In-source formation of N-acetyl- p-benzoquinone imine (NAPQI), the putatively toxic acetaminophen (paracetamol) metabolite, after derivatization with pentafluorobenzyl bromide and GC–ECNICI-MS analysis

Abstract

Pentafluorobenzyl (PFB) bromide (PFB-Br) is a versatile derivatization reagent for numerous classes of compounds. Under electron-capture negative-ion chemical ionization (ECNICI) conditions PFB derivatives of acidic compounds readily and abundantly ionize to produce intense anions due to [M−PFB] −. In the present article we investigated the PFB-Br derivatization of unlabelled acetaminophen ( N-acetyl- p-aminophenol, NAPAP-d 0; paracetamol; MW 151) and tetradeuterated acetaminophen (NAPAP-d 4; MW 155) in anhydrous acetonitrile and their GC–ECNICI-MS behavior using methane as the buffer gas. In addition to the expected anions [M−PFB] − at m/ z 150 from NAPAP-d 0 and m/ z 154 from NAPAP-d 4, we observed highly reproducibly almost equally intense anions at m/ z 149 and m/ z 153, respectively. Selected ion monitoring of these ions is suitable for specific and sensitive quantification of acetaminophen in human plasma and urine. Detailed investigations suggest in-source formation of N-acetyl- p-benzoquinone imine (NAPQI; MW 149), the putatively toxic acetaminophen metabolite, from the PFB ether derivative of NAPAP. GC–ECNICI-MS of non-derivatized NAPAP did not produce NAPQI. The peak area ratio of m/ z 149 to m/ z 150 and of m/ z 153 to m/ z 154 decreased with increasing ion-source temperature in the range 100–250 °C. Most likely, NAPQI formed in the ion-source captures secondary electrons to become negatively charged (i.e., [NAPQI] −) and thus detectable. Formation of NAPQI was not observed under electron ionization (EI) conditions, i.e., by GC–EI-MS, from derivatized and non-derivatized NAPAP. NAPQI was not detectable in flow injection analysis LC–MS of native NAPAP in positive electrospray ionization (ESI) mode, whereas in negative ESI mode low extent NAPQI formation was observed (<5%). Our results suggest that oxidation of drug derivatives in the ion-sources of mass spectrometers may form intermediates that are produced from activated drugs in enzyme-catalyzed reactions.