lohmann 2006
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ORIGINAL PAPER
Simulation of the detoxification of paracetamol using on-line
electrochemistry/liquid chromatography/mass spectrometry
Wiebke Lohmann & Uwe Karst
Received: 26 May 2006 / Revised: 17 August 2006 / Accepted: 21 August 2006 / Published online: 13 October 2006# Springer-Verlag 2006
Abstract On-line electrochemistry/liquid chromatography/
mass spectrometry was used to simulate the detoxification
mechanism of paracetamol in the body. In an electrochem-
ical flow-through cell, paracetamol was oxidized at a
porous glassy carbon working electrode at a potential of
600 mV vs. Pd/H2 with formation of a quinoneimine
intermediate. The quinoneimine further reacted with gluta-
thione and/or N -acetylcysteine to form isomeric adducts via
the thiol function. The adducts were characterized on-line
by liquid chromatography/mass spectrometry. These reac-
tions are similar to those occurring between paracetamol
and glutathione under catalysis by cytochrome P450
enzymes in the body.
Keywords Paracetamol (acetaminophen,
p-acetamidophenol) . Detoxification . N -Acetylcysteine .
Glutathione . Electrochemistry . Liquid chromatography/
mass spectrometry
Introduction
Paracetamol (acetaminophen, p-acetamidophenol, APAP)
has been used since the 1950s as an antipyretic and
analgesic drug, and it can now be obtained as an over-the-counter (OTC) remedy without prescription. The metabol-
ic pathway of APAP in man has been investigated in
numerous studies. Prescott [1] summarized the available
knowledge of APAP metabolism in adults. The majority of
APAP is metabolized and excreted in a nontoxic pathway
via glucuronidation (50 – 60%) and sulfate conjugation
(25 – 35%), whereas only a small amount (2 – 10%) is
subjected to an oxidative metabolic pathway resulting in
the toxic metabolite N -acetyl- p-benzoquinoneimine
(NAPQI) and 3-hydroxy-paracetamol [1]. Apart from the
listed APAP conjugates and cysteine and mercapturic acid
metabolites resulting from NAPQI conjugation, APAP
itself is found unchanged in urine of patients. Generally,
APAP is a harmless drug as long as the intake does not
exceed therapeutic doses. However, an APAP overdose
causes hepatotoxicity [2, 3]. The metabolite evoking
hepatotoxicity has been identified by Dahlin et al. [4] as
the highly reactive electrophile intermediate NAPQI. The
formation of NAPQI occurs in the mixed-function oxidase
system cytochrome P450 (CYP). CYP2E1, CYP1A2, and
CYP3A4 are the liver microsomal enzymes, which are
mostly involved in the conversion of APAP into NAPQI
[5, 6]. As a soft electrophile, NAPQI is normally
detoxified by conjugation with reduced glutathione
(GSH), finally leading to cysteine and mercapturic acid
metabolites [7]. At high doses of APAP, the nontoxicmetabolic pathway via glucuronidation and sulfate conju-
gation becomes saturated. As a result, APAP is converted
into higher amounts of NAPQI than usual. Consequently,
the formed NAPQI has to be detoxified by conjugation
with GSH in the liver, so that the total hepatic GSH is
finally depleted [8]. The only remaining reaction partners
for the reactive NAPQI are thiol groups of cellular
macromolecules to which NAPQI is bound covalently [9,
10]. The loss of protein thiol groups [11] ultimately leads
to liver cell necrosis [12], for which the threshold is
Anal Bioanal Chem (2006) 386:1701 – 1708
DOI 10.1007/s00216-006-0801-y
Awarded a Poster Prize on the occasion of the Conference of the German
Mass Spectrometric Society (DGMS) in Mainz, March 5 – 8, 2006.
W. Lohmann : U. Karst (*)
Institut für Anorganische und Analytische Chemie,
Westfälische Wilhelms-Universität Münster,
Corrensstr. 30,
48149 Münster, Germany
e-mail: [email protected]
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250 mg/kg in man [13]. The hepatotoxicity of APAP and
its metabolites is reviewed by Black [14] and more
recently by James et al. [15] and Sumioka et al. [16].
The last mentioned review also covers a summary of
protection mechanisms against APAP-induced hepatotox-
icity. One of these is the administration of cysteine
prodrugs like N -acetylcysteine (NAC), which is the most
widely used antidote in the case of an APAP overdose.The function of NAC as an antidote is (a) to increase the
availability of GSH by synthesis of GSH via cysteine, (b)
to supplement the substrate for sulfate conjugation, so that
the nontoxic metabolic pathway can be re-established, and
(c) to allow direct substitution of GSH by directly binding
and thus detoxifying NAPQI via thioether formation [17].
Since APAP has been known to be an electroactive
molecule for a long time [18] and the mechanism of
electrochemical oxidation has been studied systematically
[19], on-line electrochemistry/mass spectrometry (EC/MS)
seems to be a valuable tool for early-stage metabolism
studies. Mimicking the CYP-induced metabolism byelectrochemistry as a purely instrumental technique with
a simple setup represents a much cheaper and faster
technique than the commonly used in vivo and in vitro
methods using liver cells or isolated enzymes in metabo-
lite discovery. On-line EC/MS is therefore well suited for
the detection and identification of possible metabolites in
an early stage of the development process of pharmaceu-
ticals. Especially in high-throughput screening, EC/MS is
superior to the conventional methods, as it can be
completely automated. Due to these aspects, on-line EC/
MS is of particular importance for pharmaceutical indus-
try. Even though the EC/MS results are not unrestrictedly
transferable to the situation in the human liver, this
methodology may provide first clues to the metabolism
of pharmaceuticals in the human body.
First achievements in simulating CYP-induced reac-
tions, which represent the main pathway in enzymatically
eliminating xenobiotics from the body, by electrochemistry
were obtained by Hanzlik et al. several years ago [20, 21].
N -Demethylation was the major focus in these studies, in
which the EC model was compared to enzymatic mech-
anisms. However, this method is not suitable to clarify the
processes occurring in complex reaction mixtures. The on-
line combination of electrochemistry with mass spectrom-
etry (EC/MS) was presented by Getek et al. [22] at the
same time. They used a coulometric flow-through cell
with a glassy carbon working electrode with large surface
area hyphenated to a thermospray MS with the aim to
study the phase I and II metabolism of APAP. Apart from
phase I metabolites NAPQI and p-benzoquinone, APAP
conjugates with the bioavailable thiols GSH and cysteine
were detected. The EC mechanism of NAPQI formation
via a two-electron oxidation step followed by thiol
conjugation was comparable to the enzymatic reaction
pathway induced by CYP. The redox behavior of dopa-
mine and its conjugation reactions with GSH and NAC
were targets in studies using off-line EC setups [23, 24].
The dopamine redox system and following reactions with
benzene thiol as model system for biogenic nucleophiles
were further investigated by Deng and Van Berkel [25]
using a home-made thin-layer EC flow cell coupled on-line to electrospray (ESI) MS. A strong increase in the
research field of EC/MS on-line coupling was observed in
the last few years: Gamache et al. [26] studied metabolic
phase I and II reactions using on-line hyphenation of
coulometric flow-through cells with MS as a rapid
detection technique for different biologically relevant
conversions. The focus on N -demethylation, hydroxyl-
ation, oxidation, and thioether conjugation in that work
was extended by Bruins and coworkers [27, 28] by
examining of the whole potential of electrochemistry
concerning the simulation of oxidative CYP phase I
metabolism in more detail.These studies are valuable tools in the investigation of
the oxidative metabolism of pharmaceuticals in the early
phase of drug discovery. However, important information
about the polarity of the oxidation products or formation of
different isomers, which often show similar fragmentation
patterns in tandem MS, cannot be obtained by only EC/
MS. A practical approach to get further insight into the
nature of oxidation products is coupling EC to liquid
chromatography/mass spectrometry (LC/MS). With this
EC/LC/MS setup, much broader information about the
electrochemically generated products can be obtained.
Brajter-Toth and coworkers [29] reviewed studies on this
field of work for different analytes, but with exclusion of
pharmaceuticals. More recent overviews were published
by Hayen and Karst [30] and Karst [31]. The same authors
recently described an EC/LC/MS setup for the analysis of
the metabolites of phenothiazine and its derivatives.
However, this work focused on the quantification of these
substances [32]. A first paper on the real simulation of
phase I and II metabolism of clozapine based on on-line-
LC/EC/MS was released recently in cooperation of the
groups of Karst and Kauffmann [33]. Different phase I
oxidation products and phase II GSH conjugates were
generated by EC, separated by LC, and identified by
means of tandem MS. Since the EC behavior of APAP has
been thoroughly investigated in different studies, APAP is
well suited as model compound for further research on the
potential of the EC/LC/MS setup. Furthermore, no inves-
tigations concerning detoxification metabolism pathways
of pharmaceuticals and studies about the toxication/
detoxification competition in the body have been carried
out by on-line-EC/LC/MS yet. Therefore, the respective
work is described within this paper.
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Experimental
Chemicals
4-Acetamidophenol was obtained from Acros Organics
(Geel, Belgium). Reduced L-glutathione and N -acetyl-L-
cysteine were purchased from Sigma-Aldrich Chemie
GmbH (Steinheim, Germany). Ammonium acetate andacetic acid were ordered from Fluka Chemie GmbH
(Buchs, Switzerland) in the highest quality available.
Methanol for HPLC was obtained from Merck KGaA
(Darmstadt, Germany). Water used for HPLC was purified
using a Milli-Q Gradient A 10 system and filtered through a
0.22-μ m Millipak 40 (Millipore, Billerica, MA, USA).
Instruments
The equipment used for the EC oxidations was obtained
from ESA Biosciences Inc. (Chelmsford, MA, USA). It
consisted of a model 5020 guard cell and a model 70 –
2170GuardStat. The guard cell contained a glassy carbon
working electrode, a Pd counter electrode, and a Pd/H2
reference electrode. A PEEK in-line filter was placed in
front of the guard cell inlet to protect the working electrode.
The LC/MS setup comprised a Shimadzu (Duisburg,
Germany) LC system and an API 2000 mass spectrometer
(Applied Biosystems, Darmstadt, Germany), equipped with
an electrospray ionization (ESI) source. The LC system
consisted of two LC-10ADVP pumps, a DGC-14A degasser,
a SIL-HTVP autosampler, a CTO-10AVP column oven, and a
SPD-10AVVP UV detector. The software used for control-
ling LC and MS was Analyst 1.4.1 (Applied Biosystems).
Analysis
The mobile phases for the EC/(LC)/MS measurements were
methanol and aqueous buffer. The buffer was prepared in
20 mM ammonium acetate, and the pH was adjusted to pH
7 with acetic acid. EC conversions were carried out at a
potential of 600 mV vs. Pd/H2. The flow rate for the flow-
injection EC/MS measurements was 0.3 mL/min. The
electrochemically generated products were separated on a
ProntoSIL phenyl column (Bischoff, Leonberg, Germany).
Column dimensions were 250×2.0 mm; the particle size
was 5 μ m. A gradient system with methanol (B) and
aqueous buffer (A) was used; the time program is presented
in Table 1.
The injection volume was set to 10 μ L. The eluting
analytes were ionized in the ESI interface in the negative
ion mode with 30 psi nebulizer gas, 50 psi dry gas/heating
gas with a temperature of 400 °C, and an ionspray voltage
of −4,500 V. The declustering potential was set to −20 V,
the focusing potential to −400 V, and the entrance potential
to −2 V. The mass range for full scan experiments was m/z
100 –
1,000. Tandem MS experiments were carried out witha collision energy of −25 V, a cell exit potential of −35 V,
and a collision-activated dissociation (CAD) gas pressure of
2 psi.
Results and discussion
Initially, the behavior of APAP in the presence of GSH or
NAC at app lie d potential s of 0 and 600 mV was
investigated. These experiments were carried out only with
flow-injection EC/MS without using an LC column to
reduce the complexity of the system. In addition, the MS parameters could be optimized more rapidly. A solution
containing 10−4 M APAP and 5×10−4 M of the respective
thiol was injected into a stream of 5% methanol and 95%
buffer. The solvent stream containing the analytes was
conducted through an EC flow-through cell containing a
working electrode of porous glassy carbon. The porous
material allows for an almost quantitative conversion of the
analytes in the EC flow-through cell. The conversion rate is
affected by flow rate and pH of the mobile phase as well as
the nature of the analyte. In Fig. 1, the mass spectra of
APAP in the presence of a fivefold excess of GSH at EC
potentials of 0 mV and 600 mV vs. Pd/H2 are shown. As
expected, no additional signals besides those associated
with APAP and GSH were observed at 0 mV (Fig. 1). The
strongest signal at m/z 306 was traced back to the [M−H]−
of GSH. The isotopic pattern correlates well with theoret-
ical calculations. The signal at m/z 613 originated from a
non-covalent dimer [M2−H]− of GSH. This assumption was
confirmed by the fact that the m/z 613 peak decreased with
increasing declustering potential, while the m/z 306 peak
increased. Furthermore, later measurements using an LC
column showed that the two peaks with these m/z ratios
coelute at any selected gradient. After applying a potential
of 600 mV, at which APAP shows an oxidation wave in
cyclovoltammetric measurements carried out in past studies
[22], three major additional signals were observed (Fig. 1).
The peaks with m/z 338 and 611 derived from oxidation of
GSH itself, since the formation of these analytes also was
observed when GSH alone is exposed to a potential of
600 mV (data not shown). Although an unambiguous
identification of the m/z 338 peak could not be achieved,
the mass gain of 32 with respect to GSH itself possibly
indicates oxidation at the free thiol group resulting in the
Table 1 Gradient profile used for HPLC separation
Parameters Values
Time (min) 0 10 11 12 13 14
c(B) (%) 5 5 30 30 5 Stop
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formation of a – SO2H group. The peak at m/z 611 was
generated by the GSH disulfide. The highest additional
signal at m/z 455 indicated the formation of an APAP – GSH
adduct. Notably, neither hydroxylated APAP products nor
methanol adducts were observed in this experiment.
Analogous experiments were carried out with a solution
containing 10−4 M APAP and 5×10−4 M NAC as potential
coupling partner. Coupling reactions with APAP and NAC
are interesting, because NAC is a commonly administeredantidote in the case of an APAP overdose [8]. At 0 mV,
only three relevant signals were observed (Fig. 2): the peak
at m/z 162 is the [M−H]− of NAC; a signal for APAP itself
occurs at m/z 150, i.e. [M−H]−, with a weak intensity; and
the signal at m/z 325 is traced back to a non-covalent dimer
of NAC, corresponding to the m/z 613 in the measurements
concerning GSH. As for GSH, formation of the NAC dimer
could be reduced by increasing the declustering potential.
When the EC cell was switched on and a potential of
600 mV vs. Pd/H2 was applied, some additional peaks were
observed in the mass spectrum. The signals at m/z 194 and
323 represented the oxidation of NAC itself, corresponding
to the m/z 338 and 611 at GSH oxidation, and were also
observed when a NAC solution without APAP was injected
into the EC cell. No further identification of the signal at
m/z 194 was possible. A gain of two oxygen atoms is likely
due to a mass increment of 32, corresponding to NAC,
which possibly indicates the formation of a – SO2H group
analogous to the situation with GSH as described above.
The peak at m/z 323 originated from the [M−
H]
−
of the NAC disulfide. As in the case of the GSH – APAP system, a
NAC – APAP adduct was observed in these experiments
with a [M−H]− at m/z 311.
As a final flow-injection experiment, a solution contain-
ing both GSH and NAC at concentrations of 5×10−4
M
each and APAP at 10−4 M was injected into the solvent
stream leading through the EC cell (data not shown).
Interestingly, in addition to the signals already identified at
a potential of 600 mV, a new signal at m/z 467 was
observed. This signal derived from the [M−H]− of a mixed
Fig. 1 Mass spectra obtained after electrochemical oxidation of
APAP in the presence of a fivefold excess of GSH in 5% MeOH/
95% 20 mM aqueous ammonium acetate buffer of pH 7 at 0 mV and600 mV vs. Pd/H2
Fig. 2 Mass spectra of electrochemical oxidation of APAP in the
presence of a fivefold excess of NAC in 5% MeOH/95% 20 mM
aqueous ammonium acetate buffer of pH 7 at 0 mV and 600 mV
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dimer of GSH and NAC. Experiments regarding the
behavior of both GSH and NAC in the presence of APAP
offer an interesting approach to metabolism studies of
APAP, because NAC is administered as an antidote in the
case of APAP overdoses followed by GSH depletion in the
liver and replaces GSH in its role in the APAP degradation
process.
The chemical reactions occurring in the electrochemicalcell are summarized in Fig. 3. The oxidation of APAP is
induced by the loss of one electron and one proton,
resulting in an intermediate radical. As a follow-up
reaction, a second electron and proton are transferred
leading to formation of NAPQI. The formation of the
highly conjugated NAPQI and follow-up reactions in the
course of EC oxidation are well known in the literature
[22], but NAPQI itself could not be observed in the mass
spectra of the flow-injection experiments, which were
carried out in the negative ion mode. NAPQI lacks of an
acidic proton and therefore should not be ionized to a large
extent in the negative ion mode. By further reaction withone molecule of water, NAPQI is hydroxylated at the para-
position of the aromatic ring, resulting in an intermediate,
which can be deacetylated to 1,4-benzoquinone [22]. In the
presence of thiol-containing molecules like GSH or NAC,
NAPQI is quenched by adduct formation. This mechanism
is the common detoxification pathway of NAPQI in the
human liver [7]. The mass spectrometric measurements
showed only formation of GSH and NAC monoadducts,
meaning that either the oxidation potential of the formed
adducts was too high to undergo a second oxidation cycle
or the used thiols were not reactive enough to quench a
benzoquinoneimine molecule already substituted by GSH
or NAC. One reason for this might be the steric hindrance
which would occur in the case of a disubstitution of one
APAP molecule, since disubstitution is in principle possi-
ble, as was demonstrated in additional experiments with the
sterically less demanding 1-propanethiol as quencher for
NAPQI.
Experiments including separation of the reaction prod-
ucts on an LC phenyl column were carried out using asolution containing GSH and NAC both at concentrations
of 5×10−4 M, and APAP at a concentration of 10−4 M. The
mobile phase consisted of an aqueous ammonium acetate
buffer with low amounts of methanol. With the EC cell
switched off, only three peaks resulting from APAP, GSH,
and NAC were observed (Fig. 4). For comparison to the
chromatogram at 0 mV, the chromatogram resulting from
an oxidation of the analytes at 600 mV vs. Pd/H2 is shown
in Fig. 5. The m/z 150 peak for APAP was much smaller at
600 mV, indicating that APAP was converted almost
completely into different reaction products. The mass traces
for m/z 162 and 306, representing NAC and GSH, alsoshowed that the thiols were partly depleted in or after the
EC cell. The m/z 311 mass trace for the APAP – NAC adduct
showed two resolved peaks at retention times of 7.0 and
8.7 min. The first eluting peak was much smaller than the
second one. It can be concluded from the appearance of two
peaks instead of one single peak that different isomers of
the NAC – APAP conjugate were formed in the EC cell. In
general, NAC has two possible reaction sites in the NAPQI
molecule, namely, the 2- and 3-substitution positions. In the
human body, only the 2- N -acetylcysteinyl paracetamol is
formed, which is depicted in Fig. 3. Since a selective
enzymatic system containing CYP is involved in the adduct
Fig. 3 Reaction scheme for
electrochemical oxidation of
APAP in the presence of GSH
and APAP
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formation in vivo, the selectivity of the conjugate formation
in vitro, resulting in two different substitution isomers, is
reduced compared to the enzyme-catalyzed reaction, which
yields only the 2-substitution product. The assignment of
the two different adducts to the substitution positions was
not possible based on these measurements, but would rather require the use of NMR spectroscopy. The mass trace for
m/z 455 represents the APAP conjugate with GSH and
exhibited a similar picture as the previous mass trace. Two
peaks were well resolved, presumably corresponding to
2- and 3-glutathionyl paracetamol. As for the NAC adduct,
the first eluting peak at 4.1 min was smaller than the second
eluting peak at a retention time of 6.6 min. The retention
time difference between these two peaks is more than
2 min, which is surprising for a set of two isomers in a
compound of this size. The nature of the NAC and the GSH
adducts was further confirmed by tandem MS experiments.
The daughter ion spectra of all conjugates mostly showed afragment ion at m/z 182, representing the loss of the NAC
or the GSH moiety without the sulfur atom. The last three
mass traces in Fig. 5 for m/z 323, 611, and 467 represent
the dimers of NAC, GSH, and the mixed dimer of NAC and
GSH, respectively, which were generated in the EC cell as
well. Each of them eluted at very low retention times and it
Fig. 4 LC/MS chromatograms
of a mixture containing APAP
(10−4 M), GSH (5×10−4 M), and
NAC (5×10−4 M) without elec-
trochemical conversion with
mass traces of APAP (m/z 150),
NAC (m/z 162), and GSH (m/z
306) and combined TIC from
these mass traces
Fig. 5 LC/MS chromatograms
of a mixture containing APAP
(10−4 M), GSH (5×10−4 M), and
NAC (5×10−4 M) at 600 mV
with mass traces of APAP (m/z
150), NAC (m/z 162), GSH (m/z
306), NAC conjugates of APAP
(m/z 311), GSH conjugates of
APAP (m/z 455), NAC dimer
(m/z 323), GSH dimer (m/z 611),
and a mixed dimer of NAC and
GSH (m/z 467) as well as the
combined TIC from these mass
traces
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was not possible to improve the resolution of these three
peaks under reversed-phase conditions due to the rather high
polarity of the respective compounds.
To investigate the reactivity differences between GSH
and NAC, further EC/LC/MS experiments were carried out
using the same conditions as before. Initially, the influence
of the excess of the respective thiol on the formation of the
APAP adducts was investigated. Solutions containing anexcess of the thiol in the range from 0.1 to 100 relative to
APAP were injected into the EC cell at a potential of
600 mV vs. Pd/H2. After the LC separation step and MS
detection in the selected ion monitoring mode, the peak
area of the formed APAP conjugates was plotted versus the
excess of the respective thiol (Fig. 6). For this purpose, the
peak areas of the two different isomers of the NAC and
the GSH conjugate were summarized. During the increase
of the excess of thiol, the relative contribution of the two
isomers changed in such a way that the amount of the first
eluting isomer slightly increased compared to the second
eluting isomer. All measurements were carried out threetimes to investigate the reproducibility of the experiments.
The relative standard deviations were generally less than
10%, which is acceptable for this experiment, which
should only estimate the reactivity differences between
GSH and NAC. As the ionization properties of the
conjugates are unknown, no conclusions concerning the
absolute amount of the reaction products could be drawn.
Presumably, the response of the GSH adduct is lower than
the response of the NAC adduct, since the same amounts of
GSH and NAC give a signal which is approximately half as
high as the signal for NAC (see Fig. 4) and the adducts
differ only in the side chain. For the NAC adduct, a
saturation of adduct formation was observed for a tenfold
and larger excess over APAP. Only for a hundredfold
excess of NAC, the amount of formed adducts was slightly
reduced. For the GSH conjugates, a decrease of the
concentration of the formed adducts was observed with
increasing GSH concentration starting from a fivefold
excess over APAP. The decline of the peak areas was
possibly a result of the simultaneous oxidation of APAP
and the respective thiol, which may be regarded as
competitive processes occurring in the EC cell. In the
presence of a highly concentrated electroactive compoundsuch as the thiols, the oxidation capacity of the EC cell can
get saturated, thus resulting in a lower amount of NAPQI
and the respective conjugate being formed.
The following experiments were carried out to compare
the reactivity of NAC and GSH with NAPQI. A solution of
10−4 M APAP containing an excess of either NAC or GSH
in the range from 0 to 10 was conducted through the EC
cell at a potential of 600 mV. In this experiment, all the
formed NAPQI ended up as either the NAC or the GSH
adduct in all subsequent reactions. The saturation already
described in the previous paragraph was observed here,
too. In a second experiment, both NAC and GSH weresimultaneously added to a 10−
4M solution of APAP so
that the total excess of thiol was 10. In this setup, NAC
and GSH have to compete for the NAPQI molecules in the
EC cell to form their respective adducts. The peak areas of
the isomeric adducts are summarized in Fig. 7. The ratio
of the peak area of the GSH adducts derived when no
NAC was present to the peak area of the GSH adducts
derived when NAC was present was calculated and plotted
against the excess of GSH relatively to APAP. Accord-
ingly, the ratio of the peak area of the NAC adducts
derived when no GSH was present to the peak area of the
NAC add ucts derived whe n GSH was pre sen t was
Fig. 6 Peak areas of GSH and NAC conjugates of APAP as a
function of the excess of the respective thiol. Data points represent the
mean of three measurements (n=3). The error bars represent standard
deviations
Fig. 7 Ratio of the peak areas in the absence and in presence of the
respective other thiol of the GSH and NAC conjugates of APAP as a
function of the excess of the respective thiol. Data points represent the
mean of three measurements (n=3). The error bars represent standard
deviations
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calculated and plotted against the excess of NAC
relatively to APAP. Obviously, the values for the ratios
are equal or higher than 1, as in the presence of the
respective other thiol, a competition for the available
NAPQI molecules takes place between GSH and NAC. As
in the experiment before, all measurements were carried
out three times to investigate the reproducibility of the
experiments. Here, the relative standard deviations weregenerally less than 10%, too, which is acceptable for an
estimation of the reactivity differences between GSH and
NAC. The graph for the GSH adducts rises only slowly
when adding NAC. Up to a sixfold excess of NAC and
only a fourfold excess of GSH relative to APAP, the ratio
of the peak areas of the GSH adducts was lower than 2.
Even if the amount of NAC was nine times as high as the
amount of GSH, the peak area of the GSH conjugate still
is a third of the peak area obtained without NAC. The
graph for the NAC adduct showed a different course: it
rises much faster when adding GSH to the NAC/APAP
system compared to the graph for the GSH adducts. Thismeans that addition of even a small amount of GSH had a
strong influence on the NAC conjugate formation and that
the amount of the GSH conjugates is higher than it would
be if GSH and NAC had the same reactivity towards
NAPQI. From these experiments it could be concluded
that GSH had a higher reactivity than NAC towards the
electrochemically generated NAPQI. Even small amounts
of GSH added to an NAC/APAP system resulted in a
significant decrease in the formation of the NAC adduct,
whereas addition of a small amount of NAC to the GSH/
APAP system affected the formation of a GSH conjugate
to a lesser extent.
Conclusions
The oxidative metabolic detoxification pathway of APAP in
the human liver was successfully mimicked by EC/MS and
EC/LC/MS experiments. Phase I and II metabolites, which
were already known from the literature as detoxification
products in vivo, were generated in an EC flow-through cell
and identified by LC/MS using either APAP alone or in the
presence of glutathione and/or N -acetylcysteine. In contrast to in vivo experiments, different isomers of the formed
adducts were observed. The competition of NAC and GSH
for the substitution of electrochemically generated NAPQI
indicates that GSH is slightly more reactive towards
NAPQI. Future work shall include the further identification
of the different isomers by means of NMR spectroscopy
and on-line enzymatic conversion by coupling enzymatic or
living cell microreactors to LC/MS.
Acknowledgements Financial support by the Deutsche Forschungs-
gemeinschaft (DFG, Bonn, Germany) and the Fonds der Chemischen
Industrie (Frankfurt, Germany) is gratefully acknowledged.
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