Accepted Manuscript

Title: Trapping of NAPQI, the intermediate toxic paracetamol

metabolite, by aqueous sulphide (S2− ) and analysis by
GC-MS/MS

Author: Arne Trettin Sandor Batkai Thomas Thum Jens

Jordan Dimitrios Tsikas

PII: S1570-0232(14)00365-1
DOI: http://dx.doi.org/doi:10.1016/j.jchromb.2014.05.050
Reference: CHROMB 18973

To appear in: Journal of Chromatography B

Received date: 10-1-2014

Revised date: 19-5-2014
Accepted date: 23-5-2014

Please cite this article as: A. Trettin, S. Batkai, T. Thum, J. Jordan, D. Tsikas,
Trapping of NAPQI, the intermediate toxic paracetamol metabolite, by aqueous
sulphide (S2minus ) and analysis by GC-MS/MS, Journal of Chromatography B (2014),
http://dx.doi.org/10.1016/j.jchromb.2014.05.050

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Trapping of NAPQI, the intermediate toxic paracetamol metabolite, by aqueous

sulphide (S2−) and analysis by GC-MS/MS

Arne Trettin,1 Sandor Batkai,2 Thomas Thum,2,3 Jens Jordan,1 Dimitrios Tsikas1,*

t
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1
Institute of Clinical Pharmacology, Hannover Medical School, 30625 Hannover, Germany

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2
Institute of Molecular and Translational Therapeutic Strategies, IFB-TX, Hannover

Medical School, 30625 Hannover, Germany

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2,3
Excellence Cluster REBIRTH, Hannover Medical School, 30625 Hannover, Germany

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Correspondence address
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Prof. Dimitrios Tsikas

Institute of Clinical Pharmacology

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Hannover Medical School

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Carl-Neuberg-Strasse 1

30625 Hannover
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Germany

Phone: 49-511-532-3984

Fax: 49-511-532-2750

E-mail: tsikas.dimitros@mh-hannover.de

*
Corresponding author. Tel.: +49 511 532 3984; fax: +49 511 532 2750. E-mail address:
tsikas.dimitros@mh-hannover.de (D. Tsikas)

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Page 1 of 27
ABSTRACT

NAPQI, i.e., N-acetyl-p-benzoquinone imine, is considered the toxic metabolite of the

widely used analgesic drug paracetamol (acetaminophen, APAP). Due to its high

reactivity towards nucleophiles both in low- and in high-molecular-mass biomolecules,

t
ip
NAPQI is hardly detectable in its native form. Upon conjugation with glutathione, NAPQI

is finally excreted in the urine as the paracetamol mercapturic acid. Thus, determination

cr
of paracetamol mercapturate may provide a measure of in vivo NAPQI formation. In this

work, we propose the use of Na2S in aqueous solution to trap NAPQI and to analyze the

us
reaction product, i.e., 3-thio-paracetamol, together with paracetamol by GC-MS/MS in

the electron-capture negative-ion chemical ionization mode after solvent extraction with

an
ethyl acetate and derivatization with pentafluorobenzyl bromide. In mechanistic studies,

we used newly synthesized N-acetyl-p-[2,3,5,6-2H4]benzoquinone imine (d4-NAPQI). In

M
quantitative analyses, N-(4-hydroxyphenyl)-[2,3,5,6-2H4]acetamide (d4-APAP) was used

as the internal standard both for NAPQI and APAP. 3-Thio-d3-paracetamol, prepared from
d

d4-NAPQI and Na2S, may also be useful as an internal standard. We showed NAPQI in
te

vitro formation from APAP by recombinant cyclooxygenase-1 as well as by dog liver

homogenate. In vivo formation of NAPQI was demonstrated in mice given paracetamol

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intraperitoneally (about 150 mg/kg).

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Keywords: Acetaminophen; Liver; Mice; NAPQI; Quantification; 3-Thio-paracetamol

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Page 2 of 27
1. Introduction

Paracetamol (acetaminophen; N-(4-hydroxyphenyl)acetamide; APAP) is among

the most frequently used analgesic drugs. Paracetamol’s chemistry, biochemistry and

pharmacology are complex [1-5]. Native paracetamol is rapidly and extensively

t
ip
metabolized via conjugation reactions to its glucuronic and sulfuric acids and is

eliminated by the kidney. Paracetamol is also oxidized by the cytochrome P450 (CYP450)

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family to N-acetyl-p-benzoquinone imine (NAPQI; Scheme 1). NAPQI is a chemically very

reactive electrophilic species and is therefore considered the intermediate toxic

us
metabolite of paracetamol. NAPQI is inactivated by conjugation with the tripeptide

glutathione (GSH) and is excreted after metabolization as the mercapturic acid in urine

covalent binding to nucleophilic

an
[1-5]. NAPQI undergoes numerous reactions in biological systems. These reactions

include sites of biomolecules, autoreduction to

M
paracetamol, and dimerisation and/or polymerisation. NAPQI formation in biological

samples is evidenced by measuring the stable GSH conjugate and/or its metabolites
d

including mercapturic acid. Given the high electrophilicity of NAPQI and its affinity to
te

biothiols such as GSH and N-acetylcysteine (NAC), we reasoned that use of inorganic

thiols, notably the small thiol sulphide (S2−) should be a much better alternative, both, to
p

trap NAPQI during its formation and to quantify the resulting thio-paracetamol (N-(3-
ce

thiol-4-hydroxyphenyl)acetamide) by gas chromatography-mass spectrometry (GC-MS)

or gas chromatography-tandem mass spectrometry (GC-MS/MS). In the present article,

Ac

we demonstrate that aqueous Na2S is a useful reagent for high-extent trapping of NAPQI

and subsequent highly sensitive GC-MS/MS quantification of 3-thio-paracetamol as

pentafluorobenzyl derivative using N-(4-hydroxyphenyl-[2,3,5,6-2H4])acetamide (d4-

paracetamol) as the internal standard (Scheme 1).

2. Experimental

2.1. Chemicals and materials

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Page 3 of 27
 Unlabelled paracetamol (N-(4-hydroxyphenyl)-acetamide; d0-APAP) and Na2S

were obtained from Merck (Darmstadt, germany). N-(4-Hydroxyphenyl)-[2,3,5,6-

2
H4]acetamide (d4-APAP; 95%; 99.4 atom% 2H) was purchased from CDN Isotopes

(Quebec, Canada). N-Acetyl-p-benzoquinone imine (NAPQI), arachidonic acid, 2,3,4,5,6-

t
ip
pentafluorobenzyl bromide (99%), N,N-diisopropylethylamine (95%) and reduced

glutathione (GSH, 99%) were bought from Sigma-Aldrich (Munich, Germany). Silver

cr
nitrate (99.9%) came from Carl Roth (Karlsruhe, Germany). Recombinant ovine

cyclooxygenase-1 (COX-1) was obtained from Cayman Chemicals (Ann Arbor, MI, USA).

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NADPH tetrasodium salt (98%) was received from Roche (Rotkreuz, Schwitzerland).

2.2.

d3-paracetamol
an
Synthesis of N-acetyl-p-[2,3,5,6-2H4]benzoquinone imine (d4-NAPQI) and 3-thio-
M
N-Acetyl-p-[2,3,5,6-2H4]benzoquinone imine (d4-NAPQI) was newly synthesized
d

following procedures originally reported for unlabelled NAPQI [6, 7]. Briefly, silver oxide
te

(Ag2O) was freshly prepared from a 10 wt.% silver nitrate solution and precipitation with

dropwise addition of 1 M NaOH. The supernatant was decanted and the obtained silver
p

oxide was washed over filter paper several times with small amounts of water. Water was
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removed by adding a few drops of absolute ethanol and subsequent evaporation under a

nitrogen stream. The dry powder obtained was then stored in a desiccator over silica gel
Ac

for 24 h at room temperature. The washed and dried silver oxide (150 mg) together with

d4-APAP (100 mg) and a small amount of activated charcoal (three spatula tips) was

incubated in chloroform (5 mL), under constant stirring at room temperature for 25 min.

d4-NAPQI formed during this procedure was isolated by silica chromatography and elution

with ethyl acetate. Following solvent evaporation, the yellow residue was dissolved in

acetonitrile (dried over molsieve) and stored at -20 °C. d4-NAPQI yield was 21 ± 5 % (n

= 3) as determined by GC-MS analysis (see below) using commercially available

unlabelled NAPQI (d0-NAPQI). For the preparation of 3-thio-d3-paracetamol, newly

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Page 4 of 27
prepared d4-NAPQI was added to a 1 mM Na2S solution in 100 mM phosphate buffered

saline (PBS), pH 7.4, to reach a final concentration of 100 µM, and the mixture was

incubated for 15 min at room temperature. Assuming complete reaction, the 3-thio-d3-

paracetamol concentration in the reaction mixture would be 100 µM.

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2.3. Experiments with recombinant COX-1

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COX-1-catalyzed reactions and respective controls were performed under aerobic

conditions at 37 °C in 100 mM sodium/potassium phosphate buffer (pH 8.0) as described

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elsewhere [8]. The buffer contained 5 U COX-1, 10 µM arachidonic acid, 2 mM phenol, 5

mM EDTA and 1 µM haematin. Reaction mixtures were incubated with paracetamol (100

an
µM) for 10 min. As a positive control for NAPQI formation, commercially available NAPQI

was added to the COX-1 incubation mixture in the absence and in the presence of COX-1.
M
To trap potentially formed NAPQI, samples were taken after 10 min or as appropriate,

treated with Na2S (1 mM in 100 mM PBS), derivatized with PFB-Br and analyzed by GC-
d

MS as described below.
te

2.4. Dog experiment

p
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NAPQI formation from APAP was investigated in dog liver homogenate. Frozen dog

liver were thawed and homogenized by a Precellys 24 peglab® (three times for 20 s,
Ac

5500 rpm, 2-8 °C) with PBS. 1 mM NADPH as coenzyme for CYP450 was added and

incubated for 5 min at 37 °C. After addition of 100 µM APAP samples were taken at

different times, treated with Na2S (1 mM in 100 mM PBS), derivatized with PFB-Br and

analyzed by GC-MS as described below. Dog liver tissue was kindly donated for research

purposes by the Hannover Veterinary School.

2.5. Mouse experiment

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Page 5 of 27
 Paracetamol was injected intraperitoneally (1 mL of a 25 mM APAP solution in

physiological saline, i.e., 3.8 mg), into two 10-weeks old male mice (C57Bl/6N; mouse A,

27 g; mouse B, 23 g), resulting in dosages of 140 mg/kg and 164 mg/kg, respectively.

At different time points (0, 10, 20, 30, 60, 90, 120, 180 and 240 min), a small blood

droplet (about 10 µL) was collected from the tail in 2-mL Eppendorf tubes. The time point

t
ip
0 min corresponds to the sampling immediately before paracetamol administration. At

time point 240 min, venous blood was collected after euthanasia by cervical dislocation.

cr
Blood samples were treated immediately with Na2S (10 µL, 10 mM) to trap potentially

formed NAPQI, incubated at room temperature for 15 min, and reaction products were

us
extracted by rigorous vortexing with ethyl acetate (300 µL). After centrifugation (800×g,

5 min, 4 °C), the organic phase was separated and the solvent was evaporated to

an
dryness. Subsequently, PFB-Br derivatization was performed as described below. This

study was performed at the Institute of Molecular and Translational Therapeutic

M
Strategies of the Hannover Medical School and was approved by the local supervisory

committee for studies in animals (Hannover, Germany).

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2.6. Sample preparation and derivatization with PFB-Br for GC-MS and GC-MS/MS

analysis
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Reactions of NAPQI and Na2S were performed in 100 mM sodium/potassium

phosphate buffer (pH 7.4.) at room temperature (incubation for 15 min). Ethyl acetate
Ac

was added to the reaction product and extraction was performed by rigorous vortexing

for 2 min. Subsequently, sample was centrifuged (800×g, 5 min, 4 °C) and the

supernatant was transferred into glass vials. Ethyl acetate was completely evaporated

under a nitrogen stream. PFB-Br derivatization was performed by taking up the sample in

anhydrous acetonitrile (100 µL) in the presence of N,N-diisopropylethylamine (10 µL)

which served as the catalyst and PFB-Br (10 µL of a 30 vol.% solution in anhydrous

acetonitrile) as described recently for paracetamol [9, 10]. The reaction mixture was

incubated at 30 °C for 1 h. Then, solvent and reagent were evaporated to dryness under

6
Page 6 of 27
a stream of nitrogen gas. For GC-MS analysis the residues were taken up with toluene

(100 µL). All samples were stored at 6 °C.

2.7. GC-MS and GC-MS/MS conditions

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GC-MS and GC-MS/MS analyses were performed on a triple-stage quadrupole

(TSQ) mass spectrometer ThermoElectron TSQ 7000 (Finnigan MAT, San Jose, CA) as

cr
described previously for paracetamol [9, 10]. Electron-capture negative-ion chemical

ionisation (ECNICI) was performed. MS spectra were generated by scanning m/z 40 to

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m/z 800 at 1 s/scan. MS/MS and spectra were generated by subjecting selected parent

ions to collision-induced dissociation (CID) with argon as the collision gas (2 mTorr) and

an
scanning the third quadrupole m/z 40 to m/z 800 at 1 s/scan. Quantification was

performed by selected-reaction monitoring (SRM) of suitable mass transitions as

M
described below. GC separation was performed on the column Optima 17 MS (30 m ×

0.25 mm i.d., 0.25-µm film thickness) from Macherey-Nagel (Düren, Germany). Other
d

conditions were as described previously for paracetamol [9, 10].

p te

3. Results
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3.1. Reaction of NAPQI and d4-NAPQI with Na2S in aqueous buffer to form 3-thio-
Ac

paracetamol respectively 3-thio-d3-paracetamol

Incubation of NAPQI in aqueous buffer in the absence of any other reagents

resulted in formation of paracetamol and other unidentified minor reaction products as

analyzed by HPLC with UV absorbance detection at 254 nm (data not shown). GC-MS

analysis of derivatized extracts from the treatment of NAPQI with Na2S in aqueous buffer

resulted in three major GC peaks. One of this peak was identified as the paracetamol O-

PFB derivative (data not shown). The GC-MS spectra of the other two GC peaks assigned

7
Page 7 of 27
as NAPQI-I and NAPQI-II are shown in Suppl. Fig. 1A and Suppl. Fig. 2A. Under ECNICI

conditions, PFB derivatives of acidic compounds readily ionize to form anions due to

[M−PFB]− by loosing a PFB radical [11]. The most intense ions were m/z 182 [M−H]− due

to authentic 3-thio-paracetamol (molecular mass, 183) or [M−PFB]− due to S-PFB-3-thio-

paracetamol or O-PFB-3-thio-paracetamol for NAPQI-I and m/z 362 [M(PFB)2−PFB−H]−

t
ip
for NAPQI-II (see also below). These precursor ions were subjected to CID with argon as

the collision gas and generated the product ion mass spectra shown in Suppl. Fig. 1B and

cr
Suppl. Fig. 2B. Formation of the product ion m/z 342 from m/z 362 is likely to be formed

by neutral loss of HF (20 Da) presumably initiated by the nucleophilic attack of the

us
thiolate anion on the PFB ring, indicating that the lost H atom originates from the

methylene group of the PFB moiety but not from the aromatic ring of NAPQI-II. For

an
NAPQI-I, the corresponding major product ion of m/z 182 was m/z 139 due to neutral

loss of ketene (CH2=C=O, 42 Da) from the unlabelled acetyl and of a H atom (1 Da).
M
These observations are supported by similar findings obtained from the O-PFB derivative

of the catechol 2,3-dihydroxyphenylglycol [12].

d

The GC-MS and GC-MS/MS spectra discussed above likely result from two forms
te

of a common reaction product, i.e., 3-thio-paracetamol. Based on the retention times

and GC-MS spectra, NAPQI-I is presumably non-derivatized 3-thio-paracetamol or a

p

simply derivatized 3-thio-paracetamol, i.e., S-PFB-3-thio-paracetamol or O-PFB-3-thio-

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paracetamol (Suppl. Fig. 1). The latter derivatives are likely to be thermally labile and to

decompose to 3-thio-paracetamol before GC separation, e.g., during sample injection

Ac

into the hot injector (280 °C). This idea is supported by the observation that NAPQI-I is

detectable with and without PFB-Br derivatization. Yet, the GC peak is considerably larger

after PFB-Br derivatization. The large ion at m/z 522 could result from neutral loss of the

leaving group HF (20 Da) and of a H atom from the doubly pentafluorobenzylated 3-thio-

paracetamol derivative S,O-diPFB-3-thio-paracetamol (NAPQI-II; molecular mass, 543)

(Suppl. Fig. 2).

For quantitative analyses the following mass transitions were used in the SRM

mode: m/z 149  m/z 107 for APAP, m/z 153  m/z 111 for the internal standard d4-

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Page 8 of 27
APAP, m/z 182  m/z 139 for NAPQI-I and m/z 362  m/z 342 for NAPQI-II (i.e., 3-

thio-paracetamol). As specified in the respective experiments, in some quantitative

analyses d4-APAP was used as internal standard for both, paracetamol and 3-thio-

paracetamol. In other analyses, newly prepared 3-thio-d3-paracetamol was used as

internal standard for NAPQI-I and NAPQI-II. In these analyses, the mass transitions of

t
ip
m/z 185  m/z 142 for d3-NAPQI-I and m/z 364  m/z 344 for d2-NAPQI-II were used

in the SRM mode. When used as internal standard, 3-thio-d3-paracetamol was added to

cr
samples at a final concentration of 10 µM just before derivatization.

PFB-Br is known to react in aqueous solution with inorganic anions such as nitrate

us
and nitrite [13], as well as with organic anions such as those formed from

catecholamines [12]. In freshly prepared solutions of Na2S (1 mM) in 100 mM

an
sodium/potassium phosphate buffer (pH 7.4.) we found that S2− reacts with PFB-Br. Fig.

1 indicates that the reaction product is the thioether PFB-S-PFB (molecular mass, 394).
M
Under the GC conditions described above, the retention time of this thioether was 6.7

min, suggesting that PFB-S-PFB is a very volative species. From this analysis no peak
d

was obtained that would correspond to PFB-S-S-PFB (data not shown). N,N-
te

Diisopropylethylamine-catalyzed PFB-Br derivatization of ethyl acetate extracts of Na2S-

containing aqueous buffers did not reveal formation of PFB-S-PFB (data not shown).
p

Remaining PFB-Br and other possible PFB derivatives such as PFB-S-PFB and PFB-S-S-
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PFB are unlikely to interfer with the analysis of NAPQI-I and NAPQI-II derivatives.

Fig. 2 and Fig. 3 show that paracetamol and 3-thio-paracetamol formation from
Ac

NAPQI depends upon NAPQI and Na2S concentrations. In the absence of Na2S, about

45% of the initial NAPQI are spontaneously converted to paracetamol, whereas neither

NAPQI-I nor NAPQI-II were detectable. Thus, the NAPQI-I and NAPQI-II concentrations

in the absence of Na2S were set to 0 µM. We were not able to identify the species which

account for the remaining 55% of NAPQI at 0 mM Na2S. At an initial nominal NAPQI

concentration of 100 µM, addition of Na2S decreased paracetamol formation to about 20

%. Interestingly, higher Na2S concentrations did not further decrease the formation of

paracetamol from NAPQI. The concentration curves of 3-thio-paracetamol show broad

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Page 9 of 27
maxima in the range 1 mM to 2 mM Na2S. At higher Na2S concentrations, 3-thio-

paracetamol formation decreases considerably, possibly due to formation of additional

unknown reaction products. These results suggest that 1 mM Na2S is optimal to reach

maximum formation of 3-thio-paracetamol from NAPQI.

Due to the lack of a stable-isotope labelled internal standard for 3-thio-

t
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paracetamol, the authentic concentrations of NAPQI-I and NAPQI-II are actually

unknown. Also, because of the instability of NAPQI, even in its stock solution in

cr
acetonitrile, the actual amount of commercially acquired NAPQI added to the buffer is

also unknown. Fig. 3A shows almost linear relationships between the concentration (y) of

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paracetamol and 3-thio-paracetamol, i.e., NAPQI-I or NAPQI-II, and the initially added

nominal concentration of NAPQI (x) in the range 0 to 200 µM upon its treatment with the

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fixed Na2S concentration of 1 mM. The slope values of the regression equations indicate

mean yields of 21 % for paracetamol, 11 % for NAPQI-II and 57 % for NAPQI-I, i.e., a
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total yield of 89 % with respect to the nominal NAPQI concentration used. Fig. 3B

indicates an average 5-fold higher concentration of NAPQI-II compared to NAPQI-I.

d

These values are approximate because of the lack of an internal standard for 3-thio-
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paracetamol.

The results shown in Fig. 1 and Fig. 2 suggest that NAPQI can be almost
p

completely converted by Na2S to 3-thio-paracetamol and paracetamol in aqueous buffer

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of neutral pH. It is worth mentioning that APAP and Na2S did not react to form any

reaction products (data not shown). Thus, Na2S is a useful reagent to trap the short-lived
Ac

NAPQI in the presence of high molar excess of APAP and to convert it to 3-thio-

paracetamol which is more stable and has favourable chromatographic and mass

spectrometric properties than NAPQI. High Na2S-to-NAPQI molar ratios, e.g., higher than

10:1, seem to be not useful because high excess of Na2S over NAPQI decrease 3-thio-

paracetamol formation. Presumably, the nucleophilic attack of the sulphide anion on the

electrophilic ring of NAPQI is accompanied by additional not yet known secondary

reactions.

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Page 10 of 27
3.2. Effect of GSH on the reaction of NAPQI with Na2S in aqueous buffer

As outlined in the Introduction, GSH plays the most important role in the

detoxification of NAPQI in vivo. Intracellular GSH concentrations may reach values as

high as 10 mM such as in rat hepatocytes [14], whereas extracellular GSH concentrations

t
ip
such as in human plasma are almost three orders of magnitude lower [15]. Thus, GSH

from intercellular sources is likely to respresent the main competitor for Na2S against

cr
NAPQI. Indeed, Fig. 4 indicates that GSH competes with Na2S for the nucleophilic attack

on NAPQI in aqueous buffered solution. When Na2S and GSH were present at equimolar

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concentrations (1 mM), total 3-thio-paracetamol (NAPQI-I plus NAPQI-II) concentration

was about 41 % of that formed in the absence of GSH. At a GSH:Na2S molar ratio of

an
10:1, total 3-thio-paracetamol formation was only about 10 %, indicating that GSH,

despite its several times larger size compared to S2−, is a strong competitor for Na2S
M
against NAPQI. Thus, for samples reach in GSH such as lyzed hepatocytes or

erythrocytes, Na2S concentrations higher than 1 mM would be required.

d
te

3.3. Recombinant COX-1-mediated NAPQI formation from paracetamol in vitro

p

Prostaglandin H synthase (PGH), generally known as cyclooxygenases (COX),

ce

have been reported to oxidize paracetamol to NAPQI too, albeit to a very low extent [16,

17]. We applied the Na2S-based procedure to trap potentially formed NAPQI, and to
Ac

detect NAPQI formation upon incubation of paracetamol with a recombinant COX-1

isoform in the presence of its substrate arachidonic acid. At the therapeutically relevant

paracetamol concentration of 100 µM, NAPQI was hardly detectable in the incubation

mixtures, whereas paracetamol concentration did not change upon incubation time

(Suppl. Fig. 3 and Suppl. Fig. 4). Addition of commercially available NAPQI to the

arachidonic acid/COX-1-containing mixture and incubation for various times resulted in

formation of paracetamol and 3-thio-paracetamol upon addition of Na2S (Suppl. Figs. 3,

4, 5). The concentration of NAPQI-I and NAPQI-II derivatives decreased with increasing

11
Page 11 of 27
incubation time, whereas paracetamol concentration did not change remarkably. As thiols

including GSH inhibit recombinant COX-1 and COX-2 activity by about 50 to 90 % at 100

µM [18], it is likely that addition of Na2S to the incubation mixture at a final

concentration of 1 mM would have inhibited completely COX-1 activity in our

experiments.

t
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3.4. Formation of NAPQI from paracetamol in vitro in dog liver homogenate

cr
To test the general utility of the method, we investigated NAPQI formation from

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paracetamol in dog liver homogenates. As shown in Fig. 5, NAPQI is rapidly formed in

liver homogenates, indicating that hepatic CYP450 enzymes are able to quickly form

an
large amounts of NAPQI from paracetamol. We also tested NAPQI and 3-thio-

paracetamol stability in liver homogenate over 10 min. Based on GC-MS analyses, NAPQI
M
half-life was 5.4 min and that of 3-thio-paracetamol 18.3 min. Generally, NAPQI is

considered a very unstable intermediate. The quite high half-life of NAPQI in our study is
d

likely to be due to the high concentration of NAPQI added to the homogenate.

te

3.5. Formation of NAPQI from paracetamol in vivo in mice

p
ce

In mice, we investigated the utility of the present method to trap and quantify

NAPQI upon paracetamol administration at the toxicologically relevant dosage of about

Ac

150 mg/kg. This study was performed analogous to a previous study by Dickinson et al.

[19]. Samples were analyzed without an internal standard. In both mice, we observed

significant increases in NAPQI concentration after 180 min (mouse A) and 120 min

(mouse B) (Fig. 6). In comparison to other experiments which involved use of Na2S and

were performed in aqueous buffers, in the mice experiments we used Na2S at a final

concentration of 5 mM. This is required in order to account for loss of Na2S due its

reactions with biomolecules such as low- and high-molecular-mass disulphides, as well as

for a stronger competition of Na2S against erythrocytic GSH.

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Page 12 of 27
4. Discussion

The goal of the present study was to develop a GC-MS/MS method for the analysis

of NAPQI, the putative toxic intermediate metabolite of the paracetamol which is one of

the most widely used analgesic drugs. As NAPQI, i.e., N-acetyl-p-benzoquinone imine, is

t
ip
a chemically highly reactive electrophilic species, nucleophilic agents appear to be best

suitable for the stabilization of NAPQI prior to its qualitative and/or quantitative analysis.

cr
Indeed, the most important and effective route to inactivate and eliminate NAPQI in cells,

predominantly hepatocytes, involves conjugation of NAPQI with reduced GSH which is

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present at 1 to 10 mM-concentrations in the cytosol of many cells, notably of

hepatocytes [14]. Due to the acidicity of its sulphydryl group (pKa, 9.2), which is strongly

an
increased by the action of GSH S-transferases, GSH is the most important nucleophilic

agent to trap NAPQI in vivo. In theory, GSH and other SH-containing molecules such as
M
cysteine and N-acetylcysteine (NAC) are useful the nucleophilics to trap NAPQI and to

form stable thioethers. However, because conjugates of NAPQI with GSH, cysteine and
d

NAC are regularly formed upon paracetamol ingestion, such endogenous substances
te

appear not useful as trapping reagents in biological systems. In addition, thioethers of

NAPQI with GSH, cysteine and NAC have no favourable physicochemical properties in GC-
p

based analyses. For these reasons, we thought that another, small, SH-containing
ce

species may be better useful for trapping and analyzing NAPQI by GC-MS, and we

decided to use the simplest S-containing species, i.e., Na2S.

Ac

Na2S is freely soluble in aqueous phase and upon dilution releases the sulfide

dianion S2− in high abundance. It is expected that the chemical reaction between the

strong electrophilic NAPQI with the strong nucleophilic S2− would instanstenously form 3-

thio-paracetamol. Indeed, our study shows that Na2S is a quite useful reagent to trap

and quantify NAPQI in biological samples by GC-MS/MS. The reaction of NAPQI with the

sulfide dianion S2− results in abundant formation of 3-thio-paracetamol (Scheme 1) in

addition to paracetamol and other not yet identified reaction products. It is worthy of

mention that native 3-thio-paracetamol has not been identified in biological samples thus

13
Page 13 of 27
far. Yet, its intermediate formation is evidenced by the excretion of paracetamol-S-S-

paracetamol in rats [4] and of 3-methylthio-paracetamol, 3-methylthio-paracetamol

glucuronide and 3-methylsulfoxide-paracetamol in mice [20]. It is assummed that these

species are formed from paracetamol mercapturate metabolites by intestinal bacterial

activity [20]. Although the occurrence in biological samples of authentic 3-thio-

t
ip
paracetamol derived from paracetamol mercapturate metabolites can not be fully

excluded, detection of 3-thio-paracetamol by using the procedure described in this work

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is likely to directly derive from intermediately formed NAPQI and added Na2S.

Our results indicate that GSH is a strong competitor for Na2S against NAPQI

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despite the by far larger size of the thiolate anion of GSH compared to the very small

S2−. To overcome the competition of GSH from endogenous sources, use of Na2S at

an
concentrations as high as 10 mM would be required. Nevertheless, the concentration of

NAPQI formed from paracetamol can not be determined quantitatively but can only be
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roughly estimated. Another issue that is likely to complicate the measurement of NAPQI

in vivo is the time lag required to obtain the biological sample.

d

Paracetamol can be analyzed by GC-MS and GC-MS/MS without derivatization;

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however, conversion of paracetamol to its O-PFB ether derivative greatly improves the

analytical performance [9]. Because of thermally labil SH group of 3-thio-paracetamol, its

p

derivatization with PFB-Br for GC-MS and GC-MS/MS analysis is also advantageous. Yet,
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due to the acidity of the OH and SH groups of 3-thio-paracetamol, it is expected that its

derivatization with PFB-Br may result in formation of a mixture of three PFB derivatives,
Ac

i.e., O-PFB-3-thio-paracetamol, S-PFB-paracetamol and S,O-diPFB-paracetamol, in

addition to unreacted 3-thio-paracetamol (Scheme 1). In fact, we obtained two GC peaks

with distinctly different mass spectra, which we named NAPQI-I and NAPQI-II. NAPQI-II

is likely to be S,O-diPFB-paracetamol or the O-di-PFB-disulfide derivative of 3-thio-

paracetamol. NAPQI-I is likely to be 3-thio-paracetamol due to remaining non-derivatized

3-thio-paracetamol and/or due to decomposed O-PFB-3-thio-paracetamol and S-PFB-

paracetamol. Although the structure of NAPQI-I and NAPQI-II was not fully elucidated, in

part due to unavailability of synthetic unlabelled and stable-isotope labelled reference

14
Page 14 of 27
compounds, our study indicates that 3-thio-paracetamol can be quantified by GC-MS/MS

after derivatization with PFB-Br. Commercially available d4-paracetamol and newly

synthesized 3-thio-d3-paracetamol were found to be useful as internal standard for 3-

thio-paracetamol. The linear relationships observed between NAPQI-I or NAPQI-II

measured and NAPQI added underscore the utility of the method for quantitative

t
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measurements. Yet, accurate quantitative analysis requires method validation with

structurally characterized pure reference compounds.

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We demonstrated the utility of the method by capturing NAPQI formed in

incubation mixtures of paracetamol and recombinant COX-1, liver homogenate and in

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vivo in mice. Our results suggest that NAPQI can be formed and trapped in incubation

mixtures of COX-1, but the extent of its formation is very low. Our finding confirms

an
results reported by others [12, 16], although NAPQI formation of the order of 0.5% with

respect to the paracetamol concentration used in those stuies (i.e., 1 mM) was observed
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at COX concentrations being about 50 times higher than that used in the present study.

NAPQI formation from APAP was evident in dog liver homogenate in vitro and in blood ex
d

vivo in mice treated with high paracetamol doses.

p te

References
ce

[1] D.W. Potter, D.W. Miller, J.A. Hinson, J. Biol. Chem. 260 (1985) 12174-12180.
Ac

[2] D.W. Potter, D.W. Miller, J.A. Hinson, Mol. Pharmacol. 29 (1986) 155-162.

[3] L.P. James, P.R. Mayeux, J.A. Hinson, Drug Metab. Dispos. 31 (2003) 1499-1506.

[4] J. Sun, L.K. Schnackenberg, R.D. Holland, T.C. Schmitt, G.H. Contor, Y.P. Dragan,

R.D. Beger, J. Chromatogr. B 871 (2008) 328-340.

[5] J.E. Laine, S. Auriola, M. Pasanen, R.O. Juvonen, Xenobiotica 39 (2009) 11-21.

[6] I.A. Blair, A.R. Boobis, D.S. Davies, T.M. Cresp, Tetrahydro Lett. 21 (1980) 4947-

4950.

[7] D.C. Dahlin, S.D. Nelson, J. Med. Chem. 25 (1982) 885-886.

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Page 15 of 27
[8] D. Tsikas, M.T. Suchy, J. Niemann, P. Tossios, Y. Schneider, S. Rothmann, F.M.

Gutzki, J.C. Frölich, D.O. Gutzki, FEBS Lett. 586 (2012) 3723-3730.

[9] A. Trettin, A.A. Zoerner, A. Böhmer, F.M. Gutzki, D.O. Stichtenoth, J. Jordan, D.

Tsikas, J. Chromatogr. B 879 (2011) 2274-2280.

[10] D. Tsikas, A. Trettin, A.A. Zoerner, F.M. Gutzki, J. Chromatogr. B 879 (2011)

t
ip
1476-1484.

[11] S.H. Lee, I.A. Blair, Methods Enzymol. 433 (2007) 159-174.

cr
[12] A. Zoerner, K. Heusser, F.M. Gutzki, A. Mitschke, J. Tank, D.O. Stichtenoth, J.

Jordan, D. Tsikas, J. Chromatogr. B 879 (2011) 1444-1456.

us
[13] D. Tsikas, Anal. Chem. 72 (2000) 4064-4072.

[14] B. Ketterer, B. Coles, D.J. Meyer, Environ. Healthy Perspect. 49 (1983) 59-69.

[15]

[16]
an
M.A. Mansoor, A.M. Svardal, P.M. Ueland, Anal. Biochem. 200 (1992) 218-229.

D.W. Potter, J.A. Hinson, J. Biol. Chem. 262 (1987) 974-980.

M
[17] P.J. Harvison, R.W. Egan, P.H. Gale, G.D. Christian, B.S. Hill, S.D. Nelson, Chem.

Biol. Interact. 64 (1988) 251-166.

d

[18] D. Tsikas, J. Niemann, Nitric Oxide 26 (2012) 192-194.

te

[19] A.L. Dickinson, M.C. Leach, P.A. Flecknell, Lab. Anim. (2009) 357-361.

[20] M. Mikov, J. Caldwell, C.T. Dolphin, R.L. Smith, Biochem. Pharmacol. 37 (1988)
p

1445-1449.
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Page 16 of 27
Scheme and Figure legends

Scheme 1. Proposed reactions of NAPQI in aqueous solutions of Na2S. Addition of NAPQI

to an aqueous buffered Na2S solution results in formation of paracetamol (A) and 3-thio-

paracetamol (B). Derivatization of ethyl acetate extracts with pentafluorobenzyl bromide

t
ip
(PFB-Br) yields a single PFB-paracetamol derivative and three PFB derivatives of 3-thio-

paracetamol, i.e., O-mono-PFB derivative (named NAPQI-I) and S-mono-PFB derivative,

cr
and the O- and S-di-PFB derivative (named NAPQI-II). The O-mono-PFB derivative of 3-

thio-paracetamol may oxidize/dimerize to form O-di-PFB-disulfide derivative. For GC-MS

us
analysis, 3-thio-paracetamol, and the internal standard d4-paracetamol are derivatized

with PFB-Br. Ac, acetyl.

an
M
Fig. 1. ECNICI GC-MS (upper panel) and GC-MS/MS (lower panel) spectra of the GC

peak with the retention time of 6.7 min obtained by reacting a freshly prepared solution

of Na2S (1 mM) in 100 mM sodium/potassium buffer (pH 7.4) with PFB-Br as described
d

elsewhere for nitrite [13]. Briefly, an aliquot (100 µL) of the Na2S solution was treated
te

with acetone (400 µL) and pure PFB-Br (10 µL), and the mixture was allowed to react for

5 min at room temperature. After acetone evaporation under a stream of nitrogen, the
p

residue was extracted with toluene (1 mL) by vortex-mixing. An aliquot (1 µL) was
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injected into the GC-MS/MS instrument in the splitless mode. The GC-MS/MS spectrum

was obtained by subjecting the ion m/z 213 to CID with argon at a collision energy of 10
Ac

eV. Inserts show proposed structures for the derivative and major ions.

Fig. 2. Formation of paracetamol (APAP), 3-thio-paracetamol (NAPQI-I and NAPQI-II)

upon reaction of NAPQI (100 µM) with Na2S (0 – 5 mM) in 100 mM sodium phosphate

buffer, pH 7.4. Data are shown as mean ± SD from triplicate analyses for each Na2S

concentration used. The horizontal dashed line indicates the initial nominal NAPQI

concentration (n = 3). d4-APAP was used as internal standard.

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Page 17 of 27
Fig. 3. Formation of paracetamol (APAP), 3-thio-paracetamol (NAPQI-I and NAPQI-II

peaks) upon reaction of NAPQI (0 - 200 µM) with 1 mM Na2S in 100 mM sodium

phosphate buffer. (A) Illustration of used NAPQI concentrations (x axis) and the

measured concentrations (y axis). Data are shown as mean ± SD from triplicate

analyses. The diagonal dashed line indicates a sum concentration corresponding to yield

t
ip
of 100%. (B) Linear regression of the measured NAPQI-I against NAPQI-II

concentrations. (n = 3). d4-APAP was used as internal standard.

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Fig. 4. Na2S and GSH compete for the reaction with NAPQI. NAPQI (100 µM) reacted

us
with Na2S (1 mM) in the presence of 0, 0.1, 1 or 10 mM GSH. Freshly prepared 3-thio-d3-

paracetamol was used as the internal standard. After PFB derivatization NAPQI and 3-

an
thio-d3-paracetamol were measured as NAPQI-I and NAPQI-II, respectively, d3-NAPQI-I

and d2-NAPQI-II. Total means the sum of NAPQI-I and NAPQI-II. Horizontal lines indicate
M
the mean from triplicate analyses.

Fig. 5. NAPQI formation from APAP after incubation for 5 min at 37 °C with dog liver
d

homogenate prepared in PBS that contained 1 mM NADPH as coenzyme for CYP450. After
te

addition of APAP at a final concentration of 100 µM, samples were taken at different

times, derivatized with PFB-Br and measured as NAPQI-I and NAPQI-II. Data are shown
p

from three independent experiments.

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Fig. 6. Two mice were intraperitoneally administered 1 ml of 25 mM APAP solved in aqua

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ad iniectabilia. At different time points, small droplets of blood were collected via the tail

scarify. The time point zero minutes corresponded to the sampling before APAP

administration. Blood was mixed with 10 µL of a 10 mM sodium sulphide solution. After

PFB derivatization, NAPQI was measured by GC-MS/MS as NAPQI-I and NAPQI-II. (A)

Mouse A, 27 g body weight. (B) Mouse B, 23 g body weight.

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Page 18 of 27
*Highlights (for review)

Highlights

• Na2S is a useful reagent to trap NAPQI

• NAPQI and Na2S react in aqueous buffer to form 3-thio-paracetamol

• 3-Thio-paracetamol is derivatized by pentafluorobenzyl (PFB) bromide

• The PFB derivatives of 3-thio-paracetamol are analyzed by GC-MS/MS

t
ip
• NAPQI formation in vitro and in vivo is demonstrated by this method

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Scheme 1

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Figure 1

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Figure 2

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Figure 3A

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Figure 3B

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Figure 4

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Figure 5

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Figure 6A

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Page 27 of 27