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.