Background and ObjectivesTreosulfan is a prodrug applied in the treatment of ovarian cancer and conditioning prior to stem cell transplantation. So far, the bioanalysis of treosulfan in either whole blood or red blood cells (RBC) has not been carried out. In this work, the RBC/plasma partition coefficient (Ke/p) of treosulfan and its active monoepoxide was determined for the first time.MethodsMale and female 10-week-old Wistar rats (n = 6/6) received an intraperitoneal injection of treosulfan at the dose of 500 mg/kg body weight. The concentrations of treosulfan and its monoepoxide in plasma (Cp) and RBC were analyzed with a validated HPLC–MS/MS method.ResultsThe mean Ke/p of treosulfan and its monoepoxide were 0.74 and 0.60, respectively, corresponding to the blood/plasma partition coefficient of 0.88 and 0.82. The Spearman test demonstrated that the Ke/p of the prodrug correlated with its Cp, but no correlation between the Ke/p and Cp of the active monoepoxide was observed.ConclusionsTreosulfan and its monoepoxide achieve higher concentrations in plasma than in RBC; therefore, the choice of plasma for bioanalysis is rational as compared to whole blood. The distribution of treosulfan into RBC may be a saturable process at therapeutic concentrations.