Simultaneous determination of doxorubicin and its dipeptide prodrug in mice plasma by HPLC with fluorescence detection

A simple and sensitive high performance liquid chromatography with fluorescence detection (HPLC–FD) has been developed for simultaneous quantification of doxorubicin (DOX) and its dipeptide conjugate prodrug (PDOX) in mice plasma. The chromatographic separation was carried out on an Amethyst C18–H column with gradient mobile phase of 0.1% formic acid and 0.1% formic acid in acetonitrile at a flow rate of 1.0 mL/min. The excitation and emission wavelengths were set at 490 and 550 nm, respectively. The method was comprehensively validated. The limits of detection were low up to 5.0 ng/mL for DOX and 25.0 ng/mL for PDOX. And the limits of quantification were low up to 12.5 ng/mL for DOX and 50 ng/mL for PDOX, which were lower than those for most of the current methods. The calibration curves showed good linearity (R2>0.999) over the concentration ranges. The extraction recoveries ranged from 84.0% to 88.2% for DOX and from 85.4% to 89.2% for PDOX. Satisfactory intra-day and inter-day precisions were achieved with RSDs less than 9.1%. The results show that the developed HPLC–FD method is accurate, reliable and will be helpful for preclinical pharmacokinetic study of DOX and PDOX.