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Journal of Chromatography B, 963 (2014) 99–105

Contents lists available at ScienceDirect

Journal of Chromatography B

jou rn al hom ep age: www.elsev ier .com/ locate /chromb

rapping of NAPQI, the intermediate toxic paracetamol metabolite, byqueous sulfide (S2−) and analysis by GC–MS/MS

rne Trettina, Sandor Batkaib, Thomas Thumb,c, Jens Jordana, Dimitrios Tsikasa,∗

Institute of Clinical Pharmacology, Hannover Medical School, 30625 Hannover, GermanyInstitute of Molecular and Translational Therapeutic Strategies, IFB-TX, Hannover Medical School, 30625 Hannover, GermanyExcellence Cluster REBIRTH, Hannover Medical School, 30625 Hannover, Germany

r t i c l e i n f o

rticle history:eceived 10 January 2014ccepted 23 May 2014vailable online 2 June 2014

eywords:cetaminophenivericeAPQIuantification

a b s t r a c t

NAPQI, i.e., N-acetyl-p-benzoquinone imine, is considered the toxic metabolite of the widely usedanalgesic drug paracetamol (acetaminophen, APAP). Due to its high reactivity towards nucleophilesboth in low- and high-molecular-mass biomolecules, NAPQI is hardly detectable in its native form.Upon conjugation with glutathione, NAPQI is finally excreted in the urine as the paracetamol mer-capturic acid. Thus, determination of paracetamol mercapturate may provide a measure of in vivoNAPQI formation. In this work, we propose the use of Na2S in aqueous solution to trap NAPQI andto analyze the reaction product, i.e., 3-thio-paracetamol, together with paracetamol by GC–MS/MSin the electron-capture negative-ion chemical ionization mode after solvent extraction with ethylacetate and derivatization with pentafluorobenzyl bromide. In mechanistic studies, we used newlysynthesized N-acetyl-p-[2,3,5,6-2H4]benzoquinone imine (d4-NAPQI). In quantitative analyses, N-(4-

2

-Thio-paracetamol hydroxyphenyl)-[2,3,5,6- H4]acetamide (d4-APAP) was used as the internal standard both for NAPQIand APAP. 3-Thio-d3-paracetamol, prepared from d4-NAPQI and Na2S, may also be useful as an internalstandard. We showed NAPQI in vitro formation from APAP by recombinant cyclooxygenase-1 as wellas by dog liver homogenate. In vivo formation of NAPQI was demonstrated in mice given paracetamolintraperitoneally (about 150 mg/kg).

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Paracetamol (acetaminophen; N-(4-hydroxyphenyl)acetamide;PAP) is among the most frequently used analgesic drugs. Parac-tamol’s chemistry, biochemistry and pharmacology are complex1–5]. Native paracetamol is rapidly and extensively metabolizedia conjugation reactions to its glucuronic and sulfuric acids ands eliminated by the kidney. Paracetamol is also oxidized by theytochrome P450 (CYP450) family to N-acetyl-p-benzoquinone

mine (NAPQI; Scheme 1). NAPQI is a chemically very reactivelectrophilic species and is therefore considered the intermediateoxic metabolite of paracetamol. NAPQI is inactivated by conjuga-ion with the tripeptide glutathione (GSH) and is excreted after

∗ Corresponding author at: Institute of Clinical Pharmacology, Hannover Medicalchool, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. Tel.: +49 511 532 3984;ax: +49 511 532 2750.

E-mail address: tsikas.dimitros@mh-hannover.de (D. Tsikas).

ttp://dx.doi.org/10.1016/j.jchromb.2014.05.050570-0232/© 2014 Elsevier B.V. All rights reserved.

metabolization as the mercapturic acid in urine [1–5]. NAPQIundergoes numerous reactions in biological systems. Thesereactions include covalent binding to nucleophilic sites ofbiomolecules, autoreduction to paracetamol, and dimerizationand/or polymerization. NAPQI formation in biological samplesis evidenced by measuring the stable GSH conjugate and/or itsmetabolites including mercapturic acid. Given the high elec-trophilicity of NAPQI and its affinity to biothiols such as GSHand N-acetylcysteine (NAC), we reasoned that use of inor-ganic thiols, notably the small thiol sulfide (S2−) should bea much better alternative, both, to trap NAPQI during itsformation and to quantify the resulting thio-paracetamol (N-(3-thiol-4-hydroxyphenyl)acetamide) by gas chromatography–massspectrometry (GC–MS) or gas chromatography–tandem mass spec-trometry (GC–MS/MS). In the present article, we demonstratethat aqueous Na2S is a useful reagent for high-extent trapping

of NAPQI and subsequent highly sensitive GC–MS/MS quantifica-tion of 3-thio-paracetamol as pentafluorobenzyl derivative usingN-(4-hydroxyphenyl-[2,3,5,6-2H4])acetamide (d4-paracetamol) asthe internal standard (Scheme 1).

100 A. Trettin et al. / J. Chromatogr. B 963 (2014) 99–105

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 (PFB-Br) yields a single PFB-paracetamol derivative and three PFBd d S-mm -disuld

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bated for 5 min at 37 ◦C. After addition of 100 �M APAP samples

erivatives of 3-thio-paracetamol, i.e., O-mono-PFB derivative (named NAPQI-I) anono-PFB derivative of 3-thio-paracetamol may oxidize/dimerize to form O-di-PFB

4-paracetamol are derivatized with PFB-Br. Ac, acetyl.

. Experimental

.1. Chemicals and materials

Unlabelled paracetamol (N-(4-hydroxyphenyl)-acetamide;0-APAP) and Na2S were obtained from Merck (Darmstadt,ermany). N-(4-Hydroxyphenyl)-[2,3,5,6-2H4]acetamide (d4-PAP; 95%; 99.4 atom% 2H) was purchased from CDN Isotopes

Quebec, Canada). N-Acetyl-p-benzoquinone imine (NAPQI),rachidonic acid, 2,3,4,5,6-pentafluorobenzyl bromide (99%),,N-diisopropylethylamine (95%) and reduced glutathione (GSH,9%) were bought from Sigma–Aldrich (Munich, Germany). Sil-er nitrate (99.9%) came from Carl Roth (Karlsruhe, Germany).ecombinant ovine cyclooxygenase-1 (COX-1) was obtained fromayman Chemicals (Ann Arbor, MI, USA). NADPH tetrasodium salt98%) was received from Roche (Rotkreuz, Switzerland).

.2. Synthesis of N-acetyl-p-[2,3,5,6-2H4]benzoquinone imined4-NAPQI) and 3-thio-d3-paracetamol

N-Acetyl-p-[2,3,5,6-2H4]benzoquinone imine (d4-NAPQI) wasewly synthesized following procedures originally reported fornlabelled NAPQI [6,7]. Briefly, silver oxide (Ag2O) was freshly pre-ared from a 10 wt.% silver nitrate solution and precipitation withropwise addition of 1 M NaOH. The supernatant was decantednd the obtained silver oxide was washed over filter paper sev-ral times with small amounts of water. Water was removed bydding a few drops of absolute ethanol and subsequent evapora-ion under a nitrogen stream. The dry powder obtained was thentored in a desiccator over silica gel for 24 h at room temperature.he washed and dried silver oxide (150 mg) together with d4-APAP100 mg) and a small amount of activated charcoal (three spatulaips) was incubated in chloroform (5 mL), under constant stirring at

oom temperature for 25 min. d4-NAPQI formed during this proce-ure was isolated by silica chromatography and elution with ethylcetate. Following solvent evaporation, the yellow residue was dis-olved in acetonitrile (dried over molsieve) and stored at -20 ◦C.

ono-PFB derivative, and the O- and S-di-PFB derivative (named NAPQI-II). The O-fide derivative. For GC–MS analysis, 3-thio-paracetamol, and the internal standard

d4-NAPQI yield was 21 ± 5% (n = 3) as determined by GC–MS anal-ysis (see below) using commercially available unlabelled NAPQI(d0-NAPQI). For the preparation of 3-thio-d3-paracetamol, newlyprepared d4-NAPQI was added to a 1 mM Na2S solution in 100 mMphosphate buffered saline (PBS), pH 7.4, to reach a final con-centration of 100 �M, and the mixture was incubated for 15 minat room temperature. Assuming complete reaction, the 3-thio-d3-paracetamol concentration in the reaction mixture would be100 �M.

2.3. Experiments with recombinant COX-1

COX-1-catalyzed reactions and respective controls wereperformed under aerobic conditions at 37 ◦C in 100 mMsodium/potassium phosphate buffer (pH 8.0) as describedelsewhere [8]. The buffer contained 5 U COX-1, 10 �M arachidonicacid, 2 mM phenol, 5 mM EDTA and 1 �M haematin. Reactionmixtures were incubated with paracetamol (100 �M) for 10 min.As a positive control for NAPQI formation, commercially availableNAPQI was added to the COX-1 incubation mixture in the absenceand in the presence of COX-1. To trap potentially formed NAPQI,samples were taken after 10 min or as appropriate, treated withNa2S (1 mM in 100 mM PBS), derivatized with PFB-Br and analyzedby GC–MS as described below.

2.4. Dog experiment

NAPQI formation from APAP was investigated in dog liverhomogenate. Frozen dog liver were thawed and homogenized by aPrecellys 24 Peglab® (three times for 20 s, 5500 rpm, 2–8 ◦C) withPBS. 1 mM NADPH as coenzyme for CYP450 was added and incu-

were taken at different times, treated with Na2S (1 mM in 100 mMPBS), derivatized with PFB-Br and analyzed by GC–MS as describedbelow. Dog liver tissue was kindly donated for research purposesby the Hannover Veterinary School.

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A. Trettin et al. / J. Chrom

.5. Mouse experiment

Paracetamol was injected intraperitoneally (1 mL of a 25 mMPAP solution in physiological saline, i.e., 3.8 mg), into two 10-eek-old male mice (C57Bl/6N; mouse A, 27 g; mouse B, 23 g),

esulting in dosages of 140 mg/kg and 164 mg/kg, respectively. Atifferent time points (0, 10, 20, 30, 60, 90, 120, 180 and 240 min),

small blood droplet (about 10 �L) was collected from the tailn 2-mL Eppendorf tubes. The time point 0 min corresponds tohe sampling immediately before paracetamol administration. Atime point 240 min, venous blood was collected after euthanasiay cervical dislocation. Blood samples were treated immediatelyith Na2S (10 �L, 10 mM) to trap potentially formed NAPQI, incu-

ated at room temperature for 15 min, and reaction products werextracted by rigorous vortexing with ethyl acetate (300 �L). Afterentrifugation (800 × g, 5 min, 4 ◦C), the organic phase was sepa-ated and the solvent was evaporated to dryness. Subsequently,FB-Br derivatization was performed as described below. Thistudy was performed at the Institute of Molecular and Transla-ional Therapeutic Strategies of the Hannover Medical School andas approved by the local supervisory committee for studies in

nimals (Hannover, Germany).

.6. Sample preparation and derivatization with PFB-Br forC–MS and GC–MS/MS analysis

Reactions of NAPQI and Na2S were performed in 100 mModium/potassium phosphate buffer (pH 7.4.) at room temperatureincubation for 15 min). Ethyl acetate was added to the reactionroduct and extraction was performed by rigorous vortexing for

min. Subsequently, sample was centrifuged (800 × g, 5 min, 4 ◦C)nd the supernatant was transferred into glass vials. Ethyl acetateas completely evaporated under a nitrogen stream. PFB-Br deriva-

ization was performed by taking up the sample in anhydrouscetonitrile (100 �L) in the presence of N,N-diisopropylethylamine10 �L) which served as the catalyst and PFB-Br (10 �L of a 30 vol.%olution in anhydrous acetonitrile) as described recently for parac-tamol [9,10]. The reaction mixture was incubated at 30 ◦C for 1 h.hen, solvent and reagent were evaporated to dryness under atream of nitrogen gas. For GC–MS analysis the residues were takenp with toluene (100 �L). All samples were stored at 6 ◦C.

.7. GC–MS and GC–MS/MS conditions

GC–MS and GC–MS/MS analyses were performed on a triple-tage quadrupole (TSQ) mass spectrometer ThermoElectron TSQ000 (Finnigan MAT, San Jose, CA, USA) as described previously foraracetamol [9,10]. Electron-capture negative-ion chemical ion-

zation (ECNICI) was performed. MS spectra were generated bycanning m/z 40 to m/z 800 at 1 s/scan. MS/MS and spectra wereenerated by subjecting selected parent ions to collision-inducedissociation (CID) with argon as the collision gas (2 mTorr) andcanning the third quadrupole m/z 40 to m/z 800 at 1 s/scan. Quan-ification was performed by selected-reaction monitoring (SRM)f suitable mass transitions as described below. GC separation waserformed on the column Optima 17 MS (30 m × 0.25 mm i.d., 0.25-m film thickness) from Macherey-Nagel (Düren, Germany). Other

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

. Results

.1. Reaction of NAPQI and d4-NAPQI with Na2S in aqueous buffer

o form 3-thio-paracetamol, respectively, 3-thio-d3-paracetamol

Incubation of NAPQI in aqueous buffer in the absence of anyther reagents resulted in formation of paracetamol and other

r. B 963 (2014) 99–105 101

unidentified minor reaction products as analyzed by HPLC with UVabsorbance detection at 254 nm (data not shown). GC–MS analy-sis of derivatized extracts from the treatment of NAPQI with Na2Sin aqueous buffer resulted in three major GC peaks. One of thispeak was identified as the paracetamol O-PFB derivative (data notshown). The GC–MS spectra of the other two GC peaks assigned asNAPQI-I and NAPQI-II are shown in Suppl. Figs. 1A and 2A. UnderECNICI conditions, PFB derivatives of acidic compounds readily ion-ize 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 authentic3-thio-paracetamol (molecular mass, 183) or [M−PFB]− due to S-PFB-3-thio-paracetamol or O-PFB-3-thio-paracetamol for NAPQI-Iand m/z 362 [M(PFB)2−PFB−H]− for NAPQI-II (see also below).These precursor ions were subjected to CID with argon as the col-lision gas and generated the product ion mass spectra shown inSuppl. Figs. 1B and 2B. The product ion m/z 342 from m/z 362 islikely to be formed by neutral loss of HF (20 Da) presumably ini-tiated by the nucleophilic attack of the thiolate anion on the PFBring, indicating that the lost H atom originates from the methylenegroup of the PFB moiety but not from the aromatic ring of NAPQI-II.For NAPQI-I, the corresponding major product ion of m/z 182 wasm/z 139 due to neutral loss of ketene (CH2 C O, 42 Da) from theunlabelled acetyl and of a H atom (1 Da). These observations aresupported by similar findings obtained from the O-PFB derivativeof the catechol 2,3-dihydroxyphenylglycol [12].

The GC–MS and GC–MS/MS spectra discussed above likelyresult from two forms of a common reaction product, i.e.,3-thio-paracetamol. Based on the retention times and GC–MS spec-tra, NAPQI-I is presumably non-derivatized 3-thio-paracetamolor a simply derivatized 3-thio-paracetamol, i.e., S-PFB-3-thio-paracetamol or O-PFB-3-thio-paracetamol (Suppl. Fig. 1). The latterderivatives are likely to be thermally labile and to decompose to3-thio-paracetamol before GC separation, e.g., during sample injec-tion into the hot injector (280 ◦C). This idea is supported by theobservation that NAPQI-I is detectable with and without PFB-Brderivatization. Yet, the GC peak is considerably larger after PFB-Brderivatization. The large ion at m/z 522 could result from neutralloss of the leaving group HF (20 Da) and of a H atom from thedoubly 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 wereused in the SRM mode: m/z 149 → m/z 107 for APAP, m/z153 → m/z 111 for the internal standard d4-APAP, m/z 182 → m/z139 for NAPQI-I and m/z 362 → m/z 342 for NAPQI-II (i.e., 3-thio-paracetamol). As specified in the respective experiments, insome quantitative analyses d4-APAP was used as internal standardfor both, paracetamol and 3-thio-paracetamol. In other analy-ses, newly prepared 3-thio-d3-paracetamol was used as internalstandard for NAPQI-I and NAPQI-II. In these analyses, the mass tran-sitions of m/z 185 → m/z 142 for d3-NAPQI-I and m/z 364 → m/z 344for d2-NAPQI-II were used in the SRM mode. When used as internalstandard, 3-thio-d3-paracetamol was added to samples at a finalconcentration of 10 �M just before derivatization.

PFB-Br is known to react in aqueous solution with inorganicanions such as nitrate and nitrite [13], as well as with organicanions such as those formed from catecholamines [12]. In freshlyprepared solutions of Na2S (1 mM) in 100 mM sodium/potassiumphosphate 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). Under the GC conditions describedabove, the retention time of this thioether was 6.7 min, suggest-

ing that PFB-S-PFB is a very volatile species. From this analysis nopeak was obtained that would correspond to PFB-S-S-PFB (data notshown). N,N-Diisopropylethylamine-catalyzed PFB-Br derivatiza-tion of ethyl acetate extracts of Na2S-containing aqueous buffers

102 A. Trettin et al. / J. Chromatogr. B 963 (2014) 99–105

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 solutionof Na2S (1 mM) in 100 mM sodium/potassium buffer (pH 7.4) with PFB-Br as described elsewhere for nitrite [13]. Briefly, an aliquot (100 �L) of the Na2S solution was treatedwith acetone (400 �L) and pure PFB-Br (10 �L), and the mixture was allowed to react for 5 min at room temperature. After acetone evaporation under a stream of nitrogen, theresidue was extracted with toluene (1 mL) by vortex-mixing. An aliquot (1 �L) was injected into the GC–MS/MS instrument in the splitless mode. The GC–MS/MS spectrumw of 10 e

diPa

mInNtaooa

as obtained by subjecting the ion m/z 213 to CID with argon at a collision energy

id not reveal formation of PFB-S-PFB (data not shown). Remain-ng PFB-Br and other possible PFB derivatives such as PFB-S-PFB andFB-S-S-PFB are unlikely to interfere with the analysis of NAPQI-Ind NAPQI-II derivatives.

Figs. 2 and 3 show that paracetamol and 3-thio-paracetamol for-ation from NAPQI depends upon NAPQI and Na2S concentrations.

n the absence of Na2S, about 45% of the initial NAPQI are sponta-eously converted to paracetamol, whereas neither NAPQI-I norAPQI-II were detectable. Thus, the NAPQI-I and NAPQI-II concen-

rations in the absence of Na2S were set to 0 �M. We were not

ble to identify the species which account for the remaining 55%f NAPQI at 0 mM Na2S. At an initial nominal NAPQI concentrationf 100 �M, addition of Na2S decreased paracetamol formation tobout 20%. Interestingly, higher Na2S concentrations did not further

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

decrease the formation of paracetamol from NAPQI. The concen-tration curves of 3-thio-paracetamol show broad maxima in therange 1 mM to 2 mM Na2S. At higher Na2S concentrations, 3-thio-paracetamol formation decreases considerably, possibly due toformation of additional unknown reaction products. These resultssuggest that 1 mM Na2S is optimal to reach maximum formation of3-thio-paracetamol from NAPQI.

Due to the lack of a stable-isotope labelled internal standardfor 3-thio-paracetamol, the authentic concentrations of NAPQI-Iand NAPQI-II are actually unknown. Also, because of the instabil-

ity of NAPQI, even in its stock solution in acetonitrile, the actualamount of commercially acquired NAPQI added to the buffer isalso unknown. Fig. 3A shows almost linear relationships betweenthe concentration (y) of paracetamol and 3-thio-paracetamol, i.e.,

A. Trettin et al. / J. Chromatog

Fig. 2. Formation of paracetamol (APAP), 3-thio-paracetamol (NAPQI-I and NAPQI-II) upon reaction of NAPQI (100 �M) with Na2S (0–5 mM) in 100 mM sodiumphosphate buffer, pH 7.4. Data are shown as mean ± SD from triplicate analysesfor each Na2S concentration used. The horizontal dashed line indicates the initialnominal NAPQI concentration (n = 3). d4-APAP was used as internal standard.

Fig. 3. Formation of paracetamol (APAP), 3-thio-paracetamol (NAPQI-I and NAPQI-II peaks) upon reaction of NAPQI (0–200 �M) with 1 mM Na2S in 100 mM sodiumphosphate buffer. (A) Illustration of used NAPQI concentrations (x axis) and the mea-sured concentrations (y axis). Data are shown as mean ± SD from triplicate analyses.The diagonal dashed line indicates a sum concentration corresponding to yield of100%. (B) Linear regression of the measured NAPQI-I against NAPQI-II concentrations(n = 3). d4-APAP was used as internal standard.

r. B 963 (2014) 99–105 103

NAPQI-I or NAPQI-II, and the initially added nominal concentrationof NAPQI (x) in the range 0–200 �M upon its treatment with thefixed Na2S concentration of 1 mM. The slope values of the regres-sion equations indicate mean yields of 21% for paracetamol, 11% forNAPQI-II and 57% for NAPQI-I, i.e., a total yield of 89% with respectto the nominal NAPQI concentration used. Fig. 3B indicates an aver-age 5-fold higher concentration of NAPQI-II compared to NAPQI-I.These values are approximate because of the lack of an internalstandard for 3-thio-paracetamol.

The results shown in Figs. 1 and 2 suggest that NAPQI can bealmost completely converted by Na2S to 3-thio-paracetamol andparacetamol in aqueous buffer of neutral pH. It is worth men-tioning that APAP and Na2S did not react to form any reactionproducts (data not shown). Thus, Na2S is a useful reagent to trapthe short-lived NAPQI in the presence of high molar excess of APAPand to convert it to 3-thio-paracetamol which is more stable andhas favourable chromatographic and mass spectrometric proper-ties than NAPQI. High Na2S-to-NAPQI molar ratios, e.g., higher than10:1, seem to be not useful because high excess of Na2S over NAPQIdecrease 3-thio-paracetamol formation. Presumably, the nucle-ophilic attack of the sulfide anion on the electrophilic ring of NAPQIis accompanied by additional not yet known secondary reactions.

3.2. Effect of GSH on the reaction of NAPQI with Na2S in aqueousbuffer

As outlined in Section 1, GSH plays the most important role inthe detoxification of NAPQI in vivo. Intracellular GSH concentra-tions may reach values as high as 10 mM such as in rat hepatocytes[14], whereas extracellular GSH concentrations such as in humanplasma are almost three orders of magnitude lower [15]. Thus, GSHfrom intercellular sources is likely to represent the main competitorfor Na2S against NAPQI. Indeed, Fig. 4 indicates that GSH competeswith Na2S for the nucleophilic attack on NAPQI in aqueous bufferedsolution. When Na2S and GSH were present at equimolar concen-trations (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 10:1, total 3-thio-paracetamol forma-tion was only about 10%, indicating that GSH, despite its severaltimes larger size compared to S2−, is a strong competitor for Na S

2against NAPQI. Thus, for samples reach in GSH such as lysed hep-atocytes or erythrocytes, Na2S concentrations higher than 1 mMwould be required.

Fig. 4. Na2S and GSH compete for the reaction with NAPQI. NAPQI (100 �M) reactedwith 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 derivatizationNAPQI and 3-thio-d3-paracetamol were measured as NAPQI-I and NAPQI-II, respec-tively, d3-NAPQI-I and d2-NAPQI-II. Total means the sum of NAPQI-I and NAPQI-II.Horizontal lines indicate the mean from triplicate analyses.

104 A. Trettin et al. / J. Chromatogr. B 963 (2014) 99–105

Fig. 5. NAPQI formation from APAP after incubation for 5 min at 37 ◦C with dog liverhomogenate prepared in PBS that contained 1 mM NADPH as coenzyme for CYP450.AdD

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Fig. 6. Two mice were intraperitoneally administered 1 mL of 25 mM APAP solved inaqua ad iniectabilia. At different time points, small droplets of blood were collectedvia the tail scarify. The time point zero minutes corresponded to the sampling before

fter addition of APAP at a final concentration of 100 �M, samples were taken atifferent times, derivatized with PFB-Br and measured as NAPQI-I and NAPQI-II.ata are shown from three independent experiments.

.3. Recombinant COX-1-mediated NAPQI formation fromaracetamol in vitro

Prostaglandin H synthases (PGH), generally known as cyclooxy-enases (COX), have been reported to oxidize paracetamol to NAPQIoo, albeit to a very low extent [16,17]. We applied the Na2S-ased procedure to trap potentially formed NAPQI, and to detectAPQI formation upon incubation of paracetamol with a recom-inant COX-1 isoform in the presence of its substrate arachidoniccid. At the therapeutically relevant paracetamol concentration of00 �M, NAPQI was hardly detectable in the incubation mixtures,hereas paracetamol concentration did not change upon incuba-

ion time (Suppl. Figs. 3 and 4). Addition of commercially availableAPQI to the arachidonic acid/COX-1-containing mixture and incu-ation for various times resulted in formation of paracetamol and-thio-paracetamol upon addition of Na2S (Suppl. Figs. 3–5). Theoncentration of NAPQI-I and NAPQI-II derivatives decreased withncreasing incubation time, whereas paracetamol concentrationid not change remarkably. As thiols including GSH inhibit recom-inant COX-1 and COX-2 activity by about 50–90% at 100 �M [18],

t is likely that addition of Na2S to the incubation mixture at a finaloncentration of 1 mM would have inhibited completely COX-1ctivity in our experiments.

.4. Formation of NAPQI from paracetamol in vitro in dog liveromogenate

To test the general utility of the method, we investigated NAPQIormation from paracetamol in dog liver homogenates. As shownn Fig. 5, NAPQI is rapidly formed in liver homogenates, indicat-ng that hepatic CYP450 enzymes are able to quickly form largemounts of NAPQI from paracetamol. We also tested NAPQI and 3-hio-paracetamol stability in liver homogenate over 10 min. Basedn GC–MS analyses, NAPQI half-life was 5.4 min and that of 3-hio-paracetamol 18.3 min. Generally, NAPQI is considered a verynstable intermediate. The quite high half-life of NAPQI in our study

s likely to be due to the high concentration of NAPQI added to theomogenate.

.5. Formation of NAPQI from paracetamol in vivo in mice

In mice, we investigated the utility of the present method torap and quantify NAPQI upon paracetamol administration at theoxicologically relevant dosage of about 150 mg/kg. This study waserformed analogous to a previous study by Dickinson et al. [19].

APAP administration. Blood was mixed with 10 �L of a 10 mM sodium sulfide solu-tion. After PFB derivatization, NAPQI was measured by GC–MS/MS as NAPQI-I andNAPQI-II. (A) Mouse A, 27 g body weight. (B) Mouse B, 23 g body weight.

Samples were analyzed without an internal standard. In both mice,we observed significant increases in NAPQI concentration after180 min (mouse A) and 120 min (mouse B) (Fig. 6). In comparison toother experiments which involved use of Na2S and were performedin aqueous buffers, in the mice experiments we used Na2S at a finalconcentration of 5 mM. This is required in order to account for lossof Na2S due its reactions with biomolecules such as low- and high-molecular-mass disulfides, as well as for a stronger competition ofNa2S against erythrocytic GSH.

4. Discussion

The goal of the present study was to develop a GC–MS/MSmethod for the analysis of NAPQI, the putative toxic intermediatemetabolite of the paracetamol which is one of the most widely usedanalgesic drugs. As NAPQI, i.e., N-acetyl-p-benzoquinone imine,is a chemically highly reactive electrophilic species, nucleophilicagents appear to be best suitable for the stabilization of NAPQI priorto its qualitative and/or quantitative analysis. Indeed, the mostimportant and effective route to inactivate and eliminate NAPQI incells, predominantly hepatocytes, involves conjugation of NAPQIwith reduced GSH which is present at 1 to 10 mM-concentrationsin the cytosol of many cells, notably of hepatocytes [14]. Due to theacidity of its sulfhydryl group (pKa, 9.2), which is strongly increasedby the action of GSH S-transferases, GSH is the most importantnucleophilic agent to trap NAPQI in vivo. In theory, GSH and otherSH-containing molecules such as cysteine and N-acetylcysteine

(NAC) are useful nucleophilics to trap NAPQI and to form stablethioethers. However, because conjugates of NAPQI with GSH, cys-teine and NAC are regularly formed upon paracetamol ingestion,such endogenous substances appear not useful as trapping reagents

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n biological systems. In addition, thioethers of NAPQI with GSH,ysteine and NAC have no favourable physicochemical propertiesn GC-based analyses. For these reasons, we thought that another,mall, SH-containing species may be better useful for trapping andnalyzing NAPQI by GC–MS, and we decided to use the simplest-containing species, i.e., Na2S.

Na2S is freely soluble in aqueous phase and upon dilutioneleases the sulfide dianion S2− in high abundance. It is expectedhat the chemical reaction between the strong electrophilic NAPQIith the strong nucleophilic S2− would instantaneously form 3-

hio-paracetamol. Indeed, our study shows that Na2S is a quiteseful reagent to trap and quantify NAPQI in biological samplesy GC–MS/MS. The reaction of NAPQI with the sulfide dian-

on S2− results in abundant formation of 3-thio-paracetamolScheme 1) in addition to paracetamol and other not yet iden-ified reaction products. It is worthy of mention that native-thio-paracetamol has not been identified in biological sam-les thus far. Yet, its intermediate formation is evidenced by thexcretion of paracetamol-S-S-paracetamol in rats [4] and of 3-ethylthio-paracetamol, 3-methylthio-paracetamol glucuronide

nd 3-methylsulfoxide-paracetamol in mice [20]. It is assumed thathese species are formed from paracetamol mercapturate metabo-ites by intestinal bacterial activity [20]. Although the occurrencen biological samples of authentic 3-thio-paracetamol derived fromaracetamol mercapturate metabolites cannot be fully excluded,etection of 3-thio-paracetamol by using the procedure described

n this work is likely to directly derive from intermediately formedAPQI and added Na2S.

Our results indicate that GSH is a strong competitor for Na2Sgainst NAPQI despite the by far larger size of the thiolate anion ofSH compared to the very small S2−. To overcome the competitionf GSH from endogenous sources, use of Na2S at concentrations asigh as 10 mM would be required. Nevertheless, the concentrationf NAPQI formed from paracetamol cannot be determined quan-itatively but can only be roughly estimated. Another issue that isikely to complicate the measurement of NAPQI in vivo is the timeag required to obtain the biological sample.

Paracetamol can be analyzed by GC–MS and GC–MS/MS with-ut derivatization; however, conversion of paracetamol to its O-PFBther derivative greatly improves the analytical performance [9].ecause of the thermally labile SH group of 3-thio-paracetamol,

ts derivatization with PFB-Br for GC–MS and GC–MS/MS anal-sis is also advantageous. Yet, due to the acidity of the OHnd SH groups of 3-thio-paracetamol, it is expected that itserivatization with PFB-Br may result in formation of a mixturef three PFB derivatives, i.e., O-PFB-3-thio-paracetamol, S-PFB-aracetamol and S,O-diPFB-paracetamol, in addition to unreacted-thio-paracetamol (Scheme 1). In fact, we obtained two GC peaksith distinctly different mass spectra, which we named NAPQI-

and NAPQI-II. NAPQI-II is likely to be S,O-diPFB-paracetamol or

he O-di-PFB-disulfide derivative of 3-thio-paracetamol. NAPQI-I isikely to be 3-thio-paracetamol due to remaining non-derivatized-thio-paracetamol and/or due to decomposed O-PFB-3-thio-aracetamol and S-PFB-paracetamol. Although the structure of

[[[

r. B 963 (2014) 99–105 105

NAPQI-I and NAPQI-II was not fully elucidated, in part due tounavailability of synthetic unlabelled and stable-isotope labelledreference compounds, our study indicates that 3-thio-paracetamolcan 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 standardfor 3-thio-paracetamol. The linear relationships observed betweenNAPQI-I or NAPQI-II measured and NAPQI added underscore theutility of the method for quantitative measurements. Yet, accuratequantitative analysis requires method validation with structurallycharacterized pure reference compounds.

We demonstrated the utility of the method by capturing NAPQIformed in incubation mixtures of paracetamol and recombinantCOX-1, liver homogenate and in vivo in mice. Our results suggestthat NAPQI can be formed and trapped in incubation mixturesof COX-1, but the extent of its formation is very low. Our find-ing confirms results reported by others [12,16], although NAPQIformation of the order of 0.5% with respect to the paracetamol con-centration used in those studies (i.e., 1 mM) was observed at COXconcentrations being about 50 times higher than that used in thepresent study. NAPQI formation from APAP was evident in dog liverhomogenate in vitro and in blood ex vivo in mice treated with highparacetamol doses.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.jchromb.2014.05.050.

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