(PDF) 26th International Symosium on Pharmaceutical and Biomedical Analysis 2015 Tbilisi Book of Abstracts

26th International Symposium on

Pharmaceutical and Biomedical

Analysis (PBA 2015)

July 5-8, Tbilisi, Georgia

BOOK OF ABSTRACTS

PBA 2015

26th International Symposium on

Pharmaceutical and Biomedical Analysis

July 5-8, 2015

Marriott Courtyard Tbilisi Hotel

Tbilisi, Georgia

Chair of Physical and Analytical Chemistry

School of Exact and Natural Sciences

Chaired by:

Prof. Bezhan Chankvetadze

http://pba2015tbilisi.tsu.ge/

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Book of Abstracts

Most important conference in the world focusing on cutting-edge areas of

scientific research related to pharmaceutical and biomedical analysis

PBA 2015

26th International Symposium on

Pharmaceutical and Biomedical

Analysis

www.pba2015tbilisi.tsu.ge

Chaired by:

Prof. Bezhan Chankvetadze

Chair of Physical and Analytical Chemistry

School of Exact and Natural Sciences

Iv. Javakhishvili Tbilisi State University

Tbilisi, Georgia

July 5-8, 2015

Marriott Courtyard Tbilisi Hotel

Tbilisi, Georgia

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3

Table of Contents

Table of Contents 3

Committees 4

General Information 5

Workshop 6

Guidelines for Speakers 6

Guidelines for Poster Presentations 7

Symposium Proceedings 7

Poster Presentation Award 7

Young Scientists Award 8

Exhibition 9

Symposium Sponsors 9

Symposium Supporting Organizations 9

Plenary Lectures 10

Keynote Lectures 12

Oral Presentations 47

Young Scientist Session Presentations 95

Poster Presentations 101

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Committees

Chairman

Bezhan Chankvetadze

Tbilisi State University

Tbilisi, Georgia

Tel: +995 32 290648 or +49 251 2006047

Fax: +995 32 913369 or +49 251 2006048

E-mail: bezhan_chankvetadze@yahoo.com

Scientific Committee

Coral Barbas (Madrid, Spain)

Carlo Bertucci (Bologna, Italy)

Quezia Cass (São Carlos, Brazil)

Ziad El-Rassi (Stillwater, USA)

Salvatore Fanali (Rome, Italy)

Sandor Görög (Gedeon Richter, Budapest, Hungary)

Jun Haginaka (Hyogo, Japan)

Ulrike Holzgrabe (Wuerzburg, Germany)

Roman Kaliszan (Gdansk, Poland)

Wolfgang Lindner (Vienna, Austria)

Gabriella Massolini (Pavia, Italy)

Ruin Moaddel (Baltimore, USA)

Koji Otsuka (Kyoto, Japan)

Sergio Pinzauti (Sesto Fiorentino, Italy)

Mojtaba Shamsipur (Kermanshah, Iran)

Yvan Vander Heyden (Brussels, Belgium)

Jean-Luc Veuthey (Geneva, Switzerland)

Kelly Zhang (San-Francisco, USA)

Guowang Xu (Dalian, China)

Honorary Scientific Committee

Givi Tsintsadze (Georgian Technical University, Tbilisi, Georgia)

Giorgi Kvesitadze (Georgian National Academy of Sciences, Tbilisi, Georgia)

Ivan Kiguradze (Georgian National Academy of Sciences, Tbilisi, Georgia)

Irakli Zhordania (Georgian National Academy of Sciences, Tbilisi, Georgia)

Temur Naneishvili (Georgian National Academy of Sciences, Tbilisi, Georgia)

Etheri Kemertelidze (Georgian National Academy of Sciences, Tbilisi, Georgia)

Shota Samsonia (Tbilisi State University, Tbilisi, Georgia)

Giorgi Tavadze (Georgian National Academy of Sciences, Tbilisi, Georgia)

Shota Sidamonidze (Tbilisi State University, Tbilisi, Georgia)

Organizing Committee

Vladimer Papava (Rector, Tbilisi State University, Honorary Chairman)

Bezhan Chankvetadze (Chairman)

Ramaz Botchorishvili

Tamar Tsintsadze

Lali Chankvetadze

Maia Merlani

Iza Matarashvili

Giorgi Jibuti

Rusudan Kakava

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General Information

Venue: Courtyard by Marriott Hotel Tbilisi

4 Freedom Square, Tbilisi, 0105 Georgia

Tel: 995 322 779100

Fax: 995 322 779110

www.marriott.com/TBSCY

Meeting Rooms Symposium opening plenary session and concert on

Sunday, July 5th will take place in the Assembly Hall of I.

Javakhishvili Tbilisi State University (I. Chavchavadze

Ave 1, Tbilisi 0179). The path leading you from the major

entrance to the lecture room will be marked with the

symposium logo. From July 6th to 8th the symposium

sessions will take place in the Courtyard by Marriott Hotel

Tbilisi. Meeting rooms are located on the 2nd floor of the

hotel.

Poster and Exhibition Area Poster and Exhibition area is located on the 2nd floor in

the conference hotel in front of the lecture rooms.

Badges A name badge must be worn by each registered

symposium participant and accompanying person in order

to gain admittance to the meeting and social gatherings.

Registration Symposium Registration Desk will be located on July 5th

in front of the plenary session room (Assembly Hall) on

the second floor in the Major Building of I. Javakhishvili

Tbilisi State University (I. Chavchavadze Ave 1, Tbilisi

0179) on Sunday, July 5th 14.00 to 19.00 and in the main

lobby of the Courtyard by Marriott Hotel from Monday,

July 6th to Wednesday July 8th (09.00-18.00).

Transfer from the airport After passing the customs gate in the arrival area of Tbilisi

International Airport please keep to the right and look for

PBA 2015 booth in front of cafe Cappadocia inside the

terminal. The representatives of the organizing committee

of the PBA 2015 symposium will be waiting for you there

to organize your transfer to the symposium and other

downtown hotels free of charge.

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Workshop on Pharmaceutical and Biomedical Analysis

Sunday, July 5, 2015

10.00-12.00 Prof. Attila Felinger (University of Pecs, Pecs, Hungary)

High-performance liquid chromatography: Fundamentals and method

development

12.00-12.30 Lunch

12.30-14.30 Prof. Janusz Pawliszyn (University of Waterloo, Ontario, Canada)

Sample preparation: Fundamentals and recent developments

14.30-15.45 Dr. Barbara Bojko (University of Waterloo, Ontario, Canada)

Sample preparation for bioanalysis

15.45-16.00 Discussion and closing

Guidelines for Speakers

Authors are kindly requested to give their presentation file to the technicians in their lecture

room, preferably a half day before the beginning of the corresponding session.

Power Point software will be installed on all the computers. Preferred file format is in PPT for

MS Office (Office XP, Office 2003 or 2007, etc.). Please do not use MAC file format, if

possible. There will be a technician in the lecture room to assist you with your presentation.

For optimal presentation please use Microsoft PowerPoint file with the resolution 1024 x 768

GA. If you use animations please embed them. If it is a special animation please bring all the

files to the symposium. If you have a Macintosh computer, it can be plugged in having a

conventional, 15-pin socket. If your computer does not have this connection, please bring an

appropriate converter with you.

Speakers should be aware of the time limit of their talks and due to parallel sessions strictly

follow the schedule. The plenary talks are scheduled according to 40+5 (talk+discussion),

keynote lecture according to 25+5 minutes, oral presentation according to 17+3 minutes and for

Young Scientists session 12+3 minutes formats.

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Guidelines for Poster Presentations

Posters will be presented during 3 days on July 6 (PP1-PP25), 7 (PP26-PP50) and 8 (PP51-

PP77).

The posters for the given a day must be put up before starting the morning oral session (before

8.00am) and removed from the board after finishing the last oral session of that day.

Poster boards are labeled with the number corresponding to the abstract number in the Scientific

Programme.

Presenters must be present at their poster board between 2.00 pm and 3.00 pm on the day of

their poster session.

The useful area of the poster board is 60 cm at width and 85 cm at height. Pins and adhesive

tapes will be provided to fix the posters.

Symposium Proceedings

Contributors to PBA 2015 are cordially invited to submit their manuscripts for possible

publication in a special issue of the Journal of Pharmaceutical and Biomedical Analysis,

devoted to the conference. All manuscript will be subject to the usual selection process

including the peer review procedure. Therefore, acceptance for presentation at the conference

is not a warranty of inclusion in the special issue.

To enable us to deal with the manuscripts as fast and efficiently as possible, authors are kindly

invited to submit their manuscripts via the journal´s online submission and reviewing system

which is accessible at: http://ees.elsevier.com/jpba.

Please also follow the instructions received by each registered conference participant from

Elsevier directly. The deadline for manuscript submission to this special issue of Journal of

Pharmaceutical and Biomedical Analysis is August 31, 2015.

Poster Presentation Award

The award will be given for the best poster presented at PBA 2015.

The Best Poster Award (USD 300.00) is sponsored by the Organizing Committee of the 26-th

International Symposium on Pharmaceutical and Biomedical Analysis and the Institute of

Physical and Analytical Chemistry at I. Javakhishvili Tbilisi State University (Tbilisi, Georgia).

International Scientific Committee of PBA 2015 will select the best poster based on the content,

actuality, presentation quality and discussion with the poster presenter.

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Petre Melikishvili Award for the Best Oral Presentation by Young

Scientist at PBA 2015

(1850-1927)

Petre Melikishvili was an internationally well-known Georgian chemist, one of the co-founders

and the first rector of Tbilisi State University.

In 1872 Petre Melikishvili graduated from the Faculty of Physics and Mathematics at the

Department of Natural Sciences of Novorossiya (now Odessa in Ukraine) University. In 1873

he joined the laboratory of the well-known German chemist Julius Lothar Meyer (1830 – 1895)

in Karlsruhe and later Johannes Wislicenus (1835 – 1902) in Würzburg. In 1876 he returned to

Novorossiya University and in 1881 he became Magister and in 1885 Doctor of Chemistry at

the same university. Between 1885 and 1917, P. Melikishvili was appointed as a Professor of

Agricultural Chemistry at Novorossiya University. P. Melikishvili greatly contributed to the

foundation of the first university in Transcaucasia and became the first rector of Tbilisi State

University in 1918. Prof. P. Melikishvili has contributed significantly to the development of

stereochemistry and the chemistry of superoxides and superacids. He was the first to synthesize

well-known and widely used chemicals such as ammonium peroxide, sodium perborate, etc. In

July, 2015 Tbilisi State University is celebrating 165-th anniversary of his first rector Prof. Petre

Melikishvili.

It is our great pleasure to announce that “The Best Oral Presentation by Young Scientist on

PBA 2015” Award named in the honor of Petre Melikishvili will be given to one of the scientist

under the age of 35 years presenting her/his lecture at the Young Scientists Session also named

in the honor of Petre Melikishvili at the 26-th International Symposium on Pharmaceutical and

Biomedical Analysis. The winner of the price who will receive an USD 500 cash award and a

free registration for the PBA 2016 Symposium to be held in Guangzhou, China, will be

determined by an international jury of the leading experts in the field of pharmaceutical and

biomedical analysis.

The Award is funded by the Institute of Physical and Analytical Chemistry at Tbilisi State

University.

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Exhibition

The small exhibition is an important component of the meeting. Please take the time to thank

the exhibitors for their support of the programme. The area for exhibition, poster sessions,

welcome reception and coffee breaks are located on the 2nd floor of the Courtyard by Marriott

Hotel in front of the main lecture hall.

Exhibit Hours:

Monday 9:00 AM – 4:00 PM

Tuesday 9:00 AM – 4:00 PM

Wednesday 9:00 AM – 1:00 PM

Symposium Sponsors

Geolabinstruments

Elsevier

Institute of Physical and Analytical Chemistry I. Javakhishvili Tbilisi State University

Agilent Technologies

Symposium Supporting Organizations

I. Javakhishvili Tbilisi State University

Georgian National Academy of Sciences

Georgian Chemical Society

Shota Rustaveli Georgia National Science Foundation

Tbilisi City Government

Georgian National Tourism Administration

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Plenary Lectures

PL-1 MIXED MODE ION EXCHANGE CHROMATOGRAPHY, A NICHE

TECHNIQUE WITH POTENTIAL

Wolfgang Lindner, Michael Lämmerhofer

University of Vienna, Vienna, Austria

University of Tübingen, Tübingen, Germany

Classical ion exchange chromatography (IXC) is primarily based on charge interactions

of ionised analytes (SAs) with ionised functional groups (SOs) of the stationary phase.

Depending on the acidity and basicity and the pka values of these ionisable or ionised sites one

speaks of weak and strong cation and anion exchangers (WCX, SCX, WAX and SAX) which

can be based on porous organic or inorganic (silica) support materials. For organic molecules

with ionisable sites their change state will be a function of the pka and thus of the pH of the

environment (mobile phase). This way we have two variables to control the electrostatic

interaction between cationic and anionic sites, the pH of the mobile phase and its buffer salt

concentration.

In a simplified form in IXC retention follows a stoichiometric displacement model and

chromatographic selectivity is in the first place related to pka differences. In reality, however,

additional interactions besides the so-called ionised sites within a more or less hydrophobic

selector unit and the ionised selectand moieties will come into play. Most dominantly are these

of hydrophobic, - and hydrogen binding character leading to additional retention causing

increments triggered by the type and content of the organic modifier of a hydroorganic and

buffered mobile phase. The overall chemical structure of the SO unit grafted on the support

material and of the SAs become dominant descriptors.

As a result one deals essentially with a “mixed mode” type of ion exchange

chromatography which offers various variants of retention and selectivity tuning via the

structure of SOs and mobile phase optimisation. As ultimate concept such “mixed mode” ion

exchange materials can be even designed as chiral cationic or anionic ion exchangers enabling

stereoselective retention of acidic and basic SAs. Even ampholytes can be retained with chiral

ampholytic ion exchangers which can be per se characterized as mixed mode systems..

Based on representative examples this lecture will focus on the increased possibilities

of retention, selectivity and even loadability tuning of modern mixed mode ion exchange

chromatographic materials.

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PL-2 METABOLOMICS: ANALYTICAL AND BIOMEDICAL

CHALLENGES

Joanna Godzien; Emily Hooper; Annalaura Mastrangelo; Angeles Lopez-Gonzalvez; Coral

Barbas

CEMBIO (Centre of Excellence in Metabolomics and Bioanalysis): Facultad de Farmacia.

Universidad San Pablo CEU; Campus Monteprincipe; Boadilla del Monte. Madrid 28668

(Spain)

Metabolomics is considered the comprehensive and quantitative measurement of the

metabolic response of an organism to stimuli; although in practice, non-targeted metabolomics,

also termed metabolic fingerprinting, is a global analysis focusing on the compounds which

differentiate two or more situations without a priori hypothesis which could somehow pre-

define the list of metabolites of interest. A discovery based approach is very attractive, opening

the study to unexpected or unpredicted findings. To fulfil the concept of an untargeted approach,

the analysis has to be carried out with the main focus on optimising metabolite coverage in

order to reveal as much information about the system as possible. From an analytical

perspective, metabolite coverage refers to the number of metabolites extracted from a sample

that can be separated and detected. These two aspects play a crucial role in metabolomics studies

because they directly affect the amount of information that can be revealed.

Multi-platform analysis can greatly increase metabolite coverage because each

separation technique is dedicated to the analysis of different groups of molecules; however very

often the amount of sample available is very small and in that case extracting as much

information as possible from one single platform is critical.

Analytical strategies developed in our group for “in vial dual extraction” as well as

metabolite coverage in aspects of computational strategies will be presented. Metabolite

identification plays a crucial role within the entire metabolomics workflow and therefore special

attention should also be paid to it. This is because only identified signals, assigned to the

biological molecules can give valuable information about the changes occurring in an organism.

Applications based on searching pathophysiological mechanisms under different

diseases, markers of disease, effect of a drug or mechanism of resistance will be described.

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Keynote Lectures

KN-1 IDENTIFICATION IN DRUG QUALITY CONTROL AND DRUG

RESEARCH

Sándor Görög

Chemical Works of Gedeon Richter Plc., Budapest, Hungary; gor4745@t-online.hu

Identification is an important step in the quality control of drugs and the research for

new drugs. In this lecture at first the identification of bulk drugs and the active ingredients in

their formulations is discussed based mainly on pharmacopoeias. The test tube colour reactions

have greatly lost their importance and nowadays the most important methods for this purpose

are infrared and at a lesser extent ultraviolet spectroscopy as well as retention matching with

standards using high-performance liquid chromatography (HPLC) and thin-layer

chromatography (TLC). The identification of impurities and degradants is based mainly on

HPLC-UV and HPLC-mass spectrometry (HPLC-MS) as well as nuclear magnetic resonance

(NMR) spectroscopies.

The above listed methods are used for identification purposes also in drug research. The

use of MS and NMR in the research for large-molecule drugs of biotechnological origin as well

as natural products, mainly of plant origin, with special respect to traditional Chinese (and

Indian) medicines is also discussed. The lecture is concluded with the identification aspects of

the fight against counterfeit drugs.

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KN-2 SEPARATION AND DETERMINATION OF INSULINS AND

ANALOGS BY MICELLAR ELECTROKINETIC CHROMATOGRAPHY

Caroline Lamalle1, Anne-Catherine Servais1, Jacques Crommen1, Marianne Fillet1

1Laboratory of Analytical Pharmaceutical Chemistry, Department of Pharmaceutical Sciences,

University of Liege, CHU B36, B-4000 Liege, Belgium; jcrommen@ulg.ac.be

The prevalence of diabetes is increasing every year and insulin preparations are often

prescribed for treatment of both type 1 and type 2 diabetes. Since these biopharmaceutical

formulations are expensive and require a prescription, they are an important target for

counterfeiting in developing countries. Therefore there is a need for fast and efficient methods

for the quality control of biopharmaceutical products such as insulin formulations.

A micellar electrokinetic chromatography (MEKC) method was developed for the

simultaneous analysis of human insulin and its five analogs (lispro, aspart, glulisin, glargin and

detemir) [1]. After optimization of the BGE composition, the six insulins and the two major

excipients in pharmaceutical formulations could be separated within 15 min, using a BGE made

of 50 mM ammonium acetate (pH 9.0), 50 mM SDS and 15 % (v/v) acetonitrile. This method

also exhibited selectivity toward the main degradation products. A faster method with an

analysis time of 3 min was also derived by introducing the samples at the short capillary end

and using the following BGE: 50 mM ammonium acetate (pH 9.0), 20 mM SDS and 13 % (v/v)

acetonitrile. This method was fully validated for human insulin and the quality control of

formulations containing human insulin, including those with protamine, was successfully

performed.

The initial sources of insulin for clinical use were pancreases from pigs and cows. Due

to occasional allergic reactions, these insulins of animal origin were replaced later with human

insulin, produced by recombinant DNA technology. However, they might still be present in

counterfeited insulin preparations. Human, porcine and bovine insulins have very closely

related structures, which makes their separation challenging. A highly selective MEKC method

was developed for their separation, using perfluorooctanoic acid ammonium salt (PFOA) as

surfactant. The BGE composition was optimized by DoE, using a central composite design. The

three insulins could be separated with a satisfactory resolution, using a BGE made of 50 mM

ammonium acetate (pH 9.0), 65 mM PFOA and 5 % (v/v) methanol.

References:

[1] C. Lamalle, A.C. Servais, R.P. Radermecker, J. Crommen, M. Fillet. Simultaneous

determination of insulin and its analogues in pharmaceutical formulations by micellar

electrokinetic chromatography. J. Pharm. Biomed. Anal. (2015) in press.

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KN-3 FROM ENANTIOSELECTIVE HIGH PERFORMANCE TO

ENANTIOSELECTIVE ULTRA HIGH PERFORMANCE

CHROMATOGRAPHY

Francesco Gasparrini1;

1Dip. di Chimica e tecnologie del farmaco, Sapienza Università di Roma;

francesco.gasparrini@uniroma1.it

The extraordinary progress in developing highly efficient packing materials for liquid

chromatography (LC) has only partly touched chiral separations due, in part, to practical

problems in synthesis and functionalization of sub-2-μm particles with chiral selectors, and, in

part, to the lack of clear understanding of mass-transfer mechanisms in chiral chromatography.

However, there is an increasing demand for ultrafast chiral separations, mainly from

fine chemical and pharmaceutical companies.

This talk revisits the most important achievements in enantioselective Ultra High

Performance Chromatography (eUHPC) focused on brush-type chiral stationary phases, as they

represents, essentially for kinetic reasons, a very promising material for the transition from the

traditional enantioselective High Performance LC (eHPLC) to the Ultra-High Speed and Ultra-

High Pressure regimes that means enantioselective Ultra High Performance Chromatography

(eUHPC).

In particular, the results obtained by using the well-known broad-spectrum Whelk-O1

selector will be discussed. This selector, introduced by Pirkle and Welch on 1992 for

enantioselective HPLC, was recently chemically-bonded on the surface of sub-2μm totally

porous silica microparticles with high surface area. The potential of ultra-fast chiral separations

by employing the sub-2µm Whelk-O1 chiral stationary phase has been then investigated in both

UHPLC and UHPSFC.

The evaluation of column kinetic performances demonstrated that higher linear

velocities can be used in liquid- and supercritical- ultra high performance chromatography. The

benefit of UHPSFC conditions over UHPLC normal-phase ones with sub-2μm Whelk-O1

particles for ultra-fast separations is evident as, in the regime of very fast separations, they

permit to achieve larger efficiency and, consequently, even larger resolution. In addition, due

to the intrinsic instrumental limitations of the available SFC system (in terms of maximum flow

rate and pressure sustainable) the true minimum of the van Deemter curve cannot be today

reached in UHPSFC mode. Finally, focusing on development of high-throughput screening

approaches, the sub-2µm Whelk-O1 has been successfully employed in the enantioresolution

of a large group of racemates with noticeable results even for basic compounds. In many cases,

isocratic ultra-fast separations with analysis time smaller than 60 sec have been achieved with

very efficient and short column (up to 2 cm length).

References:

[1] D. Kotoni, A. Ciogli, C. Molinaro et al. Introducing Enantioselective Ultrahigh-Pressure

Liquid Chromatography (eUHPLC): Theoretical Inspections and Ultrafast Separations on a

New Sub-2-μm Whelk-O1 Stationary Phase. Anal. Chem. 84 (2012) 6805−6813.

[2] Sciascera L. Ismail O., Ciogli A. Expanding the potential of chiral chromatography

forhigh-throughput screening of large compound libraries by means of sub–2 µm Whelk-O 1

stationary phase in supercritical fluid conditions. J. Chrom. A 1383 (2015) 160–168.

15

KN-4 RETENTION PHENOMENA OF MEFLOQUINE ENANTIOMERS ON

ZWITTERIONIC STATIONARY PHASE

Attila Felinger1, Borbála Boros1, Ivett Bacskay1, Csaba Szmolnik1, Wolfgang Lindner2

1Department of Analytical and Environmental Chemistry, University of Pécs, Pécs, Ifjúság útja

6. H-7624 Pécs, Hungary; felinger@ttk.pte.hu

2Institute of Analytical Chemistry, University of Vienna, Vienna, Austria

Mefloquine is a well-known medicine successfully applied in the treatment of malaria.

The anti-malarian effect is only responsible for one of the enantiomers, thus it is important to

separate them with high efficiency. A zwitterionic (Chiralpak ZWIX+) analytical column was

used for the enantiomer separation and high selectivity and loadability can be achieved with

this column. Our preliminary study showed that the touching band scenario is reached when 20

mg of rac-mefloquine is loaded onto the column. The (+)-(11S, 12R) enantiomer shows weak

retention and strange peak shape in contrast with (-)-(11R, 12S) enantiomer to which retention

phenomena can be described well by using bi-Langmuir adsorption isotherm.

Our study has demonstrated that the formic-acid, ammonium-formate buffer plays a

substantial role in the enantiomer separation. The weak retention is caused by the displacement

effect of the ammonium ion. The aim of this study is to understand better the applied buffer –

stationary phase interactions by the help of mass spectrometry.

Overloading studies show peculiar peak shapes for both the less and the more retained

enantiomers. The competition of the counter ions and mefloquine molecules for adsorption on

the stationary phase leads to irregular band profiles under column overload.

The overloaded separation was modelled with numerical calculations and there is a good

agreement between experiments and calculations.

16

KN-5 QUANTITATIVE STRUCTURE-RETENTION RELATIONSHIPS

(QSRR) FOR PREDICTION OF BIORELEVANT PROPERTIES OF

ANALYTES AND THEIR IDENTIFICATION

Roman Kaliszan, Michał J. Markuszewski, Tomasz Bączek, Paweł Wiczling, Łukasz Kubik,

Emilia Daghir-Wojtkowiak

Medical University of Gdańsk, Department of Biopharmaceutics and Pharmacodynamics, 80-

416 Gdańsk, Gen. J. Hallera 107, Poland; roman.kaliszan@gumed.edu.pl

It is commonly acknowledged that at the basis of drug action are physicochemical

interactions, which do not lead to the formation of the new or breaking of the existing covalent

bonds between the molecules of a drug and the components of biological environment. The

same fundamental, non-bonding intermolecular interactions normally determine

chromatographic retention. Hence, information on chromatographic behavior of drug analytes

can be used for modeling of their activity in biological systems. However, the extracting of

systematic information from large diverse retention indices requires appropriate chemometric

processing of both the chromatographic and the structural data. The approach widely applied

since late 1970s consists in analysis of Quantitative Structure-Retention Relationships (QSRR).

Example QSRR will be presented allowing prediction of physicochemical properties of

compounds, which determine their “druglikeness”, i.e. which are important for their

pharmacokinetics (ADMETox) and pharmacodynamics, including blood/brain barrier’s

permeation and binding to pharmacological receptor proteins. Special attention will be put on

application of QSRR for bioanalyte identification in proteomics, metabolomics and laboratory

diagnostics. QSRR models will be presented regarding the prediction of retention of peptides

and verification of the correctness of their identification, based on semiempirical structural

descriptors requiring the experimental retention parameters of only 7 out of 20 existing natural

amino acids. Another QSRR model will be shown to support procedure of identification of

bioanalytes of relevance for control of doping in sport, based on HPLC retention parameters in

combination with the molecular descriptors of hypothetical compounds, generated exclusively

by the calculation chemistry methods. New own research will be reported on LASSO (Least

Absolute Shrinkage and Selection Operator) and dimensionality reduction techniques in QSRR

modeling of hydrophilic interaction chromatography data. Maximum a posteriori Bayesian

estimation will be described, which account for retention coefficients of ionogenic analytes in

reversed-phase HPLC and their changes with all possible combinations of organic modifier

content and mobile phase pH.

17

KN-6 CHARACTERIZATION AND CLASSIFICATION OF STATIONARY

PHASES IN SFC (AND HPLC)

Charlene Galea, Debby Mangelings, Yvan Vander Heyden

Department of Analytical Chemistry and Pharmaceutical Technology, Centre for

Pharmaceutical Research, Vrije Universiteit Brussel - VUB, Laarbeeklaan 103, 1090 Brussels,

Belgium

Packed column supercritical fluid chromatography (pSFC) is an attractive technique in

drug discovery related analysis because it offers several advantages over the more commonly

used high-performance liquid chromatography (HPLC) technique. Column characterization

aims to obtain a quantitative understanding of the properties of a column that influence the

selectivity of a separation. Determining column properties allows the rapid selection of

dissimilar columns for method development for a particular application. Columns have been

extensively characterized in HPLC using several approaches. However, limited column

characterization has been done in SFC.

Common methods used to characterize stationary phases in HPLC include

thermodynamic methods, spectroscopic techniques, and chromatographic test methods. The

linear solvation energy relationship (LSER) model (Abraham’s model) and the carotenoid

method are two methods which are used to characterize columns in both HPLC and SFC. LSER

is a quantitative structure retention relationship (QSRR) model in which solute parameters, such

as polarizability, dipolarity, steric and hydrogen-bonding properties are linked to the solute’s

retention through linear regression in order to get a better understanding of the applied

stationary-phase properties. A closer look at the LSER coefficients will help understanding the

retention differences observed between SFC and HPLC conditions, for columns with similar

chemistries. The carotenoid test consists of the analysis of carotenoid pigments and evaluates

polar surface activity, absolute hydrophobicity and the steric separation factor of octadecylsilica

(ODS) stationary phases.

The aim of this presentation is to give an overview of the approaches used to characterize

stationary phases in SFC (and HPLC), and to highlight topics that may still need to be

researched further.

18

KN-7 MULTI-DIMENSIONAL HPLC AND HYPHENATED DETECTIONS

FOR TARGETED AND NON-TARGETED PHARMACEUTICAL

ANALYSIS

Kelly Zhang

Genentech-Roche; zhang.kelly@gene.com

Complete profiling of components and impurities in pharmaceutical samples is

extremely important for drug research and development as well as for patient safety. Targeted

and non-targeted impurities can exist in final drug from raw materials, manufacturing process,

contaminations, degradation, packaging and dosing devices, etc. The traditional way of

demonstrating analytical method specificity by showing the separation of known components

and impurities is not suitable for process change and unexpected impurities, such as

contamination and degradations.

Some components and impurities may have distinct differences and one HPLC method

frequently cannot separate all of the peaks. For instance, a standard RP HPLC method may not

able to retain the highly polar components, chiral impurities and counter ions. Frequently,

multiple methods are required to get the complete profile of a sample. Additionally, some

compounds may not have UV chromophores or not ionizable by MS.

In our study, we used multi-dimensional HPLC with UV-CAD-MS detection to analyze

complex pharmaceutical samples, including small molecule drugs with multiple chiral centers

and monoclonal antibody (mAb) drug formulations with polysorbates. We used orthogonal

methods in each dimension to maximize the peak capacity and thus maximize the method

resolution. A primary column was used in the first dimension, and multiple orthogonal columns

were used in the second dimension. On-line heart-cutting was used as the interface between

different dimensions. Different separation mechanisms were applied such as HILIC, mixed-

mode, ion-exchange, size exclusion and normal phase. The hyphenated detections by UV-CAD-

MS allow complete impurity profile of the samples, not matter the compounds have UV

chromophores or MS signal.

Acknowledgments: The contribution from Yi Li, Midco Tsang, Lulu Dai and Jenny Wang at

Genentech is appreciated. Thank Dr. Pete Yehl and Dr. Nik Chetwyn at Genentech for

supporting this work.

References:

[1] K. Zhang, Y. Li, M. Tsang, N. Chetwyn. Analysis of pharmaceutical impurities using

multi-heartcutting 2D LC coupled with UV-charged aerosol MS detection. J. Sep. Sci. 36

(2013) 2986-2992.

[2] Y. Li, D. Hewitt, Y. Lentz, J. Ji, T. Zhang, K. Zhang. Characterization and Stability

Study of Polysorbate 20 in Therapeutic Monoclonal Antibody Formulation by

Multidimensional Ultrahigh-Performance Liquid Chromatography–Charged Aerosol

Detection–Mass Spectrometry. Anal Chem 86 (2014) 5150-5157.

[3] K. Zhang, J. Wang, M. Tsang, L. Wigman, N. Chetwyn. Two-dimensional HPLC in

Pharmaceutical Analysis. Am. Pharm. Rev. 16 (2013) 39-44.

19

KN-8 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY –

DETECTION BEYOND UV/VIS

Ulrike Holzgrabe1; Stefan Almeling2; Oliver Wahl1; and David Ilko1

1Institute of Pharmacy, University of Wuerzburg, Germany; u.holzgrabe@pharmazie.uni-

wuerzburg.de

2European Directorate for the Quality of Medicine and HealthCare, Strasbourg, France

In the international pharmacopoeias (PhEur, USP, JP) quality assessment of active

pharmaceutical ingredients (APIs) and excipients are mainly performed by high performance

liquid chromatography using UV/Vis detection. However, a lot of APIs, such as

aminoglycosides, and excipients do not provide a chromophore. In these cases charged aerosol

detectors are a valid alternative. Whereas the evaporative light scattering detector (ELSD) is

especially appropriate for the characterization of the composition of excipients, impurity

profiling with this detector is difficult because spike peaks appear on the tails of the main peak

when applying high API concentrations which are necessary for detection of low level

impurities.

In contrast, the charged aerosol detector (CAD) does not show such problems. In

addition, the CAD response is far more linear in a small concentration range than the ELSD [1].

Thus, it can be used for characterization of excipients and impurity assessments of APIs. This

will be demonstrated for polysorbates [2], polidocanol, and counter ions of APIs on the one

hand and for amino acids [3], carbocisteine [4], topiramate, ibandronate, and streptomycin [5]

on the other hand. The pros and cons of the CAD will be discussed.

Acknowledgments: Thanks are due to the Federal Institute for Drugs and Medical Devices for

financial support.

References:

[1] S. Almeling, D. Ilko, U. Holzgrabe. Charged aerosol detection in pharmaceutical

analysis. J. Pharm. Biomed. Anal. 69 (2012) 50-63.

[2] A. Braun, D. Ilko, U. Holzgrabe, H. Gieseler, O. Germershaus, L. Meinel. Predicting

critical micelle concentration and micelle molecular weight of polysorbate 80 using compendial

methods. Eur. J. Pharmaceutics Biopharmaceutics, in press.

[3] U. Holzgrabe, C-J. Nap, T. Beyer, S. Almeling. Alternatives to amino acid analysis for

the purity control of pharmaceutical grade L-alanine. J. Sep. Sci. 33 (2010) 2402-2410.

[4] O. Wahl, U. Holzgrabe. Impurity profiling of carbocisteine by HPLC-CAD, qNMR and

UV/Vis spectroscopy. J. Pharm. Biomed. Anal. 95 (2014) 1-10.

[5] U. Holzgrabe, C.-J. Nap, N. Kunz, S. Almeling. Identification and control of impurities

in streptomycin sulfate by high-performance liquid chromatography coupled with mass

detection and corona charged-aerosol detection. J. Pharm. Biomed. Anal. 56 (2011) 271-279.

20

KN-9 ANALYTICAL APPROACHES FOR THE IDENTIFICATION OF

NEW LIGANDS OF PEROXISOME PROLIFERATOR-ACTIVATED

RECEPTORS (PPARS) A COMPLEX JOURNEY

G. Massolini1, E. Calleri1, G. Brusotti1, G. Pochetti2, R. Moaddel3, F. Loiodice4

1.Dipartimento di Scienze del Farmaco, Università degli Studi di Pavia, 27100 Pavia, Italy;

g.massolini@unipv.it

2.Istituto di Cristallografia, Consiglio Nazionale delle Ricerche, Montelibretti, 00015

Monterotondo Stazione, Roma, Italy.

3 Biomedical Research Center, National Institute on Aging, National Institutes of Health, 251

Bayview Boulevard, Suite 100, Baltimore, Maryland 21224, USA.

4 Dipartimento di Farmacia-Scienze del Farmaco, Universit degli Studi di Bari “Aldo Moro”,

70126 Bari, Italy.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear

receptor supergene family that control many cellular and metabolic processes and are targets

with a broad therapeutic potential for human diseases including type 2 diabetes and obesity.

There are currently three known subtypes of PPAR: alpha, delta and gamma. The receptor

subtype alpha is the target of the known fibrates which are used in therapy in the treatment of

dyslipidemia while the gamma isoform is the target of the thiazolidinediones for the treatment

of type 2diabetes.

The discovery of PPARs as a target of metabolic syndrome, type 2 diabetes and

cardiovascular disorders has attracted remarkable scientific interest and had a great impact on

the pharmaceutical industry. In this contest, efforts have been made to develop high-throughput

screening processes enable to test a large numbers of molecules for activity in an assay that

examines their biological interaction with the target. Among the different available screening

methods, biochromatography represents an innovative analytical tool [1-3].

This presentation covers the role that analytical methods play in the discovery of PPAR-

alpha and gamma ligands and provides an overview of analytical approaches for the

determination of the binding parameters of new potential PPARs ligands.

As the traditional use of plant preparations can often give hits with high chemical

scaffold diversity, in this work medicinal plants and purified bioactive pure compounds have

been screened with a new bioaffinity support in order to identify new leads for PPARs.

References:

[1] E. Calleri, G. Fracchiolla, R. Montanari, G. Pochetti, A. Lavecchia, F. Loiodice, A.

Laghezza, I. Piemontese, G. Massolini, C. Temporini. Journal of Chromatography A

(ISSN:0021-9673), 2012, Vol. 1232, p. 84 - 92.

[2] C. Temporini, G. Pochetti, G. Fracchiolla, L. Piemontese, R. Montanari, R. Moaddel, A.

Laghezza, F. Altieri, L. Cervoni, D. Ubiali, E. Prada, F. Loiodice, G. Massolini, E. Calleri.

Journal of Chromatography A (ISSN:0021-9673), 2013, Vol. 1284, p. 36 - 43.

[3] E. Calleri, G. Pochetti, K.S.S. Dossou, A. Laghezza, R. Montanari, D. Capelli, E. Prada,

F. Loiodice, G. Massolini, M. Bernier, R. Moaddel. ChemBioChem (ISSN:1439-4227), 2014,

Vol. 15(8), p. 1154 - 1160.

21

KN-10 THE IDENTIFICATION OF NOVEL MODULATORS OF THE SIRT6

PROTEIN

Ruin. Moaddel1; M.Yasuda; N.Singh; S.Ravichandran; D.Wilson; S.Fugmann

1. Biomedical Research Center, National Institute on Aging, National Institutes of Health, 251

Bayview Boulevard, Suite 100, Baltimore, Maryland 21224-6825, USA;

moaddelru@mail.nih.gov

SIRT6 is a histone deacetylase that has been proposed as a potential therapeutic target

for metabolic disorders and the prevention of age-associated diseases. Thus the identification

of compounds that modulate SIRT6 activity could be of great therapeutic importance. We have

developed an assay with SIRT6 coated magnetic beads (SIRT6-MB) in order to identify novel

modulators of the SIRT6 protein. Herein, we report the application of the SIRT6-MB for

‘fishing’ experiments utilizing T. foenum-graecum seed extract, where several compounds were

identified as SIRT6 binders.

22

KN-11 DIRECT QUANTITATIVE ANALYSIS OF MULTIPLE MIRNAS

(DQAMMIR)

Sergey N. Krylov; David W. Wegman; Farhad Ghasemi

Department of Chemistry, York University, Toronto, Ontario, Canada; skrylov@yorku.ca

This lecture will introduce the concept and practical aspects of Direct Quantitative

Analysis of Multiple miRNAs (DQAMmiR). DQAMmiR detects miRNAs directly, without

chemical or enzymatic modification, and accurately determines concentrations of multiple

miRNAs without the need to build calibration curves. In essence, a sample containing multiple

miRNAs is mixed with fluorescently-labeled DNA probes complimentary to the miRNAs. After

the probes hybridize with miRNAs, the mixture is subjected to gel-free capillary electrophoresis

(CE), in which different miRNA-probe hybrids are separated from each other due to different

drag tags on the probes, and the separation of the unbound probes from the hybrids is mediated

by single strand DNA binding protein that binds the probes but not the hybrids. The

concentrations of the miRNAs are calculated using a simple mathematical approach that utilizes

the fluorescent signals from the separated hybrids and all unbound probes. Recent advances in

making DQAMmiR a practical clinical technique will be discussed along with the remaining

challenges.

Acknowledgments: Funding was provided by the Natural Sciences and Engineering Research

Council of Canada (NSERC) and the Canadian Institutes of Health Research (CIHR).

References:

[1] Wegman, D.W.; Krylov, S.N. Direct quantitative analysis of multiple miRNAs

(DQAMmiR). Angew. Chem. Int. Ed. 2011, 50, 10335-10339.

[2] Dodgson, B.J.; Mazouchi, A.; Wegman, D.W.; Gradinaru, C.C.; Krylov, S.N. Detection

of a thousand copies of miRNA without enrichment or modification. Anal. Chem. 2012, 84,

5470-5474.

[3] Wegman, D.W.; Cherney, L.T; Yousef, G.; Krylov, S.N. Universal drag tag for direct

quantitative analysis of multiple microRNAs. Anal. Chem. 2013, 85, 6518-6523.

[4] Ghasemi, F.; Wegman, D.W.; Kanoatov, M.; Yang, B.B.; Liu, S.K.; Yousef, G.M.;

Krylov, S.N. Improvements to direct quantitative analysis of multiple microRNAs facilitating

faster analysis. Anal. Chem. 2013, 85, 10062-10066.

[5] Ghasemi, F.; Wegman, D.W.; Kanoatov, M.; Yang, B.B.; Liu, S.K.; Yousef, G.M.;

Krylov, S.N. Improvements to direct quantitative analysis of multiple microRNAs facilitating

faster analysis. Anal. Chem. 2013, 85, 10062-10066.

[6] Wegman, D.W.; Ghasemi, F.; Khorshidi, A.; Yang, B.B.; Liu, S.K.; Yousef, G.M.;

Krylov, S.N. Highly-sensitive amplification-free analysis of multiple miRNAs by combining

capillary isotachophoresis and electrophoresis. Anal. Chem. 2015, 87, 1404-1410.

23

KN-12 METABOLOMICS OF HUMAN INFECTIOUS DISEASES

Oleg A. Mayboroda

Center for Proteomics and Metabolomics, Leiden University Medical Center (LUMC), Leiden,

The Netherlads; O.A.Mayboroda@lumc.nl

Metabolomics is a post-genomic discipline aiming at the study of metabolites: the end

points and the intermediate products of the metabolism. It has its roots in the technological

revolutions of the sixties, the breakthrough of the computer industry of the seventies and the re-

birth of the holistic views in biology triggered by such a major undertaking as the human

genome project. Historically, the understanding of intracellular metabolic pathways and fluxes

in model organisms has been a dominant trend in the field. However, clinical metabolomics,

which has emerged as a spin-off of the ‘mainstream application’, focuses on the research of the

metabolic regulation of the entire human organism and aims at the development of novel

diagnostic/prognostic tools, prediction of patients responses to the treatment and exploration

of the human metabolic individuality.

Among all the clinical conditions, infectious diseases deserve a special attention: the

metabolome of an infected organism represents not only the metabolic phenotype of the host

and/or the pathogen but it also depicts their cross-talk. Furthermore, for a comprehensive view

of the metabolic pattern, multiple factors have to be taken into account such as the risk of

exposure and the host’s individual susceptibility to a given infection. However, an

implementation of “metabolomics thinking” into a practical clinical work of an infectologist is

far from being an everyday reality. Here we present an overview of our ongoing projects on

metabolomics of the infectious diseases demonstrating a value of metabolomics as an

exploratory, discovery tool and trying to identify whether the clinical metabolomics can reach

a level of technical confidence to be accepted as an independent measure in epidemiological

studies.

24

KN-13 METABOLOMICS IN HYPERTENSION AND CANCER DISEASES

Michał J. Markuszewski1, Wiktoria Struck-Lewicka1, Renata Bujak1, Marcin Markuszewski2,

Katarzyna Polonis3, Michał Hoffmann3, Marta Kordalewska1, Arlette Yumba Mpanga1, Julia

Jacyna1, Małgorzata Patejko1, Marcin Matuszewski2, Krzysztof Narkiewicz, Roman Kaliszan1

1Medical University of Gdańsk; Department of Biopharmaceutics and Pharmacodynamics,

Hallera 107, 80-416 Gdańsk, Poland; markusz@gumed.edu.pl

2Department of Urology, Medical University of Gdańsk, M. Smoluchowskiego 17, 80-214

Gdańsk, Poland

3Department of Hypertension and Diabetology, Medical University of Gdańsk, Debinki 7c, 80-

211 Gdańsk, Poland

Metabolomics is an emerging approach in a systems biology field. Due to continuous

development in advanced analytical techniques and in bioinformatics, metabolomics has been

extensively applied as a novel, holistic diagnostic tool in clinical and biomedical studies.

Metabolome’s measurement, as a chemical reflection of a current phenotype of a particular

biological system, is nowadays frequently implemented to understand pathophysiological

processes involved in disease progression as well as to search for new diagnostic or prognostic

biomarkers of various organism’s disorders. Urine and serum samples from patients and healthy

volunteers were analyzed by use of HPLC-TOF/MS in positive and negative ionization modes

as well as GC-QqQ/MS in a scan mode. Next, univariate statistical analysis (t-test or U Mann-

Whitney test) was applied for obtained data sets to select statistically significant metabolites

between studied groups. Finally, the advanced multivariate chemometric methods such as

principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA)

were carried out in order to determine metabolites that were contributed the most into group

classification. The identification of selected metabolites using commonly available databases

and libraries allows for creation of a list of putative biomarkers and related biochemical

pathways they are involved in. The selected pathways, like urea and tricarboxylic acid cycle,

amino acid and purine metabolism, can play crucial role in pathogenesis of prostate cancer

disease. In case of resistant hypertension study the selected metabolites were linked to

endothelium dysfunction, vasoconstriction, cell proliferation, oxidative stress and inflammation

processes, which could be relevant in explanation of pathomechanisms leading to drug-resistant

hypertension.

Acknowledgments: The project funded by the National Science Centre, Poland, allocated on

the basis of the decision number 2012/07/E/NZ7/04411.

References:

[1] W. Struck-Lewicka et al., Urine metabolic fingerprinting using LC-MS and GC-MS

reveals metabolite changes in prostate cancer: A pilot study. J. Pharm. Biomed. Anal. (2015)

http://dx.doi.org/10. 1016/j.jpba.2014.12.026

[2] M. Kordalewska, M.J. Markuszewski. Metabolomics in cardiovascular diseases. J.

Pharm. Biomed. Anal. (2015) DOI: 10.1016/j.jpba.2015.04.021

[3] R. Bujak et al. Untargeted metabolomic approach to understand mechanisms underlying

resistant hypertension. J Hypertens, submitted.

25

KN-14 CHIRAL AMINO ACID METABOLOMICS FOR DRUG

DISCOVERY, CLINICAL DIAGNOSIS AND DESIGNING FUNCTIONAL

FOODS/ BEVERAGES

Kenji Hamase1; Yurika Miyoshi1; Masashi Mita2; Wolfgang Lindner3

1Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku,

Fukuoka 812-8582, JAPAN; hamase@phar.kyushu-u.ac.jp

2Shiseido Co., Ltd., 1-6-2 Higashi-shimbashi, Minato-ku, Tokyo 105-8310, Japan

3Institute of Analytical Chemistry, University of Vienna, Wahringerstrasse 38, A-1090 Vienna,

Austria

Most of the amino acids have chiral center(s) and D- and L-enantiomers are present.

Although L-amino acids are widely observed in higher animals, D-amino acids have long been

thought to be rare, especially as the physiologically active substances. However, along with the

progress of analytical technologies, various D-amino acids were found in mammals including

human beings, and these D-amino acids are expected as bio-functional molecules and/or

biomarkers. Because the amounts of these D-amino acids are extremely trace in most cases,

the establishment of a highly sensitive and selective analytical method is practically essential.

In the present study, we have designed a two-dimensional HPLC method combining a reversed-

phase column (a capillary monolithic ODS column, 0.53 mm ID x 1000 mm as the first

dimension) and enantioselective columns (narrowbore Pirkle-type or cinchona alkaloid based

chiral stationary phases, 1.5 mm ID x 150-250 mm as the second dimension). Prior to the 2D-

HPLC analysis, amino acids were pre-column derivatized with 4-fluoro-7-nitro-2,1,3-

benzoxadiazole (NBD-F). These NBD-amino acids were detected by fluorescence detectors

and also by a tandem mass spectrometer.

Using the 2D-HPLC system, comprehensive and enantioselective determination of all

proteinogenic amino acids (chiral amino acid metabolomics) could be performed. For example,

in the spinal cord of amyotrophic lateral sclerosis model mouse, the amount of D-Ser

significantly increased. In the serum of chronic kidney disease patients, the amounts of D-Ala,

D-Pro and D-Ser increased. Various clinical applications are currently ongoing.

For designing functional products, the amounts of D-amino acids in natural materials

were determined. In the fermented foods and beverages, high amounts of various D-amino acids

including D-Ala, D-Asp and D-Ser were observed. These D-amino acids have crucial

physiological activities such as an anti-oxidative effect and a skin barrier effect, and the

development of functional foods, beverages and cosmetics is in progress.

Acknowledgments: This work was partly supported by JSPS KAKENHI Grant Number

25293007, Japan.

References:

[1] K. Hamase, Y. Miyoshi, K. Ueno, H. Han, J. Hirano, A. Morikawa, M. Mita, T. Kaneko,

W. Lindner, K. Zaitsu. Simultaneous determination of hydrophilic amino acid enantiomers in

mammalian tissues and physiological fluids applying a fully automated micro-two-dimensional

high-performance liquid chromatographic concept, J. Chromatogr. A, 1217, 1056-1062 (2010).

[2] Y. Miyoshi, T. Oyama, Y. Itoh, K. Hamase. Enantioselective two-dimensional high-

performance liquid chromatographic determination of amino acids; analysis and physiological

significance of D-amino acids in mammals, Chromatography, 35, 49-57 (2014)

26

KN-15 THE NEED IN ORTHOGONAL CHROMATOGRAPHIC METHODS

FOR A SMALL MOLECULE PROJECT IN INNOVATIVE DRUG

DEVELOPMENT

Vladimir. Ioffe; Roxanne Schwartz; Maher Kaadan; Lev Keisar; Gregory Verba

Innovative Analytical Development, Global R&D, Teva Pharmaceutical Industries, Ltd., Israel;

vladimir.ioffe@teva.co.il

The aim of this talk is to provide an example of a mindset of the developer of

chromatographic methods and to explain the need in “orthogonal” complementary (or

alternative) chromatographic procedures and the use of diverse detection techniques to ensure

a complex coverage of all the possible characteristics of a biologically active (pharmaceutical)

material. TV-3606 is a novel molecule aimed at MS therapy as an oral drug form, having a

chemical structure close to a known drug, Riluzole, for treatment of amyotrophic lateral

sclerosis (ALS). An RP HPLC method has been developed for testing the drug substance,

capable of analyzing the main substance, as well as to quantitatively determine synthetic

impurities and degradation products, thus being a discriminative stability indicating analytical

procedure. A reverse phase HPLC method for lipophilic API required a high content of organic

solvent both in eluent and diluent. Due to water insolubility, a lipid-based formulation has been

developed, which demanded to either develop a complex extraction procedure, or chose an

alternative analytical technique – normal phase HPLC – where the entire drug product may be

dissolved in eluent-compatible solvent. For testing the formulated drug product, which is hard

gelatin capsules filled with a solution of TV-3606 in medium chain triglyceride, Miglyol, an

alternative technique, NP HPLC method has been worked out. On the example of the case, the

principles of HPLC method development, both for RP and NP chromatography, are discussed

in terms of various retention mechanisms and their combinations in chromatographic

separations. Special attention is given to available tools in development of NP HPLC methods.

Since NP HPLC provides fewer possibilities in mobile phase composition for optimization of

separation, a “scouting of columns” had to be applied. Understanding of usefulness of the use

of less volatile solvents in NP HPLC has been gained.

27

KN-16 ANALYTICAL CHALLENGES IN DERMATOPHARMACEUTICS

Reinhard H. H. Neubert

Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany;

reinhard.neubert@pharmazie.uni-halle.de

In Dermatopharmaceutics, the analytical challenges are facing the following two directions:

1. Skin Penetration Studies

In the past, the penetration profiles of biological active molecules were characterised into

human skin using radiolabeled molecules. However; this method is facing some disadvantages.

Today, modern methods are used such as LC-MS hyphenation methods. The challenges are the

penetration profiles of extremely hydrophilic compounds such as peptides as well as of

extremely lipophilic actives such as ceramides. In this lecture, some examples are presented in

order to demonstrate their advantages as well as their limitations [1].

2. Elucidation of the Ceramide Profiles in the Stratum corneum (SC)

To date nineteen ceramide subclasses are known for human SC. All of the ceramide

subclasses include several subspecies resulting from variation of chain lengths of the sphingoid

base and particularly from the chain length of the fatty acid. The chain length of the sphingoid

base most frequent is C18. The chain length of the amide-bound fatty acid varies from C16 to

C40.

The profiles of the several ceramide subspecies in each ceramide subclass are of main

interest both in healthy and diseased skin.

The characterisation of two ceramide subclasses, CER[NP] and CER[EOS], of human SC

is shown concerning the identification of the chemical structures of their subspecies. Normal

phase HPLC was used for the separation of CER fractions of interest, Nano-ESI-MS/MS was

applied to identify the chemical structures in detail. Thus, CER[EOS] fragmentation revealed

that in addition to linoleic acid other esterified fatty acids occur in the ω-hydroxylated-position

(part E). Of particular interest is the identification of a 17:2 fatty acid located in this part of the

molecule.

Several subspecies of CER[NP], including subspecies with odd numbers of carbon atoms

in both chains, the non-α-hydroxylated fatty acid moiety (part N) and the phytosphingosine

(part P) were detected. Furthermore, more than 15% of CER[NP] subspecies with an odd

number of carbon atoms in more than one chain for one molecule were detected.

Similar results were obtained for CER[EOS]. Both, the esterified fatty acid (part E) and the

sphingosine base (part S) were found to contain odd-numbered chain lengths [2].

The combination of the analytical techniques presented allows complete new insights into

the molecular structure of the SC ceramides. Next step is the identification of the profiles of the

several ceramide species of the subclasses in healthy and diseased skin.

References

[1] F.F. Sahle, S. Lange, B. Dobner, J. Wohlrab, R.H.H. Neubert. Development and

validation of LC/ESI-MS method for the detection and quantification of exogenous ceramide

NP in Stratum corneum and othe rskin layers. J. Pharm. Biomed. Anal. 60 (2012) 7-13.

[2] A. Hinder, C.E.H. Schmelzer, A.V. Rawlings, R.H.H. Neubert. Investigation of the

molecular structure of human stratum corneum ceramides [NP] and [EOS] by mass

spectrometry. Skin Pharmacol. Physiol. 24 (2011) 127-35.

28

KN-17 NOVEL SILICA-BASED STATIONARY PHASES APPLIED TO THE

SEPARATION OF ENANTIOMERS BY NANO-LC AND CEC

Salvatore Fanali

Istituto di Metodologie Chimiche, Consiglio Nazionale delle Ricerche, 00015 Monterotondo,

Italy.

The separation and analysis of enantiomers has been in the past and is continuing to be

a challenging issue in several research fields such as biomedicine, agrochemical,

pharmaceutical industry, forensic etc. It is known that several compounds possess chiral

center/s originating two or more enantiomers. Two enantiomers could exhibit different

bioactivity, e.g., one of them could be pharmacologically more active that its antipode and in

some cases even dangerous for health.

Therefore analysis of chiral compounds is carried out for several studies, e.g., food

safety, metabolism, drug composition etc. Appropriate analytical methods are continuously

requested for these purposes. Analytical techniques so far utilized include high-performance

liquid chromatography (HPLC), thin layer chromatography (TLC), supercritical fluid

chromatography (SFC), gas chromatography (GC). In addition miniaturized techniques such as

electromigration (CE, CEC) and chromatographic (CLC and nano-LC) have also been studied

and proposed.

Miniaturized techniques because their advantages over conventional ones, e.g., reduced

volumes of mobile phases and samples, high efficiency and resolution, perfect coupling with

MS (due to low flow rates), reduced costs and waste have been developed and also applied to

enantiomers separation. Along the years the interest was focused in studying new stationary

phases with different chiral selectors such as glycopeptide antibiotics, cyclodextrins,

polysaccharides etc.

In this communication recent results achieved utilizing novel stationary phases prepared

by either bonding or coating the chiral selector onto silica particles packed on capillary column

-LC and CEC will be shown. The effect of particles type (silica and

its diameter), chiral selector and mobile phase type on enantioresolution, enantioselectivity and

efficiency will also be discussed.

29

KN-18 CHIRAL SEPARATIONS IN CAPILLARY

ELECTROCHROMATOGRAPHY USING CHLORINATED AND NON-

CHLORINATED POLYSACCHARIDE-BASED SELECTORS

Debby Mangelings1, Dima Albals1, Ans Hendrickx1, Sven Declerck1, Bezhan Chankvetadze2,

Yvan Vander Heyden1

1Department of Analytical Chemistry and Pharmaceutical Technology, Vrije Universiteit

Brussel –VUB, Laarbeeklaan 103, B -1090 Brussels, Belgium; debby.mangelings@vub.ac.be

2Institute of Physical and Analytical Chemistry, Molecular Recognition and Separation Science

Laboratory, School of Exact and Natural Sciences, Tbilisi State University, Tbilisi, Georgia

Capillary electrochromatography (CEC) is a hybrid separation technique that combines

the separation principles of two techniques, capillary electrophoresis (CE) and high-

performance liquid chromatography (HPLC). Advantages of CEC are highly efficient

separations due to the plug-like electro osmotic flow (EOF) as driving force in the capillary

column, a higher sample loading capacity compared with CE due to the presence of a stationary

phase, and an overall low mobile-phase and sample consumption.

Chiral drug molecules often display different pharmacological properties. Because most

pharmaceutical compounds possess chiral properties, the development of chiral separation

methods is an important research topic the pharmaceutical industry to isolate the therapeutically

active enantiomer, the eutomer, from the other enantiomers. Consequently, the possibility to

commercialize safer medicines with less or no side effects will be achieved.

The development of chiral separation methods is often a trial-and-error procedure. For

that reason, generic chiral separation strategies are a useful tool to facilitate method

development. Such strategy was proposed earlier using CEC as separation technique. The final

CEC strategy was composed of two sub-strategies, i.e. one for acidic and one for non-acidic

compounds. For the separation of acidic compounds, a low pH mobile phase was needed while

for the separation of non-acidic substances, a high pH was needed.

In this study, an update of the existing CEC strategy was conducted by evaluating the

potential of newer types of chiral stationary phases (CSP) that use chlorinated polysaccharide

derivatives as chiral selector. In a first part, the earlier defined screening conditions were tested

on the newer types of CSP. Then the most complementary CSP were chosen to update the

screening steps for acidic and non-acidic compounds. In a second phase, the applicability of the

existing optimisation steps was verified and they were adapted where necessary. The final result

was an updated CEC strategy with a higher success rate, which uses both chlorinated and non-

chlorinated polysaccharide-based CSP.

30

KN-19 CHEMICAL BIOPSY BASED ON SPME APPROACH: A NEW

MEDICAL TOOL

Janusz Pawliszyn1; Barbara Bojko1; German Augusto Gomez-Rios1; Krzysztof Gorynski1; Jan

Matthias Knaak2; Tiago Machuca3; Erasmus Cudjoe1; Vinzent Spetzler2; Michael Hsin3;

Markus Selzner2; Mingyao Liu3; Marcelo Cypel3; Shaf Keshavjee3

1University of Waterloo, Waterloo, Canada; 2Department of Surgery, Toronto General Hospital,

Toronto, Canada; 3University Health Network, University of Toronto, Toronto, Canada;

janusz@uwaterloo.ca

Patient monitoring, metabolomics and biomarkers discovery are an integral part of

medical diagnosis and biomedical research. However, tissue analysis remains as the bottleneck

of such studies due to the invasiveness of presently used sample collection, as well as the

laborious and time-consuming sample preparation protocols. In the proposed technology we

have integrated in vivo sampling, sample preparation and global extraction of metabolites. This

approach based on a small coated metal fiber format of solid phase microextraction was

presented and evaluated during liver and lung transplantation in pig model s well as single cell

analysis. Direct coupling facilitates rapid quantification of the extracted components resulting

in possibility of close to real time monitoring. This approach has potential in biological and

clinical investigations including medical diagnosis.

31

KN-20 NOVEL SAMPLE PRETREATMENT MATERIALS AND THEIR

APPLICATIONS IN ANALYSIS OF TRACE METABOLITES

Hua Li; Yuanhong Shan; Qian Qin; Xianzhe Shi; Guowang Xu

Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical

Physics, Chinese Academy of Sciences, Dalian 116023, China; xugw@dicp.ac.cn

Many low abundant biomolecules play important roles in physiological function and

process. However, their detection is very difficult for inherently trace concentration and

complex matrix interference. The development of new pretreatment materials is very significant

for selective enrichment of low abundant biomolecules or removal of interferences with high

concentrations. In this presentation, we would like to report our recent results of pretreatment

materials and their applications in bioanalysis of small molecules:

1) A new type of echinus-like Fe3O4@TiO2 core-shell-structured magnetic

microspheres was synthesized. The microspheres can be used to not only selectively enrich

phosphopeptides from complex peptide mixtures but also selectively remove phospholipids

which are the main matrix interferences for the detection of metabolites of low abundance in

plasma.

2) Novel magnetic nanoparticles Fe3O4@SiO2@PEI-FPBA were synthesized by

grafting polyethyleneimine (PEI) onto the surface of Fe3O4@SiO2 before modification by

boronate groups. The nanoparticles exhibited large adsorption capacity and high selectivity

towards 1,2-cis-diol compounds and 60 modified nucleosides and ribosylated metabolites were

enriched from human urine.

3) A novel monolithic column modified with cuprous sulfide nanoparticles was

developed and the affinity characteristics towards low-molecular-weight electron-rich analytes

were investigated. The SPME column presented good permeability and stability in a wide pH

range from 2 to 10. Compared with the commercial Cu2+ and Ni2+ based affinity sorbents, a

larger binding capacity of cuprous sulfide modified column towards kanamycin A was obtained

under basic condition.

References

[1] Hua Li, Xianzhe Shi*, Lizhen Qiao, Xin Lu, Guowang Xu* J. Chromatogr. A 1275

(2013): 9-16

[2] Hua Li, Yuanhong Shan, Lizhen Qiao, Abo Dou, Xianzhe Shi,* Guowang Xu*, Anal.

Chem., 85 (2013):11585-92

[3] Yuanhong Shan, Xianzhe Shi, * Guowang Xu *, J. Sep. Sci. 2015 in press

32

KN-21 SINGLY AND MULTILAYERED POLAR SILICA STATIONARY

PHASES FOR HILIC

Ziad El Rassi; Renuka Rathnasekara

Oklahoma State University, Department of Chemistry, Stillwater, OK, USA;

elrassi@okstate.edu

In this talk, we will present our recent investigations on singly and multilayered polar

coated silica microparticles for hydrophilic interaction liquid chromatography (HILIC) of a

wide range of polar and slightly polar solutes. In all cases, the basic silica support was coated

with an epoxy active layer via -glycidoxypropyl trimethoxysilane.

Thereafter, the activated epoxy silica thus obtained was covered with a layer of either

tris(hydroxymethyl)aminomethane (TRIS) or sorbitol (SOR) yielding the singly layered silica

stationary phase. The TRIS-silica was further coated with a layer of chondroitin sulfate A (CSA)

by passing over the TRIS-silica column a 10 mL solution of 1% (w/v) of CSA in 50% ACN:

50% (v/v) 10 mM ammonium acetate buffer, pH 3, at 0.5 mL/min thus yielding the multi-

layered hydrophilic silica stationary phase referred to as CSA-TRIS-silica sorbent. Solid-state

infrared was used to evaluate and characterize the various coating. In addition, an extensive

chromatographic characterization was conducted to assess the extent of each coating step in

achieving the singly and multilayered polar coating the silica microparticles. This included the

effect of mobile phase composition, e.g., ACN, buffer and pH on retention factor, selectivity,

efficiency and peak resolution. As expected each coating yielded unique retention pattern and

selectivity towards the polar and slightly polar solutes tested. Among the various solutes and

test mixtures investigated over a wide range of elution conditions, included phenols, phenolic

acids, aromatic carboxylic acids, nucleobases, nucleosides, nucleotides, cyclic nucleotides,

amino acids, derivatized mono- and oligosaccharides and slightly environmental contaminants.

33

KN-22 CAPILLARY ELECTROPHORESIS-BASED ENZYME ASSAYS

FOR METHIONINE SULFOXIDE REDUCTASE

Gerhard K. E. Scriba1; Qingfu Zhu1; Roland Schönherr2; Stefan H. Heinemann2

1Friedrich Schiller University, Department of Pharmaceutical Chemistry, 07743 Jena,

Germany; gerhard.scriba@uni-jena.de

21Friedrich Schiller University, Department of Biochemistry, 07743 Jena, Germany

Under conditions of oxidative stress, L-methionine (Met) residues in peptides and

proteins are easily oxidized to L-methionine sulfoxide [Met(O)] by reactive oxygen species [1].

Protein-bound as well as free Met(O) can be reduced by methionine sulfoxide reductase (Msr)

enzymes, a group of thiol oxidoreductases, protecting cells against oxidative damage [2].

Because of the chirality of the sulfoxide moiety, Met(O) exists as the pair of diastereomers

L-methionine-(S)-sulfoxide [Met-S-(O)] and L-methionine-(R)-sulfoxide [Met-R-(O)]. For the

reduction of the diastereomers, stereospecific Msr enzymes exist, which share little sequence

homology but possess mirror-like relationships of their active sites. MsrA catalyzes the

reduction of Met-S-(O) either in the free form or bound in proteins. In contrast, MsrB reduces

Met-R-(O) in proteins but displays only low activity for free Met-R-(O). In addition, an enzyme

named free methionine-(R)-sulfoxide reductase (fRMsr), which specifically reduces free Met-

R-(O), has been found in bacteria [2, 3].

In order to determine the stereospecific activity of Msr enzymes, capillary

electrophoresis-based assays including pre-capillary and in-capillary assays were developed. In

an offline assay, the separation of the Met(O) diastereomers as well as the reduction product

Met after derivatization with dabsyl chloride was achieved in a multiple ionic polymer-coated

capillary using a 35 mM phosphate buffer, pH 8.0, containing 25 mM sodium dodecyl sulfate

as background electrolyte. An electrophoretically mediated microanalysis (EMMA) assay used

Fmoc-Met(O) as substrate. The separation of the diastereomers as well as the product Fmoc-

Met was performed in a successive multiple ionic polymer-coated capillary using a 50 mM Tris

buffer, pH 8.0, containing 30 mM sodium dodecyl sulfate as background electrolyte. Finally,

an assay employing the pentapeptide ac-Lys-Ile-Phe-Met(O)-Lys-DNP as substrate was

developed which utilized a dual system consisting of sulfated β-cyclodextrin and 15-crown-5

as selectors for the separation of the peptide sulfoxide diastereomers. The optimized conditions

were obtained by experimental design resulting in a 50 mM Tris buffer, pH 7.85, which

contained 5 mM 15-crown-5 and 14.3 mg/mL sulfated β-cyclodextrin. All assays were

subsequently validated and applied to the determination of the stereospecificity of recombinant

human and fungal Msr enzymes as well as the determination of the Michaelis-Menten kinetic

data of the enzymes.

Acknowledgments: The financial support of Q. Zhu by the China Scholarship Council (CSC)

is gratefully acknowledged.

References:

[1] W. Voigt. Oxidation of methionyl residues in proteins: Tools, targets and reversal. Free

Radic. Biol. Med. 18 (1995) 93–105.

[2] S. Boschi-Muller, A. Gand, G. Branlant. The methionine sulfoxide reductases: Catalysis

and substrate specificities. Ann. Biochem. Biophys. 474 (2008) 266–273.

[3] B. C. Lee, V. Y. Gladyshev. The biological significance of methionine sulfoxide

stereochemistry. Free Radic. Biol. Med. 50 (2011) 211–227.

34

KN-23 FROM MERCURY TO NANOSENSORS: PAST, PRESENT AND

THE FUTURE PERSPECTIVE OF ELECTROCHEMISTRY ON

PHARMACEUTICAL AND BIOMEDICAL ANALYSIS

Sibel A Ozkan

Ankara University, Faculty of Pharmacy, Department of Analytical Chemistry, 06100

Tandogan-Ankara, Turkey; ozkan@pharmacy.ankara.edu.tr

Electroanalytical methods are routinely used in pharmaceutical and biomedical analysis.

They are powerful and versatile analytical techniques that offer high sensitivity, accuracy, and

precision as well as a large linear dynamic range, with relatively low-cost instrumentation.

Polarography was the first developed automated method of voltage-controlled

electrolysis with dropping mercury electrode (DME). Then, hanging mercury drop and static

mercury drop electrodes were added as an alternative indicator electrode. In this way,

polarography turned formally into voltammetry with mercury electrodes in the electro-

reduction way. In the modern pharmaceutical and biological analysis a large number of

electroanalytical applications with different techniques are available for the sensitive

quantification of drug analysis. They have been developed for measurements in the laboratory,

mostly for fundamental research. Solid electrodes such as noble metal and carbon based

electrodes can be used for the investigation of the compounds for both oxidation and reduction

directions which named is voltammetry.

Recent trends and challenges in the electrochemical methods for the detection of DNA

hybridization and pathogens are available. Low cost, small sample requirement and possibility

of miniaturization justifies their increasing development. There is great importance in meeting

the challenges of real-time electrochemical measurements and in miniaturization. The

approaches will become increasingly important with schemes in real time in vivo experiments

becoming widespread as diagnostic tools. Important complementary studies regarding

pharmaceutical analyses involve investigating which chemical species are assimilated by living

organisms and the mechanism of uptake. Such research will show what is the real value and

extent of the role of “real-time'' electroanalytical diagnostic experiments. Then the

electroanalytical methods would be played very useful roles in drug analysis and monitoring in

near future.

References:

[1] J. Heyrovsky, P. Zuman, Introduction to Practical Polarography, Academic Press,

London, UK, 1968.

[2] P. Zuman, Past, present, and future of applications of electroanalytical techniques in

analytical and physical organic chemistry, J. Solid. State. Electrochem. 15 (2011) 1753–1759.

[3] S.A. Ozkan (Ed.), Electroanalytical methods in pharmaceutical analysis and their

validation, first ed., HNB Pub., New York, 2012.

[4] L. Meites, P. Zuman (Eds.), Handbook Series in Organic Electrochemistry, CRC Press,

Boca Raton, Florida, USA, Vols. I-VI, 1977–1983.

[5] G.J. Patriarche, J.-C. Vire, Applications of polarography and voltammetry in analysis

for drugs, Anal. Chim. Acta. 196 (1987) 193–204.

35

KN-24 NANO-LIQUID CHROMATOGRAPHY IN PHYTOCHEMOMIC

STUDIES

Chiara Fanali1, Anna Rocco2, Laura Dugo1, Paola Dugo1,3, Luigi Mondello1,3

1Campus Bio-Medico University, Centro Integrato di Ricerca, Via Alvaro del Portillo 21, 00128

Roma, Italy; c.fanali@unicampus.it

2Istituto di Metodologie Chimiche, Consiglio Nazionale delle Ricerche, 00015 Monterotondo,

Italy.

3Dipartimento di Scienze del Farmaco e dei Prodotti per la Salute, University of Messina, Viale

Annunziata, 98168, Messina, Italy.

Phytochemomics is a novel concept recently proposed del Castillo et al. which combines

different areas of knowledge on phytochemical compounds. Phytochemome includes samples

phytochemical composition, as well as, its structure, activity and mechanism of action. Several

phytochemicals have been already approved as health promoters by EFSA and FDA, among

them phenolic compounds [1]. These compounds can be naturally present or added in food as

functional ingredients. In fact, the healthy properties of such molecules have been largely

promoted by the introduction in the market of so-called functional foods, designer foods,

pharmafoods, and nutraceuticals.

Several methods for qualitative and quantitative analysis of these compounds in raw

plant, agronomical by-products, nutraceuticals and dietary supplements employing

chromatographic techniques have been developed. Among them nano-liquid chromatography

(nano-LC) has been recently proposed for the analysis of phytochemicals in food matrices, as

alternative to conventional methods. Advantages of this methodology are to offer: i) good

efficiency, ii) short analysis time and iii) low sample dilution, by using capillary columns of id

≤ 100 µm for analytes separation [2]. Moreover, the small inner diameter allows relatively low

flow rates, reducing costs analysis and making this technique eco-friendly.

Nano-LC based methods for phytochemicals analysis, particularly phenolic compounds,

in different food matrices and dietary supplements have been developed and validated. Many

parameters, including the choice of the stationary phase and on-column focusing to improving

sensitivity were evaluated for methods optimization. An on-column UV-Visible detector was

used for qualitative and quantitative analysis. Mass spectrometry was also utilized for

identification of compounds in analyzed samples. The methods were fully validated evaluating

their repeatability, linear dynamic range and sensitivity. The methods were applied for

qualitative and quantitative analyses of both food matrices and dietary supplements.

References:

[1] M.D. del Castillo, N. Martinez-Saez, M. Amigo-Benavent, J.M. Silvan.

Phytochemomics and other omics for permitting health claims made on foods. Food Res. Int.

54 (2013) 1237–1249.

[2] C. Fanali, L. Dugo, P. Dugo, L. Mondello. Capillary-liquid chromatography (CLC) and

nano-LC in food analysis. TrAC - Trends Anal. Chem. 52 (2013) 226-238.

36

KN-25 TARGETING PROTEIN-PROTEIN INTERACTIONS IN CANCER:

FROM ASSAY DEVELOPMENT TO INHIBITOR IDENTIFICATION.

J. Jose1; B. Bopp1; K.-H. Klempnauer2; S. Uttaka2; H. Gohlke3; E. Ciglia3; I.; K. Niefind4

1Institute of Pharmaceutical and Medicinal Chemistry, PharmaCampus, and 2Institute of

Biochemistry, Westphalian Wilhelms-University Münster, Germany; joachim.jose@uni-

muenster .de

3Institute of Pharmaceutical and Medicinal Chemistry, HHU Düsseldorf, and 4Institute of

Biochemistry, University of Cologne, Germany.

Protein-protein interactions (PPIs) are involved in numerous physiological mechanisms with

fundamental roles in signaling processes and regulatory pathways. Moreover, PPIs are crucial

in a large number of pathogenic processes, notably in cancer and neurodegenerative diseases.

In consequence, the vast number of PPIs, present in human cells, opens a promising perspective

for identifying novel drug targets. Because of the high selectivity and high specificity of PPIs,

drug discovery on such targets bears the option to identify more selective and specific novel

entities. Therefore more recently, research efforts have been intensified to explore and validate

the concept of modulating of PPIs as therapeutic intervention in a number of diseases. However,

the discovery of small molecule protein-protein interaction modulators (PPIMs) remains a

challenge.

We recently developed a screening assay for PPIMs addressing the interface of the  and 

subunit of human protein kinase CK2, as an emerging cancer target, by applying bacterial

surface display and fluorescence activated cells sorting (FACS) [1]. Similar screening assays

could be established for the identification of PPIMs for the human cancer targets Myb [2] and

HSP90 [3]. In this strategy, one protein was displayed on the surface of E. coli, its binding

partner was coupled to a fluorescence label and added to the cells, allowing to quantify binding

by FACS. After screening and IC50 values determination in these assays, KD values for the best

PPIM candidates identified, were determined by microscale thermophoresis (MST). In addition

MST could also be used to determine the KD of the corresponding PPIs. Whereas for Myb, the

interaction with the co-activator p300 was blocked, resulting in a loss of transcriptional

activation in cancer cells, for HSP90, inhibitors of dimerization were identified, that abolished

the binding to the chaperon’s client proteins. In case of CK2 and HSP90, the first inhibitors

obtained were peptides, derived from the PPI interface. In case of Myb, Naphtol AS-E

phosphate was identified as a first potent small molecule inhibitor of PPI, which was able to

suppress the expression of Myb target genes and induce myeloid differentiation and apoptosis.

The strategy as applied here, using bacterial surface display and flow cytometry for the rapid

screening for PPIMs and IC50 value determination, followed by MST for KD measurement of

the best candidates appears to be a convenient route for the identification of novel small

molecule inhibitors of PPIs.

References:

[1] J. Raaf, B. Guerra, I. Neundorf, B. Bopp,O.-G. Issinger, J. Jose, M. Pietsch, K. Niefind (2013)

First structure of protein kinase Ck2 catalytic subunit with an effective CK2β-competitive ligand. ACS

Chem. Biol., 8:901-907.

[2] S. Uttarkar, S. Dukare, B. Bopp, M. Goblirsch, J. Jose, K.-H. Klempnauer (2015) Naphtol AS-

E phosphate inhibits the activity of the transcription factor Myb by blocking the interaction with the KIX

domain the coactivator p300. Mol. Cancer Ther., DOI: 10.1158/1535-7163.MCT-14-0662.

[3] B. Bopp, E. Ciglia, A. Ouald-Chaib, G. Groth, H. Gohlke, J. Jose (2015) Design and biological

testing of peptidic dimerization inhibitors of human Hsp90 that target the C-terminal domain. submitted.

37

KN-26 BIO-RAMAN SPECTROSCOPY: A POWERFUL TOOL FOR

PATHOLOGICAL RESEARCH AND CLINICAL DIAGNOSIS

Aiguo Shen, Xiaodong Zhou, and Jiming Hu

Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education),

College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, 430072, China

As a vibrational spectroscopic technique, Raman spectroscopy is sensitive to the

structural and conformational changes of biomolecules. Currently, the structural and

conformational changes of biomolecules within tissue may initiate pathological changes and

occur as a result of the development of the disease. Most biomelecules, such as proteins, lipids,

nucleic acids and chromophoric-molecules, exhibit sharp features of Raman spectra. These

Raman bands are characteristic for specific structures and conformations of biomolecules

within tissues and cells, and provide more specific molecular information about normal and

diseased tissue. As a rapid, nondestructive and noninvasive technique, Bio-Raman spectroscopy

is widely used in pathological research and clinical diagnosis [1] .

In general, single Raman band of characteristic biomolecule is used as Raman marker for

diagnosis of diseased tissue. But only one Raman marker can’t be satisfactorily used for

pathological research and clinical diagnosis of tissue. My group developed a “Raman beacon”

for diagnosis of diseased tissue. The Raman beacon is consisted of two or more Raman marker

bands of two or more biomolecules in tissue. The use of Raman beacon increases greatly the

sensitivity and specificity of diagnosis of diseased tissue.

Till now, my group has used Raman beacon to develop a series of Bio-Raman

spectroscopic studies of organic tissues and cells and to realize pathological research and early,

nondestructive and noninvasive of significant diseases. We use this method for pathological

research of Alzheimer’s disease, pterygium, tooth discoloration, spinal injury, liver injury,

tumors of oral cavity, bladder and upper gastrointestinal tract early diagnosis of Alzheimer’s

disease and gastric cancer, differential diagnosis among Alzheimer’s disease, Parkinson’s

disease and vascular dementia. In combination with morphological evaluation tests, we employ

Bio-Raman spectroscopy and Raman beacon methodology to select higher potentially

reproductive embryos during in vitro fertilization (IVF) based on chemical composition of

embryos culture medium. With the help of optical tweezer, we also realize real-time molecular

profiling of photochemically induced rat thrombosis in vivo through Raman analysis of both

arterial and venous blood.

References:

[1] P. Chen, A. G. Shen, X. D. Zhou and J. M. Hu, Anal. Meth., 2011, 3, 1257-1269.

38

KN-27 HPLC SEPARATION OF CONFORMATIONAL ENANTIOMERS

Claudio Villani

Department of drug chemistry and technologies, Sapienza University of Rome;

claudio.villani@uniroma1.it

Conformational enantiomers, unlike chiral compounds with stereogenic centers,

interconvert by dynamic processes that are related to single bond rotations. Several drugs show

this interesting aspect of molecular chirality, and depending on the easy with which the two

enantiomers interconvert at room temperature, they are amenable to be physically separated and

tested individually for their overall biological activity.

Dynamic high performance liquid chromatography (DHPLC) on chiral stationary phases

is a well-known technique that allows the physical separation of interconverting enantiomers at

low temperature, and allows for the kinetic investigation of chiral molecules with labile

stereogenic elements that interconvert during chromatography. Kinetic parameters for on-

column interconversions can be extracted by computer simulation of the exchange-deformed

experimental peak profiles.

Recent results on the low temperature HPLC separation and kinetic determinations for a

series of benzodiazepines featuring conformational chirality will be presented and discussed.

References:

[1] C. Wolf, Chem. Soc. Rev. 2005, 34, 595-608

[2] R. Sabia et al, J. Chromatogr A 1362, 2014, 144-14

39

KN-28 CONFORMATIONAL CHANGE AND IRREGULAR RETENTION

BEHAVIOR OF PHARMACEUTICAL COMPOUNDS IN REVERSED-

PHASE CHIRAL SEPARATION

Brian Lingfeng He, Yande Huang, Qinggang Wang, Bent Kleintop

Research & Development, Bristol-Myers Squibb Co., New Brunswick, NJ, 08903, USA;

brian.he@bms.com

Non-linear van't Hoff plots in reversed-phase chromatography have been reported in

literature, but are relatively rare. This phenomenon may be attributed to a phase-ratio change,

exsitance of secondary equilibria, or a conformational change of stationary phase or analytes

structure. In a study of the temperature dependance of a basic pharmceutical compound with

three stereogenic centers, non-linear van’t Hoff plots were observed under reversed-phase

conditions for the compound and its diastereomers on a chiral stationary phase (Chiralcel OD-

RH) and a C18 phase (XBridge C18). The studied temperatures ranged from 5 ˚C - 65 ˚C. This

unusual retention behavior was investigated under a wide range of conditions, including

mobile phase composition, organic additives, buffer’s nature, pH and concentration, and

modification of the analyte’s structure. Spectroscopic methods, such as NMR and Circular

Dichroism, were employed to study the dependance of analytes’ molecular conformation on

tempature and diluent composition. Evidance from NMR experiments revealed that

conformational change of these analytes is a key factor to produce a non-linear van't Hoff plot.

40

KN-29 THE APPLICATION OF SPR BIOSENSOR IN THE STUDY OF

BIORECOGNITION PHENOMENA

Carlo Bertucci

Department of Pharmacy and Biotechnology, University of Bologna, via Belmeloro 6, 40126

Bologna, Italy; carlo.bertucci@unibo.it

Optical biosensors are well suited to monitor specific molecular interactions and

measure the related binding parameters, which are fundamental for the definition of the

mechanism of bio-recognition processes, as well as for their modulation [1]. This analytical

technique allows the interactions between analytes and immobilized ligands to be studied

without labelling the interacting compounds. Label-free assays are valuable and easy-to-use

tools in bio-recognition process monitoring, since they confer higher efficiency, flexibility and

lower exposure to experimental artefacts.

Here we report the use of surface plasmon resonance (SPR) based optical biosensors in

the investigation of different biomolecular systems. This technique was first used for the

investigation of drugs and natural compounds binding to plasma proteins, giving valuable

information on the distribution of the analysed compounds through quantitative determination

of the affinity and kinetics parameters. The optical biosensor was then applied to the screening

of new synthesised bicalutamide analogues for their binding to the androgen receptor [2].

Furthermore, optical biosensors can be successfully applied to obtain more basic information

on the mechanism involved in the drug-protein and protein-protein interactions. As relevant

example, the optical biosensor was applied to get further insight into the mechanisms underlying

SMYD3 inhibition [3]. SMYD3 is a histone lysine methyltransferase that plays an important

role in transcriptional activation as a member of an RNA polymerase complex, and its

oncogenic role has been described in different cancer types. The target protein SMYD3 was

immobilized through an affinity capture method to ensure that the histone binding site of

SMYD3 was accessible for interaction. H4 histone binding to SMYD3 was analysed at different

analyte concentrations, and an affinity KD of 1.18x10-5 M was determined based on the

association and dissociation rate constants (kon 357.7 ± 28.0 M-1s-1; koff 4.23x10-3 ± 2.9x10-5 s-

1; KD= koff/kon). Then, potential inhibitors were proved to modulate the SMYD3/histone binding,

supporting molecular modelling and biological data.

Optical biosensors are particularly useful for medium-high throughput screening of

binding events, for the identification of biomarkers and target proteins, and for an information-

rich analysis to characterize the mechanisms governing the biorecognition processes. These

peculiarities, together with miniaturization, automation of the system and the short analysis time,

make the optical biosensors of high potential in the discovery and development of new drugs.

Acknowledgments: This project was supported by the University of Bologna.

References:

[1] W. David Wilson, Analyzing biomolecular interactions. Science 295 (2002) 2103-2105.

[2] C. Fortugno, G. Varchi, A. Guerrini, P.A. Carrupt, C. Bertucci. Optical biosensor analysis

in studying new synthesized bicalutamide analogs binding to androgen receptor. J Pharm Biomed

Anal. 95 (2014) 151-157.

[3] A. Peserico, A. Germani, P. Sanese, A.J. Barbosa, V. Di Virgilio, R. Fittipaldi, E. Fabini,

C. Bertucci, G. Varchi, M.P. Moyer, G. Caretti, A. Del Rio, C. Simone. A SMYD3 Small-Molecule

Inhibitor Impairing Cancer Cell Growth. Journal of Cellular Physiology, 2015,

DOI 10.1002/jcp.24975.

41

KN-30 DESIGN OF NOVEL ANTIBODY-ANTIGEN BASED

VOLTAMMETRIC AND IMPEDIMETRIC IMMUNOSENSORS FOR LOW

LEVEL DETECTION OF HER2 IN SERUM SAMPLES OF BREAST

CANCER PATIENTS VIA MODIFICATION OF NANOPARTICLES’

DECORATED GLASSY CARBON AND MULTIWALL CARBON

NANOTUBE-IONIC LIQUID ELECTRODES

Mojtaba Shamsipur

Department of Chemistry, Razi University, Kermanshah, Iran; mshamsipur@yahoo.com

Realization of low-levels of tumor biomarkers is vital for early awareness of cancers

and commencing the appropriate treatment processes [1]. Human epidermal growth factor

receptor (HER2), as a key prognostic marker, is over-expressed in 10–25% of breast cancers,

which are some of the most common malignant types of tumors in women. Establishment of a

fast technique sensitive to the low-levels of HER2 biomarker which results in early diagnosis

of the cancer is of great significance not only for increasing the survival rate, but also for saving

cost and time in successful prognosis of the disease [2]. The blood HER2 extracellular domain

content of breast cancer patients is usually >15 ng mL-1, which requires assay methods capable

to precisely measure such low concentration levels in blood serum or possibly in saliva of

patients. Thus, in recent years, we have been involved in preparation of novel antibody-antigen

based differential pulse voltammetric and impedimetric immunosensors for low level detection

of HER2 in serum samples of non-metastatic and metastatic breast cancer patients at different

stages [3,4]. In this seminar, three different successful strategies based on the effective

immobilization of anti-HER2 on gold nanopareticles, iron(III) oxide nanoparticles, and a

sandwich type gold nanopareticles-iron(III) oxide nanoparticles bioconjugate will be presented.

In this way, by the advantage of their long terminals, the designed bioconjugates provided the

most possible spaces for the immuno-reaction between biomolecules, which resulted in highly

sensitive and specific detection of HER2 in serum samples.

Acknowledgments: The support of this work by Iran National Elite Foundation (INEF) is

acknowledged.

References:

[1] R. Etzioni, N. Urban, S. Ramsey, M. McIntosh, S. Schwartz, B. Reid, J. Radich, G.

Anderson, L. Hartwell. Early detection: The case for early detection. Nature Rev. Cancer 3

(2003) 243-252.

[2] C. Tse, D. Brault, J. Gligorov, M. Antoine, R. Neumann, J.-P. Lotz. Evaluation of the

quantitative analytical methods real-time PCR for HER-2 gene quantification and ELISA of

serum HER-2 protein and comparison with fluorescence in situ hybridization and

immunohistochemistry for determining HER-2 status in breast cancer patients, Clin. Chem. 51

(2005) 1093-1101.

[3] M. Emami, M. Shamsipur R. Saber, R. Irajirad. An electrochemical immunosensor for

detection of a breast cancer biomarker based on antiHER2–iron oxide nanoparticle

bioconjugates. Analyst 139 (2014) 2858-2866.

[4] E. Arkan R. Saber, Z. Karimi, M. Shamsipur. A novel antibody–antigen based

impedimetric immunosensor for low level detection of HER2 in serum samples of breast cancer

patients via modification of a gold nanoparticles decorated multiwall carbon nanotube-ionic

liquid electrode. Anal. Chi. Acta 874 (2015) 66–74.

42

KN-31 QUALITY BY DESIGN APPROACH: WHY TO USE IT?

S. Furlanetto, S. Orlandini, B. Pasquini, C. Caprini, S. Pinzauti

Department of Chemistry “U. Schiff”, University of Florence, Via U. Schiff 6, 50019 Sesto F.no,

Italy; sandra.furlanetto@unifi.it

Recent regulatory documents in the pharmaceutical field [1] strongly recommend the

implementation of Quality by Design (QbD) approach in pharmaceutical product development.

QbD strategy has been gaining increased interest in drug analysis, as it can be effectively

applied to the development of analytical methods, leading to enhanced knowledge, better

analytical performances and higher flexibility from the regulatory point of view. QbD

framework for the development of analytical separation methods has been recently presented

[2,3] and is mainly based on risk assessment and multivariate tools. It begins with the definition

of the goal of the method, systematic scouting of alternative methods, assessment of the method

for risks using structured tools, evaluation of the effects of critical process parameters on critical

quality attributes by applying experimental design, identification of the design space and finally

development of a control strategy to ensure method performances. The design space represents

the core of QbD approach and is defined as the multidimensional region where the analytical

method provides quality outputs with selected probability. DS concept marks the difference

with the classical analytical method development, where the target of the optimization process

was only the definition of a single optimum point. Experimental design strongly underpins QbD

approach, providing a greater opportunity to find optimum conditions from a limited number of

experiments, allowing significant interactions between variables to be found and predictive

maps of the considered responses to be obtained. Thus, QbD includes all aspects and advantages

of multivariate techniques, but it also adds a concept of probability which allows the

achievement of analytical assurance of quality. Examples illustrating the usefulness of QbD

approach in the optimization of capillary electrophoresis methods for drug analysis will be

presented.

References:

[1] ICH Harmonised Tripartite Guideline, Q8(R2), Pharmaceutical Development 2009.

[2] S. Orlandini, S. Pinzauti, S. Furlanetto. Application of quality by design to the

development of analytical separation methods. Anal. Bioanal. Chem. 405 (2013) 443-450.

[3] E. Rozet, P. Lebrun, B. Debrus, B. Boulanger, Ph. Hubert. Design Spaces for analytical

methods. Trens Anal. Chem. 42 (2013) 157-167.

43

KN-32 DESIGN OF EXPERIMENT APPROACH FOR ROBUSTNESS

STUDIES DURING METHOD VALIDATION- A CASE STUDY FOR FAST

HPLC METHOD DEVELOPMENT

Pravin Karmuse1, Josan Thomas1, Abhay Gupta1, Zhenming Zhong2

1Novartis healthcare private limited, Hyderabad, India; pravin.karmuse@novartis.com

2Novartis Pharmaceuticals Corporation, East Hanover, USA.

A fast HPLC method was developed for separation and quantitative determination of a

drug substance and its twenty two related substances including process impurities and

degradation product. A chromatographic separation was achieved using stationary phase

Agilent Zorbax Eclipse plus C18, 50 x 2.1mm, 1.8µm particle size. The newly developed

method was validated according to ICH guidelines. The robustness was performed

comprehensively by Design of Experiment (DoE) approach. To examine potential sources of

variability, a number of factors are selected from the operating procedure. The robustness

results were evaluated extensively for all SST parameters viz. repeatability, resolution, tailing

factor and detectability. The DoE studies also include the assessment of quantitative aspects of

the HPLC method i.e. assay and impurity content results.

Acknowledgments: Authors are thankful to Dr. Michael Kramer, Dr. Venugopal Korlam and

Arun Mathur (Novartis Healthcare) for their support and encouragement

References:

[1] Y. Vander Heyden, A. Nijhuis b, J. Smeyers-Verbeke a, B.G.M. Vandeginste b,

D.L.Massart: J. Pharm. Biomed. Anal. 24 (2001) 723–753

[2] D.B. Hibbert / J. Chromatogr. B 910 (2012) 2– 13

44

KN-33 PEPTIDES: THERAPEUTICS OF THE FUTURE

Natia Tsomaia1

1Ipsen Bioscience, Inc; Natia.Tsomaia@IPSEN.com

Rapid advancement in genomics has brought a better understanding of molecular

mechanisms for various pathologies and identified a number of highly attractive target classes.

Many essential cellular pathways, which are implicated in human diseases, are controlled by

intracellular protein-protein interactions (PPIs). Such PPIs could be potential drug targets, and

thus the ability of molecules to inhibit specific PPIs has remarkable therapeutic value. PPI

surfaces are part of a diverse and unexplored biological space, where traditional small molecule

scaffolds are not always successful. While large biologics can effectively modulate PPIs in the

extracellular region, their limitation in crossing the cellular membrane leaves intracellular

protein targets outside of their reach. There is a growing need in the pharmaceutical field to

push the boundaries of traditional drug design and discover innovative molecules that are able

to modulate key biological pathways by inhibiting intracellular PPIs. Peptides are one of the

most promising classes of molecules that could deliver such therapeutics in the near future. This

talk will address novel drug design strategies that carry the promise of opening the doors for

peptide therapeutics to reach the currently “undruggable” space.

Acknowledgments: I would like to thank IPSEN for supporting this talk.

References:

[1] Tsomaia, N. (2015). Peptide therapeutics: Targeting the undruggable space. Eur J Med

Chem. 94, 459-470. doi: 10.1016/j.ejmech.2015.01.014

[2] Watkins, A. M., & Arora, P. S. (2015). Structure-based inhibition of protein-protein

interactions. Eur. J. Med. Chem. 94, 480-488. doi: 10.1016/j.ejmech.2014.09.047

[3] Bhat, A., Roberts, L. R., & Dwyer, J. J. (2015). Lead discovery and optimization

strategies for peptide macrocycles Eur J Med Chem. 94, 471-479. doi:

10.1016/j.ejmech.2014.07.083

45

KN-34 ANALYTICAL STRATEGIES FOR SENSITIVE PEPTIDE

QUANTITATION FROM LOW BLOOD SAMPLE VOLUMES

M. Fillet; G. Cobraiville, J. Crommen, A.-C. Servais, V. Houbart

Laboratory for the Analysis of Medicines, Department of Pharmacy, CIRM, University of Liege,

Belgium; Marianne.fillet@ulg.ac.be

In bioanalysis, samples are often available in limited volumes. For example,

pharmacokinetic studies on rodents only provide a few dozens microliters of material at a time.

In this context, analytical techniques requesting the smallest sample volume possible while

keeping a high sensitivity are therefore preferred. To meet these requirements, LC

miniaturisation (micro- and nano-LC) presents undeniable advantages such as a small injection

volume (down to 0.1 µL), low peak dispersion and reduced flow rates, both favorable to MS

sensitivity.

The concept of the 3Rs (refine, reduce, replace) is widely followed for in vivo testing,

with the aim of reducing the use of animals. In the context of pharmacokinetic or toxicokinetic

studies, 3Rs principle is mainly carried out by reducing the sampling volume to perform serial

collection on the same animal over the whole study, while preserving animal health and welfare.

This evolution requires the development of robust sampling and analytical approaches that

handle very small sample volumes.

At the same time, some challenges remain for low abundance peptide absolute

quantitation: the chromatographic behaviour of small molecules is well known but it is not

likely to be fully transposed to peptides or proteins; moreover, the target concentration is often

in the pg-ng/mL range requiring high sensitivity. In addition, the tendency of peptides and

proteins to adsorb on surfaces as well as their limited stability may lead to a lack of method

robustness due to carry-over and sample degradation.

In this presentation, we will discuss the potential of Dried Blood Spot (DBS) and

Volumetric Absorptive MicroSampling (VAMS) compared to classical micro-Solid Phase

Extraction (SPE) for the quantitation of hepcidin, chosen as model peptide, in blood samples.

This low-concentration peptide was analysed by miniaturised liquid chromatography coupled

to tandem MS (LC-Chip-MS/MS) to reach the appropriate sensitivity.

To compare the performance of the sample preparation methods, protocols were

carefully optimized using the Design Of Experiment (DoE) methodology. A special attention

was also paid to phospholipid removal (PR) using 96 well-plates. Since whole blood is probably

one of the most complex biological matrices than can be analysed, matrix effect was expected

to occur during blood analysis, especially when using an aspecific sample preparation technique.

Matrix effects was thus carefully investigated and quantified.

Finally, our results confirmed the importance of the choice of an adequate ion-pairing

reagent at an appropriate concentration in the mobile phase, but the results also underlined the

key role of sample composition, an experimental parameter that is seldom if ever optimized as

it should be in quantitative or qualitative method development.

46

KN-35 DEVELOPMENT OF A MOLECULARLY IMPRINTED PEG-BASED

HYDROGEL FOR PROTEIN RECOGNITION

Takuya Kubo, Shunsuke Arimura, Toyohiro Naito, Koji Otsuka*

Graduate School of Engineering, Kyoto University, Nishikyo-ku, Kyoto 615-8510, Japan;

otsuka@anchem.mc.kyoto-u.ac.jp

Molecularly imprinting technique allows the preparation of the artificial molecular-

recognition materials. Molecularly imprinted polymers (MIPs) can be simply synthesized in a

short time and have a chemical/physical stability, so that MIPs have been applied to sensors,

separation media, and biomimetic membranes like as receptors. In recent studies, polyacryl-

amide-based hydrogels have mainly used for the protein imprinted (PI) gel. However,

polyacrylamide leads some drawbacks including high toxicity of monomers, nonspecific

adsorption for biomolecules, and low bio-compatibility. On the other hand, poly(ethylene

glycol) (PEG) has high biocompatibility, hydrophilicity, and high chemical stability to change

in temperature, pH etc. In this study, to develop a selective adsorption medium for proteins, we

prepared a molecularly imprinted hydrogel using a PEG-based cross-linker, and the molecular

recognition ability of the gel was evaluated by batch adsorption of proteins [1].

We chose a PEG 600 dimethacrylate (14G) as a cross-linker, lysozyme or cytochrome

c as template molecules, and sodium allylsulfonate (AS) or sodium p-styrenesulfonate (SS) or

2-acrylamido-2-methylpropanesulfonic acid (AMPS) as functional monomers, respectively.

We synthesized PI gels and non-imprinted gels (NIP gels) by photo polymerization (at 365 nm

for 3 h). The selective adsorption abilities of the gels were examined by the comparison of the

adsorption rate (AR) for proteins and imprinting factor (IF). The AR and IF were calculated

from the following equations: AR (%) = nafter (μmol) / nbefore (μmol)

IF = adsorption rate of PI gel / adsorption rate of NIP gel

where nafter is the adsorption amount of each protein containing lysozyme, cytochrome c, trypsin,

and bovine serum albumin by the gels, and nbefore is the amount of template proteins, lysozyme

or cytochrome c, respectively.

As results of the adsorption evaluations, the PI gel prepared with SS and AMPS using

lysozyme as a template showed higher adsorption capability to lysozyme. The result suggests

that the hydrophobic interaction and/or hydrogen bonding contributed to the adsorption of

lysozyme as well as the ionic interaction by sulfo groups. Also, the adsorption rate of lysozyme

was the highest compared to other proteins in the PI gel. Furthermore, when a nonspecific ionic

interaction was suppressed by adding NaCl to the protein solution, IF for lysozyme was

significantly increased. Additionally, the PI gel absorbed more lysozyme than cytochrome c.

Since lysozyme and cytochrome c are similar in terms of the isoelectric point and molecular

size, the PI gel adsorbed lysozyme by recognizing three-dimensional structure. Furthermore,

the PI gel prepared with AS using cytochrome c as a template showed higher adsorption

capability to cytochrome c. According to these results, the generality of the method was

confirmed for the selective adsorption of proteins.

Acknowledgments: This research has been partly supported by the Environment Research and

Technology Development Fund (5-1552) from the Ministry of the Environment, Japan and the Grant-

in-Aid for Scientific Research (No. 15K13756) from the Japan Society for the Promotion of Science.

References:

[1] Kubo, T.; Arimura, S.; Tominaga, Y.; Naito, T.; Hosoya, K.; Otsuka, K.: Molecularly Imprinted

Polymers for Selective Adsorption of Lysozyme and Cytochrome c Using a PEG-Based Hydrogel:

Selective Recognition for Different Conformations due to pH Conditions, Macromolecules 2015, in

press.

47

Oral Presentations

OP-1 ISOLATION AND CHARACTERIZATION OF A NOVEL ACID

DEGRADATION IMPURITY OF AMLODIPINE BESYLATE USING Q-

TOF, NMR, IR AND SINGLE CRYSTAL X-RAY

Ravi Rapolu, Ch. Krishnam Raju, Kolupula Srinivas, Atul Awasthi

Sameer G. Navalgund, Koduru V. Surendranath

atulawasthi@outlook.com

Identification of an impurity is paramount for the quality of a pharmaceutical product

and safety of the patients. Impurities and DPs could arise from the synthetic process of active

pharmaceutical ingredient and/or during storage of pharmaceutical products under the influence

of temperature, humidity and light. Forced degradation studies of the APIs using acid, base,

oxidative, photolytic, and thermal stress are a common way to simulate impurities which may

appear during shelf time. Forced degradation of Amlodipine (AMD) in acidic condition resulted

into a potential unknown impurity. This unknown acid degradation product (ADP), analyzed

using a new reverse-phase high performance liquid chromatography (HPLC), eluted at RRT

1.24 with respect to AMD peak. ADP was isolated by preparative HPLC and extracted to solid

form using rotary evaporator. The structure of ADP was elucidated using high resolution MS,

multidimensional NMR and FTIR spectroscopic techniques, and later characterized as ethyl-6-

(2-chlorophenyl)-8-methyl-3,4,6,7-tetrahydro-2H-benzo[b][1,4]oxazine-5-carboxylate. The

presence of ADP recemic mixture was confirmed by polarimeter and chiral HPLC. Given the

complexity associated with ADP formation, single crystal X-ray crystallography was used to

confirm proposed structure and degradation pathway proposed using Gaussian 09. In addition,

reaction mechanism was proposed and confirmed with Computational chemistry. To our

knowledge, this was a novel impurity from compendial perspective.

48

OP-2 TRENDS AND CHALLENGES IN THE ANALYSIS OF ILLEGAL

PHOSPHODIESTERASE TYPE-5 INHIBITORS (PDE-5) FOUND IN

HEALTH SUPPLEMENTS

Chee Leong Kee1, Xiaowei Ge1, Min Yong Low1

1Pharmaceutical Laboratory, Applied Sciences Group, Health Sciences Authority, 11 Outram

Road, Singapore 169078, Singapore; email: Kee_Chee_Leong@hsa.gov.sg

One of the greatest safety concerns in health supplements is the adulteration of these

products with undeclared pharmaceutical drugs or their analogues, in illicit attempts to evade

detection. The synthetic phosphodiesterase type 5 inhibitors (PDE-5), mainly the analogues of

sildenafil, tadalafil and vardenafil have been widely reported as adulterants in health

supplements [1]. It represents a problem in product quality and is one of the major causes for

adverse events. Indeed, these are high health risk products because the toxicity and potency are

often not studied. As mandatory requirements, regulating the manufacture and sale of herbal

medicines are much less stringent than those related to pharmaceuticals, these herbal products

are heavily advertised on the internet with exaggerated claims and are freely available for

purchase without prescription. Recent survey has also indicated that 61% of 150 health

supplements advertised for enhancing sexual performance were adulterated with these kinds of

illegal drugs [2].

The HSA’s experiences in screening, detection and structural elucidation of unknown

drug substances and their analogues in adulterated health products will be shared in this

presentation. It will also highlight the challenges and opportunities faced by researches in the

finding of new analogous compounds.

Acknowledgments: Health Sciences Authority (HSA) Research and Development Fund

References:

[1] B. J. Venhuis, D. Kaste. Towards a decade of detecting new analogues of sildenafil,

tadalafil and vardenafil in food supplements: a history, analytical aspects and health risks. J.

Pharm. Biomed. Anal. 69 (2012) 196-208

[2] V. Gilard, S. Balayssac, A. Tinaugus, N. Martins, R. Martino, M. Malet-Martino.

Detection, identification and quantification by 1H NMR of adulterants in 150 herbal dietary

supplements marketed for improving sexual performance. J. Pharm. Biomed. Anal. 102(2015)

476–493

49

OP-3 PERSPECTIVES OF SUPERCRITICAL FLUID CHROMATOGRAPHY

(SFC) IN DRUG ANALYSES

Maria Kristina Parr1; Alexander Schmidt1,2

1Institute of Pharmacy, Freie Universität Berlin, Germany

2Chromicent GmbH, Berlin, Germany

Since years HPLC-MS/MS gained importance for the detection of various classes of

drugs. In contrast to the classical GC-MS technique it allows for separation of analytes with

different functional properties without derivatization. However some analytes are still

challenging as HPLC-MS/MS shows limited resolution capabilities and highly polar analytes

interact only insufficiently on the conventional analytical columns.

HPLC-MS/MS of some highly polar stimulants proved hard due to very low interaction

with the reversed phase (RP) columns generally used for HPLC-MS(/MS). Even on the more

polar RP phases like C6-phenyl as well as HILIC columns their analysis remained challenging

or even impossible.

Supercritical fluid chromatography (SFC) as orthogonal separation technique to HPLC

may help to overcome these issues.

To check for the general potential of SFC-MS/MS separation as alternative a mixture of

selected polar drugs, was injected onto a SFC-system. All compounds showed sharp peaks,

good retention and resulted in retention times clearly separated from the dead time. Compared

to the HILIC method all compounds are detected in considerably shorter retention times. Also

accounting the very long between-run equilibration times in HILIC separation by SFC allows

for a significant reduction in cycle time.

Acknowledgments: The World Anti-Doping Agency is acknowledged for financial support

(Grant 14A03KP).

50

OP-4 SUPERCRITICAL FLUID CHROMATOGRAPHY FOR

ANALYTICAL AND SEMI-PREPARATIVE ENANTIOSEPARATIONS

Emmanuelle Lipka

Inserm U995-LIRIC- Laboratoire de Chimie Analytique, Faculté de Pharmacie de Lille- 59000-

F- France

The context of this study is the development of new agonists of type 2 cannabinoid

receptor (CB2). These molecules belonging to the chemical series 3-carboxamido-5-aryl

isoxazole have potential anti-inflammatory action in the case of the Inflammatory Bowel

Disease (IBD), a pathology that affects six million people in the world and still has no cure to

this day.

These molecules having a chiral center, the separation of enantiomers in order to study

their pharmacological activities is an essential step in the development of any potential drug.

The aim of this work was to separate four pairs of enantiomers by a rapid and an

ecological technique: the Supercritical Fluid Chromatography (SFC). This technique uses a

mobile phase at a supercritical state, composed mainly of carbon dioxide and under thirty per

cent of organic solvent.

First, we have optimized the analytical parameters of the separation of our molecules

(chiral stationary phase, mobile phase composition). Then in a second step, we performed a

semi-preparative separation of the compounds to obtain a sufficient quantity to implement the

pharmacological study.

1 2 3 4

51

OP-5 HPLC SEPARATION OF UNUSUAL AMINO ACID ENANTIOMERS

ON CINCHONA ALKALOID-BASED CHIRAL STATIONARY PHASES

Antal Péter1; István Ilisz1, Ferenc Fülöp2, Gábor Tóth3, Wolfgang Lindner4

1Department of Inorganic and Analytical Chemistry, University of Szeged, H-6720 Szeged,

Dóm tér 7, Hungary; apeter@chem.u-szeged.hu

2Institute of Pharmaceutical Chemistry, University of Szeged, H-6720 Szeged, Eötvös u. 6,

Hungary

3Department of Medical Chemistry, University of Szeged, H-6720 Szeged, Dóm tér 8, Hungary

4Department of Analytical Chemistry, University of Vienna, Währingerstrasse 38, 1090 Vienna,

Austria

Peptides involved in the chemistry in living organisms have traditionally been

considered unsuitable as drugs for they have been classified as metabolically unstable, with

poor gastrointestinal and blood-brain barrier penetration properties, and are rapidly excreted

through the liver and kidney. However, these shortcomings may be overcome if unusual amino

acids are incorporated into the peptide chain, and research in this direction therefore demands

a better knowledge of the three-dimensional and conformational structure and hence the

underlying molecular recognition phenomena. Several unusual primary and cyclic secondary

α-, ß- and



-amino acids have recently been designed with a view to increasing the metabolic

stability of peptides and stabilizing their structures by constraining the side-chain functional

groups of the amino acids. Peptidomimetics that may serve as agonists or antagonists of

enzymes have been sought for decades [1].

Since the biological and physicochemical properties of amino acids are strongly related

to their stereochemistry, enantioselective high-performance liquid chromatography (HPLC) is

routinely used for the discrimination of enantiomers.

The present work describes the separation of enantiomers of unusual secondary α- and

several ß-amino acids through the application of novel Cinchona-alkaloid-based zwitterionic

chiral stationary phases. With the use of polar-ionic or reversed phase conditions, the effects of

the solvent compositions, acid and base additives, the specific structural features of the analytes

(selectands), selectors and temperature on the retention and selectivity are discussed on the basis

of the experimental data. The elution sequence was determined in almost all cases.

References:

[1] D. Seebach, A. Lukaszuk, K. Patora-Komisarska, D. Podwisocka, J. Gardiner, M.-O.

Ebert, Chem. Biodiversity 8 (2011) 711.

52

OP-6 ON THE MINIATURIZATION OF CHIRAL SEPARATIONS WITH

CAPILLARY COLUMNS AND NANO-HPLC

Ashraf Ghanem

Chirality Program, Biomedical Science, Faculty of ESTEM, University of Canberra, Australian

Capital Territory (ACT), Australia; ashraf.ghanem@canberra.edu.au

Miniaturization of conventional HPLC to nano-HPLC enables high throughput, reduced

sample size and less consumption of hazardous solvents. Consequently, the chiral separation

can be achieved under environmentally friendly conditions. In this presentation, new

bonded/immobilized chiral silica and polymer-based monolithic phases are developed in thin-

hair capillary format and investigated for the enantioselective separation of different classes of

chemicals and pharmaceutical-related compounds. The expected outcome will be

environmentally responsible, cost effective and efficient analytical sample preparation and

separation technologies for analytical laboratories throughout the world. Some featured benefits

include but not limed to 1) Up to 4X increase in sensitivity; 2) Improved turn-around-time with

up to 5X faster separations; 3) Up to a 95% reduction in mobile phase consumption and 4)

Improved robustness – less sample introduced into the MS and, ultimately, less instrument

downtime.

Schematic diagram showing the preparation of β-CD-based silica monolith via one pot

approach

References:

[1] A. Ghanem, M. Ahmed, H. Ishii, T. Ikegami, Talanta, 132 (2015) 301-314.

[2] M. Ahmed, A. Ghanem, J. Chromatogr. A, 1345 (2014) 115-127.

[3] A. Ghanem, N. Berova, J. Caldwell “Chirality research in Australia special issue 2014”

Chirality, 26 (2014) 675-676.

53

OP-7 CHAOTROPIC CHROMATOGRAPHY IN PHARMACEUTICAL

ANALYSIS

A. Malenović, A. Vemić

University of Belgrade – Faculty of Pharmacy, Department of Drug Analysis, Vojvode Stepe

450, Belgrade, Serbia; andja@pharmacy.bg.ac.rs

In pharmaceutical analysis we are often faced with the challenge of simultaneous

separation of complex mixtures. Since the main factors that govern the separation are analytes

structure and nature of the chromatographic system, under certain conditions the analytes can

be ionized, so their retention, peak symmetry and separation efficiency in the reversed phase

systems may be compromised. Recently, chaotropic agents gained popularity as interesting and

useful mobile phase additives for overcoming the mentioned chromatographic problems.

However, the complexity of the chaotropic system requires special attention and detailed

understanding of the chaotropic agents’ effects. Description of the separation phenomena can

be achieved through the retention modeling from the theoretical and empirical aspect. The

extended thermodynamic approach enables a comprehensive consideration of the mechanisms

underlying the separation [1]. Namely, it examines the contribution of both the double layer

formation and its electrostatic influence on the analytes retention, and the ion-pair formation

between the chaotropic agent and the analyte, thus comprising the findings of both electrostatic

and stoichiometric retention models.

The empirical approach can take two paths. One path enables an overview of analytes

molecular properties on their retention following their direct influence on the retention factors.

Moreover, these findings can be combined and expanded with the results obtained through the

above mentioned thermodynamic insight in the separation mechanism. The second path

includes chemometric approach in the examination of chromatographic system with the final

aim of method development suitable for routine laboratory use. Furthermore, in order to

improve the understanding and control of processes in chaotropic chromatography, and hence

improve its overall quality, a quality-by-design approach (QbD) can be applied [2]. The main

goal of QbD is to compute a design space (DS) of critical process parameters (CPPs) that show

no negative effect on the set of selected critical quality attributes (CQAs). The most efficient

way to establish a relationship, gain the understanding of the effects and interactions between

CPPs and CQAs is the application of design of experiments (DoE) methodology. The DS is not

only the zone of the desired quality level represented by CQAs, but the domain within which

these critical responses fulfill predefined limits with an acceptable level of probability. This

quality assurance can be achieved by Monte Carlo simulations propagating either the error equal

to the variance of the model residuals or the error in model coefficients’ calculation. The

working point selected among DS points is robust and reliable. Eventually, the suitability of the

developed method for the intended purpose should be confirmed by method validation.

References:

[1] T. Cecchi, P. Passamonti, Retention mechanism for ion-pair chromatography with

chaotropic reagents, J. Chromatogr. A 1216 (2009) 1789–1797.

[2] E. Rozet, P. Lebrun, B. Debrus, B. Boulanger, P. Hubert, Design Spaces for analytical

methods, Trends Anal. Chem. 42 (2013) 157-167.

54

OP-8 INFORMATIONAL MAINTENANCE (DATA BASES AND

ALGORITHMS) FOR PHARMACEUTICAL AND BIOMEDICAL

CHROMATOGRAPHIC ANALYSES

Igor G. Zenkevich

St. Petersburg State University, St. Petersburg, Russia; izenkevich@yandex.ru

Reliable gas chromatographic (GC) and/or GC-MS identification of organic compounds

of different classes, including analytes of pharmaceutical and biochemical origin, requires using

both comprehensive informational maintenance and appropriate algorithms of data interpre-

tation. One of the most important problems is to complete mass spectrometric data by consi-

dering the chromatographic retention parameters, namely retention indices (RI) on standard

non-polar and polar stationary phases. The example of such software is NIST MS database,

completed with RI database since 2005. The last version (2014) includes RI data for approx.

83,000 organic compounds 1. The condition of identification using single dimensional

analytical parameters like GC RIs is the following relation:

RIexp  <RIref>  ksRI (1)

where RIexp is the experimentally measured GC retention index, <RIref> is the average interlabo-

ratory reference RI value, sRI is its standard deviations, 2  k  4 is the coefficient which regula-

tes the unambiguousness and reliability of the results (the number and ratio of so-called 1st and

2nd order errors). The content and features of interlaboratory RI data sets for different organic

compounds can be illustrated by histograms 2 (different examples are considered). Single-

mode symmetric histograms permit us to attribute RI data to the individual structures. At the

same time, histograms for a lot of analytes (especially polar) appeared to be asymmetric and

multi-mode. The last case indicates the unification into the single RI data set the data for few

compounds with similar mass spectra, but different retention indices; there are different ways

of the interpretation of these effects:

– Target analyte exists as the mixture of few diastereomers. It is typical for numerous

compounds of natural origin (e.g., sesquiterpenes);

– The chemical origin of the target analyte implies the existence of few structural isomers (that

is typical for some technical substances and mixtures, e.g., dibutyl phthalate);

– Not the total multitude of analytes with similar mass-spectra, but their restricted quantity is

represented in a database (e.g., identification of 2,2,4,6,6-pentamethyl heptane);

– The analytes are “sensitive” to the differences in the properties of standard non-polar and

semi-standard non-polar stationary phases (e.g., carbonyl compounds of different series).

The operations with asymmetric reference RI data sets require modification of the rela-

tion (1). Instead of the single sRI value, two different standard deviations for (RIref) > <RIref>

and (RIref) < <RIref> separately [usually sRI(+) > sRI(-)] should be used. Hence, the quality

control of RI databases appears to be the most important and complex time- and labor-

consuming operations.

References:

1 The NIST 14 Mass Spectral Library (NIST14/2014/EPA/NIH). Software/Data Version

(NIST14); NIST Standard Reference Database, Number 69, August 2014. National Institute of

Standards and Technology, Gaithersburg, MD 20899: http://webbook.nist.gov (accessed June 2015).

2 Zenkevich, I.G.; Babushok, V.I.; Linstrom, P.J.; White, E.; Stein, S.E. Application of histograms

in evaluation of large collections of gas chromatographic retention indices. J. Chromatogr. A. 2009. V.

1216. P. 6651-6661.

55

OP-9 DEVELOPMENT OF NOVEL MULTIFUNCTIONAL

BIOCONJUGATES FOR TDD

Gary Gellerman, Lena Regozin, Boris Redko, Yosi Gilad and Andrei Bazilevich

Department of Biological Chemistry, Ariel University, Ariel, 40700, Israel

Targeting drugs through small-molecule carriers with a high affinity to receptors on

cancer cells can overcome the lack of target cell specificity of most anticancer drugs. Although

many targeted drug delivery approaches are being tested, the linkage of several and different

drugs to a single carrier molecule might further enhance their therapeutic efficacy, particularly

if the drugs are engineered for variable time release. Therefore, we decided to develop novel

amino acid platforms for targeted delivery and controlled release of chemotherapeutic

"cocktails". These multi-functional amino acid platforms (MAAP) are loaded with various

drugs for conjugation to a peptide carrier. Each arm of the platform carries a different anticancer

agent linked though the same or different functional group, providing discrete chemo- and bio-

release profiles for each drug, and also enabling “switch off/switch on" regulation of drug

cytotoxicity by conjugation to the platform and to a cell targeting peptide. The versatility of this

approach enables efficient production of drug-loaded platforms and determination of favorable

drug combinations/modes of linkage for subsequent conjugation to a carrier moiety for targeted

cancer cell therapy.

References:

[1] M. Firer, G. Gellerman, Targeted drug delivery for cancer therapy: the other side of

antibodies. J. Hematol. Oncol. 5, (2012), 70.

[2] G. Gellerman, S. Baskin, L. Galia, Y. Gilad, M.A. Firer, Drug resistance to chlorambucil

in murine B-cell leukemic cells is overcome by its conjugation to a targeting peptide, Anti-

Cancer Drugs 24, (2013), 112-119.

[3] Y. Gilad, M. A. Firer, A. Rozovsky, E. Ragozin, B. Redko, A. Albeck, G.

Gellerman,"Switch off/switch on” regulation of drug cytotoxicity by conjugation to a cell

targeting peptide" Eur. J. Med. Chem., 85, (2014), 139-146.

56

OP-10 VOLUMETRIC ABSORPTIVE MICROSAMPLING (VAMS)

AND DRIED MATRIX SPOTS (DMS) FOR SPORT DRUG TESTING

Laura Mercolini1; Maria A. Saracino1; Michele Protti1; Angelo E. Sberna2

1Laboratory of Pharmaco-Toxicological Analysis, Department of Pharmacy and

Biotechnology, Alma Mater Studiorum - University of Bologna, Via Belmeloro 6, 40126-

Bologna (Italy); laura.mercolini@unibo.it

2Commission for the Monitoring and Control of Doping and the Protection of Health in

Sporting Activities, Italian Ministry of Health, Viale Ribotta 5, 00144-Rome (Italy).

In recent times, many efforts have been made by researchers, together with doping

control authorities, with the purpose of enhancing and simplifying sport drug testing activities,

thus providing high-throughput reliable results. However, there are still few studies, which aim

to radically renew the approach for athlete monitoring starting from basic protocols, such as

biological fluid sampling of from both in- and out-of-competition athletes. The use of dried

matrix spots (DMS) for the analysis of both large and small molecules has recently drew

considerable attention within bioanalysis and drug testing. This approach has been widely

studied lately, driven by its advantages over conventional tube sampling and mainly: reduced

biological sample volumes; simplified sample collection workflows; increased analytes

stability; no requirement for frozen storage and refrigerated transport; cost savings associated

with the shipping and storage.

The approach by volumetric absorptive microsampling (VAMS) devices involves the

absorption of a biological sample (e.g. blood) onto a porous polymeric substrate by wicking,

amount of substrate. VAMS offers a simpler sample collection workflow than other currently

available approaches (venipuncture, DBS), since the sampler can be directly applied to the

pricking site and does not require further processing and transfers, as for blood or plasma [1].

Bioanalytical methods based on liquid chromatography-tandem mass spectrometry (LC-

MS/MS) for the analysis of steroids (glucocorticoids, synthetic anabolic steroids) and

stimulants (amphetamine-like compounds, cocaine, catecholamines, synthetic cathinones) in

dried matrix spots and dried matrices adsorbed on VAMS samplers has been developed and

tested to be further applied in specific doping control protocols. Drying time, temperature,

humidity, light exposure and storage materials are being investigated in order to ensure samples

stability and develop standardized protocols. Moreover, all sample pretreatment steps

(extraction, purification and preconcentration) will be studied thoroughly and optimized in

terms of appropriate extraction solvents and miniaturized techniques suitable for extract clean-

up.

The promising results obtained from preliminary assays proved that the proposed

innovative sampling approaches (DMS and VAMS) could be suitable for the quali-quantitative

analysis of the main classes of drugs included in the World Anti-Doping Agency (WADA) and

in the Commission for the Monitoring and Control of Doping and the Protection of Health in

Sporting Activities (CVD) lists of prohibited substances, within in-competition and out-of-

competition doping control activities.

References:

[1] N. Spooner, P. Denniff, L. Michielsen, R. De Vries, Q.C. Ji, M.E. Arnold, K. Woods,

E.J. Woolf, Y. Xu, V. Boutet, P. Zane, S. Kushon, J.B. Rudge. A device for dried blood

microsampling in quantitative bioanalysis: overcoming the issues associated blood hematocrit.

Bioanalysis 7 (2015) 653-659.

57

OP-11 CHALLENGES AND CHANCES FOR BIOANALYSIS OF

BISPECIFIC ANTIBODIES

Kay Stubenrauch*1; Markus Zadak1, Klaus Mackeben1, Uwe Wessels1, Mirko Ritter2, Ulrich

Mohn3, Rudolf Vogel2, Julia Heinrich1

1Roche Innovation Center Penzberg, Pharma Research & Early Development (pRED),

Pharmaceutical Sciences, Large Molecule Bioanalytical Research & Assay Development,

Nonnenwald 2, 82377 Penzberg, Germany; *e-mail: kay-gunnar.stubenrauch@roche.com

2Roche Diagnostics GmbH, Roche Professional Diagnostics R&D, Early Development & Rare

Reagents, 82377 Penzberg, Nonnenwald 2, Germany

3Roche Diagnostics GmbH, Diagnostics Operations, Immunology/Farm, 82377 Penzberg,

Nonnenwald 2, Germany

While the emerging class of bispecific monoclonal antibodies is characterized by the

diversity of the molecular constructs, it has in common two sets of different complementarity

determining regions (CDRs). Pharmacokinetic (PK) assays can exploit the bispecificity of the

analyte for binding to the two different antigens or anti-idiotype antibodies used as capture

and detection reagents. This allows to determine in one assay run the concentration of the

analyte as well as the two specificities, thereby also confirming the structural integrity of the

analyte. Generic or monospecific PK assay may quantitate the analyte, but do not proof both

specificities in one run. The classical anti-drug antibody (ADA) assay based on use of the

drug for capture and detection of the analyte is an appropriate format to detect ADAs against

both sets of CDRs, the human IgG framework and engineered part(s) of the molecule. The

potential interference of oligomeric target antigens in the ADA assay may be avoided by

addition of alternative antibodies with the same epitope but different CDRs as drug. The

bispecific antibodies themselves can be utilized in target assays for quantification of two

different soluble target antigens. Further differentiation between free and drug-bound target

will have to be defined case by case. A good understanding of the target biology and of the

nature of the bispecific antibody combined with timely availability of specific assay reagents

will facilitate more complex bioanalytical methods for analysis of bispecific antibodies where

necessary.

Acknowledgments: The authors thank all involved Large Molecule Research, DTA, RPD

and PS sub-team colleagues for inspiring discussion and collaboration

References:

[1] Staack RF, Stracke JO, Stubenrauch K, Vogel R, Schleypen J, Papadimitriou A.:

Quality requirements for critical assay reagents used in bioanalysis of therapeutic proteins:

what bioanalysts should know about their reagents.

Bioanalysis. 2011 Mar;3(5):523-34

[2] Stubenrauch K, Wessels U, Essig U, Kowalewsky F, Vogel R, Heinrich J.:

Characterization of murine anti-human Fab antibodies for use in an immunoassay for generic

quantification of human Fab fragments in non-human serum samples including cynomolgus

monkey samples. J Pharm Biomed Anal. 2013 Jan; 72:208-15

[3] Stubenrauch K, Wessels U, Essig U, Vogel R, Waltenberger H, Hansbauer A, Koehler

A, Heinrich J. An immunodepletion procedure advances free angiopoietin-2 determination in

human plasma samples during anti-cancer therapy with bispecific anti-Ang2/VEGF

CrossMab. J Pharm Biomed Anal. 2015 Jan;102:459-67

58

OP-12 CHARACTERIZATION OF INTACT VIRUS-LIKE PARTICLES OF

HUMAN PAPILLOMAVIRUS 16 (HPV16-VLP) IN CAPILLARY

ELECTROPHORESIS

V. Bettonville1, J. Nicol2, N. Thelen3, M. Thiry3, M. Fillet1, N. Jacobs2, A.-C. Servais1

1Laboratory for the Analysis of Medicines, Dept. of Pharmaceutical Sciences, CIRM, University

of Liège, Liège, Belgium; acservais@ulg.ac.be

2Cellular and Molecular Immunology, University of Liège, Liège, Belgium;

3Cellular and Tissular Biology, GIGA-Neurosciences, University of Liège, Liège, Belgium;

Virus-like particles of human papillomavirus (HPV-VLP), resulting from the self-

assembly of the capsid proteins (L1 or L1 and L2), have been widely used to study HPV as they

are similar to the native virion. Moreover, two prophylactic vaccines, Gardasil® and Cervarix®,

are based on HPV-VLP L1. Analytical techniques currently used to characterize HPV-VLP,

such as SDS-PAGE, Western blot, ELISA, are time-consuming and semi-quantitative. In this

study, capillary electrophoresis (CE) was evaluated for the analysis of intact HPV16-VLP. The

first challenge was to prevent HPV-VLP adsorption onto the capillary wall and aggregation.

Indeed, HPV-VLP have a tendency to adsorb to container surface, leading to their aggregation

[1]. In this context, the use of coated capillaries was found to play a key role in CE analysis of

HPV-VLP. Nevertheless, this was not sufficient to achieve reproducible migration times.

Therefore, several ionic and non-ionic surfactants were investigated as BGE additives. The

nature and the concentration of the surfactant were found to have a deep impact on the

electrophoretic behavior of VLP. The developed method enables HPV-VLP detection in less

than 10 min (migration times RSD: 1.6 %). The identity of HPV-VLP peak was confirmed by

comparison with a sample obtained from a wild-type baculovirus. Finally, we applied the

developed methodology to VLP-based vaccines, demonstrating that CE could be successfully

used for vaccine quality control.

Acknowledgments: Many thanks are due to FNRS, “Fonds spéciaux pour la recherche” of the

University of Liège and Fonds Leon Fredericq for their financial support. The authors are

grateful to Dr. P. Coursaget and Pr. A. Touzé for the provision of baculovirus expressing

HPV16 L1 and L2.

References:

[1] L. Shi, G. Sanyal, A. Ni, Z. Luo, S. Doshna, B. Wang, T.L. Graham, N. Wang, D.B.

Volkin, Stabilization of human papillomavirus virus-like particles by non-ionic surfactants,

Journal of Pharmaceutical Sciences, 94 (2005) 1538-1551.

59

OP-13 METABOLIC STABILITY STUDIES SUPPORTED BY LC-MS AND

CHEMOMETRICS ANALYSIS

Tomasz Bączek, Mariusz Belka, Szymon Ulenberg

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Medical University of

Gdańsk, Hallera 107, 80-416 Gdańsk, Poland

Drug metabolism is a major determinant governing both pharmacokinetics and clinical

response and a great deal of efforts is directed to assess the key metabolic parameters in the

early stages of drug development. Within the ADME processes, metabolism certainly covers

currently the largest area of interest and there is still challenging to evaluate and to predict it. A

large number of drug candidates have undesirable properties, including low metabolic stability

that could result in poor bioavailability or weak efficacy. Metabolic stability is defined as the

susceptibility of a chemical compound to biotransformation, and could be expressed for

example as a half-life (t1/2) determined in vitro with the use of microsomal preparations

obtained from human or other species livers. That experimental step, included in drug

development process, could allow for the determination of metabolic profiles of drug candidates

early on pre-clinical stage, and therefore, this information could be also useful to plan the

synthesis of new chemical entities with improved properties more rationally.

Two examples of the metabolic stability studies of the novel derivatives of potent

anticancer or antidepressant activity are provided. In the studies, metabolic stability of the

selected benzenesulfonamide and arylpiperazine derivatives was measured with particular

emphasis on elucidation of metabolites’ structures applying LC-MS and LC-MS/MS

techniques. Human liver microsomes were chosen as a model enzymatic systems to provide an

approximation of phase I metabolism. LC-MS analyses were performed at different time points.

Poroshell EC120 C18 column (3 mm x 100 mm, 2.7 μm) was used to obtain high selectivity

and sufficient resolution for all of studied compounds. Differences in metabolic stability

between particular benzenesulfonamides and arylpiperazines derivatives were discussed in

view of their chemical structures.

With the support of advanced statistical and chemometrics analysis it was possible to

provide quantitative relationships between structure and metabolic stability. Referring to the

methodology of QSAR (quantitative structure-activity relationships), novel procedure is

proposed, namely QSMSR (quantitative structure-metabolic-stability relationships).

References:

[1] M. Belka, W. Hewelt-Belka, J. Sławiński, T. Bączek, Mass spectrometry based

identification of geometric isomers during metabolic stability study of a new cytotoxic

sulfonamide derivatives supported by quantitative structure-retention relationships, PLOS

ONE, 9 (2014) 1-14.

[2] A. Chodkowski, M.Z. Wróbel, J. Turło, J. Kleps, A. Siwek, G. Nowak, M. Belka, T.

Bączek, A.P. Mazurek, F. Herold, Novel 4-aryl-pyrido[1,2-c ]pyrimidines with dual SSRI and

5-HT1A activity. Part 4, Eur. J. Med. Chem., 90 (2015) 21-32.

60

OP-14 THE EFFECTS OF NEWLY SYNTHESIZED INDOLE-3-AMINE

DERIVATIVES ON MMP-2 ACTIVITY IN BREAST CANCER CELLS

Filiz Bakar; M.Gökhan Çağlayan2, Zuhal Kilic-Kurt3, Murat Palabıyık2, Süreyya Ölgen4

1Ankara University Faculty of Pharmacy, Department of Biochemistry; fbakar@ankara.edu.tr

2Ankara University Faculty of Pharmacy, Department of Analytical Chemistry

3Ankara University Faculty of Pharmacy, Department of Pharmaceutical Chemistry

4Kemerburgaz University Faculty of Pharmacy, Department of Pharmaceutical Chemistry

Matrix metalloproteinases (MMPs), a group of extracellular matrix remodeling

proteinases, are the principle mediators for the alterations of microenvironment during cancer

development [1]. The MMP protein family has been considered as biomarkers in many types

and stages of cancer [2]. MMP-2, a 36 kDa member of the family has been overexpressed in

breast cancer and plays important role on extracellular matrix degradation [3]. On the basis of

some indole derivatives have MMP inhibitory effects [4], we aimed to synthesize new indole

derivatives and evaluate their effects on MMP-2 enzyme activity and expression in human

breast adenocarcinoma cells.

Human MCF-7 breast carcinoma cells were cultured in DMEM medium and treated

with the synthesized 1,5-substitued-indole-3-amine derivatives at a concentration range of

compounds between 1mM to 0.1 μM. Cell growth, proliferation and apoptotic assays were

performed within the study. The enzymatic activity and expression of MMP-2 protein were also

evaluated.

Compounds [1-3] inhibited cell growth more than 50% even at 0.1uM concentration.

Although the BrdU-incorporation decreased at all treated cells, compound [4] was the most

potent anti-proliferative agent that the proliferating cell amount was measured as 23.66% when

compared with control. Compound [4] also inhibited MMP-2 activity and gene expression

significantly, when compared to control and other indole derivatives.

In conclusion, the synthesized indole derivatives has potent inhibitory effects on MMP-

2 activity. The further studies are needed to elucidate the potential effects of these compounds.

Acknowledgments: The study was supported by Scientific and Technological Research

Council of Turkey Grant, Tubitak-SBAG-113S254.

References:

[1] K. Kessenbrock, V. Plaks, Z. Werb. Matrix metalloproteinases: regulators of the tumor

microenviroment. Cell 141 (2010) 52-67.

[2] R. Roy, J. Yang, A.M. Moses, Matrix Metalloproteinases as novel biomarkers and

potential therapeutic targets in human cancer. J. Clin. Oncol. 27 (2009) 5287-5297.

[3] M. Egeblad, Z. Werb. New functions for the matrix metalloproteinases in cancer

progression. Nat. Rev. Cancer 2 (2002) 161-174.

[4] C. Gialeli, A.D. Theocharis, N.K. Karamanos. Roles of matrix metalloproteinases in

cancer progression and their pharmacological targeting. FEBS J. 278 (2011) 16-27.

61

OP-15 UHPLC-MS/MS INVESTIGATION OF METABOLISM AND

DISPOSITION OF NOVEL THIOSEMICARBAZONE ANTI-CANCER

DRUGS

Petra Kovarikova1, Vit Sestak1, Jan Stariat1, Eliska Potuckova1, Stanislav Micuda2, Jaroslav

Chladek2, Des R. Richardson3

1Department of Pharmaceutical Chemistry and Drug Analysis and Department of Biochemistry,

Faculty of Pharmacy, Charles University, Heyrovskeho 1203, 50005 Hradec Kralove, Czech

Republic; petra.kovarikova@faf.cuni.cz

2Department of Pharmacology, Faculty of Medicine in Hradec Kralove, Charles University,

Simkova 870, 50038 Hradec Kralove, Czech Republic

3Department of Pathology and Bosch Institute, University of Sydney, NSW 2031, Sydney,

Australia

Thiosemicarbazones derived from di(2-pyridyl)ketone are novel anti-cancer drugs.

Comprehensive chemical and pharmacological research on these new compounds has

demonstrated their unique efficacy against variety of cancer. Di-2-pyridylketone-4,4-dimethyl-

3-thiosemicarbazone (Dp44mT) and di-2-pyridylketone-4-cyclohexyl-4-methyl-3-

thiosemicarbazone (DpC) represent lead compounds of two generations of the novel

thiosemicarbazones that are structurally close but differ in their toxicity. However, difficulties

with bioanalysis have so far hampered thorough investigation of their metabolism and

disposition in vivo and thus hindered their advanced preclinical development. In addition, it is

not clear whether pharmacokinetics (PK) might have a connection to the toxicity of the

compounds.

This communication summarizes results of our work aimed at UHPLC-MS/MS analysis

of Dp44mT and DpC in biological materials and its application to investigation of disposition

of these former and new lead compounds in vivo in rats. Besides comparison of PK of both

compounds, the propensity towards in vivo metabolism was studied and pharmacodynamic and

toxicodynamic effects of the detected as well as predicted metabolites were tested in vitro.

UHPLC coupled with LCMS-8030 triple-quadrupole and Acquity® BEH C18 column

were used for analysis of biological samples. Dp44mT and DpC were administered to rats

(2 mg/kg, i.v.) and the concentration-time profiles of both compounds and metabolites in plasma

were determined. PK parameters were estimated by both non-compartmental and two-

compartmental analyses with population modeling. In addition cytotoxicity and intracellular

iron binding and mobilization abilities of the parent compounds and metabolites were

characterized in vitro. This investigation showed remarkable differences in metabolism and

disposition between the two structurally related thiosemicarbazones and highlighted favorable

pharmacokinetics in the new lead compound – DpC. These findings may foster advanced

preclinical development of DpC.

Acknowledgments: This study was supported by Grant Agency of Charles University for the

financial support (GAUK project no. 903113 and the Project SVV 260 183).

62

OP-16 LC-MS/MS METHOD DEVELOPMENT FOR PHARMACOKINETIC

STUDY OF DEXMEDETOMIDINE IN PEDIATRIC PATIENTS

Danuta Siluk1; Oliwia Szerkus1; Wiktoria Struck-Lewicka1; Ewa Bartosińska1, Marta

Kordalewska1, Renata Bujak1, Agnieszka Borsuk1, Agnieszka Bienert2, Alicja Bartkowska-

Śniatkowska3, Justyna Warzybok2, Lidia Andrzejczuk1;Paweł Wiczling1, Michał J. Markuszewski1,

Antoni Nasal1, Roman Kaliszan1

1Department of Biopharmaceutics and Pharmacodynamics, Medical University of Gdańsk,

Gdańsk, Poland; dsiluk@gumed.edu.pl

2Department of Clinical Pharmacy and Biopharmacy, Poznań University of Medical Sciences,

Poznań, Poland

3Department of Pediatric Anesthesiology and Intensive Therapy, Poznań University of Medical

Sciences, Poland

Dexmedetomidine is a highly selective α2-adrenergic agonist with analgesic and hypnotic

actions when used intravenously. It is mainly used for short-term sedation in intensive care unit

settings. The main objective of the present study was an HPLC-QqQ/MS method development

for quantitative dexmedetomidine determination in plasma obtained from pediatric patients

admitted to the Pediatric Anesthesiology and Intensive Therapy hospital ward in order to study

drug pharmacokinetics in 24 h drug infusion. A secondary objective of the project was a study

of dexmedetomidine metabolism in pediatric patients using HPLC-ESI-TOF/MS technique.

Sample pretreatment method (0.5 ml plasma) was carried out with the use of solid phase

extraction technique. For that purpose polymeric SPE cartridges were applied (Bond Elut Plexa

30mg/1ml, Agilent Technologies Inc., USA). Chromatographic conditions were as follows:

Zorbax Eclipse Plus C18 (4.6×100mm; 3.5 µm), mobile phase composed of 0.1% formic acid

in water and 0.1% formic acid in methanol (20:80; v/v) in an isocratic mode at a flow rate 0.5

ml/min. Detection was performed with the use of ESI as an ion source and triple quadrupole in

MRM mode as selected mass analyzer. Ion source parameters were optimized with Design of

Experiments approach (DoE) with Central Composite Design (CCD). Determination of

dexmedetomidine was performed with detomidine as internal standard. The linearity of the

method ranged from 5 to 2500 pg/ml. The chromatographic run lasted 4 min. Optimized and

validated method was successfully applied to determination of dexmedetomidine in ca. 600

pediatric plasma samples. Basing on analytical data population pharmacokinetic study was

performed. In order to analyze dexmedetomidine metabolites in plasma extracts, RP-HPLC

coupled with time-of –flight mass spectrometer technique was employed. Putative metabolites

were determined with the use of MetaboliteID computer program (Agilent Technologies Inc.,

USA). In pediatric plasma samples hydroxylated, N-methylated, carboxylated metabolites and

glucuronide conjugates were identified with the highest intensities which depended on time of

infusion as well as on patients’ age.

Acknowledgments: The project was supported by the Ministry of Science and Higher

Education of the Republic of Poland, from the quality-promoting subsidy, under the Leading

National Research Centre (KNOW) program for the years 2012-2017.

63

OP-17 METABOLOMICS & FOOD CRAVINGS QUESTIONNAIRE FOR

UNDERSTANDING OUTCOMES AFTER A BARIATRIC SURGERY

Antonia García1, Annalaura Mastrangelo1, José Ramón Muñoz-Rodríguez2, Pilar Ramos1, Luis

Beato2, Jesús Martín2, Esperanza Segura2, Alberto León2, Carmen González-Martín1,2, Luis F.

Alguacil1,2 and Coral Barbas1

1CEMBIO (Center for Metabolomics and Bioanalysis), Facultad de Farmacia, Campus

Montepríncipe Universidad San Pablo CEU Madrid (Spain); antogar@ceu.es

2 Hospital General Universitario de Ciudad Real (Spain)

Bariatric surgery (BS) is the most effective and durable treatment for obesity and its

associated comorbidities. Although the underlying mechanisms of its remarkable efficacy are

still unclear, it has been highlighted that the anatomical restriction imposed by the surgery might

not be the unique effector, but rather a contributing factor in conjunction with metabolic

alterations taken place in the body at different levels (i.e. food absorption, microbiota and brain

activity). Metabolomics offers the ability to examine global changes in metabolites associated

with physiological conditions. In the present study we have applied a multiplatform

metabolomics approach to gain a better understanding of the extent to which other factors are

associated with bariatric surgery efficacy in order to identify potential outcome predictors,

thereby uncovering new targets for less-invasive treatments.

Metabolomics analysis was performed in serum samples from healthy volunteers (group

1 n=23, BMI<25 kg/m2), obese patients before surgery (group 2 n=23, BMI>40 kg/m2) and

one year after surgery (group 3 n=23, average BMI=31 kg/m2) by UHPLC-ESI-QTOF-MS,

GC-EI-MS-Q and CE-ESI-TOF-MS (Agilent, USA). In addition, food craving, following the

Food Cravings Questionnaire trait scale (FCQ-T), was evaluated for groups 1 and 2. To estimate

the strength of the linear dependence between the observed metabolites and food-craving, linear

correlation was measured by the Pearson correlation coefficient. Finally, group 2 was divided

in two subgroups according to high (score ≥ 15, n = 13) and low food craving (score < 15, n =

10) to predict a possible discrimination among subjects after surgery.

The multiplatform approach including complementary high resolution techniques,

allowed the detection of 190,330 potential compounds. Once mined the valuable information

from the data matrix, chemometric tools (correlation, univariate and multivariate statistical data

analyses) were applied to investigate the specific metabotype of the groups. Among these

metabolites, bile acids and gut microbiota by-products (secondary bile acids, p-cresol, benzoate,

indolelactate) presented the most remarkable changes, reflecting the great influence of the

microbial community on the surgery outcome. Moreover, we observed that after surgery, the

lipid profile (fatty acids, monoacylglycerides and phospholipids) was modified with a recovery

toward normal condition, supporting BS efficacy in promoting metabolic improvements.

Regarding the recovery evaluation model, we noted some differences in the profiles pointing

out the necessity to explore other factors that might influence the outcome of the bariatric

procedure. Therefore, we have correlated the psychological factor, food-craving, with the

metabolic profile of normal weight and obese subjects. Among all metabolites that were highly

correlated between these parameters, we have selected those specific of each group, as they

might reflect a different brain behavior associated to a specific metabotype. Finally, we have

investigated the food-craving predictive capability for the discrimination of the individuals after

surgery. Metabolomics points out microbiota metabolites and food-craving evaluation as

potential contributor factors to predict the surgical outcome of BS.

Acknowledgments: This work was supported by Spanish Ministerio de Economía y

Competitividad (MINECO, previously MICINN) grant CTQ2014-55279-R.

64

OP-18 MASS SPECTROMETRY BASED LIPIDOMICS IN THE SEARCH

FOR LIPID CANCER BIOMARKERS

Michal Holčapek, Miroslav Lísa, Eva Cífková and Robert Jirásko

University of Pardubice, Faculty of Chemical Technology, Department of Analytical Chemistry,

Studentská 573, 53210 Pardubice, Czech Republic; Michal.Holcapek@upce.cz

Lipidomics deals with the complex lipidomic characterization of dynamic biological

systems. The development of new quantitative methods is important for a better coverage of

lipids applicable for high-throughput clinical analysis. Shotgun ESI-MS/MS quantitation is

based on the direct infusion of total lipid extracts containing internal standards per each

quantified lipid class. The ion suppression effects could reduce the sensitivity for the

determination of trace lipids. LC/MS is useful for the chromatographic separation of isobaric

lipids and their quantitation [1,2]. MALDI-MS is typically used without the chromatographic

separation, but it is less convenient for the quantitation due to the lower signal reproducibility.

Three basic mass spectrometric platforms (shotgun MS, separation - MS and MALDI-MS) are

used for the development of quantitative lipidomic assays. The shotgun approach is based on

well-known characteristic neutral loss and precursor ion scans characteristic for individual lipid

classes together with internal standards used for each lipid class. Two separation - MS

approaches (HILIC-UHPLC and UHPSFC) are compared for the lipid class separation followed

by their ESI-MS detection and quantitation using internal standards per each lipid class. HILIC

is applicable only for several polar lipid classes with the analysis time of 20 min, while UHSFC

enables the quantitation of up to 30 lipid classes within only 6 min [3]. The great potential of

UHPSFC/ESI-MS for high-throughput lipidomic quantitation will be demonstrated on clinical

analyses of tumor vs. normal tissues, patients body fluids and cancer cell lines. The large data

sets of lipid species are statistically evaluated by multivariate data analysis. Lipids with the

highest impact on the differentiation of tumor and normal tissues are identified [4,5].

Acknowledgments: This work was supported by ERC CZ LL1302 (MSMT, Czech Republic).

References:

[1] E. Cífková, M. Holčapek, M. Lísa, M. Ovčačíková, A. Lyčka, F. Lynen, P. Sandra. Non-

targeted quantitation of lipid classes using hydrophilic interaction liquid chromatography -

electrospray ionization mass spectrometry using single internal standard and response factor

approach. Anal. Chem. 84 (2012) 10064-10070.

[2] M. Lísa, E. Cífková, M. Holčapek. Lipidomic profiling of biological tissues using off-

line two-dimensional high-performance liquid chromatography - mass spectrometry. J.

Chromatogr. A 1218 (2011) 5146-5156.

[3] M. Lísa, M. Holčapek. New High-Throughput and Comprehensive Approach for

Lipidomic Analysis Using Ultrahigh-Performance Supercritical Fluid

Chromatography/Electrospray Ionization - Mass Spectrometry, Anal. Chem. (2015) submitted.

[4] E. Cífková, M. Holčapek, M. Lísa, D. Vrána, J. Gatěk, B. Melichar. Determination of

lipidomic differences between human breast cancer and surrounding normal tissues using

HILIC-HPLC/ESI-MS and multivariate data analysis. Anal. Bioanal. Chem. 407 (2015) 991-

1002.

[5] E. Cífková, M. Holčapek, M. Lísa, D. Vrána, B. Melichar, V. Študent. Lipidomic

differentiation of human kidney tumor and surrounding normal tissues using HILIC-

HPLC/ESI-MS and multivariate data analysis. J. Chromatogr. B (2015) revision submitted.

65

OP-19 NMR-BASED METABOLOMICS ANALYSIS TO EXPLORE THE

TISSUE NONSPECIFIC ALKALINE PHOSPHATASE (TNAP) ROLE IN

THE BRAIN

Vronique Gilard1, Thomas Cruz1, Myriam Malet-Martino1, Marie Gleizes2, Lionel Nowak2,

Stéphane Balayssac1, Jose Luis Millan3, Caroline Fonta2

1Groupe de RMN Biomdicale, Laboratoire SPCMIB UMR CNRS 5068, Universit de

Toulouse, 118 route de Narbonne, 31062 Toulouse cedex 9, France; gilard@chimie.ups-tlse.fr

2 Centre de Recherche Cerveau et Cognition, Université de Toulouse UPS, CNRS-UMR 5549,

Toulouse, France

3 Medical Research Institute, Sandford-Burnham, La Jolla, USA

The functions of TNAP have been extensively explored in the skeletal tissue where this

enzyme is involved in ossification, as shown by abnormal bone mineralization in patients with

hypophosphatasia. Although the severe forms of hypophosphatasia are associated with

epilepsy, the functions of TNAP in the nervous tissue are poorly documented. Previous studies

suggested that TNAP plays a role in neurotransmission (adenosine synthesis through TNAP

ectonucleotidase activity, and GABA synthesis through the regulation of pyridoxal phosphate

(PLP) availability, a natural TNAP substrate and a glutamate decarboxylase cofactor) [1,2,3].

To tackle this hypothesis we used a NMR metabolomics approach for the characterization of

brain metabolites variations associated with TNAP deficiency.

Metabolome was investigated using a global approach through 1H NMR profiling of