Toni Lassila1,2, Sampo Mattila1, Miia Turpeinen2,3, Olavi Pelkonen2, Ari Tolonen4. 1. Department of Chemistry, University of Oulu, P.O. Box 3000, 90014, Oulu, Finland. 2. Research Unit of Biomedicine, Department of Pharmacology and Toxicology and Medical Research Center Oulu, P.O. Box 5000, 90014, University of Oulu, Finland. 3. Oulu University Hospital, P.O. Box 10, 90029, OYS, Finland. 4. Admescope Ltd, Typpitie 1, 90620, Oulu, Finland.
Abstract
RATIONALE: Menthofuran is a hepatotoxin and a major metabolite of pulegone, a monoterpene found in the essential oils of many mint species. It is bioactivated by cytochrome P450 (CYP) enzymes to reactive metabolites, which may further react with glutathione to form S-linked and N-linked conjugates. The tandem mass spectrometric (MS/MS) fragmentation pathways of rarely observed N-linked conjugates, and the differences to fragmentation of S-linked conjugates, have not been reported in the literature previously, although this information is essential to enable comprehensive MS/MS-based screening methods covering the both types of conjugates. METHODS: (R)-(+)-Pulegone, (S)-(-)-pulegone, and menthofuran were incubated with a human liver S9 fraction with glutathione (GSH) as the trapping agent. Conjugates were searched with ultra-performance liquid chromatography (UPLC)/orbitrap MS and their MS/MS spectra were measured both in the negative and positive ionization polarities. Menthofuran was also incubated with recombinant human CYP enzymes and GSH to elucidate the CYPs responsible for the formation of the reactive metabolites. RESULTS: Four GSH conjugates of menthofuran were detected and identified as S- and N-linked conjugates based on MS/MS spectra. N-linked conjugates lacked the characteristic fragments of S-linked conjugates and commonly produced fragments that retained parts of glutamic acid. CYP1A2, 2B6 and 3A4 were observed to produce more GSH conjugates than other CYP isoforms. CONLUSIONS: Furans can form reactive aldehydes that react in Schiff-base fashion with the free glutamyl-amine of GSH to form N-linked conjugates that have distinct MS/MS spectra from S-linked adducts. This should be taken into account when setting up LC/MS/MS-based detection of glutathione conjugates to screen for reactive metabolites, at least for compounds with a furan moiety. Neutral loss scanning of 178.0412 Da and 290.0573 Da in the positive ionization mode, or neutral loss scanning of 256.0695 Da and 290.0573 Da and precursor ion scanning of m/z 143.0462 in the negative ionization mode, is recommended.
RATIONALE: Menthofuran is a hepatotoxin and a major metabolite of pulegone, a monoterpene found in the essential oils of many mint species. It is bioactivated by cytochrome P450 (CYP) enzymes to reactive metabolites, which may further react with glutathione to form S-linked and N-linked conjugates. The tandem mass spectrometric (MS/MS) fragmentation pathways of rarely observed N-linked conjugates, and the differences to fragmentation of S-linked conjugates, have not been reported in the literature previously, although this information is essential to enable comprehensive MS/MS-based screening methods covering the both types of conjugates. METHODS: (R)-(+)-Pulegone, (S)-(-)-pulegone, and menthofuran were incubated with a human liver S9 fraction with glutathione (GSH) as the trapping agent. Conjugates were searched with ultra-performance liquid chromatography (UPLC)/orbitrap MS and their MS/MS spectra were measured both in the negative and positive ionization polarities. Menthofuran was also incubated with recombinant human CYP enzymes and GSH to elucidate the CYPs responsible for the formation of the reactive metabolites. RESULTS: Four GSH conjugates of menthofuran were detected and identified as S- and N-linked conjugates based on MS/MS spectra. N-linked conjugates lacked the characteristic fragments of S-linked conjugates and commonly produced fragments that retained parts of glutamic acid. CYP1A2, 2B6 and 3A4 were observed to produce more GSH conjugates than other CYP isoforms. CONLUSIONS: Furans can form reactive aldehydes that react in Schiff-base fashion with the free glutamyl-amine of GSH to form N-linked conjugates that have distinct MS/MS spectra from S-linked adducts. This should be taken into account when setting up LC/MS/MS-based detection of glutathione conjugates to screen for reactive metabolites, at least for compounds with a furan moiety. Neutral loss scanning of 178.0412 Da and 290.0573 Da in the positive ionization mode, or neutral loss scanning of 256.0695 Da and 290.0573 Da and precursor ion scanning of m/z 143.0462 in the negative ionization mode, is recommended.