Quantification of dimethyl-ifosfamide and its N-deschloropropylated metabolites in mouse plasma by liquid chromatography–tandem mass spectrometry

Among antitumor oxazaphosphorine drugs, the prodrug ifosfamide (IFO) and its analogs require metabolic activation by specific liver cytochrome P450 (CYP) enzymes to become therapeutically active. New 7,9-dimethyl-ifosfamide analogs have shown greater cytotoxic activity than IFO, whereas side-chain oxidation still occurred leading to monochloroacetone after N-dechloropropylation. A sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay was developed and validated for the simultaneous quantitation of the prodrug 7S,9S-dimethyl-ifosfamide (diMeIFO) and its two inactive metabolites, N2- and N3-deschloropropyl-dimethylifosfamide (N2-DCP-diMeIFO and N3-DCP-diMeIFO) in mouse plasma. After protein precipitation with methanol, the analytes were separated by isocratic reversed-phase chromatography with (methanol/ammonium formate pH 5.5, 60:40, v/v) and detected by tandem mass spectrometry using multiple reaction monitoring of transitions ions m/z 289 → 168 for diMeIFO, m/z 213 → 168 for N2-DCP-diMeIFO, m/z 213 → 92 for N3-DCP-diMeIFO and m/z 261 → 154 for IFO (internal standard). The calibration curves were linear over the concentration range of 20–10,000 ng/mL for the three analytes. Mean extraction recoveries from mouse plasma were 99, 96, 99 and 100% for diMeIFO, N2-DCP-diMeIFO, N3-DCP-diMeIFO and IFO, respectively. The lower limit of quantitation for diMeIFO and its metabolites was 20 ng/mL in 50 μL plasma. The method was accurate with calculated bias from −5.8 to 4.0% for diMeIFO, from −1.1 to 10.6% for N2-DCP-diMeIFO and from −6.9 to 9.8% for N3-DCP-diMeIFO, and precise with coefficients of variation lower than 6.8%, 7.8% and 14.3%, respectively. The assay was successfully applied to a preliminary pharmacokinetic study of diMeIFO and of its metabolites in mice.