Preparation for HPLC

1 μL of daunorubicin was added to plasma and tumor samples (100 μL mouse blood and the entire tumor liquid volume) as a standard control, and 10 μL of Triton-X was added to rupture the liposomes and release the doxorubicin for chemical evaluation. Acetonitrile (600 μL; HPLC grade, Sigma) was then added and vials were vigorously rocked for 30 min. Samples were then centrifuged for 10 min at 7000 rpm, and the supernatant extracted and subsequently dried under flow of argon in a water bath at 37 °C. When the sample appeared dry, it was placed in a vacuum chamber for 3 h or overnight to ensure complete removal of liquid. The sample was then resuspended in 30% methanol (HPLC grade, Fisher), 70% water for HPLC analysis. These samples were run at 1 mL min⁻¹ using 70% methanol and 30% water with 0.1% formic acid through a C18 BDS Hypersil column. The output from these experiments yields doxorubicin concentration in blood and the tumor.