Identification of biliary metabolites of ifosfamide using ³¹P magnetic resonance spectroscopy and mass spectrometry

Biliary excretion is a significant component in the metabolism of many drugs, but remains difficult to detect and characterise non-invasively. A previous publication recently described the detection of metabolites of ifosfamide in gall bladder in a guinea pig model using in vivo ¹H-decoupled ³¹P 3-D magnetic resonance spectroscopic imaging and a clinical 1.5 T MR scanner.. Here high-resolution ³¹P magnetic resonance spectroscopy (MRS) of extracted bile identifies peaks as parent ifosfamide (1.19±1.47 mM; mean ± sd), carboxyifosfamide (2.04±1.04 mM) and a major contribution from a previously unreported peak at 16.0 ppm (4.05±2.38 mM). The unknown resonance was identified using liquid chromatography - mass spectrometry (LCMS) as the glutathione conjugate of ifosfamide (MW = 531). This was confirmed by analysing products from the reaction of glutathione with ifosfamide using LCMS and MRS. These results demonstrate how combined in vivo and analytical MRS, together with mass spectrometry, can help identify visceral routes of drug metabolism, thereby aiding understanding of ±drug disposition and mechanisms of action and toxicity. In particular, the distribution of ifosfamide and its metabolites into bile may be related to oxazophosphorine-related cholecystitis reported in patients.