,2, Rainer Bischoff2, Hilde Bakker1, Jourik Gietema3 and Nico van de Merbel1,2
1)
PRA Health Sciences, Assen, the Netherlands
2)
Department of Analytical Biochemistry, University of Groningen, the Netherlands
3)
Department of Medical Oncology, University Medical Center Groningen, the Netherlands

With this poster we present an LC-MS/MS-based method for quantitatively monitoring the in vivo
deamidation of the biopharmaceutical monoclonal antibody trastuzumab at a crucial position in one of its
complementarity determining regions (CDR) [1].

The Asn55 residue in the heavy chain of trastuzumab is susceptible to deamidation in vitro and in vivo. As this
amino acid is located at the top of the CDR2 loop of trastuzumab, which is involved in the binding of
trastuzumab to the HER2/neu receptor, it is not unlikely that deamidation of Asn55 may result in a
conformational change in the CDR2, thus leading to changes in binding affinity of trastuzumab to its receptor
and, potentially, to an altered pharmacological effect.

The multiplexed LC-MS/MS assay using selected reaction monitoring (SRM) allows simultaneous quantitation
of five molecular species derived from trastuzumab after tryptic digestion: a stable signature peptide
(FTISADTSK), a deamidation-sensitive signature peptide (IYPTNGYTR), its deamidated products (IYPTDGYTR
and IYPTisoDGYTR), and a succinimide intermediate (IYPTsuccGYTR). Digestion of a 50 μL plasma sample is
performed at pH 7 for 3 h at 37 °C, which combines a reasonable (>80%) digestion efficiency with a minimal
(<1%) formation of deamidation products during digestion.

The LC-MS/MS method was validated in accordance with international bioanalytical guidelines over the
clinically relevant range of 0.5 to 500 μg/mL with bias and CV values well below 15%. Deamidation of
trastuzumab was observed in plasma both in a 56 day in vitro forced degradation study (up to 37% of the total
drug concentration) and in samples (in vivo) obtained from breast cancer patients after treatment with the
drug for several months (up to 25%). Comparison with a validated ELISA method for trastuzumab showed that
deamidation of the drug at the CDR leads to a loss of recognition by the antibodies used in the ELISA assay.

[1] Bults et al, Anal. Chem., 88 (2016) 1871-1877.

Figure: LC-MS/MS and ELISA results in plasma obtained from a breast cancer patient on long-term treatment
with trastuzumab. LC-MS/MS concentrations obtained for peptides FTISADTSK, IYPTNGYTR,
IYPTDGYTR, and IYPTisoDGYTR.

41
Title:
Efficient, Effective, and Proven Approach to Chiral Method Development
for Purification Scale Up

Authors:
J Preston, Michael McCoy, Sean Orlowicz and Ramkumar Dhandapani

Locations:
Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA

Presenter at the symposium:

Theis Kirkhoff Guldbech

Abstract:
With the majority of today’s pharmaceuticals being chiral compounds, and a renewed focus on the
differences in biological activity of isomers, chiral separations and isolations have continued to grow in
importance. Our lab screened hundreds of compounds on various Chiral Stationary Phases (CSPs), and over
the years has developed an efficient experimental strategy for identifying the appropriate CSP for a unique
chiral separation. Due to the success of this chiral screening service, many customers have returned asking
for the isolation of pure enantiomers. As a result, we have explored the additional challenges of chiral
separations as we scale up to preparative HPLC. In this poster, we demonstrate through case studies, a
unique and effective approach to both CSP screening and subsequent scale up for chiral small molecules.
When screening compounds for the appropriate CSP, the analyte’s physical properties often define the mode
of chromatography most likely to be successful for any given CSP. Screening Normal Phase, Reversed Phase
, Polar Organic, and SFC modes of chromatography on a selection of six orthogonal polysaccharide-type
phases has proven to be successful for more than 80 % of the unique submissions. Mobile phase composition
in each mode is optimized to achieve resolution. When isolating enantiomers, we must further consider the
physical and chemical properties of the analytes. Preparative isolations on various CSPs can be drastically
affected by scale up phenomena such as; loading capacity of the CSP itself, stability of the sorbent, and
nonlinear chromatography. In summary, through real world examples we have developed an effective
strategy for identifying chromatographic conditions for chiral separation of small molecules with moderate
chiral complexity. We have successfully demonstrated the ability to transition those analytical separations to
preparative isolations.

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