Lassila Toni, Mattila Sampo, Turpeinen Miia, Pelkonen Olavi, Tolonen Ari
Department of Chemistry, University of Oulu, P.O. Box 3000, 90014, Oulu, Finland.
Research Unit of Biomedicine, Department of Pharmacology and Toxicology and Medical Research Center Oulu, P.O. Box 5000, 90014, University of Oulu, Finland.
Rapid Commun Mass Spectrom. 2016 Apr 15;30(7):917-26. doi: 10.1002/rcm.7518.
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.
(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.
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.
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. Copyright © 2016 John Wiley & Sons, Ltd.
薄荷呋喃是一种肝毒素，也是长叶薄荷酮的主要代谢产物，长叶薄荷酮是一种在许多薄荷品种精油中发现的单萜。它通过细胞色素P450（CYP）酶生物活化形成反应性代谢物，这些代谢物可能进一步与谷胱甘肽反应形成S-连接和N-连接的共轭物。尽管这些信息对于基于串联质谱（MS/MS）的涵盖这两种共轭物的综合筛选方法至关重要，但此前文献中尚未报道罕见的N-连接共轭物的MS/MS裂解途径以及与S-连接共轭物裂解的差异。
将（R）-（+）-长叶薄荷酮、（S）-（-）-长叶薄荷酮和薄荷呋喃与人肝S9组分一起孵育，以谷胱甘肽（GSH）作为捕获剂。用超高效液相色谱（UPLC）/轨道阱质谱搜索共轭物，并在负离子和正离子极性下测量其MS/MS光谱。还将薄荷呋喃与重组人CYP酶和GSH一起孵育，以阐明负责形成反应性代谢物的CYP。
检测到四种薄荷呋喃的GSH共轭物，并根据MS/MS光谱鉴定为S-连接和N-连接的共轭物。N-连接的共轭物缺乏S-连接共轭物的特征性碎片，通常产生保留部分谷氨酸的碎片。观察到CYP1A2、2B6和3A4比其他CYP同工型产生更多的GSH共轭物。
呋喃可形成反应性醛，其以席夫碱方式与GSH的游离谷氨酰胺反应形成N-连接的共轭物，其MS/MS光谱与S-连接的加合物不同。在建立基于LC/MS/MS的谷胱甘肽共轭物检测以筛选反应性代谢物时，至少对于具有呋喃部分的化合物，应考虑到这一点。建议在正离子模式下进行178.0412 Da和290.0573 Da的中性丢失扫描，或在负离子模式下进行256.0695 Da和290.0573 Da的中性丢失扫描以及m/z 143.0462的前体离子扫描。版权所有©2016 John Wiley & Sons, Ltd.
