Journal of Chromatography B, 963 (2014) 99–105 Contents lists available at ScienceDirect Journal of Chromatography B jou rn al hom ep age: www.elsevier.com/locate/chromb Trapping of NAPQI, the intermediate toxic paracetamol metabolite, by aqueous sulfide (S 2- ) and analysis by GC–MS/MS Arne Trettin a , Sandor Batkai b , Thomas Thum b,c , Jens Jordan a , Dimitrios Tsikas a,∗ a Institute of Clinical Pharmacology, Hannover Medical School, 30625 Hannover, Germany b Institute of Molecular and Translational Therapeutic Strategies, IFB-TX, Hannover Medical School, 30625 Hannover, Germany c Excellence Cluster REBIRTH, Hannover Medical School, 30625 Hannover, Germany a r t i c l e i n f o Article history: Received 10 January 2014 Accepted 23 May 2014 Available online 2 June 2014 Keywords: Acetaminophen Liver Mice NAPQI Quantification 3-Thio-paracetamol a b s t r a c t 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 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 vivo NAPQI formation. In this work, we propose the use of Na 2 S in aqueous solution to trap NAPQI and to analyze the 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 ethyl acetate and derivatization with pentafluorobenzyl bromide. In mechanistic studies, we used newly synthesized N-acetyl-p-[2,3,5,6- 2 H 4 ]benzoquinone imine (d 4 -NAPQI). In quantitative analyses, N-(4- hydroxyphenyl)-[2,3,5,6- 2 H 4 ]acetamide (d 4 -APAP) was used as the internal standard both for NAPQI and APAP. 3-Thio-d 3 -paracetamol, prepared from d 4 -NAPQI and Na 2 S, may also be useful as an internal standard. We showed NAPQI in 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 intraperitoneally (about 150 mg/kg). © 2014 Elsevier B.V. All rights reserved. 1. Introduction Paracetamol (acetaminophen; N-(4-hydroxyphenyl)acetamide; APAP) is among the most frequently used analgesic drugs. Parac- etamol’s chemistry, biochemistry and pharmacology are complex [1–5]. Native paracetamol is rapidly and extensively 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) 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 metabolite of paracetamol. NAPQI is inactivated by conjuga- tion with the tripeptide glutathione (GSH) and is excreted after ∗ Corresponding author at: Institute of Clinical Pharmacology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. Tel.: +49 511 532 3984; fax: +49 511 532 2750. E-mail address: tsikas.dimitros@mh-hannover.de (D. Tsikas). metabolization as the mercapturic acid in urine [1–5]. NAPQI undergoes numerous reactions in biological systems. These reactions include covalent binding to nucleophilic sites of biomolecules, autoreduction to paracetamol, and dimerization and/or polymerization. NAPQI formation in biological samples is evidenced by measuring the stable GSH conjugate and/or its metabolites including mercapturic acid. Given the high elec- trophilicity of NAPQI and its affinity to biothiols such as GSH and N-acetylcysteine (NAC), we reasoned that use of inor- ganic thiols, notably the small thiol sulfide (S 2- ) should be a much better alternative, both, to trap NAPQI during its formation and to quantify the resulting thio-paracetamol (N-(3- thiol-4-hydroxyphenyl)acetamide) by gas chromatography–mass spectrometry (GC–MS) or gas chromatography–tandem mass spec- trometry (GC–MS/MS). In the present article, we demonstrate that aqueous Na 2 S 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 using N-(4-hydroxyphenyl-[2,3,5,6- 2 H 4 ])acetamide (d 4 -paracetamol) as the internal standard (Scheme 1). http://dx.doi.org/10.1016/j.jchromb.2014.05.050 1570-0232/© 2014 Elsevier B.V. All rights reserved.