rising stars of separation science - unime europe poster supplem… · rising stars of separation...

Rising Stars of Separation Science

Exclusive Supplement fromDecember 2019

Volume 32 Number s12www.chromatographyonline.com

LC•GC Europe December 20192

ContentsISC 2018 Poster WinnersGenzo Shimadzu – Best Poster Award Winners

7 Influence of the Immobilization Chemistry on Chromatographic Features of Reversed-Phase/Weak Anion Exchange Mixed-Mode Silica Gel Stefanie Bäurer, University Tübingen, Germany. The objective of this poster was to decrease column bleeding and develop a better MS-compatible surface chemistry with increased longevity with selectivity for polar and charged analytes. A reversed phase–weak anion exchange stationary phase was synthesized and characterized.

9 Dilute-and-Shoot Analysis of Therapeutic Monoclonal Antibody Glycosylation from Fermentation Broth: A Method Capability Study Yoric Gagnebin, University of Geneva, Switzerland. This poster presents an original metabolomics workflow offering an improved metabolome coverage based on complementary chromatographic conditions hyphenated to mass spectrometry. A processing strategy was developed for data fusion to simplify the biological interpretation. More than 250 metabolites were investigated in plasma samples from a chronic kidney disease (CKD) cohort study. This workflow allowed patient stratification according to CKD stages and helped to generate biological hypotheses based on the metabolomic profiles.

10 Superhydrophobic Capillary Coatings for Electrophoretic Separations Charly Renard, University of Montpellier, France. One important issue in capillary electrophoresis separation is the loss of efficiency as a result of adsorption of the analytes on the capillary walls. One solution to this issue is to make superhydrophobic capillaries to reduce the interaction between the capillary and the analytes in aqueous solution. In this poster, a method was discovered to make a superhydrophobic capillary and then characterize their electrophoretic and hydrodynamic behaviour.

11 Preparative Comprehensive Two-Dimensional Chromatography: Comparison of CPC×LC and PrepLC×LC for the Isolation of Multiple Targets from Edelweiss Plant. Léa Marlot, University of Lyon, France. This poster describes a novel preparative 2D LC method to isolate multiple targets from the Edelweiss plant.

12 Dilute-and-Shoot Analysis of Therapeutic Monoclonal Antibody Glycosylation from Fermentation Broth: A Method Capability Study Therese Wohlschlager, University of Salzburg, Germany. The development and production of therapeutic monoclonal antibodies requires stringent control to ensure a specific product profile. The monitoring of product attributes involves the analysis of differential glycosylation, oxidation, deamidation and glycation, which is conventionally performed at the peptide level after purification and proteolytic digestion of the target protein. This poster presents a fast and robust analytical workflow for comprehensive characterization of antibody variants at the intact protein level directly in fermentation broth.

cccTA Best Poster Award

13 Off-Line Comprehensive Two-Dimensional Separation of Petroleum Sample by SEC×RPLC–ICP-MS/MS Marie Bernardin, Universite Claude Bernard Lyon, France This poster is a collaboration between the Institute of Analytical Sciences and IFPEN. The aim is to show potential and limitations of off-line comprehensive two-dimensional separation of petroleum sample by SEC×reversed-phase LC–ICP-MS in the specific case of organic matrices.

Chromatographia and ABC Best Poster Award

14 Native Asymmetrical Flow Field-Flow Fractionation and Size-Exclusion Chromatography for Studying the Aggregation of Beta-D-Galactosidase Iro Ventouri, University of Amsterdam, The Netherlands. This poster focuses on the evaluation of non-destructive liquid-phase separation techniques asymmetrical flow-field flow fractionation (AF4) and size-exclusion chromatography (SEC) to study the enzyme Beta-galactosidase. Beta-galactosidase is a rather large enzyme and its analysis requires techniques that can provide structural information, which can be further correlated with its catalytic activity.

3www.chromatographyonline.com

15 Influence of Primary Amine Structure in Hyperbranched Functional Layer on the Performance of Anion Exchangers for Ion Chromatography Anna Uzhel, Lomonosov Moscow State University, Russia. The purpose of this poster was to propose a way of manipulating selectivity of covalently-bonded hyperbranched anion exchangers towards weakly-retained carboxylic acids. The influence of primary amine structure in the internal part of the hyperbranched functional layer on the column performance and selectivity, was evaluated and also the estimated temperature effect on the retention of organic acids.

17 Quantification of Four Gaucher Disease Biomarkers in Plasma and Red Blood Cells: Correlation with Disease Severity and Response to Therapeutic Intervention Caroline Chipeaux, University of Paris-Sud, France. In this poster a UHPLC–MS/MS method for the simultaneous quantification of four biomarker candidates of Gaucher disease (GD) was developed. When applied to RBC extracts from GD patients, this method could validate one of the four biomarker candidates and also confirm the correlation between the rheological abnormalities of RBC and its SL composition.

18 Liquid Chromatography – High – Resolution Mass Spectrometry of Polydisperse Surfactants Used in Oil Industry Alizee Dufour, ESPCI Paris, France. In the oil industries, the control of the water–oil emulsion is mandatory. In this context, it is essential to have analytical tools to quantify surfactants used. The complexity of the matrix and the polydispersity of surfactants requires the development of improved analytical strategies involving high-resolution chromatography hyphenated to high-resolution mass spectrometry, which are described in this poster.

18 Innovative Screening Approaches for the Determination of Non-Intentionally Added Substances in Food Contact Materials Chrysoula Kanakaki, OFI Austrian Research Institute for Chemistry and Technology, Austria. This poster established an analytical basis for the identification and semi-quantification of volatile NIAS in FCMs by developing and comparing several analytical methods, for example, DHS-GC–MS, SPME–GC–MS, SBSE–GC–MS, GC×GC–MS, GC–MS/MS and GCO, and targeting the optimization of food packaging materials and their processing conditions for different sterilization procedures. The combined data from the analytical approaches was applied to original samples and products of migration studies.

AFSEP Awards (oral poster presentations)

19 Application of Linear Retention Indices in Liquid Chromatography for Reliable Characterization of Oxygen Heterocyclic Compounds in Cosmetics Adriana Arigò, University of Messina, Italy. This poster provides analytical strategies to determine furocoumarins in Citrus essential oils by overcoming the low selectivity and sensitivity of PDA detection. A new HPLC–MS/MS method is proposed to characterize furocoumarins at trace level in finished cosmetic products. A linear retention index system is used in combination with PDA, MS, and MS/MS libraries to allow a reliable characterization of several oxygen heterocyclic compounds in complex samples.

21 An Online Four-Dimensional SEC×SEC-IM×MS Methodology for In-Depth Characterization of Forced Degraded Monoclonal Antibodies Anthony Ehkirch, University of Strasbourg, France. The main technique for mAb size-variant assessment is size-exclusion chromatography (SEC) for the detection and quantitation of HMWS and LMWS, but this does not enable accurate determination of the molecular mass of the different species. This poster presents an innovative multidimensional analytical approach combining comprehensive online two-dimensional chromatography. The method involves SEC with a nonvolatile mobile phase and SEC with a volatile mobile phase connected to ion mobility and MS (IM-MS) for analytical characterization of forced degraded mAbs under nondenaturing conditions.

22 Enantioselective Chromatography for the Determination of Histidine Dipeptides in Food and Food Supplements Ettore Gilardoni, Università degli studi di Milano, Italy. Histidine dipeptides are natural products present in mammals with several biological functions. Carnosine is the most studied one because it is endogenous and carnosine-based food supplements are on the market. A quick-and-easy method for the separation of a mixture of histidine dipeptides and the quantification of carnosine was developed, allowing stereo-, chemo-, and regio-selectivity for impurity identification is described.

LC•GC Europe December 20194

HPLC 2019 Poster Winners

Agilent – Best Poster Award Winners

23 Three-Dimensional Chiral HPLC Analysis of Extraterrestrial Amino Acids in Carbonaceous Chondrites Aogu Furusho, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan. A highly selective three-dimensional chiral HPLC system was developed for the determination of extraterrestrial amino acids in the carbonaceous chondrites. The system was applied to the Murchison meteorite and the Antarctic meteorite, and all of the target amino acids were found in both meteorites. The D/L ratio of the non- proteinogenic amino acids were almost equal.

24 Comprehensive UHPLC–MS/MS Lipidomics Profiling to Study Effects of Betulin on Keratinocytes Carlos Calderon, University of Tübingen, Germany. This project is a comprehensive description of the lipidome of human immortalized keratinocytes and its changes following treatment with betulin. Our approach for identification of lipids combines information from precursor and product ions obtained from analyses in both polarities under same chromatographic conditions and from characteristic elution patterns of each lipid class in reversed phase-LC of each lipid class.

25 Screening Therapeutics According to Their Uptake Across the Blood-Brain Barrier: A High Throughput Method Based on Immobilized Artificial Membrane Liquid Chromatography-Diode-Array-Detection Coupled to Electrospray-Time-of-Flight Mass Spectrometry Giacomo Russo, Ghent University, Belgium. In this work, 79 neutral, basic, acidic and amphoteric structurally unrelated analytes were considered and their chromatographic retention coefficients on immobilized artificial membrane (IAM) stationary phase were determined employing a mass spectrometry (MS)-compatible buffer based on ammonium acetate.

25 Enantioselective Chromatography for the Determination of Histidine Dipeptides in Food and Food Supplements Jelle De Vos, Department of Chemical Engineering, Vrije Universiteit Brussel, Belgium. This project investigates the performance of columns packed with 1.7µm particles at high-pressure conditions (ΔPmax= 1034 bar) and links it to particle and column-bed integrity.

26 Chromatographic Behavior Of Bivalent Bispecific Antibodies on Cation Exchange Columns Lucas Kimerer, University of Virginia, USA. Investigating chromatographic behavior of bivalent bispecific antibodies which contain flexibly linked domains that can be present in extreme conformations with different chromatographic binding strength and interconvert with time scales on the order of chromatographic elution.

26 A New Gastro-Intestinal System to Evaluate the Effect of Food Methylglyoxal Lucia Ferron, Department of Drug Sciences, University of Pavia, Italy. A novel modular bioreactor for dynamic in vitro studies has been set-up, connecting two-dimensional (2D) scaffolds and mimicking a multi-organ model, to study the absorption/metabolization of compounds. The effect of dietary methylglyoxal, a potentially exogenous and endogenous toxic compound, on a dynamic gastro- intestinal system has been evaluated. Bioreactors represent a powerful advance in comparison with conventional in vitro static assays and could be a potential alternative to animal testing in the future.

27 Toolbox for Studying the Chemistry Of Light-induced Degradation (TooCOLD): Towards an Online Degradation System Mimi den Uijl, Universiteit van Amsterdam, Amsterdam, The Netherlands. In this project, degradation research has been performed to support the developments in the TooCOLD project. By analyzing the effect of parameters on the degradation of Annatto, a food dye mixture, with an off-line degradation system, the on-line degradation cell will be adapted and compared.

28 Retention Mechanism Study in Hydrodynamic Chromatography Noor Abdulhussain, Universiteit van Amsterdam, Amsterdam, The Netherlands. Hydrodynamic chromatography (HDC) is one of the most straightforward methods to achieve reliable size separations. In this project we revisit the existing HDC theory using computational fluid dynamics (CFD) simulations to gain more understanding of the principles of this separation technique.

30 Mixed Mode Stationary Phases and their Benefit in LC x LC Stefanie Bäurer, University of Tübingen, Germany. This poster presents an online full comprehensive impurity profiling method for the analysis of amino acids and possible impurities and degradation products with comprehensive detection.


Group Vice PresidentMike [email protected]

Editorial DirectorLaura [email protected]

Editor-in-ChiefAlasdair [email protected]

Managing EditorKate [email protected]

Associate EditorLewis [email protected]

Associate PublisherOliver [email protected]

Sales ExecutiveLiz [email protected]

Sales Operations ExecutiveSarah [email protected]

Senior Director, Digital MediaMichael [email protected]

Webcast Operations ManagerKristen [email protected]

Project ManagerVania [email protected]

Digital Production ManagerSabina [email protected]

Managing Editor, Special ProjectsKaylynn [email protected]

Art DirectorGwendolyn [email protected]

Graphic DesignerCourtney [email protected]

Corporate Chairman & Founder Mike Hennessy Sr

Vice ChairmanJack Lepping

President & CEOMike Hennessy Jr

Chief Financial OfficerNeil Glasser, CPA/CFE

Executive Vice President, OperationsTom Tolvé

Senior Vice President, ContentSilas Inman

Senior Vice President, I.T. & Enterprise SystemsJohn Moricone

Senior Vice President, Development & Enterprise SystemsJohn Paul Uva

Senior Vice President, Audience Generation & Product FulfillmentJoy Puzzo

Vice President, Human Resources & AdministrationShari Lundenberg

Vice President, Business IntelligenceChris Hennessy

Vice President, Corporate Branding & B2B MarketingAmy Erdman

Executive Creative Director, Creative ServicesJeff Brown

Subscriber Customer ServiceVisit chromatographyonline.com to request or change a subscription or call our customer service department on +001 218 740 6877

Sycamore House, Suite 2 Ground Floor, Lloyd Drive, Cheshire Oaks Business Park, Ellesmere Port, CH65 9HQ, UK Tel: +44 (0)151 353 3503

Daniel W. ArmstrongUniversity of Texas, Arlington, Texas, USAGünther K. BonnInstitute of Analytical Chemistry and Radiochemistry, University of Innsbruck, AustriaDeirdre CabooterDepartment of Pharmaceutical and Pharmacological Sciences, University of Leuven, BelgiumPeter CarrDepartment of Chemistry, University of Minnesota, Minneapolis, Minnesota, USAJean-Pierre ChervetAntec Scientific, Zoeterwoude, The NetherlandsJan H. ChristensenDepartment of Plant and Environmental Sciences, University of Copenhagen, Copenhagen, DenmarkDanilo CorradiniIstituto di Cromatografia del CNR, Rome, ItalyGert DesmetTransport Modelling and Analytical Separation Science, Vrije Universiteit, Brussels, BelgiumJohn W. DolanLC Resources, McMinnville, Oregon, USAAnthony F. FellPharmaceutical Chemistry, University of Bradford, Bradford, UKAttila FelingerProfessor of Chemistry, Department of Analytical and Environmental Chemistry, University of Pécs, Pécs, HungaryFrancesco GasparriniDipartimento di Studi di Chimica e Tecnologia delle Sostanze Biologicamente Attive, Università “La Sapienza”, Rome, ItalyJoseph L. GlajchMomenta Pharmaceuticals, Cambridge, Massachusetts, USADavy GuillarmeSchool of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Geneva, SwitzerlandJun HaginakaSchool of Pharmacy and Pharmaceutical Sciences, Mukogawa Women’s University, Nishinomiya, JapanJavier Hernández-BorgesDepartment of Chemistry (Analytical Chemistry Division), University of La Laguna Canary Islands, SpainJohn V. HinshawServeron Corp., Beaverton, Oregon, USATuulia HyötyläinenVVT Technical Research of Finland, FinlandHans-Gerd JanssenVan’t Hoff Institute for the Molecular Sciences, Amsterdam, The NetherlandsKiyokatsu JinnoSchool of Materials Sciences, Toyohasi University of Technology, JapanHuba KalászSemmelweis University of Medicine, Budapest, Hungary Hian Kee LeeNational University of Singapore, Singapore

Wolfgang LindnerInstitute of Analytical Chemistry, University of Vienna, AustriaHenk LingemanFaculteit der Scheikunde, Free University, Amsterdam, The NetherlandsTom LynchAnalytical consultant, Newbury, UKRonald E. MajorsAnalytical consultant, West Chester, Pennsylvania, USADebby MangelingsDepartment of Analytical Chemistry and Pharmaceutical Technology, Vrije Universiteit, Brussels, BelgiumPhillip MarriotMonash University, School of Chemistry, Victoria, AustraliaDavid McCalleyDepartment of Applied Sciences, University of West of England, Bristol, UKRobert D. McDowallMcDowall Consulting, Bromley, Kent, UKMary Ellen McNallyDuPont Crop Protection, Newark, Delaware, USAImre MolnárMolnar Research Institute, Berlin, GermanyLuigi MondelloDipartimento Farmaco-chimico, Facoltà di Farmacia, Università di Messina, Messina, ItalyPeter MyersDepartment of Chemistry, University of Liverpool, Liverpool, UKJanusz PawliszynDepartment of Chemistry, University of Waterloo, Ontario, Canada Colin Poole Wayne State University, Detroit, Michigan, USAFred E. RegnierDepartment of Biochemistry, Purdue University, West Lafayette, Indiana, USAHarald RitchieAdvanced Materials Technology, Chester, UKKoen SandraResearch Institute for Chromatography, Kortrijk, BelgiumPat SandraResearch Institute for Chromatography, Kortrijk, BelgiumPeter SchoenmakersDepartment of Chemical Engineering, Universiteit van Amsterdam, Amsterdam, The NetherlandsRobert ShellieDeakin University, Melbourne, AustraliaYvan Vander HeydenVrije Universiteit Brussel, Brussels, Belgium

The Publishers of LCGC Europe would like to thank the members of the Editorial Advisory Board for their continuing support and expert advice. The high standards and editorial quality associated with LCGC Europe are maintained largely through the tireless efforts of these individuals. LCGC Europe provides troubleshooting information and application solutions on all aspects of separation science so that laboratory-based analytical chemists can enhance their practical knowledge to gain competitive advantage. Our scientific quality and commercial objectivity provide readers with the tools necessary to deal with real-world analysis issues, thereby increasing their efficiency, productivity and value to their employer.

EDITORIAL ADVISORY BOARD

Follow us @ LC_GC

‘Like’ our page LCGC

Join the LCGC LinkedIn group

SUBSCRIPTIONS: LCGC Europe is free to qualified readers in Europe.To apply for a free subscription, or to change your name or address, go to www.chromatographyonline.com, click on Subscribe, and follow the prompts. To cancel your subscription or to order back issues, please email your request to: [email protected], putting LCE in the subject line. Please quote your subscription number if you have it.MANUSCRIPTS: For manuscript preparation guidelines, visit www.chromatographyonline.com or call the Editor, +44 (0)151 353 3503. All submissions will be handled with reasonable care, but the publisher assumes no responsibility for safety of artwork, photographs or manuscripts. Every precaution is taken to ensure accuracy, but the publisher cannot accept responsibility for the accuracy of information supplied herein or for any opinion expressed.DIRECT MAIL LIST: Telephone: +44 (0)151 353 3503.Reprints: Reprints of all articles in this issue and past issues of this publication are available (250 minimum). Licensing and Reuse of Content: Contact Mike Tessalone at MJH Life Sciences. Telephone: (732) 346 3016. E-mail: [email protected]© 2019 Multimedia Healthcare LLC (UK) all rights reserved. No part of the publication may be reproduced in any material form (including photocopying or storing it in any medium by electronic means and whether or not transiently or incidentally to some other use of this publication) without the written permission of the copyright owner except in accordance with the provisions of the Copyright Designs & Patents Act (UK) 1988 or under the terms of the license issued by the Copyright License Agency’s 90 Tottenham Court Road, London W1P 0LP, UK. Applications for the copyright owner’s permission to reproduce any part of this publication outside of the Copyright Designs & Patents Act (UK) 1988 provisions, should be forwarded in writing to Permission Dept. email: [email protected]. Warning: the doing of an unauthorized act in relation to a copyright work may result in both a civil claim for damages and criminal prosecution 10% Post

ConsumerWaste

Posters play a pivotal role at conferences and this supplement, a collaboraton between the scientific committee of ISC 2018, HPLC 2018 and LCGC Europe, celebrates the winners from the ISC 2018 conference, which was held in Cannes-Mandelieu in the South of France and the HPLC 2019 congress in Milan.

Poster presentations are a vital part of any congress because they catalyse debate between participants and allow innovative results to be show-cased and discussed during breaks.

Presenting a poster allows the younger generation of separation scientists to make themselves known to their peer group and exchange ideas with renowned scientists.

This special supplement interviews poster winners form the past year to recognize the innovativion of the posters presented at both of hese conferences and identify novel areas of research and emerging trends. LCGC Europe would like to thank the organisers of both conferences for their support and also Gerard Rozing for his dedication to organsing the Poster Awards at both events and ensuring the assessment process runs smoothly.

The next edition of ISC, the 33rd, will be organized in Hungary, in Budapest, from 20 to 24 September 2020 under the chairmanship of Professor Attila Felinger.

HPLC 2020 will be organized by Mary Wirth from 20–25 June 2020 in San Diego, CA, USA.

We wish them every success — and look forward to the poster sessions!

Rising Stars of Separation ScienceLCGC Europe presents a special supplement profiling poster winners from ISC 2018 and HPLC 2019.

Poster presentations are a vital part of any congress because they catalyse debate between participants and allow innovative results to be show-cased and discussed during breaks.

Didier Thiebaut, ESPCI Paris, Paris, France.

Jean-Luc Veuthey, School of Pharmaceutical Sciences, University of Geneva, Switzerland.

Valérie Pichon, Sorbonne University, Paris, France; Institute of Chemistry, Biology, and Innovation, ESPCI Paris, Paris, France.

Alberto Cavazzini, Università degli Studi di Ferrara, Ferrara, Italy.

Massimo Morbidelli, Politecnico di Milano, Milan, Italy.

ISC 2018

HPLC 2019

Alasdair Matheson, Editor-in-Chief, LCGC Europe.

LC•GC Europe Supplement December 20196

Poster Award Winners

increasingly important.Moreover, the column lifetime is limited because the

selector detachment leads to retention time shifts or changes in selectivity.

Therefore, we looked at developing a more stable and MS-compatible surface chemistry by immobilization of the chromatographic ligand to a multiple crosslinked polymeric layer on the silica surface. Furthermore, an additional separation material was developed with optimized ionic interactions on the reversed phase/weak anion exchange stationary phase.

What are you doing in this poster that is novel?To overcome and characterize this problem, we synthesized two new stationary phases. The first synthesized stationary phase was a polymer-coated reversed-phase/weak anion exchange stationary phase. The selector N -(10-undecenoyl)-3-aminoquinuclidine was immobilized by simultaneous double thiol-ene click reaction of poly(3-mercaptopropyl)methylsiloxane (PMPMS) (thiol component) to vinyl silica (-ene component) and the selector (-ene component). In this reaction, the mixed mode selector is attached on a polysiloxane layer, which is formed on the surface of the silica particle. We used a “one-pot” approach that included the immobilization of the ligand and the immobilization of the PMPMS layer on the surface. We also used this procedure for the synthesis of chiral stationary phases (1,2,3). Theoretically, the layer showed multiple linkages to the silica surface, which prevents fast selector hydrolysis. The successful reaction was confirmed by elemental analysis and solid-state 29Si cross-polarization-magic angle spinning nuclear magnetic resonance (CP-MAS NMR). For further verification of this theory of enhanced stability, we treated the stationary phase with heat (60 °C) under buffered highly aqueous conditions. After several stress intervals, the retention of hydrophobic and acidic compounds was tested under reversed-phase conditions and a strongly improved stability was confirmed.

Furthermore, an additional optimization approach was investigated concerning the surface charge properties of the modified silica. Therefore, the newly synthesized polymer coated reversed phase/weak anion exchange silica gel was treated with performic acid, which oxidized the thiol groups to sulfonic acid groups. As a consequence, the strong cation exchange (SCX) sites were introduced to its surface. The results of the elemental analysis showed only a slight loss of the nitrogen amount after this treatment. Also, the solid-state 29Si CP-MAS NMR was used for elucidation of the surface structure. The characterization of the surface charge properties showed that the additional acidic co-ligands lead to decreased

Compiled by Alasdair Matheson, Kate Jones, and Lewis Botcherby

A compilation of interviews with poster winners from ISC 2018 and HPLC 2019.

Influence of the Immobilization Chemistry on Chromatographic Features of Reversed-Phase/Weak Anion Exchange Mixed-Mode Silica Gel

Stefanie Bäurer, Institute of Pharmaceutical Sciences, University of Tübingen, Germany

How did the idea for this project arise and what were the aims of this research?The need for separation methods complementary to the well-established and dominant reversed-phase liquid chromatography (LC) technique is increasing in pharmaceutical quality control, as well as for two-dimensional liquid chromatography (2D-LC). The introduction of additional interaction sites on the surface of the separation material leads to additional selectivities. This is known as mixed mode chromatography (MMC). In addition, mass spectrometric (MS) detection has become increasingly important. This limits the variety of buffers and concentrations that can be used.

The use of mixed mode stationary phases can exhibit excellent, complementary separations under MS-compatible conditions, while ion exchange chromatography (IEX) and ion-pair reversed-phase chromatography (IP reversed-phase) have problems relating to the MS compatibility of eluents. Another important point that should be considered when discussing MS compatibility is column bleeding. Usually, chromatographic ligands are bonded to silica via siloxane bonds. Siloxane bonds are prone to the detachment and hydrolysis of the chromatographic selectors. The better their accessibility for water in the mobile phase, the higher the instability. Reversed-phase selectors, usually alkyl chains of different length, show a poor ionization when using electrospray ionization (ESI).

The polar groups that are introduced in MMC–ligands increase the ionization efficiency. This usually results in increased noise and simultaneously in reduced signal intensity in LC–MS. The limit of detection (LOD) and limit of quantification (LOQ) of the investigated analytes will suffer from this phenomenon. In the case of the impurity analysis in pharmaceutical formulations, low LODs and LOQs are of the utmost importance. Because of the additional sample dilution in the second dimension when comprehensive two-dimensional chromatography (LC×LC) is used, overcoming the issue of low signal sensitivity becomes


ISC 2018 Poster Winners

better MS compatibility are considered, this is a price that can easily be paid.

What were your main findings and why are they useful?The first objective of this study was to evaluate if the new immobilization strategy is more stable than the previously investigated one of the analoque brush-type stationary phases. The improved MS compatibility has already been demonstrated in our previously published work (1). In this case, we flushed the stationary phase with aqueous acidic conditions buffered at pH 5 at 60 °C. After several time intervals the retention factors of acidic and hydrophobic compounds under reversed-phase conditions were monitored. The retention time of the investigated compound DETP, a polar acid, was mostly affected since the retention mechanism is based on electrostatic interactions. In the case of the brush-type stationary phases, the retention dropped quickly after 8 h and 20 h flushing under stress conditions. The polymer-coated material was stable. The stress test had to be aborted for the brush-type stationary phase after 80 h because of insufficient retention. It was prolonged to almost 140 h for the polymer coated column, which maintained most of its retention after this time.

The second objective was to illustrate the chromatographic potential of the new separation material with enhanced stability. It was therefore necessary to characterize the stationary phase chromatographically. Consequently, we investigated in comparative separations phosphorylated carbohydrates, peptides, and oligonucleotides. The obtained resolutions were equivalent to the brush-type stationary phases, while the stationary phase with additional acidic co-ligands showed a significantly lower retention with only slightly decreased selectivity.

In case of the oxidized stationary phase, the acidic co-ligands acted like surface anchored counter-ions, which were also recognized for chiral ion exchangers (2). This offered the possibility to use lower buffer concentrations. Independent of the investigated pH, this stationary phase showed a positive (low pH) and negative (higher pH) surface charge. This phenomenon can be used to govern the retention of the charged analytes by changing the polarity of the surface.

How are you planning to develop this research further?To complete this project, we need to perform stress tests. Furthermore, we also intend to offer alternatives to ion-pair reversed-phase chromatography or ion exchange chromatography that are MS-compatible (6).

Additionally, we want to evaluate the use of mixed mode stationary phases in 2D-LC.

References(1) A. Zimmermann et al., J. Chromatogr. A 1436, 73–83 (2016).(2) U. Woiwode et al., J. Chromatogr. A 1503, 21–31 (2017).(3) K. Schmitt et al., J. Sep. Sci. 41(6), 1–8 (2018).(4) M. Lämmerhofer et al., J. Sep. Sci. 31, 2572–2588 (2008).(5) A. Zimmermann, Ph.D. thesis, University of Tübingen, Germany

(2016).(6) S. Bäurer et al., J. Chromatogr. A 1593, 110–118 (2019).

values (negative offset of ζ-potentials of around -10 mV), while the selectivity for the investigated analytes was only slightly decreased.

Moreover, the new separation material was characterized concerning the chromatographic properties under hydrophilic interaction liquid chromatography (HILIC) and reversed-phase conditions. Therefore, we used the already well-established set of analytes (4). For evaluation of the selectivity for hydrophobic compounds, we investigated butylbenzene and pentylbenzene. The separation of these analytes served for the calculation of the methylene selectivity. While the polymer-coated reversed-phase/weak anion-exchange stationary phase showed comparable results, the methylene selectivity of the additionally oxidized stationary phase was slightly decreased. The retention of a polar acid (diethylthiophosphate [DETP]) and a hydrophobic acid (N -tert-butoxycarbonyl-prolylphenylalanine [BocProPhe]) showed the anion exchange properties. The selectivity decreased slightly in this case for the oxidized separation material, but it has to be highlighted that the analysis time could be shortened significantly. Amongst other analytes, nucleosides were separated under HILIC conditions. The result confirmed that the selectivity for these structures was conserved in the new oxidized MMC phase. The results of the reversed-phase test, as well as the results of the HILIC test, which also included the separation of vitamins and xanthines, served for classification by principal component analysis. The resulting score plot fits well to already made observations and the stationary phases were located in between reversed-phase and HILIC phases.

In summary, it can be stated that the combination of the new attachment chemistry with the further developed surface charge ratios represents a clear step towards MS-compatible ion exchange chromatography, especially in the case of (multiple) negatively charged analytes.

What were the main analytical challenges you had to overcome?Polymeric immobilization strategies often suffer from enhanced mass transfer resistances. Thus, in a preliminary earlier study the thickness of the coating film and other synthesis parameters required a thorough optimization (5).Another issue was the formation of disulfide bonds, which are unstable and not available for the thiol-ene click reaction. The preliminary experiments showed that a cleavage of the bindings before immobilization is not necessary and we investigated a “one-pot” approach for this modified silica gel. It was possible to form the polymeric layer and simultaneously immobilize the mixed mode selector.

The absence of disulfide bonds was further substantiated by the low selector load loss determined by elemental analysis after performic acid oxidation.

From a chromatographic point of view, the combination of the polymeric- and silica-based separation material combines advantages as well as disadvantages. It should be taken into account that the use of aqueous mobile phases required a slightly prolonged initial equilibration of the column compared to brush-type stationary phases. This is a general known problem of HILIC stationary phases. If the overall column lifetime and the benefit from



advantages to extensively explore the metabolome. This was used to combine the biochemical information from multiple data sources and better understand the links between specific metabolic pathways and the pathology.

What were the main analytical challenges you had to overcome?In metabolomics, we must face many analytical challenges, from the sample preparation to metabolite identification. First and foremost, metabolomic workflows must confirm the link between disease and metabolites concentration variations by ensuring similar analytical conditions for all samples and avoiding analytical confounding factors. To overcome this challenge, we have set up an automated procedure using a robot for every step of the sample preparation, thus offering normalized conditions. Furthermore, acquisition stability was monitored using quality control (QC), prepared by pooling equivolume of all samples and injected at regular interval in the sequence. This constitutes an efficient approach to evaluate analytical variations in the dataset. An overall decrease of the peak area can be caused by the contamination of the electrospray ionization source. The latter is generally low after system conditioning but becomes significant after a large number of injections without a washing step. When dealing with a large number of samples involving multi-batch analysis, specific normalization must be implemented. In that context, we developed a software for feature selection and normalization based on LOESS regression fitted to the QCs and interpolated to the samples inducing intra- and inter-batch correction of all samples for each dataset.

Identification is another major challenge in metabolomics. A common approach in untargeted metabolomics is to analyze the whole dataset and consider only discriminant features for identification. This approach is efficient but can become complex and time-consuming. We choose to use another strategy consisting of rapidly annotating features at the highest level of identification, that is, level 1, based on an in-house database before multivariate analysis instead of accounting for unknown features. Our in-house database covers most metabolic pathways thanks to the combination of the three orthogonal analytical conditions. Metabolite annotation based on standard compounds helps to focus on relevant signal and rapidly generates biological hypotheses using metabolite enrichment analysis and pathway overrepresentation. The metabolic picture obtained is therefore limited to known compounds, but, thanks to the untargeted acquisition, retrospective data reprocessing is made possible for additional investigations without new data acquisitions.

Handling multiple datasets in metabolomics could be critical and complicate biological interpretation. A lot of efficient approaches have already been developed in data fusion and multi-block analysis with different degrees of complexity. In this study, we used the same HRMS for every chromatographic condition and only well-defined signals were kept, allowing a simple approach to data fusion. We simply concatenated the data matrices into a synthetic dataset from the three analyses and applied classical multivariate models, thus simplifying biological interpretation. Despite the orthogonality of the

Metabolomics in Chronic Kidney Disease: Towards a Better Understanding of Disease Progression and Hemodialysis

Yoric Gagnebin, School of Pharmaceutical Sciences, University of Geneva, Switzerland

How did the idea of this project arise and what were the aims of this research?Chronic kidney disease (CKD) is becoming a major public health issue and new medical care approaches are necessary for diagnostic and therapeutic treatment. CKD is characterized by the progressive loss of kidney function leading to high risk of cardiovascular morbidity and mortality. As a result, the irreversibility of the disease progression, the lack of specificity, and sensibility of actual biomarkers in early stages delay the medical care. Furthermore, an improved monitoring of end-stage patients is mandatory, when hemodialysis and transplantation are the only solutions. For that purpose, metabolomics could offer deeper insights into CKD biochemical mechanisms and improve our understanding of the disease to discover new therapeutic targets preventing or reducing kidney degradation. In that context, a collaboration started with Service of Nephrology of the Geneva University Hospitals (HUG), Switzerland to conduct a study for the evaluation of metabolic profile alterations in plasma associated with different CKD stages and hemodialysis effects on the metabolome.

What are you doing in this poster that is novel?Thanks to the study design involving four different groups of patients, we were able to evaluate the alterations related to disease progression and the effect of hemodialysis on the plasma metabolome in a single analysis. From an analytical point of view, blood metabolites are characterized by diverse chemical structures and physicochemical properties, including both very polar molecules, such as amino acids or sugars, and very apolar compounds such as lipids. Since no single analytical technique can offer an exhaustive coverage of all metabolites, we developed a strategy involving multiple complementary chromatography conditions hyphenated to high-resolution mass spectrometry (HRMS). The implementation of a multiplatform analysis involving reverse-phase liquid chromatography (LC), but also hydrophilic interaction liquid chromatography (HILIC) with amide and polymeric zwitterionic stationary phases, helps to improve coverage of both apolar and polar metabolites. It has to be noted that despite its prominent contribution to several metabolomic applications, HILIC remains rarely used in the context of CKD research. This new approach also includes data fusion and post-targeted annotation from an in-house library of standard compounds offering several



chosen three platforms, several signals were common to multiple datasets. When signals collected from different sources are combined, one specific metabolite can be detected using two or more analytical platforms, generating redundant information that may alter multivariate data analysis. In that context, we selected one single feature metabolite according to the best signal quality in terms of intensity, retention time, and peak shape.

What were your main findings and why are they useful?More than 250 metabolites were monitored to evaluate metabolic profile alterations related to the different stages of the disease. We were able to define a list of potential biomarkers that could help diagnosis. This list of metabolites was used for enrichment analysis showing major alterations of specific metabolic pathways, for example, tryptophan metabolism. The study also brings out the beneficial effect of dialysis for stage 5 patients, but also highlights the possible interest of metabolite supplementation for the patient. The monitoring of all these metabolites opens the discussion with clinicians involved in the project to find how these results could help patients.

How are you planning to develop this research further?Investigating the complementarity of analytical platforms constitutes a very promising approach for improving the metabolite coverage. We are now working on the integration of other analytical platforms such as capillary electrophoresis (CE) and supercritical fluid chromatography (SFC) hyphenated to HRMS. Furthermore, targeted analysis is planned to refine biomarker selection based on absolute concentration of the most interesting metabolite and confirm the results to provide potent diagnostic tools for clinical routine.

Superhydrophobic Capillary Coatings for Electrophoretic Separations

Charly Renard, Institut des Biomolécules Max Mousseron (IBMM), Université de Montpellier, CNRS, ENSCM, Montpellier, France

How did the idea for this research project arise and what were the aims of this research? The aim is to reduce capillary adsorption that causes separation efficiency loss in capillary electrophoresis. The idea to use superhydrophoby in a capillary came from the recent expansion of superhydrophoby used in many different fields. A superhydrophobic coating gives a surface superhydrophobic properties. They are useful because they allow water to flow easily, even in narrow

capillaries. Examples of superhydrophoby coatings would be car windshield treatments against rain, hospital wall protection from bacteria, or waterproof textiles. Essentially situations where preventing water, or water solute, from touching a surface is important.

Superhydrophoby is a property of a surface that makes it so water-repellent that if water is deposited on the surface an air layer will appear between the surface and the water, preventing any direct interaction between the surface and the water. Superhydrophoby is a result of a precise combination of some hydrophobic chemicals and nanostructuration of the surface.

The material is commercially used as fabric waterproof treatment for items such as shoes, clothes, or camping tents, and also to prevent droplet formation on car windshields.

Scientifically, superhydrophobic coatings can be used to prevent bacteria deposition on hospital walls or in a pipe to reduce friction between the pipe wall and the water running in it.

What are you doing in this poster that is novel? First, we are investigating a superhydrophobic capillary, which has not been done before. Then, we are using this totally new capillary to separate peptides by capillary electrophoresis, and compare the efficiency of separation in those conditions of this superhydrophobic capillary with the efficiency of separation of a silica capillary.

What were the main analytical challenges you had to overcome? There are currently two challenges: First, to maintain a stable electrical field on our capillary during the analysis, which is complicated by the existence of a nanobubble air layer in the capillary. The second challenge is the durability of the capillaries, because the coating is not covalent.

What were your main findings? We found that it was possible to have superhydrophoby in a narrow space, such as 50-µm-internal-diameter capillaries. It will be useful in our work to enhance electrophoresis performances, but it could be used in many other fields, such as hydrodynamic flow in microfluidic studies. In the preliminary experiences shown in the poster, the peptides separation efficiency is roughly two times better on a superhydrophobic capillary than on a bare silica capillary.

This type of coating can be used with every type of analyte, as long as they are in solution in water. Even hydrophobic analytes can be separated, because the air layer between the wall and the water will prevent any interaction between the analytes and the wall.

How are you planning to develop this research further? We aim to resolve the two challenges brought up earlier, and to separate molecules and even macromolecules, because it is with macromolecules that the adsorption effect is the strongest.



Preparative Comprehensive Two-Dimensional Chromatography: Comparison of CPC×LC and Preparative LC×LC to isolate Multiple Targets from Edelweiss Plant

Léa Marlot, Institut des Sciences Analytiques, France Techniques

How did the idea for this project arise and what were the aims of this research?The purification of newly discovered molecules in complex matrices, such as natural products, allow their structure to be identified by NMR and to produce reference substances to help develop analytical methods. Usually, a multi-step purification is performed, followed by a one-dimensional preparative chromatographic separation. This slows down the research for new molecules. To avoid tedious steps and loss of sample, especially for expensive or rare samples, we investigated a two-dimensional separation with the aim of isolating multiple targets simultaneously. For this purpose, we developed comprehensive separations to increase the number of molecules that can be isolated. In the preparative field, liquid chromatography (LC) is commonly used because the scale-up process to larger apparatus is well-known.

Centrifugal partition chromatography (CPC) is another preparative technique based on liquid–liquid partition of the compounds between mobile and stationary phases. As a result of the presence of a liquid stationary phase, this technique has several advantages, including a high amount of sample load and a total recovery of the sample. Therefore, for an efficient two-dimensional separation, it seemed interesting to implement CPC as a first dimension and LC as a second dimension. In the literature, CPC and LC have already been compared in one-dimensional separations for preparative objectives, but the study has never been performed for two-dimensional separations. We were interested in comparing the specificities and the performances of LC and CPC as the first dimensions of an off-line two-dimensional separation with the same LC separation implemented in the second dimension.

To illustrate our work, we were glad to be provided with a sample of the rare plant Edelweiss from a company that manufactures phytochemical substances as analytical standards. As this mountain plant generates bioactive compounds to protect itself from the high-altitude environment, they were interested in the isolation of several antioxidant molecules from it for therapeutic uses. This was a great opportunity to evaluate our 2D preparative separation process.

What are you doing in this poster that is novel?Two-dimensional separations for preparative purposes have just emerged, with either LC or CPC in the first

dimension. CPC and LC have already been compared in one-dimensional separations for preparative purposes but the performances of these techniques in the first dimension in a two-dimensional process are unknown. In this poster, we looked at the distance between peaks in the 2D separation spaces and the loading capacity for both CPC×LC and LC×LC configurations for high purity and high recovery requirements. The impact of the transferred volumes on the second dimension were compared and finally these volumes were optimized in both configurations to achieve the best 2D separation quality. The quality of separation was compared using 2D-contour plots.

What were the main analytical challenges you had to overcome?The main challenge in this study is the transfer of high volumes between the two dimensions without resolution loss. In preparative chromatography the entire sample has to go through both dimensions, so the entire fractions from the first dimension have to be transferred to the second dimension. Consequently, peak overloading and deformations can easily occur. Thus, the challenge here was to optimize the volume transferred in the second dimension while maintaining the resolution of compounds in both dimensions and by finding the best compromise between the number of transferred fractions and the total duration.

For a minimal duration, the number of transferred fractions should be reduced. This will result in an increase of the volume of the fractions produced. By increasing this volume, the injection volume in the second dimension is also increased, leading to peak deformation because of phase incompatibility or overloading, and to the loss of resolution in the second dimension. Additionally, by increasing the fraction volume, the peaks separated in the first dimension can be mixed again and the first-dimension resolution can also be lost. Thus, optimizing the transferred volume between both dimensions means reaching a suitable sampling time in the first dimension so that a suitable fraction volume is transferred to the second dimension to maintain the resolution in the first and second dimensions. Peak deformation at high load and solvent incompatibility were a great concern despite the fact that these effects are very different if LC or CPC is implemented as the first dimension. We overcame this challenge by applying a new method that relies on the scale-down of the second dimension to study the transfer process. The first step allows the evaluation of the peaks position in the separation space by injecting aliquots of the first dimension on a smaller second dimension column. With the simulation of the entire transfer of fractions on the small second dimension, the second step allows the evaluation of the loading capacity while maintaining resolution. The suitable transferred volume can then be optimized.

What were your main findings and why are they useful?By injecting 1 mL of Edelweiss sample in the first dimension using CPC or LC, 50 fractions were recovered in both cases. By applying our development method on



a smaller column, the fractions were analyzed on the second dimension with an injection volume of 1% of the column dead volume. The preparative LC×LC separation showed a high sample peak capacity with a high occupation rate. As a result of weak selectivity between peaks, the strategy with this 2D configuration is to spread the solutes over a large separation space to get better chance to separate compounds. Conversely, the CPC×LC separation showed a low sample peak capacity with a high occupation rate. As a result of the high selectivity between peaks coming from non-correlated mechanisms, the strategy with this 2D configuration is to selectively separate targeted compounds from the rest of the matrix by finely tuning the solvent system of the first-dimension CPC. To simulate a full transfer of the fractions on the second dimension, a volume of 65% of the second-dimension column dead volume was then transferred for both configurations. This step highlighted the solvent compatibility issue between dimensions for the preparative LC×LC approach, leading to peak deformations whereas for the CPC×LC separation, positive dilution effects were underlined on solvent compatibility. Consequently, the injection volume had to be decreased to 19% for the preparative LC×LC separation whereas for the CPC×LC separation, the injection volume was increased to 313% without any peak distortions on the second dimension and with peak resolution maintained on the first dimension. These findings are useful for industrial scientists who want to purify multiple compounds but are unsure about which technique to use This study highlighted that both techniques, LC and CPC, implemented as the first dimension of a 2D separation, are attractive techniques with advantages and drawbacks. The two configurations offer a great deal of possibilities for the development of 2D preparative separations for rare and complex samples, the preparative LC×LC providing a very limited amount of a large number of collected molecules, the CPC×LC provided a large amount of selected molecules.

How are you planning to develop this research further?This study is a comparative study to help researchers or industrial scientists develop two-dimensional separations with the most appropriate technique to isolate the molecules, depending on their objective. This comparison can be explored more deeply by selecting and comparing different elution modes or solvents, focusing more on solvent compatibility. We decided to develop the CPC×LC separation further by proposing an in silico method to easily select the 2D systems for the best performances in terms of distances between peaks for enhancing purity and recovery rate. Studying peak deformation as well as the position and the spread of the peaks in the 2D separation space based on simulated separations allows a faster column screening. We hope these tools can speed up the 2D method development and convince more users to apply this great tool for molecules isolation.

Dilute-and-Shoot Analysis of Therapeutic Monoclonal Antibody Glycosylation from Fermentation Broth: A Method Capability Study

Therese Wohlschlager, Department of Biosciences, Bioanalytical Research Labs, University of Salzburg, Austria

How did the idea of this research project arise and what are the aims of this research?Therapeutic monoclonal antibodies (mAbs) are produced in cellular systems resulting in a multitude of molecular variants. As the production of a therapeutic mAb aims at a defined product profile, process development and optimization involve in-depth characterization of the target protein. In this context, our collaboration partner Novartis approached us with the following question: “Could you develop a fast analytical method providing information on the glycosylation pattern of the expressed mAb?” We received fermentation broth samples and set up a workflow for mAb characterization at the intact protein level. In addition to revealing glycosylation patterns of the target product, our approach allows relative quantification of mAb truncation variants, which are not easily amenable to conventional bottom-up analysis involving proteolytic digestion. Thanks to minimal sample preparation, a fast analytical method and straight-forward data evaluation, the whole procedure from sampling to data analysis takes less than one hour. The dilute-and-shoot approach may thus facilitate monitoring of multiple molecular attributes in the course of a mAb fermentation process in real-time, which is usually hampered by long analysis times arising from lengthy sample preparation.

What are you doing in this poster that is novel?Before in-depth protein characterization of mAb variants in the course of process monitoring, mAb species are conventionally purified from fermentation samples using protein A affinity chromatography. Here, we eliminate this purification step and directly analyze mAb variants in fermentation broth at the intact protein level. Sample preparation consists of a brief centrifugation step to remove undissolved cell components followed by simple dilution. The analytical workflow involves ion pair reversed-phase HPLC with online UV- and MS-detection employing a 15 minute HPLC-method. In a single analysis, we can detect N-glycosylation patterns of the intact mAb and relatively quantify mAb truncation variants. In addition, we determined glycation levels and subsequently performed relative quantification of N-glycoforms. Simultaneously, we absolutely quantified the target protein based on online UV-detection. Our workflow therefore represents the first multiple attribute monitoring (MAM) approach at the intact mAb level.



Off-Line Comprehensive Two-Dimensional Separation of Petroleum Sample by SEC×RPLC–ICP-MS/MS

Marie Bernardin, Univ Lyon, CNRS, Universite Claude Bernard Lyon, Institut des Sciences Analytiques, Villeurbanne, France

How did the idea of this project arise and what were the aims of this research? The industry of fuels is changing rapidly with more heavy products coming as a source of hydrocarbons and new fuels based on biomass. Both of these sources are complex organic materials that require deep characterization to improve the process of purification that are bringing fuel to their final specification. Crude oil and oil issued from biomass are complex mixtures containing heteroatoms, such as alkalines, sulphur, and heavy metals like nickel and vanadium. They are known to be present in these matrices up to few hundred parts per million. These compounds significantly impact the processing of the materials in which they occur. Therefore, the knowledge of metal speciation is necessary to fully understand how these elements impact refining processes. For example, metal complexes decompose during hydrodesulfurization and catalytic cracking operations, resulting in a metal deposition on catalysts.

Vanadium and nickel issued from crude oil deposits cause reduction in catalysts activities and lifetime. It has been shown than vanadium degrades catalyst selectivity, blocks active sites on catalysts used in cracking, increases coke formation, reduces gasoline yields, and forms sodium vanadates that induce the corrosion of metal surfaces. On the other side, sulphur is widely present in crude oil and needs to be carefully handled. Indeed, sulphur contributes to the increasing problem of atmospheric pollution when sulphur oxides are emitted into the atmosphere by combustion of high sulphur fuel oil. As a result of the complexity of such matrices, fractionation is often needed before speciation analysis. One of the main methods for this is a liquid chromatography (LC) method known as saturates, aromatics, resins, and asphaltenes (SARA). This method is extensively used in the petroleum industry but may differ slightly from one laboratory to another. The aim of the fractionation is to obtain four different SARA hydrocarbon fractions with different properties. Saturates comprise paraffin, iso-paraffins, and napthenes and are the most valuable fraction of crude oil. Aromatics comprise at least one aromatic ring or thiophenic ring. Resins and asphaltenes contain the heaviest part of the oil and constitute the most polar and aromatic fraction of a crude sample. Understanding the composition of a SARA fraction can give valuable insights into the heavy oil. One-dimensional liquid chromatography is commonly used for heavy oil analysis, but because of broad and unresolved peaks it is impossible to identify every compound of such

What were the main analytical challenges you had to overcome?The main assets of our method are minimal sample preparation and fast analysis using a standard HPLC–MS setup. However, relative quantification of N-glycosylation variants turned out to be affected by glycation, which is the non-enzymatic addition of hexose-residues to lysine. At the intact protein level, it is therefore not possible to discern a hexose arising from glycation from a hexose present on an N-glycan, for example a galactose. In order to derive unbiased glycoform levels, we determined glycation levels upon enzymatic removal of N-glycans and corrected the fractional abundances of N-glycans for this bias using a custom algorithm.

What were your main findings and why are they useful?To ensure the safety and efficacy of a therapeutic protein, the manufacturing process requires stringent control. Our workflow provides comprehensive information on multiple attributes including N-glycosylation, glycation, and truncation variants. Remarkably, we were able to relatively quantify glycoforms lacking core-fucose, which are an important molecular attribute that may be relevant for the antibody-dependent cell-mediated cytotoxicity (ADCC) potency of the mAb. Moreover, we simultaneously performed absolute quantification of the intact mAb based on UV-detection. In addition to providing information on multiple molecular attributes, our analytical strategy involves minimal sample preparation and fast analysis, which is a requirement for real-time monitoring of fermentation processes. Thus, our strategy may facilitate characterization of mAb variants in the course of fermentation to enable instant tuning of process parameters. We therefore consider the dilute-and-shoot approach a powerful tool for clone selection and process optimization procedures during the development of originator and biosimilar drugs.

How are you planning to develop this research further?In the future we will expand the workflow to metabolite screening in fermentation broth in addition to determination of mAb variants. Comprehensive information on fermentation broth composition would allow targeted supplementation of media compounds in addition to tuning of process parameters. Furthermore, we will apply our dilute-and-shoot method to investigate the impact of different process parameters on the glycosylation profile of the mAb being expressed.

How has your research progressed since you received the award?Since receiving the award, we have applied our dilute-and-shoot approach to study the effect of fermentation process conditions, such as pH and shear stress on mAb glycosylation patterns. The obtained data provides new insights into product quality that was previously not amenable to bioprocess engineers. In upcoming steps, we will establish links between molecular attributes and process conditions aiming at process optimization via digital twins. Once this is established, realtime monitoring of fermentation products via HPLC–MS analysis can be implemented in order to facilitate fine-tuning of process conditions to tailor molecular attributes of therapeutic proteins.



asphaltene fractions need to be better characterized and such a method is the first step towards a better understanding of the behaviour of sulphur, vanadium, and nickel in these fractions.

How are you planning to develop this research further? The method can be easily applied to compare refining process samples. Additional analyses are planned in our laboratory to compare different heavy crude oils to demonstrate that this method can be used as a tool for routine sample comparison. Further investigations are ongoing to optimize the signal intensity, depending on plasma instabilities throughout the second dimension gradient.

Native Asymmetrical Flow Field-Flow Fractionation and Size-Exclusion Chromatography for Studying the Aggregation of Beta-D-Galactosidase

Iro K. Ventouri, University of Amsterdam, Analytical Chemistry Group, The Netherlands

How did the idea of this project arise and what were the aims of this research?In many domains of chemistry and life sciences there is a trend to produce and study larger and more complex molecules, including their supramolecular assemblies. For example, aggregation in protein therapeutics brings a great safety concern for biopharmaceutical companies, because it can potentially lead to adverse effects regarding the drug activity, immunogenicity, and pharmacokinetic profile. As a result, the availability of appropriate analytical methods to detect and monitor the presence of aggregate formation throughout the production process is of great importance. This poses great challenges to analytical chemists, who are confronted with molecules of increasing size, complexity, and structural diversity within one sample. Analysis of intact macromolecules—whether synthetic, natural, or biotechnological—is essential if we are to fully characterize their (supra)molecular structure and function. In this project we sought maximum analytical synergy by exploring new combinations of advanced separations and state-of-the-art spectrometric techniques that in a single analysis will provide detailed and orthogonal information on size, chemical composition, conformation, and supramolecular structure of intact macromolecules.

Efficient analysis of intact macromolecules may be challenging. This not only encompasses the currently often limited selectivity, resolution, and sensitivity in macromolecular separation and detection methodologies, but also the potential lack of conservation of structural integrity, molecular conformation, and activity during analysis. In other words, we do not know for sure whether our analytical results are an accurate reflection of the

matrices. Therefore, the aim of this research is to present an off-line comprehensive two-dimensional separation of petroleum sample by size-exclusion chromatography × reversed-phase liquid chromatography inductively coupled plasma tandem mass spectrometry (SEC × reversed-phase LC–ICP-MS/MS).

What are you doing in this poster that is novel? The novelty of this approach is the coupling of a high resolution chromatographic technique with a specific detection such as ICP-MS/MS in the special case of organic matrices. This approach has never been developed in the literature and is presented in this poster for the first time. In comprehensive two-dimensional liquid chromatography (LC×LC), the whole sample is subjected to two separations. This makes two-dimensional liquid chromatography (2D-LC) very attractive for the separation of complex samples. In off-line comprehensive 2D-LC, fractions of the first dimension (1D) are sent to the second dimension (2D) to obtain a 2D contour plot after processing the data that will be used for comparison of samples.

What were the main analytical challenges you had to overcome? First of all, the main challenge we faced for this coupling was from an instrumental point of view. Indeed, LC hyphenated to a specific detector such as ICP-MS is a technique of choice for elemental speciation analysis. However, various instrumental limitations may considerably reduce the expected sensitivity of the technique. The interface consists of sample introduction system (SIS) and a possible flow-splitter prior to SIS. Among those, we were interested by the solute dispersion into the interface located between LC and ICP-MS. The objective was to find a suitable interface to easily handle the introduction of organic solvent into the plasma without causing any plasma instabilities while maintaining the separation performance. A large set of interfaces were tested to evaluate their impact on the separation, for example, sensitivity and peak band broadening. Once the interface had been chosen, the coupling between LC and ICP-MS/MS was allowed. Indeed, as heavy crude oils are such complex matrices, the aim of this study was to find different retention mechanisms allowing the separation of metal components, depending on the size, the polarity, and the aromaticity of the molecules containing sulphur, vanadium, and nickel. Finally, the introduction of an unstable amount of organic solvent throughout the second dimension gradient in the plasma is a real challenge concerning sensitivity and we are currently working on this particular aspect.

What were your main findings and why are they useful? Off-line comprehensive LC×LC–ICP-MS/MS appears to be an attractive method for the analysis of vanadium in heavy crude oils. The obtained 2D contour plots show significant difference between asphaltene and resin fractions. Indeed, the porphyrinic peak is more intense in resin fractions than in asphaltenes. This study clearly highlights the advantage of using two dimensions, large mass being coeluted in SEC while well separated in reversed-phase LC. These results are useful for the petroleum industry because the knowledge on these specific materials is growing. Indeed,



How has your research progressed since you received the award?The focus of the work presented in HPLC2019 was the evaluation of the size-based separation techniques, mainly AF4 and SEC coupled to various detectors for studying labile biomacromolecules. Overloading effects with increasing salt concentration in AF4 are discussed, giving certain guidelines for the AF4 method development of proteins. Additionally, in this study, we demonstrated a comparison between AF4 and SEC separation under similar conditions. Results highlighted the limitations of SEC regarding the interactions with the stationary phase and shear forces and their subsequent significant impact on the native protein structure. Contrarily, AF4 was able to preserve the supramolecular agglomerates of the investigated enzyme regardless of the strength of the flow field. Although SEC and AF4 are considered non-destructive liquid phase separation techniques, in both careful optimization and a multiple detection system is necessary for accurate insights of the native conformational structure and intact folding of labile proteins. These results also show the importance of using complementary techniques for accurate quantitation of the agglomerated species especially of biomacromolecules.

How are you planning to develop this research further?In the present project we aim to address the structural-integrity challenge by first evaluating established analytical technologies and methodologies for a representative array of compounds, including natural (bio-)macromolecules, synthetic polymers, and pharmaceutical proteins. Given the challenge of macromolecular size, shape, and aggregation, we aim to evaluate, implement, and integrate SEC, hydrodynamic chromatography (HDC), and FFF, in combination with light-scattering detection, fluorescence spectroscopy, IMS, and HRMS. This diverse set of methods ensures efficient information-rich analysis. With such setups, the main focus will be on answering the crucial questions on whether—and to what extent—macromolecules and supramolecular agglomerates stay intact during analysis, and whether the molecular conformations and (absence of) molecular associations observed in IMS and HRMS reflect the state of molecules in solution.

Influence of Primary Amine Structure in Hyperbranched Functional Layer on the Performance of Anion Exchangers for Ion Chromatography

Anna Uzhel, Chemistry Department, Lomonosov Moscow State University, Russia

How did the idea for this project arise and what were the aims of this research?Increasingly difficult analytical tasks solved by ion chromatography (IC) generate a large amount of interest in the

structure and composition of the sample.In this project, we focused on the optimization of separation,

interfacing, and detection associated with native size-exclusion chromatography (SEC), flow field-flow fractionation (FFF) coupled to size specific optical detection, such as light scattering, and ion-mobility–high-resolution mass spectrometry (IM–HRMS). The goal is to establish nondestructive separations, preserving conformation, and supramolecular structure, which also allows the analysis of chemically labile macromolecules, such as enzymes, where three-dimensional structure and activity are highly interdependent.

What are you doing in this poster that is novel?The three-dimensional structure of an enzyme is of fundamental importance to almost every function it performs. In this study, we aim to investigate how the structure of enzymes can provide direct information on its stability and, most importantly, its activity. To this end the main goal is to evaluate the suitability of two important size-based separation techniques, namely asymmetrical flow field-flow fractionation (AF4) and SEC coupled to multi-angle light scattering detection (MALS) for native protein analysis. Proposing nondestructive analytical platforms can enrich our understanding of these important biological molecules. Deeper insights can help in developing new strategies for annotating enzyme structures of unknown function and for designing novel enzymes.

What were the main analytical challenges you had to overcome?The main challenges during this study are imposed on the one hand by the necessity of near-native analytical conditions that can ensure structural integrity of the analyzed enzyme and also provide sufficient selectivity and resolution of the higher order structures. On the other hand, they arise from the intrinsic complexity and size of the enzyme. AF4 covers a large range of detectable protein sizes, from nano- up to micro-sized species, which makes it a good candidate for protein-aggregation studies. Additionally, the absence of a stationary phase makes it a very advantageous candidate for native analysis, especially compared with the widely used SEC where possible interactions with the stationary-phase material may feasibly induce structural alterations. Nevertheless, both techniques have advantages and limitations that had to be addressed to ensure near-native separation conditions that are indispensable for unravelling reliable and useful structural information, which can be further correlated with the enzymatic activity.

What were your main findings and why are they useful?Aiming not only to a complete characterization of the enzyme, but also highlighting structural differences between species obtained from different sources and further evaluation of their complexity, agglomeration behaviour, and enzymatic activity, native analysis was essential. Results confirmed the different stability ranges and agglomeration processes of enzymes, depending on their natural source. AF4 and native SEC coupled to MALS detection are proven to be useful complementary techniques, able to provide information on the intact enzyme and its supramolecular agglomerates.



exchangers prepared using glutamic and aspartic acids provided baseline resolution of glycolic, acetic, lactic, and formic acids.

We also studied the impact of temperature on the separation of organic acids and found that temperature increase results in the growth of retention times for all examined mono- and di-valent organic acids and also leads to improved resolution for some pairs of analytes. Significant selectivity changes noted for some pairs of organic acids make temperature a useful parameter for tailoring selectivity of covalently-bonded hyperbranched phases.

In the final part of this research we demonstrated the ability of novel stationary phases to separate a large set of organic acids together with inorganic anions and polyphosphates. Finally, we used the most selective stationary phase prepared with aspartic acids for the analysis of orange and apple juice. We showed that our new resin is suitable for determining full organic acid profiles in fruit juice, and provides baseline resolution of key weakly retained organic acids, even in samples with such complex matrices. This validated method of juice analysis demonstrated good linearity, low detection limits, and intra- and inter-day precision appropriate for commercial anion exchangers.

What were the main analytical challenges you had to overcome?Currently, there are several challenges in suppressed IC connected with the separation of organic acids. This includes, for example, the resolution of glycolic, acetic, lactic, and formic acids, and also a pair of malic and succinic acids, which are difficult to resolve to baseline. Another challenge was to achieve good reproducibility of the developed preparation procedure, which included twelve modification steps in total.

What were your main findings and why are they useful?We developed new types of covalently-bonded hyperbranched anion exchangers and found a way of manipulating their selectivity towards organic acids by using primary amines with various substitutes in the internal part of the functional layer. Established trends of selectivity shifts for organic acids and also investigated temperature effects lead us to the better understanding of how to prepare anion exchangers for particular analytical tasks and how to optimize the separation conditions.

The main breakthrough of the research is that the proposed approach for preparing covalently-bonded hyperbranched anion exchangers with negatively charged groups allowed us to separate to baseline the most problematic set of organic acids containing glycolic, acetic, lactic, and formic acids. Many available anion exchangers do not provide baseline separation of those analytes in real samples with complex matrices. Thus, in the food and beverage industry the determination of the full organic acid profile in fruit juices is usually performed by using electrochemical and enzymatic methods in addition to suppressed IC. The possibility to resolve these acids in real samples can significantly simplify the analysis of fruit juices and other products in the food and beverage industry and eliminate the need to use additional methods of chemical analysis for determining organic acids separately.

How are you planning to develop this research further?We have already started investigating the influence of the external part of the functional layer structure on the selectivity of hyperbranched anion exchangers. This is done

creation of novel stationary phases with improved selectivity toward mono- and di-valent organic acids. Our scientific group has been working in the area of stationary phase design for more than 10 years, and our goal was to develop novel anion exchangers for IC, which will be able to solve the most challenging tasks of the food, beverage, and pharmaceutical industry with regards to the determination of organic acids.

Currently, one of the most selective commercially available anion exchangers are electrostatically-bonded hyperbranched stationary phases and latex agglomerated resins. We decided to adapt a hyperbranching technique to our chemically derivatized aminated polystyrene-divinylbenzene (PS-DVB) substrates to prepare more stable stationary phases with covalently-bonded hyperbranched layers. Introduced in 2008 by Chris Pohl (1), a hyperbranching technique traditionally includes repeating steps of alkylation with diglycidyl ether and amination with primary amine. Such an approach provides a lot of possibilities to tailor the selectivity of the stationary phase by varying the structure of ethers and amines in all steps. We noted that both types of the most selective commercially available anion exchangers contain negatively charged sulfonic groups on the substrate surface, which may lead to additional repulsion of analytes, preventing their diffusion into the substrate pores and resulting in improved chromatographic performance of the column. In the case of chemically-derivatized stationary phases, there is no possibility of introducing negatively charged groups directly onto the substrate surface, therefore, we devised an idea to use amino acids as primary amines for the creation of a hyperbranched layer. Being used in the first modification cycle, an amino acid allows the creation of a layer of carboxylic groups, which are negatively charged in alkaline media of eluent (KOH), close to the substrate surface.

In our first trials we used glycine in different reaction cycles instead of methylamine and estimated how the amount and position of negatively charged fragments in the functional layer affected selectivity. We noted that the introduction of glycine into the internal part of the functional layer (the first modification cycle in hyperbranching) resulted in a significant improvement in selectivity towards weakly retained organic acids. The main aim of our current research was to broaden the range of primary amines, to evaluate the influence of their structure on selectivity of the anion exchangers, and to achieve baseline resolution of organic acids, such as glycolic, acetic, lactic, and formic acids, which are difficult to separate well.

What are you doing in this poster that is novel?In this work, for the first time, we inserted novel types of primary amines with various substitutes into the internal part of a covalently bonded hyperbranched functional layer. In addition to glycine, we also used 2-aminoethanol, 2-aminoethanesulfonic acid (taurine), and some divalent amino acids, namely, glutamic and aspartic acids in the first modification cycle of hyperbranching. This allowed us to study the effect of particular negatively charged or polar groups, as well as the impact of their amount and hydrophilicity, on selectivity towards mono- and di-valent organic acids. Furthermore, the performance of anion exchangers was studied. The obtained data showed that inserting primary amines with negatively charged groups, especially carboxylic ones, provides much better selectivity towards weakly retained organic acids than using amines with hydroxyl groups. From all classes of amino acids examined, the best results were achieved using di-valent ones—anion



What are you doing in this poster that is novel?In this poster our main objective was to validate both in plasma and in RBCs four Gaucher disease (GD) biomarker candidates: glucosylceramide (GLC), glucosylsphingosine (GLS), sphingosine (Sph), and sphingosine-1-phosphate (S1P).

As a result of the wide diversity of polarity between these sphingolipids, up until now, at least two liquid chromatographic (LC) runs were needed for the determination of the four targeted sphingolipids in plasma.

On the other hand, except for GLC, nothing is known concerning the RBCs sphingolipid content. GLC accumulation has already been noted in RBCs, but with a cumbersome and obsolete procedure. Indeed, GLC RBC concentration was determined by quantitative densitometry after a high-performance thin-layer chromatography (HPTLC) run of 40 min or by high performance liquid chromatography (HPLC) after 60 min of sample preparation.

Here, we have developed and validated a rapid ultrahigh-pressure liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) method for the simultaneous quantification of the four GD biomarker candidates in both plasma and RBCs.

What were the main analytical challenges you had to overcome?The main challenge with the simultaneous separation of the four targeted sphingolipids relies on the large difference in polarity between GLC and the three other sphingolipids.

Taking into account the large variability of these lipids, we showed that polar embedded RP18 phases are the most appropriate for gradient separation and quantification of such compounds in less than 7 min.

What were your main findings and why are they useful?The main findings are the development and validation of the method of simultaneous determination of the targeted sphingolipids, the validation of one biomarker among the four biomarker candidates, and, lastly, the establishment of a strong correlation between sphingolipids accumulation and RBC abnormalities in GD patients.

These results are of important for the early diagnosis and monitoring of GD patients, and the understanding of the physiopathology of GD. The developed method can also be used after minor modification for the exploration of the whole of sphingolipids and phospholipids content of an RBC.

How are you planning to develop this research further?Our main objective is to apply this method to the exploration of the whole RBC lipidome, mainly sphingolipids and phospholipids RBC content. This is important for the understanding of RBC abnormalities in inborn errors of metabolism, such as Minkowski–Chauffard syndrome, glucose-6-phosphate dehydrogenase deficiency, and sickle cell disease.

What else are you using this method for?We are currently applying this method for the evaluation of enzyme replacement therapy on GD patients.

How has your research progressed since you received the award?A systematic study of the operating conditions allowed us to generalize the proposed method to the determination

by using different types of di- and poly-amines instead of methylamine in the last modification cycle.

The next step will be to optimize functional layer structure by combining various amines in the external and internal part of the functional layer. At the moment our resins cannot provide the separation of more than three divalent organic acids in one run even with temperature variation, and this is another problem we are going to tackle. So, we know where to move and hope that our approach will help us to achieve better results and overcome some other current challenges in IC.

References(1) C. Pohl and C. Saini, J. Chromatogr. A 1213, 37–44 (2008).

Quantification of Four Gaucher Disease Biomarkers in Plasma and Red Blood Cells: Correlation with Disease Severity and Response to Therapeutic Intervention

Caroline Chipeaux, Lip(Sys)², LETIAM, Univ. Paris Sud, Université Paris-Saclay, IUT d’Orsay, Orsay, France

How did the idea for this research project arise and what were the aims of this research?Recently it has been shown that clinical manifestations of several hereditary metabolic diseases such as sickle cell disease, Minkowski–Chauffard syndrome, Gaucher disease, and lysosomal diseases in general, were related to red blood cell (RBC) abnormalities.

According to a widely accepted hypothesis, the common point of all these clinical manifestations could be linked to a lack of deformability of the RBCs. The partial or complete stiffening of the RBCs could be linked to an accumulation of lipid metabolites caused by an enzyme deficiency. For example, it was recently demonstrated that the β-glucocerebrosidase (GBA) deficiency is accompanied by an accumulation of sphingolipids in the plasma. Meanwhile, rheological abnormalities of RBCs were observed in patients with the same type of deficit. The accumulation of sphingolipids in blood is now considered a biomarker of GBA deficiency. This discovery has contributed to the recent development of new enzymatic treatments, enzyme replacement therapy (ERT).

The RBCs normal lipid profile remains unknown and the effect of ERT on the lipid profile of an abnormal RBC remains unproven. Clearly, the exploration of a RBCs lipidome became necessary both for understanding the pathophysiology of these diseases and for the diagnosis and therapeutic monitoring of abnormalities of RBCs related to its lipid content. This is the overall objective of this project built in the frame of a collaboration between the Laboratoire d’Etudes des Techniques et Instruments d’Analyse Moléculaire (LETIAM), the Institut National de Transfusion Sanguine (INTS), and Shire company.



What were your main findings and why are they useful? The comparison of the performances of different liquid chromatographic modes, performed with standard surfactants, has highlighted excellent candidate stationary phases for the separation. Indeed, surfactant types were well separated using a reversed-phase approach (PFP, C4, and C30 columns). In the case of HILIC, the bare silica column provided excellent separation of homologues. Concerning the HRMS characterization of the polydisperse surfactants, infusions of whole families have been made in both positive and negative ionization modes and reprocessed according to a homemade Excel spreadsheet.

How are you planning to develop this research further?Owing to the complexity of these polydisperse surfactants and the difficulty of the water–oil matrix emulsion, a multidimensional approach will be developed. It is all the more justified as complementary selectivity was observed in HILIC and reversed-phase modes.

Innovative Screening Approaches for the Determination of Non-Intentionally Added Substances in Food Contact Materials

Chrysoula Kanakaki, OFI Austrian Research Institute for Chemistry and Technology, Vienna, Austria

How did the idea of researching non-intentionally added substances (NIAS) arise, why is this an issue, and what were the aims of this research?NIAS can cause sensory impairments of the packaged foods or even lead to health risks, especially if they are low-molecular-weight volatile compounds. Therefore, screening the food contact materials (FCMs) for volatile NIAS is of particular importance for the development of safe and organoleptically acceptable products. This fact, together with the wide experience of my colleagues with packaging materials and the broad spectrum of tests performed for several years at our company, motivated us to start a project dedicated exclusively to NIAS screening.

What are you presenting in this poster that is novel?This poster summarizes the analytical approaches we have investigated in the frame of the Coin Senses Project, both at OFI (Austrian Research Institute for Chemistry and Technology) and at the research group of Professor Leitner, at the Institute of Analytical Chemistry and Food Chemistry at the Technical University of Graz, Austria. As a result of our three year occupation with untargeted NIAS screening, several analytical techniques have been investigated and compared, checking which technique works best for each type of packaging material. Original raw and composite materials of plastic or paper origin and

of not only all the sphingolipids present in RBC but also all phospholipids, which are the major constituents of its membrane.

The application of the later method to the simultaneous quantification of thirty sphingolipids and phospholipids in normal and GD RBCs, allowed us to validate it and to unravel the involvement of other candidate biomarkers of GD, different from the four previous sphingolipids.

Liquid Chromatography High Resolution Mass Spectrometry for Polydisperse Surfactants Used in the Oil Industry

Alizée Dufour, Total Exploration & Production, Lacq Research Center (PERL), Lacq, France and Department of Analytical, Bioanalytical Sciences, and Miniaturization (LSABM), UMR CBI 8231 CNRS – ESPCI Paris, PSL Research University, France

How did the idea of this project arise about and what were the aims of this research?In the oil industries, the control of emulsion of oil–water is mandatory in both surface and the sub-surface part of crude oil production. In the context of the highly volatile crude oil market, it is very important to monitor surfactants throughout the crude oil production process. So, the aim of this research project was to develop advanced analytical tools to improve the reliability of the quantification of different families of surfactants used for this purpose.

What are you doing in this poster that is novel?The poster presents a detailed study of the retention behaviour of commercially available standard surfactants on different stationary phases. Two retention modes were studied: reversed-phase and hydrophilic interaction liquid chromatography (HILIC). It appeared that both modes were complementary. In fact, using reversed-phase mode, each family was separated from each other, whereas using HILIC mode the separation was according to the hydrophilic character. The great novelty of this study was related to the intended matrix that is oil matrix.

What were the main analytical challenges you had to overcome?Liquid chromatography (LC) and mass spectrometry (MS) are the most prominent techniques to perform the analysis of surfactants in oil–water emulsion. However, present analytical methods do not allow a reliable quantitative analysis of the surfactants in the oil matrix. The complexity of the matrix and the polydispersity of surfactants required the development of improved analytical strategies involving ultrahigh-pressure liquid chromatography (UHPLC) hyphenated to high-resolution mass spectrometry (HRMS).



to a wider range of volatilities and polarities for the expected NIAS. Consequently, characterization of FCMs with additional analytical approaches will enable their more complete evaluation and contribute significantly to a further enhancement in the properties of these materials.

Anything else you would like to add?A big step towards the production of safer FCMs has already been made, with the current guidelines of the European Regulation obliging the manufacturers of such articles to evaluate their products with respect to NIAS. However, this is an active area of research, requiring the support of not only the manufacturers of FCMs, but also the funding authorities. The manufacturers should trust the scientists and provide them with the recipes of their products, facilitating the evaluation of their results. On the other hand, the necessary resources should be provided to motivate scientists, allowing them to work undistracted and with up-to-date instrumentation, which could enhance the quantity and quality of their data even further.

Application of Linear Retention Indices in Liquid Chromatography for Reliable Characterization of Oxygen Heterocyclic Compounds in Cosmetics

Adriana Arigò, Department of Chemical, Biological, Pharmaceutical, and Environmental Sciences, University of Messina, Italy

How did the idea of this research project arise and what were the aims of this research?Under the denomination of oxygen heterocyclic compounds (OHC) are coumarins (C), furocoumarins (FC), and polymethoxyflavones (PMF), which are naturally occurring compounds in the nonvolatile fraction of Citrus essential oils. These molecules exert several beneficial effects on human health, but furocoumarins form molecular complexes with DNA if activated with UVA light.

Citrus oils are widely used in cosmetic products by determining the content of furocoumarins and consequently potential phototoxic effects. For this reason, Regulation (EC) No. 1223/2009 of the European Parliament limits the content of FC in cosmetics; moreover, several associations propose different maximum amounts of psoralens in cosmetics, to ensure the absence of phototoxic reactions.

The official regulation includes furocoumarins in the list of prohibited substances “except for normal content in natural essences used” and sets the limit of 1 mg/kg in sun protection and bronzing products. Whereas, the Scientific Committee on Consumer Products suggests 1 ppm in any finished cosmetic product and

products of different migration studies have been intensively investigated. With the focus on odour-active compounds, additional research was performed using alternative analytical approaches and moving towards targeted screening for compounds that proved to have a high impact on the organoleptic properties of the products, however, their identification and quantification was still a challenge, requiring the application of highly selective and sensitive techniques. The investigation of NIAS in FCMs using such a wide spectrum of analytical techniques is, to our knowledge, performed here for the first time, and has led to the development of a fully functional NIAS database, already comprising of several hundreds of identified volatile organic compounds (VOCs).

What were the main analytical challenges you had to overcome?Dealing with unknown screening, we developed our methods using a wide range of VOCs and matrices for better simulations of real sample conditions. However, working with actual samples required compromises in the method optimization and allowed only semi-quantification of the detected and identified NIAS. Concerning the data evaluation and interpretation, knowing the composition of the examined materials is of paramount importance, particularly when the effects of various sterilization procedures were investigated. Sadly, this information was only seldom obtained, making the evaluation of the origin of identified NIAS an almost impossible task. Last but not least, when working with odour-active substances, enhanced sensitivity is often required, as a result of the particularly low-odour thresholds of many frequently occurring NIAS, and dictating the use of alternative analytical approaches.

What were your main findings and why are they useful?During this research project, several analytical techniques were evaluated for unknown screening of NIAS, as well as for targeted analysis of particular odour-active compounds. Numerous FCMs were analyzed using the migration procedures and methods of analysis that proved to be most appropriate for each sample type and material investigated. Summarizing these results, we developed a NIAS database with more than 500 entries. The effects of different sterilization processes on FCMs were also observed, categorizing them in terms of suitability for the materials of different origins selected for further examination. Combining this information, the manufacturers of FCMs can optimize their procedures and provide their customers with safe products.

How are you planning to develop this research further?Our follow-up project in the field of FCMs is called Migratox and utilizes the findings of the Coin Senses Project. This project is primarily concerned with the determination of the toxic components of plastic migration by means of bioassays, while chemical characterization of the samples will be further expanded



repeatability of the LRI values in different instruments showed how they can be used as parameters for unambiguous identification, especially in combination with libraries, which work only on the basis of the spectral similarity.

Companies obliged to certify their products for the presence of OHC could be interested in LRI and libraries as useful tools to make the quality control process more automated and rapid.

The validity of calibration curves created by spiking distilled oils also represents a great result, considering what it means in terms of applicability of this strategy. Distilled essential oils have the same volatile composition as the respective cold pressed oils, but are OHC-free, making them the most suitable samples to be used as matrices. This approach resulted in the possibility to quantify FC in cold pressed Citrus oils, even at concentrations lower than 10 mg/L, without the need to possess expensive instrumentations. By applying this method, the overestimation of FC as a result of interfering compounds is avoided, and the quality control of OHC in cold pressed Citrus oils by HPLC–PDA is absolutely correct. On the other hand, PDA detection is not appropriate for the analysis of FC in finished cosmetic products.

The sensitivity of tandem MS (MS/MS) in multiple reaction monitoring (MRM) mode is the second finding and it is proposed as an analytical tool to determine OHC, especially FC, when they are contained at trace levels, for example, in finished products. In this case, LOQ results were very low, providing a new sensitive analytical approach—useful for industries involved in this field to guarantee the limits imposed by the official regulations and to be ready in case of future restrictions.

How are you planning to develop this research further?This research offers many suggestions to improve the method performances and its applications. Currently there is a lack of LC software that can process data for the automatic calculation and the use as filter of LRI, so currently we are working to make the LRI system faster and easily applicable in LC. The HPLC–MS/MS method developed is widely usable to characterize 35 OHCs in several matrices. This method provided a detailed profile of the OHC content in the Citrus essential oils analyzed to date, and we plan to perform a screening of several samples, which differ in species and geographical origin.

I believe that this method could also be applied for the analysis of furocoumarins in food which is not currently a restricted class of compound according to the current European regulation. However, the European Parliament has indicated different maximum levels of coumarin (Regulation EC No. 1334/2008), depending on the type of product, and the European Food Safety Authority suggests a total daily intake of 0.1 mg/kg. However, in recent years researchers have been working more on the toxicity evaluation related to the dietary intake of furocoumarins, and are less focused on the already well known topical effects. This method can be applied to characterize C, FC, and PMF simultaneously

the International Fragrance Association proposes a maximum 5 ppm of any combination of 6 markers and 50 ppm in leave-on and rinse-off products, respectively.

On the basis of these considerations, analytical strategies for the correct determination of these compounds in essential oils and especially in finished products are needed. The aim of my research was to develop solutions to meet these needs, as required by cosmetic industries and regulatory bodies, considering the importance of cosmetics in the current global market.

What are you doing in this poster that is novel?The main novelty of this research is the application of a linear retention index (LRI) in liquid chromatography (LC). This system has been used for a long time in gas chromatography (GC) because it was developed for the first time by Kováts (1), but its use in LC has been hampered as a result of the variability of chromatographic conditions, mainly related to the mobile phase, and the low repeatability of old LC systems and columns.

We propose the use of LRI in both high performance liquid chromatography photodiode-array detection (HPLC–PDA) and HPLC–tandem mass spectrometry (MS/MS) methods, in combination with UV–vis, MS, and MS/MS libraries, created with 35 targets compounds among C, FC, and PMF. LRI ensures the reliable identification of OHC by working as an additional criterion to the spectral similarity.

Additionally, the PDA detection of OHC in Citrus essences was evaluated from a different point of view, trying to make the HPLC–PDA method suitable for the reliable characterization of FC.

What were the main analytical challenges you had to overcome?The LRI system applied to both HPLC–PDA and HPLC–MS/MS methods is the main innovation of this research, and so the main challenge was to demonstrate that it is properly applicable to LC. The LRI stability and repeatability has been proven, showing how this system could be useful for the quality control of FC, needed for cosmetic industries, according to the official regulation.

Moreover, in 2012 the Analytical Working Group of the International Fragrance Association published a scientific article, concluding that the LOQ of 15 FC targets in Citrus essential oils is equal to 10 mg/L as a result of the limits of PDA detection (2). As LC coupled to a PDA detector is still the main analytical approach used for the quality control of FC in essential oil, another challenge was to demonstrate the use of calibration curves, created spiking suitable matrices, and for the LRI approach to overcome the low sensitivity and selectivity of PDA detection, respectively. We also tried to find the relation between high limits of quantification and interfering compounds of the matrix.

What were your main findings and why are they useful?The main finding is inevitably related to the main challenge of the project, which is the use of LRI. The



An Online Four-Dimensional SEC×SEC-IM×MS Methodology for In-Depth Characterization of Forced Degraded Monoclonal Antibodies

Anthony Ehkirch, Laboratoire de Spectrométrie de Masse BioOrganique, Université de Strasbourg, France

How did the idea for this project arise and what were the aims of this research?The analytical characterization of mAbs requires the investigation of multiple critical quality attributes (CQAs), among which the determination of high-molecular-weight species (HMWS) and low-molecular-weight species (LMWS) are very important. In particular, HMWS (aggregates) can inhibit the product efficacy or cause hypersensitivity responses such as anaphylaxis.

Size-exclusion chromatography (SEC) appears as a reference method for measuring HMWS. Nevertheless, the use of nonvolatile mobile phases to attain the best SEC performance is necessary, however, these are not directly amenable to native ion-mobility–mass spectrometry (IM-MS). Moreover, SEC using volatile mobile phase (MS-compatible) leads to poor-quality chromatograms with less accurate HMW and LMW separation and quantitation. The goal of this research is to hyphenate classical SEC to native IM-MS for simultaneous online identification of HMWS and LMWS, and obtain the best SEC performances currently possible.

What are you doing in this poster that is novel?We have online coupled 2D-LC in nondenaturing conditions to native MS and ion mobility. The direct hyphenation of SEC with a classical nonvolatile salt mobile phase to native IM-MS is an innovative strategy to keep the best SEC performances and to simultaneously identify each separated species by native MS with conformational information using IM.

What were the main analytical challenges you had to overcome?The main analytical challenge to overcome was to make SEC with classical nonvolatile salts (phosphate buffer) compatible with native mass spectrometry (performed in ammonium acetate buffer). A multidimensional LC strategy was developed using a 2D comprehensive SEC×SEC setup, where the 1D SEC used a classical nonvolatile mobile phase to maintain the best SEC performances and 2D SEC used a volatile and MS-compatible mobile phase (ammonium acetate) to provide a fast and online desalting.

What were your main findings?Adalimumab is difficult to analyze by SEC because of its high pI and suspected presence of solvent-accessible basic patches. This first comparative SEC×SEC-native IM×MS experiment emphasized the ability of our 4D setup

in multiple food matrices and dietary supplements, even if contained at trace levels, thereby helping the researchers involved in this field.

In particular, we would like to investigate the amount of FC contained in Citrus alcoholic beverages, and make a comparison between commercial and homemade production. This interest stems from the wide use of Citrus alcoholic macerates in southern Italy (“limoncello”). For example, the food use of bergamot, which is a fruit native and specific of Calabria, may cause a high dietary intake of FC, especially bergamottin.

Last but not least, going back to the main aim of the study, we are working to develop a suitable procedure for the exhaustive extraction of FC from complex cosmetic samples, such as creams, soaps, and makeup products.

Anything else you would like to add? Working on this project I really realized the key role of analytical chemistry in the current global regulatory and commercial landscape. Legislation regarding the maximum content of a specific substance is strongly affected by the availability of methods able to quantify these substances at trace level. Moreover, in many cases industries do not dispose of techniques for the quality control of finished products. The interest that some companies have already shown towards this study highlights this point.

We hope that both developed methods may represent a tool for industries and a suggestion for the regulatory bodies, in the issue of new opinions regarding cosmetics and food.

How has your research progressed since you received the award?Since I received the award, the project has been presented at several conferences. As a result, many cosmetic and food companies were interested in this research and provided us samples to be analyzed, especially through the new HPLC–MS/MS method.

The idea of developing a standard procedure for the extraction of complex cosmetic samples is going to practice, by using a new fully automated online preparative station connected to the HPLC–MS/MS system. This system is going to be used to optimize the extraction of OHC in food matrices.

Currently, the HPLC–MS/MS method previously developed in combination with the library and the LRI system is being used for the analysis of citrus beverages, starting from homemade and commercial liqueurs to flavoured beers. Non-alcoholic samples, such as commercial juices and infusions, for example the Earl Grey tea, characterized by the presence of bergamot essential oil, are also in evaluation.

The results will be useful to evaluate the authenticity of commercial products, and the influence of the production process and the ingredients on the content of FC in the final product.

References(1) E. Kováts, Helv. Chim. Acta. 41, 1915–1932 (1958).

(2) A.P. Macmaster et al., J. Chromatogr. A 1257, 34–40 (2012).



Carnosine-based food supplements are found on the market, and because small structural changes can lead to the loss of its function, an easy, quick, and unique method for the quantification of carnosine and the identification of other histidine dipeptides was developed.

What are you doing in this poster that is novel?Methods that are able to separate histidine dipeptides in food have been already described in the literature, another has been developed for the separation of carnosine enantiomers (5). Therefore, we developed a new method with stereo-, chemo-, and regio-selectivity for the separation of histidine peptides and their enantiomers.

What were the main analytical challenges you had to overcome?The main challenge was to find the right column, eluents, and instrumental conditions for both the separation of carnosine analogues and carnosine enantiomers. Moreover, we were also looking at multiple detector hyphenation options, such as UV and mass spectrometer (MS)-friendly conditions. For this task we collaborated with the group of Prof. Sardella from the University of Perugia (Italy).

What were your main findings?The teicoplanin-based column offered an excellent retention factor, selectivity, and resolution of peaks, with UV- and MS-friendly eluents. The enantiomeric separation of carnosine was completely achieved with both a UV and an MS detector. The method was validated for the quantification of L-carnosine with the UV detector, demonstrating a good reproducibility, repeatability, and linearity.

The column also exhibited a good response in the separation of a standard mixture of L,D-carnosine and methyl-analogues of L-carnosine, that is, balenine and anserine. Although in UV an incomplete resolution was observed, the selectivity of MS working in multiple reacting monitoring (MRM) mode allowed the complete peak resolution in sub-optimal chromatographic conditions.

Moreover, MRM allowed the identification of an unpredicted impurity of balenine (D-balenine), which confirmed the method selectivity.

How are you planning to develop this research further?The next step is to enhance the separation of the peaks, with the UV detector trying to reach the complete resolution of the peptides from the mixtures and validate the quantitative analysis of all of them. The method must be tested on complex matrices, such as food extracts, to assess the sensitivity and selectivity of MS analyzers.

How has your research progressed since you received the award?In the past year we have worked to resolve the peak completely via HPLC–UV. Analyzing different conditions, we identified the best conditions for an isocratic-flow chromatography method. We further validated this

to simultaneously identify HMWS and LMWS under similar native MS/IM-MS conditions, providing a comprehensive characterization of forced degraded mAb samples within a single run. The benefits of the 4D SEC×SEC-nativeIM×MS are the following: maintaining optimal SEC performance (under classical nonvolatile salt conditions), performing online native MS identification, and providing IM-MS conformational characterization of all separated size variants.

To illustrate the benefits of the SEC×SEC-native IM×MS approach compared to standalone SEC-UV or native IM-MS techniques for size variant characterization, we focused on forced degraded studies of pembrolizumab, which is challenging to analyze in SEC with volatile mobile phase because of its basic pI (7.5) leading to severe peak broadening, tailing, and strong adsorption.

According to SEC–UV standalone, peaks eluting before the main monomer peak would be classified as HMWS. Surprisingly, native MS allowed the identification of peaks as monomeric species instead of multimers. At this stage, IM-MS analyses were of utmost importance, considering the different measured TWCCSN2 values of the 26+ charge state. TWCCSN2 values of the main monomer were significantly different from the minor monomers, providing a significantly different conformation between main monomer and first monomer.

How are you planning to develop this research further?The ability to have a straight online coupling of SEC with nonvolatile mobile phase to nondenaturing MS using our 4D technique might have a strong impact on the analytical characterization of mAbs-related compounds, not only for mAb development purposes and forced degradation studies, but also more generally for comparability and biosimilarity assessment. We believe that 2D LC×LC-nativeIM×MS setups will also be of interest for the broad diversity of next generation empowered mAb formats like antibody–drug conjugates (ADCs) or multispecific mAbs, such as bispecific or trispecific, as well as for protein oligomerization state assessment.

Enantioselective Chromatography for the Determination of Histidine Dipeptides in Food and Food Supplements

Ettore Gilardoni, Department of Pharmaceutical Sciences, University of Milan, Italy

How did the idea for this research project arise and what were the aims of this research?Histidine dipeptides are natural products present in mammals (1). In the past decades they have gained attention for their antioxidant, metal-chelating, buffering, and carbonyl mitigator activities. Carnosine, because it is endogenous, is the most studied for sport nutrition, pharmacological, and cosmetic uses (2–4).



method following ICH guidelines and the work was recently published on Journal of Chromatography B: doi: 10.1016/j.jchromb.2019.121747

References(1) A.A. Boldyrev et al., Physiol. Rev. 93(4), 1803–1845 (2013).

DOI: 10.1152/physrev.00039.2012(2) W. Derave et al., Sports Med. 40(3), 247–263 (2010). DOI:

10.2165/11530310-000000000-00000(3) L. Regazzoni et al., Sci. Rep. 6, 27224 (2016). DOI: 10.1038/

srep27224(4) A.R. Hipkiss et al., Maturitas. 93, 28–33 (2016). DOI: 10.1016/j.

maturitas.2016.06.002(5) L. Fumagalli et al., J. Sep. Sci. 41(6), 1240–1246 (2018).

DOI:10.1002/jssc201701308

HPLC 2019 Poster WinnersThree-Dimensional Chiral HPLC Analysis of Extraterrestrial Amino Acids in Carbonaceous Chondrites

Aogu Furusho, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan

How did the idea of this research project arise and what are the aims of this research?Amino acids have their enantiomers, D- and L-forms, and they are thought to have been synthesized by chemical reaction on the primitive Earth. Though the D/L ratio of amino acids on the primitive Earth might be racemates, higher organisms now mainly use L-amino acids as bioactive substances. This situation is called homochirality and the origin of this is still unclear. Various amino acids have been found in the extraterrestrial samples (meteorites), and their enantiomeric excesses of L-forms have been reported. Amino acids from the cosmos could be the primitive substances of life, so chiral amino acid properties in the extraterrestrial samples are gathering attention. However, the amount of sample is limited and the amount of amino acids in the cosmic samples are trace-level. In addition, various structural isomers are present, so a highly sensitive and selective analytical method is required. We have developed a three-dimensional (3D) high performance liquid chromatography (HPLC) system combining three different separation modes, reversed-phase, anion-exchange, and enantioselective separations. Amino acids in the samples were first derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) for a sensitive fluorescence detection and introduced into the 3D-HPLC system. We have determined chiral amino acids in various biological samples including clinical samples. Since the 3D-HPLC

system has high sensitivity and selectivity, we thought it would be a suitable tool for the determination of chiral amino acids in the cosmic samples. The aim of this research was to develop a novel 3D-HPLC system for the enantioselective determination of extraterrestrial amino acids in carbonaceous chondrites.

What are you doing in this poster that is novel?This is the first work determining chiral amino acids in the extraterrestrial samples using a 3D-HPLC system. For the determination of chiral amino acids in the extraterrestrial samples, gas chromatography (GC) and LC methods have been used. We previously developed a two-dimensional HPLC system combining reversed-phase and enantioselective separations. However, it was sometimes difficult to precisely determine the trace amounts of chiral amino acids because of the disturbance by uncountable nontarget compounds. In this research, we added an anion-exchange separation in the second dimension. With an anion-exchange column, compounds were separated by the difference in the anionic strength, which is quite different from reversed-phase mode. The interfering compounds not separated from amino acids in the reversed-phase column were separated in the second dimension, and the selectivity of the system was significantly improved. The present 3D-HPLC system could separate the target amino acids from interfering compounds in the extraterrestrial samples, and the peaks of the target amino acids were clearly observed without interferences in the third dimension. The determination of chiral amino acids in the Murchison meteorite and the Antarctic meteorite were successfully accomplished.

What were the main analytical challenges you had to overcome?The most difficult challenge was the chiral separation of isovaline (iVal). iVal has two similar side chains, a methyl group and an ethyl group at α-carbon. In this research, we used the original Pirkle type enantioselective column, KSAACSP-001S, which has L-leucine at the chiral centre. This column can separate all proteinogenic amino acid enantiomers, and D-forms always elute faster than L-forms. The mixed solution of methanol and acetonitrile containing formic acid was selected as the mobile phase, and various mobile phase conditions were tested. We tried different concentrations of formic acid and different ratios of methanol and acetonitrile, however, the separation was not sufficient. So, we tandemly connected the same two enantioselective columns, and the best separation was finally obtained.

What were your main findings and why are they useful?By using the developed 3D-HPLC system, the Murchison meteorite and the Antarctic meteorite were analyzed. These meteorite samples were provided by Dr Naraoka, who is a professor at the Department of Earth and Planetary Sciences, at Kyushu University. Amino acids in the samples were extracted with hot water and derivatized with NBD-F. All five target amino acid enantiomers, alanine (Ala), valine (Val), 2-aminobutylic



acid (2AB), norvaline (nVal), and iVal, were found in both meteorites. The amount of amino acids in the meteorites were determined, and %L values were calculated. In the Murchison meteorite, the D/L ratios of non-proteinogenic amino acids (2AB, nVal, and iVal) were almost equal. In the Antarctic meteorite, the D/L ratios of all target amino acids were almost equal. Regarding the proteinogenic amino acids, Ala and Val, the amounts of L-forms were higher than those of D-forms in the Murchison meteorite. It might be caused by the contamination of L-amino acids, which are abundantly present on the Earth. The Murchison meteorite fell at Murchison village in Australia, while the Antarctic meteorite fell in the Antarctic. The place where the meteorites fell could affect the contamination of the L-forms.

How are you planning to develop this research further?To avoid the contamination of amino acids on the Earth, sampling in the cosmic space is one of the best ways. The two sample return missions Hayabusa2 and OSIRIS-REx are ongoing, and our team belongs to the Hayabusa2 project. We will apply the current 3D-HPLC system to the samples collected in the cosmic space to clarify the enantiomeric excess of amino acids. In addition, some amino acids that were not the targets of this research have been found in the cosmic samples. Therefore, we would like to add other amino acids to the analytical targets.

Comprehensive UHPLC–MS/MS Lipidomics Profiling to Study Effects of Betulin on Keratinocytes

Carlos Calderon, University of Tübingen, Germany.

How did the idea of this research project arise and what are the aims of this research? Our research collaboration partner, Prof. Dr. Irmgard Merfort from the University of Freiburg, Germany, has previously investigated the bioactivity of the natural compound betulin, a pentacyclic triterpene, which has shown to exert a variety of different biological effects including anti-cancer, anti-inflammatory, anti-fungal, and anti-viral activities. Studies have been conducted in her laboratory to explain the clinically proven wound-healing effects of betulin (1–3). Those studies found that betulin influences the inflammatory phase and the new-tissue -formation phase in the wound-healing process, which enhances keratinocytes migration and stimulates their differentiation.

In our laboratory, we decided to undertake a pharmaco-lipidomics analysis of betulin in human primary keratinocytes to monitor alterations in the lipid profiles induced by treatment with betulin.

Our goal was to clarify whether, or in which way, the lipophilic betulin may act on cell membranes, permeate

membranes to enter the cell, or have an impact on the lipid profile of the cell.

What are you doing in this poster that is novel?We performed a comprehensive description of the lipidome of human immortalized keratinocytes and their changes following treatment with betulin.

The novel aspect of this research is the results detailing the changes in upregulation and downregulation of lipid species following betulin treatment. Additionally, for the identification of lipids we have employed a novel targeted data-processing approach combining the confidence given from the comparison of mass spectrometry (MS) and tandem mass spectrometry (MS/MS) fragmentation patterns, with the confidence given from the analysis of reverse phase-liquid chromatography (LC) elution patterns of lipids belonging to the same lipid class. Most lipidomic studies are based on data-dependent acquisition (DDA) which acquires MS data comprehensively but only MS/MS data from the most abundant signals and for structural confirmation only. This means that when an MS signal is above a certain threshold an MS/MS experiment is triggered. It provides high-quality spectra for identification but no comprehensive MS/MS data across the peak and chromatogram which means no extracted ion chromatograms (EICs) can be generated on the MS/MS level. In our study based on independent data acquisition (IDA), EICs can be extracted on a MS/MS level, and can also be used for fragment ions, which provides additional options for data processing, higher confidence, and assay specificity (4).

What were the main analytical challenges you had to overcome?Our main analytical challenge was the identification of lipid species in the keratinocytes. Our untargeted LC–MS approach with DIA using SWATH generates large amount of data. Tools for processing the data and performing identification of lipids are available. However, many misidentifications are also obtained. These can insert bias into the findings of the study. For this reason, the aforementioned targeted MS data-processing was employed.

What were your main findings and why are they useful?We made a detailed characterization of the lipidome in keratinocytes. At the same time, we showed that there are significant differences in keratinocytes after treatment with betulin. In total, 611 lipids were identified at the “lipid species level” and 440 of them were shown to be significantly changed. Changes can be described in terms of lipid classes. Cholesteryl esters and diacylglycerides were significantly down-regulated in botulin-treated samples and glycerophospholipids, sphingolipids, and diacylglycerides were up-regulated.

Additionally, it was observed that the presence of betulin in extracts of keratinocytes, which were previously treated with betulin and washed, indicated that betulin may be incorporated into the membrane of keratinocytes. Also, the presence of other triterpenes in samples treated with betulin and absent in control samples could represent betulin metabolized within the keratinocytes (4).



How are you planning to develop this research further?Based on the obtained results, we would like to develop more targeted experiments to determine how those changes in specific lipid classes following betulin treatment are related with the healing properties of the compound in the skin.

References1. U. Woelfle et al., J. Invest. Dermatol. 130, 113–123

(2010). doi:10.1038/jid.2009.248.2. S. Ebeling et al., PLoS One. 9 (2014) e86147.

doi:10.1371/journal.pone.0086147.3. T. Wardecki et al., J. Nat. Prod. 79, 1112–1123 (2016).

doi:10.1021/acs.jnatprod.6b00027.4. C. Calderón et al., Proteomics. (2019) 1900113.

doi:10.1002/pmic.201900113.

Screening Therapeutics According to Their Uptake Across the Blood–Brain Barrier: A High Throughput Method Based on Immobilized Artificial Membrane Liquid Chromatography-Diode-Array-Detection Coupled to Electrospray-Time-of-Flight Mass Spectrometry

Giacomo Russo, Ghent University, Belgium

How did the idea of this research project come about and what are the aims of this research?This idea is actually an expansion of a previous project focused on the development of analytical methodologies based on immobilized artificial membranes- and micellar- liquid chromatography (LC). The approach of combining in-silico physicochemical descriptors, achieved by molecular docking and conformational analysis with the chromatographic data, was indeed a winning strategy. However, after an insightful talk with my supervisor, Frederic Lynen, we agreed that, albeit really promising, that methodology would not appeal to pharmaceutical companies because of the limited throughput and the low selectivity of UV detection. From there, the idea arose of moving to mass spectrometry/diode array detection (MS/DAD) and setting this research in a large-scale industrial-like scenario. The aim of this research is to provide a much faster screening method than those already published to profile compound libraries for their likelihood to cross the blood–brain barrier and exert central nervous system activity.

What are you doing in this poster that is novel?The development of a new analytical method, based on organic buffers rather than on phosphate salts, widely employed in biopartitioning studies and its validation on a data set as wide as nearly 80 compounds is unprecedented. The integration of in vitro methods with molecular dynamics simulation also brought some consistent advances in the field of biological activity prediction and allowed molecular mechanism elucidation.

What were the main analytical challenges you had to overcome?Working on very short columns can easily lead to overloading, especially when the analysis of compounds is accomplished in mixtures. Moreover, the use of a biomimetic stationary phase brings some constraints in method development. Indeed, the organic modifier in operating conditions should be preferably as much as, or below, 30% v/v to mirror biological phenomena that occur in aqueous medium. Gradient elution should also be avoided because the affinity for the phospholipid membranes has to be measured in identical conditions to ensure flawless quantitative structure activity relationship (QSAR) study.

What were your main findings and why are they useful?A method 554% faster than the previous one and far more environmentally friendly was developed. In fact, the miniaturization of the LC set-up meant only 0.5 L of mobile phase was consumed of which 150 mL was a methanolic fraction for screening 79 compounds in triplicate plus a blank sample every five runs. The analyses were completed in only 1.1 days. Using an organic buffer, in this case ammonium acetate, rather than the phosphate buffers used in biopartitioning studies, only negligibly affected the accuracy of the screening method. This seemed a reasonable price to pay for faster analyses and to place the research in a pharmaceutical industry-like context.

How are you planning to develop this research further?By exploring the undeniable advantages in terms of selectivity and peak capacity offered by multidimensional LC exploited for biopartitioning purpose with the aim of making this method appealing to the pharmaceutical industry.

Assessment of Bed and Particle Integrity of Size-Exclusion Columns Packed with Sub-2-µm Particles

Jelle De Vos, Department of Chemical Engineering, Vrije Universiteit, Brussel, Belgium

How did the idea of this research project arise and what are the aims of this research?Back in 2016 we published a paper on the effects of operating pressure on selectivity and efficiency in aqueous size-exclusion mode for the separation of intact proteins (1). We used kinetic plots to describe the possibilities of faster methods in size-exclusion chromatography (SEC) mode when applying ultrahigh pressures, which laid the foundation for the work presented in the poster.

What are you doing in this poster that is novel?The poster explores the potential of sub-2-µm particulate SEC columns operated under ultrahigh-pressure liquid chromatography (UHPLC) conditions. There are currently no studies that investigate the effects of high operating



pressures on packing stability and particle integrity, and, hence, the selectivity of aqueous size-exclusion columns. These are essential aspects when method speed-up is envisioned.

What were the main analytical challenges you had to overcome?The experiments were performed using SEC columns packed with 3.5-µm and 1.7-µm particles. These columns were subjected to extreme pressure stress-tests and their degradation was monitored by separating a mixture of protein standards. At first, we were not able to explain the cause of the impaired chromatography when higher backpressures were applied, as this is usually not the case in reversed-phase LC mode. Hence, following the stress tests, the columns were opened and the particles were subjected to physical analytical techniques (scanning electron microscopy, argon adsorption measurements, and mercury intrusion porosimetry). The results were verified using columns originating from different batches. All these results were combined to comprehensively describe the issues that arise at higher operating pressures when using columns packed with sub-2-µm particles.

What were your main findings and why are they useful?The improved mass transfer characteristics of sub-2-µm particles was demonstrated by a sub-min separation of intact proteins by applying a flow rate of 1.8 mL/min, corresponding to a column pressure of 530 bar. High pressure operation typically results in bed compression, which impairs chromatography. Improved column packing is essential to obtain robust high performance SEC separations.

How are you planning to develop this research further?This study demonstrates the potential of sub-2-µm particles in aqueous SEC, and can be expanded towards exploring the potential of even smaller particle diameters ranging around 1 µm.

References(1) J. De Vos, E. R. Kaal, R. Swart, M. Baca, Y. Vander Heyden, and S.

Eeltink, J. Sep. Sci. 39, 689–695 (2016).

Chromatographic Behaviour of Bivalent Bispecific Antibodies on Cation Exchange Columns

Lucas Kimerer, University of Virginia, USA.

How did the idea of this research project arise and what are the aims of this research?In a collaboration with AstraZeneca we are investigating how bivalent bispecific antibodies (BiSAb) behave on chromatographic media. Bivalent bispecific antibodies contain an entire framework mAb with a pair of flexibly linked single chain variable fragments (scFvs) which are responsible for binding the second targeted epitope.

What are you doing in this poster that is novel?We investigated three peak elution behaviour on cation exchange columns where a pure feed of BiSAbs elute as three reversible peaks. Each peak when collected, diluted and reinjected elute the same three peak profile. At very long residence times or increased temperature of about 45 °C the binding conformations interconvert sufficiently to reach an equilibrium and elute as a single, merged peak (1). The framework antibody without flexibly linked scFvs elutes as a single peak. Enzymatically cleaved fragments and intramolecular crosslinking indicated that the multiple peaks are directly caused by the number of linked scFvs and their subsequent conformational flexibility. The three peaks correspond to each scFv being either collapsed and able to interact with the framework mAb or outstretched and able to more strongly bind the column (2).

We found a novel multiple peak elution mechanism where the peaks correspond to reversible tertiary structure conformation changes due to flexibly linked domains.

What were the main analytical challenges you had to overcome?As a result of the reversibility of the conformation states it was not possible to characterize the different peaks offline. We used in-line measurements of hydrodynamic radius from dynamic light scattering and tryptophan autofluorescence to see that all three peaks are monomer sized and slightly differ in solvent exposure due to different conformations of the linked scFvs.

What were your main findings and why are they useful?Our findings explain that in analytical CEX runs the BiSAb protein feed which appeared to contain three peaks and high levels of impurities is not highly impure, but that the same protein is able to elute at three different retentions depending on the linked scFv’s conformation.

How are you planning to develop this research further?Study the same BiSAb homologous series on hydrophobic interaction chromatography resins and at high load.

References(1) L.K. Kimerer et al., J. Chromatogr. A 1601, 121–132 (2019).

(2) L.K. Kimerer et al., J. Chromatogr. A 1601, 133–144 (2019).

A New Gastro-Intestinal System to Evaluate the Effect of Food Methylglyoxal

Lucia Ferron, Nutraceutical and Food Chem Toxicol Analysis Laboratory, Department of Drug Sciences, University of Pavia, Italy, FlaNat Research Italia Srl, Rho (MI), Italy

How did the idea of this research project arise and what are the aims of this research?This project aims to develop a new dynamic and multi-compartmental millifluidic bioreactor simulating a gastro-intestinal system suitable for the evaluation of



the effect (absorption/metabolization and cytotoxicity) of exogenous bioactives derived from food or drug intake.

By using this new dynamic protocol, we tested methylglyoxal (MGO), which is a potentially toxic compound, produced both in food and endogenously, responsible for the formation of the advanced glycation end-products involved in many pathological chronic implications, such as diabetes, cardiovascular diseases, and ageing-associated disorders (1–4).

The experiments were also performed with the standardized Minekus’ enzymatic digestion protocol to investigate the potential of this new dynamic system compared with static traditional assays (5).

What are you doing in this poster that is novel?The novelty aspect of this work is the setup of a new platform that gives the opportunity to simulate continuous flow conditions, reproducing different simil-physiological flows and connecting different compartments to recreate a multi-organ model. This is the first setup of a dynamic gastro-intestinal platform and such a system represents a potential advance in the study of kinetic, metabolic, and cytotoxic profiles of different substances.

What were the main analytical challenges you had to overcome?First of all, it was necessary to carefully optimize the flow conditions to treat gastric (GIST-882) and intestinal (Caco-2) cells in the dynamic system when the two compartments were connected, in order to reproduce a simil-physiological digestive process.

Then, to monitor MGO metabolic fate by using a millifluidic system, it is mandatory to collect samples at different monitoring times and to analyze them by a proper analytical technique. To detect and quantify MGO in the cell medium during gastric and intestinal phases, we used a reversed-phase high performance liquid chromatography diode-array detector (HPLC-DAD) method, slightly modified from the literature and validated by us. The choice of UV detector required a sample preparation step, consisting of a derivatization procedure to obtain a UV-detectable MGO derivative (the derivatization procedure required a setup, too).

What were your main findings and why are they useful?The obtained results highlight that exogenous MGO is rapidly metabolized by a safe digestive process, and this is very important for human health in relation to widespread diseases, such as diabetes and ageing-associated disorders. Furthermore, by comparing the dynamic and the static processes, it emerged that the two protocols provided complementary data, and in particular the dynamic system elucidated a new role of gastric cells, which should be further investigated in the metabolization of toxic compounds.

How are you planning to develop this research further?We are going to submit to this dynamic digestion protocol other molecules, both toxic and active compounds, also in association. Moreover, we will probably use this platform for complex matrices, such as food samples, to test the effects of the interactions among the different food components, on the absorption/metabolization of a specific molecule.

Anything else you would like to add?This platform could reduce in vivo experiments, in particular during preliminary investigations, and find a very promising application in high-throughput cell-based compound screening for food analysis, drug discovery, and toxicity tests.

References(1) M. Hellwig, S. Gensberger-Reigl, T. Henle, and M.

Pischetsrieder, Semin. Cancer Biol. 49, 1–8 (2018).(2) H.J. Chun, Y. Lee, A.H. Kim, and J. Lee, Neurotox. Res. 29,

419–431 (2016).(3) N.M.J. Hanssen et al., Diabetes 66, 2278–2283 (2017).(4) T.M. Jensen, D. Vistisen, T. Fleming, et al., Diabet. Med. 33,

1625–1631 (2016).

(5) M. Minekus et al., Food Funct. 5, 1113–1124 (2014).

Toolbox for Studying the Chemistry Of Light-induced Degradation (TooCOLD): Towards an Online Degradation System

Mimi den Uijl, University of Amsterdam, Amsterdam, The Netherlands

How did the idea of this research project arise and what are the aims of this research?A lot of organic compounds change under the influence of UV light. Within the cultural heritage field, this is undesirable. For that reason, the idea to analyze light degradation in a more comprehensive way originated from this discipline. However, cultural heritage is not the only discipline that deals with light-sensitive organic compounds. For example, in water-purification processes, high-energy UV is used to degrade contaminants in water. Another example is the food industry, where companies want to prevent degradation of healthy food ingredients and prevent the formation of toxic by-products. When these disciplines came together, the TooCOLD project (Toolbox for studying the Chemistry Of Light-induced Degradation) was started under the supervision of Maarten van Bommel (professor at the Faculty of Sciences and the Faculty of Humanities at the University of Amsterdam).

The aim of TooCOLD is to develop a fast, robust, and fully integrated analytical system to study degradation mechanisms of individual components and complex mixtures. A sample of a historical object consists of multiple components. In the first step, these are separated. Next, the separated compounds eluting from the column are sent to a degradation cell. In this degradation cell, the sample is illuminated and tracked at the same time. When the degradation is sufficient, the degradation mixture is sent to another dimension separation and analyzed with UV and mass spectrometry (MS). This project is divided into three



parts: chromatography (that’s me), spectroscopy (Iris Groeneveld), and data analysis (Carl Emil Eskildsen).

The TooCOLD project started in September 2018 and the degradation cell is fully in development, as well as the two-dimensional setup for this degradation research. In this poster, I presented the preliminary results. The initial aim was to develop an off-line degradation system to compare future on-line work to a reference. After the development of this off-line cell, other parameters were changed to assess their influence on the degradation. Parameters that are found to greatly affect degradation can be implemented in that way in the on-line degradation cell.

What are you doing in this poster that is novel?For the study encompassed by this poster, we took an extract of the seeds of the Achiote tree. This mixture is known as Annatto and is extensively used in the food industry to colour dairy products, such as cheese and margarine, and to a lesser extent as a textile dye in cultural heritage. For this reason, this mixture has been characterized and its thermal degradation mechanism has been studied in previous studies. The light stability of the compound has already been analyzed, but not in the way that we did in our research. Other analytical techniques were applied for the analysis and most of the work was performed 30 years ago on analytical systems from that time. Annatto has not yet been used as a model compound to measure the degree of degradation, and not from the perspective of comprehensive degradation research.

What were the main analytical challenges you had to overcome?The first challenge we had to overcome was to create the degradation system. In particular, the repeatability of the system was pivotal. We decided to work with two degradation-wavelength spectra, one that contained only visible light and one that contained both visible and UV light. This led to the use of two different lamps and thus two different setups. Another problem was the complexity of the Annatto sample. While bixin is the main component, there are also many other similar compounds that only differ in stereochemistry or in methylation. The third problem we encountered was the ostensible inability to obtain reference data from undegraded annatto. When the extraction is performed on the annatto seeds, you retrieve a mixture of compounds that could have, and most likely has, already been degraded by light, either in the tree or in the process of shipping. Because you don’t have the clear undegraded benchmark, it is difficult to say something about these by-products. The last problem we encountered was concerning the detection after the liquid chromatography (LC) analysis. While the annatto sample, which contained all kinds of colouring components, can be analyzed with UV, the degradants often did not allow UV detection.

What were your main findings and why are they useful?In this research, the goal was to set the boundaries for the online degradation cell and to see what parameters have an influence on the degradation. In this poster, there were three main focuses: the effect

of different degradation-wavelength spectra, the degradation speed of similar compounds, and the effect of solvent composition. We have found that when different wavelength spectra are used, the difference in degradation is enormous. Not only does the high-energy spectrum degrade much faster but the degradation products are also completely different. This also led to a different problem because these degradation products cannot be analyzed by a diode-array detector (DAD). The degradation products of the lower energy spectrum could be detected in the same way as the original mixture. We have found that very similar compounds have different reaction speeds, which is something that will be taken with us into the next phase of the project. The most important finding of the poster was that the composition of the solvent has an influence on the degradation speed. This might seem logical, but it has consequences on the setup of the TooCOLD project.

How are you planning to develop this research further?The main findings presented in this poster are linked to the degradation of Annatto. While the degradation cell is being developed at the moment, we are thinking about the implementation of the cell. The challenges that we need to overcome are how we will get the effluent in the cell and how to make sure we have the right matrix during degradation. Another challenge is how to get the sample into the second-dimension separation. Since we will change the matrix before degradation, we might need to alter the solvent composition or the concentration before starting our second-dimension separation.

The degradation research performed on the poster will be used to compare the results of the on-line degradation and to compare it to different standards for degradation research, such as the Xenotest (a standard light degradation test used extensively in cultural heritage).

Retention Mechanism Study in Hydrodynamic Chromatography

Noor Abdulhussain, Universiteit van Amsterdam, Amsterdam, The Netherlands

How did the idea of this research project arise and what are the aims of this research?Several years ago, I worked on the application of comprehensive two-dimensional liquid chromatography (LC×LC) as part of my master’s graduation project. In this project, we sought to characterize polymeric nanoparticles based on their size and polymer composition. To characterize the particles based on their hydrodynamic volume, we applied hydrodynamic chromatography (HDC) as a first-dimension separation. After the HDC separation, the effluent was then



combined with an organic solvent to achieve dissolution of the particles into their constituting polymers. Using stationary-phase-assisted modulation, the polymers were filtered out from the effluent and injected in the second dimension. Here, the polymers were characterized based on their size (1,2). We observed that our experimental data concerning the HDC separation did not agree with established theory. I consulted the scientific literature to gain more fundamental knowledge about hydrodynamic chromatography.

Theory describing the retention in hydrodynamic chromatography was proposed in the early 1970s. Since the interparticular channels are exploited in packed-column HDC, most of these theories have been derived from models used to describe retention in open capillaries as a result of laminar flow. To some extent, the packed column used in HDC can be considered as a collection of capillaries. Typically, significant approximations were needed. The influence of various factors, such as hydrodynamic wall interaction, the apparent slip-velocity of solute particles, stress-induced trans-streamline migration, and deformation-induced chain-stretching of flexible analytes, such as polymers, has often been ignored. Despite these various studies throughout the years, no definite and clear understanding of retention in HDC has been obtained. In fact, recent studies have suggested that the theoretical models for HDC are insufficiently capable to account for the above non-idealities (3).

The main aim of my current research is to revisit the existing HDC theory using computational fluid dynamics (CFD) simulations. CFD applies fluid-flow-simulation software, which allows analysis of turbulent and laminar flow profiles in various geometries that we design. We aim to gain a better understanding of HDC theory using CFD. More specifically, the aim of this study is to investigate the effect of the packing order on the flow profiles. In addition, the relationship between the physical parameters of the packing material and the above-mentioned approximations is investigated.

What are you doing in this poster that is novel?The main novelty is that we apply CFD simulations to investigate hydrodynamic chromatography; older works relied solely on the use of experimental data. As a result of the increased computational power and the commercialization of ready-to-use software packages, CFD has become widely accepted as a design tool in many fields to help with understanding flow behaviours in complex geometries. As HDC is a flow-based technique, CFD can be useful for the study of the separation aspects. Using CFD in combination with Matlab, simulation data are translated into graphs that represent the practical result, which allows us to compare the theoretical, experimental, and simulated data all at once.

What were the main analytical challenges you had to overcome?One of the first challenges we encountered was to decide the best geometry that fits the ideal conditions for

particle packing. The ordering of particle packing has been previously investigated in the literature (1). The main particle arrangements are the face-centred cubic (FCC), the body-centered cubic (BCC), and the simple cubic (SC) configurations.

The different ordering of the particles in these packing arrangements can have a large impact on flow (profile) in the channel. The flow channel is the interstitial space between the particles. Consequently, it is the domain where the fluid can flow, and thus the main computational domain. The size and structure of the flow domain depends on the size, packing arrangement, and the ratio between the channel diameter and the analyte radius. These can influence the velocity profile. Separation in HDC arises from a nonturbulent Poiseuille flow profile, therefore the packing order is important. Based on the literature, it is found that zone broadening is less for the FCC structure compared to the other configurations (2). Researchers also found that by investigating other aspects, such as the efficiency and plate height, the FCC configuration is really the best fit for our research. Therefore, we created a domain structure that can be regarded as an array of 3D “boxes” known as unit cells in the FCC configuration.

Another critical challenge is the interpretation of data obtained from the simulations. It is critical that insights into the fundamental separation mechanism of HDC are accurately distilled from the simulations.

What were your main findings and why are they useful?We are in the initial stages of our research. From the computational results we indeed observed a Poiseuille flow profile, which is in agreement with HDC theoretical principles. The simulation data suggest that the velocity decreases when approaching the wall, which is important because small particles tend to migrate with the slower streamlines close to the walls. A higher velocity was observed at the near wall region, meaning that large particles will migrate at the average velocities of the faster streamlines.

How are you planning to develop this research further?We are now evaluating average velocities for specific analytes. This allows us to predict the velocity needed to separate different size particles in a given unit cell. The final step at this stage is linking these velocity profiles to the required diameter needed for analytes to benefit from the HDC effect.

Looking further on the horizon, we will shift our focus to another interesting phenomenon currently known in HDC, which is the extreme band broadening as has been observed by multiple groups (2).

Furthermore, there are several other parameters of interest that need to be further considered in the simulation. The structures that we designed are highly ordered and therefore do not represent the situation in practice. For example, packed particles in chromatographic columns feature a size distribution. This must be included in the simulation. Moreover, the eluent for packed-column HDC typically comprises deionized water, with additives, salts, and surfactants. These are added to the eluent to optimize electrostatic



repulsion between the analytes and the beads of the packing (4). Variation in the ionic strength creates a difference in the thickness of the electrical double layer. Assuming this layer is stagnant, this will cause the flow channels to change and the HDC separation range to shift. Therefore, the addition of this layer must also be considered in the simulation. To investigate these points, the currently developed framework using CFD simulations will be needed.

References(1) M.R. Schure, R.S. Maier, D.M. Kroll, H.T. Davis, J. Chromatogr.

A 1031(1–2), 79–86 (2004). https://doi.org/10.1016/j.chro-ma.2003.12.030.

(2) L. Li, X. Yan, J. Yang, Q. Wang, Energy Procedia 75, 3322–

3327 (2015). https://doi.org/10.1016/j.egypro.2015.07.719.

Mixed Mode Stationary Phases and their Benefit in LC×LC

Stefanie Bäurer, University of Tübingen, Germany.

How did the idea of this research project arise and what are the aims of this research?The requirements in pharmaceutical quality control are steadily increasing and the trend tends to a comprehensive characterization of the pharmaceutical formulation. Thus, separation methods complementary to the well-known and widespread reversed-phase liquid chromatography (LC) methods are needed to elucidate coelution of impurities with the active pharmaceutical ingredient(s) (API). Furthermore, the analysis of very polar analytes like amino acids is only possible when, for example, ion pair reagents are added to the mobile phase otherwise it lacks sufficient retention and selectivity. A drawback of this chromatographic mode is the incompatibility with mass spectrometry (MS).

As an orthogonal alternative, hydrophilic interaction chromatography (HILIC) enjoys great popularity. It is well suited to the separation of analytes of this kind and offers an excellent compatibility with MS. The retention mechanism was discussed as a partitioning process of the analytes into a water enriched layer on the surface of the separation material. Recent discussions also consider contributions of hydrophilic and electrostatic interactions.

Another chromatographic alternative of rising importance is mixed mode chromatography (MMC) (1–3). The utilized stationary phases possess multiple different kinds of interaction sites. Therefore, multiple different interactions like hydrophilic, hydrophobic, and electrostatic ones influence the retention of the

analytes (1–5). The dominant occurring interactions can be governed by the choice of the chromatographic conditions. This leads to a great flexibility in the choice of the chromatographic mode which is a valuable chromatographic tool and offers new possibilities especially in combination with the two-dimensional separations, as well as in the field of pharmaceutical analysis (3,6–10).

Since offline separations are time consuming and error prone, we developed an online fully comprehensive two-dimensional impurity-profiling screening method (LC×LC) for pharmaceutical drug formulations containing very polar analytes like amino acid infusion solutions. Besides the chromatographic separation we focused on simultaneous complementary detection methods realized by the hyphenation of UV detection, charged aerosol detection, and high-resolution mass spectrometry (QTOF-MS/MS).

What are you doing in this poster that is novel?Chiral stationary phases are only rarely considered to be used for achiral separations. However, many chiral stationary phases offer multiple interaction sites (11) and can be defined as mixed-mode stationary phases. Herein, the chiral stationary phase was used for the separation of underivatized amino acids. Additionally, a range of HILIC phases were screened under HILIC conditions for evaluation of the most promising stationary phase combinations for online full comprehensive two dimensional separations (LC×LC). The chiral stationary phase was used in the first dimension under polar organic elution conditions. This prevented a solvent mismatch in the second dimension when HILIC conditions were utilized.

Furthermore, complementary detectors were hyphenated for an impurity profiling set up less error prone than offline separations. The spectroscopic characterization was done by a diode array detector to get information about the precursor of the impurity peak. For universal quantification a charged aerosol detector (CAD) and, for identification, a high-resolution mass spectrometer (QTOF-MS/MS) were coupled. Since the latter ones are both destructive detection methods, we used a flow splitter to offer simultaneous use of both detectors in a single analysis.

What were the main analytical challenges you had to overcome?For the orthogonality determination of two dimensional separation systems many models were developed. This approach presents a universal screening approach covering the APIs as well as possible impurities. This includes degradation products, excipients as well as compounds from the primary packing material. Therefore, the analyte set is unknown and the orthogonality of the present chromatographic system is hard to predict. Hence, we spent special attention to the not covered retention space which should leave enough space for the elution of impurities.

Since amino acids are very similar compounds, many HILIC separations showed quite similar retention



patterns. The use of a chiral stationary phase under polar organic elution conditions offered useful additional interactions and in consequence beneficial selectivity.

What were your main findings and why are they useful?The first part presented in this poster was the column screen of commercially available stationary phases including ionic and zwitterionic HILIC and MMC surface chemistries under HILIC conditions using proteinogenic amino acids as analytes. The orthogonality plots can help other chromatographers analyzing similar analytes by the choice of suitable stationary phase to face their individual separation issue.

Furthermore, solvent mismatch is a problem which often occurs when highly orthogonal separation methods are coupled in two dimensional separations. For example, reversed-phase×HILIC separations usually show peak broadening and distortion when solvent modulation systems, such as fixed solvent modulation (FSM), active solvent modulation (ASM), stationary phase assisted modulation (SPAM), or other modulation strategies are not used (12). The observed poor peak shape is a consequence of the fact that the first dimension eluent with high water amount has high elution strength under HILIC conditions resulting in peak broadening and distortion. This means in other words, if the first-dimension eluent has a weaker elution strength in the second dimension than the utilized second dimension mobile phase, this issue can be overcome. In former studies, we also observed this solvent incompatibility phenomenon for HILIC×HILIC amino acid separations, especially in case of the strongly retained amino acids. To overcome this problem, we used acetonitrile-rich conditions combined with methanol as a polar component and only a very small amount of water, also known as polar organic conditions, in the first dimension. For elution of the stronger retained amino acids, the methanol amount was significantly increased while the water amount was moderately increased after an initial isocratic period. The resulting online full comprehensive two-dimensional separations showed a nice peak shape without peak broadening and distortion.

Impurity profiling usually requires the analysis of the samples under complementary chromatographic modes to ensure that degradation products are not coeluting with active pharmaceutical ingredient (API). Also, two dimensional separations with offline coupling are popular. Therefore, the first dimension eluent can be analyzed directly after collection of the fractions or after evaporation and reconstitution in another solvent in a second chromatographic dimension. Using this approach, the treatment of the sample during the sample preparation process needs to be evaluated. For instance, oxidation during the collection and/or the evaporation process can occur. Also, volatile compounds can evaporate during the collection or evaporation if used. By using an online coupling of the two dimensions these problems can be easily overcome.

Additionally, the hyphenation of complementary detectors offers the possibility to characterize the sample comprehensively. Therefore, we used a diode array detector (DAD), a charged aerosol detector (CAD), and a

high-resolution mass spectrometer (QTOF-MS/MS). The UV detector, including UV spectra of the analytes, can give information of the precursor of the degradation product. The charge aerosol detector can be used for a universal quantification. Furthermore, the analytes can be identified by the high-resolution mass spectrometer.

How are you planning to develop this research further?The presented online full comprehensive two-dimensional chromatographic method is well suited to very polar analytes. To cover moderate polar and apolar analytes, a two dimensional separation method could be developed and hyphenated to UV, charged aerosol detection (CAD), and high-resolution mass spectrometry (QTOF-MS/MS) (13). In this case, the screening of these samples with the herein presented methods and the to be developed method would cover a broad polarity range and give useful information about the impurities and degradation products (13).

References(1) D. Sýkora et al., J. Sep. Sci. 42, 89–129 (2019).(2) L. Zhang, TrAC Trends in Analytical Chemistry 82, 143–163

(2016).(3) E. Lemasson et al., Mixed Mode Chromatography - A Review,

Suppl. to LCGC 22–33 (2017).(4) S. Bäurer et al., J. Chromatogr. A. 1560, 45–54 (2018).(5) S. Bäurer et al., J. Chromatogr. A 1593, 110–118 (2019).(6) K. Zhang et al., J. Pharm. Biomed. Anal. 128, 73–88 (2016).(7) D.R. Stoll, LCGC North Am. 35, 680–387 (2017).(8) M. Iguiniz & S. Heinisch, J. Pharm. Biomed. Anal. 145, 482–

503 (2017).(9) V. D’Atri et al., Anal. Chem. 91, 210–239 (2019).(10) S. Bäurer et al., Chromatographia 82, (167–180) (2019).(11) M. Lämmerhofer, J. Chromatogr. A 1217, 814–85 (2010).(12) B.W.J. Pirok et al., Anal. Chem. 91, 240–263 (2019).(13) S. Bäurer et al., Anal. Chim. Acta. In Press (https://doi.

org/10.1016/j.aca.2019.09.068)

COMPILED BY:

Lewis Botcherby, Associate Editor

Kate Jones, Managing Editor

Alasdair Matheson, Editor-in-Chief



rising stars of separation science - unime europe poster supplem… · rising stars of separation...

Documents