Lohmann 2006

7/17/2019 Lohmann 2006

http://slidepdf.com/reader/full/lohmann-2006 1/8

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]

7/17/2019 Lohmann 2006


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.

1702 Anal Bioanal Chem (2006) 386:1701 – 1708

7/17/2019 Lohmann 2006


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

Anal Bioanal Chem (2006) 386:1701 – 1708 1703

7/17/2019 Lohmann 2006


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


7/17/2019 Lohmann 2006


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


7/17/2019 Lohmann 2006


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


7/17/2019 Lohmann 2006


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


7/17/2019 Lohmann 2006


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.

References

1. Prescott LF (1996) Paracetamol (Acetaminophen): a critical

bibliographic review. Taylor and Francis, London

2. Davidson DG, Eastham WN (1966) Br Med J 5512:497

3. Mitchell JR (1988) N Engl J Med 319:1601

4. Dahlin DC, Miwa GT, Lu AYH, Nelson SD (1984) Proc Nat Acad

Sci USA 81:1327

5. Raucy JL, Lasker JM, Lieber CS, Black M (1989) Arch Biochem

Biophys 271:270

6. Thummel KE, Lee CA, Kunze KL, Nelson SD, Slattery JT (1993)

Biochem Pharmacol 45:1563

7. Mitchell JR, Jollow DJ, Potter WZ, Gillette JR, Brodie BB (1973)

J Pharmacol Exp Ther 187:211

8. Prescott LF, Park J, Ballantyne A (1977) Lancet 2:4329. Jollow DJ, Mitchell JR, Potter WZ, Davis DC, Gillette JR, Brodie

BB (1973) J Pharmacol Exp Ther 187:195

10. Potter DW, Hinson JA (1986) Mol Pharmacol 30:33

11. Moore M, Thor H, Moore G, Nelson S, Moldéus P, Orrenius S

(1985) J Biol Chem 260:13035

12. Kyle ME, Sakaida I, Serroni A, Farber JL (1990) Biochem

Pharmacol 40:1211

13. Thomas SHL (1993) Pharmac Ther 60:91

14. Black M (1984) Ann Rev Med 35:577

15. James LP, Mayeux PR, Hinson JA (2003) Drug Metab Dispos

31:1499

16. Sumioka I, Matsura T, Yamada K (2004) Yonago Acta Med 47:14

17. Marzullo L (2005) Curr Opin Pediatr 17:239

18. Nash RA, Skuden DM, Purdy WC (1958) J Am Pharm Assoc

47:43319. Garcia R, Váquez MD, Barrado E, Tascón ML, Velasco E,

Sánchez-Batanero P (1989) Bull Soc Chim 1:19

20. Hall LR, Iwamoto RT, Hanzlik RP (1989) J Org Chem 54:2446

21. Hall LR, Hanzlik RP (1990) J Biol Chem 265:12349

22. Getek TA, Korfmacher WA, McRae TA, Hinson JA (1989)

J Chromatogr 474:245

23. Zhang F, Dryhurst G (1995) J Electroanal Chem 398:117

24. Xu R, Huang X, Kramer KJ, Hawley MD (1996) Bioorg Chem

24:110

25. Deng H, Van Berkel GJ (1999) Electroanalysis 11:857

26. Gamache P, Smith R, McCarthy R, Waraska J, Acworth I (2003)

Spectroscopy 18:14

27. Jurva U, Wikström HV, Bruins AP (2000) Rapid Commun Mass

Spectrom 14:529

28. Jurva U, Wikström HV, Weidolf L, Bruins AP (2003) RapidCommun Mass Spectrom 17:800

29. Volk KJ, Yost RA, Brajter-Toth A (1992) Anal Chem 64:21A

30. Hayen H, Karst U (2003) J Chromatogr A 1000:549

31. Karst U (2004) Angew Chem Int Ed 43:2476

32. Hayen H, Karst U (2003) Anal Chem 75:4833

33. Van Leeuwen SM, Blankert B, Kauffmann JM, Karst U (2005)

Anal Bioanal Chem 382:742


Documents

Lohmann 2006