Tandem mass spectrometric analysis of cyclophosphamide, ifosfamide and their metabolites

Abstract

A detailed multi-stage (MSn) fragmentation study of cyclophosphamide (CP), ifosfamide (IF) and their major metabolites, using an ion-trap mass spectrometer and a Q-TOF mass spectrometer, was performed with the aid of specifically deuterium-labeled analogs. The analytes showed good responses in positive-ion electrospray mass spectrometry as [MH]+ ions. Tandem mass spectra revealed a wealth of structurally specific ions, allowing characterization of the fragmentation pathways of these analytes. The major fragmentation pathways of the protonated CP and IF are elimination of ethylene from C5 and C6 of 1,3,2-oxazaphosphorine-2-oxide via a McLafferty rearrangement, and cleavage of the P-N bond. However, their activated 4-OOH and 4-OH metabolites primarily underwent hydrogen peroxide elimination and dehydration, respectively, followed by fragmentation pathways similar to those of CP and IF. These results should prove useful in structural elucidation of future analogs of CP and IF, and/or of their metabolites.

Simultaneous determination of cyclophosphamide, ifosfamide, doxorubicin, epirubicin and daunorubicin in human urine using high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry: bioanalytical method validation

“…The structures of the fragment ions reported in Figs. 4 and 5 are based on the multi‐stage fragmentation studies 37. The transition from m/z 261 to 92 was used in our MRM analysis.…”

“…4 and 5, respectively. Based on the results proposed by other authors,37 the major fragmentation pathways of the protonated CP and IF are elimination of ethylene from C5 and C6 of 1,3,2‐oxazaphosphorine‐2‐oxide via a McLafferty rearrangement, and cleavage of the P–N bond. As regards protonated CP, elimination of ethylene yielded the fragment ion at m/z 233 and cleavage of the P–N bond gave the fragment ion at m/z 140 (Fig.…”

New Ifosfamide Analogs Designed for Lower Associated Neurotoxicity and Nephrotoxicity with Modified Alkylating Kinetics Leading to Enhanced in Vitro Anticancer Activity

“…2A) were performed to identify IFO metabolites by means of their mass spectra and to evaluate the metabolism yield of the prodrug into N-deschloroethylated metabolites (toxic pathway) and into 4-hydroxylated metabolite (active pathway). ϩ -CHϭCH-Cl (m/z 92) with a difference of 14 mass units, corresponding to a methyl group, confirming fragments ions described previously for IFO, whose interpretation was facilitated by various deuterated IFO analogs (Liu et al, 2005). …”

Post-column hydrogen–deuterium exchange technique to assist in the identification of small organic molecules by mass spectrometry

“…High deuteration levels are obtained in the online technique in which a deuterium-96 enriched mobile phase is used to induce HDX during the liquid chromatography separation 97 D r a f t process. Online HDX has been successfully applied for the identification of metabolites, 98 pharmaceutical compounds as well as other small molecules 2,8 . As an example, online HDX was 99 shown helpful in reducing the number of potential isomers during the characterization of 100 sulfadiazine metabolites present in pig manure 10 .…”