Simultaneous quantification of preactivated ifosfamide derivatives and of 4-hydroxyifosfamide by high performance liquid chromatography–tandem mass spectrometry in mouse plasma and its application to a pharmacokinetic study

• A LC–MS/MS assay for quantification of ifosfamide conjugates and hydroxy metabolite.
• The hydroxyifosfamide is stabilized as semicarbazone derivative in plasma.
• The sensitivity for hydroxyifosfamide is improved five-fold compared to analogues.
• The assay is validated and applied to a comparative pharmacokinetic study in mice.
• The active metabolite is more produced by some of antitumor ifosfamide conjugates.

The antitumor drug, ifosfamide (IFO), requires activation by cytochrome P450 (CYP) to form the active metabolite, 4-hydroxyisfosfamide (4-OHIFO), leading to toxic by-products at high dose. In order to overcome these drawbacks, preactivated ifosfamide derivatives (RXIFO) were designed to release 4-OHIFO without CYP involvement. A high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was developed for the simultaneous quantification of 4-OHIFO, IFO and four derivatives RXIFO in mouse plasma using multiple reaction monitoring. Because of its instability in plasma, 4-OHIFO was immediately converted to the semi-carbazone derivative, 4-OHIFO-SCZ. For the six analytes, the calibration curves were linear from 20 to 5000 ng/mL in 50 μL plasma and the lower limit of quantitation was determined at 20 ng/mL with accuracies within ±10% of nominal and precisions less than 12%. Their recoveries ranged from 62 to 96% by using liquid–liquid extraction. With an improved assay sensitivity compared to analogues, the derivative 4-OHIFO-SCZ was stable in plasma at 4 °C for 24 h and at −20 °C for three months. For all compounds, the assay was validated with accuracies within ±13% and precisions less than 15%. This method was applied to a comparative pharmacokinetic study of 4-OHIFO from IFO and three derivatives RXIFO in mice. This active metabolite was produced by some of the novel conjugates with good pharmacokinetic properties.