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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
www.markes.com
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2015PerkinElme
r,Inc.400217A_01.Allrightsreserved.PerkinElmerisaregisteredtrademarkofPerkinElmer,Inc.
Together we can change the world.At Perki nEl mer, we share your com mitment
to making the world a beer place. Our detection solutions are used to analyze more
than two billion environmental samples every year, safeguarding the ai r, water and soil
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7/23/2019 2015-03A Single Reversed-Phase UPLC Method for Quantification of Levofloxacin in Aqueous Humour and Pharma
4/68-$r($ &VSPQF March 201532
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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*/(
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John W. Dolan
A stepwise process is described to help isolate and identify the
cause of a method failure.
($ $0//&$5*0/4
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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 $PVQMFE UP.BTT 4QFDUSPNFUSZ " 3FWJFXHerbert Oberacher, Florian Pitterl,and Jean-Pierre ChervetRedox reactions are integral partsof many cellular processes. Theyare therefore extensively studiedJOWJUSP andJO WJWP. Electrochemistry(EC) represents a purely instrumentalapproach to characterize direct andindirect effects of redox reactions onbioorganic molecules. This reviewhighlights important trends and recentdevelopments.
.BSDI | 2015
7PMVNF /VNCFS
7/23/2019 2015-03A Single Reversed-Phase UPLC Method for Quantification of Levofloxacin in Aqueous Humour and Pharma
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Think of these instruments as yourTools for Macromolecular Characterization
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7/23/2019 2015-03A Single Reversed-Phase UPLC Method for Quantification of Levofloxacin in Aqueous Humour and Pharma
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5IF 1VCMJTIFST PG -$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 + $PSUFTH.J. Cortes Consulting,
Midland, Michigan, USA
(FSU %FTNFUTransport Modelling and Analytical
Separation Science, Vrije Universiteit,
Brussels, Belgium
+PIO 8 %PMBOLC Resources, Walnut Creek, California,
USA
3PZ <UFFOSigma-Aldrich/Supelco, Bellefonte,
Pennsylvania, USA
"OUIPOZ ' 'FMMPharmaceutical Chemistry,
University of Bradford, Bradford, UK
"UUJMB 'FMJOHFSProfessor of Chemistry, Department of
Analytical and Environmental Chemistry,
University of Pcs, Pcs, Hungary
'SBODFTDP (BTQBSSJOJDipartimento di Studi di Chimica e
Tecnologia delle Sostanze Biologica-mente Attive, Universit La Sapienza,
Rome, Italy
+PTFQI - (MBKDIMomenta Pharmaceuticals, Cambridge,
Massachusetts, USA
+VO )BHJOBLBSchool of Pharmacy and PharmaceuticalSciences, Mukogawa Womens
University, Nishinomiya, Japan
+BWJFS )FSOOEF[#PSHFTDepartment of Analytical Chemistry,Nutrition and Food Science University of
Laguna, Canary Islands, Spain
+PIO 7 )JOTIBXServeron Corp., Hillsboro, Oregon, USA
5VVMJB )ZUZMJOFOVVT Technical Research of Finland,
Finland
)BOT(FSE +BOTTFOVant Hoff Institute for the Molecular
Sciences, Amsterdam, The Netherlands
,JZPLBUTV +JOOPSchool of Materials Sciences, ToyohasiUniversity of Technology, Japan
)VCB ,BMT[Semmelweis University of Medicine,Budapest, Hungary
)JBO ,FF -FFNational University of Singapore,
Singapore
8PMGHBOH -JOEOFSInstitute of Analytical Chemistry,
University of Vienna, Austria
)FOL -JOHFNBOFaculteit der Scheikunde, Free University,
Amsterdam, The Netherlands
5PN -ZODIBP Technology Centre, Pangbourne, UK
3POBME & .BKPSTAgilent Technologies,
Wilmington, Delaware, USA
1IJMMJQ .BSSJPUMonash University, School of Chemistry,
Victoria, Australia
%BWJE .D$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
$PMJO 1PPMFWayne State University, Detroit,
Michigan, USA
'SFE & 3FHOJFSDepartment of Biochemistry, Purdue
University, West Lafayette, Indiana, USA
)BSBME 3JUDIJFTrajan Scientific and Medical. Milton
Keynes, UK
1BU 4BOESBResearch Institute for Chromatography,Kortrijk, Belgium
1FUFS 4DIPFONBLFSTDepartment of Chemical Engineering,Universiteit van Amsterdam, Amsterdam,
The Netherlands
3PCFSU 4IFMMJFAustralian Centre for Research onSeparation Science (ACROSS), University
of Tasmania, Hobart, Australia:WBO 7BOEFS )FZEFOVrije Universiteit Brussel,
Brussels, Belgium
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Scientists have published a novel
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)1-$ *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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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
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For product information: www.vwr.com
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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 (
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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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7/23/2019 2015-03A Single Reversed-Phase UPLC Method for Quantification of Levofloxacin in Aqueous Humour and Pharma
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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7/23/2019 2015-03A Single Reversed-Phase UPLC Method for Quantification of Levofloxacin in Aqueous Humour and Pharma
15/68143XXXDISPNBUPHSBQIZPOMJOFDPN
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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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
7/23/2019 2015-03A Single Reversed-Phase UPLC Method for Quantification of Levofloxacin in Aqueous Humour and Pharma
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analysis requirements. We offer a range of products
and services from instruction courses and training
thru contract analysis, consulting, method develop-
ment and qualifi cation services all the way up to
supplying turnkey GPC/SEC and LC/2D systems.
All this comes with the personal and direct supportof our dedicated team of innovative and pioneering
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7/23/2019 2015-03A Single Reversed-Phase UPLC Method for Quantification of Levofloxacin in Aqueous Humour and Pharma
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0CFSBDIFS et al
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Mizrahi, P.V. Pridkhochenko, A. Modestov, and O. Lev, Isr. J. Chem.
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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.
7/23/2019 2015-03A Single Reversed-Phase UPLC Method for Quantification of Levofloxacin in Aqueous Humour and Pharma
21/68149XXXDISPNBUPHSBQIZPOMJOFDPN
0CFSBDIFS et al
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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.
7/23/2019 2015-03A Single Reversed-Phase UPLC Method for Quantification of Levofloxacin in Aqueous Humour and Pharma
23/68151www.chromatographyonline.com
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OVNCFS PG BWBJMBCMF $41T JU JT OPU QPTTJCMF UP GJOE BVOJWFSTBM TUBUJPOBSZ QIBTF GPS B SBOHF PG EJGGFSFOU D1"$T
5IJT JT B SFTVMU PG UIF EJGGFSFOU NFDIBOJTNT JOWPMWFE
BOE UIF IJHI TFMFDUJWJUZ PG DIJSBM TFMFDUPST SFRVJSFE UP
BDIJFWF DIJSBM SFDPHOJUJPO 'PS QPMZTBDDIBSJEFCBTFE
$41T NBEF CZ WBSJPVT EFSJWBUJWFT PG DFMMVMPTF BOE
BNZMPTF NPMFDVMFT )CPOEJOH EJQPMF BOE TUFSJD
JOUFSBDUJPOT BSF UIF NBJO NFDIBOJTNT JOWPMWFE XIJMF GPS
QSPUFJOCBTFE BDJE HMZDPQSPUFJO DFMMPCJPIZESPMBTF
PS BMCVNJO BOE BOUJCJPUJDCBTFE WBODPNZDJO UFJDPQMBOJO
PS SJTUPUFDJO " $41T )CPOEJOH IZESPQIPCJD BOE
JPOJD JOUFSBDUJPOT BOE JODMVTJPO DPNQMFYFT BSF UIF NBJO
JOUFSBDUJPOT UIBU EFUFSNJOF DIJSBM TFQBSBUJPO 'JHVSF
$IJSBM BOBMZTJT DBO QSPWJEF VTFGVM JOGPSNBUJPO BCPVUUIF PDDVSSFODF GBUF BOE USBOTGPSNBUJPO PG D1"$T BOE
UIFJS NFUBCPMJUFT JO UIF FOWJSPONFOU CFDBVTF FOBOUJPNFST
PG UIF TBNF D1"$ DBO JOUFSBDU JO EJGGFSFOU XBZT XIFO
FYQPTFE UP B DIJSBM FOWJSPONFOU TVDI BT JO CJPMPHJDBM
TZTUFNT FO[ZNFT QSPUFJOT 5IFSF BSF GPVS NBJO BSFBT
XIFSF DIJSBM BOBMZTJT PG 1"$T JO UIF FOWJSPONFOU JT
TUVEJFE
Fate of cPACs during Wastewater Treatment: 'PMMPXJOH
DPOTVNQUJPO CZ UIF IVNBO QPQVMBUJPO D1"$T SFBDI
XBTUFXBUFS USFBUNFOU QMBOUT 8851T JO B NPEJGJFE GPSN
GPS FYBNQMF BT NFUBCPMJUFT PS XJUI UIFJS &' BMUFSFE
%VSJOH XBTUFXBUFS USFBUNFOU UIFZ BSF TVCKFDUFE UP CJPUJD
QSPDFTTFT UIBU MFBE UP GVSUIFS DIBOHFT JO FOBOUJPNFSJD
DPNQPTJUJPO 5IJT TUFSFPTFMFDUJWF EFHSBEBUJPO OFFET
BUUFOUJPO CFDBVTF UIF EJTDIBSHFE FGGMVFOU NJHIU CF
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
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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
TBNQMJOH NPEFT IBT CFFO DPOEVDUFE CZ 0SU et al.
5IF DPMMFDUJPO PG HSBCTQPU TBNQMFT IBT PCWJPVT
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
F10:MRM of 2 channels, ES +278.15 > 260.10
9.031e+004
F2:MRM of 2 channels, ES +150.2 > 119.05
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
F5:MRM of 2 channels, ES +180.03 >163.10
1.269e+004
F20:MRM of 2 channels, ES +266.9 >190.1
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 .%."
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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
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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
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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
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