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ElectrochemistryMass Spectrometry (ECMS)ЧA New Tool To Study Drug Metabolism and Reaction Mechanisms.

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Highlights
Analytical Methods
Electrochemistry/Mass Spectrometry (EC/MS)—A New
Tool To Study Drug Metabolism and Reaction
Mechanisms
Uwe Karst*
Keywords:
derivatization · electrochemistry · liquid
chromatography · mass spectrometry · metabolism
The possibility of simulating the oxidative metabolism of drugs is only one
major reason for the strongly increasing
attraction in the on-line coupling between electrochemistry and electrospray mass spectrometry (EC/MS) for
both industry and academia. Although
this combination has been known from a
few publications for a long time and has
been covered by recent reviews,[1] it is
now leaving its hibernation phase for
good reasons. Some very attractive new
applications for EC/MS, partly in combination with liquid chromatography
(LC), have recently been published.
Herein, the major characteristics of
EC/MS are summarized, the most interesting recent applications are discussed,
and future perspectives are presented.
In addition to the clear fact that
electrospray mass spectrometric (ESIMS) detection provides a larger wealth
of information than electrochemical detection, some other known drawbacks of
electrochemical detection are avoided
when using EC/MS: As quantification is
performed on the basis of the integration of the mass spectrometric signals
and not on current measurements, unstable baselines can more easily be
avoided and small changes in solvent
composition do not automatically lead
to quantification problems. This is most
[*] Prof. Dr. U. Karst
University of Twente
Department of Chemical Analysis and
MESA+ Institute for Nanotechnology
P.O. Box 217, 7500 AE Enschede
(The Netherlands)
Fax: (+ 31) 53-489-4645
E-mail: u.karst@utwente.nl
2476
important for coupling with gradientelution liquid chromatography.
The additional coupling of EC/MS
with LC is useful for two different
situations. On the one hand, the investigation of the electrochemical conversion of any single substance out of a
complex mixture requires a separation.
The favored procedure consists first of a
reversed-phase LC separation of the
individual constituents followed by electrochemical on-line treatment and
mass spectrometric detection (Figure 1,
route a). In this way, only the reaction
products of one single compound are
detected at one time. On the other hand,
the separation of the oxidation products
may provide useful information, for
example, on the polarity of the individual products. In this case, a pure starting
material is first oxidized electrochemically, and the products are subsequently
separated by liquid chromatography and
then detected by mass spectrometry
(Figure 1, route b). The integration of
an additional UV/Vis detector prior to
the mass spectrometer is possible in
both cases. An interesting aspect is the
fact that, in contrast to electrochemical
detection, LC/EC/MS detection is unrestrictedly compatible with gradient elution in LC, thus allowing for improved
LC separations to be obtained.
The most widely applied cells for
electrochemical detection, for example,
thin-layer or wall-jet cells, have the
reputation of being contaminated very
easily by reaction side products (for
example, polymerization products), thus
requiring frequent maintenance. This
severely hampers the use of electrochemical detection in routine analysis.
Pulse techniques, which involve measuring and cleaning steps, are advantageous
for increasing the mean time between
maintenance intervals. However, a
quantitative turnover of the target compound would allow mass spectra to be
obtained which contain only a few
different peaks and are easy to interprete. This is not possible with pulsed
electrochemical techniques. Electrochemical cells with large surface areas,
however, are advantageous for combining quantitative turnover with long
maintenance intervals, thus rendering
the method suitable for the routine
analysis of a large number of samples.
Figure 1. Schematic set-up of LC/EC/MS (route a) and EC/LC/MS (route b), including an
optional UV/Vis detector prior to the mass spectrometer.
2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/anie.200301763
Angew. Chem. Int. Ed. 2004, 43, 2476 –2478
Angewandte
Chemie
For these reasons, most of the research
groups who are active in this field use
commercially available electrodes with
an extremely large surface area glassy
carbon working electrode, a Pd counterelectrode, and a Pd/H2 reference electrode. Significant progress is currently
being made by academic groups as well
as by industry towards making electrodes available which combine a large
surface area with other working electrode materials (for example, Pt) and
low flow rates.
Bruins and co-workers[2] have recently described an interesting approach
to mimic oxidations catalyzed by cytochrome P450 by using on-line electrochemistry/mass spectrometry. Although
not all the reactions catalyzed by cytochrome P450 were observed in the EC/
MS system, those initiated by a oneelectron oxidation, including dehydrogenation, alcohol oxidation, S and P oxidation, and N dealkylation were found.
In contrast, reactions initiated by direct
hydrogen abstraction, for example, hydroxylation of unsubstituted aromatic
rings and O dealkylation, were not detected because their oxidation potential
was too high.[2] Nevertheless, the method may become an extremely valuable
tool in the early phases of drug discovery, as it provides a rapid and simple
means to investigate the metabolic stability of possible drug candidates, which
may be derived in large numbers from
combinatorial substance libraries.
The same research group has used
an EC/MS system to rapidly analyze the
oxidation products of peptides.[3] The
results of this study confirm earlier
literature data obtained using much
more laborious methods in which tyrosine-containing peptides were readily
oxidized and resulted in the formation
of various products, including peptide
fragments caused by hydrolysis at the Cterminus of tyrosine (Scheme 1). It became evident in a comparison of phos-
phorylated and non-phosphorylated tyrosine residues that cleavage only occurs
at native tyrosine residues. This result
allows an easy distinction between phosphorylated and non-phosphorylated
peptides. It may be expected that an
attractive EC/MS-based protein digestion and peptide-mapping method could
be developed from this work in the
future.
The application of dedicated derivatizing agents for EC/MS has been introduced by Van Berkel et al.[4] The oxidation process inherent in the electrospray
interface was used to selectively detect
the ferrocene-derivatized analytes. In
this pioneering article, no additional
electrochemical cell was used, and good
results were achieved without LC separation of the samples. Applications are
presented for the detection of alcohols
(with ferrocenoyl azide) and for the
detection of diols (with ferroceneboronic acid).[4] In more recent work, ferrocenoyl azide was used in combination with
the same detection technique for the
analysis of mixtures of natural products,
and the fragmentation pathways were
investigated for the derivatives of primary, secondary, and tertiary alcohols.[5]
Karst et al. used ferrocenoyl chloride for
the determination of phenols and alcohols. A reversed-phase separation was
used, the derivatives were oxidized in an
electrochemical cell and an atmospheric
pressure chemical ionization (APCI)
interface was applied with the corona
discharge needle switched off.[6] It can
be expected that these techniques will
strongly gain importance for quantitative analysis, as a result of the excellent
selectivity and sensitivity; in addition
new dedicated derivatizing agents for
EC/MS based on ferrocenes and other
redox systems will undoubtedly soon be
introduced.
Gun et al. investigated the reduction
of [(C5Me5)2Mo2O5] and related complexes by using on-line EC/MS based on
Scheme 1. Electrochemically induced C-terminal peptide cleavage at a tyrosine residue.[3]
Angew. Chem. Int. Ed. 2004, 43, 2476 –2478
www.angewandte.org
the conversion in an electrochemical
flow cell. The authors succeeded in
identifying mono- to tetranuclear organometallic molybdenum oxides.[7] This
work may open up new pathways for the
analysis of metal complexes, because the
different oxidation states of the analytes
can be identified on-line by varying the
the applied electrochemical potential.
As already indicated in earlier work by
Amster and co-workers,[8] the use of
high-resolution mass spectrometry may
even allow insight into the redox status
of metalloproteins to be obtained. The
oxidation state of the metal centers was
found to be stable in an electrospray
interface (both in the positive and
negative ion modes). More applications
for the analysis of metalloproteins are
likely to follow in the near future,
because this technique is able to provide
information which is not available using
any other analytical method.
The large number of EC/MS applications developed within the last few
years clearly shows that this technique
may now find rapidly increasing application in different scientific areas. The
use of EC/MS for metabolic studies and
peptide or protein digestion is particularly promising because of the large
number of current challenges in bioanalysis. However, it is important that
appropriate flow cells suitable for very
low flow rates are soon developed to
make the technique compatible with
micro- and nano-LC separations and
minute amounts of available analytes.
Different working electrode materials
(for example, Pt) in combination with
large surface areas would also help to
expand the range of applications. The
full potential of EC/MS is only just
starting to be discovered, and significant
new applications in this field can certainly be expected in the near future.
[1] a) G. Hambitzer, J. Heitbaum, Anal.
Chem. 1986, 58, 1067 – 1070; b) K. J.
Volk, M. S. Lee, R. A. Yost, A. BrajterToth, Anal. Chem. 1988, 60, 722 – 724;
c) K. J. Volk, R. A. Yost, A. Brajter-Toth,
J. Chromatogr. 1989, 474, 231 – 243; d) G.
Diehl, U. Karst, Anal. Bioanal. Chem.
2002, 373, 390 – 398; e) H. Hayen, U.
Karst, J. Chromatogr. A 2003, 1000,
549 – 565.
[2] a) U. Jurva, H. V. WikstrGm, A. P. Bruins,
Rapid Commun. Mass Spectrom. 2000,
2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2477
Highlights
14, 529 – 533; b) U. Jurva, H. V. WikstrGm, L. Weidolf, A. P. Bruins, Rapid
Commun. Mass Spectrom. 2003, 17, 800 –
810.
[3] H. P. Permentier, U. Jurva, B. Barroso,
A. P. Bruins, Rapid Commun. Mass Spectrom. 2003, 17, 1585 – 1592.
[4] G. J. Van Berkel, J. M. E. Quirke, R. A.
Tigani, A. S. Dilley, T. R. Covey, Anal.
Chem. 1998, 70, 1544 – 1554.
2478
[5] a) J. M. E. Quirke, Y. L. Hsu, G. J. Van
Berkel, J. Nat. Prod. 2000, 63, 230 – 237;
b) J. M. E. Quirke, G. J. Van Berkel, J.
Mass Spectrom. 2001, 36, 179 – 187.
[6] a) G. Diehl, A. Liesener, U. Karst, Analyst 2001, 126, 288 – 290; b) G. Diehl, U.
Karst, J. Chromatogr. A 2002, 974, 103 –
109.
[7] a) J. Gun, A. Modestov, O. Lev, D.
Saurenz, M. A. Vorotyntsew, R. Poli,
2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
Eur. J. Inorg. Chem. 2003, 482 – 492;
b) J. Gun, A. Modestov, O. Lev, R. Poli,
Eur. J. Inorg. Chem. 2003, 2264 – 2272.
[8] K. A. Johnson, B. A. Shira, J. L. Anderson, I. J. Amster, Anal. Chem. 2001, 73,
803 – 808.
Angew. Chem. Int. Ed. 2004, 43, 2476 –2478
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