Isolation from pig liver microsomes, identification by tandem mass spectrometry and in vitro immunosuppressive activity of an SDZ-RAD 17,18,19,20,21,22-tris-epoxide

Lhoëst, Georges; Gougnard, Thierry; Maton, N.; Dehoux, J. P.; Schüler, W.

doi:10.1002/(sici)1096-9888(200003)35:3<454::aid-jms959>3.3.co;2-b

Cited by 5 publications

(8 citation statements)

References 7 publications

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“…Moreover, open-chain tautomeric forms involving the lactone function were identified for the tacrolimus epoxides, and these have already been reported for SDZ-RAD. 21 These effects are very similar to the ring-chain tautomers described for certain pentenoic acids. 22 In addition, we describe in this report the identification of an SDZ-RAD C 16 , C 27 O-demethyl 17, 18-19, 20-21, 22 trisepoxide new metabolite isolated from pig liver microsomes.…”

Section: Introductionsupporting

confidence: 67%

In vitro immunosuppressive activity of tacrolimus dihydrodiol precursors obtained by chemical oxidation and identification of a new metabolite of SDZ‐RAD by electrospray and electrospray‐linked scan mass spectrometry

Lhoëst¹,

Hertsens²,

Verbeeck³

et al. 2001

J. Mass Spectrom.

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Different tacrolimus epoxides and dihydrodiol epoxides arising from the chemical oxidation of the parent drug are described. Open-chain tautomeric forms involving the lactone function were identified for the tacrolimus epoxides. Moreover, the identification by electrospray and electrospray linked scan mass spectrometry of an SDZ-RAD C 16 -C 27 O-demethyl 17, 18-19, 20-21, 22 tris-epoxide new metabolite isolated from pig liver microsomes is reported. The in vitro immunosuppressive activity, using mixed lymphocyte reactions of the two macrolide reported oxidation compounds are discussed.

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Section: Introductionsupporting

confidence: 67%

In vitro immunosuppressive activity of tacrolimus dihydrodiol precursors obtained by chemical oxidation and identification of a new metabolite of SDZ‐RAD by electrospray and electrospray‐linked scan mass spectrometry

Lhoëst¹,

Hertsens²,

Verbeeck³

et al. 2001

J. Mass Spectrom.

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“…Because of the lack of detailed structural information of everolimus fragments, previous attempts to identify everolimus structures using LC/MS were not only of limited success,22 but also led to incorrect conclusions 15, 16. We would like to emphasize again that the H/D experiments are critical to prove the number of hydroxylations, and clearly excluded epoxy metabolites of sirolimus and everolimus,15, 16 which have been proposed based on nonvalidated hypothetical fragmentation patterns.…”

Section: Discussionmentioning

confidence: 90%

“…This is the only experiment that distinguished between an epoxide and a hydroxyl group as a result of two possible biotransformation reactions. Because epoxy metabolites of sirolimus and everolimus have been proposed,15, 16 this experiment was important for determining the correct structures of all metabolites.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2007

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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.

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“…However, a detailed analysis of the closely related compound SDZ-RAD (40-O-(2-hydroxyethyl) rapamycin) has been reported (16). However, a detailed analysis of the closely related compound SDZ-RAD (40-O-(2-hydroxyethyl) rapamycin) has been reported (16).…”

Section: Analysis Ofmentioning

confidence: 99%

High-Throughput Liquid Chromatography-Tandem Mass Spectrometric Analysis of Sirolimus in Whole Blood

Wallemacq

Vanbinst²,

Asta³

et al. 2003

Clinical Chemistry and Laboratory Medicine

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Sirolimus appears as a new potent immunosuppressive agent taking advantage of therapeutic drug monitoring to optimize its use in organ transplantation. In the absence of any available commercial immunoassay it was mandatory to develop chromatographic assays. Some methods have already been proposed to quantify sirolimus in whole blood, based either on HPLC-UV, liquid chromatography-mass spectrometry (LC-MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS). We have developed a new faster and simpler LC-MS/MS method to quantify sirolimus in blood using ascomycin as an internal standard and multiple reaction monitoring (MRM) acquisition mode. This method displays a limit of detection of 0.3 microg/l, and the intra-assay reproducibility ranges from 4.1-7.9%. The pre-analytical preparation steps are quite similar to those required for semi-automated immunoassays. Ascomycin and sirolimus present retention times of 0.89 and 0.93 min, respectively, and the turnaround time for a result (2.5 min) is also similar to that observed using a clinical analyzer. The comparison performed between HPLC-UV and LC-MS/MS displays good correlation (r = 0.949). The LC-MS/MS method described above has been used routinely for more than 2000 patient blood specimens and may present several advantages over existing methods, e.g., specificity with sufficient sensitivity, rapidity, and small blood sampling (10 microl), making it particularly adapted for routine clinical use.

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Isolation from pig liver microsomes, identification by tandem mass spectrometry and in vitro immunosuppressive activity of an SDZ-RAD 17,18,19,20,21,22-tris-epoxide

Cited by 5 publications

References 7 publications

In vitro immunosuppressive activity of tacrolimus dihydrodiol precursors obtained by chemical oxidation and identification of a new metabolite of SDZ‐RAD by electrospray and electrospray‐linked scan mass spectrometry

In vitro immunosuppressive activity of tacrolimus dihydrodiol precursors obtained by chemical oxidation and identification of a new metabolite of SDZ‐RAD by electrospray and electrospray‐linked scan mass spectrometry

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

High-Throughput Liquid Chromatography-Tandem Mass Spectrometric Analysis of Sirolimus in Whole Blood

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