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Assessment and validation of the MS/MS fragmentation patterns of the macrolide immunosuppressant everolimus
Author(s) -
Boernsen K. Olaf,
EggeJacobsen Wolfgang,
Inverardi Bruno,
Strom Tobin,
Streit Frank,
Schiebel HansMartin,
Benet Leslie Z.,
Christians Uwe
Publication year - 2007
Publication title -
journal of mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 1076-5174
DOI - 10.1002/jms.1215
Subject(s) - chemistry , fragmentation (computing) , everolimus , chromatography , medicine , computer science , operating system
Everolimus (40‐ O ‐(2‐hydroxyethyl)rapamycin, Certican) is a 31‐membered macrolide lactone. In lymphocytes, it inhibits the mammalian target of rapamycin (mTOR) and is used as an immunosuppressant after organ transplantation. Due to its instability in pure organic solvents and insufficient HPLC separation, NMR spectroscopy analysis of its metabolite structures is nearly impossible. Therefore, structural identification based on tandem mass spectrometry (MS/MS) and MS n fragmentation patterns is critical. Here, we have systematically assessed the fragmentation pattern of everolimus during liquid chromatography (LC)‐electrospray ionization (ESI)‐MS/MS and validated the fragment structures by (1) comparison with structurally identified derivatives (sirolimus), (2) high‐resolution mass spectrometry, (3) elucidation of fragmentation pathways using ion trap mass spectrometry (up to MS 5 ) and (4) H/D exchange. In comparison with the structurally related immunosuppressants tacrolimus and sirolimus, our study was complicated by the low ionization efficiency of everolimus. Detection of positive ions gave the best sensitivity, and everolimus and its fragments were mainly detected as sodium adducts. LC‐ESI‐MS/MS of everolimus in combination with collision‐induced dissociation (CID) resulted in a complex fragmentation pattern and the structures of 53 fragments were identified. These detailed fragmentation pathways of everolimus provided the basis for structural elucidation of all everolimus metabolites generated in vivo und in vitro . Copyright © 2007 John Wiley & Sons, Ltd.