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

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 mercapturic acid. Thus, determination 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 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-[2,3,5,6-2H4]benzoquinone imine (d4-NAPQI). In 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 d4-NAPQI and Na2S, 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).