2015-03a single reversed-phase uplc method for quantification of levofloxacin in aqueous humour and...

7/23/2019 2015-03A Single Reversed-Phase UPLC Method for Quantification of Levofloxacin in Aqueous Humour and Pharma

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March 2015 Volume 28 Number 3

www.chromatographyonline.com

Advances inElectrochemistry

A review of electrochemistry coupled to

LCMS in omics applications

THE ESSENTIALS

Selecting the right HPLC

stationary phase

LC TROUBLESHOOTING

Identifying the causes of

method failure

GC CONNECTIONS

The fundamentals of GC ovens

Innovative Technology ForTrace Organic Analysis

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2015PerkinElme

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4/68-$r($ &VSPQF March 201532

&EJUPSJBM 1PMJDZ

All articles submitted to -$t($ &VSPQF

are subject to a peer-review process in association

with the magazines Editorial Advisory Board.

$PWFS

Original materials: Hong Li/Getty Images

'FBUVSFT $IJSBM -JRVJE $ISPNBUPHSBQIZ $PVQMFE XJUI 5BOEFN .BTT

4QFDUSPNFUSZ GPS &OWJSPONFOUBM "OBMZTJT PG

1IBSNBDPMPHJDBMMZ "DUJWF $PNQPVOET

Bruce Petrie, Dolores Camacho-Muoz, Erika Castrignan, Sian

Evans, and Barbara Kasprzyk-Hordern

This article gives an up-to-date commentary on chiral liquid

chromatography coupled with mass spectrometry for the

determination of pharmacologically active chiral compounds

(cPACs) (including illicit drugs) in environmental matrices.

Several applications are discussed to demonstrate the benefits of

performing environmental analysis of cPACs at the enantiomeric

level. Finally, future perspectives in this rapidly developing field of

research are outlined.

$PMVNOT -$ 5306#-&4)005*/(

-JTUFO UP UIF %BUB

John W. Dolan

A stepwise process is described to help isolate and identify the

cause of a method failure.

($ $0//&$5*0/4

(BT $ISPNBUPHSBQIZ 0WFOT

John V. Hinshaw

This instalment examines ovens for GC in several forms plus how

oven thermals affect peak retention behaviour.

5)& &44&/5*"-4

$IPPTJOH UIF 3JHIU )1-$ 4UBUJPOBSZ 1IBTF

A guide to selecting the correct HPLC stationary phase.

%FQBSUNFOUT 1SPEVDUT

&WFOUT

5IF "QQMJDBUJPOT #PPL

$07&3 4503: i0NJDTu "QQMJDBUJPOT PG

&MFDUSPDIFNJTUSZ $PVQMFE UP.BTT 4QFDUSPNFUSZ " 3FWJFXHerbert Oberacher, Florian Pitterl,and Jean-Pierre ChervetRedox reactions are integral partsof many cellular processes. Theyare therefore extensively studiedJOWJUSP andJO WJWP. Electrochemistry(EC) represents a purely instrumentalapproach to characterize direct andindirect effects of redox reactions onbioorganic molecules. This reviewhighlights important trends and recentdevelopments.

.BSDI | 2015

7PMVNF /VNCFS


5/68

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6/68-$r($ &VSPQF March 201534

5IF 1VCMJTIFST PG -$t($ &VSPQF XPVME MJLF UP UIBOL UIF NFNCFST PG UIF &EJUPSJBM "EWJTPSZ #PBSE GPS

their continuing support and expert advice. The high standards and editorial quality associated with-$t($ &VSPQF BSF NBJOUBJOFE MBSHFMZ UISPVHI UIF UJSFMFTT FGGPSUT PG UIFTF JOEJWJEVBMT

LCGC Europe provides troubleshooting information and application solutions on all aspects of

separation science so that laboratory-based analytical chemists can enhance their practicalknowledge to gain competitive advantage. Our scientific quality and commercial objectivity provide

readers with the tools necessary to deal with real-world analysis issues, thereby increasing theirefficiency, productivity and value to their employer.

Editorial Advisory Board

,FWJO "MUSJBGlaxoSmithKline, Harlow, Essex, UK

%BOJFM 8 "SNTUSPOHUniversity of Texas, Arlington, Texas, USA

.JDIBFM 1 #BMPHIWaters Corp., Milford, Massachusetts, USA

#SJBO " #JEMJOHNFZFSAgilent Technologies, Wilmington,Delaware, USA

(OUIFS , #POOInstitute of Analytical Chemistry andRadiochemistry, University of Innsbruck,

Austria

1FUFS $BSSDepartment of Chemistry, University

of Minnesota, Minneapolis, Minnesota, USA

+FBO1JFSSF $IFSWFUAntec Leyden, Zoeterwoude, TheNetherlands

+BO ) $ISJTUFOTFODepartment of Plant and EnvironmentalSciences, University of Copenhagen,

Copenhagen, Denmark

%BOJMP $PSSBEJOJIstituto di Cromatografia del CNR, Rome,

Italy)FSOBO + $PSUFTH.J. Cortes Consulting,

Midland, Michigan, USA

(FSU %FTNFUTransport Modelling and Analytical

Separation Science, Vrije Universiteit,

Brussels, Belgium

+PIO 8 %PMBOLC Resources, Walnut Creek, California,

USA

3PZ &LTUFFOSigma-Aldrich/Supelco, Bellefonte,

Pennsylvania, USA

"OUIPOZ ' 'FMMPharmaceutical Chemistry,

University of Bradford, Bradford, UK

"UUJMB 'FMJOHFSProfessor of Chemistry, Department of

Analytical and Environmental Chemistry,

University of Pcs, Pcs, Hungary

'SBODFTDP (BTQBSSJOJDipartimento di Studi di Chimica e

Tecnologia delle Sostanze Biologica-mente Attive, Universit La Sapienza,

Rome, Italy

+PTFQI - (MBKDIMomenta Pharmaceuticals, Cambridge,

Massachusetts, USA

+VO )BHJOBLBSchool of Pharmacy and PharmaceuticalSciences, Mukogawa Womens

University, Nishinomiya, Japan

+BWJFS )FSOOEF[#PSHFTDepartment of Analytical Chemistry,Nutrition and Food Science University of

Laguna, Canary Islands, Spain

+PIO 7 )JOTIBXServeron Corp., Hillsboro, Oregon, USA

5VVMJB )ZUZMJOFOVVT Technical Research of Finland,

Finland

)BOT(FSE +BOTTFOVant Hoff Institute for the Molecular

Sciences, Amsterdam, The Netherlands

,JZPLBUTV +JOOPSchool of Materials Sciences, ToyohasiUniversity of Technology, Japan

)VCB ,BMT[Semmelweis University of Medicine,Budapest, Hungary

)JBO ,FF -FFNational University of Singapore,

Singapore

8PMGHBOH -JOEOFSInstitute of Analytical Chemistry,

University of Vienna, Austria

)FOL -JOHFNBOFaculteit der Scheikunde, Free University,

Amsterdam, The Netherlands

5PN -ZODIBP Technology Centre, Pangbourne, UK

3POBME & .BKPSTAgilent Technologies,

Wilmington, Delaware, USA

1IJMMJQ .BSSJPUMonash University, School of Chemistry,

Victoria, Australia

%BWJE .D$BMMFZDepartment of Applied Sciences,

University of West of England, Bristol, UK3PCFSU % .D%PXBMMMcDowall Consulting, Bromley, Kent, UK

.BSZ &MMFO .D/BMMZDuPont Crop Protection,Newark,Delaware, USA

*NSF .PMOSMolnar Research Institute, Berlin, Germany

-VJHJ .POEFMMPDipartimento Farmaco-chimico, Facoltdi Farmacia, Universit di Messina,

Messina, Italy

1FUFS .ZFSTDepartment of Chemistry,

University of Liverpool, Liverpool, UK

+BOVT[ 1BXMJT[ZODepartment of Chemistry, University of

Waterloo, Ontario, Canada

$PMJO 1PPMFWayne State University, Detroit,

Michigan, USA

'SFE & 3FHOJFSDepartment of Biochemistry, Purdue

University, West Lafayette, Indiana, USA

)BSBME 3JUDIJFTrajan Scientific and Medical. Milton

Keynes, UK

1BU 4BOESBResearch Institute for Chromatography,Kortrijk, Belgium

1FUFS 4DIPFONBLFSTDepartment of Chemical Engineering,Universiteit van Amsterdam, Amsterdam,

The Netherlands

3PCFSU 4IFMMJFAustralian Centre for Research onSeparation Science (ACROSS), University

of Tasmania, Hobart, Australia:WBO 7BOEFS )FZEFOVrije Universiteit Brussel,

Brussels, Belgium

Published by

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The Column spoke to Rosa

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Scientists have published a novel

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of nonvolatile methyl-branched

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)1-$ *OTUSVNFOUTWhat does risk assessment in the contextof the life cycle of a high performanceliquid chromatography (HPLC) instrumentreally mean? This instalment of Questionsof Quality will look at problems with anoperational liquid chromatograph to see ifthey can be picked up in the performancequalification (PQ) or prevented in the operational

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Hydrophilic interaction liquid

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Instrument manufacturers try to convince us that mass

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3FBE )FSF HPPHMMM$X

LCGC57-VJHJ .POEFMMP PO 'PPE

"OBMZTJT 6TJOH % ($Comprehensive GC is a

multi-dimensional technique that

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Redox reactions are essential for life. There are two important

groups of biologically important redox reactions: enzymatic

and non-enzymatic reactions. Enzymatic redox reactions

often involve complex mechanisms of several enzymes.

The electrons are transported by flavin- or heme-containing

coenzymes from one reaction to another. These reactions

represent an integral part of many metabolic pathways.

Biological energy, for instance, is stored and released

by means of redox reactions. Cellular respiration is the

oxidation of glucose to carbon dioxide and the reduction of

oxygen to water; photosynthesis involves the reduction of

carbon dioxide into sugars and the oxidation of water into

molecular oxygen. Furthermore, oxidoreductases such as

the members of the cytochrome P450 superfamily generate

structural complexity during natural product biosynthesis,

and they play a central role in the biotransformation of

drugs and toxins (phase I metabolisms). Biotransformation

reactions involving xenobiotics are usually intended to

make them more polar and, thus, enable a more rapid

excretion. Biotranformation can lead to loss or gain of activity.

Furthermore, in some cases reactive intermediates are

produced that can bind to proteins, lipids, and nucleic acidsgiving rise to cellular damage.

Biologically important redox reactions can also involve

nonenzymatic processes. Reactive oxygen species (ROS)

are natural by-products of the normal metabolism of oxygen.

However, during times of environmental stress (for example,

UV or heat exposure), ROS levels can increase dramatically.

The availability of ROS can be of importance for an organism,

because they are used by the immune system as a way

to attack and kill pathogens. Furthermore, some ROS

function as physiological regulators of intracellular signalling

pathways. In the majority of cases, however, ROS production

is considered to be harmful. ROS can react with proteins,

lipids, and nucleic acids, which usually gives rise to celldamage called oxidative stress. In humans oxidative stress

is thought to be involved in the pathogenesis of dif ferent

kinds of disease, including neurodegenerative disease,

cardiovascular disease, and cancer.

Because of their importance, biological redox reactions

are extensively studied in in vitroand in vivomodels.

Electrochemistry (EC) was found to be a powerful approach

to complement existing assays in characterizing direct and

indirect effects of redox reactions on bioorganic molecules.

EC was particularly useful in generating oxidation products

as well as reactive intermediates, which can be trapped by

different kinds of electrophiles and nucleophiles. Moreover,

EC is a purely instrumental approach. Experimental

conditions, including the electrochemical potential, the

electrode material, pH, as well as the kind and concentration

of reactants, can be precisely controlled. In addition, the use

of EC means costly and often non-specific enzymes and the

use of harsh chemicals can be made obsolete (117). Thus,

EC can be regarded as a greenor sustainable chemistry

approach.

Redox reactions may give rise to the formation of complex

mixtures of intermediates, products, and by-products.

Different kinds of analytical techniques can be used for

comprehensive analysis. Mass spectrometry (MS) inparticular has found widespread application for that purpose.

Mass spectrometric techniques enable qualitative (that is,

3FEPY SFBDUJPOT BSF JOUFHSBM QBSUT PG NBOZ DFMMVMBS QSPDFTTFT 5IFZ BSF UIFSFGPSF FYUFOTJWFMZ TUVEJFEinvitroBOE in vivo &MFDUSPDIFNJTUSZ &$ SFQSFTFOUT B QVSFMZ JOTUSVNFOUBM BQQSPBDI UP DIBSBDUFSJ[F EJSFDU BOEJOEJSFDU FGGFDUT PG SFEPY SFBDUJPOT PO CJPPSHBOJD NPMFDVMFT TVDI BT QFQUJEFT QSPUFJOT BOE FOEPHFOPVTBOE FYPHFOPVT TNBMM NPMFDVMFT BT XFMM BT OVDMFJD BDJET *O BEEJUJPO UP EJSFDU JOGVTJPO FMFDUSPTQSBZJPOJ[BUJPO &4*mNBTT TQFDUSPNFUSZ .4 IZQIFOBUFE UFDIOJRVFT TVDI BT MJRVJE DISPNBUPHSBQIZmNBTT

TQFDUSPNFUSZ -$m.4 BSF PGUFO BQQMJFE GPS DPNQSFIFOTJWF DIBSBDUFSJ[BUJPO PG SFBDUJPO NJYUVSFT HFOFSBUFECZ &$ FYQFSJNFOUT &$m-$m.4 SFQSFTFOUT B GBTU BVUPNBUBCMF BOE iHSFFOu BQQSPBDI UIBU DBO CF VTFE JO BWBSJFUZ PG iPNJDTu EJTDJQMJOFT 5IJT SFWJFX IJHIMJHIUT JNQPSUBOU USFOET BOE SFDFOU EFWFMPQNFOUT

,&: 10*/54t Redox reactions are extensively studied in omics

because of their importance in many cellular processes.

t Electrochemistry (in electro) represents a powerful

alternative to in vivoand in vitroassays to study redox

reactions.

t MS in combination with LC enables comprehensive

characterization of the reaction mixtures generated by ECexperiments.

)FSCFSU 0CFSBDIFS1 'MPSJBO 1JUUFSM1 BOE +FBO1JFSSF $IFSWFU2 1Institute of Legal Medicine and Core Facility

Metabolomics, Medical University of Innsbruck, Innsbruck, Austria, 2Antec, Zoeterwoude, The Netherlands.

Omics Applications of

Electrochemistry Coupled to MassSpectrometry " 3FWJFX

PhotoCred

it:HongLi/GettyImages


11/68

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0CFSBDIFS et al

identification and structure elucidation) and quantitative

analysis. Among the different ionization techniques available,

electrospray ionization (ESI) is the most commonly applied

technique. ESI allows the MS characterization of a large

variety of compounds ranging from small molecules to largebiopolymers. The importance of ECESIMS techniques is

emphasized by the large number of reviews that have been

published in recent years (117). Particularly in life sciences,

ECESIMS has found widespread application. This

review will give a short overview on recent advances in that

important field of research (Figure 1).

&YQFSJNFOUBM 4FUVQDifferent experimental setups have been developed that

enable ECESIMS experiments. In the simplest setup, the

inherent EC of ESI is used (14). From the electrochemical

point of view (1820), the ESI source represents a

controlled-current cell consisting of two electrodes. One

electrode is the capillary emitter; the mass spectrometer acts

as the counter electrode. The two electrodes are connected

on the one hand by the power supply and on the other by a

series of resistors consisting of the electrochemical contact

to the solution, the solution resistance, the resistance at the

solution-air interface and in the gas-phase, and the charge

neutralization at the counter electrode. During operation,

charges are transported from the emitter via the gas phase

to the mass spectrometer. The loss of charges needs

to be balanced in solution, and this is accomplished by

electrochemical processes at the emitter electrode. These

redox reactions may involve analytes (2123), the solvent(24,25), or the electrode material (18,26). Practitioners have

learned to control the electrochemical part of ESI in a way

that the impact of EC on the mass spectra observed can

be very much tuned. In the majority of cases, experimental

conditions are chosen that prevent the mass spectrometric

detection of ESI-inherent electrochemical reactions.

However, in some situations EC can be used as an analytical

advantage. A clear limitation of this setup is its inability to

precisely control the electrochemical potential at the emitter

electrode. Thus, experimental setups allowing separation of

the electrochemical processes studied from the ESI-inherent

EC are more commonly used.

Discrete electrochemical cells can be on-line hyphenatedto ESIMS (Figure 2[a]). The electrochemical cells used

are typically controlled-potential cells consisting of three

electrodes (that is, working electrode, reference electrode,

and auxiliary electrode). The cells contain either porous

flow-through or planar, thin-layer, flow-by working electrodes.

Cells with porous electrodes are considered to provide good

conversion rates even at high flow rates because of the large

surface area provided. Typically, glassy carbon is used as

electrode material. A clear disadvantage of glassy carbon

is the occurrence of analyte adsorption on the electrode

surface. Thus, the cells are usually operated with solventscontaining high organic contents. Other problems of this cell

type are limited robustness and reproducibility. Life history

or age of the electrochemical cell can sometimes have an

impact on the oxidation reactions observed (27). For the

planar thin-layer cells, adsorption on the working electrode

is usually a less common problem (28). Another advantage

of this cell type is the possibility to use different kinds of

electrode materials, such as glassy carbon, boron-doped

diamond, platinum, and titanium. For obtaining high

conversion rates, thin-layer cells are usually operated at very

low flow rates (


13/68141XXXDISPNBUPHSBQIZPOMJOFDPN

0CFSBDIFS et al

chromatography is the preferred chromatographic mode

of operation. The electrochemical cell can be integrated at

different positions into the LCMS system.

Post-column EC (Figure 2[b]) can be used to study the

redox chemistry of selected compounds within complex

mixtures (43,44). Pre-column EC (Figure 2[c]) enables the

comprehensive characterization of redox reaction products

via LC fractionation and subsequent MS detection (45,46).

As the commonly applied chromatographic systems areoperated at flow rates of several hundred microlitres per

minute, electrochemical cells with porous flow-through

working electrodes with very high conversion efficiencies are

exclusively used for both types of experimental setups. To

enable the use of thin-layer cells, the EC cell can be integrated

into the injection device (29,47). In such a setup, the sample

solution is delivered through the electrochemical cell into the

injection loop and subsequently transferred into the LC flow

path.

Despite considerable success of the currently available

EC instrumentation in converting bioorganic molecules,

there is still a need for improved experimental conditions. A

lot of research is focused on increasing predictability, yield,and reproducibility of electrochemical reactions. The yield

can be improved by optimization of reaction parameters

including solvent composition, pH, and electrode material (29).

Another strategy to increase the rate of conversion is based

on the application of square-wave potential pulses (48,49).

The superior performance with pulsing was attributed to an

increased desorption of reaction products and continuous

particularly useful to reduce the complexity of the sample

submitted either to the EC cell or to ESIMS. Because

of its compatibility with EC and ESI, reversed-phase

'JHVSF Commonly used setups for combining EC with LC and

ESIMS: (a) ECMS, (b) LCECMS, and (c) ECLCMS.

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14/68-$r($ &VSPQF March 2015

0CFSBDIFS et al

renewal of the electrode surface. The

potential pulses were also found to

give rise to the formation of products

that were not seen in direct EC alone.

Other approaches developed to

expand the reactivity of EC include

enzyme-modified systems (50) and

hydroxyl radical-producing systems

(5154).

.FUBCPMPNJDT -JQJEPNJDT BOE

3FMBUFE "QQMJDBUJPOTBased on the number of published

articles, one of the most important

fields of application of ECESIMS lies

in studying the redox chemistry of small

bioorganic molecules. In this context,

ECESIMS techniques are particularly

useful to mimic biotransformation

reactions.

The first attempts at using ECMS

to characterize oxidation processes

of organic molecules date back to the

pioneering work of Hambitzer and

Heitbaum as well as Yost, Brajter-Toth,

and colleagues in the late 1980s(5559). However, Bruins and

colleagues were the first who

'JHVSF Putative products of electrochemical cholesterol oxidation identified by

LCMSMS. The carbon numbering at the cholesterol backbone is shown in blue. The

sites of oxidation are highlighted by red circles. Indicated are the free radical driven and

possible consecutive reactions leading to the formation of the identified compounds, as well

as enzymes (in brackets) involved in the generation of oxysterols in vivo. Courtesy of Maria

Fedorova and Dieter Weber, Institute of Bioanalytical Chemistry, Faculty of Chemistry and

Mineralogy, Universitt Leipzig, Leipzig, Germany.

5(a)

(b)

4

3

2

1

0

3

Light chain

Abs.

intensity(106)

Abs.

intensity(107)

Heavy chain

Intact antibody

Intact antibodyEC CELLOFF

EC CELLOFF

12+11+

10+

50+48+

49+47+

46+45+

44+

43+

42+

41+

40+

39+ 36+

35+

34+

32+

33+

31+

30+

9+

8+

7+6+

29+30+

31+32+33+

34+35+

36+

13+

14+

15+ 37+

2

1

01500 2000 2500 3000 3500 4000 4500 m/z

'JHVSF EC reduction of the disulphide bonds of a commercially available monoclonal

antibody (mAb) monitored by on-line ESIFTICRMS. Cleavage of the four inter-disulphide

bonds yields two light and two heavy chains. Courtesy of Simone Nicolardi and Yuri E.

M. van der Burgt, Leiden University Medical Center (LUMC), Center for Proteomics and

Metabolomics, Leiden, The Netherlands.

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0CFSBDIFS et al

The applicability of ECESIMS in drug metabolism

studies has been demonstrated for a number of important

pharmaceutical compounds, including amodiaquine (50,

6365), diclofenac (66,67), lidocaine (48,53,54),

acetaminophen (63), haloperidol (68), troglitazone (6971),

clozapine (63), trimethoprim (63), flunitrazepam (72),

clonazepam (72), chlorpromazine (72), zotepine (73,74),

acebutolol (75,76), alprenolol (75), albendazole (77),

tetrazepam (47), verapamil (78), and galantamin (79).Phase I metabolism of drug compounds often gives

rise to the formation of reactive species that are generally

short-lived and unstable. These species can react with

lipids, proteins, and nucleic acids giving rise to substantial

damage. Within living organisms reactive intermediates

are usually detoxified by binding to glutathione (phase

II reaction). Reactive species are also produced in EC

experiments. For the detection of these compounds, and to

mimic phase II metabolism, trapping agents are commonly

applied. Appropriate nucleophiles include glutathione (65,80),

glutathione derivatives (81), cysteine (82), and proteins (83).

These compounds can be added to the sample solution

after or before EC. The study of the skin-sensitizing potentialof chemicals is a very interesting application of the ability

of EC to produce electrophiles that subsequently react with

proteins (8486). Allergic reactions are most often caused by

haptenation of proteins. A presupposition for this reaction is

the ability of chemical allergens (small organic molecules and

their metabolites) to form covalent bonds to nucleophilic sites

in proteins (for example, cysteine and lysine). These activation

systematically studied the redox reactivity of pharmaceutical

compounds with ECESIMS (60,61). Their work laid the

foundation for the use of ECESIMS techniques to mimic

phase I oxidative reactions in drug metabolism studies.

Because valuable information concerning the sensitivity of a

substrate towards oxidation can be obtained from ECESI

MS experiments, the technique is regarded as an efficient

tool in the drug development process that complements

existing in vitroand in vivoscreening techniques. ECwas found to be particularly useful in cases where P450

enzyme catalyzed reactions are supposed to proceed

via a mechanism initiated by a one-electron oxidation.

Typically, direct EC oxidation successfully mimics benzylic

hydroxylation, hydroxylation of aromatic rings containing

electron-donating groups, N-dealkylation, S-oxidation,

dehydrogenation, and, less eff iciently, N-oxidation and

O-dealkylation. The range of oxidizable moieties was

extended by the application of hydroxyl radical-producing

systems (5154). For instance, with electrochemically

assisted Fenton chemistry aliphatic hydroxylation, aromatic

hydroxylation, N-oxidation, and O-dealkylation were also

efficiently mimicked. Furthermore, combining EC withenzyme-modified systems holds the promise to enable

simulation of the full range of biotransformation reactions

occurring in vivo(50).

Although ECMS has been used extensively to simulate

oxidation reactions, it can also be applied to mimic reductive

metabolism reactions, and this has been demonstrated for

nitro aromatics (62).

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18/68-$r($ &VSPQF March 201546

0CFSBDIFS et al

is a protein that is involved in the development of Alzheimers

disease (119).

(FOPNJDT "QQMJDBUJPOTThe conical nucleosides adenosine, cytidine, guanosine,

uridine, and thymidine are the main building blocks of all

naturally- occurring nucleic acid polymers (DNA and RNA).

Nevertheless, a large number of nucleoside derivatives have

been identified in DNA and RNA of living organisms, as wellas of viruses, mitochondria, and chloroplasts. The variety of

modification reactions is enormous; often oxidation reactions

are involved either as part of enzymatic or non-enzymatic

processes.

Non-enzymatic oxidation reactions are typically involved

in processes leading to DNA damage. Oxidative stress can

lead to the production of ROS, which react with nucleic

acids. Furthermore, exogenous chemical agents, such as

toxins, pharmaceuticals, or pollutants, are often activated

by oxidation to react with the genetic material. Produced

lesions include base and sugar damages, strand breaks,

and crosslinks with proteins as well as the formation of

bulky adducts. The cellular response to damage involvesseveral processes, such as DNA repair, cell cycle arrest,

and apoptosis, while irreversible mutations contribute to

oncogenesis.

To enable the development of strategies to protect the

genetic material from oxidative stress, mechanistic aspects

of nucleic acids oxidation have been extensively studied.

The electroactivity of nucleic acids was discovered by

Palecek in the 1960s (120). In the following years, a number

of oxidation products of nucleobases, nucleosides, and

nucleotides were identified using EC combined off-line with

analytical techniques, such as UVvis spectroscopy, gas

chromatography (GC)MS after derivatization, or LCMS

(121123). In this context, ECESIMS holds the promise

to facilitate and accelerate the identification process by

eliminating laborious and time-consuming isolation and

derivatization steps (29,99,124129).

ECESIMS was applied to study oxidation of

guanine-containing species. Guanine exhibits the lowest

oxidation potential among nucleic acids components and is,

therefore, the preferential target of oxidation within nucleic

acids. The primary products of guanine oxidation were

identified as 8-hydroxyguanine and cross-linked guanines.

The product 8-hydroxyguanine represents the most important

biomarker to indicate oxidative damage of genetic material,

and the formation of this compound clearly demonstrates thatEC is able to mimic in vivooxidation of nucleic acids induced

by oxidative stress.

The formation of bulky adducts is an alternative mode of

nucleic acids alteration (130). Here, oxidation reactions are

involved in activation processes; the produced electrophiles

react with nucleophilic sites within the genetic material.

ECMS represented a useful tool to study the formation of

adducts between nucleic acids and environmental pollutants

(131), amino acids (132), and pharmaceutical compounds

(124,125).

The most important enzymatic modification in genomic

DNA is methylation of the C5 atom of cytosine (C).

Production of 5-methylcytosine (5mC) is catalyzed byDNA methyltransferase enzymes. Methylation of C is an

important epigenetic DNA modification that is essential for

with immediate application in bottom-up and top-down

proteomics (72,108114). A clear advantage of EC is the

possibility to analyze the reaction products directly by MS.

EC is a reagent-free cleavage approach. There is no need

for any kind of sample preparation between cleavage and

MS analysis. High conversion yields can be obtained with

proper selection of experimental conditions (that is, Ti-based

electrode, 1% formic acid in the solvent). In peptide mapping,

EC enables the identification of disulphide-bridged peptideswithin enzymatic digest mixtures by inducing changes in ion

abundance. Furthermore, in combination with tandem MS

analysis, disulphide linkage pattern and sequence information

for the examined peptides can be determined. At the protein

level, electrochemical reduction of disulphide bonds enables

tandem MS sequencing with high sequence coverage.

For instance, the van der Burgt group reported sequence

coverage of over 80% for oxytocin and hepcidin after

electrochemical reduction (111). In the case of hepcidin, 21

of the 24 peptide bonds were cleaved after full EC reduction

of the four disulphide bonds, while only seven peptide bonds

were fragmented in the native hepcidin. Recently, EC has

also been applied for the reduction of large proteins such asmonoclonal antibodies (mAbs) (113), thereby generating light

and heavy chains with high selectivity (Figure 4). Besides the

reduction of the four inter-chain disulphide bonds, it was also

possible to reduce most of the intra-chain disulphide bonds.

Another promising field of EC-based cleavage of disulphide

bonds is hydrogen/deuterium exchange monitored mass

spectrometry (HDXMS) (114). HDXMS is increasingly being

used to characterize the dynamic properties of proteins.

When a protein is incubated in D2O, the backbone amide

hydrogen/deuterium exchange kinetics directly reflect the

conformational dynamics of the polypeptide backbone.

HDXMS experiments usually involve digestion of proteins.

A critical step in sample preparation is the reduction of

disulphide bonds. Cleavage has to be performed under cold

and acidic conditions where the amide hydrogen exchange

reaction is quenched (pH 2.5, 0 C). In addition, the reduction

must be performed as quickly as possible (within a few

minutes) to minimize artifactual deuterium loss or gain in

the backbone amides. Here, EC is used for the controlled

reduction of the disulphide bonds, replacing the chemical

reducing agent Tris(2-carboxyethyl)-phosphine (TCEP), which

often causes serious adverse effects on H/D exchange,

chromatography, and MS.

EC can also indirectly be used for peptide and protein

characterization. Mass tags or labels for specific aminoacid residues can be generated electrochemically

(9,115119). Usually, the inherent EC of ESI is applied for

activation. Metal ions are used for non-covalent labelling,

copper ions for targeting cysteine residues, and zinc ions

for targeting histidine residues and phosphorylation sites.

Covalent labelling usually involves activated hydroquinone

species, which selectively react with cysteine residues.

Electrochemically-assisted labelling was found to be useful to

determine the number of cysteine residues, which improves

protein identification by database searching. Furthermore,

based on the labelling extent of different residues within

peptide and proteins, the accessibility, reactivity, or affinity

of these sites can be tested. The usefulness of this approachhas, for instance, been demonstrated by identifying the

preferred binding sites of copper ions to -amyloid 16, which


19/68

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20/68-$r($ &VSPQF March 201548

0CFSBDIFS et al

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and U. Karst, Angew. Chem. Int. Ed. Engl., A52A58 (2011).

7. A. Baumann, and U. Karst, Expert Opin. Drug Metab. Toxicol.6,

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9. C. Roussel, L. Dayon, N. Lion, T.C. Rohner, J. Josserand, J.S.

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8, 4656 (2008).

11. E. Nouri-Nigjeh, R. Bischoff, A.P. Bruins, and H.P. Permentier, Curr.

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26. G.J. Van Berkel, K.G. Asano, and P.D. Schnier, J. Am. Soc. Mass

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17071716 (2004).

28. A. Baumann, W. Lohmann, T. Rose, K.C. Ahn, B.D. Hammock,

U. Karst, and N.H. Schebb, Drug Metab. Dispos.38, 21302138

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14931499 (2004).

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development and normal function in all mammals (133). In

2009 it was demonstrated that genomic 5mC is transformed

to 5-(hydroxymethyl)cytosine (5hmC) (134,135). Oxidation was

accomplished by the ten-eleven translocation enzymes. These

iron and 2-oxoglutarate-dependent enzymes were furthermore

able to convert 5hmC to 5-formylcytosine (5fC) and

5-carboxylcytosine (5caC) (136). The later oxidation products

are considered to represent important intermediates in active

demethylation of 5mC. They are recognized and excized bythe mammalian thymine-DNA glycosylase, and subsequently

converted to C through base excision repair (137,138). There

is increasing evidence that 5hmC may also serve as a stable

epigenetic mark that possesses unique regulatory functions

(139,140). It binds to specific regulatory proteins and is mainly

present at actively transcribed genes (141,142).

In vitroexperiments employing different sources of radicals

demonstrated that 5hmC and 5fC can also be formed by

one-electron oxidation of 5mC (143,144). However, both

species were considered to represent only minor products

of non-enzymatic 5mC oxidation (145). The 5,6-double bond

was considered to be more reactive than the methyl group.

Thus, oxidation should mainly lead to 5,6-dihydroxy-5,6-dihydro-5-methylcytosine (glycolmC).

However, there is convincing evidence from studying the

electrochemical oxidation of cytidine and 5-methylcytidine

that 5hmC and 5fC are major products of 5mC oxidation

(Figure 5)(126). Simulation experiments have further revealed

that C5-methylation reduces stability and increases reactivity

of pyrimidine bases. Based on these results, it can be

speculated that oxidative stress may induce epigenetic

alterations by influencing the previoualy described equilibrium

of the oxidized forms of 5mC.

$PODMVTJPOTECESIMS represents a versatile tool for studying redox

processes involving different kinds of bioorganic molecules,

such as peptides, proteins, endogenous, and exogenous

small molecules as well as nucleic acids. ECESIMS

is a purely instrumental approach. EC enables the fast,

automated, and cost-effective generation of redox reaction

products, and is a green chemistry technique. ECESI

MS, often in combination with LC separation, allows the

comprehensive characterization of the reaction mixture

obtained. ECESIMS represents a very useful complement

to in vitroand in vivotechniques in metabolomics, proteomics,

genomics, and related areas of application. Despite the

applicability of EC in general and ECMS in particular toomics this approach is relatively new and therefore unknown

to analytical chemists. Nevertheless, the fast growing number

of ECMS publications illustrates the power of this technology

and will help to create a broader acceptance and use in life

science applications.

"DLOPXMFEHFNFOUTThis work was funded by the Austrian Science Fund (FWF):

P 22526-B11. Furthermore, the authors want to thank

Maria Fedorova and Dieter Weber (Institute of Bioanalytical

Chemistry, Faculty of Chemistry and Mineralogy, Universitt

Leipzig, Leipzig, Germany) as well as Simone Nicolardi and

Yuri E. M. van der Burgt (Leiden University Medical Center(LUMC), Center for Proteomics and Metabolomics, Leiden,

The Netherlands) for providing Figures 3 and 4.



0CFSBDIFS et al

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Karst, J. Chromatogr. A1216, 31923198 (2009).

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Analytical Chemistry83, 55195525 (2011).

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Spectrom.16, 19341940 (2002).

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Spectrom.21, 23232331 (2007).

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Chem.82, 76257633 (2010).

54. E. Nouri-Nigjeh, A.P. Bruins, R. Bischoff, and H.P. Permentier,

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)FSCFSU 0CFSBDIFS is associate professor for

bioanalytical chemistry at the Medical University of

Innsbruck (Innsbruck, Austria). His research focuses on

the development and application of analytical techniquesfor the analysis of bioorganic molecules with special

emphasis on nucleic acids and small molecules. Please

direct any correspondence to: herbert.oberacher@i-med.

ac.at

'MPSJBO 1JUUFSMis research associate at the Institute of

Legal Medicine of the Medical University of Innsbruck

(Innsbruck, Austria). His main research interests are

related to the development and application of LCMS

methods for the qualitative and quantitative analysis of

small molecules.

+FBO1JFSSF $IFSWFUis President and CEO of Antec BV,

headquartered in Zoeterwoude, The Netherlands, and with

a subsidiary in Boston, USA. His main research interest isthe development of electrochemistrymass spectrometry

as a new, sustainable analytical approach.


23/68151www.chromatographyonline.com

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FOSJDIFE XJUI POF PG UIF FOBOUJPNFST BOE DVSSFOU SJTL

BTTFTTNFOU XJMM OPU QSPWJEF B SFBMJTUJD WJFX 5P EBUF UIFSF

BSF SFMBUJWFMZ GFX TUVEJFT UIBU IBWF SFQPSUFE FOBOUJPNFSJD

DPODFOUSBUJPOT PG D1"$T JO JOGMVFOU BOE FGGMVFOU

XBTUFXBUFST BOE JO TFXBHF TMVEHF o

Wastewater-Based Epidemiology: 8BTUFXBUFSCBTFE

FQJEFNJPMPHZ 8#& JT B OFX BQQSPBDI UP FTUJNBUF

UIF DPOTVNQUJPO PG JMMJDJU ESVHT CZ B DPNNVOJUZ CZ

EFUFSNJOJOH UIFJS DPODFOUSBUJPO JO JOGMVFOU XBTUFXBUFS

5IF JODPSQPSBUJPO PG FOBOUJPNFSTQFDJGJD BOBMZTJT JO UIJT

BQQSPBDI DBO IFMQ BEESFTT DVSSFOU EJGGJDVMUJFT TVDI BTEJTUJOHVJTIJOH CFUXFFO MFHBM BOE OPOMFHBM VTF PG ESVHT

WFSJGJDBUJPO PG UIF NFUIPE PG TZOUIFTJT PG JMMJDJU ESVHT SPVUF

PG BENJOJTUSBUJPO JEFOUJGJDBUJPO PG XIFUIFS ESVH SFTJEVF

SFTVMUT GSPN DPOTVNQUJPO PG BO JMMJDJU ESVH PS NFUBCPMJTN

PG PUIFS ESVHT BOE NPOJUPSJOH USFOET JO ESVH BCVTF

cPACs as Chemical Markers of Water Contamination

with Wastewater: " TNBMM OVNCFS PG TUVEJFT IBWF

SFWFBMFE UIF QPUFOUJBM GPS D1"$T UP CF VTFE BT FGGFDUJWF

JOEJDBUPST PG IVNBO TFXBHF DPOUBNJOBUJPO JO XBUFS

DPVSTFT EJGGFSFOUJBUJOH CFUXFFO TPVSDFT PG VOUSFBUFE

FGGMVFOUT BOE USFBUFE FGGMVFOUT EJTDIBSHFE GSPN 8 851T

"T &WBOT et al. TUBUFE TVDI B NBSLFS NVTU CF

DPOTJTUFOU JO TBNQMFT XIFSF XBTUFXBUFS JT GPVOE BOENVTU DPOTJTUFOUMZ BOE TJHOJGJDBOUMZ DIBOHF JUT &' EVSJOH

JUT SFTJEFODZ JO 8851T 5IJT NVTU CF JO B XBZ UIBU JT OPU

D

D

B B

B

A A

B

C C

R R

C

C

A A

Figure 2:5IF iUISFF QPJOU JOUFSBDUJPO NPEFMw "EBQUFE BOE

SFQSPEVDFE XJUI QFSNJTTJPO GSPN Trends in Environmental

Analytical Chemistry1 4JBO & &WBOT BOE #BSCBSB

,BTQS[ZL)PSEFSO Applications of Chiral Chromatography

Coupled with Mass Spectrometry in the Analysis of Chiral

Pharmaceuticals in the Environment FoF &MTFWJFSO

O OH HO O

O

(S)-Ketoprofen(R)-Ketoprofen

Figure 1:4USVDUVSF PG LFUPQSPGFO FOBOUJPNFST


25/68


26/68-$r($ &VSPQF .BSDI 54


DIBSBDUFSJTUJD XJUI BUUFOVBUJPO JO UIF FOWJSPONFOU BOE

QSPQSBOPMPM XBT JEFOUJGJFE BT B QPTTJCMF NBSLFS

Environmental Risk Assessment and Fate in the

Environment: 4UFSFPTFMFDUJWJUZ SFRVJSFT BUUFOUJPO

CFDBVTF DVSSFOU FOWJSPONFOUBM SJTL BTTFTTNFOU EPFT OPU

BDDPVOU GPS JOEJWJEVBM FOBOUJPNFST BOE DPOTJEFSJOH UIBU

FOBOUJPNFSJD TQFDJGJD UPYJDJUZ DBO PDDVS UIF SJTL QPTFE

NBZ CF VOEFSFTUJNBUFE 0ODF QSFTFOU JO UIF FOWJSPONFOU

DIBOHFT JO UIF &' DPVME CF VTFE UP EJGGFSFOUJBUF

CJPUSBOTGPSNBUJPO GSPN PUIFS SFNPWBM QSPDFTTFT 6OMJLF

BCJPUJD NFDIBOJTNT UIBU JT TPSQUJPO QIPUPDIFNJDBM

USBOTGPSNBUJPO BJSXBUFS BOE TPJMXBUFS FYDIBOHF

XIJDI BSF CFMJFWFE UP CF UIF TBNF GPS CPUI FOBOUJPNFST

CJPUJD NFDIBOJTNT DBO CF FOBOUJPTFMFDUJWF )PXFWFS UIFBCTFODF PG FOBOUJPTFMFDUJWJUZ EPFT OPU FYQMJDJUMZ NFBO

BDIJSBM EFHSBEBUJPO CFDBVTF OPU BMM CJPUJD QSPDFTTFT TIPX

QSFGFSFODF GPS UIF USBOTGPSNBUJPO PG POF FOBOUJPNFS PWFS

UIF PUIFS

Critical Aspects of Chiral EnvironmentalAnalysisSampling Strategy: "MUIPVHI PGUFO PWFSMPPLFE TBNQMJOH

JT B DSJUJDBM BTQFDU PG BOZ FOWJSPONFOUBM NPOJUPSJOH

QSPDFEVSF "O FYDFMMFOU DSJUJDBM FWBMVBUJPO PG EJGGFSFOU

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MJNJUBUJPOT CFDBVTF JU DBO POMZ HJWF JOGPSNBUJPO GPS BTQFDJGJD QPJOU JO UJNF 'JOEJOHT UIFSFGPSF NBZ OPU CF

SFQSFTFOUBUJWF BT D1"$ DPODFOUSBUJPO BOE XBTUFXBUFS

Figure 3:B 1PUFOUJBM CJOEJOH TJUFT BOE NFDIBOJTNT JO

UFJDPQMBOJO BOE C DFMMVMPTF USJTEJNFUIZMQIFOZMDBSCBNBUF

"EBQUFE BOE SFQSPEVDFE XJUI QFSNJTTJPO GSPN Trends in

Environmental Analytical Chemistry1 4JBO & &WBOT BOE

#BSCBSB ,BTQS[ZL)PSEFSO Applications of Chiral

Chromatography Coupled with Mass Spectrometry in the

Analysis of Chiral Pharmaceuticals in the Environment FoF

&MTFWJFS

F4:MRM of 2 channels, ES +166.09 > 133.00

8.300e+004


9.031e+004


2.102e+003

F1:MRM of 2 channels, ES +

136.16 > 119.12.139e+004

F6:MRM of 2 channels, ES +194.09 >163.1

4.443e+005


1.269e+004


58.19e+005

1R,2S(-)-Ephedrine

E1-Venlafaxine

E2-Venlafaxine

S(+)-METH

S(+)-AMPH

S(+)-MDMA

S(+)-MDA

S(+)-Atenolol

R(-)-METH

R

(-

)-

AMPH

R(-)-MDMA

R(-)-MDA

R(-)-Atenolol

1S,2S(+)-Pseudoephedrine23.89

31.57

24.45

10.02

27.27

25.7531.66

32.3345.48

26.04

33.01

36.94

44.71

43.94

51.16

66.6945.3941.5022.09

28.09

16.85

20.0 30.0 40.0 50.0 60.0

20.0 30.0 40.0 50.0 60.0

56.57

52.64 56.3463.1224.24

20.0 30.0 40.0 50.0 60.0

30.66

Time (min)

Time (min)

Time (min)

Time (min)

Time (min)

Time (min)

Time (min)

100

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

0

Figure 4:&YUSBDUFE JOnVFOU XBTUFXBUFS TIPXJOH

D1"$T TFQBSBUFE VTJOH B DIJSBM DPMVNO .&5)

NFUIBNQIFUBNJOF ".1) BNQIFUBNJOF .%."

NFUIZMFOFEJPYZNFUIBNQIFUBNJOF .%"

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& Technology46 #BSCBSB ,BTQS[ZL)PSEFSO BOE %BWJE 4

#BLFS Enantiomeric Profiling of Chiral Drugs in Wastewater

and Receiving Waters o "NFSJDBO

$IFNJDBM 4PDJFUZ


27/68

HPLC Columns

Asahipak ODP-50

t QPMZNFSCBTFE 31 $ DPMVNOt CFUUFS TFQBSBUJPO PG CBTJD TVCTUBODFTt TJMBOPM GSFFt Q) TUBCJMJUZ GSPN UP t MPXFS CMFFEJOH BOE IJHIFS 4/ SBUJPt SFDPNNFOEFE GPS .4 EFUFDUJPOt XBUFS PS CVFSt UJNFT MPOHFS MJGF UJNF

(1$ DPMVNOTt GPS TZOUIFUJD QPMZNFST QMBTUJDT SFTJOT SVCCFST

TJMJDPOFT DPQPMZNFSTt TJOHMF QPSF MJOFBS BOE NJYFECFE DPMVNOTt IVHF SBOHF PG FYDMVTJPO MJNJUTt QSFMMFE XJUI 5)' $IMPSPGPSN %.' )'*1t TQFDJBM IJHI UFNQFSBUVSF (1$ DPMVNOTt CFTU TUBCJMJUZ BOE SFQSPEVDJCJMJUZ1PMZTUZSFOF 14 BOE 1.." 4UBOEBSE GPS DBMJCSBUJPO

"TBIJQBL /)1t QPMZNFSCBTFE BNJOP DPMVNO /)t GPS TVHBST BOE QPMBS DPNQPVOETt TJMBOPM GSFFt Q) TUBCJMJUZ GSPN UP t SFDPNNFOEFE GPS .4 $"% BOE MJHIUTDBUUFSJOH EFUFDUPST

t CFTUTFMMJOH DPMVNO GPS )*-*$t &YDFMMFOU MPOHFWJUZ MBTUT UJNFT

MPOHFS UIBO TJMJDB DPMVNOT

1305&*/ ,8t GPS QSPUFJOT QFQUJEFT FO[ZNFT BOUJCPEJFTt TJMJDBCBTFEt GPS XBUFS CVFS TBMU BOE PSHBOJD TPMWFOUTt NBOZ BQQMJDBUJPOT GPS CJPQPMZNFST

0)QBL 4#t GPS NPEJFE QSPUFJOT QPMZTBDDIBSJEFTXBUFSTPMVCMF QPMZNFST

t QPMZNFSCBTFEt NBOZ FYDMVTJPO MJNJUT BWBJMBCMF

1VMMVMBO 4UBOEBSE GPS DBMJCSBUJPO

*PO $ISPNBUPHSBQIZ DPMVNOTt GPS JOPSHBOJD BOJPOT BOE DBUJPOTt GPS OPOTVQQSFTTFE PS TVQQSFTTPS NFUIPETt XJUI DBSCPOBUF CVFS BOJPOT BOE PYZIBMJEFTt DPNQBUJCMF XJUI BMM JOTUSVNFOUT

46("3 DPMVNOTt MJHBOE FYDIBOHF XJUI /B+ ;O $BBOE 1C

BT DPVOUFS JPOt GPS NPOP EJ BOE PMJHPTBDDIBSJEFTt DPTU BOE FDPGSJFOEMZ QVSF XBUFS BT TPMWFOUt IJHIFS FYDMVTJPO MJNJUT GPS QPMZTBDDIBSJEFT

0SHBOJD BDJET DPMVNOTt JPO FYDMVTJPO XJUI )+

t GPS PSHBOJD BDJET PS NJYUVSFT PG TVHBST BOE BMDPIPMT

Reversed Phase

HILIC

IC

SEC (organic GPC)

SEC (aqueous GFC)SUGAR

t $PNQSFIFOTJWF UFDIOJDBM TVQQPSUt 4QFDJBMJTUT JO IJHIRVBMJUZ MPOHMBTUJOH QPMZNFSCBTFE DPMVNOTt )1-$ DPMVNOT NBEF JO +BQBO

t ZFBST PG FYQFSJFODF

] JOGP!TIPEFYEF ] XXXTIPEFYEF

:PV DBO UFTU BMM PVS DPMVNOT GPS GSFF 7JTJU PVS XFCTJUF GPS NPSF JOGPSNBUJPO BCPVU EFNP DPMVNOT


28/68-$r($ &VSPQF .BSDI 56


PS SJWFS GMPX DBO WBSZ UISPVHIPVU UIF EBZ "MUFSOBUJWFMZ

BVUPNBUFE TBNQMFST BSF VTFE UP DPMMFDU B DPNQPTJUF

TBNQMF UIBU DBO CF FJUIFS UJNF PS WPMVNF QSPQPSUJPOBM PWFS

B I QFSJPE 7PMVNF QSPQPSUJPOBM TBNQMJOH JT DPOTJEFSFE

UP CF UIF NPTU SFQSFTFOUBUJWF NPEF PG TBNQMJOH CFDBVTF

JU BDDPVOUT GPS WBSJBUJPOT JO GMPX )PXFWFS UIF TUBCJMJUZ PG

D1"$T JO DPNQPTJUF TBNQMFST JT VOLOPXO GPS B WBSJFUZ

PG NBUSJDFT 4JHOJGJDBOU EFHSBEBUJPO IBT CFFO

PCTFSWFE GPS TPNF 1"$T JO XBTUFXBUFS PWFS B I QFSJPEEFTQJUF TUPSBHF BU

2015-03a single reversed-phase uplc method for quantification of levofloxacin in aqueous humour and...

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