Abstract
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 7 S,9 S-dimethyl-ifosfamide (diMeIFO) and its two inactive metabolites, N 2- and N 3-deschloropropyl-dimethylifosfamide ( N 2-DCP-diMeIFO and N 3-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 N 2-DCP-diMeIFO, m/ z 213 → 92 for N 3-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, N 2-DCP-diMeIFO, N 3-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 N 2-DCP-diMeIFO and from −6.9 to 9.8% for N 3-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.
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