Analytical Chemistry
Article
in Figure 2a, since they are not needed for monitoring the
electrochemical conversion of APAP. The complete spectrum
of the electrochemical oxidation of APAP is supplied in the
Supporting Information (Figure S-3).
concentrations due to its inherently low sensitivity. Especially
for substances that show a low electrochemical conversion rate,
this could result in more lengthy infusion time to achieve a
reasonable signal-to-noise ratio. This could lead to the
decomposition of highly reactive oxidation products and hinder
their detection by NMR. EC coupled online to MS will thus
remain an important tool in the electrochemical mimicry of
oxidative drug metabolism. Its high sensitivity makes it
irreplaceable for the detection of oxidation products generated
in low yields and complements the data obtained by EC/NMR.
Fast screening by EC/MS and presentation of the obtained
results in three-dimensional mass voltammograms deliver a
precise overview on the generated oxidation products. There-
fore, EC/NMR and EC/MS can ideally be used as
complementary techniques in the simulation of oxidative
metabolism.
An oxidation potential of 600 mV was selected to obtain a 1H
NMR spectrum of the reactive metabolite NAPQI in the on-
flow mode (Figure 2b). Under these conditions, very little
oxidation has occurred as 1H NMR signals can be observed for
the parent compound with only very small additional signals for
a second species (6.64 and 7.02 ppm). The upfield chemical
shift changes for the aromatic protons of the second species
and the downfield chemical shift change for the methyl signal
(not shown) are consistent with the electrochemical formation
of NAPQI. An increase of the oxidation potential to 1200 mV
led to a higher yield of oxidation product (Figure 2c).
Integration of the signals for NAPQI showed that approx-
imately 5% of the parent compound was converted at 600 mV,
while doubling the potential leads to approximately 15%
conversion of APAP. At 1700 mV (Figure 2d), the level of this
product remains at approximately 15% conversion of parent
compound. However, at this higher potential, the formation of
an additional signal, which was putatively identified as BQ, was
observed at 6.78 ppm. NMR signal integration indicates that
approximately 4% of the parent has been oxidized to BQ. To
confirm the formation of BQ at 1700 mV, a spiking experiment
was performed in a standard NMR tube in the same solvent
system as used in the flow cell. A 10 mM solution of BQ was
added to a 2 mM solution of APAP, resulting in final
concentrations of 1.97 mM for APAP and 0.14 mM for BQ.
Figure 3 shows a comparison of APAP spiked with BQ (a) and
APAP electrochemically oxidized at 1700 mV (b).
ASSOCIATED CONTENT
* Supporting Information
Additional information as noted in text. This material is
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S
AUTHOR INFORMATION
Corresponding Author
+49 251 83-36013.
■
Notes
The authors declare no competing financial interest.
REFERENCES
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(1) Davidson, D. G.; Eastham, W. N. Br. Med. J. 1966, 2, 497−499.
(2) Dahlin, D. C.; Miwa, G. T.; Lu, A. Y. H.; Nelson, S. D. Proc. Natl.
Acad. Sci. U.S.A. 1984, 81, 1327−1331.
(3) Mitchell, J. R.; Jollow, D. J.; Potter, W. Z.; Gillette, J. R.; Brodie,
B. B. J. Pharmacol. Exp. Ther. 1973, 187, 211−217.
The signal resulting from the spiking with BQ (Figure 3a)
showed the same chemical shift as the singlet observed in
Figure 3b and thus confirms the oxidation of NAPQI to BQ.
The oxidation pathway of APAP, which was summarized in
Scheme 1, was therefore confirmed by the results gained from
online EC/MS and EC/NMR experiments. Upon electro-
chemical oxidation of APAP (151.1 Da), one electron and one
proton are abstracted in the first step, resulting in the formation
of an APAP radical. Further loss of one electron and one
proton leads to the generation of NAPQI (149.1 Da). Finally,
after hydrolysis with water from the buffer solution and the loss
of the acetyl moiety, the p-benzoquinone (108.1 Da) is formed.
However, if GSH is added to the oxidized solution as a trapping
agent, adduct formation with NAPQI can be observed (456.1
Da).
(4) Jollow, D. J.; Mitchell, J. R.; Potter, W. Z.; Davis, D. C.; Gillette, J.
R.; Brodie, B. B. J. Pharmacol. Exp. Ther. 1973, 187, 195−202.
(5) Thummel, K. E.; Lee, C. A.; Kunze, K. L.; Nelson, S. D.; Slattery,
J. T. Biochem. Pharmacol. 1993, 45, 1563−1569.
(6) Jurva, U.; Wikstrom, H. V.; Weidolf, L.; Bruins, A. P. Rapid
̈
Commun. Mass Spectrom. 2003, 17, 800−810.
(7) Faber, H.; Melles, D.; Brauckmann, C.; Wehe, C.; Wentker, K.;
Karst, U. Anal. Bioanal. Chem. 2012, 403, 345−354.
(8) Lohmann, W.; Karst, U. Anal. Bioanal. Chem. 2006, 386, 1701−
1708.
(9) Lohmann, W.; Hayen, H.; Karst, U. Anal. Chem. 2008, 80, 9714−
9719.
(10) Richards, J. A.; Evans, D. H. Anal. Chem. 1975, 47, 964−966.
(11) Mincey, D. W.; Popovich, M. J.; Faustino, P. J.; Hurst, M. M.;
Caruso, J. A. Anal. Chem. 1990, 62, 1197−1200.
(12) Sandifer, M. E.; Zhao, M.; Kim, S. H.; Scherson, D. A. Anal.
Chem. 1993, 65, 2093−2095.
CONCLUSIONS
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Online EC/NMR is presented here as a simple and elegant way
for the generation, detection, and structure elucidation of
potential reactive intermediates that might be formed in vivo.
NMR analysis provides not only detailed structure elucidation
but also quantification of generated oxidation products. It offers
precise knowledge about the oxidation sites, which could help
to predict toxic side reactions of potential active molecules.
Extension of the instrumental setup with HPLC could make the
presented method available to more complex systems, where
various products are generated upon oxidation. The use of a
flow probe allows the flow to be stopped and single peaks to be
“parked” in the probe head of the NMR. This would ensure
enough analysis time to obtain spectra of very small amounts of
oxidation products with a sufficient signal-to-noise ratio.
However, it has to be noted that NMR is limited to higher
(13) Mairanovsky, V. G.; Yusefovich, L. Y.; Filippova, T. M. J. Magn.
Reson. 1983, 54, 19−35.
(14) Prenzler, P. D.; Bramley, R.; Downing, S. R.; Heath, G. A.
Electrochem. Commun. 2000, 2, 516−521.
(15) Webster, R. D. Anal. Chem. 2004, 76, 1603−1610.
(16) Zhang, X.; Zwanziger, J. W. J. Magn. Reson. 2011, 208, 136−147.
(17) Klod, S.; Ziegs, F.; Dunsch, L. Anal. Chem. 2009, 81, 10262−
10267.
(18) Albert, K.; Dreher, E. L.; Straub, H.; Rieker, A. Magn. Reson.
Chem. 1987, 25, 919−922.
(19) Madsen, K. G.; Skonberg, C.; Jurva, U.; Cornett, C.; Hansen, S.
H.; Johansen, T. N.; Olsen, J. Chem. Res. Toxicol. 2008, 21, 1107−19.
(20) Thevis, M.; Lohmann, W.; Schrader, Y.; Kohler, M.; Bornatsch,
W.; Karst, U.; Schanzer, W. Eur. J. Mass Spectrom. 2008, 14, 163−170.
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dx.doi.org/10.1021/ac302152a | Anal. Chem. XXXX, XXX, XXX−XXX