Assessment and validation of the MS/MS fragmentation patterns of the macrolide immunosuppressant everolimus

Abstract

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.

Age-Dependent Changes in Sirolimus Metabolite Formation in Patients With Neurofibromatosis Type 1

“…15 The sirolimus metabolites were structurally identified based on detailed analysis of their MS/MS and MS n fragmentation patterns as previously described. 16 Key assay performance parameters for sirolimus were as follows: lower limit of quantitation, 0.1 ng/mL; range of linear response, 0.1-100 ng/mL (r . 0.99, n = 6); mean absolute recovery during extraction, 87.3%; total imprecision, 6.7%-9.8%; and interday accuracy, 103.7%-108.0%.…”

The Macrolide Everolimus Forms an Unusual Metabolite in Animals and Humans: Identification of a Phosphocholine Ester

“…The fragment ions of protonated ATG181 at m/z 532 and 829 (Fig. 2B) appeared to correspond to the Fragments a and C of everolimus at m/z 389 and 686 (Boernsen et al, 2007), differing from their precursor ions by 591 and 294 mass units, respectively. These two fragment ions and a few ions in the low mass region, all containing the trimethylammonium function, were the only ones observed in the product ion spectrum of protonated ATG181, in contrast to the product ion spectrum of sodiated everolimus, which showed fragment ions containing all parts of the molecule ( Fig.…”

The Specific Cleavage of Lactone Linkage to Open-Loop in Cyclic Lipopeptide during Negative ESI Tandem Mass Spectrometry: The Hydrogen Bond Interaction Effect of 4-Ethyl Guaiacol

“…Next the hydrogen from the methylene in the middle of aliphatic chain and carbonyl (colored in red), transfers to the oxygen of ester group via 1,3 hydrogen migration. Thereafter, the carbon (from aliphatic chain) - oxygen (from ester group) bond is broken, sequentially opening the loop and forming a carboxyl and ethylene at both ends respectively [24]–[28]. Finally, the compound, which loses water after ring opening, forms the [M-H-H 2 O] − ( m/z 1016).…”